Nonalcoholic Fatty Liver Disease and Its Associated Parameters

**29**

**Chapter 3**

**Abstract**

*Ian James Martins*

relevance to NAFLD and diabetes management.

mitophagy, curcumin, cinnamon

**1. Introduction**

(NAFLD) epidemic.

Insulin Therapy and Autoimmune

The diabetes epidemic is now expected by the year 2050 to become a global pandemic with approx. 592 million affected in both the developed and developing world. The treatment of diabetes by insulin therapy has been the focus for many diabetics with the improvement and prevention of various diseases such as cardiovascular disease, kidney disease and neurodegeneration. The global nonalcoholic fatty liver disease (NAFLD) epidemic has now become of major concern to diabetes with critical interest in insulin therapy to reverse and stabilize autoimmune disease with relevance to NAFLD and the diabetes pandemic. Dietary components that activate anti-aging genes improve insulin therapy and should be assessed with specific amounts and doses of Indian spices consumed that may not interfere with insulin therapy and induce mitophagy in various diseases. Food quality, appetite control and core body temperature are critical to maintain insulin therapy with unhealthy diets linked to NAFLD and diabetes. Genomic medicine and dietary activators are essential to maintain insulin therapy and prevent toxic immune reactions with

**Keywords:** insulin therapy, genomic, autoimmune disease, diabetes, global,

The diabetes epidemic is expected to affect approx. 592 people by the year 2035. The urgency to prevent the largest diabetes epidemic in history has now assessed multiple risk factors involved with induction of Type 3 diabetes connected to various chronic diseases. Insulin resistance and brain aging now indicate neuron vulnerability to mitophagy associated with the diabetes pandemic expected in 2050 [1, 2]. Diabetes and its connections autoimmunity [3] have become important to mitophagy, metabolic disease with relevance to the nonalcoholic fatty liver disease

An association between various genes and the immune system [4, 5] has been proposed to be involved with the regulation of life-span in various species. Immune gene activation has been associated with brain aging [6] with the critical involvement of inflammation in the development of neuro-degeneration. Autoimmune disease, drugs and immunosenescence are related to the chronic disease epidemic with uncontrolled release of inflammatory cytokines such as tumor necrosis factor

Disease with Relevance to Non

Alchoholic Fatty Liver Disease

#### **Chapter 3**

## Insulin Therapy and Autoimmune Disease with Relevance to Non Alchoholic Fatty Liver Disease

*Ian James Martins*

#### **Abstract**

The diabetes epidemic is now expected by the year 2050 to become a global pandemic with approx. 592 million affected in both the developed and developing world. The treatment of diabetes by insulin therapy has been the focus for many diabetics with the improvement and prevention of various diseases such as cardiovascular disease, kidney disease and neurodegeneration. The global nonalcoholic fatty liver disease (NAFLD) epidemic has now become of major concern to diabetes with critical interest in insulin therapy to reverse and stabilize autoimmune disease with relevance to NAFLD and the diabetes pandemic. Dietary components that activate anti-aging genes improve insulin therapy and should be assessed with specific amounts and doses of Indian spices consumed that may not interfere with insulin therapy and induce mitophagy in various diseases. Food quality, appetite control and core body temperature are critical to maintain insulin therapy with unhealthy diets linked to NAFLD and diabetes. Genomic medicine and dietary activators are essential to maintain insulin therapy and prevent toxic immune reactions with relevance to NAFLD and diabetes management.

**Keywords:** insulin therapy, genomic, autoimmune disease, diabetes, global, mitophagy, curcumin, cinnamon

#### **1. Introduction**

The diabetes epidemic is expected to affect approx. 592 people by the year 2035. The urgency to prevent the largest diabetes epidemic in history has now assessed multiple risk factors involved with induction of Type 3 diabetes connected to various chronic diseases. Insulin resistance and brain aging now indicate neuron vulnerability to mitophagy associated with the diabetes pandemic expected in 2050 [1, 2]. Diabetes and its connections autoimmunity [3] have become important to mitophagy, metabolic disease with relevance to the nonalcoholic fatty liver disease (NAFLD) epidemic.

An association between various genes and the immune system [4, 5] has been proposed to be involved with the regulation of life-span in various species. Immune gene activation has been associated with brain aging [6] with the critical involvement of inflammation in the development of neuro-degeneration. Autoimmune disease, drugs and immunosenescence are related to the chronic disease epidemic with uncontrolled release of inflammatory cytokines such as tumor necrosis factor

α and interleukin-6 [7, 8]. Major interests to determine human longevity require the assessment of nutrition and diet with relevance to the control of inflammatory cytokines that are associated with age-related changes in the immune system and the induction of diabetes, NAFLD and neurodegeneration.

Appetite control with relevance to immuno-metabolism has become critical to the treatment of NAFLD. The major defect in global chronic disease is autoimmune disease with defective adipose tissue and liver interaction involved with the release of inflammatory cytokines and adipo-cytokines relevant to toxic immune reactions that involve the pancreas, brain, heart, thyroid, kidneys and reproductive organs. Appetite control and autoimmune disease are connected to anti-aging genes with relevance to irreversible programmed cell death in various cells and tissues. Immune competence changes over a human's life span with a process known as immunosenescence [9, 10]. In man multiple theories of aging have been proposed with the immune theory of aging that involve abnormal inflammatory responses that contribute to the induction of chronic diseases [11].

In various communities in the developing and developed world the understanding of the ingestion of a healthy diet and hepatic fat metabolism has become of critical importance to the treatment diabetes that is now linked to various organ diseases. In the world [12] transition to healthy diets has become urgent to prevent insulin resistance, autoimmune disease and NAFLD. The liver is the major organ for the metabolism of dietary fat and after consumption of a meal in healthy individuals the fat is rapidly metabolized by the mitochondria in the liver.

A diet rich in fat and sugar that lead to fat deposition in the liver can be referred to as liver steatosis. The defect in the liver fatty acid metabolism is possibly related to mitochondrial dysfunction and a careful calorie controlled diet may reverse liver steatosis. As mitochondrial apoptosis occurs steatohepatitis may be associated with liver inflammation. Steatohepatitis may induce NAFLD that may then progress to severe inflammation and liver cirrhosis. In obesity and diabetes the metabolism of a fat meal by the liver is defective with associated hyperglycemia and hyperinsulinemia. Food restriction [13] and appetite control are vital to the treatment of NAFLD with hepatic fat metabolism connected to insulin resistance, autoimmune disease and mitophagy [14].

#### **2. Diabetes and pathogenetic loop complications**

Insulin treatment in diabetes has provided information that approx. 30% of patients are involved with insulin treatment or plan to start insulin with insulin regimens [15] associated with various insulin doses and failure of oral anti-diabetic medications. Type 2 diabetes mellitus is characterized by hyperglycemia, insulin resistance, and impairment of insulin secretion [16]. The impairment of insulin secretion is related to hyperglycemia, high serum low-density lipoprotein cholesterol concentrations and low serum high-density lipoprotein cholesterol concentrations with relevance to cardiovascular disease [17]. The relative importance of impaired insulin release and insulin resistance in the pathogenesis of Type 2 diabetes has been evaluated and may be connected to NAFLD. NAFLD may be connected to autoimmune disease and mitophagy associated with impairment in insulin secretion and cardiovascular disease [18–20]. In Type 1 diabetes the use of insulin therapy has been assessed with the critical importance to reduce hyperglycemia, severe hypoglycemia and the development of long-term complications [21–23]. Insulin therapy should be carefully evaluated in Type 1 and Type 2 diabetes with relevance to reduction in plasma glucose levels [24]. Interference in hepatic glucose production [24, 25] or interference with increased glucose uptake by the liver may be sensitive to repression

**31**

**death**

**Figure 1.**

*species and man.*

*Insulin Therapy and Autoimmune Disease with Relevance to Non Alchoholic Fatty Liver Disease*

of glucose related genes associated with the induction of glucolipotoxicity, NAFLD and insulin resistance. Exercise and insulin therapy [26] may reduce glucolipotoxicity and NAFLD but with the aging process the pathogenetic loop [27–32] that involve hyperglycemia, hypercholesterolemia and hyperinsulinemia may be associated with autoimmune disease, mitophagy and programmed cell death of various cells and tissues [18–20]. The role of diet, lifestyle, stress, sleep and circadian disorders [33] may inactivate the anti-aging gene Sirtuin 1 (Sirt 1) with relevance to insulin therapy and induction of NAFLD associated with the pathogenetic loop (**Figure 1**) and

*Diabetes and the pathogenetic loop associated with inflammation, age related diseases and neurodegeneration involve inactivation of the anti-aging gene Sirtuin 1 (Sirt 1) associated with mitochondrial apoptosis in various* 

**3. Anti-aging genes, mitochondrial apoptosis and programmed cell** 

Insulin resistance is involved early in alterations of nuclear, subcellular and cell membrane function that lead to cell transformation without reversible changes with accelerated cell apoptosis [34]. In 2050 the predicted global diabetes pandemic [1, 2] has accelerated scientific research to determine the identification of novel genomic pathways such as the anti-aging gene Sirt 1 that may provide new knowledge with relevance to accelerated cell apoptosis and inactivated insulin therapy. In Type 2 diabetes and Type 1 diabetes various genes and genetic loci have been

uncontrolled inflammation of cells and tissues [18–20].

*DOI: http://dx.doi.org/10.5772/intechopen.81297*

*Insulin Therapy and Autoimmune Disease with Relevance to Non Alchoholic Fatty Liver Disease DOI: http://dx.doi.org/10.5772/intechopen.81297*

#### **Figure 1.**

*Nonalcoholic Fatty Liver Disease - An Update*

α and interleukin-6 [7, 8]. Major interests to determine human longevity require the assessment of nutrition and diet with relevance to the control of inflammatory cytokines that are associated with age-related changes in the immune system and

Appetite control with relevance to immuno-metabolism has become critical to the treatment of NAFLD. The major defect in global chronic disease is autoimmune disease with defective adipose tissue and liver interaction involved with the release of inflammatory cytokines and adipo-cytokines relevant to toxic immune reactions that involve the pancreas, brain, heart, thyroid, kidneys and reproductive organs. Appetite control and autoimmune disease are connected to anti-aging genes with relevance to irreversible programmed cell death in various cells and tissues. Immune competence changes over a human's life span with a process known as immunosenescence [9, 10]. In man multiple theories of aging have been proposed with the immune theory of aging that involve abnormal inflammatory responses

In various communities in the developing and developed world the understanding of the ingestion of a healthy diet and hepatic fat metabolism has become of critical importance to the treatment diabetes that is now linked to various organ diseases. In the world [12] transition to healthy diets has become urgent to prevent insulin resistance, autoimmune disease and NAFLD. The liver is the major organ for the metabolism of dietary fat and after consumption of a meal in healthy individu-

A diet rich in fat and sugar that lead to fat deposition in the liver can be referred to as liver steatosis. The defect in the liver fatty acid metabolism is possibly related to mitochondrial dysfunction and a careful calorie controlled diet may reverse liver steatosis. As mitochondrial apoptosis occurs steatohepatitis may be associated with liver inflammation. Steatohepatitis may induce NAFLD that may then progress to severe inflammation and liver cirrhosis. In obesity and diabetes the metabolism of a fat meal by the liver is defective with associated hyperglycemia and hyperinsulinemia. Food restriction [13] and appetite control are vital to the treatment of NAFLD with hepatic fat metabolism connected to insulin resistance, autoimmune

Insulin treatment in diabetes has provided information that approx. 30% of patients are involved with insulin treatment or plan to start insulin with insulin regimens [15] associated with various insulin doses and failure of oral anti-diabetic medications. Type 2 diabetes mellitus is characterized by hyperglycemia, insulin resistance, and impairment of insulin secretion [16]. The impairment of insulin secretion is related to hyperglycemia, high serum low-density lipoprotein cholesterol concentrations and low serum high-density lipoprotein cholesterol concentrations with relevance to cardiovascular disease [17]. The relative importance of impaired insulin release and insulin resistance in the pathogenesis of Type 2 diabetes has been evaluated and may be connected to NAFLD. NAFLD may be connected to autoimmune disease and mitophagy associated with impairment in insulin secretion and cardiovascular disease [18–20]. In Type 1 diabetes the use of insulin therapy has been assessed with the critical importance to reduce hyperglycemia, severe hypoglycemia and the development of long-term complications [21–23]. Insulin therapy should be carefully evaluated in Type 1 and Type 2 diabetes with relevance to reduction in plasma glucose levels [24]. Interference in hepatic glucose production [24, 25] or interference with increased glucose uptake by the liver may be sensitive to repression

the induction of diabetes, NAFLD and neurodegeneration.

that contribute to the induction of chronic diseases [11].

**2. Diabetes and pathogenetic loop complications**

disease and mitophagy [14].

als the fat is rapidly metabolized by the mitochondria in the liver.

**30**

*Diabetes and the pathogenetic loop associated with inflammation, age related diseases and neurodegeneration involve inactivation of the anti-aging gene Sirtuin 1 (Sirt 1) associated with mitochondrial apoptosis in various species and man.*

of glucose related genes associated with the induction of glucolipotoxicity, NAFLD and insulin resistance. Exercise and insulin therapy [26] may reduce glucolipotoxicity and NAFLD but with the aging process the pathogenetic loop [27–32] that involve hyperglycemia, hypercholesterolemia and hyperinsulinemia may be associated with autoimmune disease, mitophagy and programmed cell death of various cells and tissues [18–20]. The role of diet, lifestyle, stress, sleep and circadian disorders [33] may inactivate the anti-aging gene Sirtuin 1 (Sirt 1) with relevance to insulin therapy and induction of NAFLD associated with the pathogenetic loop (**Figure 1**) and uncontrolled inflammation of cells and tissues [18–20].

#### **3. Anti-aging genes, mitochondrial apoptosis and programmed cell death**

Insulin resistance is involved early in alterations of nuclear, subcellular and cell membrane function that lead to cell transformation without reversible changes with accelerated cell apoptosis [34]. In 2050 the predicted global diabetes pandemic [1, 2] has accelerated scientific research to determine the identification of novel genomic pathways such as the anti-aging gene Sirt 1 that may provide new knowledge with relevance to accelerated cell apoptosis and inactivated insulin therapy. In Type 2 diabetes and Type 1 diabetes various genes and genetic loci have been

reported to be involved in the development of diabetes [35]. Novel genes [36] have been identified that are involved with autoimmune disease [18, 19, 36, 37] and glucolipotoxicity with irreversible immune complications relevant to NAFLD, diabetes [3] and the pathogenetic loop. The discovery of the anti-aging gene Sirt 1 now has become important to the treatment of diabetes with insulin therapy in Type 1 and Type 2 diabetes connected to Sirt 1 activation in the pancreas with relevance to insulin release [38] with Sirt 1 associated with mitochondrial biogenesis (**Figure 1**) and cell survival in various tissues [38, 39]. The inactivation of Sirt 1 [39] in humans leads to the pathogenetic loop in diabetes and implicates nutritional and environmental factors in the induction of programmed cell death.

Sirt 1 is a nicotinamide adenine dinucleotide (NAD+) dependent class III histone deacetylase (HDAC) that targets transcription factors such as p 53 to adapt gene expression to metabolic activity and the deacetylation of nuclear receptors indicate its critical involvement in insulin resistance and autoimmune disease [18]. In situ hybridization analysis has localized the human Sirt 1 gene to chromosome 10q21.3 [18]. Calorie restriction is essential for Sirt 1 transcriptional regulation with other factors such as diet and lifestyle critical for the prevention of insulin resistance and NAFLD. Sirt 1 is an acute phase protein involved with neuron proliferation [18] and its regulation of the suprachiasmatic nucleus is involved with control of the circadian rhythm [18]. The circadian rhythm and immune system are closely connected to the immune response. Nutritional interventions that are controlled by the consumption of a low calorie diet indicate the maintenance of connections between Sirt 1 and other anti-aging genes such as Klotho, p66shc (longevity protein) and FOXO1/FOXO3a that are connected to programmed cell death [36]. Sirt 1 and transcriptional regulation of anti-aging genes are critical to mitophagy (**Figure 1**) and neurodegenerative disease with accelerated brain aging connected to NAFLD and diabetes [19, 36].

#### **4. Insulin therapy and Indian spices with relevance to NAFLD and diabetes**

The connections between NAFLD and diabetes have become of central importance to the expected diabetes pandemic by the year 2050 [1, 2]. NAFLD in diabetic individuals may completely inactivate insulin therapy with defective insulin dose regimens and failure of oral anti-diabetic medications. The defect in the liver fatty acid metabolism is possibly related to mitochondrial dysfunction associated with severe liver inflammation and steatohepatitis that may induce NAFLD that may then progress to severe inflammation (NASH) and liver cirrhosis. Insulin therapy has been used to improve liver function but with NAFLD, high dose insulin therapy may be unsuccessful with liver inflammation [40–42] associated with uncontrolled hyperglycemia and mitochondrial apoptosis (**Figure 2**). Insulin therapy with insulin dose and oral anti-diabetic medications should be re-evaluated to improve hepatocyte mitochondrial biogenesis with relevance to reversal of liver disease connected to hyperglycemia and NAFLD in various Type 1, Type 2 and Type 3 [35, 39] diabetics.

The connections between Sirt 1 and insulin resistance have accelerated in recent years with Sirt 1 as a calorie sensitive gene is now implicated in insulin resistance and to the important to glucose dependent insulin secretion with protection of pancreatic β-cell mass [43–46]. Sirt 1 may be involved in silencing insulin resistance by regulation of specific proteins involved in insulin action [47]. Anti-inflammatory actions in adipocytes involve Sirt 1 repression and inflammation [48, 49] associated with the adipose-liver defect [49, 50] and the induction of NAFLD. Sirt 1

**33**

**Figure 2.**

*apoptosis in the brain and the periphery.*

*Insulin Therapy and Autoimmune Disease with Relevance to Non Alchoholic Fatty Liver Disease*

dysfunction in the brain leads to systemic insulin resistance [51] with close links to Type 3 diabetes and NAFLD [52, 53]. In Sirt 1 knockout mice increased adipose tissue mass has been connected to NAFLD [33]. The expression of Sirt 1 protein has a molecular weight (Mol Wt) of 81 kda with Mol Wt variation (81–110 kda). Insulin therapy to prevent NAFLD requires insulin dose/antidiabetic medication calculation to release the Sirt 1 acute phase protein [18, 37]. Sirt 1 is essential to prevent inflammation and Sirt 1 inactivation may induce NAFLD that may corrupt pancreas function. Insulin therapy and plasma Sirt 1 levels may allow mitochondrial biogenesis to be assessed with relevance to therapeutic glucose control in Type 1, 2 and 3 diabetics. It is unclear if inactivation of insulin therapy is associated with mitophagy and the induction of NAFLD and various organ diseases [52]. Appetite control [13, 18] is now critical to the maintenance of mitochondrial biogenesis and insulin therapy with overeating [13] connected to inactivation of insulin therapy

*Indian spices have become important as a diabetes technology and its use in diabetes has become of concern. Indian spices such as curcumin and cinnamon associated with glucose control in diabetics but excessive curcumin or piperine may inactivate insulin therapy associated with hyperglycemic induced mitochondrial* 

Indian spices have become important as a diabetes technology [54] with Indian spices such as curcumin and cinnamon associated with glucose control in diabetics (**Figure 2**). The event of insulin therapy as the primary therapy in diabetes technology has raised concern with relevance to the consumption of Indian spices as a secondary technology [55]. Indian spices consumed over many years are not cleared from the body and may bind to cells and receptors with excess Indian spices that may associate with insulin receptors related to altered insulin actions and inactivated insulin therapy. In normal individuals consumption of cinnamon and curcumin may inactivate the biological activity of insulin [54, 55] with Indian spices as the secondary treatment for glucose control in the brain and the periphery. Drugs such as anti-obese drugs [56] and novel drugs [57] are now of critical importance to NAFLD and insulin therapy. Insulin therapy and the use of various therapeutic drugs in diabetes have been linked to the treatment of organ dysfunction [35, 57–59] in diabetes. The use of Indian spices should be reassessed in various populations to

and linked to the severity of the diabetic condition.

*DOI: http://dx.doi.org/10.5772/intechopen.81297*

*Insulin Therapy and Autoimmune Disease with Relevance to Non Alchoholic Fatty Liver Disease DOI: http://dx.doi.org/10.5772/intechopen.81297*

#### **Figure 2.**

*Nonalcoholic Fatty Liver Disease - An Update*

and diabetes [19, 36].

**diabetes**

mental factors in the induction of programmed cell death.

reported to be involved in the development of diabetes [35]. Novel genes [36] have been identified that are involved with autoimmune disease [18, 19, 36, 37] and glucolipotoxicity with irreversible immune complications relevant to NAFLD, diabetes [3] and the pathogenetic loop. The discovery of the anti-aging gene Sirt 1 now has become important to the treatment of diabetes with insulin therapy in Type 1 and Type 2 diabetes connected to Sirt 1 activation in the pancreas with relevance to insulin release [38] with Sirt 1 associated with mitochondrial biogenesis (**Figure 1**) and cell survival in various tissues [38, 39]. The inactivation of Sirt 1 [39] in humans leads to the pathogenetic loop in diabetes and implicates nutritional and environ-

Sirt 1 is a nicotinamide adenine dinucleotide (NAD+) dependent class III histone

deacetylase (HDAC) that targets transcription factors such as p 53 to adapt gene expression to metabolic activity and the deacetylation of nuclear receptors indicate its critical involvement in insulin resistance and autoimmune disease [18]. In situ hybridization analysis has localized the human Sirt 1 gene to chromosome 10q21.3 [18]. Calorie restriction is essential for Sirt 1 transcriptional regulation with other factors such as diet and lifestyle critical for the prevention of insulin resistance and NAFLD. Sirt 1 is an acute phase protein involved with neuron proliferation [18] and its regulation of the suprachiasmatic nucleus is involved with control of the circadian rhythm [18]. The circadian rhythm and immune system are closely connected to the immune response. Nutritional interventions that are controlled by the consumption of a low calorie diet indicate the maintenance of connections between Sirt 1 and other anti-aging genes such as Klotho, p66shc (longevity protein) and FOXO1/FOXO3a that are connected to programmed cell death [36]. Sirt 1 and transcriptional regulation of anti-aging genes are critical to mitophagy (**Figure 1**) and neurodegenerative disease with accelerated brain aging connected to NAFLD

**4. Insulin therapy and Indian spices with relevance to NAFLD and** 

The connections between NAFLD and diabetes have become of central importance to the expected diabetes pandemic by the year 2050 [1, 2]. NAFLD in diabetic individuals may completely inactivate insulin therapy with defective insulin dose regimens and failure of oral anti-diabetic medications. The defect in the liver fatty acid metabolism is possibly related to mitochondrial dysfunction associated with severe liver inflammation and steatohepatitis that may induce NAFLD that may then progress to severe inflammation (NASH) and liver cirrhosis. Insulin therapy has been used to improve liver function but with NAFLD, high dose insulin therapy may be unsuccessful with liver inflammation [40–42] associated with uncontrolled hyperglycemia and mitochondrial apoptosis (**Figure 2**). Insulin therapy with insulin dose and oral anti-diabetic medications should be re-evaluated to improve hepatocyte mitochondrial biogenesis with relevance to reversal of liver disease connected to hyperglycemia and NAFLD in various Type 1, Type 2 and Type 3 [35, 39] diabetics. The connections between Sirt 1 and insulin resistance have accelerated in recent years with Sirt 1 as a calorie sensitive gene is now implicated in insulin resistance and to the important to glucose dependent insulin secretion with protection of pancreatic β-cell mass [43–46]. Sirt 1 may be involved in silencing insulin resistance by regulation of specific proteins involved in insulin action [47]. Anti-inflammatory actions in adipocytes involve Sirt 1 repression and inflammation [48, 49] associated with the adipose-liver defect [49, 50] and the induction of NAFLD. Sirt 1

**32**

*Indian spices have become important as a diabetes technology and its use in diabetes has become of concern. Indian spices such as curcumin and cinnamon associated with glucose control in diabetics but excessive curcumin or piperine may inactivate insulin therapy associated with hyperglycemic induced mitochondrial apoptosis in the brain and the periphery.*

dysfunction in the brain leads to systemic insulin resistance [51] with close links to Type 3 diabetes and NAFLD [52, 53]. In Sirt 1 knockout mice increased adipose tissue mass has been connected to NAFLD [33]. The expression of Sirt 1 protein has a molecular weight (Mol Wt) of 81 kda with Mol Wt variation (81–110 kda). Insulin therapy to prevent NAFLD requires insulin dose/antidiabetic medication calculation to release the Sirt 1 acute phase protein [18, 37]. Sirt 1 is essential to prevent inflammation and Sirt 1 inactivation may induce NAFLD that may corrupt pancreas function. Insulin therapy and plasma Sirt 1 levels may allow mitochondrial biogenesis to be assessed with relevance to therapeutic glucose control in Type 1, 2 and 3 diabetics. It is unclear if inactivation of insulin therapy is associated with mitophagy and the induction of NAFLD and various organ diseases [52]. Appetite control [13, 18] is now critical to the maintenance of mitochondrial biogenesis and insulin therapy with overeating [13] connected to inactivation of insulin therapy and linked to the severity of the diabetic condition.

Indian spices have become important as a diabetes technology [54] with Indian spices such as curcumin and cinnamon associated with glucose control in diabetics (**Figure 2**). The event of insulin therapy as the primary therapy in diabetes technology has raised concern with relevance to the consumption of Indian spices as a secondary technology [55]. Indian spices consumed over many years are not cleared from the body and may bind to cells and receptors with excess Indian spices that may associate with insulin receptors related to altered insulin actions and inactivated insulin therapy. In normal individuals consumption of cinnamon and curcumin may inactivate the biological activity of insulin [54, 55] with Indian spices as the secondary treatment for glucose control in the brain and the periphery. Drugs such as anti-obese drugs [56] and novel drugs [57] are now of critical importance to NAFLD and insulin therapy. Insulin therapy and the use of various therapeutic drugs in diabetes have been linked to the treatment of organ dysfunction [35, 57–59] in diabetes. The use of Indian spices should be reassessed in various populations to

prevent interference with drug/insulin therapy (**Figure 2**) or with caffeine effects [60] relevant to the treatment of NAFLD and diabetes. The mixing of spices such as curcumin, turmeric and black pepper in coffee should be discouraged and may contribute to the transcriptional dysregulation of Sirt 1 and induction of mitochondrial apoptosis relevant to diabetes and the pathogenetic loop [27–32].

#### **5. Genomic medicine and Sirt 1 activators reverse immune reactions in global chronic disease**

Genomic medicine in the treatment of cardiovascular disease and diabetes [19, 37] has now accelerated in various communities. Peripheral nutrition is essential early to prevent neurodegeneration (Type 3 diabetes) that lead to uncontrolled peripheral glucose homeostasis. Type 3 diabetes is associated with suprachiasmatic nucleus defects with the abnormal maintenance of brain and whole body glucose metabolism in various species and man [20]. Nutritional therapy in diabetics now need to involve the use of Sirt 1 activators [61] to prevent the effects of various Sirt 1 inhibitors that accumulate in the blood plasma that repress Sirt 1 expression in cells and tissues. A dose of 4 g/day of phosphatidylinositol [62] is essential with insulin therapy to prevent hyperglycemia, NAFLD and other neurodegenerative diseases. Sirt 1 inhibitors such as excess palmitic acid (cream, cheese), alcohol and drugs (suramin and sirtinol) should be carefully controlled to prevent inactivation of insulin therapy. Sirt 1 activators such as pyruvic acid, leucine and magnesium are critical with relevance to insulin therapy. Diabetic individuals with Indian spice consumption (**Figure 3**) over years need to be carefully evaluated with relevance to plasma Sirt 1 inhibitors, xenobiotics [63], caffeine content [60], drug therapy, bacterial lipopolysaccharides (LPS) and mycotoxins [62] that may interfere with insulin/oral medication therapy. The importance of genomic medicine may indicate that the immune system may malfunction [37] early with relevance to poor nutrition of food quality with irreversible organ disease manifestations. Biotherapy and the immune system [37, 61] may be critical to insulin therapy and connected to insulin resistance and NAFLD. Appetite control and essential food components [64] may be essential to maintain the immune system with autoimmune disease

#### **Figure 3.**

*Poor food quality and core body temperature defects will inactivate Sirt 1 and induce insulin resistance and NAFLD. Sirt 1 inhibitors such as xenobiotics, caffeine/Indian spice over-consumption and magnesium deficiency may lead to the diabetes pandemic with high doses of phosphatidylinositol essential to maintain insulin therapy and prevent the induction of NAFLD.*

**35**

immune reactions [80].

*Insulin Therapy and Autoimmune Disease with Relevance to Non Alchoholic Fatty Liver Disease*

associated with appetite dysregulation and poor food quality. Specific mitochondrial nutrients [65] with insulin therapy need to be consumed to prevent severe

Food quality with relevance to stroke, synaptic plasticity and neurological diseases has become important to diabetic individuals with essential maintenance and prevention of brain diseases by insulin therapy. Unhealthy diets that contain LPS, mycotoxins and xenobiotics can induce NAFLD with inactivation of insulin therapy. In the developing world increased plasma LPS levels (**Figure 3**) have raised concern with relevance to induction of metabolic and neurodegenerative diseases [66, 67]. Antibiotic resistance with relevance to antimicrobial drug use should be carefully controlled to prevent excessive release of LPS from the debris of gram negative bacteria [68]. Food preparation should be carefully assessed to prevent end

products such as LPS and patulin that may persist in contaminated food

kidney disease and neurodegeneration in diabetes.

[63, 69]. LPS and patulin may inactivate Sirt 1 [62] with relevance to insulin resistance and NAFLD. Xenobiotics [63] in air, food and water may inactivate insulin therapy (**Figure 3**) with increased xenobiotic levels associated with mitochondrial

Core body temperature (**Figure 3**) and insulin therapy are closely connected and dysregulation of core body temperature may induce NAFLD. The discovery of the heat shock gene Sirt 1 [70] has indicated that careful body temperature control is critical to prevent autoimmune disease and mitochondrial apoptosis. Sirt 1 and its inactivation are associated with increased heat shock protein 70 with relevance to natural killer cell activation and mitochondrial apoptosis. Nutritional therapy and core body temperature are essential to maintain insulin therapy in diabetics with relevance to mitophagy and programmed cell death. The event of heat shock protein 70 disturbances may lead to kidney injury [71] and associated with chronic

**6. Novel biomarkers and insulin therapy may reverse NAFLD and** 

The analysis of various plasma biomarkers with insulin therapy [72] has become of major interest to NAFLD development, therapeutic strategies [73–77] and diabetes research. Essential measurements of plasma Sirt 1 and heat shock protein levels need to be determined to indicate core body temperature defects with relevance to inactivation of insulin therapy. Tissue analysis of anti-aging genes [18, 33, 54] need to be conducted to determine the role of insulin therapy with relevance to reversal of NAFLD [18, 33, 35, 49, 55, 68, 69] with connections to inflammation and metabolic diseases. Plasma assays of inflammatory cytokines such as tumor necrosis factor alpha, interleukin-1 and interleukin-6 [10, 11] need to be assayed with effective insulin therapy. The major limitation with insulin therapy is to correlate the dose of insulin injected with plasma biomarkers [78] that maintain mitochondrial biogenesis associated with the prevention of NAFLD (**Figure 4**). The use of antimicrobials [79] with insulin therapy should be carefully controlled to prevent increased release of gram negative bacteria LPS end products that may interfere with glucose homeostasis and induce NAFLD. Plasma LPS should be measured with antimicrobial use in individuals on insulin therapy. The connections between the antimicrobial activity, immune system and nitric oxide homeostasis involve Sirt 1 and connected to toxic

The geriatric population in many communities is associated with insulin resistance, Sirt 1 repression and nuclear-mitochondria defects relevant to NAFLD. Sirt 1 measurement in the plasma, cytoplasm and nucleus are essential to determine

*DOI: http://dx.doi.org/10.5772/intechopen.81297*

mitophagy and organ disease.

apoptosis.

**diabetes**

*Insulin Therapy and Autoimmune Disease with Relevance to Non Alchoholic Fatty Liver Disease DOI: http://dx.doi.org/10.5772/intechopen.81297*

associated with appetite dysregulation and poor food quality. Specific mitochondrial nutrients [65] with insulin therapy need to be consumed to prevent severe mitophagy and organ disease.

Food quality with relevance to stroke, synaptic plasticity and neurological diseases has become important to diabetic individuals with essential maintenance and prevention of brain diseases by insulin therapy. Unhealthy diets that contain LPS, mycotoxins and xenobiotics can induce NAFLD with inactivation of insulin therapy. In the developing world increased plasma LPS levels (**Figure 3**) have raised concern with relevance to induction of metabolic and neurodegenerative diseases [66, 67]. Antibiotic resistance with relevance to antimicrobial drug use should be carefully controlled to prevent excessive release of LPS from the debris of gram negative bacteria [68]. Food preparation should be carefully assessed to prevent end products such as LPS and patulin that may persist in contaminated food [63, 69]. LPS and patulin may inactivate Sirt 1 [62] with relevance to insulin resistance and NAFLD. Xenobiotics [63] in air, food and water may inactivate insulin therapy (**Figure 3**) with increased xenobiotic levels associated with mitochondrial apoptosis.

Core body temperature (**Figure 3**) and insulin therapy are closely connected and dysregulation of core body temperature may induce NAFLD. The discovery of the heat shock gene Sirt 1 [70] has indicated that careful body temperature control is critical to prevent autoimmune disease and mitochondrial apoptosis. Sirt 1 and its inactivation are associated with increased heat shock protein 70 with relevance to natural killer cell activation and mitochondrial apoptosis. Nutritional therapy and core body temperature are essential to maintain insulin therapy in diabetics with relevance to mitophagy and programmed cell death. The event of heat shock protein 70 disturbances may lead to kidney injury [71] and associated with chronic kidney disease and neurodegeneration in diabetes.

#### **6. Novel biomarkers and insulin therapy may reverse NAFLD and diabetes**

The analysis of various plasma biomarkers with insulin therapy [72] has become of major interest to NAFLD development, therapeutic strategies [73–77] and diabetes research. Essential measurements of plasma Sirt 1 and heat shock protein levels need to be determined to indicate core body temperature defects with relevance to inactivation of insulin therapy. Tissue analysis of anti-aging genes [18, 33, 54] need to be conducted to determine the role of insulin therapy with relevance to reversal of NAFLD [18, 33, 35, 49, 55, 68, 69] with connections to inflammation and metabolic diseases. Plasma assays of inflammatory cytokines such as tumor necrosis factor alpha, interleukin-1 and interleukin-6 [10, 11] need to be assayed with effective insulin therapy. The major limitation with insulin therapy is to correlate the dose of insulin injected with plasma biomarkers [78] that maintain mitochondrial biogenesis associated with the prevention of NAFLD (**Figure 4**). The use of antimicrobials [79] with insulin therapy should be carefully controlled to prevent increased release of gram negative bacteria LPS end products that may interfere with glucose homeostasis and induce NAFLD. Plasma LPS should be measured with antimicrobial use in individuals on insulin therapy. The connections between the antimicrobial activity, immune system and nitric oxide homeostasis involve Sirt 1 and connected to toxic immune reactions [80].

The geriatric population in many communities is associated with insulin resistance, Sirt 1 repression and nuclear-mitochondria defects relevant to NAFLD. Sirt 1 measurement in the plasma, cytoplasm and nucleus are essential to determine

*Nonalcoholic Fatty Liver Disease - An Update*

**in global chronic disease**

prevent interference with drug/insulin therapy (**Figure 2**) or with caffeine effects [60] relevant to the treatment of NAFLD and diabetes. The mixing of spices such as curcumin, turmeric and black pepper in coffee should be discouraged and may contribute to the transcriptional dysregulation of Sirt 1 and induction of mitochon-

**5. Genomic medicine and Sirt 1 activators reverse immune reactions** 

Genomic medicine in the treatment of cardiovascular disease and diabetes [19, 37] has now accelerated in various communities. Peripheral nutrition is essential early to prevent neurodegeneration (Type 3 diabetes) that lead to uncontrolled peripheral glucose homeostasis. Type 3 diabetes is associated with suprachiasmatic nucleus defects with the abnormal maintenance of brain and whole body glucose metabolism in various species and man [20]. Nutritional therapy in diabetics now need to involve the use of Sirt 1 activators [61] to prevent the effects of various Sirt 1 inhibitors that accumulate in the blood plasma that repress Sirt 1 expression in cells and tissues. A dose of 4 g/day of phosphatidylinositol [62] is essential with insulin therapy to prevent hyperglycemia, NAFLD and other neurodegenerative diseases. Sirt 1 inhibitors such as excess palmitic acid (cream, cheese), alcohol and drugs (suramin and sirtinol) should be carefully controlled to prevent inactivation of insulin therapy. Sirt 1 activators such as pyruvic acid, leucine and magnesium are critical with relevance to insulin therapy. Diabetic individuals with Indian spice consumption (**Figure 3**) over years need to be carefully evaluated with relevance to plasma Sirt 1 inhibitors, xenobiotics [63], caffeine content [60], drug therapy, bacterial lipopolysaccharides (LPS) and mycotoxins [62] that may interfere with insulin/oral medication therapy. The importance of genomic medicine may indicate that the immune system may malfunction [37] early with relevance to poor nutrition of food quality with irreversible organ disease manifestations. Biotherapy and the immune system [37, 61] may be critical to insulin therapy and connected to insulin resistance and NAFLD. Appetite control and essential food components [64] may be essential to maintain the immune system with autoimmune disease

*Poor food quality and core body temperature defects will inactivate Sirt 1 and induce insulin resistance and NAFLD. Sirt 1 inhibitors such as xenobiotics, caffeine/Indian spice over-consumption and magnesium deficiency may lead to the diabetes pandemic with high doses of phosphatidylinositol essential to maintain* 

drial apoptosis relevant to diabetes and the pathogenetic loop [27–32].

**34**

**Figure 3.**

*insulin therapy and prevent the induction of NAFLD.*

**Figure 4.**

*Complications of insulin therapy in diabetes lead to irreversible mitophagy and programmed cell death with relevance to defective Sirt 1 expression in diabetic individuals. Conventional clinical biochemistry tests do not indicate nuclear-mitochondria defects associated with autoimmune disease and mitophagy but lipidomic tests may be relevant to insulin therapy and Sirt 1 analysis.*

the relevance of insulin therapy and mitochondrial apoptosis when compared to the validity of various diagnostic tests and plasma analytic measurements. In many biomarker laboratories the comprehensive assessment of various biomarkers may not be correlated with insulin therapy with mitophagy the inevitable cellular defect in geriatric individuals. Analysis of plasma biomarkers (**Figure 4**) and tissue samples may indicate a primary autoimmune reaction related to a defective nuclearmitochondria interaction.

Insulin therapy and its use should be carefully revised with relevance to conventional plasma tests that do not indicate cellular mitophagy and toxic immune reactions associated with diabetes [81, 82]. Previous studies [83, 84] with the assessment of the role of insulin on cytokines, lymphocytes and macrophages do not assess Sirt 1's role in toxic immune reactions and mitophagy. Recent studies have shown that molecular lipid biomarkers from lipidomic analysis [85–88] may determine diabetes severity. The role of insulin therapy with relevance to lipidomic biomarkers may integrate routine plasma biomarker testing with relevance to cellular Sirt 1 expression and plasma Sirt 1 analysis (**Figure 4**).

#### **7. Conclusion**

Insulin treatment has been evaluated in diabetes but the global NAFLD epidemic that is expected to reach between 20 and 30% of the worldwide communities will now be connected to diabetes pandemic and the pathogenetic loop. Insulin therapy has been assessed with relevance to improvement in inflammatory conditions but the defect in the anti-aging gene Sirt 1 and diabetic mitophagy still persists with the induction of NAFLD and various organ diseases. Insulin therapy with Indian spice consumption requires reassessment to avoid over-consumption of Indian spices that may inactivate insulin therapy and mitochondrial biogenesis. Food quality, appetite control and core body temperature are critical to maintain insulin therapy with unhealthy diets linked to NAFLD and diabetes. Genomic medicine and Sirt 1 activators are essential to maintain insulin therapy in the developing world with toxic immune reactions important to NAFLD. Insulin therapy may not reverse the nuclear-mitochondria defect that is relevant to global organ disease and various plasma biomarkers.

**37**

provided the original work is properly cited.

\*Address all correspondence to: i.martins@ecu.edu.au

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Centre of Excellence in Alzheimer's Disease Research and Care,

Sarich Neuroscience Research Institute, Edith Cowan University, Nedlands,

2 School of Psychiatry and Clinical Neurosciences, The University of Western

3 McCusker Alzheimer's Research Foundation, Hollywood Medical Centre,

*Insulin Therapy and Autoimmune Disease with Relevance to Non Alchoholic Fatty Liver Disease*

This work was supported by grants from Edith Cowan University, the McCusker Alzheimer's Research Foundation and the National Health and Medical Research

*DOI: http://dx.doi.org/10.5772/intechopen.81297*

NAFLD nonalcoholic fatty liver disease

LPS bacterial lipopolysaccharides

**Acknowledgements**

Council.

**Abbreviations**

**Author details**

Ian James Martins1,2,3\*

Nedlands, Australia

Western Australia, Australia

Australia, Nedlands, Australia

Sirt 1 Sirtuin 1

*Insulin Therapy and Autoimmune Disease with Relevance to Non Alchoholic Fatty Liver Disease DOI: http://dx.doi.org/10.5772/intechopen.81297*

#### **Acknowledgements**

*Nonalcoholic Fatty Liver Disease - An Update*

*may be relevant to insulin therapy and Sirt 1 analysis.*

mitochondria interaction.

**Figure 4.**

**7. Conclusion**

the relevance of insulin therapy and mitochondrial apoptosis when compared to the validity of various diagnostic tests and plasma analytic measurements. In many biomarker laboratories the comprehensive assessment of various biomarkers may not be correlated with insulin therapy with mitophagy the inevitable cellular defect in geriatric individuals. Analysis of plasma biomarkers (**Figure 4**) and tissue samples may indicate a primary autoimmune reaction related to a defective nuclear-

*Complications of insulin therapy in diabetes lead to irreversible mitophagy and programmed cell death with relevance to defective Sirt 1 expression in diabetic individuals. Conventional clinical biochemistry tests do not indicate nuclear-mitochondria defects associated with autoimmune disease and mitophagy but lipidomic tests* 

Insulin therapy and its use should be carefully revised with relevance to conventional plasma tests that do not indicate cellular mitophagy and toxic immune reactions associated with diabetes [81, 82]. Previous studies [83, 84] with the assessment of the role of insulin on cytokines, lymphocytes and macrophages do not assess Sirt 1's role in toxic immune reactions and mitophagy. Recent studies have shown that molecular lipid biomarkers from lipidomic analysis [85–88] may determine diabetes severity. The role of insulin therapy with relevance to lipidomic biomarkers may integrate routine plasma biomarker testing with relevance to cel-

Insulin treatment has been evaluated in diabetes but the global NAFLD epidemic that is expected to reach between 20 and 30% of the worldwide communities will now be connected to diabetes pandemic and the pathogenetic loop. Insulin therapy has been assessed with relevance to improvement in inflammatory conditions but the defect in the anti-aging gene Sirt 1 and diabetic mitophagy still persists with the induction of NAFLD and various organ diseases. Insulin therapy with Indian spice consumption requires reassessment to avoid over-consumption of Indian spices that may inactivate insulin therapy and mitochondrial biogenesis. Food quality, appetite control and core body temperature are critical to maintain insulin therapy with unhealthy diets linked to NAFLD and diabetes. Genomic medicine and Sirt 1 activators are essential to maintain insulin therapy in the developing world with toxic immune reactions important to NAFLD. Insulin therapy may not reverse the nuclear-mitochondria defect that is relevant to global organ disease and various

lular Sirt 1 expression and plasma Sirt 1 analysis (**Figure 4**).

**36**

plasma biomarkers.

This work was supported by grants from Edith Cowan University, the McCusker Alzheimer's Research Foundation and the National Health and Medical Research Council.

### **Abbreviations**


### **Author details**

Ian James Martins1,2,3\*

1 Centre of Excellence in Alzheimer's Disease Research and Care, Sarich Neuroscience Research Institute, Edith Cowan University, Nedlands, Western Australia, Australia

2 School of Psychiatry and Clinical Neurosciences, The University of Western Australia, Nedlands, Australia

3 McCusker Alzheimer's Research Foundation, Hollywood Medical Centre, Nedlands, Australia

\*Address all correspondence to: i.martins@ecu.edu.au

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Pharmacy and Pharmaceutical Sciences.

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[16] Kramer CK, Zinman B, Retnakaran

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[18] Martins IJ. Appetite control and biotherapy in the Management of Autoimmune Induced Global Chronic Diseases. Journal of Clinical Immunology and Research. 2018;**2**:1-4

[19] Martins IJ. Genomic medicine and acute cardiovascular disease progression

R. Short-term intensive insulin therapy in type 2 diabetes mellitus: A systematic review and meta-analysis. Lancet Diabetes and Endocrinology.

[11] Lai Y, Dong C. Therapeutic antibodies that target inflammatory cytokines in autoimmune diseases.

International Immunology.

2005;**8**:602-625

2016;**28**:181-188

2016;**7**:211-217

978-3-659-40372-9

2017;**1**:10-12

2015;**33**:123-135

2013;**1**:28-34

2018;**9**:449-492

Epidemiology of diabetes-status of a pandemic and issues around metabolic surgery. Diabetes Care. 2016;**39**:878-883

[4] Gruver AL, Hudson LL, Sempowski GD. Immunosenescence of ageing. The Journal of Pathology. 2007;**211**:144-156

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[6] Fulop T, Witkowski JM, Pawelec G, Alan C, Larbi A. On the immunological theory of aging. Interdisciplinary Topics

[7] Passarino G, De Rango F, Montesanto

[8] Doria G, Frasca D. Genetic factors in immunity and aging. Vaccine.

[9] Cribbs DH, Berchtold NC, Perreau V, Coleman PD, Rogers J, Tenner AJ, et al. Extensive innate immune gene activation accompanies brain aging, increasing vulnerability to cognitive decline and neurodegeneration: A microarray study. Journal of Neuroinflammation. 2012;**9**:179

[10] Kulmatycki KM, Jamali F. Drug

inflammatory mediators in disease and variability in drug response. Journal of

disease interactions: Role of

in Gerontology. 2014;**39**:163-176

A. Human longevity: Genetics or lifestyle? It takes two to tango. Immunity and Ageing. 2016;**13**:12

2000;**18**:1591-1595

[2] Zimmet PZ, Alberti KG.

[3] Michels AW, Eisenbarth GS. Immunologic endocrine disorders. Journal of Allergy and Clinical Immunology. 2010;**125**:S226-S237

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[59] Martins IJ. Indian spices and unhealthy diets interfere with drug therapy in diabetes and neurodegenerative diseases. Novel Approaches in Drug Designing and Development. 2018;**3**:555616

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*Nonalcoholic Fatty Liver Disease - An Update*

[37] Martins IJ. Genomic medicine and endocrine autoimmunity as key to mitochondrial disease. Global Journal of Endocrinological Metabolism.

[46] Zabolotny JM, Kim YB. Silencing insulin resistance through SIRT1. Cell

Inflammation and insulin resistance. FEBS Letters. 2008;**582**:97-105

[48] Wieser V, Moschen AR, Tilg H. Inflammation, cytokines and insulin resistance: A clinical perspective. Archivum Immunologiae et Therapia Experimentalis (Warsz).

[49] Martins IJ. Unhealthy Nutrigenomic diets accelerate NAFLD and adiposity in global communities. Journal of Molecular and Genetic Medicine.

[50] Martins IJ. Defective interplay between adipose tissue and immune system induces non alcoholic fatty liver disease. Updates in Nutritional Disorders and Therapy. 2017;**1**:1-6

[51] Lu M, Sarruf DA, Li P, Osborn O, Sanchez-Alavez M, Talukdar S, et al. Neuronal Sirt1 deficiency increases insulin sensitivity in both brain and peripheral tissues. Journal of Biological Chemistry. 2013;**288**:10722-10735

[52] Martins IJ. Type 3 diabetes with links to NAFLD and other chronic diseases in the Western world. International Journal of Diabetes.

[53] Martins IJ. Heat shock gene Sirtuin 1 regulates post-prandial lipid metabolism with relevance to nutrition and appetite regulation in diabetes. International Journal of Diabetes and Clinical Diagnosis. International Journal of Diabetes and Clinical Diagnosis.

[54] Martins IJ. Indian spices and insulin therapy in diabetes and

neurodegenerative diseases. Journal of Diabetes and Clinical Studies. 2018;**1**:1-3

Metabolism. 2007;**6**:247-249

[47] de Luca C, Olefsky JM.

2013;**61**:119-125

2015;**9**:1

2016;**1**:1-5

2016;**3**:20

[38] Bordone L, Motta MC, Picard F, Robinson A, Jhala US, Apfeld J, et al. Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic β cells.

[39] Martins IJ. Anti-aging gene linked to appetite regulation determines longevity in humans and animals. International Journal of Aging Research.

[40] Jeschke MG, Klein D, Herndon DH. Insulin treatment improves the systemic inflammatory reaction to severe trauma. Annal of Surgery. 2004;**239**:553-560

[41] Chen Z, Yu R, Xiong Y, Du F, Zhu S.

A vicious circle between insulin resistance and inflammation in nonalcoholic fatty liver disease. Lipids in Health and Disease. 2017;**16**:203

[42] Dal S, Jeandidier N, Seyfritz E, Bietiger W, Péronet C, Moreau F, et al. Featured article: Oxidative stress status and liver tissue defenses in diabetic rats during intensive subcutaneous insulin therapy. Experimental Biology and Medicine (Maywood). 2016;**241**:184-192

[43] Cao Y, Jiang X, Ma H, Wang Y, Xue P, Liu Y. SIRT1 and insulin resistance. The Journal of Diabetic Complications.

[44] Liang F, Kume S, Koya D. SIRT1 and insulin resistance. Nature Reveiws in Endocrinology. 2009;**5**:367-373

[45] Yoshizaki T, Milne JC, Imamura T, Schenk S, Sonoda N, Babendure JL, et al. SIRT1 exerts anti-inflammatory effects and improves insulin sensitivity in adipocytes. Molecular and Cellular

Biology. 2009;**29**:1363-1374

2016;**30**:178-183

PLoS Biology. 2006;**4**:e31

2018;**2**:1-3

2018;**1**:1-4

**40**

[56] Martins IJ. Drug therapy for obesity with anti-aging genes modification. Annals of Obesity and Disorders. 2016;**1**:1001

[57] Cole BK, Feaver RE, Wamhoff BR, Dash A. Non-alcoholic fatty liver disease (NAFLD) models in drug discovery. Expert Opinion in Drug Discovery. 2018;**13**:193-205

[58] Martins IJ. Sirtuin 1, a diagnostic protein marker and its relevance to chronic disease and therapeutic drug interventions. EC Pharmacology and Toxicology. 2018;**6.4**:209-215

[59] Martins IJ. Indian spices and unhealthy diets interfere with drug therapy in diabetes and neurodegenerative diseases. Novel Approaches in Drug Designing and Development. 2018;**3**:555616

[60] Martins IJ. Indian spices and caffeine treatment for obesity and cardiovascular disease. Annals of Clinical Endocrinology and Metabolism. 2018;**2**:010-014

[61] Martins IJ. Biotherapy and the immune system in ageing science. Acta Scientific Nutritional Health. 2018;**2**(**4**):29-31

[62] Martins IJ. Overnutrition determines LPS regulation of mycotoxin induced neurotoxicity in neurodegenerative diseases. International Journal of Molecular Sciences. 2015;**16**:29554-29573

[63] Martins IJ. Chapter 01. Increased risk for obesity and diabetes with neurodegeneration in developing countries. In: Top 10 Contribution on Genetics. Book Chapter. Avid Science.2018. www.avid.science.com

[64] Martins IJ. Functional foods and active molecules with relevance to health and chronic disease: Editorial. Functional Foods in Health and Disease. 2017;**7**:833-836

[65] Martins IJ. The global obesity epidemic is related to stroke, dementia and Alzheimer's disease. JSM Alzheimer's Disease and Related Dementia. 2014;**1**:1010

[66] Martins IJ. Bacterial lipopolysaccharides and neuron toxicity in neurodegenerative diseases. Neurology and Neurosurgery. 2018;**1**:1-3

[67] Martins IJ. Bacterial lipopolysaccharides change membrane fluidity with relevance to phospholipid and amyloid Beta dynamics in Alzheimer's disease. Journal of Microbial and Biochemical Technology. 2016;**8**:322-324

[68] Martins IJ. Antibiotic resistance involves antimicrobial inactivation in global communities. SAJ Pharmaacy and Pharmacology. 2017;**2**:102

[69] Martins IJ. Food quality induces a miscible disease with relevance to Alzheimer's disease and neurological diseases. Journal of Food Research. 2016;**5**:45-52

[70] Martins IJ. Heat shock gene inactivation and protein aggregation with links to chronic diseases. Diseases. 2018;**6**:1-5

[71] Martins IJ. Heat shock protein aggregation and chronic kidney disease. Research on Chronic Diseases. 2018;**2**:42-44

[72] Martins IJ. Advances in biomarkers and insulin therapy with relevance to reversal of diabetes. Journal of Studies on Diabetes. 2018;**1**:9-14

[73] Kitade H, Chen G, Ni Y, Ota T. Nonalcoholic fatty liver disease and insulin resistance: New insights and potential new treatments. Nutrients. 2017;**9**:pii: E387

[74] Mills EP, Brown KPD, Smith JD, Vang PW, Trotta K. Treating nonalcoholic fatty liver disease in patients with type 2 diabetes mellitus: A review of efficacy and safety. Therapeutic Advances in Endocrinology and Metabolism. 2018;**9**:15-28

[75] Issa D, Patel V, Sanyal AJ. Future therapy for non-alcoholic fatty liver disease. Liver International. 2018;**38**:56-63

[76] Friedman SL, Neuschwander-Tetri BA, Rinella M, Sanyal AJ. Mechanisms of NAFLD development and therapeutic strategies. Nature Medicine. 2018;**24**:908-922

[77] Sumida Y, Yoneda M. Current and future pharmacological therapies for NAFLD/NASH. Journal of Gastroenterology. 2018;**53**:362-376

[78] Lees T, Nassif N, Simpson A, Shad-Kaneez F, Martiniello-Wilks R, Lin Y, et al. Recent advances in molecular biomarkers for diabetes mellitus: A systematic review. Biomarkers. 2017;**22**:604-613

[79] Dos Reis SA, do Carmo Gouveia Peluzio M, Bressan J. The use of antimicrobials as adjuvant therapy for the treatment of obesity and insulin resistance: Effects and associated mechanisms. Diabetes/Metabolism Research and Reveiws. 2018 Apr 16:e3014. [Epub ahead of print]

[80] Martins IJ. Antimicrobial activity inactivation and toxic immune reactions induce epilepsy in human. Journal of Medical Discovery. 2017;**2**:jmd17040

[81] Itariu BK, Stulnig TM. Autoimmune aspects of type 2 diabetes mellitus—A mini-review. Gerontology. 2014;**60**: 189-196

[82] Hemminki K, Liu X, Försti A, Sundquist J, Sundquist K, Ji J. Subsequent type 2 diabetes in patients with autoimmune disease. Scientific Reports. 2015;**5**:13871

[83] Nell LJ, Thomas JW. The human immune response to insulin. I. Kinetic and cellular aspects of lymphocyte proliferative responses in diabetics. Journal of Immunology. 1983;**131**:701-705

[84] Watters C, Everett JA, Haley C, Clinton A, Rumbaugh KP. Insulin treatment modulates the host immune system to enhance *Pseudomonas aeruginosa* wound biofilms. Infection and Immunity. 2014;**82**:92-100

[85] Markgraf DF, Al-Hasani H, Lehr S. Lipidomics—Reshaping the analysis and perception of type 2 diabetes. International Journal of Molecular Sciences. 2016;**17**:1841

[86] Suvitaival T, Bondia-Pons I, Yetukuri L, Pöhö P, Nolan JJ, Hyötyläinen T, et al. Lipidome as a predictive. Tool in progression to type 2 diabetes in Finnish men. Metabolism. 2018;**78**:1-12

[87] Kopprasch S, Dheban S, Schuhmann K, Xu A, Schulte KM, Simeonovic CJ, et al. Detection of independent associations of plasma lipidomic parameters with insulin sensitivity indices using data mining methodology. PLoS One. 2016;**11**:e0164173

[88] Lydic TA, Goo Y-H. Lipidomics unveils the complexity of the lipidome in metabolic diseases. Clinical and Translational Medicine. 2018;**7**:4

**43**

**Chapter 4**

**Abstract**

patients with NAFLD.

**1. Introduction**

ment of NAFLD [8–13].

Dysregulation of Bile Acids in

Bile acids are synthesized in the liver and tightly regulated through the enterohepatic circulation. Recent studies reveal that bile acids serve as hormone-like signaling molecules to activate nuclear receptors, notably farnesoid X receptor (FXR), regulating metabolic homeostasis of bile acids, cholesterol, lipids, and glucose. A connection between bile acids and nonalcoholic fatty liver disease (NAFLD) has long been recognized. Although inconsistent or even contradictory results are reported, a large body of evidence from clinical as well as preclinical studies demonstrates that bile acid homeostasis is disrupted in patients with NAFLD. The bile acid dysregulation gets worsening as NAFLD progresses from early stage simple steatosis to late stage nonalcoholic steatohepatitis (NASH) and NASH with fibrosis. As the risk factors for NAFLD, obesity and insulin resistance, which are often associated with NAFLD, contribute to the dysregulation of bile acids in patients with NAFLD. Total serum and fecal bile acid concentrations are mostly elevated in patients with NAFLD as a result of increased bile acid synthesis, elevated hepatic bile acids, and upregulation of bile acid transporters. The two negative feedback regulatory pathways for bile acid synthesis, FXR/SHP (small heterodimer partner) and fibroblast growth factor-19 (FGF19)/FGF receptor-4 (FGFR4), are impaired in

**Keywords:** NAFLD, steatosis, fatty liver, NASH, bile acids, FXR, bile acid synthesis,

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic

Bile acids are the metabolites of cholesterol and synthesized in the liver. It is well known that bile acids act as biological detergents to solubilize cholesterol and lipids in the bile and intestine, play important roles in cholesterol and lipid absorption and transport. Recent studies have revealed that bile acids can serve

liver disease worldwide. It affects about 30% of the population in the United States [1, 2] and 10% of adolescents and children [3, 4]. NAFLD is a spectrum of metabolic disorders starting with simple steatosis characterized with excessive accumulation of triglycerides in the hepatocytes, progressing to nonalcoholic steatohepatitis (NASH) characterized with inflammation, to fibrosis and cirrhosis, and eventually to liver failure and hepatocellular carcinoma (HCC) [5–7]. Obesity and insulin resistance or diabetes are the most prevalent risk factors for develop-

enterohepatic circulation, bile acid transporters, FGF19

Patients with NAFLD

*Xinmu Zhang and Ruitang Deng*

#### **Chapter 4**

*Nonalcoholic Fatty Liver Disease - An Update*

insulin resistance: New insights and potential new treatments. Nutrients. [82] Hemminki K, Liu X, Försti A, Sundquist J, Sundquist K, Ji J.

Reports. 2015;**5**:13871

1983;**131**:701-705

Sciences. 2016;**17**:1841

2018;**78**:1-12

[86] Suvitaival T, Bondia-Pons I, Yetukuri L, Pöhö P, Nolan JJ, Hyötyläinen T, et al. Lipidome as a predictive. Tool in progression to type 2 diabetes in Finnish men. Metabolism.

PLoS One. 2016;**11**:e0164173

[88] Lydic TA, Goo Y-H. Lipidomics unveils the complexity of the lipidome in metabolic diseases. Clinical and Translational Medicine. 2018;**7**:4

Subsequent type 2 diabetes in patients with autoimmune disease. Scientific

[83] Nell LJ, Thomas JW. The human immune response to insulin. I. Kinetic and cellular aspects of lymphocyte proliferative responses in diabetics. Journal of Immunology.

[84] Watters C, Everett JA, Haley C, Clinton A, Rumbaugh KP. Insulin treatment modulates the host immune system to enhance *Pseudomonas aeruginosa* wound biofilms. Infection and Immunity. 2014;**82**:92-100

[85] Markgraf DF, Al-Hasani H, Lehr S. Lipidomics—Reshaping the analysis and perception of type 2 diabetes. International Journal of Molecular

[87] Kopprasch S, Dheban S, Schuhmann K, Xu A, Schulte KM, Simeonovic CJ, et al. Detection of independent associations of plasma lipidomic parameters with insulin sensitivity indices using data mining methodology.

[74] Mills EP, Brown KPD, Smith JD, Vang PW, Trotta K. Treating nonalcoholic fatty liver disease in patients with type 2 diabetes mellitus: A review of efficacy and safety.

Therapeutic Advances in Endocrinology

[75] Issa D, Patel V, Sanyal AJ. Future therapy for non-alcoholic fatty liver disease. Liver International.

[76] Friedman SL, Neuschwander-Tetri BA, Rinella M, Sanyal AJ. Mechanisms of NAFLD development and therapeutic strategies. Nature Medicine. 2018;**24**:908-922

[77] Sumida Y, Yoneda M. Current and future pharmacological therapies

[78] Lees T, Nassif N, Simpson A,

[79] Dos Reis SA, do Carmo Gouveia Peluzio M, Bressan J. The use of antimicrobials as adjuvant therapy for the treatment of obesity and insulin resistance: Effects and associated mechanisms. Diabetes/Metabolism Research and Reveiws. 2018 Apr 16:e3014. [Epub ahead of print]

[80] Martins IJ. Antimicrobial activity inactivation and toxic immune reactions induce epilepsy in human. Journal of Medical Discovery. 2017;**2**:jmd17040

[81] Itariu BK, Stulnig TM. Autoimmune aspects of type 2 diabetes mellitus—A mini-review. Gerontology. 2014;**60**:

2017;**22**:604-613

Shad-Kaneez F, Martiniello-Wilks R, Lin Y, et al. Recent advances in molecular biomarkers for diabetes mellitus: A systematic review. Biomarkers.

for NAFLD/NASH. Journal of Gastroenterology. 2018;**53**:362-376

and Metabolism. 2018;**9**:15-28

2017;**9**:pii: E387

2018;**38**:56-63

**42**

189-196

## Dysregulation of Bile Acids in Patients with NAFLD

*Xinmu Zhang and Ruitang Deng*

#### **Abstract**

Bile acids are synthesized in the liver and tightly regulated through the enterohepatic circulation. Recent studies reveal that bile acids serve as hormone-like signaling molecules to activate nuclear receptors, notably farnesoid X receptor (FXR), regulating metabolic homeostasis of bile acids, cholesterol, lipids, and glucose. A connection between bile acids and nonalcoholic fatty liver disease (NAFLD) has long been recognized. Although inconsistent or even contradictory results are reported, a large body of evidence from clinical as well as preclinical studies demonstrates that bile acid homeostasis is disrupted in patients with NAFLD. The bile acid dysregulation gets worsening as NAFLD progresses from early stage simple steatosis to late stage nonalcoholic steatohepatitis (NASH) and NASH with fibrosis. As the risk factors for NAFLD, obesity and insulin resistance, which are often associated with NAFLD, contribute to the dysregulation of bile acids in patients with NAFLD. Total serum and fecal bile acid concentrations are mostly elevated in patients with NAFLD as a result of increased bile acid synthesis, elevated hepatic bile acids, and upregulation of bile acid transporters. The two negative feedback regulatory pathways for bile acid synthesis, FXR/SHP (small heterodimer partner) and fibroblast growth factor-19 (FGF19)/FGF receptor-4 (FGFR4), are impaired in patients with NAFLD.

**Keywords:** NAFLD, steatosis, fatty liver, NASH, bile acids, FXR, bile acid synthesis, enterohepatic circulation, bile acid transporters, FGF19

#### **1. Introduction**

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver disease worldwide. It affects about 30% of the population in the United States [1, 2] and 10% of adolescents and children [3, 4]. NAFLD is a spectrum of metabolic disorders starting with simple steatosis characterized with excessive accumulation of triglycerides in the hepatocytes, progressing to nonalcoholic steatohepatitis (NASH) characterized with inflammation, to fibrosis and cirrhosis, and eventually to liver failure and hepatocellular carcinoma (HCC) [5–7]. Obesity and insulin resistance or diabetes are the most prevalent risk factors for development of NAFLD [8–13].

Bile acids are the metabolites of cholesterol and synthesized in the liver. It is well known that bile acids act as biological detergents to solubilize cholesterol and lipids in the bile and intestine, play important roles in cholesterol and lipid absorption and transport. Recent studies have revealed that bile acids can serve as hormone-like signaling molecules to activate several nuclear receptors, notably the farnesoid X receptor (FXR) [14, 15]. The bile acids/FXR signaling plays critical roles in regulating a myriad of metabolic homeostasis including bile acids, cholesterol, lipids, and glucose [16–19], as well as inflammation/immunity [20–24] and liver regeneration [25–27].

Under physiological condition, bile acid homeostasis is maintained through multiple negative feedback loops for bile acid synthesis [18, 28–30] and a tightly regulated enterohepatic circulation of bile acids [31–34]. Since liver is the organ for bile acid synthesis and metabolism and biliary excretion of bile acids is the limiting step for the enterohepatic circulation [35, 36], impairment of liver function as a result of various liver disorders leads to dysregulation of bile acids. Indeed, the measurement of bile acids is considered a biomarker of liver function and serves as an indicator of hepatobiliary impairment or diseases [37–41]. On the other hand, excessive accumulation of bile acids in the liver causes liver damages by multiple mechanisms including disrupting the integrity of cell membranes through their detergent property [42–44], causing mitochondrial stress and promoting the generation of reactive oxygen species [45–48], and inducing endoplasmic reticulum stress [49–51] and inflammatory responses [52–54], resulting in cell death via apoptosis and/or necrosis [55–58].

Because of the reciprocal effects between liver damage and bile acid dysregulation, it is often difficult, if not impossible, to determine the cause-and-effect relation between liver damage and bile acid dysregulation for many liver disorders. In one hand, liver damage causes bile acid dysregulation. On the other hand, bile acid dysregulation potentially causes liver damage. The connection between NAFLD and bile acid dysregulation has long been recognized and reported [59–67]. It is well established that liver function is compromised in patients with NAFLD, especially advanced stages of NAFLD, such as NASH and NASH-associated fibrosis and cirrhosis, due to pathological and structural damages to the liver. Research interests and emphasis are recently condensed on investigating the contribution of bile acid dysregulation to the pathogenesis of NAFLD and developing therapeutic interventions for NAFLD by manipulating the bile acid signaling pathway [66–73]. However, the outcomes of clinical trials targeting bile acid signaling using ursodeoxycholic acid (UDCA) and obeticholic acid (OCA) to treat NASH patients are not very encouraging [74–79], indicating that our understanding on the relationship between bile acids and NAFLD is not complete or even may be misinterpreted.

Taken together, the link between bile acids and NAFLD has been firmly established. However, certain fundamental questions remain to be answered. How bile acid homeostasis is disrupted in patients with NAFLD? Whether dysregulation of bile acids is one of the manifestations of NAFLD or actually contributes to the development and/or progression of NAFLD? It only becomes possible to develop rationalized approaches to treat patients with NAFLD until those fundamental questions are fully addressed. In this chapter, the effects of NAFLD on bile acid homeostasis are reviewed and discussed.

#### **2. Altered bile acid profiles in subjects with NAFLD**

In human, cholic acids (CAs) and chenodeoxycholic acid (CDCA) are two primary bile acids synthesized in the liver and account for majority of bile acids in the bile acid pool. Upon excretion into intestine, primary bile acids can be converted into secondary bile acids by gut bacteria. Specifically, CA is converted into deoxycholic acid (DCA), while CDCA is converted into lithocholic acid (LCA) or UDCA in the intestine by dehydroxylation [80, 81] or 7β epimerization [82–84]. Majority

**45**

*Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

**2.1 Altered bile acid profiles in patients with NAFLD**

separately described in the following sections.

*2.1.1.1 Serum bile acid profiling in adults with NAFLD*

rodent NAFLD models.

*2.1.1 Serum bile acids*

of primary and secondary bile acids are conjugated by either glycine or taurine in the liver, generating glycine- or taurine-conjugated bile acids [80, 81]. Under physiological conditions, total bile acid levels, as well as the composition of the bile acid pool, are regulated and maintained. However, under various pathological conditions, especially liver disorders, the bile acid pool size or total bile acids and bile acid pool compositions are altered. A large number of clinical and preclinical studies have revealed that bile acid profiles are altered in patients with NAFLD and

Under the physiological condition, serum bile acid concentrations are much lower than those in the enterohepatic system. However, when the enterohepatic cycling of bile acids is compromised due to hepatic injuries or intestine disorders, bile acids are spilled into the blood circulation system, altering serum bile acid concentrations, as well as compositions. Bile acid profiling in healthy populations has revealed that serum bile acid concentrations and compositions are age dependent [85]. Therefore, the serum bile acid profiles in adult and children with NAFLD are

Currently, there are total nine clinical studies investigating serum bile acid levels

and compositions in adults with NAFLD. In study 1 with 25 healthy subjects, 11 patients with steatosis, and 24 patients with NASH, it was found that serum bile acid profiles after overnight fasting were significantly altered in both steatotic and NASH patients, especially in patients with NASH [86]. The most prominent alteration is the markedly increased conjugated CA concentration. Taurine-conjugated CAs (TCAs) were elevated 4- and 2.2-fold, while glycine-conjugated CAs (GCAs) were increased 4.3- and 3.1-fold in patients with NASH and steatosis, respectively. Similarly, GCDCA levels were also elevated by 2- and 2.4-fold in patients with NASH and steatosis, respectively. Other bile acid species, including CA, GDCA, TDCA, and TCDCA, exhibited a trend of increase but their levels did not reach a statistical significance. It should be noted that patients with steatosis or NASH had significantly elevated insulin levels and exhibited insulin resistance, although the blood glucose levels were within the normal range. The patients, especially those with NASH, also had elevated serum alanine aminotransferase (ALT) and aspartate

aminotransferase (AST) levels, indicating liver damage in those patients.

In study 2 with 15 healthy controls and 7 NASH patients, both fasting and postprandial bile acids were altered [87]. Total fasting serum bile acid levels were increase by more than twofold. Such increases in total bile acids are mainly due to significantly increased conjugated bile acids with both glycine and taurine, while unconjugated bile acids were not significantly altered. Both primary (CDCA and CA and their conjugated) and secondary (DCA and LCA and their conjugated) bile acids were markedly elevated. Similarly, postprandial serum bile acid levels were also markedly increased in patients with NASH, including total, conjugated and unconjugated, primary, and secondary bile acids. However, the relative ratios or the compositions of the serum bile acid pools were not significantly altered in both fasting and postprandial levels. Significant elevations in individual bile acids including DCA, GCA, GCDCA, and TCA were also noted. Other bile acid species including CA and CDCA were either not altered or slightly increased without reaching a

*Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

of primary and secondary bile acids are conjugated by either glycine or taurine in the liver, generating glycine- or taurine-conjugated bile acids [80, 81]. Under physiological conditions, total bile acid levels, as well as the composition of the bile acid pool, are regulated and maintained. However, under various pathological conditions, especially liver disorders, the bile acid pool size or total bile acids and bile acid pool compositions are altered. A large number of clinical and preclinical studies have revealed that bile acid profiles are altered in patients with NAFLD and rodent NAFLD models.

#### **2.1 Altered bile acid profiles in patients with NAFLD**

#### *2.1.1 Serum bile acids*

*Nonalcoholic Fatty Liver Disease - An Update*

liver regeneration [25–27].

apoptosis and/or necrosis [55–58].

homeostasis are reviewed and discussed.

**2. Altered bile acid profiles in subjects with NAFLD**

In human, cholic acids (CAs) and chenodeoxycholic acid (CDCA) are two primary bile acids synthesized in the liver and account for majority of bile acids in the bile acid pool. Upon excretion into intestine, primary bile acids can be converted into secondary bile acids by gut bacteria. Specifically, CA is converted into deoxycholic acid (DCA), while CDCA is converted into lithocholic acid (LCA) or UDCA in the intestine by dehydroxylation [80, 81] or 7β epimerization [82–84]. Majority

as hormone-like signaling molecules to activate several nuclear receptors, notably the farnesoid X receptor (FXR) [14, 15]. The bile acids/FXR signaling plays critical roles in regulating a myriad of metabolic homeostasis including bile acids, cholesterol, lipids, and glucose [16–19], as well as inflammation/immunity [20–24] and

Under physiological condition, bile acid homeostasis is maintained through multiple negative feedback loops for bile acid synthesis [18, 28–30] and a tightly regulated enterohepatic circulation of bile acids [31–34]. Since liver is the organ for bile acid synthesis and metabolism and biliary excretion of bile acids is the limiting step for the enterohepatic circulation [35, 36], impairment of liver function as a result of various liver disorders leads to dysregulation of bile acids. Indeed, the measurement of bile acids is considered a biomarker of liver function and serves as an indicator of hepatobiliary impairment or diseases [37–41]. On the other hand, excessive accumulation of bile acids in the liver causes liver damages by multiple mechanisms including disrupting the integrity of cell membranes through their detergent property [42–44], causing mitochondrial stress and promoting the generation of reactive oxygen species [45–48], and inducing endoplasmic reticulum stress [49–51] and inflammatory responses [52–54], resulting in cell death via

Because of the reciprocal effects between liver damage and bile acid dysregula-

tion, it is often difficult, if not impossible, to determine the cause-and-effect relation between liver damage and bile acid dysregulation for many liver disorders. In one hand, liver damage causes bile acid dysregulation. On the other hand, bile acid dysregulation potentially causes liver damage. The connection between NAFLD and bile acid dysregulation has long been recognized and reported [59–67]. It is well established that liver function is compromised in patients with NAFLD, especially advanced stages of NAFLD, such as NASH and NASH-associated fibrosis and cirrhosis, due to pathological and structural damages to the liver. Research interests and emphasis are recently condensed on investigating the contribution of bile acid dysregulation to the pathogenesis of NAFLD and developing therapeutic interventions for NAFLD by manipulating the bile acid signaling pathway [66–73]. However, the outcomes of clinical trials targeting bile acid signaling using ursodeoxycholic acid (UDCA) and obeticholic acid (OCA) to treat NASH patients are not very encouraging [74–79], indicating that our understanding on the relationship between bile acids and NAFLD is not complete or even may be misinterpreted. Taken together, the link between bile acids and NAFLD has been firmly established. However, certain fundamental questions remain to be answered. How bile acid homeostasis is disrupted in patients with NAFLD? Whether dysregulation of bile acids is one of the manifestations of NAFLD or actually contributes to the development and/or progression of NAFLD? It only becomes possible to develop rationalized approaches to treat patients with NAFLD until those fundamental questions are fully addressed. In this chapter, the effects of NAFLD on bile acid

**44**

Under the physiological condition, serum bile acid concentrations are much lower than those in the enterohepatic system. However, when the enterohepatic cycling of bile acids is compromised due to hepatic injuries or intestine disorders, bile acids are spilled into the blood circulation system, altering serum bile acid concentrations, as well as compositions. Bile acid profiling in healthy populations has revealed that serum bile acid concentrations and compositions are age dependent [85]. Therefore, the serum bile acid profiles in adult and children with NAFLD are separately described in the following sections.

#### *2.1.1.1 Serum bile acid profiling in adults with NAFLD*

Currently, there are total nine clinical studies investigating serum bile acid levels and compositions in adults with NAFLD. In study 1 with 25 healthy subjects, 11 patients with steatosis, and 24 patients with NASH, it was found that serum bile acid profiles after overnight fasting were significantly altered in both steatotic and NASH patients, especially in patients with NASH [86]. The most prominent alteration is the markedly increased conjugated CA concentration. Taurine-conjugated CAs (TCAs) were elevated 4- and 2.2-fold, while glycine-conjugated CAs (GCAs) were increased 4.3- and 3.1-fold in patients with NASH and steatosis, respectively. Similarly, GCDCA levels were also elevated by 2- and 2.4-fold in patients with NASH and steatosis, respectively. Other bile acid species, including CA, GDCA, TDCA, and TCDCA, exhibited a trend of increase but their levels did not reach a statistical significance. It should be noted that patients with steatosis or NASH had significantly elevated insulin levels and exhibited insulin resistance, although the blood glucose levels were within the normal range. The patients, especially those with NASH, also had elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, indicating liver damage in those patients.

In study 2 with 15 healthy controls and 7 NASH patients, both fasting and postprandial bile acids were altered [87]. Total fasting serum bile acid levels were increase by more than twofold. Such increases in total bile acids are mainly due to significantly increased conjugated bile acids with both glycine and taurine, while unconjugated bile acids were not significantly altered. Both primary (CDCA and CA and their conjugated) and secondary (DCA and LCA and their conjugated) bile acids were markedly elevated. Similarly, postprandial serum bile acid levels were also markedly increased in patients with NASH, including total, conjugated and unconjugated, primary, and secondary bile acids. However, the relative ratios or the compositions of the serum bile acid pools were not significantly altered in both fasting and postprandial levels. Significant elevations in individual bile acids including DCA, GCA, GCDCA, and TCA were also noted. Other bile acid species including CA and CDCA were either not altered or slightly increased without reaching a

statistical significance. Patients with NASH had significantly elevated alkaline phosphatase (ALP), ALT, insulin, and homeostatic model assessment (HOMA) levels accompanied with significantly higher fast blood glucose levels when compared to the control subjects.

In study 3 with 24 healthy subjects, 25 steatotic, and 37 NASH patients, plasma bile acids after fasting were measured [88]. Total plasma primary bile acids (CA and CDCA) were gradually increased from controls to steatotic to NASH patients. On the contrary, total secondary bile acids (DCA and LCA) were gradually decreased from controls to steatotic to NASH patients. The increases in primary bile acids are mainly resulted from elevation of the conjugated bile acids, while the unconjugated primary bile acids (CA and CDCA) were comparable to those in the control subjects. Comparison between the two NAFLD groups, total conjugated CA and conjugated primary bile acids, was significantly higher in subjects with NASH compared to steatotic subjects. In addition, the compositions of the primary bile acid pools were also changed with significant increase in the ratios of total primary CA to CDCA, regardless of the status of diabetes. Although total secondary bile acids were lower in NASH patients, most of the individual secondary bile acids including GDAC, TDCA, TLCA, and GLCA were comparable among the three groups except for unconjugated DCA, which was significantly higher in NASH patients. Unconjugated UDCA levels were comparable among the three groups, while conjugated UDCA was significantly higher in NASH patients compared to steatotic and control subjects. It should be mentioned that AST and ALT levels were significantly elevated in both steatotic and NASH patients, indicating hepatic injury under the steatotic and NASH conditions. In addition, a large percentage of NASH patients (62.2%) were diabetic, while 20% of steatotic patients were diabetic with only one subject (4.2%) being diabetic in the control group.

In study 4 with 14 healthy controls and 7 patients with NASH, serum total bile acids were significantly elevated by 2.5-fold in patients with NASH compared to healthy control subjects [89]. Individual bile acids including GCA and TCA were markedly increased by 3.1- and 5.7-fold in patients with NASH, respectively. In addition, linear regression analysis revealed a significant association between NAFLD activity scores (NAS) and fasting total serum bile acid, GCA, and TCA concentrations. It should be mentioned that the fasting total bile acids, GCA and TCA serum, concentrations in healthy controls in the study were comparable to those reported previously for healthy adults [90].

In study 5 with 46 healthy control subjects and 13 patients with NAFLD, serum bile acids were dysregulated in patients with NAFLD [38]. Total serum bile acid levels were significantly increased by 4.7-fold from 2.8 μM in control subjects to 13.0 μM in NAFLD patients. Primary and secondary bile acids were elevated by 3.8- and 1.9-fold, respectively, in NAFLD patients. These increases in total, primary, and secondary bile acids are mainly due to much higher concentrations of conjugated bile acids in NAFLD patients (5.0 μM) than in control subjects (1.2 μM). Unconjugated bile acids were also slightly increased from 0.88 μM in control subjects to 1.30 μM in NAFLD patients without reaching a statistical significance.

In contrast to most of the previous studies, a recent study 6 with 32 patients with NASH and 26 non-NASH controls reported that plasma total, primary, secondary, unconjugated, and conjugated bile acids were not significantly different between the two groups [91]. The compositions of the plasma bile acid pools were also not altered either in patients with NASH when compared to non-NASH subjects. However, when the subjects were subcategorized into insulin resistance and insulin sensitive groups, significant changes in bile acid profiles were detected. Total serum CA (CA + GCA and TCA), unconjugated CA, total CDCA (CDCA + GCDCA + TCDCA), unconjugated CDCA, total primary and unconjugated primary, total

**47**

patients.

patients with NAFLD [94].

concentrations [85, 95, 96].

*Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

unconjugated, and non-12α bile acids were all significantly elevated in subjects with insulin resistance compared to insulin sensitive subjects. The authors therefore concluded that bile acid alterations were associated with insulin resistance but not NASH. The study also showed that body mass index (BMI), fasting plasma insulin concentrations, and HOMA values positively correlated with plasma CA and CDCA levels. It should be mentioned that the BMI and HOMA values were matched between the NASH patients and the non-NASH control subjects in this study. The average BMI was 40.2 for NASH and 39.4 for non-Nash subjects, indicating that both groups are severely obese. The average HOMA was 4.05 for NASH patients and

3.25 for non-NASH controls, indicating insulin resistance in both groups. Consistent with the findings from the sixth study, another clinical study reported that patients with NAFLD exhibited comparable serum total bile acid concentrations to those in healthy control subjects [92]. The study included 16 healthy controls with an average BMI of 24.2, 14 overweight NAFLD patients with BMI of 28.3, and 12 obese NAFLD patients with an average BMI of 35.3. No significant alterations in fasting as well as postprandial serum total bile acid levels were detected between healthy control subjects and overweight or obese NAFLD

In another study with 38 control subjects and 36 NASH patients, limited information about the characteristics of the studied subjects was provided and only data on three individual bile acid species were reported. The plasma concentrations of GCA, TCA, and TCDCA during fasting were significantly elevated in patients with NASH compared to control subjects [93]. Consistent with finding from most of the studies, another clinical study with 10 healthy controls, 39 steatotic, and 59 NASH patients reported that total serum bile acid levels were significantly elevated in

The findings from six clinical studies, which provide detailed characteristics of the studied subjects as well as the corresponding bile acid profiles, are summarized in **Table 1**. The results from studies 1 to 5 are largely consistent. Serum total, primary, and conjugated bile acids were all significantly increased with limited changes for unconjugated bile acids. However, the secondary bile acids were significantly increased in studies 1, 2, and 5 but decreased in study 3. In contrast to the findings from studies 1 to 5, no significant alterations were detected in serum total,

Compared the characteristics of the control and NAFLD subjects, it is noticed that the control subjects in study 6 were severely obese with BMI 39.4 ± 5.9, while the control subjects in studies 1–5 have normal or close to normal body weights with BMI ranging from 24.5 ± 2.6 to 27.3 ± 5.8. The BMI values were matched between NAFLD patients and control subjects in study 6 but significantly different in studies 1–5. Compared with serum bile acid levels in a healthy population [85], the control subjects in study 6 had markedly increased total, primary, secondary, conjugated, and unconjugated bile acids. The results indicate that obesity or increased BMI is a contributing factor to the dysregulation of serum bile acids. Indeed, several studies have reported that subjects with overweight or obese had increased serum bile acid

The second characteristic of the studied subjects that is different between study 6 and studies 1–5 is the status of insulin resistance in the control subjects. The serum insulin levels and HOMA values in study 6 are markedly higher than those in the other five studies, suggesting that insulin resistance is a contributing factor for the dysregulation of serum bile acids. Indeed, when all the subjects (NAFLD and control patients) in the study 6 were separated by insulin resistance status, primary bile acids, unconjugated bile acids, and non-12α bile acids, total CA and total CDCA were significantly increased in subjects with

primary, secondary, conjugated, and unconjugated bile acids in the study 6.

#### *Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

*Nonalcoholic Fatty Liver Disease - An Update*

pared to the control subjects.

statistical significance. Patients with NASH had significantly elevated alkaline phosphatase (ALP), ALT, insulin, and homeostatic model assessment (HOMA) levels accompanied with significantly higher fast blood glucose levels when com-

only one subject (4.2%) being diabetic in the control group.

reported previously for healthy adults [90].

In study 3 with 24 healthy subjects, 25 steatotic, and 37 NASH patients, plasma bile acids after fasting were measured [88]. Total plasma primary bile acids (CA and CDCA) were gradually increased from controls to steatotic to NASH patients. On the contrary, total secondary bile acids (DCA and LCA) were gradually decreased from controls to steatotic to NASH patients. The increases in primary bile acids are mainly resulted from elevation of the conjugated bile acids, while the unconjugated primary bile acids (CA and CDCA) were comparable to those in the control subjects. Comparison between the two NAFLD groups, total conjugated CA and conjugated primary bile acids, was significantly higher in subjects with NASH compared to steatotic subjects. In addition, the compositions of the primary bile acid pools were also changed with significant increase in the ratios of total primary CA to CDCA, regardless of the status of diabetes. Although total secondary bile acids were lower in NASH patients, most of the individual secondary bile acids including GDAC, TDCA, TLCA, and GLCA were comparable among the three groups except for unconjugated DCA, which was significantly higher in NASH patients. Unconjugated UDCA levels were comparable among the three groups, while conjugated UDCA was significantly higher in NASH patients compared to steatotic and control subjects. It should be mentioned that AST and ALT levels were significantly elevated in both steatotic and NASH patients, indicating hepatic injury under the steatotic and NASH conditions. In addition, a large percentage of NASH patients (62.2%) were diabetic, while 20% of steatotic patients were diabetic with

In study 4 with 14 healthy controls and 7 patients with NASH, serum total bile acids were significantly elevated by 2.5-fold in patients with NASH compared to healthy control subjects [89]. Individual bile acids including GCA and TCA were markedly increased by 3.1- and 5.7-fold in patients with NASH, respectively. In addition, linear regression analysis revealed a significant association between NAFLD activity scores (NAS) and fasting total serum bile acid, GCA, and TCA concentrations. It should be mentioned that the fasting total bile acids, GCA and TCA serum, concentrations in healthy controls in the study were comparable to those

In study 5 with 46 healthy control subjects and 13 patients with NAFLD, serum bile acids were dysregulated in patients with NAFLD [38]. Total serum bile acid levels were significantly increased by 4.7-fold from 2.8 μM in control subjects to 13.0 μM in NAFLD patients. Primary and secondary bile acids were elevated by 3.8- and 1.9-fold, respectively, in NAFLD patients. These increases in total, primary, and secondary bile acids are mainly due to much higher concentrations of conjugated bile acids in NAFLD patients (5.0 μM) than in control subjects (1.2 μM). Unconjugated bile acids were also slightly increased from 0.88 μM in control subjects to 1.30 μM in NAFLD patients without reaching a statistical significance. In contrast to most of the previous studies, a recent study 6 with 32 patients with NASH and 26 non-NASH controls reported that plasma total, primary, secondary, unconjugated, and conjugated bile acids were not significantly different between the two groups [91]. The compositions of the plasma bile acid pools were also not altered either in patients with NASH when compared to non-NASH subjects. However, when the subjects were subcategorized into insulin resistance and insulin sensitive groups, significant changes in bile acid profiles were detected. Total serum CA (CA + GCA and TCA), unconjugated CA, total CDCA (CDCA + GCDCA + TCDCA), unconjugated CDCA, total primary and unconjugated primary, total

**46**

unconjugated, and non-12α bile acids were all significantly elevated in subjects with insulin resistance compared to insulin sensitive subjects. The authors therefore concluded that bile acid alterations were associated with insulin resistance but not NASH. The study also showed that body mass index (BMI), fasting plasma insulin concentrations, and HOMA values positively correlated with plasma CA and CDCA levels. It should be mentioned that the BMI and HOMA values were matched between the NASH patients and the non-NASH control subjects in this study. The average BMI was 40.2 for NASH and 39.4 for non-Nash subjects, indicating that both groups are severely obese. The average HOMA was 4.05 for NASH patients and 3.25 for non-NASH controls, indicating insulin resistance in both groups.

Consistent with the findings from the sixth study, another clinical study reported that patients with NAFLD exhibited comparable serum total bile acid concentrations to those in healthy control subjects [92]. The study included 16 healthy controls with an average BMI of 24.2, 14 overweight NAFLD patients with BMI of 28.3, and 12 obese NAFLD patients with an average BMI of 35.3. No significant alterations in fasting as well as postprandial serum total bile acid levels were detected between healthy control subjects and overweight or obese NAFLD patients.

In another study with 38 control subjects and 36 NASH patients, limited information about the characteristics of the studied subjects was provided and only data on three individual bile acid species were reported. The plasma concentrations of GCA, TCA, and TCDCA during fasting were significantly elevated in patients with NASH compared to control subjects [93]. Consistent with finding from most of the studies, another clinical study with 10 healthy controls, 39 steatotic, and 59 NASH patients reported that total serum bile acid levels were significantly elevated in patients with NAFLD [94].

The findings from six clinical studies, which provide detailed characteristics of the studied subjects as well as the corresponding bile acid profiles, are summarized in **Table 1**. The results from studies 1 to 5 are largely consistent. Serum total, primary, and conjugated bile acids were all significantly increased with limited changes for unconjugated bile acids. However, the secondary bile acids were significantly increased in studies 1, 2, and 5 but decreased in study 3. In contrast to the findings from studies 1 to 5, no significant alterations were detected in serum total, primary, secondary, conjugated, and unconjugated bile acids in the study 6.

Compared the characteristics of the control and NAFLD subjects, it is noticed that the control subjects in study 6 were severely obese with BMI 39.4 ± 5.9, while the control subjects in studies 1–5 have normal or close to normal body weights with BMI ranging from 24.5 ± 2.6 to 27.3 ± 5.8. The BMI values were matched between NAFLD patients and control subjects in study 6 but significantly different in studies 1–5. Compared with serum bile acid levels in a healthy population [85], the control subjects in study 6 had markedly increased total, primary, secondary, conjugated, and unconjugated bile acids. The results indicate that obesity or increased BMI is a contributing factor to the dysregulation of serum bile acids. Indeed, several studies have reported that subjects with overweight or obese had increased serum bile acid concentrations [85, 95, 96].

The second characteristic of the studied subjects that is different between study 6 and studies 1–5 is the status of insulin resistance in the control subjects. The serum insulin levels and HOMA values in study 6 are markedly higher than those in the other five studies, suggesting that insulin resistance is a contributing factor for the dysregulation of serum bile acids. Indeed, when all the subjects (NAFLD and control patients) in the study 6 were separated by insulin resistance status, primary bile acids, unconjugated bile acids, and non-12α bile acids, total CA and total CDCA were significantly increased in subjects with


#### **Table 1.**

*Dysregulation of bile acids in adults with NAFLD with characteristics of the studied subjects.*

insulin resistance compared to insulin sensitive subjects regardless of the status of NAFLD. These data strongly suggest that insulin resistance is a contributing factor to the dysregulation of bile acids, which is supported by the findings from previous studies [95–100].

**49**

those patients.

*Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

*2.1.1.2 Serum bile acid profiling in children with NAFLD*

those studies.

The third different characteristic of the NASH patients in study 6 from studies 1 to 5 is the liver injury status. The NASH patients in study 6 exhibited much lower ALT and AST levels than those in the NASH patients in studies 1–5, indicating that the NASH patients in study 6 experienced minimal liver injury, while the NASH patients in other studies exhibited hepatic injury or damage with the ALT and AST levels above the physiological values. As discussed earlier, it is well established that liver injury potentially can cause dysregulation of bile acids [38–42]. The differing liver injury statuses may provide an explanation for the discrepancy in bile acid alterations between the study 6 and the other five studies. Taken together, the characteristic variations in BMI, insulin resistance, and hepatic injury of the studied subjects may all contribute to the inconsistency in serum bile acid levels reported in

There are three studies conducted with children from ages 4 to 17 years old. In one study with 11 healthy controls (average age 12.8 years) and 16 patients with NASH (average age 13.7 years), total serum bile acid levels were significantly elevated by threefold in children with NASH compared to healthy controls [101]. More specifically, the absolute concentrations of CA, CDCA, DCA, and UDCA were all markedly increased. The percentages of CA and DCA in the total bile acid pools were significantly increased, while the percentages of CDCA in the pools were decreased with no changes in UDCA. It is noted that both ALT and AST levels in patients with NASH were increased, indicating hepatic injury in those patients. The children with NASH also exhibited insulin resistance with an average HOMA value

In the second study with 105 healthy controls at ages 9.3 ± 2.5 and 92 children with NAFLD, which were further classified into two groups based on the stages of fibrosis: NAFLD-F0 group at ages 10.9 ± 3.7 and F ≥ 1 group at ages 11.5 ± 1.9, total serum bile acids were significantly decreased from 3.6 μM in control subjects to 1.73 μM in nonfibrotic (NAFLD-F0) patients accompanied by decreased glycineconjugated bile acids and slightly increased taurine-conjugated and unconjugated

NAFLD with fibrosis (NAFLD-F ≥ 1) were also decreased to 2.45 μM. Comparison between the two NAFLD groups, the serum bile acid levels increased by 41.9% in the NAFLD-F ≥ 1 group. These data indicate that serum bile acid levels decrease in the early stage of NAFLD, followed by an increase as NAFLD progresses to fibrosis. No significant differences were detected in the compositions of serum total bile acid pools among the groups. It should be mentioned that compared with control subjects (BMI 18.8 ± 4.2), children in the NAFLD groups were overweight (BMI > 26) with significantly elevated glucose and insulin levels. In addition, NAFLD patients had elevated AST and ALT levels, indicating hepatic injury in

In a most recent third study with 35 control children at ages 12.8 ± 4.2 and 41 NAFLD children at ages 13.7 ± 2.4, which were further divided into mild and moderate/severe NAFLD groups, no significant alternations in serum total, primary, and secondary bile acids were detected in children with NAFLD compared to control subjects [103]. Most of individual bile acid species (CA, CDCA, DCA, and LCA), conjugated and unconjugated bile acids, were comparable among the groups. Significant differences were only detected for unconjugated CDCA and unconjugated primary bile acids (CDCA + CA). Unconjugated CDCA and primary bile acids increased by 1.58-fold and 1.43-fold, respectively, in NAFLD children. After adjusted for age, sex, HOMA and BMI, unconjugated DCA, conjugated DCA

of 4.3 ± 2.8 and overweight or obese with an average BMI of 33.8 ± 7.7.

bile acids [102]. The total serum bile acids in patients with more advanced

#### *Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

*Nonalcoholic Fatty Liver Disease - An Update*

**[87]**

Steatosis 11 25

Steatosis 43.5 ± 10.7 54.6 ± 10.1

Steatosis 34.0 ± 4.0 32.6 ± 5.4

Steatosis 20.6 ± 9.0 N/A

Steatosis 2.6 ± 1.1 5 (20%)

NASH 3.26 ± 1.6 13 ± 8.7 23 (62%)

Steatosis 44.4 ± 30.0 45.5 ± 24.0

Steatosis 31.4 ± 15.4 45.6 ± 51.9

Unconjugated N/A\* Increased

HOMA-IR Control 0.9 ± 0.4 1.6 ± 0.6 1 (4.2%)

**Study 2 [88]**

Control 25 15 24 14 46 26

NASH 24 7 37 7 13 32

NASH 43.6 ± 12.6 48 ± 10 58.0 ± 8.8 48 ± 10 62.5 ± 16.5 41.3 ± 11.9

NASH 34.8 ± 4.7 32.0 ± 5.2 34.4 ± 4.2 32 ± 5.2 25.5 ± 2.8 40.2 ± 5.8

NASH 26.5 ± 14.9 40 ± 27 N/A 40 ± 27 N/A 18.9 ± 11.4

diabetic

diabetic

diabetic

Age Control 42.6 ± 9.2 43 ± 12 39.2 ± 12.4 42 ± 13 20–39 40.5 ± 11.7

BMI Control 24.5 ± 2.6 25 ± 2.7 27.3 ± 5.8 26 ± 2.7 <25 39.4 ± 5.9

Insulin Control 7.6 ± 3.1 7.6 ± 2.6 N/A 8.0 ± 3.0 N/A 15.7 ± 9.1

ALT Control 17.6 ± 5.0 33 ± 11 22.7 ± 15.5 33 ± 11 Normal 27.3 ± 15.8

NASH 84.6 ± 58.6 75 ± 36 57.1 ± 29.3 75 ± 36 64.1

AST Control 22.8 ± 5.7 N/A 22.3 ± 11.3 N/A Normal 21.3 ± 11.9

NASH 63.4 ± 46.7 N/A 42.4 ± 18.7 N/A 46.2

but not significant

significant changes

N/A No

*Dysregulation of bile acids in adults with NAFLD with characteristics of the studied subjects.*

Bile acids Total Increased Increased Increased Increased Increased No changes

Primary Increased Increased Increased Increased Increased No changes Secondary Increased Increased Decreased N/A Increased No changes Conjugated Increased Increased Increased Increased Increased No changes

> Significantly altered

No changes N/A Slightly

**Study 3 [89] Study 4** 

**[90]**

**Study 5 [92]**

2.0 ± 1.0 0% DM 3.25 ± 2.05

12 ± 9.0 53.8% DM 4.05 ± 2.65

(34.2–120)

(26.8–79.8)

increased

N/A N/A No changes

36.5 ± 22.7

24.6 ± 17.5

No changes

**Study 6 [93]**

**Features Subjects Study 1** 

Sample size

**48**

previous studies [95–100].

*\*N/A, not available.*

**Table 1.**

Compositions of bile acids

insulin resistance compared to insulin sensitive subjects regardless of the status of NAFLD. These data strongly suggest that insulin resistance is a contributing factor to the dysregulation of bile acids, which is supported by the findings from

The third different characteristic of the NASH patients in study 6 from studies 1 to 5 is the liver injury status. The NASH patients in study 6 exhibited much lower ALT and AST levels than those in the NASH patients in studies 1–5, indicating that the NASH patients in study 6 experienced minimal liver injury, while the NASH patients in other studies exhibited hepatic injury or damage with the ALT and AST levels above the physiological values. As discussed earlier, it is well established that liver injury potentially can cause dysregulation of bile acids [38–42]. The differing liver injury statuses may provide an explanation for the discrepancy in bile acid alterations between the study 6 and the other five studies. Taken together, the characteristic variations in BMI, insulin resistance, and hepatic injury of the studied subjects may all contribute to the inconsistency in serum bile acid levels reported in those studies.

#### *2.1.1.2 Serum bile acid profiling in children with NAFLD*

There are three studies conducted with children from ages 4 to 17 years old. In one study with 11 healthy controls (average age 12.8 years) and 16 patients with NASH (average age 13.7 years), total serum bile acid levels were significantly elevated by threefold in children with NASH compared to healthy controls [101]. More specifically, the absolute concentrations of CA, CDCA, DCA, and UDCA were all markedly increased. The percentages of CA and DCA in the total bile acid pools were significantly increased, while the percentages of CDCA in the pools were decreased with no changes in UDCA. It is noted that both ALT and AST levels in patients with NASH were increased, indicating hepatic injury in those patients. The children with NASH also exhibited insulin resistance with an average HOMA value of 4.3 ± 2.8 and overweight or obese with an average BMI of 33.8 ± 7.7.

In the second study with 105 healthy controls at ages 9.3 ± 2.5 and 92 children with NAFLD, which were further classified into two groups based on the stages of fibrosis: NAFLD-F0 group at ages 10.9 ± 3.7 and F ≥ 1 group at ages 11.5 ± 1.9, total serum bile acids were significantly decreased from 3.6 μM in control subjects to 1.73 μM in nonfibrotic (NAFLD-F0) patients accompanied by decreased glycineconjugated bile acids and slightly increased taurine-conjugated and unconjugated bile acids [102]. The total serum bile acids in patients with more advanced NAFLD with fibrosis (NAFLD-F ≥ 1) were also decreased to 2.45 μM. Comparison between the two NAFLD groups, the serum bile acid levels increased by 41.9% in the NAFLD-F ≥ 1 group. These data indicate that serum bile acid levels decrease in the early stage of NAFLD, followed by an increase as NAFLD progresses to fibrosis. No significant differences were detected in the compositions of serum total bile acid pools among the groups. It should be mentioned that compared with control subjects (BMI 18.8 ± 4.2), children in the NAFLD groups were overweight (BMI > 26) with significantly elevated glucose and insulin levels. In addition, NAFLD patients had elevated AST and ALT levels, indicating hepatic injury in those patients.

In a most recent third study with 35 control children at ages 12.8 ± 4.2 and 41 NAFLD children at ages 13.7 ± 2.4, which were further divided into mild and moderate/severe NAFLD groups, no significant alternations in serum total, primary, and secondary bile acids were detected in children with NAFLD compared to control subjects [103]. Most of individual bile acid species (CA, CDCA, DCA, and LCA), conjugated and unconjugated bile acids, were comparable among the groups. Significant differences were only detected for unconjugated CDCA and unconjugated primary bile acids (CDCA + CA). Unconjugated CDCA and primary bile acids increased by 1.58-fold and 1.43-fold, respectively, in NAFLD children. After adjusted for age, sex, HOMA and BMI, unconjugated DCA, conjugated DCA (GDCA and TDCA), and total DCA were significantly lower in NAFLD patients than those in the control group. Meanwhile, serum GLCA and total conjugated LCA (GCA + TLCA) were significantly decreased in NAFLD patients compared to control subjects.

The findings from the three clinical studies with detailed characteristics of the studied subjects are summarized in **Table 2**. The results from the three studies are largely inconsistent. In the second study with a larger size of samples, serum bile acid levels decrease in early stage of NAFLD and then increase during progression to fibrosis, but the levels were still below that in control subjects. In the third study with a medium size of samples, no significant alternations in serum total, primary, and secondary bile acids were detected in children with NAFLD. However, in the first study with smallest size of samples, total serum bile acid levels and compositions were significantly altered in patients with NASH compared to healthy controls.

Comparing the characteristics of studied subjects, the trend of bile acid alterations from decrease to no changes to increase correlates with the trend of gradually increased BMI from slightly overweight (26.5 ± 3.59) to severe overweight (29.6 ± 5.2) to obese (33.8 ± 7.7). Such correlation indicates that NAFLD-associated overweight or obesity may play important roles in influencing the bile acid homeostasis in children as well [85, 95, 96]. Another possible factor playing a role in bile acid dysregulation under the NAFLD conditions is the HOMA values, which were increased from 3.0 ± 3.0 in study 1 to 4.1 ± 3.2 in study 2 and 4.3 ± 2.8 in study 3. The differences in sample sizes of the studies certainly also contribute to the variations of bile acid levels among the studies. The sample sizes in the control groups ranged from 105 in study 2 to 35 in study 3 to 11 in study 1. Meanwhile, the sample sizes for NAFLD subjects were decreased from 92 in study 2 to 42 in study 3 to 16 in study 1. Taken together, serum bile acid levels were differentially altered in children with NAFLD. The differences in BMI, insulin resistance, and sample sizes may contribute to the variations of serum bile acids detected among the three studies.

#### *2.1.2 Hepatic bile acids*

There are three relevant studies investigating hepatic bile acids in patients with NAFLD. In the first study with 15 NASH patients and 8 control subjects, total hepatic bile acids were significantly increased in patients with NASH compared to control subjects [104]. The concentrations of individual bile acid species including CA, CDCA, and DCA were markedly higher in patients with NASH than those in the controls. It was also found that hepatic total bile acid levels were significantly correlated with hepatic inflammation status. Meanwhile, CDCA concentrations were positively correlated with fibrosis status in patients with NASH.

In a second study with liver tissues from 17 normal control subjects, 4 patients with simple steatosis, and 37 patients with NASH, significant alterations in hepatic bile acids were detected in NASH patients [105]. Hepatic CA and GDCA concentrations were markedly decreased by 69 and 91%, respectively, in patients with NASH compared to the control subjects. In contrast, hepatic TCA, TDCA, and GCDCA were significantly increased by approximately three, five, and twofold in NASH patients, respectively. Overall, hepatic total and conjugated bile acid concentrations were significantly higher in patients with NASH than those in controls. On the other hand, unconjugated bile acids were significantly decreased in patients with NASH. In patients with simple steatosis, total, conjugated, and unconjugated bile acids were all decreased without reaching a statistical significance mainly due to a small sample size of the group.

**51**

In a relevant third study with 20 control subjects and 22 diabetic patients, hepatic bile acid concentrations were significantly altered [106]. Among the 22 diabetic patients, 77.7% patients had NAFLD with NAS score of 2 or above. Consistently, majority of patients (68%) were overweight, obese, or severe obese, with hypercholesterolemia being detected in 86.4% of the patients. Total hepatic

*Dysregulation of bile acids in children with NAFLD with characteristics of the studied subjects.*

*Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

Sample size

**Features Subjects Study 1 [104] Study 2 [105] Study 3 [106]**

Age Control 12.8 ± 4.2 16 ± 3 9.3 ± 2.5

BMI Control 19.2 ± 3.4 20.5 ± 4.2 18.8 ± 4.2

Insulin Control N/A\* 3.2 ± 0.6 6.9 ± 4.9

HOMA-IR Control N/A 1.5 ± 0.3 1.3 ± 1.0

ALT Control 19.4 ± 4.4 24 ± 28 14.0 ± 8.5

AST Control 24.4 ± 11.5 25 ± 13 25 ± 8.5

increased

increased

increased

Significantly altered

Conjugated N/A Significantly

Bile acids Total Significantly

Compositions of bile

acids

*\*N/A, not available.*

**Table 2.**

Primary Significantly

Secondary Significantly

Control 11 105 35

Steatosis 27 18 NASH 16 65 23

Steatosis 10 ± 5 10.9 ± 3.7 NASH 13.7 ± 2.4 12 ± 5 11.5 ± 1.9

Steatosis 26.8 ± 3.9 26.0 ± 5.1 NASH 33.8 ± 7.7 26.5 ± 3.5 29.6 ± 5.2

Steatosis 11.2 ± 4.8 11.0 ± 5.9 NASH N/A 12.4 ± 6.0 17.5 ± 11.4

Steatosis 2.6 ± 1.8 2.6 ± 1.5 NASH 4.3 ± 2.8 3.0 ± 3.0 4.1 ± 3.2

Steatosis 62 ± 20 25.5 ± 17 NASH 54.1 ± 29.7 87 ± 59 68.5 ± 42.0

Steatosis 40 ± 8 23 ± 6.7 NASH 33.9 ± 15.0 61 ± 29 40.9 ± 24.7

Unconjugated N/A Slightly increased No significant

Significantly decreased

Significantly decreased

decreased

Slightly decreased No significant

No changes No changes

No significant changes

No significant changes

changes

No significant changes

changes


#### *Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

*Nonalcoholic Fatty Liver Disease - An Update*

control subjects.

controls.

*2.1.2 Hepatic bile acids*

small sample size of the group.

(GDCA and TDCA), and total DCA were significantly lower in NAFLD patients than those in the control group. Meanwhile, serum GLCA and total conjugated LCA (GCA + TLCA) were significantly decreased in NAFLD patients compared to

The findings from the three clinical studies with detailed characteristics of the studied subjects are summarized in **Table 2**. The results from the three studies are largely inconsistent. In the second study with a larger size of samples, serum bile acid levels decrease in early stage of NAFLD and then increase during progression to fibrosis, but the levels were still below that in control subjects. In the third study with a medium size of samples, no significant alternations in serum total, primary, and secondary bile acids were detected in children with NAFLD. However, in the first study with smallest size of samples, total serum bile acid levels and compositions were significantly altered in patients with NASH compared to healthy

Comparing the characteristics of studied subjects, the trend of bile acid alterations from decrease to no changes to increase correlates with the trend of gradually increased BMI from slightly overweight (26.5 ± 3.59) to severe overweight (29.6 ± 5.2) to obese (33.8 ± 7.7). Such correlation indicates that NAFLD-associated overweight or obesity may play important roles in influencing the bile acid homeostasis in children as well [85, 95, 96]. Another possible factor playing a role in bile acid dysregulation under the NAFLD conditions is the HOMA values, which were increased from 3.0 ± 3.0 in study 1 to 4.1 ± 3.2 in study 2 and 4.3 ± 2.8 in study 3. The differences in sample sizes of the studies certainly also contribute to the variations of bile acid levels among the studies. The sample sizes in the control groups ranged from 105 in study 2 to 35 in study 3 to 11 in study 1. Meanwhile, the sample sizes for NAFLD subjects were decreased from 92 in study 2 to 42 in study 3 to 16 in study 1. Taken together, serum bile acid levels were differentially altered in children with NAFLD. The differences in BMI, insulin resistance, and sample sizes may contribute to the variations of serum bile acids detected among the three studies.

There are three relevant studies investigating hepatic bile acids in patients with

In a second study with liver tissues from 17 normal control subjects, 4 patients with simple steatosis, and 37 patients with NASH, significant alterations in hepatic bile acids were detected in NASH patients [105]. Hepatic CA and GDCA concentrations were markedly decreased by 69 and 91%, respectively, in patients with NASH compared to the control subjects. In contrast, hepatic TCA, TDCA, and GCDCA were significantly increased by approximately three, five, and twofold in NASH patients, respectively. Overall, hepatic total and conjugated bile acid concentrations were significantly higher in patients with NASH than those in controls. On the other hand, unconjugated bile acids were significantly decreased in patients with NASH. In patients with simple steatosis, total, conjugated, and unconjugated bile acids were all decreased without reaching a statistical significance mainly due to a

NAFLD. In the first study with 15 NASH patients and 8 control subjects, total hepatic bile acids were significantly increased in patients with NASH compared to control subjects [104]. The concentrations of individual bile acid species including CA, CDCA, and DCA were markedly higher in patients with NASH than those in the controls. It was also found that hepatic total bile acid levels were significantly correlated with hepatic inflammation status. Meanwhile, CDCA concentrations

were positively correlated with fibrosis status in patients with NASH.

**50**

#### **Table 2.**

*Dysregulation of bile acids in children with NAFLD with characteristics of the studied subjects.*

In a relevant third study with 20 control subjects and 22 diabetic patients, hepatic bile acid concentrations were significantly altered [106]. Among the 22 diabetic patients, 77.7% patients had NAFLD with NAS score of 2 or above. Consistently, majority of patients (68%) were overweight, obese, or severe obese, with hypercholesterolemia being detected in 86.4% of the patients. Total hepatic

bile acids were markedly reduced by 53% in diabetic patients compared to control subjects. The significant decrease in total bile acids is largely due to the marked reduction in conjugated bile acids. On the other hand, unconjugated bile acids were slightly increased by 33% without reaching a statistical significance. Among the conjugated bile acids, both glycine and taurine conjugated bile acids were significantly reduced in diabetic patients. However, the reductions were more severe in glycine conjugated than taurine-conjugated bile acids.

In summary, no clear consensus can be reached for hepatic bile acid profiles in patients with NAFLD. Both increases and decreases of hepatic bile acids were reported. Some specific bile acid species were markedly increased, while other species significantly decreased in the same patients. From the limited clinical studies, it can be concluded that hepatic bile acid homeostasis is dysregulated in patients with NAFLD. However, due to the complexity of bile acid regulation, variations in characteristics and stages of NAFLD patients, and lack of high quality clinical studies, it largely remains to be determined by the effects of NAFLD on hepatic bile acid homeostasis.

#### *2.1.3 Fecal and urine bile acids*

There are only one study investigating fecal bile acids in patients with NAFLD. The study has 25 healthy controls, 12 patients with steatosis, and 17 patients with NASH [107]. Total fecal bile acid levels were significantly higher in patients with NASH compared to healthy controls. Meanwhile, total fecal bile acids also showed an increased trend in steatotic patients without reaching a statistical significance. Primary, secondary, conjugated, and unconjugated bile acid concentrations all exhibited a gradual increase from healthy controls to steatotic to NASH patients. Unconjugated primary bile acids including CA and CDCA were significantly increased in NASH patients compared to healthy controls, while unconjugated secondary bile acids were not significantly different among the three groups. Patients with NASH had significantly higher concentrations of conjugated LCA compared to patients with steatosis. In addition, a higher ratio of primary to secondary bile acids in patients with NASH was also detected. However, the ratio of total conjugated over unconjugated bile acids was not significantly different among the groups. Correlation analysis revealed that fecal unconjugated primary bile acids positively correlated with steatosis, ballooning, fibrosis, NAS scores, and liver injury (AST and ALT levels). The results from the study demonstrated that fecal disposition of bile acids was enhanced in patients with NASH. However, it remains to be determined that such increase in fecal disposition of bile acids is resulted from impairment of intestine reabsorption of bile acids or enhanced biliary excretion of bile acids or both.

There is only one study with 15 healthy controls and 7 NASH patients to investigate urine bile acid profiles in patients with NAFLD. Urine total, primary, secondary, conjugated, and unconjugated bile acids all exhibited a trend of increase without reaching a statistical significance [87]. However, individual bile acid species including DCA, TCA, GCA, and GCDCA were significantly elevated in patients with NASH compared to control subjects. Consistently, total serum bile acid levels were also significantly increased by more than twofold in NASH patients compared to control subjects.

In summary, the findings from clinical studies to evaluate serum, hepatic, and urine bile acid profiles are inconsistent among the studies. The reasons for those inconsistent or even conflicting results are multiple folds. First, bile acid synthesis and serum concentrations fluctuate during the days and nights [108–112].

**53**

acid species.

control diet [119].

*Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

**2.2 Altered bile acid profiles in NAFLD animal models**

ing the alterations.

Although most of the samples were collected after fasting, there was no mentioning on exactly when the samples were collected in the studies. Second, NAFLD represents a spectrum of pathological liver conditions from simple steatosis to NASH with or without fibrosis. The severity of bile acid dysregulation appears NAFLD stage dependent. Bile acid alterations gets worsening in patients with advanced stages of NAFLD, such as NASH, compared to the patients with simple steatosis [86, 88, 101, 102, 107]. Some studies differentiate NAFLD patients into simple steatosis and NASH [86, 88, 101, 102, 107], while the others [38, 87, 89, 91, 103] do not, which certainly influences the outcomes of the studies. Third, NAFLD is often associated with various metabolic conditions, especially obesity and insulin resistance/diabetes. It has been reported that obesity and insulin resistance directly impacts bile acid homeostasis [85, 95–100]. Fourth, selection of the control groups varies from study to study [38, 86–89, 91], which certainly contributes to the discrepancy of the outcomes among the studies. Finally, the sizes of samples are relatively small in most of the studies with individual variations potentially mask-

Several rodent models for NAFLD have been developed [113–115], including high-fat cholesterol (HFC) and methionine- and choline-deficient (MCD) diet-induced or genetic deficient models, including leptin-deficient *ob/ob*, leptin receptor-deficient *db/db* mice, and *fa/fa* rats. Several preclinical studies were conducted to investigate the effects of NAFLD on bile acid homeostasis using NAFLD mouse or rat models. In one study, NAFLD was induced in rats with HFC diet [116]. Total hepatic bile acids were significantly increased in rats on HFC diet for 2 weeks. Primary, secondary, conjugated, and unconjugated bile acid concentrations were all increased after 2 weeks on HFC diet. Most bile acid species remained higher in rats on HFC diet for 8 and 14 weeks than those on regular diet. However, the levels of CA and DCA species declined from their peaks at 2 weeks, while CDCA species persistently increased for the entire treatment. In addition, CDCA species positively correlated with macrovesicular steatosis score, serum ALT levels, and quantified fibrotic area. Among the conjugated bile acids, glycine-conjugated bile acid species (GCA, GCDCA, GDCA, GLCA, and GUDCA) were predominate over taurineconjugated bile acid species and positively correlated with macrovesicular steatosis score. The finding demonstrated that bile acid homeostasis is severely disrupted in HFC diet-induced NAFLD rats, especially the CDCA and glycine-conjugated bile

In another study with MCD-induced NASH mouse model, markedly increased serum concentrations of taurine-conjugated CA and β-muricholate (βMCA) were detected in mice on MCD diet for 2 or 8 weeks compared to mice on control diet, indicating dysregulation of serum bile acid in mice with NASH [117]. Similar findings were reported with *ob/ob* mouse model. Serum total bile acid concentrations were markedly elevated by sevenfold from 1.9 ± 1.0 μM in wt control mice to 14.9 ± 5.4 μM in *ob/ob* mice [118]. In contrast to the findings from the studies described above, a more recent study showed that total serum bile acid concentrations were not significantly different in HFD-induced NAFLD mice than mice on

Taken together, bile acid homeostasis is disrupted in NAFLD rodent models. Serum bile acid levels were markedly elevated in most of the studies. However, variations in serum bile acid concentrations exist in different NAFLD rodent

models, may reflecting species difference between mouse and rat.

*Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

*Nonalcoholic Fatty Liver Disease - An Update*

acid homeostasis.

bile acids or both.

to control subjects.

*2.1.3 Fecal and urine bile acids*

glycine conjugated than taurine-conjugated bile acids.

bile acids were markedly reduced by 53% in diabetic patients compared to control subjects. The significant decrease in total bile acids is largely due to the marked reduction in conjugated bile acids. On the other hand, unconjugated bile acids were slightly increased by 33% without reaching a statistical significance. Among the conjugated bile acids, both glycine and taurine conjugated bile acids were significantly reduced in diabetic patients. However, the reductions were more severe in

In summary, no clear consensus can be reached for hepatic bile acid profiles in patients with NAFLD. Both increases and decreases of hepatic bile acids were reported. Some specific bile acid species were markedly increased, while other species significantly decreased in the same patients. From the limited clinical studies, it can be concluded that hepatic bile acid homeostasis is dysregulated in patients with NAFLD. However, due to the complexity of bile acid regulation, variations in characteristics and stages of NAFLD patients, and lack of high quality clinical studies, it largely remains to be determined by the effects of NAFLD on hepatic bile

There are only one study investigating fecal bile acids in patients with NAFLD. The study has 25 healthy controls, 12 patients with steatosis, and 17 patients with NASH [107]. Total fecal bile acid levels were significantly higher in patients with NASH compared to healthy controls. Meanwhile, total fecal bile acids also showed an increased trend in steatotic patients without reaching a statistical significance. Primary, secondary, conjugated, and unconjugated bile acid concentrations all exhibited a gradual increase from healthy controls to steatotic to NASH patients. Unconjugated primary bile acids including CA and CDCA were significantly increased in NASH patients compared to healthy controls, while unconjugated secondary bile acids were not significantly different among the three groups. Patients with NASH had significantly higher concentrations of conjugated LCA compared to patients with steatosis. In addition, a higher ratio of primary to secondary bile acids in patients with NASH was also detected. However, the ratio of total conjugated over unconjugated bile acids was not significantly different among the groups. Correlation analysis revealed that fecal unconjugated primary bile acids positively correlated with steatosis, ballooning, fibrosis, NAS scores, and liver injury (AST and ALT levels). The results from the study demonstrated that fecal disposition of bile acids was enhanced in patients with NASH. However, it remains to be determined that such increase in fecal disposition of bile acids is resulted from impairment of intestine reabsorption of bile acids or enhanced biliary excretion of

There is only one study with 15 healthy controls and 7 NASH patients to investigate urine bile acid profiles in patients with NAFLD. Urine total, primary, secondary, conjugated, and unconjugated bile acids all exhibited a trend of increase without reaching a statistical significance [87]. However, individual bile acid species including DCA, TCA, GCA, and GCDCA were significantly elevated in patients with NASH compared to control subjects. Consistently, total serum bile acid levels were also significantly increased by more than twofold in NASH patients compared

In summary, the findings from clinical studies to evaluate serum, hepatic, and urine bile acid profiles are inconsistent among the studies. The reasons for those inconsistent or even conflicting results are multiple folds. First, bile acid synthesis and serum concentrations fluctuate during the days and nights [108–112].

**52**

Although most of the samples were collected after fasting, there was no mentioning on exactly when the samples were collected in the studies. Second, NAFLD represents a spectrum of pathological liver conditions from simple steatosis to NASH with or without fibrosis. The severity of bile acid dysregulation appears NAFLD stage dependent. Bile acid alterations gets worsening in patients with advanced stages of NAFLD, such as NASH, compared to the patients with simple steatosis [86, 88, 101, 102, 107]. Some studies differentiate NAFLD patients into simple steatosis and NASH [86, 88, 101, 102, 107], while the others [38, 87, 89, 91, 103] do not, which certainly influences the outcomes of the studies. Third, NAFLD is often associated with various metabolic conditions, especially obesity and insulin resistance/diabetes. It has been reported that obesity and insulin resistance directly impacts bile acid homeostasis [85, 95–100]. Fourth, selection of the control groups varies from study to study [38, 86–89, 91], which certainly contributes to the discrepancy of the outcomes among the studies. Finally, the sizes of samples are relatively small in most of the studies with individual variations potentially masking the alterations.

#### **2.2 Altered bile acid profiles in NAFLD animal models**

Several rodent models for NAFLD have been developed [113–115], including high-fat cholesterol (HFC) and methionine- and choline-deficient (MCD) diet-induced or genetic deficient models, including leptin-deficient *ob/ob*, leptin receptor-deficient *db/db* mice, and *fa/fa* rats. Several preclinical studies were conducted to investigate the effects of NAFLD on bile acid homeostasis using NAFLD mouse or rat models. In one study, NAFLD was induced in rats with HFC diet [116]. Total hepatic bile acids were significantly increased in rats on HFC diet for 2 weeks. Primary, secondary, conjugated, and unconjugated bile acid concentrations were all increased after 2 weeks on HFC diet. Most bile acid species remained higher in rats on HFC diet for 8 and 14 weeks than those on regular diet. However, the levels of CA and DCA species declined from their peaks at 2 weeks, while CDCA species persistently increased for the entire treatment. In addition, CDCA species positively correlated with macrovesicular steatosis score, serum ALT levels, and quantified fibrotic area. Among the conjugated bile acids, glycine-conjugated bile acid species (GCA, GCDCA, GDCA, GLCA, and GUDCA) were predominate over taurineconjugated bile acid species and positively correlated with macrovesicular steatosis score. The finding demonstrated that bile acid homeostasis is severely disrupted in HFC diet-induced NAFLD rats, especially the CDCA and glycine-conjugated bile acid species.

In another study with MCD-induced NASH mouse model, markedly increased serum concentrations of taurine-conjugated CA and β-muricholate (βMCA) were detected in mice on MCD diet for 2 or 8 weeks compared to mice on control diet, indicating dysregulation of serum bile acid in mice with NASH [117]. Similar findings were reported with *ob/ob* mouse model. Serum total bile acid concentrations were markedly elevated by sevenfold from 1.9 ± 1.0 μM in wt control mice to 14.9 ± 5.4 μM in *ob/ob* mice [118]. In contrast to the findings from the studies described above, a more recent study showed that total serum bile acid concentrations were not significantly different in HFD-induced NAFLD mice than mice on control diet [119].

Taken together, bile acid homeostasis is disrupted in NAFLD rodent models. Serum bile acid levels were markedly elevated in most of the studies. However, variations in serum bile acid concentrations exist in different NAFLD rodent models, may reflecting species difference between mouse and rat.

### **3. Alterations in bile acid synthesis in subjects with NAFLD**

#### **3.1 Alterations in bile acid synthesis in patients with NAFLD**

Primary bile acids CA and CDCA are synthesized in the liver through either the classical or alternative synthesis pathways. In the intestine, CA can be converted into secondary bile acid DCA, while CDCA is converted into secondary bile acids LCA or UDCA (**Figure 1**). Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme in the classical pathway, while CYP8B1 is the rate-limiting enzyme for the production of CA. The two rate-limiting enzymes for the alternative pathway are CYP27A1 and CYP7B1 (**Figure 1**). Alterations in the expression levels of rate-limiting enzymes in the bile acid synthesis pathways result in dysregulation of bile acid homeostasis. A number of clinical studies have conducted to investigate the effects of NAFLD on bile acid synthesis.

#### *3.1.1 CYP7A1*

There are eight clinical studies investigating the expression of CYP7A1 in patients with NAFLD. Most of the studies revealed that CYP7A1 expression was dysregulated in patients with NAFLD. Among the eight studies, the results from five studies showed that the mRNA expression levels of CYP7A1 were significantly increased in patients with NAFLD [88, 91, 94, 101, 120], indicating that bile acid synthesis through the classical pathway is enhanced in patients with NAFLD. However, in a study with 17 normal control subjects, 4 patients with simple steatosis, and 37 patients with NASH, CYP7A1 expression was not altered in patients with steatosis or NASH [105]. In another study with 6 lean healthy controls, 20 obese normal controls, 20 patients with simple steatosis, and 20 patients with NASH [121], CYP7A1 mRNA expression significantly increased in obese normal control subjects, patients with steatosis, and NASH compared to healthy lean subjects. However, at the protein level, CYP7A1 expression was comparable in obese normal controls compared to healthy lean subjects. More

#### **Figure 1.**

*Primary bile acids CDCA and CA are synthesized in the liver through classical (CYP7A1) and alternative (CYP27A1) bile acid synthesis pathways and converted into secondary bile acids LCA, UDCA, and DCA in the intestine.*

**55**

*Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

CYP7A1 under the NAFLD condition.

of NAFLD on CYP27A1 expression are inclusive.

on CYP8B1 and CYP7B1 expression.

not change in patients with NAFLD.

NAFLD [122].

*3.1.2 CYP27A1*

*3.1.3 Other enzymes*

*3.1.4 C4*

strikingly, CYP7A1 protein expression was markedly decreased in patients with steatosis and especially with NASH, indicating that bile acid synthesis through the classical pathway is reduced in patients with NAFLD. In a study with 78 NAFLD patients, the subjects were divided into three groups based on the NAS scores, NAS 1–2, NAS 3–4, and NAS 5–8. The mRNA expression levels of CYP7A1 were comparable among the three groups, indicating that bile acid synthesis through the classical pathway remains unchanged during the progression of

Taken together, CYP7A1 expression was largely upregulated in patients with

There are three clinical studies evaluating the effects of NAFLD on the expression of CYP27A1. The findings from the three studies are largely inconsistent. In one study, the expression levels of CYP27A1 were significantly induced in patients with NAFLD [101]. In contrast, a second study reported that CYP27A1 expression was significantly decreased in patients with NAFLD compared to control subjects [105]. A third study showed that CYP27A1 expression was not altered in NAFLD subjects [121]. Therefore, it can be concluded that the effects

There are a couple of studies investigating other enzymes involved in bile acid synthesis, including CYP8B1 and CYP7B1. One study reported that the expression levels of CYP8B1 were decreased, while CYP7B1 levels were increased in patients with NAFLD [105]. The other study revealed that the expression levels of CYP8B1 were significantly increased in patients with NAFLD compared to control subjects [101]. Therefore, additional studies are required to determine the effects of NAFLD

7α-Hydroxy-4-cholesten-3-one (C4) is an intermediate of bile acid synthesis (**Figure 1**) and serves as an indicator for bile acid synthesis *in vivo* [123]. There are three studies investigating serum C4 levels in patients with NAFLD. In one study with 26 NAFLD patients and 16 healthy controls, the serum concentrations of C4 were not significantly different between the two groups, indicating that de novel bile acid synthesis was not changed in patients with NAFLD [38]. Consistent results were obtained with a second study which includes 26 healthy controls and 32 patients with NASH. The serum C4 concentrations were not significantly altered in patients with NASH compared to the control subjects [90]. However, in the third study with 25 healthy controls, 12 patients with steatosis, and 16 patients with NASH, serum C4 levels were significantly elevated in patients with steatosis and NASH compared to healthy control subjects, suggesting that bile acid synthesis is enhanced in patients with NAFLD. Correlation analysis revealed that the serum C4 concentrations were directly correlated with fecal total bile acid levels in the studied subjects [104]. Taken together, the serum C4 concentrations either increased or did

NAFLD, indicating that bile acid synthesis through the classical pathway is enhanced in patients with NAFLD. Discrepancy in CYP7A1 mRNA and protein levels was noted, indicating the importance of post-transcriptional regulation of

#### *Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

strikingly, CYP7A1 protein expression was markedly decreased in patients with steatosis and especially with NASH, indicating that bile acid synthesis through the classical pathway is reduced in patients with NAFLD. In a study with 78 NAFLD patients, the subjects were divided into three groups based on the NAS scores, NAS 1–2, NAS 3–4, and NAS 5–8. The mRNA expression levels of CYP7A1 were comparable among the three groups, indicating that bile acid synthesis through the classical pathway remains unchanged during the progression of NAFLD [122].

Taken together, CYP7A1 expression was largely upregulated in patients with NAFLD, indicating that bile acid synthesis through the classical pathway is enhanced in patients with NAFLD. Discrepancy in CYP7A1 mRNA and protein levels was noted, indicating the importance of post-transcriptional regulation of CYP7A1 under the NAFLD condition.

#### *3.1.2 CYP27A1*

*Nonalcoholic Fatty Liver Disease - An Update*

bile acid synthesis.

*3.1.1 CYP7A1*

**3. Alterations in bile acid synthesis in subjects with NAFLD**

Primary bile acids CA and CDCA are synthesized in the liver through either the classical or alternative synthesis pathways. In the intestine, CA can be converted into secondary bile acid DCA, while CDCA is converted into secondary bile acids LCA or UDCA (**Figure 1**). Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme in the classical pathway, while CYP8B1 is the rate-limiting enzyme for the production of CA. The two rate-limiting enzymes for the alternative pathway are CYP27A1 and CYP7B1 (**Figure 1**). Alterations in the expression levels of rate-limiting enzymes in the bile acid synthesis pathways result in dysregulation of bile acid homeostasis. A number of clinical studies have conducted to investigate the effects of NAFLD on

There are eight clinical studies investigating the expression of CYP7A1 in patients with NAFLD. Most of the studies revealed that CYP7A1 expression was dysregulated in patients with NAFLD. Among the eight studies, the results from five studies showed that the mRNA expression levels of CYP7A1 were significantly increased in patients with NAFLD [88, 91, 94, 101, 120], indicating that bile acid synthesis through the classical pathway is enhanced in patients with NAFLD. However, in a study with 17 normal control subjects, 4 patients with simple steatosis, and 37 patients with NASH, CYP7A1 expression was not altered in patients with steatosis or NASH [105]. In another study with 6 lean healthy controls, 20 obese normal controls, 20 patients with simple steatosis, and 20 patients with NASH [121], CYP7A1 mRNA expression significantly increased in obese normal control subjects, patients with steatosis, and NASH compared to healthy lean subjects. However, at the protein level, CYP7A1 expression was comparable in obese normal controls compared to healthy lean subjects. More

*Primary bile acids CDCA and CA are synthesized in the liver through classical (CYP7A1) and alternative (CYP27A1) bile acid synthesis pathways and converted into secondary bile acids LCA, UDCA, and DCA in* 

**3.1 Alterations in bile acid synthesis in patients with NAFLD**

**54**

**Figure 1.**

*the intestine.*

There are three clinical studies evaluating the effects of NAFLD on the expression of CYP27A1. The findings from the three studies are largely inconsistent. In one study, the expression levels of CYP27A1 were significantly induced in patients with NAFLD [101]. In contrast, a second study reported that CYP27A1 expression was significantly decreased in patients with NAFLD compared to control subjects [105]. A third study showed that CYP27A1 expression was not altered in NAFLD subjects [121]. Therefore, it can be concluded that the effects of NAFLD on CYP27A1 expression are inclusive.

#### *3.1.3 Other enzymes*

There are a couple of studies investigating other enzymes involved in bile acid synthesis, including CYP8B1 and CYP7B1. One study reported that the expression levels of CYP8B1 were decreased, while CYP7B1 levels were increased in patients with NAFLD [105]. The other study revealed that the expression levels of CYP8B1 were significantly increased in patients with NAFLD compared to control subjects [101]. Therefore, additional studies are required to determine the effects of NAFLD on CYP8B1 and CYP7B1 expression.

#### *3.1.4 C4*

7α-Hydroxy-4-cholesten-3-one (C4) is an intermediate of bile acid synthesis (**Figure 1**) and serves as an indicator for bile acid synthesis *in vivo* [123]. There are three studies investigating serum C4 levels in patients with NAFLD. In one study with 26 NAFLD patients and 16 healthy controls, the serum concentrations of C4 were not significantly different between the two groups, indicating that de novel bile acid synthesis was not changed in patients with NAFLD [38]. Consistent results were obtained with a second study which includes 26 healthy controls and 32 patients with NASH. The serum C4 concentrations were not significantly altered in patients with NASH compared to the control subjects [90]. However, in the third study with 25 healthy controls, 12 patients with steatosis, and 16 patients with NASH, serum C4 levels were significantly elevated in patients with steatosis and NASH compared to healthy control subjects, suggesting that bile acid synthesis is enhanced in patients with NAFLD. Correlation analysis revealed that the serum C4 concentrations were directly correlated with fecal total bile acid levels in the studied subjects [104]. Taken together, the serum C4 concentrations either increased or did not change in patients with NAFLD.

#### **3.2 Alterations in bile acid synthesis in NAFLD animal models**

There are five studies investigating the expression of enzymes involved in bile acid synthesis. In one study with high fat diet (HFD)–induced NAFLD mice, the mRNA expression levels of Cyp7a1 and Cyp8b1 were markedly decreased compared to control mice on regular diet [124], indicating that de novel bile acid synthesis through the classical pathway is reduced in NAFLD mice. Consistent with the finding, a study with *ob/ob* mice, the expression levels of Cyp7a1 were significantly decreased in *ob/ob* mice compared to lean wt mice [118]. However, in a study with HFD/streptozotocin (STZ)-induced NAFLD rats, the expression levels of Cyp7a1 were dramatically increased, while the expression levels of Cyp27a1 and Cyp7b1 were also significantly induced in NAFLD rats compared to control rats [125]. The findings indicate that bile acid synthesis through both classical and alternative pathway is increased in HFD/STZ-induced NAFLD rats. On the other hand, in one study with MCD-induced simple steatotic rats, the expression levels of Cyp7a1 were comparable between the steatotic rats and healthy control rats [126]. Consistently, a study with MCD-induced NASH in mice showed that the expression levels of Cyp7a1 were not altered in mice with NASH compared to control mice [117]. In addition, the expression levels of Cyp27a1 and Cyp8b1 were not significantly changed in steatotic mice compared to healthy control mice. The findings indicate that both classical and alternative bile acid synthesis pathways are not impaired in MCD-induced NASH mice. In summary, the effects of NAFLD on Cyp7a1, Cyp27a1, and Cyp8b1 expression are inconclusive in NAFLD rodent models, which are to a large extent different from the findings in patients with NAFLD, especially for CYP7A1.

#### **4. Alterations in bile acid transporters in subjects with NAFLD**

The enterohepatic circulation of bile acids is mediated by a series of bile acid transporters in the liver and intestine (**Figure 2**). After synthesis in the liver, bile acids are excreted into bile through the bile salt export pump (BSEP). Majority of bile acids are actively transported into enterocytes by the apical sodium-dependent bile acid transporter (ASBT). Bile acids exit the enterocytes on the basolateral side via the heterodimeric organic solute transporter α and β (OSTα/β) and then return to the liver through the Na<sup>+</sup> /taurocholate cotransporting polypeptide (NTCP), completing the circulation. In the liver, other transporters are also capable of transport bile acids, including multidrug resistance associated protein 2 (MRP2) on the canalicular membrane and multidrug resistance-associated protein 3 (MRP3), MRP4, and organic anion-transporting polypeptides (OATP1B1 and OATP1B3) on the basolateral membrane. It should be emphasized that biliary excretion through BSEP is the rate-limiting step in the circulation and bile acid spilling into blood is mediated mainly by MRP3 and MRP4. Alteration in bile acid transporter expression has significant impact on bile acid compartmenting and homeostasis.

#### **4.1 Alterations in bile acid transporters in patients with NAFLD**

#### *4.1.1 BSEP*

As the canalicular bile acid transporter, BSEP expression was dysregulated in patients with NAFLD. Three clinical studies showed that BSEP mRNA expression

**57**

of the patients.

*4.1.2 NTCP*

**Figure 2.**

*Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

was decreased in steatotic or NASH patients compared to control subjects [88, 91, 122]. A different study reported that BSEP mRNA expression levels were increased in patients with NASH compared to the patients with simple steatosis [118]. On the other hand, two studies revealed that BSEP mRNA expression was not altered in patients with NAFLD or diabetes compared to healthy control subjects [101, 106]. Finally, another study reported that BSEP mRNA levels were elevated in lean NAFLD patients but reduced in overweight or obese patients with steatosis or NASH [94], indicating that body weight of the patients influences the expression of BSEP under the NAFLD condition. Taken together, it can be cautiously concluded that BSEP expression was largely decreased in patients with NAFLD. The alterations BSEP expression may be influenced by the body weight

*synthesis by the FXR/SHP and FGF19/FGFR4 synthesis pathway.*

*Enterohepatic circulation of bile acids through a series of bile acid transporters, and regulation of bile acid* 

Three clinical studies showed that NTCP mRNA expression levels were signifi-

cantly upregulated in patients with NAFLD compared to control subjects [88, 101, 94]. However, a different study reported that NTCP mRNA expression was significantly decreased as NAFLD progressed from earlier stage (steatosis) to late stage (NASH) [122]. On the other hand, one study reported that the mRNA levels of NTCP were significantly increased in patients with NASH compared to patients with simple steatosis. However, at the protein level, NTCP expression was significantly reduced in the patients with NASH compared to patients with simple steatosis [127], indicating the dominance of post-transcriptional regulation of NTCP under the NASH condition. Another study with diabetic patients reported that NTCP mRNA expression levels were comparable between diabetic patients

*Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

**Figure 2.**

*Nonalcoholic Fatty Liver Disease - An Update*

**3.2 Alterations in bile acid synthesis in NAFLD animal models**

**4. Alterations in bile acid transporters in subjects with NAFLD**

The enterohepatic circulation of bile acids is mediated by a series of bile acid transporters in the liver and intestine (**Figure 2**). After synthesis in the liver, bile acids are excreted into bile through the bile salt export pump (BSEP). Majority of bile acids are actively transported into enterocytes by the apical sodium-dependent bile acid transporter (ASBT). Bile acids exit the enterocytes on the basolateral side via the heterodimeric organic solute transporter α and β (OSTα/β) and then return

completing the circulation. In the liver, other transporters are also capable of transport bile acids, including multidrug resistance associated protein 2 (MRP2) on the canalicular membrane and multidrug resistance-associated protein 3 (MRP3), MRP4, and organic anion-transporting polypeptides (OATP1B1 and OATP1B3) on the basolateral membrane. It should be emphasized that biliary excretion through BSEP is the rate-limiting step in the circulation and bile acid spilling into blood is mediated mainly by MRP3 and MRP4. Alteration in bile acid transporter expression has significant impact on bile acid compartmenting and

As the canalicular bile acid transporter, BSEP expression was dysregulated in patients with NAFLD. Three clinical studies showed that BSEP mRNA expression

**4.1 Alterations in bile acid transporters in patients with NAFLD**

/taurocholate cotransporting polypeptide (NTCP),

There are five studies investigating the expression of enzymes involved in bile acid synthesis. In one study with high fat diet (HFD)–induced NAFLD mice, the mRNA expression levels of Cyp7a1 and Cyp8b1 were markedly decreased compared to control mice on regular diet [124], indicating that de novel bile acid synthesis through the classical pathway is reduced in NAFLD mice. Consistent with the finding, a study with *ob/ob* mice, the expression levels of Cyp7a1 were significantly decreased in *ob/ob* mice compared to lean wt mice [118]. However, in a study with HFD/streptozotocin (STZ)-induced NAFLD rats, the expression levels of Cyp7a1 were dramatically increased, while the expression levels of Cyp27a1 and Cyp7b1 were also significantly induced in NAFLD rats compared to control rats [125]. The findings indicate that bile acid synthesis through both classical and alternative pathway is increased in HFD/STZ-induced NAFLD rats. On the other hand, in one study with MCD-induced simple steatotic rats, the expression levels of Cyp7a1 were comparable between the steatotic rats and healthy control rats [126]. Consistently, a study with MCD-induced NASH in mice showed that the expression levels of Cyp7a1 were not altered in mice with NASH compared to control mice [117]. In addition, the expression levels of Cyp27a1 and Cyp8b1 were not significantly changed in steatotic mice compared to healthy control mice. The findings indicate that both classical and alternative bile acid synthesis pathways are not impaired in MCD-induced NASH mice. In summary, the effects of NAFLD on Cyp7a1, Cyp27a1, and Cyp8b1 expression are inconclusive in NAFLD rodent models, which are to a large extent different from the findings in patients with NAFLD, especially for

**56**

homeostasis.

*4.1.1 BSEP*

CYP7A1.

to the liver through the Na<sup>+</sup>

*Enterohepatic circulation of bile acids through a series of bile acid transporters, and regulation of bile acid synthesis by the FXR/SHP and FGF19/FGFR4 synthesis pathway.*

was decreased in steatotic or NASH patients compared to control subjects [88, 91, 122]. A different study reported that BSEP mRNA expression levels were increased in patients with NASH compared to the patients with simple steatosis [118]. On the other hand, two studies revealed that BSEP mRNA expression was not altered in patients with NAFLD or diabetes compared to healthy control subjects [101, 106]. Finally, another study reported that BSEP mRNA levels were elevated in lean NAFLD patients but reduced in overweight or obese patients with steatosis or NASH [94], indicating that body weight of the patients influences the expression of BSEP under the NAFLD condition. Taken together, it can be cautiously concluded that BSEP expression was largely decreased in patients with NAFLD. The alterations BSEP expression may be influenced by the body weight of the patients.

#### *4.1.2 NTCP*

Three clinical studies showed that NTCP mRNA expression levels were significantly upregulated in patients with NAFLD compared to control subjects [88, 101, 94]. However, a different study reported that NTCP mRNA expression was significantly decreased as NAFLD progressed from earlier stage (steatosis) to late stage (NASH) [122]. On the other hand, one study reported that the mRNA levels of NTCP were significantly increased in patients with NASH compared to patients with simple steatosis. However, at the protein level, NTCP expression was significantly reduced in the patients with NASH compared to patients with simple steatosis [127], indicating the dominance of post-transcriptional regulation of NTCP under the NASH condition. Another study with diabetic patients reported that NTCP mRNA expression levels were comparable between diabetic patients

and control subjects [106]. In summary, NTCP expression was likely upregulated in NAFLD patients with certain inconsistency.

#### *4.1.3 MRPs*

In one study with NAFLD and one study with diabetic patients, MRP2 mRNA expression levels were not significantly altered in NAFLD and diabetic patients compared to control subjects [88, 106]. Another study reported that MRP2 mRNA expression levels were decreased as the NAFLD progressed from steatosis to NASH [122]. Supporting MRP2's role in NAFLD, it was found that a polymorphism in MRP2 was significantly associated with NAFLD [128]. Currently, there is only one study investigating the expression levels of MRP3 in patients with NAFLD. The MRP3 mRNA expression levels were significantly elevated in patients with NAFLD, especially with NASH, compared to the healthy control subjects [94]. In another study with diabetic patients, MRP3 and MRP4 expression levels were not significantly altered in diabetic patients compared to control subjects [106]. Taken together, the results from limited studies suggest that MRP3 and MRP4 were upregulated in patients with NAFLD, while the effects of NAFLD on MRP2 expression were minimal.

#### *4.1.4 OATPs*

There is currently only one study investigating the expression of OATPs in patients with NAFLD. Both OATP1B1 and OATP1B3 mRNA expression levels were significantly upregulated in patients with NAFLD compared to control subjects [101]. In a different study with diabetic patients, the expression levels of OATP1B1 were comparable in diabetic patients compared to control subjects [106]. Therefore, it can be cautiously concluded that OATP1B1 and OATP1b3 expression were largely induced in patients with NAFLD.

#### **4.2 Alterations in bile acid transporters in NAFLD animal models**

#### *4.2.1 Bsep*

Several studies have investigated the effects of NAFLD on Bsep expression in rodents. In one study with HFD/STZ-induced NAFLD rats, the Bsep mRNA levels were significantly downregulated in NAFLD rats compared to control rats [125], indicating reducing biliary excretion of bile acids in HFD/STZ-induced NAFLD rats. However, in two other studies with MCD-induced NAFLD rats and mice, the mRNA expression levels of Bsep were not altered in NAFLD rats or mice [126, 117]. Consistently, a study with obese zucker rats showed that Bsep expression was not significantly altered in obese ZR rats compared to control rats [129]. In another study with obese ZR rats, the expression levels of Bsep mRNA were significantly decreased in obese ZR rats, while Bsep protein levels detected by Western blot as well as immunohistochemistry were comparable between obese ZR rats and lean control rats [130]. In another study with *ob/ob* mice, the expression levels of Bsep mRNA were significantly increased in *ob/ob* mice compared to lean control mice. However, in contrast to the mRNA levels, Bsep protein levels were significantly decreased in *ob/ob* mice when compared to lean control mice [118]. Consistent with decreased Bsep expression in NAFLD mice, overexpression of Bsep increases hepatobiliary lipid secretion and reduces hepatic steatosis [131]. Taken together, Bsep expression was either not altered or decreased in NAFLD rodent models.

**59**

rodent models.

compared to the controls [125, 129, 130].

*Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

Currently, there are six studies evaluating the effects of NAFLD on Ntcp expression in rodents. In three studies, the expression levels of Ntcp were consis-

[117, 125, 132]. Two studies were conducted in rats, while one study was carried out in mice. The common feature among the three studies is that NAFLD was induced by MCD for 8 weeks. In the same study [132], Ntcp expression levels were also significantly decreased when NAFLD was induced by HFD. On the other hand, another two studies with rats showed the Ntcp expression was not altered under the NAFLD condition [126, 130]. Different effects of NAFLD on Ntcp mRNA and protein expression were reported in a study with *ob/ob* mice [118]. The expression levels of Ntcp mRNA were not changed in *ob/ob* mice compared to the lean control mice. However, Ntcp protein levels were significantly lower in *ob/ob* mice than those in lean control mice. Taken together, the effects of NAFLD on Ntcp expression were largely consistent among the six studies, either no significant changes or decreased dependent on the species and methods by which

The effects of NAFLD on Mrp expression were extensively investigated mainly

In summary, the effects of NAFLD on Mrp2 expression were inconsistent or even conflicting. The discrepancy between Mrp2 mRNA and protein levels was also noted in the studies, indicating that post-transcriptional regulation plays an important role in regulating Mrp2 expression under the NAFLD condition. On the other hand, the expression of Mrp3 and Mrp4 was largely upregulated in NAFLD

due to the fact that Mrps are important transporters for xenobiotics including drugs. Data from eight studies evaluating Mrp2 expression in NAFLD rodents are not consistent. Two studies with obese ZR rats reported consistent results that the expression levels of Mrp2 mRNA and protein were significantly downregulated in obese ZR rats compared to lean control rats [129, 130]. Consistent with downregulation of Mrp2 in obese zucker rats, Mrp2 expression levels were reduced in MCD-induced NAFLD rats compared to control rats on supplemented MCD [126]. On the other hand, other two studies showed that Mrp2 expression levels were not significantly altered in MCD-induced NAFLD rats or HFD/STZ-induced NAFLD mice compared to the control rats or mice [117, 125]. In a study with *ob/ ob* mice, Mrp2 expression levels were significantly increased at the mRNA level but decreased at the protein level [118]. In contrast, the mRNA levels of Mrp2 were decreased but protein levels were increased in MCD-induced NAFLD rats [132]. In a comprehensive study to evaluate various NAFLD models with mice and rats, Mrp2 expression was significantly increased in athrogenic diet and MCD-induced NAFLD rats and all four types of NAFLD mouse models when compared to the corresponding control rats or mice. At the protein level, Mrp2 expression was only increased in MCD-induced NAFLD rats [133]. Compared with Mrp2, the data for the effects of NAFLD on the expression of Mrp3 and Mrp4 are more consistent among the studies. Mrp3 and/or Mrp4 expression were significantly upregulated in five studies with NAFLD rats or mice [117, 124, 126, 118, 133]. On the other hand, another three studies with HFD/STZ-induced NAFLD rats or obese ZR rats reported that the expression levels of Mrp3 and/or Mrp4 were not altered in NAFLD or obese ZR rats

tently decreased in animals with NAFLD compared to control animals

*4.2.2 Ntcp*

NAFLD was induced.

*4.2.3 Mrps*

#### *4.2.2 Ntcp*

*Nonalcoholic Fatty Liver Disease - An Update*

*4.1.3 MRPs*

*4.1.4 OATPs*

*4.2.1 Bsep*

induced in patients with NAFLD.

NAFLD patients with certain inconsistency.

and control subjects [106]. In summary, NTCP expression was likely upregulated in

In one study with NAFLD and one study with diabetic patients, MRP2 mRNA expression levels were not significantly altered in NAFLD and diabetic patients compared to control subjects [88, 106]. Another study reported that MRP2 mRNA expression levels were decreased as the NAFLD progressed from steatosis to NASH [122]. Supporting MRP2's role in NAFLD, it was found that a polymorphism in MRP2 was significantly associated with NAFLD [128]. Currently, there is only one study investigating the expression levels of MRP3 in patients with NAFLD. The MRP3 mRNA expression levels were significantly elevated in patients with NAFLD, especially with NASH, compared to the healthy control subjects [94]. In another study with diabetic patients, MRP3 and MRP4 expression levels were not significantly altered in diabetic patients compared to control subjects [106]. Taken together, the results from limited studies suggest that MRP3 and MRP4 were upregulated in patients with NAFLD,

There is currently only one study investigating the expression of OATPs in patients with NAFLD. Both OATP1B1 and OATP1B3 mRNA expression levels were significantly upregulated in patients with NAFLD compared to control subjects [101]. In a different study with diabetic patients, the expression levels of OATP1B1 were comparable in diabetic patients compared to control subjects [106]. Therefore, it can be cautiously concluded that OATP1B1 and OATP1b3 expression were largely

Several studies have investigated the effects of NAFLD on Bsep expression in rodents. In one study with HFD/STZ-induced NAFLD rats, the Bsep mRNA levels were significantly downregulated in NAFLD rats compared to control rats [125], indicating reducing biliary excretion of bile acids in HFD/STZ-induced NAFLD rats. However, in two other studies with MCD-induced NAFLD rats and mice, the

while the effects of NAFLD on MRP2 expression were minimal.

**4.2 Alterations in bile acid transporters in NAFLD animal models**

mRNA expression levels of Bsep were not altered in NAFLD rats or mice

[126, 117]. Consistently, a study with obese zucker rats showed that Bsep expression was not significantly altered in obese ZR rats compared to control rats [129]. In another study with obese ZR rats, the expression levels of Bsep mRNA were significantly decreased in obese ZR rats, while Bsep protein levels detected by Western blot as well as immunohistochemistry were comparable between obese ZR rats and lean control rats [130]. In another study with *ob/ob* mice, the expression levels of Bsep mRNA were significantly increased in *ob/ob* mice compared to lean control mice. However, in contrast to the mRNA levels, Bsep protein levels were significantly decreased in *ob/ob* mice when compared to lean control mice [118]. Consistent with decreased Bsep expression in NAFLD mice, overexpression of Bsep increases hepatobiliary lipid secretion and reduces hepatic steatosis [131]. Taken together, Bsep expression was either not altered or decreased in NAFLD

**58**

rodent models.

Currently, there are six studies evaluating the effects of NAFLD on Ntcp expression in rodents. In three studies, the expression levels of Ntcp were consistently decreased in animals with NAFLD compared to control animals [117, 125, 132]. Two studies were conducted in rats, while one study was carried out in mice. The common feature among the three studies is that NAFLD was induced by MCD for 8 weeks. In the same study [132], Ntcp expression levels were also significantly decreased when NAFLD was induced by HFD. On the other hand, another two studies with rats showed the Ntcp expression was not altered under the NAFLD condition [126, 130]. Different effects of NAFLD on Ntcp mRNA and protein expression were reported in a study with *ob/ob* mice [118]. The expression levels of Ntcp mRNA were not changed in *ob/ob* mice compared to the lean control mice. However, Ntcp protein levels were significantly lower in *ob/ob* mice than those in lean control mice. Taken together, the effects of NAFLD on Ntcp expression were largely consistent among the six studies, either no significant changes or decreased dependent on the species and methods by which NAFLD was induced.

#### *4.2.3 Mrps*

The effects of NAFLD on Mrp expression were extensively investigated mainly due to the fact that Mrps are important transporters for xenobiotics including drugs. Data from eight studies evaluating Mrp2 expression in NAFLD rodents are not consistent. Two studies with obese ZR rats reported consistent results that the expression levels of Mrp2 mRNA and protein were significantly downregulated in obese ZR rats compared to lean control rats [129, 130]. Consistent with downregulation of Mrp2 in obese zucker rats, Mrp2 expression levels were reduced in MCD-induced NAFLD rats compared to control rats on supplemented MCD [126]. On the other hand, other two studies showed that Mrp2 expression levels were not significantly altered in MCD-induced NAFLD rats or HFD/STZ-induced NAFLD mice compared to the control rats or mice [117, 125]. In a study with *ob/ ob* mice, Mrp2 expression levels were significantly increased at the mRNA level but decreased at the protein level [118]. In contrast, the mRNA levels of Mrp2 were decreased but protein levels were increased in MCD-induced NAFLD rats [132]. In a comprehensive study to evaluate various NAFLD models with mice and rats, Mrp2 expression was significantly increased in athrogenic diet and MCD-induced NAFLD rats and all four types of NAFLD mouse models when compared to the corresponding control rats or mice. At the protein level, Mrp2 expression was only increased in MCD-induced NAFLD rats [133]. Compared with Mrp2, the data for the effects of NAFLD on the expression of Mrp3 and Mrp4 are more consistent among the studies. Mrp3 and/or Mrp4 expression were significantly upregulated in five studies with NAFLD rats or mice [117, 124, 126, 118, 133]. On the other hand, another three studies with HFD/STZ-induced NAFLD rats or obese ZR rats reported that the expression levels of Mrp3 and/or Mrp4 were not altered in NAFLD or obese ZR rats compared to the controls [125, 129, 130].

In summary, the effects of NAFLD on Mrp2 expression were inconsistent or even conflicting. The discrepancy between Mrp2 mRNA and protein levels was also noted in the studies, indicating that post-transcriptional regulation plays an important role in regulating Mrp2 expression under the NAFLD condition. On the other hand, the expression of Mrp3 and Mrp4 was largely upregulated in NAFLD rodent models.

#### *4.2.4 Oatps*

Currently, there are seven studies evaluating the effects of NAFLD on the expression of Oatps. The expression levels of Oatp1a1 mRNA and/or protein were consistently decreased in six studies [117, 118, 125, 129, 132, 133], while one study showed no changes [126]. There are three studies investigating Oatp1a4. In one study, the expression levels of Oatp1a4 were significantly reduced at both mRNA and protein levels in *ob/ob* mice compared to lean control mice [118]. In another study, the expression levels of Oatp1a4 mRNA were increased but its protein levels were decreased in various mouse and rat NAFLD models compared to the corresponding control mice or rats [133]. On the other hand, no alterations in Oatp1a4 expression were detected in MCD-induced NAFLD rats [126]. The effects of NAFLD on the expression of Oatp1b2 were very much consistent among the five studies. Oatp1b2 expression was significantly downregulated in four studies [117, 126, 132, 133], while no alterations in Oatp1b2 expression were detected in one study [125]. There are three studies investigating Oatp2b1. One study with *ob/ob* mice reported that Oatp2b1 mRNA levels were significantly upregulated in *ob/ob* mice compared to the lean control mice [118]. However, other two studies showed that Oatp2b1 was downregulated in obese ZR rats compared to lean control rats [129, 132]. Taken together, Oatp1a1 and Oatp1b2 were consistently downregulated, while the effects on Oatp1a4 and Oatp2b1 were inconsistent in NAFLD rodents.

#### **5. Alterations in bile acid regulators in subjects with NAFLD**

Bile acid synthesis is tightly regulated by multiple signaling pathways, mainly the FXR/SHP [134, 135] and FGF19/FGFR4 [136, 137] negative feedback loops (**Figure 2**). In the liver, activation of FXR by bile acids induces SHP expression, which in turn represses CYP7A1 expression, leading to reduced bile acid synthesis. In the intestine, activation of FXR by bile acids upregulates FGF19 (FGF15 in rodents). After entering the circulation, FGF19 binds to FGFR4 in the liver to activate the downstream signaling, which subsequently inhibits CYP7A1 expression, resulting in decreased bile acid synthesis. Those two negative feedback regulatory loops play critical roles in regulating bile acid synthesis and maintaining bile acid homeostasis. Impairment or dysregulation of the FXR/SHP and FGF19/FGFR4 signaling pathways interrupts bile acid balance.

#### **5.1 FXR/SHP signaling pathway**

#### *5.1.1 In human*

Most of the human clinical studies revealed that the FXR/SHP signaling pathway was dysregulated in patients with NAFLD. In one study with 10 healthy controls, 39 steatotic, and 59 NASH patients, both FXR and SHP mRNA levels were significantly downregulated [94]. In two studies with 20 or 11 normal control subjects and 20 NAFLD or 16 NASH patients, the expression levels of FXR were significantly deceased in NAFLD or NASH patients compared to control subjects [101, 138]. However, the expression levels of SHP remain comparable between the control subjects and NAFLD or NASH patients, indicating that the FXR signaling is impaired in NAFLD or NASH patients [101, 138]. In a study with 33 children (19 NAFLD patients and 14 control children), the FXR protein levels were gradually

**61**

NAFLD.

*Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

ing as NAFLD progresses from simple steatosis to NASH.

*5.1.2 In rodent NAFLD models*

**5.2 FGF19/FGFR4 signaling pathway**

decreased from control subjects to steatotic to NASH patients, indicating the worsening of FXR signaling as NAFLD progresses [139]. Consistently, in a study with 20 simple steatosis and 20 NASH patients, the FXR protein expression levels were significantly decreased in NASH patients compared to the patients with simple steatosis, although at the mRNA level, FXR expression was higher in patients with NASH than those in patients with simple steatosis [127]. On the other hand, in one study with 26 controls and 32 NASH patients, no differences were detected in the expression of both FXR and SHP between control and NASH subjects [91]. Finally, one study showed gender differences in FXR expression. A significant decrease in FXR expression was detected in female but not male NASH patients compared to control subjects, while SHP expression was significantly decreased in both male and female with NASH [122]. In summary, most of the studies revealed a decreased or impaired FXR signaling in patients with NAFLD, and such impairment gets worsen-

Inconsistent results have been reported regarding the status of FXR signaling in NAFLD rodent models. In two studies with HFD or fructose-induced NAFLD mice, the FXR expression levels were significantly reduced in NAFLD mice compared to control mice [124, 140]. However, SHP expression remained unchanged in fructoseinduced NAFLD mice while significantly increased in HFD-induced NAFLD mice. In another two studies with HFD/STZ or MCD-induced NAFLD rats, the FXR expression levels remained comparable between the NAFLD and control rats [125, 126]. Consistent with no changes in FXR expression, SHP expression was comparable between the two groups. In another study with *ob/ob* mice, FXR mRNA and protein were significantly increased in *ob/ob* mice compared to lean control mice, while no alterations in SHP expression was detected [118]. Finally, in a study with HFD-induced NAFLD mice, the FXR signaling status was investigated during the progression of NAFLD from simple steatosis to NASH, fibrosis, and hepatocellular carcinoma (HCC) on an HFD [141]. FXR signaling was strongly activated in the early stage of NAFLD (simple steatosis) evidenced by strong upregulation of FXR target genes including Bsep, Mrp2, and ATP-binding cassette subfamily G member 5 (Abcg5)/Abcg8. However, as NAFLD progressed, FXR signaling gradually decreased but was still higher than that in the control mice on regular diet. Taken together, the inconsistent results from the NAFLD rodent models indicate that the effects of NAFLD on the FXR signaling pathway are dependent on the methods by which NAFLD is induced as well as the species (mouse or rat).

A large number of clinical studies have demonstrated that the FGF19 signaling is dysregulated in patients with NAFLD. Serum FGF19 concentrations were significantly reduced in patients with simple steatosis or NASH compared to control subjects [88, 92, 101, 102, 139, 142–144]. The decreases in FGF19 concentrations were more severe in patients with NASH than the patients with steatosis, indicating the worsening of FGF19 signaling impairment as the NAFLD progresses from simple steatosis to NASH. On the other hand, there are two clinical studies showing that the fasting serum concentrations of FGF19 were not altered in patients with NAFLD compared to control subjects [107, 145]. Taken together, most of the clinical studies showed that the FGF19 signaling was reduced or impaired in patients with

#### *Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

*Nonalcoholic Fatty Liver Disease - An Update*

Currently, there are seven studies evaluating the effects of NAFLD on the expression of Oatps. The expression levels of Oatp1a1 mRNA and/or protein were consistently decreased in six studies [117, 118, 125, 129, 132, 133], while one study showed no changes [126]. There are three studies investigating Oatp1a4. In one study, the expression levels of Oatp1a4 were significantly reduced at both mRNA and protein levels in *ob/ob* mice compared to lean control mice [118]. In another study, the expression levels of Oatp1a4 mRNA were increased but its protein levels were decreased in various mouse and rat NAFLD models compared to the corresponding control mice or rats [133]. On the other hand, no alterations in Oatp1a4 expression were detected in MCD-induced NAFLD rats [126]. The effects of NAFLD on the expression of Oatp1b2 were very much consistent among the five studies. Oatp1b2 expression was significantly downregulated in four studies [117, 126, 132, 133], while no alterations in Oatp1b2 expression were detected in one study [125]. There are three studies investigating Oatp2b1. One study with *ob/ob* mice reported that Oatp2b1 mRNA levels were significantly upregulated in *ob/ob* mice compared to the lean control mice [118]. However, other two studies showed that Oatp2b1 was downregulated in obese ZR rats compared to lean control rats [129, 132]. Taken together, Oatp1a1 and Oatp1b2 were consistently downregulated, while the effects on Oatp1a4 and Oatp2b1 were inconsistent in

**5. Alterations in bile acid regulators in subjects with NAFLD**

signaling pathways interrupts bile acid balance.

**5.1 FXR/SHP signaling pathway**

*5.1.1 In human*

Bile acid synthesis is tightly regulated by multiple signaling pathways, mainly the FXR/SHP [134, 135] and FGF19/FGFR4 [136, 137] negative feedback loops (**Figure 2**). In the liver, activation of FXR by bile acids induces SHP expression, which in turn represses CYP7A1 expression, leading to reduced bile acid synthesis. In the intestine, activation of FXR by bile acids upregulates FGF19 (FGF15 in rodents). After entering the circulation, FGF19 binds to FGFR4 in the liver to activate the downstream signaling, which subsequently inhibits CYP7A1 expression, resulting in decreased bile acid synthesis. Those two negative feedback regulatory loops play critical roles in regulating bile acid synthesis and maintaining bile acid homeostasis. Impairment or dysregulation of the FXR/SHP and FGF19/FGFR4

Most of the human clinical studies revealed that the FXR/SHP signaling pathway was dysregulated in patients with NAFLD. In one study with 10 healthy controls, 39 steatotic, and 59 NASH patients, both FXR and SHP mRNA levels were significantly downregulated [94]. In two studies with 20 or 11 normal control subjects and 20 NAFLD or 16 NASH patients, the expression levels of FXR were significantly deceased in NAFLD or NASH patients compared to control subjects [101, 138]. However, the expression levels of SHP remain comparable between the control subjects and NAFLD or NASH patients, indicating that the FXR signaling is impaired in NAFLD or NASH patients [101, 138]. In a study with 33 children (19 NAFLD patients and 14 control children), the FXR protein levels were gradually

*4.2.4 Oatps*

NAFLD rodents.

**60**

decreased from control subjects to steatotic to NASH patients, indicating the worsening of FXR signaling as NAFLD progresses [139]. Consistently, in a study with 20 simple steatosis and 20 NASH patients, the FXR protein expression levels were significantly decreased in NASH patients compared to the patients with simple steatosis, although at the mRNA level, FXR expression was higher in patients with NASH than those in patients with simple steatosis [127]. On the other hand, in one study with 26 controls and 32 NASH patients, no differences were detected in the expression of both FXR and SHP between control and NASH subjects [91]. Finally, one study showed gender differences in FXR expression. A significant decrease in FXR expression was detected in female but not male NASH patients compared to control subjects, while SHP expression was significantly decreased in both male and female with NASH [122]. In summary, most of the studies revealed a decreased or impaired FXR signaling in patients with NAFLD, and such impairment gets worsening as NAFLD progresses from simple steatosis to NASH.

#### *5.1.2 In rodent NAFLD models*

Inconsistent results have been reported regarding the status of FXR signaling in NAFLD rodent models. In two studies with HFD or fructose-induced NAFLD mice, the FXR expression levels were significantly reduced in NAFLD mice compared to control mice [124, 140]. However, SHP expression remained unchanged in fructoseinduced NAFLD mice while significantly increased in HFD-induced NAFLD mice. In another two studies with HFD/STZ or MCD-induced NAFLD rats, the FXR expression levels remained comparable between the NAFLD and control rats [125, 126]. Consistent with no changes in FXR expression, SHP expression was comparable between the two groups. In another study with *ob/ob* mice, FXR mRNA and protein were significantly increased in *ob/ob* mice compared to lean control mice, while no alterations in SHP expression was detected [118]. Finally, in a study with HFD-induced NAFLD mice, the FXR signaling status was investigated during the progression of NAFLD from simple steatosis to NASH, fibrosis, and hepatocellular carcinoma (HCC) on an HFD [141]. FXR signaling was strongly activated in the early stage of NAFLD (simple steatosis) evidenced by strong upregulation of FXR target genes including Bsep, Mrp2, and ATP-binding cassette subfamily G member 5 (Abcg5)/Abcg8. However, as NAFLD progressed, FXR signaling gradually decreased but was still higher than that in the control mice on regular diet. Taken together, the inconsistent results from the NAFLD rodent models indicate that the effects of NAFLD on the FXR signaling pathway are dependent on the methods by which NAFLD is induced as well as the species (mouse or rat).

#### **5.2 FGF19/FGFR4 signaling pathway**

A large number of clinical studies have demonstrated that the FGF19 signaling is dysregulated in patients with NAFLD. Serum FGF19 concentrations were significantly reduced in patients with simple steatosis or NASH compared to control subjects [88, 92, 101, 102, 139, 142–144]. The decreases in FGF19 concentrations were more severe in patients with NASH than the patients with steatosis, indicating the worsening of FGF19 signaling impairment as the NAFLD progresses from simple steatosis to NASH. On the other hand, there are two clinical studies showing that the fasting serum concentrations of FGF19 were not altered in patients with NAFLD compared to control subjects [107, 145]. Taken together, most of the clinical studies showed that the FGF19 signaling was reduced or impaired in patients with NAFLD.

### **6. Conclusions**

A large body of evidence from clinical as well as preclinical studies has demonstrated that bile acid homeostasis is disrupted in subjects with NAFLD. The dysregulation of bile acids in patients with NAFLD gets worsening as the disease progresses from early stage simple steatosis to late stages NASH and NASH with fibrosis. Risk factors for NAFLD, especially obesity and insulin resistance, contribute to the dysregulation of bile acids in NAFLD patients.

Due to the complexity of bile acid regulation, small sample sizes in most of the clinical studies, variations in control subject selection, inherited differences in various rodent NAFLD models, and discrepancy in mRNA and protein levels of the target genes, inconsistent or even conflicting results, have been reported for serum and hepatic bile acid concentrations and compositions, as well as the expression levels of bile acid synthesis enzymes, transporters, and regulators. However, detailed examination and evaluation of the results from various studies, especially considering the characteristics of the studied subjects and the quality of each study, certain trends on alterations in serum and hepatic bile acid levels, bile acid synthesis, and regulation in patients with NAFLD are emerged.

As depicted in **Figure 3**, serum total bile acid concentrations are increased in patients with NAFLD, as a result of increased CYP7A1 expression and bile acid synthesis, elevated hepatic bile acids, and augment of MRP3 and MRP4 expression. Increased CYP7A1 expression and bile acid synthesis in patients with NAFLD are mainly due to the impairment of the FXR/SHP and FGF19/FGFR4 signaling pathways. Limited studies on investigating fecal and urine bile acids showed that both fecal and urine bile acid concentrations were elevated in patients with NAFLD, consistent with increased serum and hepatic bile acid levels in those patients.

#### **Figure 3.**

*Effects of NAFLD on serum, hepatic, and fecal bile acid concentrations as well as on bile acid synthesis (CYP7A1), transporters (MRP3 and MRP4), and regulators (FXR, SHP, FGF19/15).*

**63**

*Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

Future studies with high quality and large sample size are needed to solidify the trends depicted in **Figure 3**. The following points should be considered in the design of the future studies and interpretation of the findings. First, limited studies with children and adolescents revealed a different feature in bile acid dysregulation from adults with NAFLD. In contrast to the findings in adults, serum bile acid levels decrease in the early stage of NAFLD, followed by an increase as NAFLD progresses to fibrosis but the levels remain lower than those in the healthy control children. The effects of NAFLD on bile acid regulation appear different in children from adults. Second, the effects of NAFLD on bile acid homeostasis are stage dependent. No or mild effects of simple steatosis on bile acid regulation were detected, while significant alterations in bile acids are mostly detected in patients with NASH. A large percentage of previous studies did not separate the steatotic and NASH patients in the test groups, which certainly complicates the analysis and interpretation of data. Third, as risk factors for NAFLD, obesity and insulin resistance/diabetes are often associated with NAFLD. It is well documented that obesity and insulin resistance directly cause dysregulation of bile acids. Therefore, those risk factors should be adjusted or matched in the control group in order to reveal the exact effects of NAFLD on bile acid homeostasis. Among the clinical studies reported, only one study was conducted with a matched control group, in which a different conclusion was reached that NASH has no effects on bile acid regulation [93]. Fourth, in future studies using NAFLD rodent models, it should be emphasized that species differences between rodents and human and even between mouse and rat exist, especially in the effects of NAFLD on bile acid transporter expression. Finally, in the investigation of gene expression, both mRNA and protein levels should be detected and quantified for the target genes. Most of the previous studies only evaluated the mRNA levels. However, discrepancy between the mRNA and protein levels is often detected in studies investigating both levels. It appears that under the NAFLD condition, posttranscriptional regulation plays a predominant role in regulating the

genes involved in bile acid synthesis, transport, and regulation.

R01DK087755, R01CA213419 and R01GM061988.

The authors have no conflict of interest.

This work was supported by the National Institutes of Health (NIH) Grants

**Acknowledgements**

**Conflict of interest**

**7. Guidance for future studies**

### **7. Guidance for future studies**

*Nonalcoholic Fatty Liver Disease - An Update*

ute to the dysregulation of bile acids in NAFLD patients.

regulation in patients with NAFLD are emerged.

A large body of evidence from clinical as well as preclinical studies has demonstrated that bile acid homeostasis is disrupted in subjects with NAFLD. The dysregulation of bile acids in patients with NAFLD gets worsening as the disease progresses from early stage simple steatosis to late stages NASH and NASH with fibrosis. Risk factors for NAFLD, especially obesity and insulin resistance, contrib-

Due to the complexity of bile acid regulation, small sample sizes in most of the clinical studies, variations in control subject selection, inherited differences in various rodent NAFLD models, and discrepancy in mRNA and protein levels of the target genes, inconsistent or even conflicting results, have been reported for serum and hepatic bile acid concentrations and compositions, as well as the expression levels of bile acid synthesis enzymes, transporters, and regulators. However, detailed examination and evaluation of the results from various studies, especially considering the characteristics of the studied subjects and the quality of each study, certain trends on alterations in serum and hepatic bile acid levels, bile acid synthesis, and

As depicted in **Figure 3**, serum total bile acid concentrations are increased in patients with NAFLD, as a result of increased CYP7A1 expression and bile acid synthesis, elevated hepatic bile acids, and augment of MRP3 and MRP4 expression. Increased CYP7A1 expression and bile acid synthesis in patients with NAFLD are mainly due to the impairment of the FXR/SHP and FGF19/FGFR4 signaling pathways. Limited studies on investigating fecal and urine bile acids showed that both fecal and urine bile acid concentrations were elevated in patients with NAFLD, consistent with increased serum and hepatic bile acid levels in those

*Effects of NAFLD on serum, hepatic, and fecal bile acid concentrations as well as on bile acid synthesis* 

*(CYP7A1), transporters (MRP3 and MRP4), and regulators (FXR, SHP, FGF19/15).*

**6. Conclusions**

patients.

**62**

**Figure 3.**

Future studies with high quality and large sample size are needed to solidify the trends depicted in **Figure 3**. The following points should be considered in the design of the future studies and interpretation of the findings. First, limited studies with children and adolescents revealed a different feature in bile acid dysregulation from adults with NAFLD. In contrast to the findings in adults, serum bile acid levels decrease in the early stage of NAFLD, followed by an increase as NAFLD progresses to fibrosis but the levels remain lower than those in the healthy control children. The effects of NAFLD on bile acid regulation appear different in children from adults. Second, the effects of NAFLD on bile acid homeostasis are stage dependent. No or mild effects of simple steatosis on bile acid regulation were detected, while significant alterations in bile acids are mostly detected in patients with NASH. A large percentage of previous studies did not separate the steatotic and NASH patients in the test groups, which certainly complicates the analysis and interpretation of data. Third, as risk factors for NAFLD, obesity and insulin resistance/diabetes are often associated with NAFLD. It is well documented that obesity and insulin resistance directly cause dysregulation of bile acids. Therefore, those risk factors should be adjusted or matched in the control group in order to reveal the exact effects of NAFLD on bile acid homeostasis. Among the clinical studies reported, only one study was conducted with a matched control group, in which a different conclusion was reached that NASH has no effects on bile acid regulation [93]. Fourth, in future studies using NAFLD rodent models, it should be emphasized that species differences between rodents and human and even between mouse and rat exist, especially in the effects of NAFLD on bile acid transporter expression. Finally, in the investigation of gene expression, both mRNA and protein levels should be detected and quantified for the target genes. Most of the previous studies only evaluated the mRNA levels. However, discrepancy between the mRNA and protein levels is often detected in studies investigating both levels. It appears that under the NAFLD condition, posttranscriptional regulation plays a predominant role in regulating the genes involved in bile acid synthesis, transport, and regulation.

#### **Acknowledgements**

This work was supported by the National Institutes of Health (NIH) Grants R01DK087755, R01CA213419 and R01GM061988.

#### **Conflict of interest**

The authors have no conflict of interest.

*Nonalcoholic Fatty Liver Disease - An Update*

#### **Author details**

Xinmu Zhang and Ruitang Deng\* Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, RI, USA

\*Address all correspondence to: dengr@uri.edu

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**65**

*Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

Gastroenterology. 2015;**21**:4103-4110.

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10.1016/j.cgh.2015.07.024

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10.1210/jc.2014-2739

Endocrinology, Diabetes and Obesity. 2009;**16**:141-149. DOI: 10.1097/ MED.0b013e3283293015

[13] Portillo-Sanchez P, Bril F, Maximos M, Lomonaco R, Biernacki D, Orsak B, et al. High prevalence of nonalcoholic fatty liver disease in patients with type 2 diabetes mellitus and normal plasma aminotransferase levels. Bile acids: Natural ligands for an orphan. Nuclear Receptor. 2015;**100**:2231-2238. DOI:

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**Author details**

Xinmu Zhang and Ruitang Deng\*

provided the original work is properly cited.

University of Rhode Island, Kingston, RI, USA

\*Address all correspondence to: dengr@uri.edu

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy,

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Medicine. 2010;**10**:579-595. DOI: 10.2174/1566524011009060579

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Acta (BBA)—Gene Regulatory

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[104] Aranha MM, Cortez-Pinto H, Costa A, da Silva IB, Camilo ME, de Moura MC, et al. Bile acid levels are increased in the liver of patients with steatohepatitis. European Journal of Gastroenterology & Hepatology. 2008;**20**:519-525. DOI: 10.1097/ MEG.0b013e3282f4710a

[105] Lake AD, Novak P, Shipkova P, Aranibar N, Robertson D, Reily MD, et al. Decreased hepatotoxic bile acid composition and altered synthesis in progressive human nonalcoholic fatty liver disease. Toxicology and Applied Pharmacology. 2013;**268**: 132-140. DOI: 10.1016/j.taap. 2013.01.022

[106] Valanejad L, Ghareeb M, Shiffka S, Nadolny C, Chen Y, Guo L, et al. Dysregulation of Δ4-3-oxosteroid 5β-reductase in diabetic patients: Implications and mechanisms. Molecular and Cellular Endocrinology. 2018;**470**:127-141. DOI: 10.1016/j. mce.2017.10.005

[107] Mouzaki M, Wang AY, Bandsma R, Comelli EM, Arendt BM, Zhang L, et al. Bile acids and dysbiosis in nonalcoholic fatty liver disease. PLoS One. 2016;**11**:e0151829. DOI: 10.1371/journal. pone.0151829. eCollection 2016

[108] Han S, Zhang R, Jain R, Shi H, Zhang L, Zhou G, et al. Circadian control of bile acid synthesis by a KLF15-Fgf15 axis. Nature

Communications. 2015;**6**:7231. DOI: 10.1038/ncomms8231

[109] Setchell KD, Lawson AM, Blackstock EJ, Murphy GM. Diurnal changes in serum unconjugated bile acids in normal man. Gut. 1982;**23**:637-642

[110] Gälman C, Angelin B, Rudling M. Bile acid synthesis in humans has a rapid diurnal variation that is asynchronous with cholesterol synthesis. Gastroenterology. 2005;**129**:1445-1453

[111] Noshiro M, Nishimoto M, Okuda K. Rat liver cholesterol 7 alphahydroxylase. Pretranslational regulation for circadian rhythm. Journal Biology Chemistry. 1990;**265**:10036-10041

[112] Ferrell JM, Chiang JY. Short-term circadian disruption impairs bile acid and lipid homeostasis in mice. Cell Molecular Gastroenterol Hepatology. 2015;**1**:664-677. DOI: 10.1016/j. jcmgh.2015.08.003

[113] Santhekadur PK, Kumar DP, Sanyal AJ. Preclinical models of nonalcoholic fatty liver disease. The Journal of Hepatology. 2018;**68**:230-237. DOI: 10.1016/j.jhep.2017.10.031

[114] Van Herck MA, Vonghia L, Francque SM. Animal models of nonalcoholic fatty liver disease-a starter's guide. Nutrients. 2017;**9**(10). pii: E1072. DOI: 10.3390/nu9101072

[115] Rasselli E, Canesi L, Portincasa P, Voci A, Vergani L, Demori I. Models of non-alcoholic fatty liver disease and potential translational value: The effects of 3,5-L-diiodothyronine. Annals of Hepatology. 2017;**16**:707-719. DOI: 10.5604/01.3001.0010.2713

[116] Jia X, Suzuki Y, Naito H, Yetti H, Kitamori K, Hayashi Y, et al. A possible role of chenodeoxycholic acid and glycine-conjugated bile acids in fibrotic

**73**

*Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

> progression of nonalcoholic fatty liver disease. Journal of Gastroenterology. 2016;**51**:808-818. DOI: 10.1007/

[123] Axelson M, Bjorkhem I, Reihner E, Einarsson K. The plasma level of 7 alpha-hydroxy-4-cholesten-3 one reflects the activity of hepatic cholesterol 7 alpha-hydroxylase in man. FEBS Letters. 1991;**284**:216-218. DOI: 10.1016/0014-5793(91)80688-Y

[124] Wang C, Tao Q, Wang X, Wang X, Zhang X. Impact of high-fat diet on liver genes expression profiles in mice model of nonalcoholic fatty liver disease. Environmental Toxicology and Pharmacology. 2016;**45**:52-62. DOI:

[125] Pozzo L, Vornoli A, Coppola I, Croce CM, Giorgetti L, Gervasi PG, et al. Effect of HFD/STZ on expression of genes involved in lipid, cholesterol and glucose metabolism in rats. Life Sciences. 2016;**166**:149-156. DOI:

[126] Lionarons DA, Heger M, van Golen RF, Alles LK, van der Mark VA, Kloek JJ, et al. Simple steatosis sensitizes cholestatic rats to liver injury and dysregulates bile salt synthesis and transport. Scientific Reports. 2016;**6**:31829. DOI: 10.1038/srep31829

[127] Aguilar-Olivos NE, Carrillo-Córdova D, Oria-Hernández J,

Hepatology. 2015;**14**:487-493

[128] Sookoian S, Castaño G, Gianotti TF, Gemma C, Pirola CJ. Polymorphisms of MRP2 (ABCC2) are associated with susceptibility to nonalcoholic fatty liver disease. Journal of Nutritional Biochemistry. 2009;**20**:765- 770. DOI: 10.1016/j.jnutbio.2008.07.005

Sánchez-Valle V, Ponciano-Rodríguez G, Ramírez-Jaramillo M, et al. The nuclear receptor FXR, but not LXR, up-regulates bile acid transporter expression in nonalcoholic fatty liver disease. Annals of

10.1016/j.etap.2016.05.014

10.1016/j.lfs.2016.09.022

s00535-015-1148-y

steatohepatitis in a dietary rat model. Digestive Diseases and Sciences. 2014;**59**:1490-1501. DOI: 10.1007/

Krausz KW, Patterson AD, Gonzalez FJ.

[118] Martin IV, Schmitt J, Minkenberg A, Mertens JC, Stieger B, Mullhaupt B, et al. Bile acid retention and activation of endogenous hepatic farnesoid-X-receptor in the pathogenesis of fatty liver disease in ob/ob-mice. Journal of Biological Chemistry. 2010;**391**: 1441-1449. DOI: 10.1515/BC.2010.141

[119] Rasineni K, Penrice DD, Natarajan SK, McNiven MA, McVicker BL, Kharbanda KK, et al. Alcoholic vs non-alcoholic fatty liver in rats: Distinct differences in endocytosis and vesicle trafficking despite similar pathology. BMC Gastroenterology. 2016;**16**:27. DOI:

10.1186/s12876-016-0433-4

10.4254/wjh.v9.i8.443

[120] Wruck W, Adjaye J. Metaanalysis reveals up-regulation of cholesterol processes in non-alcoholic and down-regulation in alcoholic fatty liver disease. World Journal of Gastroenterology. 2017;**9**:443-454. DOI:

[121] Min HK, Kapoor A, Fuchs M, Mirshahi F, Zhou H, Maher J, et al. Increased hepatic synthesis and

is associated with the severity of nonalcoholic fatty liver disease. Cell Metabolism. 2012;**15**:665-674. DOI:

10.1016/j.cmet.2012.04.004

dysregulation of cholesterol metabolism

[122] Okushin K, Tsutsumi T, Enooku K, Fujinaga H, Kado A, Shibahara J, et al. The intrahepatic expression levels of bile acid transporters are inversely correlated with the histological

s10620-014-3028-3

10.1002/hep.25630

[117] Tanaka N, Matsubara T,

Disruption of phospholipid and bile acid homeostasis in mice with nonalcoholic steatohepatitis. Hepatology. 2012;**56**:118-129. DOI: *Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

steatohepatitis in a dietary rat model. Digestive Diseases and Sciences. 2014;**59**:1490-1501. DOI: 10.1007/ s10620-014-3028-3

*Nonalcoholic Fatty Liver Disease - An Update*

nonalcoholic fatty liver disease. Journal of Pediatric Gastroenterology and Nutrition. 2015;**61**:85-90. DOI: 10.1097/ Communications. 2015;**6**:7231. DOI:

[110] Gälman C, Angelin B, Rudling M. Bile acid synthesis in humans has a rapid diurnal variation that is asynchronous with cholesterol synthesis. Gastroenterology.

[111] Noshiro M, Nishimoto M, Okuda K. Rat liver cholesterol 7 alpha-

hydroxylase. Pretranslational regulation for circadian rhythm. Journal Biology Chemistry. 1990;**265**:10036-10041

[112] Ferrell JM, Chiang JY. Short-term circadian disruption impairs bile acid and lipid homeostasis in mice. Cell Molecular Gastroenterol Hepatology. 2015;**1**:664-677. DOI: 10.1016/j.

[113] Santhekadur PK, Kumar DP, Sanyal AJ. Preclinical models of nonalcoholic fatty liver disease. The Journal of Hepatology. 2018;**68**:230-237. DOI:

[114] Van Herck MA, Vonghia L, Francque SM. Animal models of nonalcoholic fatty liver disease-a starter's guide. Nutrients. 2017;**9**(10). pii: E1072. DOI: 10.3390/nu9101072

[115] Rasselli E, Canesi L, Portincasa P, Voci A, Vergani L, Demori I. Models of non-alcoholic fatty liver disease and potential translational value: The effects of 3,5-L-diiodothyronine. Annals of Hepatology. 2017;**16**:707-719. DOI:

[116] Jia X, Suzuki Y, Naito H, Yetti H, Kitamori K, Hayashi Y, et al. A possible role of chenodeoxycholic acid and glycine-conjugated bile acids in fibrotic

10.1016/j.jhep.2017.10.031

10.5604/01.3001.0010.2713

[109] Setchell KD, Lawson AM, Blackstock EJ, Murphy GM. Diurnal changes in serum unconjugated bile acids in normal man. Gut.

10.1038/ncomms8231

1982;**23**:637-642

2005;**129**:1445-1453

jcmgh.2015.08.003

[103] Lu LP, Wan YP, Xun PC, Zhou KJ, Chen C, Cheng SY, et al. Serum bile acid level and fatty acid composition in Chinese children with non-alcoholic fatty liver disease. Journal of Digestive Diseases. 2017;**18**:461-471. DOI:

MPG.0000000000000774

10.1111/1751-2980.12494

[104] Aranha MM, Cortez-Pinto H, Costa A, da Silva IB, Camilo ME, de Moura MC, et al. Bile acid levels are increased in the liver of patients with steatohepatitis. European Journal of Gastroenterology & Hepatology. 2008;**20**:519-525. DOI: 10.1097/ MEG.0b013e3282f4710a

[105] Lake AD, Novak P, Shipkova P, Aranibar N, Robertson D, Reily MD, et al. Decreased hepatotoxic bile acid composition and altered synthesis in progressive human nonalcoholic fatty liver disease. Toxicology and Applied Pharmacology. 2013;**268**: 132-140. DOI: 10.1016/j.taap.

[106] Valanejad L, Ghareeb M, Shiffka S, Nadolny C, Chen Y, Guo L, et al. Dysregulation of Δ4-3-oxosteroid 5β-reductase in diabetic patients: Implications and mechanisms.

Molecular and Cellular Endocrinology. 2018;**470**:127-141. DOI: 10.1016/j.

[107] Mouzaki M, Wang AY, Bandsma R, Comelli EM, Arendt BM, Zhang L, et al. Bile acids and dysbiosis in nonalcoholic fatty liver disease. PLoS One. 2016;**11**:e0151829. DOI: 10.1371/journal.

pone.0151829. eCollection 2016

[108] Han S, Zhang R, Jain R, Shi H, Zhang L, Zhou G, et al. Circadian control of bile acid synthesis by a KLF15-Fgf15 axis. Nature

2013.01.022

mce.2017.10.005

**72**

[117] Tanaka N, Matsubara T, Krausz KW, Patterson AD, Gonzalez FJ. Disruption of phospholipid and bile acid homeostasis in mice with nonalcoholic steatohepatitis. Hepatology. 2012;**56**:118-129. DOI: 10.1002/hep.25630

[118] Martin IV, Schmitt J, Minkenberg A, Mertens JC, Stieger B, Mullhaupt B, et al. Bile acid retention and activation of endogenous hepatic farnesoid-X-receptor in the pathogenesis of fatty liver disease in ob/ob-mice. Journal of Biological Chemistry. 2010;**391**: 1441-1449. DOI: 10.1515/BC.2010.141

[119] Rasineni K, Penrice DD, Natarajan SK, McNiven MA, McVicker BL, Kharbanda KK, et al. Alcoholic vs non-alcoholic fatty liver in rats: Distinct differences in endocytosis and vesicle trafficking despite similar pathology. BMC Gastroenterology. 2016;**16**:27. DOI: 10.1186/s12876-016-0433-4

[120] Wruck W, Adjaye J. Metaanalysis reveals up-regulation of cholesterol processes in non-alcoholic and down-regulation in alcoholic fatty liver disease. World Journal of Gastroenterology. 2017;**9**:443-454. DOI: 10.4254/wjh.v9.i8.443

[121] Min HK, Kapoor A, Fuchs M, Mirshahi F, Zhou H, Maher J, et al. Increased hepatic synthesis and dysregulation of cholesterol metabolism is associated with the severity of nonalcoholic fatty liver disease. Cell Metabolism. 2012;**15**:665-674. DOI: 10.1016/j.cmet.2012.04.004

[122] Okushin K, Tsutsumi T, Enooku K, Fujinaga H, Kado A, Shibahara J, et al. The intrahepatic expression levels of bile acid transporters are inversely correlated with the histological

progression of nonalcoholic fatty liver disease. Journal of Gastroenterology. 2016;**51**:808-818. DOI: 10.1007/ s00535-015-1148-y

[123] Axelson M, Bjorkhem I, Reihner E, Einarsson K. The plasma level of 7 alpha-hydroxy-4-cholesten-3 one reflects the activity of hepatic cholesterol 7 alpha-hydroxylase in man. FEBS Letters. 1991;**284**:216-218. DOI: 10.1016/0014-5793(91)80688-Y

[124] Wang C, Tao Q, Wang X, Wang X, Zhang X. Impact of high-fat diet on liver genes expression profiles in mice model of nonalcoholic fatty liver disease. Environmental Toxicology and Pharmacology. 2016;**45**:52-62. DOI: 10.1016/j.etap.2016.05.014

[125] Pozzo L, Vornoli A, Coppola I, Croce CM, Giorgetti L, Gervasi PG, et al. Effect of HFD/STZ on expression of genes involved in lipid, cholesterol and glucose metabolism in rats. Life Sciences. 2016;**166**:149-156. DOI: 10.1016/j.lfs.2016.09.022

[126] Lionarons DA, Heger M, van Golen RF, Alles LK, van der Mark VA, Kloek JJ, et al. Simple steatosis sensitizes cholestatic rats to liver injury and dysregulates bile salt synthesis and transport. Scientific Reports. 2016;**6**:31829. DOI: 10.1038/srep31829

[127] Aguilar-Olivos NE, Carrillo-Córdova D, Oria-Hernández J, Sánchez-Valle V, Ponciano-Rodríguez G, Ramírez-Jaramillo M, et al. The nuclear receptor FXR, but not LXR, up-regulates bile acid transporter expression in nonalcoholic fatty liver disease. Annals of Hepatology. 2015;**14**:487-493

[128] Sookoian S, Castaño G, Gianotti TF, Gemma C, Pirola CJ. Polymorphisms of MRP2 (ABCC2) are associated with susceptibility to nonalcoholic fatty liver disease. Journal of Nutritional Biochemistry. 2009;**20**:765- 770. DOI: 10.1016/j.jnutbio.2008.07.005

[129] Geier A, Dietrich CG, Grote T, Beuers U, Prüfer T, Fraunberger P, et al. Characterization of organic anion transporter regulation, glutathione metabolism and bile formation in the obese Zucker rat. Journal of Hepatology. 2005;**43**:1021-1130. DOI: 10.1016/j. jhep.2005.05.031

[130] Pizarro M, Balasubramaniyan N, Solís N, Solar A, Duarte I, Miquel JF, et al. Bile secretory function in the obese Zucker rat: Evidence of cholestasis and altered canalicular transport function. Gut. 2004;**53**:1837-1843. DOI: 10.1136/ gut.2003.037689

[131] Figge A, Lammert F, Paigen B, Henkel A, Matern S, Korstanje R, et al. Hepatic overexpression of murine Abcb11 increases hepatobiliary lipid secretion and reduces hepatic steatosis. Journal of Biological Chemistry. 2004;**279**:2790-2799. DOI: 10.1074/jbc. M307363200

[132] Fisher CD, Lickteig AJ, Augustine LM, Oude Elferink RP, Besselsen DG, Erickson RP, et al. Experimental non-alcoholic fatty liver disease results in decreased hepatic uptake transporter expression and function in rats. European Journal of Pharmacology. 2009;**613**:119-127. DOI: 10.1016/j.ejphar.2009.04.002

[133] Canet MJ, Hardwick RN, Lake AD, Dzierlenga AL, Clarke JD, Cherrington NJ. Modeling human nonalcoholic steatohepatitis-associated changes in drug transporter expression using experimental rodent models. Drug Metabolism and Disposition. 2014;**42**:586-695. DOI: 10.1124/ dmd.113.055996

[134] Goodwin B, Jones SA, Price RR, Watson MA, McKee DD, Moore LB, et al. A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Molecular Cell. 2000;**6**:517-526

[135] Lu TT, Makishima M, Repa JJ, Schoonjans K, Kerr TA, Auwerx J, et al. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Molecular Cell. 2000;**6**:507-515

[136] Holt JA, Luo G, Billin AN, Bisi J, McNeill YY, Kozarsky KF, et al. Definition of a novel growth factor-dependent signal cascade for the suppression of bile acid biosynthesis. Genes & Development. 2003;**17**:1581-1591

[137] Song KH, Li T, Owsley E, Strom S, Chiang JY. Bile acids activate fibroblast growth factor 19 signaling in human hepatocytes to inhibit cholesterol 7alpha-hydroxylase gene expression. Hepatology. 2009;**49**:297-305. DOI: 10.1002/hep.22627

[138] Yang ZX, Shen W, Sun H. Effects of nuclear receptor FXR on the regulation of liver lipid metabolism in patients with non-alcoholic fatty liver disease. Hepatology International. 2010;**4**:741-748. DOI: 10.1007/ s12072-010-9202-6

[139] Nobili V, Alisi A, Mosca A, Della Corte C, Veraldi S, De Vito R, et al. Hepatic farnesoid X receptor protein level and circulating fibroblast growth factor 19 concentration in children with NAFLD. International Journal. 2018;**38**:342-349. DOI: 10.1111/ liv.13531

[140] Volynets V, Spruss A, Kanuri G, Wagnerberger S, Bischoff SC, Bergheim I. Protective effect of bile acids on the onset of fructose-induced hepatic steatosis in mice. Journal of Lipid Research. 2010;**51**:3414-3424. DOI: 10.1194/jlr.M007179

[141] Cazanave S, Podtelezhnikov A, Jensen K, Seneshaw M, Kumar DP, Min HK, et al. The transcriptomic signature of disease development and progression of nonalcoholic fatty liver disease.

**75**

*Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

Scientific Reports. 2017;**7**:17193. DOI:

[142] Alisi A, Ceccarelli S, Panera N, Prono F, Petrini S, De Stefanis C, et al. Association between serum atypical fibroblast growth factors 21 and 19 and pediatric nonalcoholic fatty liver disease. PLoS One. 2013;**8**:e67160. DOI: 10.1371/journal.pone.0067160. Print

[143] Eren F, Kurt R, Ermis F, Atug O, Imeryuz N, Yilmaz Y. Preliminary evidence of a reduced serum level of fibroblast growth factor 19 in patients with biopsy-proven nonalcoholic fatty liver disease. Clinical Biochemistry. 2012;**45**:655-658. DOI: 10.1016/j. clinbiochem.2012.03.019

[144] Wojcik M, Janus D, Dolezal-Oltarzewska K, Kalicka-Kasperczyk A, Poplawska K, Drozdz D, et al. A decrease in fasting FGF19 levels is associated with the development of non-alcoholic fatty liver disease in obese adolescents. Journal of Pediatric Endocrinology and Metabolism. 2012;**25**:1089-1093. DOI: 10.1515/

[145] Schreuder TC, Marsman HA, Lenicek M, van Werven JR, Nederveen AJ, Jansen PL, et al. The hepatic response to FGF19 is impaired in patients with nonalcoholic fatty liver disease and insulin resistance. American Journal of Physiology— Gastrointestinal and Liver Physiology. 2010;**298**:G440-G445. DOI: 10.1152/

jpem-2012-0253

ajpgi.00322.2009

10.1038/s41598-017-17370-6

2013

*Dysregulation of Bile Acids in Patients with NAFLD DOI: http://dx.doi.org/10.5772/intechopen.81474*

Scientific Reports. 2017;**7**:17193. DOI: 10.1038/s41598-017-17370-6

*Nonalcoholic Fatty Liver Disease - An Update*

[129] Geier A, Dietrich CG, Grote T, Beuers U, Prüfer T, Fraunberger P, et al. Characterization of organic anion transporter regulation, glutathione metabolism and bile formation in the obese Zucker rat. Journal of Hepatology. 2005;**43**:1021-1130. DOI: 10.1016/j.

[135] Lu TT, Makishima M, Repa JJ, Schoonjans K, Kerr TA, Auwerx J, et al. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Molecular Cell.

[136] Holt JA, Luo G, Billin AN, Bisi J, McNeill YY, Kozarsky KF, et al. Definition of a novel growth factor-dependent signal cascade for the suppression of bile acid biosynthesis. Genes & Development.

[137] Song KH, Li T, Owsley E, Strom S, Chiang JY. Bile acids activate fibroblast growth factor 19 signaling in human hepatocytes to inhibit cholesterol 7alpha-hydroxylase gene expression. Hepatology. 2009;**49**:297-305. DOI:

[138] Yang ZX, Shen W, Sun H. Effects of nuclear receptor FXR on the regulation of liver lipid metabolism in patients with non-alcoholic fatty liver disease. Hepatology International. 2010;**4**:741-748. DOI: 10.1007/

[139] Nobili V, Alisi A, Mosca A, Della Corte C, Veraldi S, De Vito R, et al. Hepatic farnesoid X receptor protein level and circulating fibroblast growth factor 19 concentration in children with NAFLD. International Journal. 2018;**38**:342-349. DOI: 10.1111/

[140] Volynets V, Spruss A, Kanuri G, Wagnerberger S, Bischoff SC, Bergheim I. Protective effect of bile acids on the onset of fructose-induced hepatic steatosis in mice. Journal of Lipid Research. 2010;**51**:3414-3424. DOI:

[141] Cazanave S, Podtelezhnikov A, Jensen K, Seneshaw M, Kumar DP, Min HK, et al. The transcriptomic signature of disease development and progression of nonalcoholic fatty liver disease.

2000;**6**:507-515

2003;**17**:1581-1591

10.1002/hep.22627

s12072-010-9202-6

10.1194/jlr.M007179

liv.13531

[130] Pizarro M, Balasubramaniyan N, Solís N, Solar A, Duarte I, Miquel JF, et al. Bile secretory function in the obese Zucker rat: Evidence of cholestasis and altered canalicular transport function. Gut. 2004;**53**:1837-1843. DOI: 10.1136/

[131] Figge A, Lammert F, Paigen B, Henkel A, Matern S, Korstanje R, et al. Hepatic overexpression of murine Abcb11 increases hepatobiliary lipid secretion and reduces hepatic steatosis. Journal of Biological Chemistry. 2004;**279**:2790-2799. DOI: 10.1074/jbc.

jhep.2005.05.031

gut.2003.037689

M307363200

[132] Fisher CD, Lickteig AJ, Augustine LM, Oude Elferink RP, Besselsen DG, Erickson RP, et al. Experimental non-alcoholic fatty liver disease results in decreased hepatic uptake transporter expression and function in rats. European Journal of Pharmacology. 2009;**613**:119-127. DOI: 10.1016/j.ejphar.2009.04.002

[133] Canet MJ, Hardwick RN, Lake AD, Dzierlenga AL, Clarke JD, Cherrington NJ. Modeling human nonalcoholic steatohepatitis-associated changes in drug transporter expression using experimental rodent models. Drug Metabolism and Disposition. 2014;**42**:586-695. DOI: 10.1124/

[134] Goodwin B, Jones SA, Price RR, Watson MA, McKee DD, Moore LB, et al. A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis.

Molecular Cell. 2000;**6**:517-526

**74**

dmd.113.055996

[142] Alisi A, Ceccarelli S, Panera N, Prono F, Petrini S, De Stefanis C, et al. Association between serum atypical fibroblast growth factors 21 and 19 and pediatric nonalcoholic fatty liver disease. PLoS One. 2013;**8**:e67160. DOI: 10.1371/journal.pone.0067160. Print 2013

[143] Eren F, Kurt R, Ermis F, Atug O, Imeryuz N, Yilmaz Y. Preliminary evidence of a reduced serum level of fibroblast growth factor 19 in patients with biopsy-proven nonalcoholic fatty liver disease. Clinical Biochemistry. 2012;**45**:655-658. DOI: 10.1016/j. clinbiochem.2012.03.019

[144] Wojcik M, Janus D, Dolezal-Oltarzewska K, Kalicka-Kasperczyk A, Poplawska K, Drozdz D, et al. A decrease in fasting FGF19 levels is associated with the development of non-alcoholic fatty liver disease in obese adolescents. Journal of Pediatric Endocrinology and Metabolism. 2012;**25**:1089-1093. DOI: 10.1515/ jpem-2012-0253

[145] Schreuder TC, Marsman HA, Lenicek M, van Werven JR, Nederveen AJ, Jansen PL, et al. The hepatic response to FGF19 is impaired in patients with nonalcoholic fatty liver disease and insulin resistance. American Journal of Physiology— Gastrointestinal and Liver Physiology. 2010;**298**:G440-G445. DOI: 10.1152/ ajpgi.00322.2009

**77**

**1. Introduction**

**Chapter 5**

*Zaki A. Sherif*

nostic approaches and in therapeutic methods.

disease (NAFLD), nonalcoholic steatohepatitis (NASH)

**Abstract**

The Rise in the Prevalence of

and Hepatocellular Carcinoma

Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) affects a third of the world's population and its rapid rise parallels the increase in hepatocellular carcinoma (HCC). NAFLD replacing hepatitis C virus (HCV) infection as a leading indicator for liver transplantation (LT) in the United States. NAFLD is a spectrum of disease ranging from simple steatosis (NAFL) to nonalcoholic steatohepatitis (NASH), which can progress to advanced fibrosis (AF) and cirrhosis, culminating in HCC. The main clinical concern of public health administrators is that many patients who are unaware of NAFLD remain undiagnosed and risk developing end-stage liver disease (ESLD). Clinicians overly rely on surrogate liver enzymes to identify patients with NAFLD, allowing for substantial liver disease to go unnoticed and untreated. Furthermore, according to epidemiological studies, in patients diagnosed with NAFLD, ethnicity plays a role in complications and treatment response, and ethnic correlations with NAFLD are thoroughly underreported. Although liver biopsy is the gold standard method for appropriately diagnosing and staging NAFLD, most patients can be effectively diagnosed non-invasively with imaging modalities and integrated tests that are routinely available in the clinic today. This chapter discusses the current global rise in the rates of NAFLD and HCC; the current key findings incidences and the recommended diag-

**Keywords:** obesity, cirrhosis, hepatocellular carcinoma (HCC), insulin resistance (IR), liver transplantation (LT), metabolic syndrome (MetS), nonalcoholic fatty liver

The liver is a 1.5 kg reddish-brown biochemical processing plant of immense responsibilities that include protein synthesis, xenobiotic or drug metabolism, blood detoxification, and the release of bile acids for digestion. In short, the liver plays a key role in the hemostasis of the body by regulating the levels of sugar, protein, and fat that circulate in the blood. However, obesity, which must be carefully defined according to ethnic-specific BMI cut-off points, may alter normal liver physiology and lead to liver disease [1]. Obesity is at the intersection of the chronic liver disease pathway that includes diabetes, metabolic syndrome (MetS), nonalcoholic fatty liver disease (NAFLD), and hepatocellular carcinoma (HCC). The complex association between obesity and liver function involving NAFLD, HCC, histopathology, and genetic factors is the subject of several collaborative research investigations [2–7].

#### **Chapter 5**

## The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma

*Zaki A. Sherif*

#### **Abstract**

Nonalcoholic fatty liver disease (NAFLD) affects a third of the world's population and its rapid rise parallels the increase in hepatocellular carcinoma (HCC). NAFLD replacing hepatitis C virus (HCV) infection as a leading indicator for liver transplantation (LT) in the United States. NAFLD is a spectrum of disease ranging from simple steatosis (NAFL) to nonalcoholic steatohepatitis (NASH), which can progress to advanced fibrosis (AF) and cirrhosis, culminating in HCC. The main clinical concern of public health administrators is that many patients who are unaware of NAFLD remain undiagnosed and risk developing end-stage liver disease (ESLD). Clinicians overly rely on surrogate liver enzymes to identify patients with NAFLD, allowing for substantial liver disease to go unnoticed and untreated. Furthermore, according to epidemiological studies, in patients diagnosed with NAFLD, ethnicity plays a role in complications and treatment response, and ethnic correlations with NAFLD are thoroughly underreported. Although liver biopsy is the gold standard method for appropriately diagnosing and staging NAFLD, most patients can be effectively diagnosed non-invasively with imaging modalities and integrated tests that are routinely available in the clinic today. This chapter discusses the current global rise in the rates of NAFLD and HCC; the current key findings incidences and the recommended diagnostic approaches and in therapeutic methods.

**Keywords:** obesity, cirrhosis, hepatocellular carcinoma (HCC), insulin resistance (IR), liver transplantation (LT), metabolic syndrome (MetS), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH)

#### **1. Introduction**

The liver is a 1.5 kg reddish-brown biochemical processing plant of immense responsibilities that include protein synthesis, xenobiotic or drug metabolism, blood detoxification, and the release of bile acids for digestion. In short, the liver plays a key role in the hemostasis of the body by regulating the levels of sugar, protein, and fat that circulate in the blood. However, obesity, which must be carefully defined according to ethnic-specific BMI cut-off points, may alter normal liver physiology and lead to liver disease [1]. Obesity is at the intersection of the chronic liver disease pathway that includes diabetes, metabolic syndrome (MetS), nonalcoholic fatty liver disease (NAFLD), and hepatocellular carcinoma (HCC). The complex association between obesity and liver function involving NAFLD, HCC, histopathology, and genetic factors is the subject of several collaborative research investigations [2–7].

Over the past few decades, dramatic changes in lifestyle behaviors and health priorities have contributed to a significant rise in noncommunicable diseases such as obesity and NAFLD. Obesity is highly prevalent in the United States of America, estimated to represent between 30 and 38% of adults with a body mass index (BMI) greater than 30 kg/m2 [8, 9]. Obesity is also a risk factor for metabolic syndrome (MetS), which increases hepatic triglyceride (TGs) depositions. NAFLD is the most common cause of impaired liver function in Western countries, affecting over one quarter of the population [10, 11]. Obesity is driving the rise of NAFLD and nonalcoholic steatohepatitis (NASH), the culmination of the fatty liver disease spectrum that is manifested by ballooning, scarring, cirrhosis, and finally liver failure and HCC [12]. It is estimated that globally the prevalence of NAFLD in the general population is 24–30% [13, 14]. Accounting for errors in accuracy that may exist in indirect measurement methodologies, in the United States, the prevalence of NAFLD in adults has risen from 18% in 1988–1991, to 29% in 1999–2000, to 31% in 2011–2012 [15]. However, the prevalence of NAFLD in the United States diagnosed by ultrasonography alone was estimated to be 24.13% (95% CI 19.73–29.15%) [16].

#### **1.1 NAFLD definition**

Nonalcoholic fatty liver disease (NAFLD) is a broad-spectrum disease ranging from fat infiltration of hepatocytes with no symptoms (simple steatosis aka nonalcoholic fatty liver, NAFL) to excess intrahepatic macroglobular and macrovesicular fat accumulation (5–10% by weight of liver) with aggravated inflammation (steatohepatitis aka nonalcoholic steatohepatitis, NASH) in the presence of little ethanol (typically <30 g per day for men and <20 g per day for women) or no alcohol consumption in the last 12 months [12, 17]. It should be noted, however, there is now convincing evidence demonstrating that even "safe" levels of alcohol consumption are associated with adverse health outcomes [17–20] suggesting that future studies should include only nondrinker individuals in the "NAFLD definition" [21]. Therefore, for NAFLD classification, the patient must show evidence of hepatic fat accumulation in the absence of declared chronic alcohol consumption, or drug use that can induce steatosis, or hereditary disorders. This NAFLD designation excludes both macrovesicular and microvesicular steatosis encompassing certain drugs, toxins, viral hepatitis B (HBV), hepatitis C (HCV) or human immunodeficiency virus (HIV) infections, celiac disease, α-1 antitrypsin deficiency, hepatobiliary infectious diseases, hepatolenticular degeneration, hepatic malignancies, genetic hemochromatosis, Wilson's disease, lipodystrophy, abetalipoproteinemia, Reye's syndrome, HELLP syndrome, or decompensated cirrhosis, which may contribute to secondary causes of steatosis and elevated liver enzymes [22–24]. Additional medications targeted for exclusion are estrogen, sodium valproate, nonsteroidal anti-inflammatory drugs (NSAIDs), calcium antagonists, perhexiline-maleate, and antiretroviral drugs [25–27]. Appropriate medical history must also be taken to exclude the uncommon causes of fatty liver secondary to treatment with drugs such as amiodarone, diltiazem, steroids, synthetic estrogens, tamoxifen, and highly active antiretroviral therapy; refeeding syndrome and total parenteral nutrition; severe weight loss after jejunoileal or gastric bypass; lipodystrophy; and other rare disorders [28]. There are also strong opinions for the exclusion of "whole-body system diseases" such as inflammatory bowel syndrome, hypothyroidism, and lipoatrophy [25] from the "secondary fatty liver diseases" category because they may also induce liver steatosis.

NAFLD can be distinguished from alcoholic steatohepatitis (ASH) by the absence of alcohol consumption and on histological markers such as sclerosing hyaline necrosis, hepatocyte ballooning, portal granulocytic inflammation, lobular

**79**

is presented in **Figure 1** [44].

**1.3 NAFLD diagnosis and staging**

The general classification of NAFLD as stated above and accepted by the American Association for the Study of Liver Diseases (AASLD) is a hepatic fat accumulation exceeding 5–10% by weight of the liver [45]. Accordingly, NAFLD diagnosis in the liver is based on: (i) the presence of simple steatosis, as determined by histological or imaging procedure; (ii) a total weekly consumption of less than 140 g of ethanol for men and less than 70 g for women in the last 12 months; and (iii) the absence of competing etiologies for simple liver steatosis and the absence of coexisting causes for chronic liver disease [46]. An appropriate diagnosis of

*The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma*

inflammation, satellitosis, perisinusoidal fibrosis, Mallory-Denk bodies, and acute cholestasis among others [29–31]. However, it is important to note that NAFLD can also coexist with other liver diseases including HCV, hemochromatosis, and alcoholic liver disease, which can accelerate progression to end-stage liver disease

The pathophysiology of NAFLD and its variants is still incompletely understood thereby limiting the availability of effective diagnostic and therapeutic intervention. The ongoing persistence of obesity and the accompanying high rates of diabetes will increase the prevalence of NAFLD [33]. In many cases, the natural cause of the disease is the development of cirrhosis and ESLD as the population ages. Increased mortality rates have been reported in studies that compared NAFLD patients with a normal reference population [34–36]. The primary cause of death for NAFLD patients is cardiovascular disease followed by nonliver cancer, whereas the third leading cause of mortality is liver-related complications including cirrhosis [33]. The exact prevalence of fatty liver condition is not known, but population studies from the United States and China estimate that 28–30% of the general population has simple steatosis that carries a relatively benign prognosis and is measured using magnetic resonance spectroscopy (the most accurate imaging modality) and that 8% of the population has elevated alanine transaminase (ALT) [37, 38]. A follow-up of population-based studies examining the natural history of NAFLD patients in Minnesota revealed that 3.1% of the patients developed cirrhosis-related complications including ascites (2%), jaundice (2%), encephalopathy (2%), variceal bleeding (1%), and HCC (0.5%) [34]. Approximately 10–30% of those with steatosis develop NASH, and the development of NASH cirrhosis is associated with a poor long-term prognosis for 2.6% of them who will be at a risk of developing HCC [39–41]. Ten years following diagnosis, 45% will decompensate and the mortality rate for subjects with Child-Pugh A disease will be 20% [42]. Furthermore, besides having an increased liver-related mortality rate compared to the general population, patients with NASH also have an increased risk of cardiovascular death (15.5 vs. 7.5%, *p* = 0.04) [35]. Generally, NAFLD is a slowly progressing disease, which does not culminate in ESLD in most patients. Identifying those who will develop a complete liver failure is a difficult proposition [43]. NAFLD data are limited on predictors of clinical progression to NASH and beyond. Due to the compounding effect of obesity, prospective longitudinal studies are needed to help in the prediction of outcomes for individual patients. On the other hand, patients with NASH have a worse prognosis and attempts should be made to include them in clinical trials of novel treatments for this condition. The sequence of steps in liver disease commencing with steatosis and eventually culminating in HCC (i.e., ESLD)

*DOI: http://dx.doi.org/10.5772/intechopen.85780*

**1.2 The natural history of NAFLD**

(ESLD) [32].

#### *The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.85780*

inflammation, satellitosis, perisinusoidal fibrosis, Mallory-Denk bodies, and acute cholestasis among others [29–31]. However, it is important to note that NAFLD can also coexist with other liver diseases including HCV, hemochromatosis, and alcoholic liver disease, which can accelerate progression to end-stage liver disease (ESLD) [32].

#### **1.2 The natural history of NAFLD**

*Nonalcoholic Fatty Liver Disease - An Update*

greater than 30 kg/m2

**1.1 NAFLD definition**

Over the past few decades, dramatic changes in lifestyle behaviors and health priorities have contributed to a significant rise in noncommunicable diseases such as obesity and NAFLD. Obesity is highly prevalent in the United States of America, estimated to represent between 30 and 38% of adults with a body mass index (BMI)

(MetS), which increases hepatic triglyceride (TGs) depositions. NAFLD is the most common cause of impaired liver function in Western countries, affecting over one quarter of the population [10, 11]. Obesity is driving the rise of NAFLD and nonalcoholic steatohepatitis (NASH), the culmination of the fatty liver disease spectrum that is manifested by ballooning, scarring, cirrhosis, and finally liver failure and HCC [12]. It is estimated that globally the prevalence of NAFLD in the general population is 24–30% [13, 14]. Accounting for errors in accuracy that may exist in indirect measurement methodologies, in the United States, the prevalence of NAFLD in adults has risen from 18% in 1988–1991, to 29% in 1999–2000, to 31% in 2011–2012 [15]. However, the prevalence of NAFLD in the United States diagnosed by ultrasonography alone was estimated to be 24.13% (95% CI 19.73–29.15%) [16].

Nonalcoholic fatty liver disease (NAFLD) is a broad-spectrum disease ranging from fat infiltration of hepatocytes with no symptoms (simple steatosis aka nonalcoholic fatty liver, NAFL) to excess intrahepatic macroglobular and macrovesicular fat accumulation (5–10% by weight of liver) with aggravated inflammation (steatohepatitis aka nonalcoholic steatohepatitis, NASH) in the presence of little ethanol (typically <30 g per day for men and <20 g per day for women) or no alcohol consumption in the last 12 months [12, 17]. It should be noted, however, there is now convincing evidence demonstrating that even "safe" levels of alcohol consumption are associated with adverse health outcomes [17–20] suggesting that future studies should include only nondrinker individuals in the "NAFLD definition" [21]. Therefore, for NAFLD classification, the patient must show evidence of hepatic fat accumulation in the absence of declared chronic alcohol consumption, or drug use that can induce steatosis, or hereditary disorders. This NAFLD designation excludes both macrovesicular and microvesicular steatosis encompassing certain drugs, toxins, viral hepatitis B (HBV), hepatitis C (HCV) or human immunodeficiency virus (HIV) infections, celiac disease, α-1 antitrypsin deficiency, hepatobiliary infectious diseases, hepatolenticular degeneration, hepatic malignancies, genetic hemochromatosis, Wilson's disease, lipodystrophy, abetalipoproteinemia, Reye's syndrome, HELLP syndrome, or decompensated cirrhosis, which may contribute to secondary causes of steatosis and elevated liver enzymes [22–24]. Additional medications targeted for exclusion are estrogen, sodium valproate, nonsteroidal anti-inflammatory drugs (NSAIDs), calcium antagonists, perhexiline-maleate, and antiretroviral drugs [25–27]. Appropriate medical history must also be taken to exclude the uncommon causes of fatty liver secondary to treatment with drugs such as amiodarone, diltiazem, steroids, synthetic estrogens, tamoxifen, and highly active antiretroviral therapy; refeeding syndrome and total parenteral nutrition; severe weight loss after jejunoileal or gastric bypass; lipodystrophy; and other rare disorders [28]. There are also strong opinions for the exclusion of "whole-body system diseases" such as inflammatory bowel syndrome, hypothyroidism, and lipoatrophy [25] from the "secondary fatty liver diseases" category because they

NAFLD can be distinguished from alcoholic steatohepatitis (ASH) by the absence of alcohol consumption and on histological markers such as sclerosing hyaline necrosis, hepatocyte ballooning, portal granulocytic inflammation, lobular

[8, 9]. Obesity is also a risk factor for metabolic syndrome

**78**

may also induce liver steatosis.

The pathophysiology of NAFLD and its variants is still incompletely understood thereby limiting the availability of effective diagnostic and therapeutic intervention. The ongoing persistence of obesity and the accompanying high rates of diabetes will increase the prevalence of NAFLD [33]. In many cases, the natural cause of the disease is the development of cirrhosis and ESLD as the population ages. Increased mortality rates have been reported in studies that compared NAFLD patients with a normal reference population [34–36]. The primary cause of death for NAFLD patients is cardiovascular disease followed by nonliver cancer, whereas the third leading cause of mortality is liver-related complications including cirrhosis [33]. The exact prevalence of fatty liver condition is not known, but population studies from the United States and China estimate that 28–30% of the general population has simple steatosis that carries a relatively benign prognosis and is measured using magnetic resonance spectroscopy (the most accurate imaging modality) and that 8% of the population has elevated alanine transaminase (ALT) [37, 38]. A follow-up of population-based studies examining the natural history of NAFLD patients in Minnesota revealed that 3.1% of the patients developed cirrhosis-related complications including ascites (2%), jaundice (2%), encephalopathy (2%), variceal bleeding (1%), and HCC (0.5%) [34]. Approximately 10–30% of those with steatosis develop NASH, and the development of NASH cirrhosis is associated with a poor long-term prognosis for 2.6% of them who will be at a risk of developing HCC [39–41]. Ten years following diagnosis, 45% will decompensate and the mortality rate for subjects with Child-Pugh A disease will be 20% [42]. Furthermore, besides having an increased liver-related mortality rate compared to the general population, patients with NASH also have an increased risk of cardiovascular death (15.5 vs. 7.5%, *p* = 0.04) [35]. Generally, NAFLD is a slowly progressing disease, which does not culminate in ESLD in most patients. Identifying those who will develop a complete liver failure is a difficult proposition [43]. NAFLD data are limited on predictors of clinical progression to NASH and beyond. Due to the compounding effect of obesity, prospective longitudinal studies are needed to help in the prediction of outcomes for individual patients. On the other hand, patients with NASH have a worse prognosis and attempts should be made to include them in clinical trials of novel treatments for this condition. The sequence of steps in liver disease commencing with steatosis and eventually culminating in HCC (i.e., ESLD) is presented in **Figure 1** [44].

#### **1.3 NAFLD diagnosis and staging**

The general classification of NAFLD as stated above and accepted by the American Association for the Study of Liver Diseases (AASLD) is a hepatic fat accumulation exceeding 5–10% by weight of the liver [45]. Accordingly, NAFLD diagnosis in the liver is based on: (i) the presence of simple steatosis, as determined by histological or imaging procedure; (ii) a total weekly consumption of less than 140 g of ethanol for men and less than 70 g for women in the last 12 months; and (iii) the absence of competing etiologies for simple liver steatosis and the absence of coexisting causes for chronic liver disease [46]. An appropriate diagnosis of

**Figure 1.**

*The progression and stages of NAFLD (adapted from Baranova et al., [44]). Steatosis is the initial NAFLD stage and is characterized by excessive accumulation of fat in hepatocytes. Subsequent inflammatory conditions accelerate the progression to NASH followed by liver cirrhosis, which may lead to HCC. Both steatosis and NASH can reverse to NAFLD.*

NAFLD, which is multifaceted, requires that there is evidence of hepatic steatosis upon imaging and histology or both and that other causes of liver disease including steatosis have been excluded [23].

The increasing prevalence of obesity in the past few decades has led to a surge in NAFLD, which manifests liver cells as bloated with droplets of fat. It has been reported that 70% of centrally obese patients with diabetes and hypertension (HTN) harbor steatohepatitis on liver biopsy [47]. Imaging has enabled the observation of central obesity in 70–80% of these subjects and in 50–80% of patients with type 2 diabetes mellitus (T2DM). NAFLD is typically asymptomatic; therefore, diagnosis usually follows the subsidiary finding of abnormal liver enzymes or steatosis on imaging. Early diagnosis of NAFLD requires skilled and informed practitioners to halt fibrosis progression to more advanced stages. Liver needle puncture biopsy, although invasive, is the gold standard. Less-invasive methods of image detection tools may not provide consistent information due to the subjective interpretations of the data by radiologists [48]. But imaging tools such as abdominal ultrasound (US), computed tomography (CT), and magnetic resonance imaging (MRI) are beginning to meet this need. Ultrasound or sonography is very effective in diagnosing steatosis where greater than 33% of hepatocytes are steatotic but can be unreliable with lesser degrees of steatosis [49]. The other imaging modalities such as CT or MRI can also detect hepatic steatosis even though they are not used in the evaluation of steatosis. Currently, the combination of MRI and proton magnetic resonance spectroscopy (MRI/1 H-MRS) is the most accurate noninvasive measuring tool of steatosis [50, 51]. 1 H-MRS, which defines NAFLD as hepatic fat accumulation (steatosis) >5% of total weight of the liver, is the most reliable quantitative tool. However, due to its prohibitive cost, it is not widely available. Ultrasonography, on the other hand, is the instrument of choice for most of the clinics due to its low cost and wide availability even though it is still relatively limited in the detection of inflammation, a more important and higher risk concern than steatosis for fibrosis, cirrhosis, and HCC [52, 53].

Controlled attenuation parameter (CAP), which is a novel ultrasound-based technique that assesses liver stiffness and steatosis simultaneously by employing transient elastography (TE) [54]. This CAP technique has been shown to accurately detect steatosis although its diagnostic threshold has not yet been determined. Obesity and diabetes are the main risk factors for NAFLD [55]. It has been reported that the presence of T2DM significantly increases the prevalence

**81**

individuals [66].

risk factors are age (>45–50), BMI (>28–30 kg/m2

*The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma*

of NAFLD regardless of the diagnostic tool [56]. For example, using controlled attenuation parameter (CAP), the prevalence of NAFLD is estimated at 75% in T2DM population and 40% in the general population, whereas it is 65% and

down when assessed by liver ultrasound, computed tomography, and plasma ALT

In contrast, most global population studies base their NAFLD characterization on less sensitive and less specific surrogate markers of the disease including elevated liver-associated enzymes such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT >40 IU/L in males; >31 IU/L in females) [57, 58]. Furthermore, serum ALT levels are within the range currently considered "normal" in a sizeable proportion of NAFLD subjects [59]. Typically, depending on the reference values from different laboratories, the broad range for normal AST is reported between 10 and 40 IU/L and ALT between 7 and 56 IU/L. This is because ALT usually falls (and AST may rise) as fibrosis progresses to cirrhosis. Mild elevations, which are generally asymptomatic, are considered to be 2–3 times higher than the normal range, and drastic elevations are 5 times higher than the upper limit of normal, which varies according to gender [60]. Moreover, the very selective measurement of ALT level based on race or ethnicity underscores the lack of effective surrogate markers for NAFLD/NASH in the absence of biopsy [61]. Therefore, an innovative approach is needed to use metabolic risk factors to identify subjects with NAFLD/NASH rather than relying on liver enzyme

There is an active research that is underway to discover serum biomarkers for NASH since it is associated with increased apoptosis and therefore blood markers of apoptosis may be instrumental in distinguishing NASH from simple steatosis [62]. Apoptosis activates caspases that cleave various substrates such as cytokeratin-18 (CK-18), a key intermediate filament protein in hepatocytes, that can be detected with an ELISA test using an M30 antibody to identify patients with NASH [63, 64]. However, liver biopsy provides a superior assessment of hepatic steatosis, hepatocellular injury, inflammation, and fibrosis as well as its ability to demonstrate the presence of hepatocyte ballooning and degeneration in association with steatosis as the key histological feature that distinguishes NASH from simple steatosis. Notwithstanding its limitations such as inherent variability in histologic assessment of NAFLD stage and activity, its invasiveness, its high possibility of complications related to liver damage, its proneness to sampling error generated by the operators, and its limitations in accessibility and reproducibility, liver biopsy is still the standard criterion for the most accurate diagnosis of NAFLD and NASH. Also, because only 7–30% of NAFLD patients in the world population had an indication of biopsy for accurate measurement, Younosis et al., re-evaluated and reported the global prevalence of NASH to be between 1.5 and 6.45% and the North American rate at an average of 8.69% (between 7.2 and 14.63%) [4, 65]. Regarding obesity, reports show that NASH can be verified by histological examination in about 47% of all NAFLD cases among obese

Liver fibrosis is the inordinate accretion of extracellular matrix proteins that include collagen in most types of liver disease including NAFLD. Fibrosis stage is a crucial histological variable to predict mortality. There are well-known independent predictors of fibrosis, which is a subway to chronic liver disease state. Some of these

tes, and HTN [67]. Staging hepatic fibrosis is essential in all patients with NAFLD to identify individuals with advanced fibrosis (AF) who may later develop liverrelated complications such as hepatocellular dysfunction and portal hypertension

), insulin resistance (IR), diabe-

H-MRS. The prevalence rate goes

*DOI: http://dx.doi.org/10.5772/intechopen.85780*

about 37% respectively when measured by <sup>1</sup>

in that order [56].

abnormalities.

#### *The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.85780*

of NAFLD regardless of the diagnostic tool [56]. For example, using controlled attenuation parameter (CAP), the prevalence of NAFLD is estimated at 75% in T2DM population and 40% in the general population, whereas it is 65% and about 37% respectively when measured by <sup>1</sup> H-MRS. The prevalence rate goes down when assessed by liver ultrasound, computed tomography, and plasma ALT in that order [56].

In contrast, most global population studies base their NAFLD characterization on less sensitive and less specific surrogate markers of the disease including elevated liver-associated enzymes such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT >40 IU/L in males; >31 IU/L in females) [57, 58]. Furthermore, serum ALT levels are within the range currently considered "normal" in a sizeable proportion of NAFLD subjects [59]. Typically, depending on the reference values from different laboratories, the broad range for normal AST is reported between 10 and 40 IU/L and ALT between 7 and 56 IU/L. This is because ALT usually falls (and AST may rise) as fibrosis progresses to cirrhosis. Mild elevations, which are generally asymptomatic, are considered to be 2–3 times higher than the normal range, and drastic elevations are 5 times higher than the upper limit of normal, which varies according to gender [60]. Moreover, the very selective measurement of ALT level based on race or ethnicity underscores the lack of effective surrogate markers for NAFLD/NASH in the absence of biopsy [61]. Therefore, an innovative approach is needed to use metabolic risk factors to identify subjects with NAFLD/NASH rather than relying on liver enzyme abnormalities.

There is an active research that is underway to discover serum biomarkers for NASH since it is associated with increased apoptosis and therefore blood markers of apoptosis may be instrumental in distinguishing NASH from simple steatosis [62]. Apoptosis activates caspases that cleave various substrates such as cytokeratin-18 (CK-18), a key intermediate filament protein in hepatocytes, that can be detected with an ELISA test using an M30 antibody to identify patients with NASH [63, 64]. However, liver biopsy provides a superior assessment of hepatic steatosis, hepatocellular injury, inflammation, and fibrosis as well as its ability to demonstrate the presence of hepatocyte ballooning and degeneration in association with steatosis as the key histological feature that distinguishes NASH from simple steatosis. Notwithstanding its limitations such as inherent variability in histologic assessment of NAFLD stage and activity, its invasiveness, its high possibility of complications related to liver damage, its proneness to sampling error generated by the operators, and its limitations in accessibility and reproducibility, liver biopsy is still the standard criterion for the most accurate diagnosis of NAFLD and NASH. Also, because only 7–30% of NAFLD patients in the world population had an indication of biopsy for accurate measurement, Younosis et al., re-evaluated and reported the global prevalence of NASH to be between 1.5 and 6.45% and the North American rate at an average of 8.69% (between 7.2 and 14.63%) [4, 65]. Regarding obesity, reports show that NASH can be verified by histological examination in about 47% of all NAFLD cases among obese individuals [66].

Liver fibrosis is the inordinate accretion of extracellular matrix proteins that include collagen in most types of liver disease including NAFLD. Fibrosis stage is a crucial histological variable to predict mortality. There are well-known independent predictors of fibrosis, which is a subway to chronic liver disease state. Some of these risk factors are age (>45–50), BMI (>28–30 kg/m2 ), insulin resistance (IR), diabetes, and HTN [67]. Staging hepatic fibrosis is essential in all patients with NAFLD to identify individuals with advanced fibrosis (AF) who may later develop liverrelated complications such as hepatocellular dysfunction and portal hypertension

*Nonalcoholic Fatty Liver Disease - An Update*

steatosis have been excluded [23].

*NASH can reverse to NAFLD.*

**Figure 1.**

resonance spectroscopy (MRI/1

tool of steatosis [50, 51]. 1

cirrhosis, and HCC [52, 53].

NAFLD, which is multifaceted, requires that there is evidence of hepatic steatosis upon imaging and histology or both and that other causes of liver disease including

*The progression and stages of NAFLD (adapted from Baranova et al., [44]). Steatosis is the initial NAFLD stage and is characterized by excessive accumulation of fat in hepatocytes. Subsequent inflammatory conditions accelerate the progression to NASH followed by liver cirrhosis, which may lead to HCC. Both steatosis and* 

The increasing prevalence of obesity in the past few decades has led to a surge in NAFLD, which manifests liver cells as bloated with droplets of fat. It has been reported that 70% of centrally obese patients with diabetes and hypertension (HTN) harbor steatohepatitis on liver biopsy [47]. Imaging has enabled the observation of central obesity in 70–80% of these subjects and in 50–80% of patients with type 2 diabetes mellitus (T2DM). NAFLD is typically asymptomatic; therefore, diagnosis usually follows the subsidiary finding of abnormal liver enzymes or steatosis on imaging. Early diagnosis of NAFLD requires skilled and informed practitioners to halt fibrosis progression to more advanced stages. Liver needle puncture biopsy, although invasive, is the gold standard. Less-invasive methods of image detection tools may not provide consistent information due to the subjective interpretations of the data by radiologists [48]. But imaging tools such as abdominal ultrasound (US), computed tomography (CT), and magnetic resonance imaging (MRI) are beginning to meet this need. Ultrasound or sonography is very effective in diagnosing steatosis where greater than 33% of hepatocytes are steatotic but can be unreliable with lesser degrees of steatosis [49]. The other imaging modalities such as CT or MRI can also detect hepatic steatosis even though they are not used in the evaluation of steatosis. Currently, the combination of MRI and proton magnetic

tion (steatosis) >5% of total weight of the liver, is the most reliable quantitative tool. However, due to its prohibitive cost, it is not widely available. Ultrasonography, on the other hand, is the instrument of choice for most of the clinics due to its low cost and wide availability even though it is still relatively limited in the detection of inflammation, a more important and higher risk concern than steatosis for fibrosis,

Controlled attenuation parameter (CAP), which is a novel ultrasound-based technique that assesses liver stiffness and steatosis simultaneously by employing transient elastography (TE) [54]. This CAP technique has been shown to accurately detect steatosis although its diagnostic threshold has not yet been determined. Obesity and diabetes are the main risk factors for NAFLD [55]. It has been reported that the presence of T2DM significantly increases the prevalence

H-MRS) is the most accurate noninvasive measuring

H-MRS, which defines NAFLD as hepatic fat accumula-

**80**

(PHTN). A noninvasive and an indirect assessment, which is performed in all liver disease patients including children, may include blood tests such as liver function tests (low albumin), complete blood count (thrombocytopenia and neutropenia), and coagulation profile (prolonged prothrombin time) [68]. Among the diagnostic tools used to measure the prevalence of AF in the setting of T2DM versus the general population, vibration-controlled transient elastography shows the highest prevalence rate followed by NAFLD fibrosis score and FibroTest in that order. It should be noted that the prevalence of T2DM significantly increases the prevalence of AF in similar ways to NAFLD [56].

The most widespread clinically implemented histological grading and staging system is the 'NAFLD activity score' (NAS) [6] (see **Table 1**). More recently, the SAF score encompassing an assessment of steatosis (S), activity (A), and fibrosis (F) has been used to produce more accurate measurements of NASH [5]. These recent developments underscore the fact that NAFLD patients can be diagnosed and staged effectively using noninvasive strategies even though liver biopsy can still be applied for individuals with dubious diagnostic tests or if noninvasive staging is unspecified [69]. However, there is no widely available simple blood test or imaging modality that can differentiate simple steatosis from NASH.

In summary, early diagnosis of NAFLD is essential to halting the progression of the disease. Biopsy is intrusive and therefore cannot be routinely applied. Ultrasound (sonography) and magnetic resonance imaging tools have become alternative noninvasive detection tools that can be routinely employed in clinical practice. The NAFLD activity score is important as part of the diagnosis procedure. But the fibrosis score is just as important. **Table 2** shows the fibrosis score currently used to stage the degree of fibrosis in the liver. There are a few noninvasive fibrosis imaging tests on the market such as Fibroscan that offers a liver stiffness measurement (LSM) using pulsed-echo ultrasound as a surrogate marker of fibrosis [70] and acoustic radiation force impulse (ARFI), which uses conventional B-mode ultrasonography to produce an ultrasonic pulse and measure the response of the liver tissue as shear wave velocity [71]. The Centers for Disease Control (CDC) and Prevention projects that diabetes mellitus is likely to impact the fibrosis progression rates, given the close link between diabetes and fibrosis in those with NAFLD [72, 73].

Some commercial biomarker tests include enhanced liver fibrosis (ELF), a panel of markers of matrix turnover as tissue inhibitor of matrix metalloproteinase 1 (TIMP1), hyaluronic acid and PIIINP [74] and FibroTest (FT), a panel of markers of fibrosis widely used in France.


*NASH activity grade=total score: S + L + B range (0–8). Score of* ≥*5 is equivalent to NASH; score of 3 or 4 is borderline NASH; score of* ≤*2 denotes non-NASH NAFLD. The NAFLD activity score is based on three pathologic features: Steatosis, hepatocyte ballooning degeneration, and lobular inflammation. Higher scoring denotes severity of NASH: >5=NASH; <5=No NASH; 3–4=borderline; none (0); few (1); many (2).*

**83**

**Table 3.**

*Features of the metabolic syndrome.\**

*The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma*

**Liver injury Fibrosis score (0–4)\* Fibrosis stage** None 0 0 Mild (delicate fibrosis)/zone 3 presinusoidal fibrosis <5% (0), 5–33% = (1) 1a Moderate (dense fibrosis)/zone 3 presinusoidal fibrosis >33–66% (2), >66% = (3) 1b Periportal/portal fibrosis 0 (0), <2 (1), 2–4 (2), > (3) 1c Portal and periportal fibrosis/presinusoidal fibrosis None (0) 2 Bridging fibrosis Few (1) 3 Cirrhosis Many (2) 4

Recognizing patients with the metabolic syndrome (MetS) is key to identifying patients at risk of NAFLD. MetS is a group of risk factors that raises risk of heart disease, diabetes, stroke, etc. [75] and is diagnosed when any three of the following five clinical risk factors are present [76]: impaired fasting serum glucose, low levels of serum HDL cholesterol, elevated serum triglycerides (i.e., hypertriglyceridemia), central obesity or larger than cut-off waist circumference (varies according to

Insulin resistance is also a major risk factor for the development of steatosis. Once considered benign, NAFL (or simple steatosis), which is defined as the presence of hepatic steatosis with no evidence of hepatocellular injury in the form of ballooning of the hepatocytes, is now believed to be a serious risk factor for progression to liver disease, cardiovascular disease, and mortality [37, 77]. This is because an excess of abdominal fat is most tightly associated with the metabolic risk factors [78, 79]. The duration of obesity and the presence of MetS in an individual patient are closely tied to the risk of developing NASH-related cirrhosis and HCC [80]. Some of the characteristics of MetS are present in most NAFLD individuals, with 65–71% being obese, 57–68% having deranged lipid profiles, 36–70% suffering from HTN, and 12–37% having impaired fasting glucose tolerance [81]. Approximately a third of patients with NAFLD have the full metabolic syndrome and >90% have at least one feature [47]. There is a consensus that considers NAFLD as a hepatic manifestation of the MetS [82, 83]. On the other hand, clinical signs of the disease are manifested in 70–75% of T2DM patients and up to 95% of obese patients [84]. Thus, the development of the MetS, which is an important

gender and ethnicity), and high blood pressure (HTN) (see **Table 3**).

**Features Terms of condition** Blood glucose (sugar) Fasting, ≥100 mg/dL Blood HDL ("good") cholesterol ♂ < 40 mg/dL; ♀ < 50 mg/dL Blood triglycerides (TGs) Fasting, ≥150 mg/dL Waist circumference ♂ > 40″; ♀ > 35″ Blood pressure (HTN) ≥130/85 mm Hg

*\*The MetS is present with any three of the features shown in the table.* ♂ *= male;* ♀ *= female; HDL = high-density lipoprotein;* ″ *= inches.*

*DOI: http://dx.doi.org/10.5772/intechopen.85780*

**1.4 The metabolic syndrome (MetS)**

*NAFLD fibrosis score (NFS) and stage.*

*Fibrosis score of F1-F4 is generally considered NASH [4].*

*\**

**Table 2.**

**Table 1.** *NAFLD activity score (NAS).* *The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.85780*


#### **Table 2.**

*Nonalcoholic Fatty Liver Disease - An Update*

of AF in similar ways to NAFLD [56].

from NASH.

those with NAFLD [72, 73].

**Grade Steatosis** 

*NAFLD activity score (NAS).*

of fibrosis widely used in France.

**(%)**

(PHTN). A noninvasive and an indirect assessment, which is performed in all liver disease patients including children, may include blood tests such as liver function tests (low albumin), complete blood count (thrombocytopenia and neutropenia), and coagulation profile (prolonged prothrombin time) [68]. Among the diagnostic tools used to measure the prevalence of AF in the setting of T2DM versus the general population, vibration-controlled transient elastography shows the highest prevalence rate followed by NAFLD fibrosis score and FibroTest in that order. It should be noted that the prevalence of T2DM significantly increases the prevalence

The most widespread clinically implemented histological grading and staging

In summary, early diagnosis of NAFLD is essential to halting the progression of the disease. Biopsy is intrusive and therefore cannot be routinely applied. Ultrasound (sonography) and magnetic resonance imaging tools have become alternative noninvasive detection tools that can be routinely employed in clinical practice. The NAFLD activity score is important as part of the diagnosis procedure. But the fibrosis score is just as important. **Table 2** shows the fibrosis score currently used to stage the degree of fibrosis in the liver. There are a few noninvasive fibrosis imaging tests on the market such as Fibroscan that offers a liver stiffness measurement (LSM) using pulsed-echo ultrasound as a surrogate marker of fibrosis [70] and acoustic radiation force impulse (ARFI), which uses conventional B-mode ultrasonography to produce an ultrasonic pulse and measure the response of the liver tissue as shear wave velocity [71]. The Centers for Disease Control (CDC) and Prevention projects that diabetes mellitus is likely to impact the fibrosis progression rates, given the close link between diabetes and fibrosis in

Some commercial biomarker tests include enhanced liver fibrosis (ELF), a panel

of markers of matrix turnover as tissue inhibitor of matrix metalloproteinase 1 (TIMP1), hyaluronic acid and PIIINP [74] and FibroTest (FT), a panel of markers

**inflammation**

0 <5 0 No foci 0 None 0 1 5–33 1 <2 foci per 200 × field 1 Few cells 1 2 34–66 2 2–4 foci per 200 × filed 2 Many cells 2 3 >66 3 >4 foci per 200 × filed 3 N/A N/A *NASH activity grade=total score: S + L + B range (0–8). Score of* ≥*5 is equivalent to NASH; score of 3 or 4 is borderline NASH; score of* ≤*2 denotes non-NASH NAFLD. The NAFLD activity score is based on three pathologic features: Steatosis, hepatocyte ballooning degeneration, and lobular inflammation. Higher scoring denotes severity of* 

**L score Hepatocyte** 

**ballooning (B)**

**B score**

**S score Lobular (L)** 

*NASH: >5=NASH; <5=No NASH; 3–4=borderline; none (0); few (1); many (2).*

system is the 'NAFLD activity score' (NAS) [6] (see **Table 1**). More recently, the SAF score encompassing an assessment of steatosis (S), activity (A), and fibrosis (F) has been used to produce more accurate measurements of NASH [5]. These recent developments underscore the fact that NAFLD patients can be diagnosed and staged effectively using noninvasive strategies even though liver biopsy can still be applied for individuals with dubious diagnostic tests or if noninvasive staging is unspecified [69]. However, there is no widely available simple blood test or imaging modality that can differentiate simple steatosis

**82**

**Table 1.**

*NAFLD fibrosis score (NFS) and stage.*

#### **1.4 The metabolic syndrome (MetS)**

Recognizing patients with the metabolic syndrome (MetS) is key to identifying patients at risk of NAFLD. MetS is a group of risk factors that raises risk of heart disease, diabetes, stroke, etc. [75] and is diagnosed when any three of the following five clinical risk factors are present [76]: impaired fasting serum glucose, low levels of serum HDL cholesterol, elevated serum triglycerides (i.e., hypertriglyceridemia), central obesity or larger than cut-off waist circumference (varies according to gender and ethnicity), and high blood pressure (HTN) (see **Table 3**).

Insulin resistance is also a major risk factor for the development of steatosis. Once considered benign, NAFL (or simple steatosis), which is defined as the presence of hepatic steatosis with no evidence of hepatocellular injury in the form of ballooning of the hepatocytes, is now believed to be a serious risk factor for progression to liver disease, cardiovascular disease, and mortality [37, 77]. This is because an excess of abdominal fat is most tightly associated with the metabolic risk factors [78, 79]. The duration of obesity and the presence of MetS in an individual patient are closely tied to the risk of developing NASH-related cirrhosis and HCC [80]. Some of the characteristics of MetS are present in most NAFLD individuals, with 65–71% being obese, 57–68% having deranged lipid profiles, 36–70% suffering from HTN, and 12–37% having impaired fasting glucose tolerance [81]. Approximately a third of patients with NAFLD have the full metabolic syndrome and >90% have at least one feature [47]. There is a consensus that considers NAFLD as a hepatic manifestation of the MetS [82, 83]. On the other hand, clinical signs of the disease are manifested in 70–75% of T2DM patients and up to 95% of obese patients [84]. Thus, the development of the MetS, which is an important


### **Table 3.**

predictor of NASH in NAFLD patients, poses a sweeping and unfavorable prognosis [85]. IR is a key mediator that links NAFLD and MetS, which is a constellation of anthropometric and metabolic abnormalities (see **Table 3** above).

According to the latest data from NHANES (National Health and Nutrition Examination Survey) study conducted between 2011 and 2012, the prevalence of MetS has increased to 35% in American adults [86]. MetS is a risk factor for diabetes and cardiovascular diseases. It induces an abnormal production of hormones such as leptin, adiponectin, and cytokines such as TNF (tumor necrosis factor) alpha that regulate inflammatory responses and cause disequilibrium between the pro-inflammatory and anti-inflammatory state of the organ [86]. These are mutually antagonistic: the pro-inflammatory factors such as TNF-alpha promote pro-apoptotic processes, recruit white blood cells, and promote insulin resistance. On the other hand, adiponectin acting as an anti-inflammatory factor inhibits fatty acid uptake, stimulates fatty acid oxidation and lipid export, and enhances insulin sensitivity. Both an increase in pro-inflammatory factors and a decrease in anti-inflammatory factors cause a cytokine imbalance that would lead to steatosis (NAFL) followed by necroinflammation (NASH) and IR. There is a supporting evidence that a high TNF to adiponectin ratio promotes fatty liver and steatohepatitis in animal [87] and human [88] studies. The importance of MetS including IR is that it predicts the occurrence of diabetes and cardiovascular diseases, which can further promote the development and progression of arteriosclerosis and HTN leading to significant morbidity and mortality [89]. Also, NAFLD and obesity are risk factors for the progression to fibrosis among HCV-infected patients [90–93]. Furthermore, elevated levels of ferritin are common in NAFLD patients and typically reflect active IR or underlying inflammatory activity [68, 81, 94]. Therefore, because of many different correlates and etiological factors and an assortment of assessment tools associated with MetS, there are some unresolved uncertainties in the current estimates of the global and the United States prevalence of NAFLD.

#### **1.5 The genetics of NAFLD**

Genetic disorders of lipid metabolism can cause hepatic fat deposition. However, they are far less common than excess body weight and features of MetS as risk factors for NAFLD and NASH. Several genes have been associated with NAFLD. These include *NCAN*, which may have a protective effect for Hispanics but increases risk of steatosis for non-Hispanic blacks; *LYPLAL1*, *GCKR*, as well as *PPP1R3B*, which may confer increased risk for hepatic steatosis but the data of distinctive serum lipid profiles in all these genes are sparse [61, 95–97]. *GCKR* is reported to be closely associated with NAFLD in Chinese [98]. Another gene, Patatin-like phospholipase domain-containing 3 (*PNPLA3* or adiponutrin), has emerged as the genetic factor predisposing Hispanics more at risk for fatty liver disease [99]. This adiponutrin gene is a single variant considered responsible for increased hepatic TG levels, fibrosis, and inflammation, observed among ethnic groups [100, 101]. Homozygote patients have a twofold rise in hepatic fat content than heterozygotes, and Hispanic populations exhibit the highest frequency of this polymorphism (49%) compared to 23% in European-Americans (EAs) and 17% in African-Americans (AAs) [101]. It also shows more allelic frequency with Hispanics than other ethnic groups. Romeo and colleagues [102, 103] along with Singal and colleagues published papers in 2008 in which they reported that *PNPLA3* is strongly associated with hepatic steatosis and elevated ALT and also recently showed that *PNPLA3* is associated with NASH, fibrosis progression, and hepatocellular cancer as well [102].

A genetic marker, *TM6SF2*, discovered in an exome-wide association study of liver fat content, has also shed some light on its association with hepatic steatosis. It

**85**

*The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma*

is involved in the loss of function mutation in very low-density lipoprotein (VLDL) secretion, and its association with NASH and advanced fibrosis has been recently validated even though its precise function has not been delineated [102]. However, its mutation is associated with elevated ALT, hepatic steatosis, and lower level of alkaline phosphatase, LDL, and TGs. This gene is most prevalent in European

There is a reported 52% heritability rate of NAFLD, but evidence pertaining to specific genetic mutations is scant according to multivariable models used after adjusting for sex, age, and ethnicity [13]. Although the mechanism is not well understood, genetic mutations in hemochromatosis (HFE) gene, which is responsible for iron uptake and transferrin plasma concentration, may also be associated with NAFLD development [105, 106]. Several other factors have been indicated in the development and outcomes of NAFLD including epigenetic alterations [107, 108], maternal perinatal nutrition [109–111], and gut microbiota [107, 112–114]. A recent study also reports a novel pathway in which hepatic vitamin D receptor (VDR) expression is increased in patients with simple

steatosis (nonalcoholic fatty liver without inflammation), and the activated VDR upregulates angiopoietin-like protein 8 (ANGPTL8) expression, thus contributing to triglyceride accumulation in human hepatocytes [115]. At any rate, studies have reported that fibrosis-initiated fatty liver disease progresses over many years, thus providing a potential window for intervention by examining diseaseprogression/modifying factors in NAFLD [116–118]. It is important to note that increased BMI and insulin resistance have been associated with a more rapid

In most epidemiological studies, the prevalence of NAFLD in the general population is determined by imaging or other indirect methods. Accordingly, the epidemiology and demographic characteristics of NAFLD vary worldwide [12, 16]. In epidemiological studies, the pathophysiological aspects, the natural history, and the determinants of NAFLD are important parameters for the diagnosis and evaluation of therapeutic interventions. This section will provide global perspectives on the prevalence of NAFLD (and later HCC) with emphasis on the United States and

There are wide-ranging estimates of NAFLD prevalence in the general population of the United States. An estimated 17–51% of adults have NAFLD [23, 120, 121].

Globally, NAFLD is a growing cause of chronic liver disease and NASH is replacing HCV infection as the primary reason for LT [13, 123, 124]. The broad category of NAFLD can manifest as NAFL or NASH. Fibrosis precedes cirrhosis and is therefore used as a prognosticator of the clinical risk of progression to cirrhosis and long-term liver-related adverse outcomes and mortality [34]. Recent evidence has shown that NAFLD and NASH can progress to HCC even in the absence of cirrhosis [125–127]. In most epidemiological studies including the NHANES data set, the assumptions about NASH in the NAFLD population are based on a post-hoc application of liver enzymes (i.e., AST and ALT) and clinical measurements. In the same vein, the fibrosis stages in population-based studies reflect best estimates derived

Analysis of liver ultrasound data collected between 1988 and 1994 from the NHANES III reported that 19% of adults have NAFLD [122], whereas a meta-analysis of studies from 2006 to 2014 estimated a NAFLD prevalence of 24% (20–29%) in the general population [65]. The prevalence of NASH is difficult to estimate as biopsy is the necessary tool for screening, but it is cost-prohibitive and impractical

*DOI: http://dx.doi.org/10.5772/intechopen.85780*

ancestry and less in Hispanics and AAs [104].

progression to fibrosis [35, 119].

the possible reasons for the rapid rise.

for a population study.

**1.6 The epidemiology and prevalence of NAFLD**

*The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.85780*

is involved in the loss of function mutation in very low-density lipoprotein (VLDL) secretion, and its association with NASH and advanced fibrosis has been recently validated even though its precise function has not been delineated [102]. However, its mutation is associated with elevated ALT, hepatic steatosis, and lower level of alkaline phosphatase, LDL, and TGs. This gene is most prevalent in European ancestry and less in Hispanics and AAs [104].

There is a reported 52% heritability rate of NAFLD, but evidence pertaining to specific genetic mutations is scant according to multivariable models used after adjusting for sex, age, and ethnicity [13]. Although the mechanism is not well understood, genetic mutations in hemochromatosis (HFE) gene, which is responsible for iron uptake and transferrin plasma concentration, may also be associated with NAFLD development [105, 106]. Several other factors have been indicated in the development and outcomes of NAFLD including epigenetic alterations [107, 108], maternal perinatal nutrition [109–111], and gut microbiota [107, 112–114]. A recent study also reports a novel pathway in which hepatic vitamin D receptor (VDR) expression is increased in patients with simple steatosis (nonalcoholic fatty liver without inflammation), and the activated VDR upregulates angiopoietin-like protein 8 (ANGPTL8) expression, thus contributing to triglyceride accumulation in human hepatocytes [115]. At any rate, studies have reported that fibrosis-initiated fatty liver disease progresses over many years, thus providing a potential window for intervention by examining diseaseprogression/modifying factors in NAFLD [116–118]. It is important to note that increased BMI and insulin resistance have been associated with a more rapid progression to fibrosis [35, 119].

#### **1.6 The epidemiology and prevalence of NAFLD**

In most epidemiological studies, the prevalence of NAFLD in the general population is determined by imaging or other indirect methods. Accordingly, the epidemiology and demographic characteristics of NAFLD vary worldwide [12, 16]. In epidemiological studies, the pathophysiological aspects, the natural history, and the determinants of NAFLD are important parameters for the diagnosis and evaluation of therapeutic interventions. This section will provide global perspectives on the prevalence of NAFLD (and later HCC) with emphasis on the United States and the possible reasons for the rapid rise.

There are wide-ranging estimates of NAFLD prevalence in the general population of the United States. An estimated 17–51% of adults have NAFLD [23, 120, 121]. Analysis of liver ultrasound data collected between 1988 and 1994 from the NHANES III reported that 19% of adults have NAFLD [122], whereas a meta-analysis of studies from 2006 to 2014 estimated a NAFLD prevalence of 24% (20–29%) in the general population [65]. The prevalence of NASH is difficult to estimate as biopsy is the necessary tool for screening, but it is cost-prohibitive and impractical for a population study.

Globally, NAFLD is a growing cause of chronic liver disease and NASH is replacing HCV infection as the primary reason for LT [13, 123, 124]. The broad category of NAFLD can manifest as NAFL or NASH. Fibrosis precedes cirrhosis and is therefore used as a prognosticator of the clinical risk of progression to cirrhosis and long-term liver-related adverse outcomes and mortality [34]. Recent evidence has shown that NAFLD and NASH can progress to HCC even in the absence of cirrhosis [125–127]. In most epidemiological studies including the NHANES data set, the assumptions about NASH in the NAFLD population are based on a post-hoc application of liver enzymes (i.e., AST and ALT) and clinical measurements. In the same vein, the fibrosis stages in population-based studies reflect best estimates derived

*Nonalcoholic Fatty Liver Disease - An Update*

**1.5 The genetics of NAFLD**

predictor of NASH in NAFLD patients, poses a sweeping and unfavorable prognosis [85]. IR is a key mediator that links NAFLD and MetS, which is a constellation

According to the latest data from NHANES (National Health and Nutrition Examination Survey) study conducted between 2011 and 2012, the prevalence of MetS has increased to 35% in American adults [86]. MetS is a risk factor for diabetes and cardiovascular diseases. It induces an abnormal production of hormones such as leptin, adiponectin, and cytokines such as TNF (tumor necrosis factor) alpha that regulate inflammatory responses and cause disequilibrium between the pro-inflammatory and anti-inflammatory state of the organ [86]. These are mutually antagonistic: the pro-inflammatory factors such as TNF-alpha promote pro-apoptotic processes, recruit white blood cells, and promote insulin resistance. On the other hand, adiponectin acting as an anti-inflammatory factor inhibits fatty acid uptake, stimulates fatty acid oxidation and lipid export, and enhances insulin sensitivity. Both an increase in pro-inflammatory factors and a decrease in anti-inflammatory factors cause a cytokine imbalance that would lead to steatosis (NAFL) followed by necroinflammation (NASH) and IR. There is a supporting evidence that a high TNF to adiponectin ratio promotes fatty liver and steatohepatitis in animal [87] and human [88] studies. The importance of MetS including IR is that it predicts the occurrence of diabetes and cardiovascular diseases, which can further promote the development and progression of arteriosclerosis and HTN leading to significant morbidity and mortality [89]. Also, NAFLD and obesity are risk factors for the progression to fibrosis among HCV-infected patients [90–93]. Furthermore, elevated levels of ferritin are common in NAFLD patients and typically reflect active IR or underlying inflammatory activity [68, 81, 94]. Therefore, because of many different correlates and etiological factors and an assortment of assessment tools associated with MetS, there are some unresolved uncertainties in the current estimates of the global and the United States prevalence of NAFLD.

Genetic disorders of lipid metabolism can cause hepatic fat deposition. However, they are far less common than excess body weight and features of MetS as risk factors for NAFLD and NASH. Several genes have been associated with NAFLD. These include *NCAN*, which may have a protective effect for Hispanics but increases risk of steatosis for non-Hispanic blacks; *LYPLAL1*, *GCKR*, as well as *PPP1R3B*, which may confer increased risk for hepatic steatosis but the data of distinctive serum lipid profiles in all these genes are sparse [61, 95–97]. *GCKR* is reported to be closely associated with NAFLD in Chinese [98]. Another gene, Patatin-like phospholipase domain-containing 3 (*PNPLA3* or adiponutrin), has emerged as the genetic factor predisposing Hispanics more at risk for fatty liver disease [99]. This adiponutrin gene is a single variant considered responsible for increased hepatic TG levels, fibrosis, and inflammation, observed among ethnic groups [100, 101]. Homozygote patients have a twofold rise in hepatic fat content than heterozygotes, and Hispanic populations exhibit the highest frequency of this polymorphism (49%) compared to 23% in European-Americans (EAs) and 17% in African-Americans (AAs) [101]. It also shows more allelic frequency with Hispanics than other ethnic groups. Romeo and colleagues [102, 103] along with Singal and colleagues published papers in 2008 in which they reported that *PNPLA3* is strongly associated with hepatic steatosis and elevated ALT and also recently showed that *PNPLA3* is associated with

NASH, fibrosis progression, and hepatocellular cancer as well [102].

A genetic marker, *TM6SF2*, discovered in an exome-wide association study of liver fat content, has also shed some light on its association with hepatic steatosis. It

of anthropometric and metabolic abnormalities (see **Table 3** above).

**84**

from clinical aids (e.g., fibrosis-4, ALT to platelet ratio index, and NAFLD fibrosis scores) [128, 129]. The current prevalence rates for NAFLD, NASH, and HCC based on definitive clinical manifestations are shown in **Table 4**.

Certain risk factors such as advanced age, obesity, ethnicity, and T2DM increase the incidence and prevalence of NAFLD and NASH and have been consistently identified as salient risk factors for fibrotic progression to cirrhosis [130] (see **Table 5**).

The current global estimate is that 24–30% of the world's population is affected by NAFLD [65] and that includes between 80 and 100 million Americans (http:// www.mayoclinic.com), making it the primary etiology for liver disease in the United States; see **Figure 2**.

The increasing incidence of obesity, diabetes, and metabolic syndrome in the United States and Europe may soon catapult NAFLD/NASH to become the most common cause of HCC in developed countries. In the United States, among the more than 26 million people with diabetes, the prevalence of biopsy-proven NAFLD and NASH is as high as 74 and 11%, respectively [138, 139]*.*

#### **1.7 The rise in burden of NAFLD/NASH**

The global rise of NAFLD has exasperated the looming healthcare burden of disease. It may be difficult to accurately forecast the current and future burden of a disease that is rapidly progressing. However, there are modeling techniques and approaches that incorporate real-world surveillance data for NAFLD and NASH incidences, which are growing causes of cirrhosis and HCC. As with many models, the utility of the model is linked to the validity of the inputs into the model. One of these modeling approaches is based on the premise that public awareness and


*HCC in patients with NASH-related cirrhosis.*

**87**

**Figure 2.**

*\**

**Table 5.** *NAFLD/NASH\**

*\*\*MetS = metabolic syndrome. \*\*\*OSA = obstructive sleep apnea.*

 *risk factors.*

*The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma*

**Risk factor Description References** Age Risk increases with age [4, 40]

Higher risk of advanced fibrosis in women

MetS is an independent predictor of fibrosis

High fructose intake, low carbohydrates

OSA\*\*\* Increased risk of hepatic fibrosis [137]

Genetics Patatin-like phospholipase domain-containing 3 gene [133]

[4, 96, 131, 132]

[85, 95]

[89, 134–136]

[61]

government health policies will be able to eventually level off national obesity incidences and prevalence, which in return will level off NAFLD [4]. The interpretation of the output of this and other models attempting to analyze the burden of NAFLD is constrained by the lack of accurate diagnosis of steatohepatitis with simple epidemiologic tools. Nevertheless, the proportion of individuals with NASH in the NAFLD population will probably continue to rise through the next 15 years

Analyzing the cost and burden of disease with respect to NASH has several potential implications. First, it helps introduce strategies and treatment regimen that will stem its exponential rise in incidence and mortality rates; it will reduce the growing contribution of NASH to LT, which is expensive; and due to an oversupply of decompensated cirrhosis, matching organ availability is rare, and insurance companies have exclusive policy of qualifying subjects with NASH-induced cirrho-

The epidemiology and demographic characteristics of NAFLD vary worldwide. The rise in NAFLD and NASH will balloon the number of patients with decompensated cirrhosis and pose a major emotional and financial burden on subjects and

*Global picture of estimated prevalence of NAFLD and distribution of PNPLA3 genotypes adopted from Zobair Younossi [16]. PNPLA3 is presented as minor allele frequency in some areas (light blue section of the pie chart).*

based on the rising prevalence of diabetes mellitus [4].

sis based on whether they have associated co-morbidities.

*DOI: http://dx.doi.org/10.5772/intechopen.85780*

Gender More common in men

MetS\*\* 70–90% of patients have NAFLD

Lower risk in blacks

Diet Elevated levels of cholesterol and saturated fats

*NAFLD/NASH = nonalcoholic fatty liver disease/nonalcoholic steatohepatitis.*

Ethnicity Elevated risk in Hispanics

#### **Table 4.**

*Prevalence of NAFLD and its more progressive forms, NASH and HCC.*

*The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.85780*


**Table 5.**

*Nonalcoholic Fatty Liver Disease - An Update*

United States; see **Figure 2**.

on definitive clinical manifestations are shown in **Table 4**.

and NASH is as high as 74 and 11%, respectively [138, 139]*.*

**1.7 The rise in burden of NAFLD/NASH**

NAFLD Spectrum of fatty liver disease with <140 g for men and < 70 g for women per week of alcohol consumption

NAFL >5% simple hepatic steatosis

ballooning)

NASH >5% hepatic steatosis

*HCC in patients with NASH-related cirrhosis.*

by weight of liver without evidence of hepatocellular injury (i.e., hepatocyte

by weight of liver with inflammation and hepatocellular injury with or

Presence of cirrhosis with current or past histologic evidence of steatosis

Hepatocellular carcinoma induced by NASH

*Prevalence of NAFLD and its more progressive forms, NASH and HCC.*

without fibrosis Confirmed histologically

from clinical aids (e.g., fibrosis-4, ALT to platelet ratio index, and NAFLD fibrosis scores) [128, 129]. The current prevalence rates for NAFLD, NASH, and HCC based

Certain risk factors such as advanced age, obesity, ethnicity, and T2DM increase the incidence and prevalence of NAFLD and NASH and have been consistently identified as salient risk factors for fibrotic progression to cirrhosis [130] (see **Table 5**). The current global estimate is that 24–30% of the world's population is affected by NAFLD [65] and that includes between 80 and 100 million Americans (http:// www.mayoclinic.com), making it the primary etiology for liver disease in the

The increasing incidence of obesity, diabetes, and metabolic syndrome in the United States and Europe may soon catapult NAFLD/NASH to become the most common cause of HCC in developed countries. In the United States, among the more than 26 million people with diabetes, the prevalence of biopsy-proven NAFLD

The global rise of NAFLD has exasperated the looming healthcare burden of disease. It may be difficult to accurately forecast the current and future burden of a disease that is rapidly progressing. However, there are modeling techniques and approaches that incorporate real-world surveillance data for NAFLD and NASH incidences, which are growing causes of cirrhosis and HCC. As with many models, the utility of the model is linked to the validity of the inputs into the model. One of these modeling approaches is based on the premise that public awareness and

(7)

Estimated at 24–30% of global population [13, 14, 16] and at least 31% of US population

>80% of NAFLD patients

Estimated at up to 21–59% of patients with NAFLD Estimated at 1.5–6.45% of US general US population [40, 96, 122]

10–30% of patients with NASH

Estimated at annual rate of 2.6–12.8%\*

*The prevalence of NASH-HCC is not firmly established. Data in the table are the annual incidence rate of developing* 

—

to cirrhosis

15 years

6.5 years

disease

Low probability of progression

11% progress to cirrhosis over

About 31% have liver decompensation over 8 years; about 7% develop HCC over

Progresses to end-stage liver

**Status Definition Prevalence Prognosis**

**86**

**Table 4.**

*\**

NASH cirrhosis

NASH HCC

*NAFLD/NASH\* risk factors.*

government health policies will be able to eventually level off national obesity incidences and prevalence, which in return will level off NAFLD [4]. The interpretation of the output of this and other models attempting to analyze the burden of NAFLD is constrained by the lack of accurate diagnosis of steatohepatitis with simple epidemiologic tools. Nevertheless, the proportion of individuals with NASH in the NAFLD population will probably continue to rise through the next 15 years based on the rising prevalence of diabetes mellitus [4].

Analyzing the cost and burden of disease with respect to NASH has several potential implications. First, it helps introduce strategies and treatment regimen that will stem its exponential rise in incidence and mortality rates; it will reduce the growing contribution of NASH to LT, which is expensive; and due to an oversupply of decompensated cirrhosis, matching organ availability is rare, and insurance companies have exclusive policy of qualifying subjects with NASH-induced cirrhosis based on whether they have associated co-morbidities.

The epidemiology and demographic characteristics of NAFLD vary worldwide. The rise in NAFLD and NASH will balloon the number of patients with decompensated cirrhosis and pose a major emotional and financial burden on subjects and

#### **Figure 2.**

*Global picture of estimated prevalence of NAFLD and distribution of PNPLA3 genotypes adopted from Zobair Younossi [16]. PNPLA3 is presented as minor allele frequency in some areas (light blue section of the pie chart).*

their caregivers, thus adding to the overall cost of health care. Furthermore, the main etiologic factor adding to the burden of HCC is NAFLD [4]. In select NASHrelated HCC patients, liver resection and transplantation provide potentially curative therapeutic options; however, these procedures place a significant burden to healthcare resources and utilization [140]. Currently, NASH-related HCC has replaced HCV-related HCC as the fastest growing indication for LT in HCC candidates.

#### **2. Hepatocellular carcinoma (HCC)**

Liver cancer, which has limited therapeutic choices, has the second highest mortality rate in the world [141]. HCC, which can lead to complications such as portal vein thrombosis (PVT), accounts for the majority of primary liver malignancies and is one of the leading causes of death in patients with advanced fibrosis or cirrhosis [141–144]. HCC can be caused by chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), alcohol abuse, as well as obesity and diabetes-induced MetS. NAFLD often occurs in the setting of metabolic disorders such as obesity and T2DM. These same metabolic conditions are also risk factors for NAFLD-associated HCC, which can materialize in individuals even in the absence of advanced fibrosis or cirrhosis. NASH-HCC appears to be phenotypically different from HCC arising from other chronic liver diseases (**Table 6**). By all accounts, the formation and progression of HCC are multistep processes. Therefore, the specific and detailed molecular events that underlie HCC development remain only partially understood [143].

#### **2.1 The epidemiology and prevalence of HCC**

Primary liver cancer in 2012 was identified as the second most common cause of cancer-related death in the world. In the United States, HCC is the most common histological subtype of liver cancer that accounts for 70–85% of primary liver malignancies [145, 146]. It is also the most rapidly rising cause of cancer and


*\*Diagnosis of cirrhosis is based on the presence of the ICD-9 codes for cirrhosis or complications of cirrhosis (gastroesophageal varices, encephalopathy, and nonmalignant ascites) recorded at least twice in any inpatient or outpatient encounter.*

**89**

**2.2 The genetics of HCC**

*The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma*

cancer-related deaths with an incidence that has more than tripled over the last two decades. This high mortality reflects a poor prognosis and a poorer therapeutic intervention [147]. Compared to HCC caused by alcoholic liver disease and viral hepatitis, there is a lack of strong epidemiological data associated with the incidence and prevalence of HCC precipitating from NAFLD [140, 148]. While the prevalence of NAFLD is thought to be highest among Hispanics and Caucasians, the ethnic distribution among NAFLD-/NASH-related HCC patients has yet to be defined. Male patients are overrepresented in NASH-related HCC; however, gender has not been proven to be a statistical risk factor in NASH progression to HCC [7]. The rising incidence of NAFLD/NASH in the setting of obesity has led to a drastic growth in NASH-related HCC incidence [149]. Although NAFLD can present with HCC in the absence of NASH or cirrhosis, the cumulative annual incidence rate for developing HCC in patients with NASH-related cirrhosis is approximately 2.4–12.8% [125]. This suggests or utmost underlies that cirrhosis may be the main cause of HCC despite new emerging data suggesting that NAFLD may be an independent risk

factor for HCC, even in the absence of cirrhosis [126, 150, 151].

genetic and epigenetic alterations that are under investigation [4].

There was also a twofold increase in the incidence of HCC in the United States over the past two decades, and it is projected to double over the next two decades. Compared to HCC in alcoholic liver disease and viral hepatitis, there is a lack of strong epidemiological data regarding the incidence and prevalence of HCC in NAFLD [148]. It is projected that in just 12 more years, HCC at its current pace of growth in the United States will outstrip breast and colorectal cancers as the third leading cause of cancer-related death. This is because the prevalence of HCC is expected to increase by 149% from 10,000 to 24,900 during 2015–2030, while the incidence of HCC cases is expected to increase from 5160 to 12,240 in 2030, an increase of 137% [4]. This alarming incidence is attributed to several different

Modeling the epidemic of HCC suggests that in 2015, 3280 incident HCC cases were

estimated to have progressed from compensated cirrhosis (64% of total), with the remaining 1880 incident cases occurring among ≤F3 (fibrosis score-3) cases [4]. By 2030, 8790 incident HCC cases are predicted to occur among compensated cirrhotic cases or 72% of the annual incidence, reflecting aging and disease progression [4]. The true prevalence of NASH and NASH-related HCC is probably underestimated. This is because in 6.9–29% of HCC cases, the underlying etiology is unknown, further questioning the designation that the liver disease is secondary to cryptogenic cirrhosis [148]. Traits of NASH are more frequently observed in HCC patients with cryptogenic cirrhosis than in age- and sex-matched HCC patients of well-defined viral or alcoholic etiology [152]. In the past several years, myriad studies have tried to determine the variability of relationships between NAFLD/ NASH, cryptogenic cirrhosis, and HCC. In a recent meta-analysis, White et al. [125] estimated that 60% of HCC cases ascribed to NAFLD/NASH had cirrhosis either prior to diagnosis or at the time of diagnosis. This same analysis showed that NASHassociated cirrhosis consistently manifested an increased HCC risk. Furthermore, the study also revealed that when compared to those with chronic HCV, the risk of developing HCC is lower in patients with cirrhosis due to NAFLD/NASH (HCV, 19.7% vs. NAFLD/NASH, 26.9%) [125]. Although the prevalence of NAFLD-/ NASH-related HCC is not well delineated, the growing incidence of obesity and diabetes suggests the impact of NAFLD-/NASH-related HCC will continue to grow.

Genomic analyses promise to improve tumor characterization for the optimization of precision or personalized medicine for patients with HCC. Recent

*DOI: http://dx.doi.org/10.5772/intechopen.85780*

#### **Table 6.**

*Common risk factors for hepatocellular carcinoma.*

#### *The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.85780*

cancer-related deaths with an incidence that has more than tripled over the last two decades. This high mortality reflects a poor prognosis and a poorer therapeutic intervention [147]. Compared to HCC caused by alcoholic liver disease and viral hepatitis, there is a lack of strong epidemiological data associated with the incidence and prevalence of HCC precipitating from NAFLD [140, 148]. While the prevalence of NAFLD is thought to be highest among Hispanics and Caucasians, the ethnic distribution among NAFLD-/NASH-related HCC patients has yet to be defined. Male patients are overrepresented in NASH-related HCC; however, gender has not been proven to be a statistical risk factor in NASH progression to HCC [7]. The rising incidence of NAFLD/NASH in the setting of obesity has led to a drastic growth in NASH-related HCC incidence [149]. Although NAFLD can present with HCC in the absence of NASH or cirrhosis, the cumulative annual incidence rate for developing HCC in patients with NASH-related cirrhosis is approximately 2.4–12.8% [125]. This suggests or utmost underlies that cirrhosis may be the main cause of HCC despite new emerging data suggesting that NAFLD may be an independent risk factor for HCC, even in the absence of cirrhosis [126, 150, 151].

There was also a twofold increase in the incidence of HCC in the United States over the past two decades, and it is projected to double over the next two decades. Compared to HCC in alcoholic liver disease and viral hepatitis, there is a lack of strong epidemiological data regarding the incidence and prevalence of HCC in NAFLD [148]. It is projected that in just 12 more years, HCC at its current pace of growth in the United States will outstrip breast and colorectal cancers as the third leading cause of cancer-related death. This is because the prevalence of HCC is expected to increase by 149% from 10,000 to 24,900 during 2015–2030, while the incidence of HCC cases is expected to increase from 5160 to 12,240 in 2030, an increase of 137% [4]. This alarming incidence is attributed to several different genetic and epigenetic alterations that are under investigation [4].

Modeling the epidemic of HCC suggests that in 2015, 3280 incident HCC cases were estimated to have progressed from compensated cirrhosis (64% of total), with the remaining 1880 incident cases occurring among ≤F3 (fibrosis score-3) cases [4]. By 2030, 8790 incident HCC cases are predicted to occur among compensated cirrhotic cases or 72% of the annual incidence, reflecting aging and disease progression [4].

The true prevalence of NASH and NASH-related HCC is probably underestimated. This is because in 6.9–29% of HCC cases, the underlying etiology is unknown, further questioning the designation that the liver disease is secondary to cryptogenic cirrhosis [148]. Traits of NASH are more frequently observed in HCC patients with cryptogenic cirrhosis than in age- and sex-matched HCC patients of well-defined viral or alcoholic etiology [152]. In the past several years, myriad studies have tried to determine the variability of relationships between NAFLD/ NASH, cryptogenic cirrhosis, and HCC. In a recent meta-analysis, White et al. [125] estimated that 60% of HCC cases ascribed to NAFLD/NASH had cirrhosis either prior to diagnosis or at the time of diagnosis. This same analysis showed that NASHassociated cirrhosis consistently manifested an increased HCC risk. Furthermore, the study also revealed that when compared to those with chronic HCV, the risk of developing HCC is lower in patients with cirrhosis due to NAFLD/NASH (HCV, 19.7% vs. NAFLD/NASH, 26.9%) [125]. Although the prevalence of NAFLD-/ NASH-related HCC is not well delineated, the growing incidence of obesity and diabetes suggests the impact of NAFLD-/NASH-related HCC will continue to grow.

#### **2.2 The genetics of HCC**

Genomic analyses promise to improve tumor characterization for the optimization of precision or personalized medicine for patients with HCC. Recent

*Nonalcoholic Fatty Liver Disease - An Update*

**2. Hepatocellular carcinoma (HCC)**

**2.1 The epidemiology and prevalence of HCC**

their caregivers, thus adding to the overall cost of health care. Furthermore, the main etiologic factor adding to the burden of HCC is NAFLD [4]. In select NASHrelated HCC patients, liver resection and transplantation provide potentially curative therapeutic options; however, these procedures place a significant burden to healthcare resources and utilization [140]. Currently, NASH-related HCC has replaced HCV-related HCC as the fastest growing indication for LT in HCC candidates.

Liver cancer, which has limited therapeutic choices, has the second highest mortality rate in the world [141]. HCC, which can lead to complications such as portal vein thrombosis (PVT), accounts for the majority of primary liver malignancies and is one of the leading causes of death in patients with advanced fibrosis or cirrhosis [141–144]. HCC can be caused by chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), alcohol abuse, as well as obesity and diabetes-induced MetS. NAFLD often occurs in the setting of metabolic disorders such as obesity and T2DM. These same metabolic conditions are also risk factors for NAFLD-associated HCC, which can materialize in individuals even in the absence of advanced fibrosis or cirrhosis. NASH-HCC appears to be phenotypically different from HCC arising from other chronic liver diseases (**Table 6**). By all accounts, the formation and progression of HCC are multistep processes. Therefore, the specific and detailed molecular events

Hepatitis B infection (HBV) Hepatitis C infection (HCV) Hepatitis D infection (HDV)

Hereditary hemochromatosis

Glycogen storage disease type 1a

*Common risk factors for hepatocellular carcinoma.*

Nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH)

that underlie HCC development remain only partially understood [143].

Primary liver cancer in 2012 was identified as the second most common cause of cancer-related death in the world. In the United States, HCC is the most common histological subtype of liver cancer that accounts for 70–85% of primary liver malignancies [145, 146]. It is also the most rapidly rising cause of cancer and

*\*Diagnosis of cirrhosis is based on the presence of the ICD-9 codes for cirrhosis or complications of cirrhosis (gastroesophageal varices, encephalopathy, and nonmalignant ascites) recorded at least twice in any inpatient or* 

Alcohol (ethanol)

Obesity/diabetes

Oral contraceptives

Aflatoxin B Tyrosinemia

Smoking Cirrhosis\*

*outpatient encounter.*

**88**

**Table 6.**

developments and molecular techniques have significantly improved our understanding of the pathogenesis of HCC and its complex genetic landscape [153–156]. The integration of several profiling data from various sources may provide additional insight into the molecular mechanisms of HCC [153]. The first largescale multiplatform analysis of HCC conducted as part of The Cancer Genome Atlas (TCGA) network included valuation of somatic mutations by whole exome sequencing and DNA copy number analyses in 363 patients whose tissue and tumor specimens were obtained [157]. This high-throughput analysis also included further investigation of DNA methylation, mRNA expression, microRNA (miRNA) expression, and proteomic expression in 196 patients. To decipher the molecular landscape of HCC and extract biological insights for therapeutic targets and prognostic implications, analyses were made by integrating multiple data platforms with the available clinical data for HCC [157]. Mutational and DNA sequencing analyses identified an array of genes altered either by downregulation or by mutation. Among the significantly mutated genes were EEF1A1, SMARCA4, LZTR1, and SF3B1 [157]. Those genes downregulated by hypermethylation including ALB, APOB, and CPS1 may cause metabolic reprogramming in HCC. The analysis of integrated molecular platform also yielded the identification of a subtype linked to poorer prognosis in three HCC cohorts. This large-scale multiplatform, highthroughput analysis enabled the design of a p53 target gene expression signature correlating with poor survival. This TCGA network analysis produced potential therapeutic targets including WNT signaling, IDH1, MET, VEGFA, MCL1, MDM4, TERT, and immune checkpoint proteins PD-1, PD-L1, and CTLA-4 [157]. This is significant because effective inhibitors already exit for these targets, which alter hepatocyte energy balance [157].

In exome sequencing analysis of over 200 liver tumors, investigators identified mutational signatures that are associated with specific risk factors such as alcohol and tobacco consumption and exposure to aflatoxin B1 [158]. As a result, they found that 161 putative driver genes were associated with 11 recurrently altered pathways involving *CTNNB1* (alcohol), *TP53* (hepatitis B virus, HBV), and *AXIN1* [158]. Further analysis of tumor stage progression identified *TERT* as an early event, whereas *FGF3*, *FGF4*, *FGF19*, or *CCND1* amplification and *TP53* and *CDKN2A* alterations were prominent in aggressive tumors. The involvement of these many altered genes and pathways in the development and/ or progression of HCC leads to the extensive landscape and multifaceted nature of this lethal cancer. **Figure 3** shows the salient signaling pathways associated with HCC.

In another recent study, gene expression and DNA methylation profiles were screened to identify potential genetic biomarkers of HCC. The findings from this study suggest potential HCC biomarker roles for certain genes such as *DTL, DUSP1, NFKBIA*, and *SOCS2* [160]. Similar to TCGA Research Network analyses mentioned above [157], these investigators also suggest that the tumor protein 'p53 signaling' and 'metabolic' pathways may serve important roles in the pathogenesis of HCC [160]. Other polymorphic variants serving as potential risk factors for HCC in highrisk patients infected with HBV/HCV have also been reported [161]. As for prognostic biomarkers, recent RNA sequencing data from the Cancer Genome Atlas (TCGA) reveal that among the 12 tissue types studied, the liver had the largest number of tissue-enriched genes, which are associated with the prognosis of patients with HCC and represent distinct physiological patterns [162]. A further study of the characteristics of liver-enriched genes showed that hypermethylation might be partially responsible for the downregulation of these genes, most of which were metabolismrelated genes associated with pathological stage and dedifferentiation in patients with HCC. The authors suggest that hypermethylation might be a mechanism

**91**

**Figure 4.**

**Figure 3.**

*The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma*

*The most reported signaling pathways in HCC. Adapted from Birgani et al. [159].*

*Validation of liver-enriched genes and KEGG analysis. (A) an example of liver-enriched genes. SPP2 was exclusively expressed in the corresponding nontumor tissues of HCC. (B) Four-set Venn diagram showing the overlap of the liver-enriched genes derived from the TCGA and three other databases, including HPA, PaGenBase, and TiGER. (C) Significantly enriched KEGG pathways of 188 liver-enriched genes. −log10 (adjusted p-value) was annotated on each bar of the KEGG pathway. Adapted from Binghua et al. [162].*

*DOI: http://dx.doi.org/10.5772/intechopen.85780*

*The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.85780*

**Figure 3.** *The most reported signaling pathways in HCC. Adapted from Birgani et al. [159].*

#### **Figure 4.**

*Nonalcoholic Fatty Liver Disease - An Update*

hepatocyte energy balance [157].

developments and molecular techniques have significantly improved our understanding of the pathogenesis of HCC and its complex genetic landscape [153–156].

In exome sequencing analysis of over 200 liver tumors, investigators identified mutational signatures that are associated with specific risk factors such as alcohol and tobacco consumption and exposure to aflatoxin B1 [158]. As a result, they found that 161 putative driver genes were associated with 11 recurrently altered pathways involving *CTNNB1* (alcohol), *TP53* (hepatitis B virus, HBV), and *AXIN1* [158]. Further analysis of tumor stage progression identified *TERT* as an early event, whereas *FGF3*, *FGF4*, *FGF19*, or *CCND1* amplification and *TP53* and *CDKN2A* alterations were prominent in aggressive tumors. The involvement of these many altered genes and pathways in the development and/ or progression of HCC leads to the extensive landscape and multifaceted nature of this lethal cancer. **Figure 3** shows the salient signaling pathways associated

In another recent study, gene expression and DNA methylation profiles were screened to identify potential genetic biomarkers of HCC. The findings from this study suggest potential HCC biomarker roles for certain genes such as *DTL, DUSP1, NFKBIA*, and *SOCS2* [160]. Similar to TCGA Research Network analyses mentioned above [157], these investigators also suggest that the tumor protein 'p53 signaling' and 'metabolic' pathways may serve important roles in the pathogenesis of HCC [160]. Other polymorphic variants serving as potential risk factors for HCC in highrisk patients infected with HBV/HCV have also been reported [161]. As for prognostic biomarkers, recent RNA sequencing data from the Cancer Genome Atlas (TCGA) reveal that among the 12 tissue types studied, the liver had the largest number of tissue-enriched genes, which are associated with the prognosis of patients with HCC and represent distinct physiological patterns [162]. A further study of the characteristics of liver-enriched genes showed that hypermethylation might be partially responsible for the downregulation of these genes, most of which were metabolismrelated genes associated with pathological stage and dedifferentiation in patients with HCC. The authors suggest that hypermethylation might be a mechanism

The integration of several profiling data from various sources may provide additional insight into the molecular mechanisms of HCC [153]. The first largescale multiplatform analysis of HCC conducted as part of The Cancer Genome Atlas (TCGA) network included valuation of somatic mutations by whole exome sequencing and DNA copy number analyses in 363 patients whose tissue and tumor specimens were obtained [157]. This high-throughput analysis also included further investigation of DNA methylation, mRNA expression, microRNA (miRNA) expression, and proteomic expression in 196 patients. To decipher the molecular landscape of HCC and extract biological insights for therapeutic targets and prognostic implications, analyses were made by integrating multiple data platforms with the available clinical data for HCC [157]. Mutational and DNA sequencing analyses identified an array of genes altered either by downregulation or by mutation. Among the significantly mutated genes were EEF1A1, SMARCA4, LZTR1, and SF3B1 [157]. Those genes downregulated by hypermethylation including ALB, APOB, and CPS1 may cause metabolic reprogramming in HCC. The analysis of integrated molecular platform also yielded the identification of a subtype linked to poorer prognosis in three HCC cohorts. This large-scale multiplatform, highthroughput analysis enabled the design of a p53 target gene expression signature correlating with poor survival. This TCGA network analysis produced potential therapeutic targets including WNT signaling, IDH1, MET, VEGFA, MCL1, MDM4, TERT, and immune checkpoint proteins PD-1, PD-L1, and CTLA-4 [157]. This is significant because effective inhibitors already exit for these targets, which alter

**90**

with HCC.

*Validation of liver-enriched genes and KEGG analysis. (A) an example of liver-enriched genes. SPP2 was exclusively expressed in the corresponding nontumor tissues of HCC. (B) Four-set Venn diagram showing the overlap of the liver-enriched genes derived from the TCGA and three other databases, including HPA, PaGenBase, and TiGER. (C) Significantly enriched KEGG pathways of 188 liver-enriched genes. −log10 (adjusted p-value) was annotated on each bar of the KEGG pathway. Adapted from Binghua et al. [162].*

underlying the downregulation of these liver-enriched genes. When they overlapped the tissue-enriched and prognostic genes across cancer types, they found that, in HCC, 55% (84/188) of the liver-enriched genes were prognostic (see **Figure 4**).

Circulating regulatory nucleic acids like miRNA profiles can also reflect the pathogenic changes occurring in organs including the liver. Changes in miR-21, miR-122, and miR-223 were correlated with the histological status of the human liver and were specific for liver injury [163]. These miRNA levels were significantly higher in the serum of chronic hepatitis (i.e., HBV and HCV) and HCC patients compared to healthy controls [44]. Yet, the biological heterogeneity of HCC makes it difficult to clarify the key mechanisms of cancer initiation and progression, and thereby develop and implement effective therapies [164].

#### **3. The projection of NAFLD and HCC**

A recent Markov model was used to predict incidence of NAFLD and to forecast NAFLD disease progression in the United States. The model was based on historical and projected changes in adult prevalence of obesity and T2DM as well as national surveillance data for incidence of NAFLD-related HCC [4]. The report forecasts that prevalent NAFLD cases will increase to 21% (100.9 million) by 2030, while prevalent NASH cases will increase 63% from 16.5 million to 27.00 million cases [4]. Overall NAFLD prevalence among the adult population (aged ≥15 years) is projected at 33.5% in 2030, and the median age of the NAFLD population will increase from 50 (estimated at 2015 level) to 55 years between 2015 and 2030 [4]. In 2015, approximately 20% of NAFLD cases were classified as NASH and are expected to increase to 27% by 2030, a reflection of both disease progression and an aging population. The estimated prevalence of NASH in adults living in the United States is 3–5% [6, 23, 121, 165] and is projected to increase by 63% from 16.5 million in 2015 to 27.00 million cases in 2030 [4]. This prevalence of NASH was calculated based on published estimates and modeling of fibrosis progression. It was assumed that up to 5% of NAFLD cases without NASH could be NASH regressors, with most NASH regressors still in F0 stage [4]. Similarly, the incidence of decompensated cirrhosis will surge by 168% to 105,430 cases in 2030, while incidence of HCC will increase by 137% to 12,240 cases. Liver deaths are estimated to increase 178% to 78,300 deaths in 2030. During 2015–2030, there are projected to be nearly 800,000 excess liver deaths. The aging population, the continuing high rates of adult obesity, and T2DM will propel NAFLD-related liver disease and mortality in the United States. Immediate strategies are required to curtail new NAFLD cases and mitigate disease burden.

Currently, NAFLD is estimated to affect more than 80 million and up to 90 million Americans, making it the most common etiology for liver disease in the United States [16, 65]. In the United Kingdom, NAFLD has now become the most common cause of abnormal liver function tests (LFTs) [166]. Although NAFLD has emerged as a serious disease in affluent Western economies, its global prevalence encompasses the Middle East (32%), South America (31%), Asia (27%), the United States (24%), Europe (23%), and Africa (14%) [167]. Because of the increasing incidence of obesity and diabetes around the world, NAFLD has become a global public health concern. The prevalence of NAFLD varies according to age, sex, and the methodology used to measure the condition in each geographical location [61]. Currently, NAFLD is the most prevalent liver disease observed in patients with obesity, diabetes, and metabolic syndrome (MetS), all of which can confer insulin resistance (IR) and are known risk factors for the development of HCC, a growing indicator of LT [45, 69]. While HCV infection has been the most common indication for liver transplants to date, NASH is surpassing it as obesity reaches historic

**93**

*The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma*

highs and new direct-acting antiviral (DAA) drugs are essentially curing hepatitis C [168]. Furthermore, with the continued decline in the prevalence of HCV infection, the proportion of NASH-HCC is anticipated to increase exponentially due to the growing epidemic of obesity and diabetes [140]. Currently, NASH-related HCC is the fastest growing indication for LT in HCC candidates [140]. NAFLD and NASH

*Risk factors and proposed mechanisms for NAFLD- and NASH-related HCC, which is multifactorial. Proposed pathogenic mechanisms include obesity, peripheral and hepatic IR from T2DM, increased hepatic lipid storage and lipotoxicity, genetic mutations, and intestinal microbiota dysregulation. HCC, hepatocellular carcinoma; EMT, epithelial to mesenchymal transition; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; FFA, free fatty acid; IGF, insulin-like growth factor; LPS, lipopolysaccharide;*  PNPLA3*, patatin-like phospholipase domain-containing 3; TM6SF2, transmembrane 6 superfamily member 2.* 

Globally, Asia is leading the rise in NAFLD followed by the United States. Although our understanding of NAFLD is steadily evolving, it is not an isolated disease. It is commonly associated with the leading metabolic comorbidities such as obesity, MetS, T2DM, and dyslipidemia. The potential progression of NAFLD subtypes is from fibrosis to advanced fibrosis, ESLD, and HCC (**Figure 1**). As the incidence of obesity and concurrently diabetes and MetS continues to surge in Europe and the United States, NAFLD/NASH may become the most common cause

A 2002–2012 retrospective cohort study among adult patients revealed a fourfold increase in patients undergoing LT for NASH-related HCC in contrast to only twofold increase in number of patients undergoing transplantation for HCV-related HCC. In the United States, about 6000–7000 liver transplants are performed annually, and the rapid increase in the percentage (44.9%) of obese individuals during a 14-year period (2000–2014) is expected to escalate to 55% the number of NASH patients awaiting LT by 2030 [172]. The increased morbidity and mortality, healthcare costs, and declining health-related quality of life associated with NAFLD require more in-depth analysis. **Figure 5** depicts the proposed mechanisms that ties

Although still not fully resolved, the prevalence of NAFLD in the United States

can vary by ethnicity. Even in this context, there are several factors that could explain the reported ethnic disparities. These include access to health care, genetic factors, environmental factors, affliction with chronic diseases, and the presence of chronic diseases such as the MetS [61, 65, 173]. In this context, the prevalence of NAFLD is reported to be highest in Hispanic-Americans, followed by Americans of European descent and then African-Americans [40, 61, 65, 122, 173]. Several

of HCC in developed countries in the foreseeable future [169–171].

**4. Ethnic and gender differences in NAFLD and HCC**

*DOI: http://dx.doi.org/10.5772/intechopen.85780*

**Figure 5.**

*Adapted from Cholankeril [140].*

are a growing cause of cirrhosis and HCC.

NAFLD/NASH and HCC.

*The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.85780*

#### **Figure 5.**

*Nonalcoholic Fatty Liver Disease - An Update*

thereby develop and implement effective therapies [164].

**3. The projection of NAFLD and HCC**

new NAFLD cases and mitigate disease burden.

underlying the downregulation of these liver-enriched genes. When they overlapped the tissue-enriched and prognostic genes across cancer types, they found that, in HCC, 55% (84/188) of the liver-enriched genes were prognostic (see **Figure 4**). Circulating regulatory nucleic acids like miRNA profiles can also reflect the pathogenic changes occurring in organs including the liver. Changes in miR-21, miR-122, and miR-223 were correlated with the histological status of the human liver and were specific for liver injury [163]. These miRNA levels were significantly higher in the serum of chronic hepatitis (i.e., HBV and HCV) and HCC patients compared to healthy controls [44]. Yet, the biological heterogeneity of HCC makes it difficult to clarify the key mechanisms of cancer initiation and progression, and

A recent Markov model was used to predict incidence of NAFLD and to forecast NAFLD disease progression in the United States. The model was based on historical and projected changes in adult prevalence of obesity and T2DM as well as national surveillance data for incidence of NAFLD-related HCC [4]. The report forecasts that prevalent NAFLD cases will increase to 21% (100.9 million) by 2030, while prevalent NASH cases will increase 63% from 16.5 million to 27.00 million cases [4]. Overall NAFLD prevalence among the adult population (aged ≥15 years) is projected at 33.5% in 2030, and the median age of the NAFLD population will increase from 50 (estimated at 2015 level) to 55 years between 2015 and 2030 [4]. In 2015, approximately 20% of NAFLD cases were classified as NASH and are expected to increase to 27% by 2030, a reflection of both disease progression and an aging population. The estimated prevalence of NASH in adults living in the United States is 3–5% [6, 23, 121, 165] and is projected to increase by 63% from 16.5 million in 2015 to 27.00 million cases in 2030 [4]. This prevalence of NASH was calculated based on published estimates and modeling of fibrosis progression. It was assumed that up to 5% of NAFLD cases without NASH could be NASH regressors, with most NASH regressors still in F0 stage [4]. Similarly, the incidence of decompensated cirrhosis will surge by 168% to 105,430 cases in 2030, while incidence of HCC will increase by 137% to 12,240 cases. Liver deaths are estimated to increase 178% to 78,300 deaths in 2030. During 2015–2030, there are projected to be nearly 800,000 excess liver deaths. The aging population, the continuing high rates of adult obesity, and T2DM will propel NAFLD-related liver disease and mortality in the United States. Immediate strategies are required to curtail

Currently, NAFLD is estimated to affect more than 80 million and up to 90 million Americans, making it the most common etiology for liver disease in the United States [16, 65]. In the United Kingdom, NAFLD has now become the most common cause of abnormal liver function tests (LFTs) [166]. Although NAFLD has emerged as a serious disease in affluent Western economies, its global prevalence encompasses the Middle East (32%), South America (31%), Asia (27%), the United States (24%), Europe (23%), and Africa (14%) [167]. Because of the increasing incidence of obesity and diabetes around the world, NAFLD has become a global public health concern. The prevalence of NAFLD varies according to age, sex, and the methodology used to measure the condition in each geographical location [61]. Currently, NAFLD is the most prevalent liver disease observed in patients with obesity, diabetes, and metabolic syndrome (MetS), all of which can confer insulin resistance (IR) and are known risk factors for the development of HCC, a growing indicator of LT [45, 69]. While HCV infection has been the most common indication for liver transplants to date, NASH is surpassing it as obesity reaches historic

**92**

*Risk factors and proposed mechanisms for NAFLD- and NASH-related HCC, which is multifactorial. Proposed pathogenic mechanisms include obesity, peripheral and hepatic IR from T2DM, increased hepatic lipid storage and lipotoxicity, genetic mutations, and intestinal microbiota dysregulation. HCC, hepatocellular carcinoma; EMT, epithelial to mesenchymal transition; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; FFA, free fatty acid; IGF, insulin-like growth factor; LPS, lipopolysaccharide;*  PNPLA3*, patatin-like phospholipase domain-containing 3; TM6SF2, transmembrane 6 superfamily member 2. Adapted from Cholankeril [140].*

highs and new direct-acting antiviral (DAA) drugs are essentially curing hepatitis C [168]. Furthermore, with the continued decline in the prevalence of HCV infection, the proportion of NASH-HCC is anticipated to increase exponentially due to the growing epidemic of obesity and diabetes [140]. Currently, NASH-related HCC is the fastest growing indication for LT in HCC candidates [140]. NAFLD and NASH are a growing cause of cirrhosis and HCC.

Globally, Asia is leading the rise in NAFLD followed by the United States. Although our understanding of NAFLD is steadily evolving, it is not an isolated disease. It is commonly associated with the leading metabolic comorbidities such as obesity, MetS, T2DM, and dyslipidemia. The potential progression of NAFLD subtypes is from fibrosis to advanced fibrosis, ESLD, and HCC (**Figure 1**). As the incidence of obesity and concurrently diabetes and MetS continues to surge in Europe and the United States, NAFLD/NASH may become the most common cause of HCC in developed countries in the foreseeable future [169–171].

A 2002–2012 retrospective cohort study among adult patients revealed a fourfold increase in patients undergoing LT for NASH-related HCC in contrast to only twofold increase in number of patients undergoing transplantation for HCV-related HCC. In the United States, about 6000–7000 liver transplants are performed annually, and the rapid increase in the percentage (44.9%) of obese individuals during a 14-year period (2000–2014) is expected to escalate to 55% the number of NASH patients awaiting LT by 2030 [172]. The increased morbidity and mortality, healthcare costs, and declining health-related quality of life associated with NAFLD require more in-depth analysis. **Figure 5** depicts the proposed mechanisms that ties NAFLD/NASH and HCC.

#### **4. Ethnic and gender differences in NAFLD and HCC**

Although still not fully resolved, the prevalence of NAFLD in the United States can vary by ethnicity. Even in this context, there are several factors that could explain the reported ethnic disparities. These include access to health care, genetic factors, environmental factors, affliction with chronic diseases, and the presence of chronic diseases such as the MetS [61, 65, 173]. In this context, the prevalence of NAFLD is reported to be highest in Hispanic-Americans, followed by Americans of European descent and then African-Americans [40, 61, 65, 122, 173]. Several

studies have shown a relative sparsity of NAFLD cases among individuals of African descent living in or coming from Africa or the Caribbean region. Although the prevalence of metabolic disease and obesity is high in Afro-Caribbean ethnic groups compared to Caucasian and Hispanic groups, the frequency of NAFLD/NASH is reported to be low [61, 174]. This discrepancy might be due to an actual low number rate or biases that include low-recognition and low-referral rates in these ethnic minorities [175], as Afro-Caribbean patients are categorically less likely to be referred to other tertiary hospitals [176].

There are also ethnic differences in the incidence of HCC in the United States (see Sherif et al. for a comprehensive review) [61]. Compared to European-Americans (EAs), the incidence of HCC is higher in African-Americans (AAs) and is associated with more advanced tumor stage at diagnosis and lower survival rates overall. Assessment of changes in the levels of metabolites of samples stratified by race was made using gas chromatography-mass spectrometry in selected ion monitoring mode to identify ethnically diverse biomarkers in HCC between EA and AAs [177]. Race-specific metabolites including alpha tocopherol for AA and EA combined, glycine for EA, and valine for AA exhibited better sensitivity and specificity than the standard serological marker for HCC, alpha-fetoprotein (AFP) that is widely used for the diagnosis of HCC [177–180]. It is hypothesized that there is a variation in HCC-associated epigenetic modifications between AAs and EAs. Thus, the identification of aberrant DNA methylation and differentially modulated miRNAs can be used to better understand the mechanisms of disparities in HCC between races. Also, identifying epigenetic markers for HCC in a specific population will enhance personalized medicine that targets specific therapeutic approaches [181, 182]. This also demands the gathering together of a highly interdisciplinary team of experts to investigate changes in both DNA methylation and miRNA expression patterns between tumor, cirrhotic, and normal liver tissues from AA and EA participants. Identifying molecular cancer gene drivers and mutations may 1 day become critical for precision oncology.

Most epidemiological studies document prevalence of individual diseases in selected tertiary hospital populations [183]. This widespread practice, particularly when imaging and liver enzyme tests are involved and when the patients may be asymptomatic in the early stage of diagnosis, leads to underestimation and underdiagnosis of NAFLD. This is especially true for minority populations in whom the natural development and progression of NAFLD and NASH are understudied and underreported as reflected by the paucity of data in the literature. Furthermore, the predictive value of the MetS may not reflect the true state of NAFLD in AAs since the criteria for the syndrome were developed for non-Hispanic whites [184] thereby influencing underdiagnosis or misdiagnosis of NAFLD and NASH in Hispanics and non-Hispanic blacks (NHB). There is also a strong relationship between insulin resistance and hypertriglyceridemia, one of the crucial components of MetS. However, NHB often have normal triglycerides (TG) level [185], which is used as a diagnostic criterion of the MetS leading to underdiagnosis of the MetS in NHB [186]. This suggests that lowering the threshold for TG level in AAs will lead to grasping the true cases of NAFLD. Moreover, the racial differences in NAFLD and NASH may be a function of the differences in TGs or the differences in the distribution of adiposity (e.g., subcutaneous vs. visceral) since AAs have relatively less VAT and lower TGs than Hispanics [119, 173, 175]. In addition, AAs may be more resistant to both the accretion of TG in the abdominal visceral compartment (adipose tissue and liver) and hypertriglyceridemia associated with IR [119].

Epidemiologic studies establish the foundational framework for the control and prevention of diseases. In the case of NAFLD and NASH, it should be done by first tracking the prevalence of the disease, characterizing its natural history, and

**95**

*The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma*

identifying both its social and health determinants along ethnic lines. This type of study is critical for the proper diagnosis and early intervention of NAFLD especially

Genome-wide association studies have revealed several genetic variants that are associated with NAFLD and NASH. Yet, these variants either represent only a limited amount of variation in hepatic steatosis among ethnic groups or may just be

There is an urgent need to gain a better understanding of the underlying biological mechanisms responsible for why some people with NAFLD are more prone to developing HCC, and the causes for disparities in NAFLD-related HCC. There is also an urgent need for a less invasive method than biopsy and for a more sensitive biomarker than ALT for large-scale NAFLD screening. The lack of high-throughput studies employing proteomics or metabolomics for the discovery of novel and reliable diagnostic biomarkers for NAFLD also hampers our understanding of the

pathophysiology of the disease among the disparate ethnicities [12, 177].

One recent area of exploration is the involvement of DNA methylation and miRNA regulation. Epigenetic alterations are potentially reversible, and this possibility will facilitate the development of biomarkers and therapeutics in the prevailing disparities between AA and EA patients in HCC initiation and development. The identification and functional validation of race-specific methylation hotspots and miRNAs can be used to understand the mechanisms of disparities in HCC. This can be done by first identifying DNA methylation sites and miR-NAs with statistically significant changes between HCC cases and cirrhotic or normal controls in a race-specific manner. Then, network-based methods and hierarchical integrative models can be used to integrate epigenomic data with transcriptomic, proteomic, glycoproteomic, and metabolomic data acquired from the same cirrhotic and HCC participants to select methylation hotspots and miRNAs relevant for understanding the mechanisms of disparities in HCC [177]. The selected candidates can then be validated by independent methods using frozen and formalin-fixed, paraffin-embedded (FFPE) liver tissues collected from patients with HCC and liver cirrhosis. Finally, functional validation of race-specific epigenetic modifications discovered in this type of high-throughput study can be performed through *in vitro* experiments using established cell lines derived from racially diverse populations. These cell cultures may present unique opportunities for targeted functional validation of epigenetic modifications and

In addition to exploring the external environment and how it influences HCC disease status, it is also necessary to explore the intestinal environment of different ethnicities. Experimental data from the obesity epidemic have revealed that the composition and products of the gut microbiome, which is altered with obesity and/ or a high fat diet, are carcinogenic to the liver [187, 188]. Studies suggest that there are ethnic differences in microbial composition in a cirrhotic population at elevated risk for HCC as a result of metabolites, which can differentiate cirrhotic with HCC from those without HCC. Therefore, a case-control study can be designed to examine the contributions of race/ethnicity, fecal microbiome, fecal metabolome, and host factors (e.g., specific dietary factors and markers of body and liver fat composition) to NAFLD-related HCC. All in all, a multiethnic study of NAFLD and HCC that encompasses all racial/ethnic groups is needed to lay the groundwork for the elucidation of factors that account for health disparities across these populations. The prevalence of NAFLD is reported to be highest among Hispanics and Caucasians as mentioned above. However, NASH was the leading cause of waitlist LT registration in 2016 among Asian, Hispanic, and non-Hispanic white females,

whereas HCV is still the leading cause in AA females [189].

*DOI: http://dx.doi.org/10.5772/intechopen.85780*

markers representing a larger body of genetic variations.

in minority populations [61].

the downstream consequences.

#### *The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.85780*

identifying both its social and health determinants along ethnic lines. This type of study is critical for the proper diagnosis and early intervention of NAFLD especially in minority populations [61].

Genome-wide association studies have revealed several genetic variants that are associated with NAFLD and NASH. Yet, these variants either represent only a limited amount of variation in hepatic steatosis among ethnic groups or may just be markers representing a larger body of genetic variations.

There is an urgent need to gain a better understanding of the underlying biological mechanisms responsible for why some people with NAFLD are more prone to developing HCC, and the causes for disparities in NAFLD-related HCC. There is also an urgent need for a less invasive method than biopsy and for a more sensitive biomarker than ALT for large-scale NAFLD screening. The lack of high-throughput studies employing proteomics or metabolomics for the discovery of novel and reliable diagnostic biomarkers for NAFLD also hampers our understanding of the pathophysiology of the disease among the disparate ethnicities [12, 177].

One recent area of exploration is the involvement of DNA methylation and miRNA regulation. Epigenetic alterations are potentially reversible, and this possibility will facilitate the development of biomarkers and therapeutics in the prevailing disparities between AA and EA patients in HCC initiation and development. The identification and functional validation of race-specific methylation hotspots and miRNAs can be used to understand the mechanisms of disparities in HCC. This can be done by first identifying DNA methylation sites and miR-NAs with statistically significant changes between HCC cases and cirrhotic or normal controls in a race-specific manner. Then, network-based methods and hierarchical integrative models can be used to integrate epigenomic data with transcriptomic, proteomic, glycoproteomic, and metabolomic data acquired from the same cirrhotic and HCC participants to select methylation hotspots and miRNAs relevant for understanding the mechanisms of disparities in HCC [177]. The selected candidates can then be validated by independent methods using frozen and formalin-fixed, paraffin-embedded (FFPE) liver tissues collected from patients with HCC and liver cirrhosis. Finally, functional validation of race-specific epigenetic modifications discovered in this type of high-throughput study can be performed through *in vitro* experiments using established cell lines derived from racially diverse populations. These cell cultures may present unique opportunities for targeted functional validation of epigenetic modifications and the downstream consequences.

In addition to exploring the external environment and how it influences HCC disease status, it is also necessary to explore the intestinal environment of different ethnicities. Experimental data from the obesity epidemic have revealed that the composition and products of the gut microbiome, which is altered with obesity and/ or a high fat diet, are carcinogenic to the liver [187, 188]. Studies suggest that there are ethnic differences in microbial composition in a cirrhotic population at elevated risk for HCC as a result of metabolites, which can differentiate cirrhotic with HCC from those without HCC. Therefore, a case-control study can be designed to examine the contributions of race/ethnicity, fecal microbiome, fecal metabolome, and host factors (e.g., specific dietary factors and markers of body and liver fat composition) to NAFLD-related HCC. All in all, a multiethnic study of NAFLD and HCC that encompasses all racial/ethnic groups is needed to lay the groundwork for the elucidation of factors that account for health disparities across these populations. The prevalence of NAFLD is reported to be highest among Hispanics and Caucasians as mentioned above. However, NASH was the leading cause of waitlist LT registration in 2016 among Asian, Hispanic, and non-Hispanic white females, whereas HCV is still the leading cause in AA females [189].

*Nonalcoholic Fatty Liver Disease - An Update*

referred to other tertiary hospitals [176].

may 1 day become critical for precision oncology.

studies have shown a relative sparsity of NAFLD cases among individuals of African descent living in or coming from Africa or the Caribbean region. Although the prevalence of metabolic disease and obesity is high in Afro-Caribbean ethnic groups compared to Caucasian and Hispanic groups, the frequency of NAFLD/NASH is reported to be low [61, 174]. This discrepancy might be due to an actual low number rate or biases that include low-recognition and low-referral rates in these ethnic minorities [175], as Afro-Caribbean patients are categorically less likely to be

There are also ethnic differences in the incidence of HCC in the United States

Most epidemiological studies document prevalence of individual diseases in selected tertiary hospital populations [183]. This widespread practice, particularly when imaging and liver enzyme tests are involved and when the patients may be asymptomatic in the early stage of diagnosis, leads to underestimation and underdiagnosis of NAFLD. This is especially true for minority populations in whom the natural development and progression of NAFLD and NASH are understudied and underreported as reflected by the paucity of data in the literature. Furthermore, the predictive value of the MetS may not reflect the true state of NAFLD in AAs since the criteria for the syndrome were developed for non-Hispanic whites [184] thereby influencing underdiagnosis or misdiagnosis of NAFLD and NASH in Hispanics and non-Hispanic blacks (NHB). There is also a strong relationship between insulin resistance and hypertriglyceridemia, one of the crucial components of MetS. However, NHB often have normal triglycerides (TG) level [185], which is used as a diagnostic criterion of the MetS leading to underdiagnosis of the MetS in NHB [186]. This suggests that lowering the threshold for TG level in AAs will lead to grasping the true cases of NAFLD. Moreover, the racial differences in NAFLD and NASH may be a function of the differences in TGs or the differences in the distribution of adiposity (e.g., subcutaneous vs. visceral) since AAs have relatively less VAT and lower TGs than Hispanics [119, 173, 175]. In addition, AAs may be more resistant to both the accretion of TG in the abdominal visceral compartment (adipose tissue and liver) and hypertriglyceridemia associated with IR [119]. Epidemiologic studies establish the foundational framework for the control and prevention of diseases. In the case of NAFLD and NASH, it should be done by first tracking the prevalence of the disease, characterizing its natural history, and

(see Sherif et al. for a comprehensive review) [61]. Compared to European-Americans (EAs), the incidence of HCC is higher in African-Americans (AAs) and is associated with more advanced tumor stage at diagnosis and lower survival rates overall. Assessment of changes in the levels of metabolites of samples stratified by race was made using gas chromatography-mass spectrometry in selected ion monitoring mode to identify ethnically diverse biomarkers in HCC between EA and AAs [177]. Race-specific metabolites including alpha tocopherol for AA and EA combined, glycine for EA, and valine for AA exhibited better sensitivity and specificity than the standard serological marker for HCC, alpha-fetoprotein (AFP) that is widely used for the diagnosis of HCC [177–180]. It is hypothesized that there is a variation in HCC-associated epigenetic modifications between AAs and EAs. Thus, the identification of aberrant DNA methylation and differentially modulated miRNAs can be used to better understand the mechanisms of disparities in HCC between races. Also, identifying epigenetic markers for HCC in a specific population will enhance personalized medicine that targets specific therapeutic approaches [181, 182]. This also demands the gathering together of a highly interdisciplinary team of experts to investigate changes in both DNA methylation and miRNA expression patterns between tumor, cirrhotic, and normal liver tissues from AA and EA participants. Identifying molecular cancer gene drivers and mutations

**94**

As for gender differences in NAFLD or NASH, there are uncertainties including the role of IR in the influence of gender on NAFLD. Ruhl et al. reported that NAFLD is about 2.7 times more prevalent in men than in women [190]. One reasonable explanation for this reported gender difference in NAFLD is due to the higher waist-to-hip circumference (WHR) ratio in men [96]. Pan et al. further state that WHR is associated with visceral adipose tissue (VAT), which is correlated with both peripheral and hepatic IR. Similarly, in the Dallas Heart Study, European-American (EA) men had an approximately twofold higher prevalence of hepatic steatosis than EA women. This gender disparity has been blamed on alcohol use, sex hormones or lifestyle behaviors, and no differences in body weight or insulin sensitivity [96].

The ethnic distribution among NAFLD-/NASH-related HCC patients has yet to be defined [191]. If the increase in the number of ethnic groups waitlisted for LT from 2004 to 2016 is a good indicator of the rise in NASH-HCC, then it could be inferred from a recent retrospective study that Asian females had an 854% change in NASH waitlist registration, while Asian males had a 552% change [189]. The increase in African-American waitlist population was much less compared to the other ethnic groups. In contrast, the Hispanic females had a 3010% change in the rate of waitlist registration for NASH with HCC, while non-Hispanic white females had a 1992% change [189].

NASH-related HCC patients are primarily male even though gender is not a proven statistical risk factor in the progression of NASH to HCC. However, NASH is currently the second leading cause for LT waitlist in females, whereas in men, alcoholic liver disease (ALD) continues to be the leading cause [189]. Although old data of 698 patients from biopsy-proven NASH show that NASH patients are more likely to be female than male possibly reflecting a higher disease burden rate in women [192], it is likely that both gender and racial ethnic differences in NAFLD and NASH are attributed to interaction among genetic, environmental, and lifestyle behaviors.

#### **5. Medical therapy for NAFLD and HCC**

The biological heterogeneities of NAFLD and HCC create predicaments in deciphering the key mechanisms of development and progression from NAFLD to ESLD. Although progress is being made in understanding the molecular underpinnings of chronic liver disease and its various offshoots, there are still formidable challenges in providing effective treatment regimens. Aside from a few prophylactic agents that have shown promise in the prevention and treatment of steatohepatitis and fibrosis, there is no treatment consensus due to scarcity of data [140]. Wholesome lifestyle and behavioral changes that include regular physical activity, low caloric intake, and weight loss are the main bulwarks against NAFLD, which may progress to HCC with or without cirrhosis. However, the extent to which these modifications are effective to prevent the development of HCC is unclear. There is currently no effective chemoprevention to decrease the incidence of HCC except using nucleoside analogs to reduce viral replication for those infected with HBV [193] and direct-acting antivirals (DAAs) for those infected with HCV [194], the latter demonstrating very high cure rates but also raising concerns about the recurrence or development of HCC after the achievement of a sustained virological response [195]. Obeticholic acid (OCA), a selective agonist of the Farnesoid X receptors, was touted to be a promising pharmacological drug for the management of NAFLD. However, its low efficacy and specificity have dampened enthusiasm for its practical use. Also, the drug pioglitazone has no long-term impact on NASH. This entails a pressing need to develop more effective and safe agents for NAFLD and HCC. Several other experimental studies suggest a direct role for vitamin D in

**97**

*The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma*

modulating liver fibrosis and inflammation by enhancing hepatic response to insulin via binding to vitamin D receptor on liver cells [196–198]. Vitamin E and carotenoids are also shown to decrease plasma levels of patients with NASH [199], whereas dietary antioxidants such as vitamin C and coenzyme Q12, trace minerals such as selenium, anticholesterol medications such as statins, antidiabetic drugs such as metformin, and methyl radical donors such as S-adenosylmethionine have

Hepatic steatosis is associated with many other morbidities. Therefore, dissecting the myriad causative agents including genetic, hormonal, or environmental factors underlying the pathogenicity of simple hepatic steatosis must be a priority to avoid the maze of complications that may arise during the development of NAFLD

• Global prevalence of NAFLD is at 24% but is rising to greater than 30%; highest rates to lowest rates are found in South America, Middle East, Asia, United

• The large volume of patients sets NAFLD apart from other liver diseases; thus, clinical care must focus on discerning highest risk of progressive liver disease.

• Overweight in childhood and adolescence is associated with the risk of NAFLD

• NAFLD patients have an elevated risk of liver-related morbidity/mortality and

• NAFLD warrants that primary-care physicians, specialists, and health policy-

• Older age, being male, and HA are independent risk factors for NAFLD/NASH.

• MetS as currently defined is not a good predictor of NAFLD in non-Hispanic blacks (NHB); because in contrast to others, TG level is normal in this group.

• Proton magnetic resonance spectroscopy is currently the best proven alternative

• Most effective therapeutic strategies include lifestyle changes including diet, exercise, modifying metabolic risk factors, early screening, and intervention.

• Treatment options require more robust studies on etiology of NAFLD.

• Certain genes may be associated with disparities in lipid metabolism.

tool to biopsy for accurate diagnosis of NAFLD.

• There is no proven medical therapy for NASH.

• NAFLD is linked with higher BMI, higher HTN, and lower physical activity.

later in life and increases liver-related morbidity and/or mortality.

metabolic comorbidities, which place a strain on healthcare systems.

makers stress prevention of excessive weight gain during childhood.

• Bariatric surgery may be an alternative option to committed weight loss.

all been touted as potential prophylactic agents [169, 200–202].

**6. Key findings, future trends, and unmet needs**

and its progression to HCC. The key findings are:

States, and Europe.

*DOI: http://dx.doi.org/10.5772/intechopen.85780*

*The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.85780*

modulating liver fibrosis and inflammation by enhancing hepatic response to insulin via binding to vitamin D receptor on liver cells [196–198]. Vitamin E and carotenoids are also shown to decrease plasma levels of patients with NASH [199], whereas dietary antioxidants such as vitamin C and coenzyme Q12, trace minerals such as selenium, anticholesterol medications such as statins, antidiabetic drugs such as metformin, and methyl radical donors such as S-adenosylmethionine have all been touted as potential prophylactic agents [169, 200–202].

### **6. Key findings, future trends, and unmet needs**

Hepatic steatosis is associated with many other morbidities. Therefore, dissecting the myriad causative agents including genetic, hormonal, or environmental factors underlying the pathogenicity of simple hepatic steatosis must be a priority to avoid the maze of complications that may arise during the development of NAFLD and its progression to HCC. The key findings are:


*Nonalcoholic Fatty Liver Disease - An Update*

females had a 1992% change [189].

**5. Medical therapy for NAFLD and HCC**

As for gender differences in NAFLD or NASH, there are uncertainties including the role of IR in the influence of gender on NAFLD. Ruhl et al. reported that NAFLD is about 2.7 times more prevalent in men than in women [190]. One reasonable explanation for this reported gender difference in NAFLD is due to the higher waist-to-hip circumference (WHR) ratio in men [96]. Pan et al. further state that WHR is associated with visceral adipose tissue (VAT), which is correlated with both peripheral and hepatic IR. Similarly, in the Dallas Heart Study, European-American (EA) men had an approximately twofold higher prevalence of hepatic steatosis than EA women. This gender disparity has been blamed on alcohol use, sex hormones or lifestyle behaviors, and no differences in body weight or insulin sensitivity [96]. The ethnic distribution among NAFLD-/NASH-related HCC patients has yet to be defined [191]. If the increase in the number of ethnic groups waitlisted for LT from 2004 to 2016 is a good indicator of the rise in NASH-HCC, then it could be inferred from a recent retrospective study that Asian females had an 854% change in NASH waitlist registration, while Asian males had a 552% change [189]. The increase in African-American waitlist population was much less compared to the other ethnic groups. In contrast, the Hispanic females had a 3010% change in the rate of waitlist registration for NASH with HCC, while non-Hispanic white

NASH-related HCC patients are primarily male even though gender is not a proven statistical risk factor in the progression of NASH to HCC. However, NASH is currently the second leading cause for LT waitlist in females, whereas in men, alcoholic liver disease (ALD) continues to be the leading cause [189]. Although old data of 698 patients from biopsy-proven NASH show that NASH patients are more likely to be female than male possibly reflecting a higher disease burden rate in women [192], it is likely that both gender and racial ethnic differences in NAFLD and NASH are attributed to interaction among genetic, environmental, and lifestyle behaviors.

The biological heterogeneities of NAFLD and HCC create predicaments in deciphering the key mechanisms of development and progression from NAFLD to ESLD. Although progress is being made in understanding the molecular underpinnings of chronic liver disease and its various offshoots, there are still formidable challenges in providing effective treatment regimens. Aside from a few prophylactic agents that have shown promise in the prevention and treatment of steatohepatitis and fibrosis, there is no treatment consensus due to scarcity of data [140]. Wholesome lifestyle and behavioral changes that include regular physical activity, low caloric intake, and weight loss are the main bulwarks against NAFLD, which may progress to HCC with or without cirrhosis. However, the extent to which these modifications are effective to prevent the development of HCC is unclear. There is currently no effective chemoprevention to decrease the incidence of HCC except using nucleoside analogs to reduce viral replication for those infected with HBV [193] and direct-acting antivirals (DAAs) for those infected with HCV [194], the latter demonstrating very high cure rates but also raising concerns about the recurrence or development of HCC after the achievement of a sustained virological response [195]. Obeticholic acid (OCA), a selective agonist of the Farnesoid X receptors, was touted to be a promising pharmacological drug for the management of NAFLD. However, its low efficacy and specificity have dampened enthusiasm for its practical use. Also, the drug pioglitazone has no long-term impact on NASH. This entails a pressing need to develop more effective and safe agents for NAFLD and HCC. Several other experimental studies suggest a direct role for vitamin D in

**96**


#### **Acknowledgements**

This work was supported by the National Institutes of Health (NIH) Grant U01CA185188.

### **Conflicts of interest**

The author has no conflict of interest.

### **Abbreviations**


**99**

**Author details**

Medicine, Washington, DC, USA

provided the original work is properly cited.

\*Address all correspondence to: zaki.sherif@howard.edu

Zaki A. Sherif

*The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma*

Department of Biochemistry and Molecular Biology, Howard University College of

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*DOI: http://dx.doi.org/10.5772/intechopen.85780*

*The Rise in the Prevalence of Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.85780*

### **Author details**

*Nonalcoholic Fatty Liver Disease - An Update*

trials of new treatments.

**Acknowledgements**

**Conflicts of interest**

The author has no conflict of interest.

AA African-American AF advanced fibrosis

BMI body mass index

HTN hypertension IR insulin resistance LT liver transplantation MetS metabolic syndrome NAFL nonalcoholic fatty liver

DAA direct-acting antiviral EA European-American ESLD end-stage liver disease HA Hispanic-American HCC hepatocellular carcinoma HDL high-density lipoprotein

NAFLD nonalcoholic fatty liver disease NASH nonalcoholic steatohepatitis NFS NAFLD fibrosis score NAS NAFLD activity score

NFS NAFLD fibrosis score T2DM type 2 diabetes mellitus

TCGA the cancer genome atlas

TG triglyceride

NHANES National Health and Nutrition Examination Survey

ALT alanine aminotransferase AST aspartate aminotransferase

CDC Centers for Disease Control and Prevention

U01CA185188.

**Abbreviations**

• Alternative noninvasive markers of NASH may now be available even though there are no proven biomarkers for various stages of the NAFLD spectrum.

• Discovery of new biomolecules during clinical trials and metabolomics studies is crucial for understanding NAFLD/NASH initiation and progression.

• Patients with NASH have a worse prognosis and must be included in clinical

• The biological heterogeneity of HCC makes it difficult to assess the key mecha-

• Certain genes have been identified to be associated with progression to HCC.

This work was supported by the National Institutes of Health (NIH) Grant

nisms of cancer development and thus implement effective therapies.

**98**

Zaki A. Sherif

Department of Biochemistry and Molecular Biology, Howard University College of Medicine, Washington, DC, USA

\*Address all correspondence to: zaki.sherif@howard.edu

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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**113**

Section 4

New Diagnostic Tools for

Nonalcoholic Fatty Liver

Disease

Section 4
