**1. Introduction**

Non-alcoholic fatty liver disease (NAFLD) is currently the most prevalent chronic liver disease worldwide [1]. A subset of NAFLD patients have the progressive form of NAFLD termed non-alcoholic steatohepatitis (NASH). NASH is typically characterized by a specific pattern on liver histology, including steatosis, lobular inflammation, and ballooning with or without perisinusoidal fibrosis [2]. It can progress to advanced fibrosis, cirrhosis, hepatocellular carcinoma, and liverrelated morbidity and mortality. Liver disease is only the third leading cause of death in patients with NAFLD, following cardiovascular disease and malignancy [3].

Precise histological diagnosis of NAFLD is commonly based on liver biopsy [4]; however, biopsies present several potential problems [5]. Thus, there is a need for reliable and cost-effective noninvasive biomarkers to avoid the invasiveness of biopsy [6].

Although there are some clinical strategies to ameliorate NAFLD progression, such as treatments for obesity or type 2 diabetes mellitus (T2DM), there is no medication proven to be effective as a treatment for NASH [7]. Therefore, it is necessary to improve the research on possible therapeutic targets for NASH due to the severity of this pathological condition.

Previous evidences have linked gut dysbiosis with obesity, insulin resistance (IR), metabolic syndrome (MS), and NAFLD [8, 9]. The impact of the GM on NAFLD/NASH has been attributed to increased gut permeability, intestinal endotoxemia, endogenous alcohol production, upregulation of hepatic de novo lipogenesis and triglyceride synthesis, reduction in choline metabolism, and aggravation of IR [10]. The increased permeability of the intestinal barrier results in the release of substances such as lipopolysaccharides (LPS), bacterial components, short-chain fatty acids (SCFAs), bile acids (BAs), choline metabolites, and endogenous ethanol that reach the liver and seem to contribute to the pathogenesis of NAFLD (**Figure 1**) [11, 12]. It is important to note that some of these substances could perhaps be employed as potential noninvasive biomarkers of NAFLD progression.

Manipulation of the microbiota through probiotics, prebiotics, and antibiotic treatment yields encouraging results for the treatment of obesity, T2DM, and NASH in animal models, but data in humans are scarce. In regard to NAFLD, this

#### **Figure 1.**

*Implication of intestinal dysbiosis in NAFLD pathogenesis. Short-chain fatty acids (SCFAs), bile acids (BAs), lipopolysaccharides (LPS), trimethylamine-N-oxide (TMAO), ethanol (EtOH), non-alcoholic fatty liver (NAFL), and non-alcoholic fatty liver disease (NAFLD).*

**45**

*Intestinal Dysbiosis and Non-Alcoholic Fatty Liver Disease*

gut microbiota as a therapeutic target in NAFLD.

individuals with nondiabetic morbid obesity [14].

disease, hepatocellular carcinoma, and liver transplantation).

provides the most information without irradiation [24, 25].

**2. Non-alcoholic fatty liver disease**

therapeutic strategy seeks to prevent the endotoxicity produced by the microbiota-derived metabolites that reach the liver and promote the progression of the disease [13]. Thus, there is a need to focus research on the GM as a therapeutic

To provide a broad overview of the relationship between intestinal dysbiosis and NAFLD, we have elaborated on this subject in this book chapter. In this sense, this narrative chapter will explain (a) non-alcoholic fatty liver disease, (b) the gut microbiota, (c) gut microbiota-derived mediators involved in NAFLD, and (d) the

NAFLD has emerged as the most common form of chronic liver disease worldwide. The incidence of NAFLD has drastically increased in parallel with obesity in recent years. Currently, the global prevalence of NAFLD is approximately 25% [1], but it can increase to 58% in individuals who are overweight or as high as 98% in

NAFLD comprises a spectrum of disorders extending from simple steatosis (SS) to NASH, fibrosis, and cirrhosis [2, 15]. This pathology has potentially serious sequelae [16]. Although SS tends to develop into a favorable clinical course [3], NASH can develop into liver cirrhosis and hepatocellular carcinoma [15]. Thus, liver-related mortality increases exponentially with an advance in the fibrosis stage [17]. In this regard, NASH is a very common cause of liver transplant worldwide [1]. Although the most common cause of death in patients with NAFLD is cardiovascular disease, independent of other metabolic comorbidities, NAFLD is becoming a major cause of liver disease-related morbidity (e.g., cirrhosis, end-stage liver

NAFLD is characterized by significant lipid deposition in the hepatocytes of the liver parenchyma [18]. Obesity, T2DM, dyslipidemia, MS, and IR are the main risk factors for NAFLD [19]. Most NAFLD patients are asymptomatic, and the evidence of hepatic steatosis should be detected via a routine blood test, showing a deregulation in liver enzymes. Currently, it is not possible to diagnose NAFLD with only a blood test, but the aspartate aminotransferase (AST)-alanine aminotransferase ratio (ALT) can be used as a first step [20–22]. However, the ALT level correlation with histological findings has poor sensitivity and specificity for the diagnosis of NASH [23]. Then, it is necessary to rule out other causes of liver damage, such as alcoholic fatty liver disease, drug-induced liver injury, viral hepatitis, autoimmune liver disease, hemochromatosis, celiac disease, and Wilson's disease [1]. Finally, ultrasonography is the most common noninvasive tool used to detect NAFLD. There are also other imaging techniques used to detect liver steatosis, such as computer tomography or magnetic resonance imaging, but ultrasound is the technique that

One-third of the NAFLD-affected subjects progress to NASH. This condition is characterized by the presence of hepatocellular ballooning and inflammation and has a prevalence of 2–3% worldwide [2]. Key issues in NAFLD patients are the differentiation of NASH from SS and the identification of advanced hepatic fibrosis. To date, liver biopsy has been the *gold standard* for identifying these two critical end points but has well-known limitations, including invasiveness; rare but potentially life-threatening complications; poor tolerance; sampling variability; and cost. Furthermore, due to the epidemic proportion of individuals with NAFLD worldwide, liver biopsy evaluation is impractical, and noninvasive assessment for the diagnosis of NASH and fibrosis is needed [5]. NASH is confirmed when the hepatic

*DOI: http://dx.doi.org/10.5772/intechopen.92972*

target to ameliorate NASH.

*Intestinal Dysbiosis and Non-Alcoholic Fatty Liver Disease DOI: http://dx.doi.org/10.5772/intechopen.92972*

*Human Microbiome*

of biopsy [6].

progression.

of this pathological condition.

for reliable and cost-effective noninvasive biomarkers to avoid the invasiveness

Although there are some clinical strategies to ameliorate NAFLD progression, such as treatments for obesity or type 2 diabetes mellitus (T2DM), there is no medication proven to be effective as a treatment for NASH [7]. Therefore, it is necessary to improve the research on possible therapeutic targets for NASH due to the severity

Previous evidences have linked gut dysbiosis with obesity, insulin resistance (IR), metabolic syndrome (MS), and NAFLD [8, 9]. The impact of the GM on NAFLD/NASH has been attributed to increased gut permeability, intestinal endotoxemia, endogenous alcohol production, upregulation of hepatic de novo lipogenesis and triglyceride synthesis, reduction in choline metabolism, and aggravation of IR [10]. The increased permeability of the intestinal barrier results in the release of substances such as lipopolysaccharides (LPS), bacterial components, short-chain fatty acids (SCFAs), bile acids (BAs), choline metabolites, and endogenous ethanol that reach the liver and seem to contribute to the pathogenesis of NAFLD (**Figure 1**) [11, 12]. It is important to note that some of these substances

could perhaps be employed as potential noninvasive biomarkers of NAFLD

Manipulation of the microbiota through probiotics, prebiotics, and antibiotic treatment yields encouraging results for the treatment of obesity, T2DM, and NASH in animal models, but data in humans are scarce. In regard to NAFLD, this

*Implication of intestinal dysbiosis in NAFLD pathogenesis. Short-chain fatty acids (SCFAs), bile acids (BAs), lipopolysaccharides (LPS), trimethylamine-N-oxide (TMAO), ethanol (EtOH), non-alcoholic fatty liver* 

**44**

**Figure 1.**

*(NAFL), and non-alcoholic fatty liver disease (NAFLD).*

therapeutic strategy seeks to prevent the endotoxicity produced by the microbiota-derived metabolites that reach the liver and promote the progression of the disease [13]. Thus, there is a need to focus research on the GM as a therapeutic target to ameliorate NASH.

To provide a broad overview of the relationship between intestinal dysbiosis and NAFLD, we have elaborated on this subject in this book chapter. In this sense, this narrative chapter will explain (a) non-alcoholic fatty liver disease, (b) the gut microbiota, (c) gut microbiota-derived mediators involved in NAFLD, and (d) the gut microbiota as a therapeutic target in NAFLD.

## **2. Non-alcoholic fatty liver disease**

NAFLD has emerged as the most common form of chronic liver disease worldwide. The incidence of NAFLD has drastically increased in parallel with obesity in recent years. Currently, the global prevalence of NAFLD is approximately 25% [1], but it can increase to 58% in individuals who are overweight or as high as 98% in individuals with nondiabetic morbid obesity [14].

NAFLD comprises a spectrum of disorders extending from simple steatosis (SS) to NASH, fibrosis, and cirrhosis [2, 15]. This pathology has potentially serious sequelae [16]. Although SS tends to develop into a favorable clinical course [3], NASH can develop into liver cirrhosis and hepatocellular carcinoma [15]. Thus, liver-related mortality increases exponentially with an advance in the fibrosis stage [17]. In this regard, NASH is a very common cause of liver transplant worldwide [1]. Although the most common cause of death in patients with NAFLD is cardiovascular disease, independent of other metabolic comorbidities, NAFLD is becoming a major cause of liver disease-related morbidity (e.g., cirrhosis, end-stage liver disease, hepatocellular carcinoma, and liver transplantation).

NAFLD is characterized by significant lipid deposition in the hepatocytes of the liver parenchyma [18]. Obesity, T2DM, dyslipidemia, MS, and IR are the main risk factors for NAFLD [19]. Most NAFLD patients are asymptomatic, and the evidence of hepatic steatosis should be detected via a routine blood test, showing a deregulation in liver enzymes. Currently, it is not possible to diagnose NAFLD with only a blood test, but the aspartate aminotransferase (AST)-alanine aminotransferase ratio (ALT) can be used as a first step [20–22]. However, the ALT level correlation with histological findings has poor sensitivity and specificity for the diagnosis of NASH [23]. Then, it is necessary to rule out other causes of liver damage, such as alcoholic fatty liver disease, drug-induced liver injury, viral hepatitis, autoimmune liver disease, hemochromatosis, celiac disease, and Wilson's disease [1]. Finally, ultrasonography is the most common noninvasive tool used to detect NAFLD. There are also other imaging techniques used to detect liver steatosis, such as computer tomography or magnetic resonance imaging, but ultrasound is the technique that provides the most information without irradiation [24, 25].

One-third of the NAFLD-affected subjects progress to NASH. This condition is characterized by the presence of hepatocellular ballooning and inflammation and has a prevalence of 2–3% worldwide [2]. Key issues in NAFLD patients are the differentiation of NASH from SS and the identification of advanced hepatic fibrosis. To date, liver biopsy has been the *gold standard* for identifying these two critical end points but has well-known limitations, including invasiveness; rare but potentially life-threatening complications; poor tolerance; sampling variability; and cost. Furthermore, due to the epidemic proportion of individuals with NAFLD worldwide, liver biopsy evaluation is impractical, and noninvasive assessment for the diagnosis of NASH and fibrosis is needed [5]. NASH is confirmed when the hepatic

tissue shows the presence of perilobular inflammation, hepatocellular ballooning, Mallory's hyaline, and acidophil bodies with or without fibrosis. Although there are other noninvasive tests, such as the fatty liver index, NAFLD fibrosis score, and FibroMeter, and elastographic techniques, such as FibroScan, that can suggest the presence of NASH and detect fibrosis [15], a precise histological diagnosis of NASH is commonly based on liver biopsy [26]. The development of alternative noninvasive strategies has been an area of intensive research over the past decade and currently.

Regarding NAFLD therapeutics, all forms of treatment of metabolic disorders are able to modify liver damage. Diet and lifestyle modification and insulinsensitizing agents appear to be promisingly effective against NAFLD progression. However, these approaches may not be effective in some patients. Many other drugs are currently being studied to establish treatments for NAFLD. At present, no accepted drug treatment for NASH has been stated [24]. In this sense, it is very important to improve the knowledge of NAFLD physiopathology. Actually, the underlying precise mechanisms of NAFLD pathogenesis have just begun to be understood. The classic "multiple hit" theory states that lipid accumulation initiates hepatic steatosis and subsequently triggers multiple insults acting together (hormones/adipokines from adipose tissue, inflammation, deregulated fat metabolism, lipotoxicity, oxidative stress, mitochondrial dysfunction, and genetic and epigenetic factors), ultimately inducing NASH and cirrhosis [27]. Progression to NASH is linked to systemic inflammation, and it is associated with other pathological processes, such as innate immunity alterations, endoplasmic-reticulum stress, toll-like receptor (TLR) signaling, mitochondrial dysfunction, and intestinal dysbiosis [6, 28–32]. Regarding this last process, approximately 70–75% of blood that reaches the liver comes from the portal vein circulation that communicates the liver with the intestine [33]. The liver is continually exposed to GM-derived mediators, including bacteria and bacterial components, such as LPS, promoting an inflammatory response that contributes to liver injury [13].

## **3. The gut microbiota**

Millions of symbiotic microorganisms live on and within human beings and play an important role in human health and disease. Initial colonization occurs at the time of birth, and humans progressively acquire ∼1014 bacterial cells at equilibrium, which remain for life [13]**.**

The human microbiota, especially the GM, has even been considered to be an "essential organ," carrying approximately 150 times more genes than the human genome [34]. The GM is composed of an immense number of microorganisms (bacteria, viruses, and fungi) with several functions, such as host nutrition, bone mineralization, immune system regulation, xenobiotic metabolism, proliferation of intestinal cells, and protection against pathogens [35, 36]. This bacterial community is dominated by anaerobic bacteria and includes 500–1000 species [37]. *Firmicutes* and *Bacteroidetes* are the most important phyla among the intestinal bacteria, with a proportion of over 90% of the total community [38].

The duodenum and proximal jejunum normally contain small numbers of bacteria, usually lactobacilli and enterococci, which are facultative anaerobes. The distal ileum is a transition zone between sparse populations of aerobic bacteria of the proximal small intestine and very dense populations of anaerobic microorganisms in the large bowel. Occasional groups of bacteria can be found in low concentrations within the lumen of the small intestine. Bacteria do not form clusters, and the luminal contents are separated from the mucosa by a mucus layer [13].

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*Intestinal Dysbiosis and Non-Alcoholic Fatty Liver Disease*

The GM is specific to an individual and highly resilient to changes. However, it can be affected by several factors, intrinsic and extrinsic to the host, such as the subject's genetic makeup, dietary habits, antibiotic use, and environmental changes [13, 39, 40]. A disruption in the composition of the normal GM is known as intestinal dysbiosis [41, 42]. Generally, this process includes an unfavorable change in the bacterial composition, with a reduction in autochthonous bacteria and growth of

Intestinal dysbiosis is a process that may adversely impact metabolism and produce immune responses, favoring NAFLD progression. Important studies on the relationship of the GM with obesity have identified profound changes in the composition and metabolic function of the GM in subjects with obesity. Moreover, these studies demonstrated that the GM interacts with host epithelial cells to indirectly control energy expenditure and storage and activate inflammatory responses in NASH pathogenesis [44]. Qualitative or quantitative imbalances in the GM might have serious health consequences for the host, including small intestinal bacterial overgrowth (SIBO) syndrome [13]. Due to gut dysbiosis, there is an elevated production of toxic bacterial components and metabolic mediators, which consequently accumulate in the intestine. In addition, an increase in intestinal permeability and further disruption of the epithelial barrier lead to the release of these GM-derived mediators [42], which could reach the liver through portal circulation, favoring hepatic inflammation and the development of NAFLD [45, 46]. After disruption of the gut epithelial barrier, the liver is exposed to microbial products and metabolites resulting from bacterial metabolism [47, 48]. In this sense, it has been demonstrated that patients with NAFLD have gut dysbiosis, gut epithelial barrier dysfunction, and increased translocation of bacterial components to the liver [49]. For this reason, mediators derived from gut dysbiosis might also be related to the pathogenesis of the disease. Several previous studies in clinical settings have associated intestinal dysbiosis with the occurrence of NAFLD [50–52] and with the progression to NASH [10, 53].

Among the various factors, dietary habits are considered to be most influential on the gut microbiome in subjects with obesity and NAFLD patients. It is wellknown that a high-fat diet causes gut dysbiosis characterized by lowered species richness and changes in microbial composition, such as decreased *Bacteroidetes* and increased *Firmicutes* and *Proteobacteria* abundances [43]. On the other hand, *Prevotella*, a member of the phylum *Bacteroidetes*, is associated with plant-rich diets. *Prevotella*-dominated microbiotas have higher fiber utilizing capacity than *Bacteroides*-dominated microbiotas, producing higher amounts of SCFAs [54]. There are some studies that consider *Prevotella* to be a beneficial commensal bacterium [10, 55], but there are others that noted enriched fecal *Prevotella* in NASH or cirrhotic patients [56–58]. These contradictory results may be partly explained by the differences in populations, age, or NAFLD stages between the studies. In this sense, further studies on *Prevotella* should be directed to characterize properties at

the species level and to evaluate these species in different stages of NAFLD.

GM-derived mediators resulting from intestinal dysbiosis could play a key role in NAFLD progression through several mechanisms: (1) enhanced energy extraction from food nutrients by formation of SCFAs; (2) modulation of BA synthesis, which is crucial for fat absorption and affects metabolism of glucose via farnesoid X receptor (FXR); (3) innate immune system activation by bacterial component translocation; (4) endogenous ethanol production; and (5) reduction in choline metabolism, which reduces efflux of very-low-density lipoprotein (VLDL) from hepatocytes, promoting inflammation. These mechanisms involve translocation

*DOI: http://dx.doi.org/10.5772/intechopen.92972*

others that prejudice host health [43].

**3.1 Intestinal dysbiosis**

The GM is specific to an individual and highly resilient to changes. However, it can be affected by several factors, intrinsic and extrinsic to the host, such as the subject's genetic makeup, dietary habits, antibiotic use, and environmental changes [13, 39, 40]. A disruption in the composition of the normal GM is known as intestinal dysbiosis [41, 42]. Generally, this process includes an unfavorable change in the bacterial composition, with a reduction in autochthonous bacteria and growth of others that prejudice host health [43].
