**2. NAFLD pathophysiology aspects**

The main mechanism related to the development of hepatic steatosis are: increased supply of free fatty acids due to increased lipolysis of visceral/subcutaneous adipose tissue and/or increased lipid intake; reduction of free fatty acid oxidation; increased hepatic de novo lipogenesis; and decreased hepatic secretion of very low density lipoprotein (VLDL). In addition, during NAFLD there is an imbalance between the intrahepatic production of triglycerides (TG) (derived mainly from plasma fatty acids delivered to the liver and not oxidized as a fuel) and the removal of intrahepatic TG (mainly exported from the liver to very low density lipoproteins, VLDL-TG) [8, 9], as shown in **Figure 1**.

TGs are the lipids that accumulate most in hepatocytes in NAFLD. TGs are synthesized through various enzymatic steps of glycerol and fatty acids condensation after activation thereof to their acyl-CoA esters. The liver obtains fatty acids from the circulation from the hydrolysis (lipolysis) of triglycerides in adipocytes in the post-absorptive state and, to a lesser degree, the postprandial lipolysis of triglyceride-rich particles (chylomicrons and VLDL). Fatty acids are released from triglyceride stores in adipose tissue through the action of the sensitive hormone lipase (located in adipocytes). These fatty acids upon their release into circulation are bound to Animal Models of Obesity Characterized by Non-alcoholic Fatty Liver Disease (NAFLD) http://dx.doi.org/10.5772/intechopen.72707 89

**1. Introduction**

cardiovascular disease and fatty liver disease [1].

88 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

**2. NAFLD pathophysiology aspects**

lipoproteins, VLDL-TG) [8, 9], as shown in **Figure 1**.

The rapid increase in the rate of obesity is a health problem critical in developed countries. Obesity is associated with a number of health problems that are often summarized together as metabolic syndrome and involve the development of insulin resistance, type 2 diabetes,

Nonalcoholic Fatty Liver Disease (NAFLD) is the leading cause of chronic liver disease affecting 20–30% of the world's adult population, and is characterized by a buildup of fat, mainly in the form of triglycerides, in the hepatocyte cytoplasm, exceeding 5–10% of the cell weight, verified histologically or by imaging techniques. It requires exclusion of other causes of steatosis, such as excessive alcohol consumption, drugs or genetic diseases [2, 3]. About 20–30% of individuals with NAFLD can develop Non-alcoholic steatohepatitis (NASH) [4], a more severe disease condition related to metabolic abnormalities associated with obesity, namely hyperinsulinemia, dyslipidemia, and ectopic lipid accumulation [5]. More specifically, NASH is associated with lobular inflammation, hepatocellular damage and/or hepatic fibrosis [4].

NAFLD is common in Western countries, usually associated with the main characteristics of the metabolic syndrome, such as obesity, insulin resistance and hyperlipidemia [6]. Data from the U.S. National Health and Nutrition Examination Surveys, collected from 1988 to 2008, show a 2-fold higher prevalence of NAFLD, concomitant with the increased or increasing

In this context, obesity induction with the development of hepatic steatosis in animal models is discussed. Animal models of obesity focused on NAFLD and NASH, as well as pathophysiological aspects related to obesity and liver diseases were systematically addressed in this chapter.

The main mechanism related to the development of hepatic steatosis are: increased supply of free fatty acids due to increased lipolysis of visceral/subcutaneous adipose tissue and/or increased lipid intake; reduction of free fatty acid oxidation; increased hepatic de novo lipogenesis; and decreased hepatic secretion of very low density lipoprotein (VLDL). In addition, during NAFLD there is an imbalance between the intrahepatic production of triglycerides (TG) (derived mainly from plasma fatty acids delivered to the liver and not oxidized as a fuel) and the removal of intrahepatic TG (mainly exported from the liver to very low density

TGs are the lipids that accumulate most in hepatocytes in NAFLD. TGs are synthesized through various enzymatic steps of glycerol and fatty acids condensation after activation thereof to their acyl-CoA esters. The liver obtains fatty acids from the circulation from the hydrolysis (lipolysis) of triglycerides in adipocytes in the post-absorptive state and, to a lesser degree, the postprandial lipolysis of triglyceride-rich particles (chylomicrons and VLDL). Fatty acids are released from triglyceride stores in adipose tissue through the action of the sensitive hormone lipase (located in adipocytes). These fatty acids upon their release into circulation are bound to

prevalence of metabolic conditions such as obesity and insulin resistance [7].

**Figure 1.** NAFLD mechanisms. Associated factors such as increased lipolysis and de novo hepatic lipogenesis (DNL), decreased free fatty acid oxidation and hepatic secretion of low density lipoprotein (VLDL) aggravate the onset of the disease. The action of Mitochondrial β-oxidation contributes to the synthesis of adenosine triphosphate (ATP), however results in the increase of reactive oxygen species.

albumin. The peripheral tissues, in turn, receive fatty acids as substrates for oxidation (mainly muscle) and storage (adipose tissue), through the action of endothelial lipoprotein lipase on particles rich in circulating triglycerides, either VLDL secreted by the liver, or chylomicrons delivered to lymphatic circulation after intestinal absorption of lipids [10].

During fat digestion, medium and short chain fatty acids are absorbed directly into the portal circulation. Whereas long chain fatty acids (C > 14) are mainly reesterified in chylomicrons, but a proportion of long chain unsaturated free fatty acids (FFA) enters the portal circulation [10].

According to the hypothesis on hepatic lipotoxic lesions, certain FFAs and their metabolites flow through the liver and cause NAFLD/NASH. Dietary intake plays an important role in the generation of FFA. Excess carbohydrate consumption, particularly fructose, leads to *de novo* lipogenesis. Excess calories and fats can result in accumulation of lipids in adipose tissue, and finally, the stored fatty acids are released through lipolysis. Free fatty acids generated by lipogenesis, lipolysis or other mechanisms have three potential destinations: triglycerides formation, oxidation and elimination, or intermediate lipid formation [11].

Hepatic mitochondria play an important role in the oxidation of fatty acids and in the synthesis of ATP. Mitochondrial β-oxidation is a pathway for the elimination of fatty acids, but results in the generation of reactive oxygen species (ROS). In most circumstances, the endogenous antioxidant mechanisms are able to protect against cellular damage caused by ROS. However, in the configuration of impaired mitochondrial function related to obesity and chronic lipid overload, ROS lead to peroxidation of fatty acids, further interfering in mitochondrial function through oxidative damage to mitochondrial DNA and proteins [12].

Thus, accumulation of fat within the liver results in a blockage in insulin signaling in IRS-2. This proximal blockade in the insulin signaling cascade may limit the ability of insulin to activate hepatic glycogen synthesis and suppress hepatic glucose production, involving the role of PKC, IKK-β and/or JNK1 in the pathogenesis of hepatic insulin resistance induced by fat [17]. Based on a study of patients with diabetes, accumulation of fat in the liver may also

Animal Models of Obesity Characterized by Non-alcoholic Fatty Liver Disease (NAFLD)

http://dx.doi.org/10.5772/intechopen.72707

91

In addition, the hormonal and biological activity of adipose tissue through the secretion of adipokines by white adipose tissue (WAT) contributes to the insulin and metabolic resistance of the disease, including NASH. This is due to the release of inflammatory cytokines by macrophages in the adipose tissue and the increased release of free fatty acids from adipocytes [12]. In the context of chronic overfeeding, white adipose tissue becomes expanded with lipids, leading to adipocyte hyperplasia or hypertrophy. The remodeling of white adipose tissue helps to accommodate adipocyte hypertrophy, but, eventually, impaired innervation and vascularization result in hypoxia and adipocyte dysfunction [26]. This dysfunction would increase adipocyte secretion mediated by JNK1 - inflammatory cytokines [27], resulting in increases and changes of immune cells in adipose tissue and eventual systemic metabolic stress with mitochondrial dysfunction, lipolysis, decreased lipid storage capacity and signaling rupture of insulin [28].

In addition, the Western dietary pattern with excessive intake of high-calorie, rich in fat, sugar and cholesterol and sedentary lifestyle are risk factors for hepatic steatosis. Such factors have an impact on lipid metabolism related to liver diseases, which include decreased conversion of cholesterol to bile acids; increased hydrolysis of cholesterol esters to free cholesterol [29]; increased endogenous cholesterol synthesis [30]; increased absorption of cholesterol-rich lipoproteins [29]; and decreased cholesterol excretion [31]. Excess cholesterol influences the fluidity of the membrane, affecting the function of its proteins [32]. It is also worth mentioning that this excessive consumption activates Kupffer cells and hepatic stellate cells, exacerbating liver inflammation, increasing extracellular matrix synthesis and eventually accelerating progress for NASH [33, 34].

The progress of obesity with the presence of the NAFLD disorder is characterized as a very complex inflammatory process. Despite the search for new studies in the area, there are a number of challenges in conducting research with humans, especially to investigate genetic and dietary aspects. Controlling the diet and environment of humans becomes difficult for long periods of time, and ethical restrictions limit access to biological samples. Such problems

Animal models are extremely important for elucidating the etiology of diseases in humans, besides having an integrated view of the pathogenic mechanisms, as well as monitoring the natural evolution of the disease under controlled genetic and environmental conditions. These models constitute important resources in the identification of targets and therapeutic

The choice of the animal model for studies of obesity is comprised mostly of small animals such as mice, rats, guinea pigs, hamsters, with the genetic, neuroendocrine and dietary alterations.

**3. Developing animal models of non-alcoholic hepatic steatosis**

can be circumvented with the use of animal models of disease [35].

agents that can prevent or revert diseases [36].

be associated with increased hepatic gluconeogenesis [25].

The reason some obese individuals are able to regulate mitochondrial function and compensate lipid overload, while others are not, are still unclear. Multiple pathways involving a complex interaction between excess lipids, systemic inflammation and cellular stress probably contribute to the development and progression of NAFLD [13].

#### **2.1. Crosstalk between obesity and NAFLD**

Obesity is an important risk factor for non-alcoholic fatty liver disease (NAFLD). Although it is not a risk factor present in all obese individuals, a minority of patients with NAFLD are lean. In the survey conducted by the National Health and Nutrition Examination Survey III, 7.4% of lean adults and 27.8% of overweight/obese adults had hepatic steatosis that could be detected by ultrasound, which highlights the higher prevalence in overweight or obessed individuals. [14].

The pathophysiology of NAFLD is complex, since it is a multifactorial disease, whose disorders contribute to the metabolic syndrome, involving obesity, diabetes mellitus, hypertension and dyslipidemia [15]. Patients with NAFLD and metabolic syndrome (MS) have a higher prevalence and severity of fibrosis and necroinflammatory activity, compared to individuals of NAFLD without MS. In addition, the presence of MS is associated with a high risk of NASH among NAFLD individuals, after correction for gender, age and body mass [15].

Insulin resistance, an important feature of MS and type 2 diabetes mellitus (DM 2), is classified as peripheral insulin resistance and/or hepatic insulin resistance [16, 17]. Peripheral insulin resistance refers primarily to decreased insulin-mediated glucose uptake in skeletal muscle and adipocytes, whereas hepatic insulin resistance relates to the inability of insulin to decrease hepatic glucose production. The accumulation of fat in skeletal muscle has been considered the main pathogenic event leading to resistance to peripheral insulin. Briefly, the accumulation of arachidonic acid fatty acid metabolites in the muscle initiates a series of signaling reactions that increase the phosphorylation of specific serine residues (e.g, S307) on the insulin-1 receptor substrate (IRS-1) in the muscle. However, several researchers have begun to focus on the mechanism of hepatic insulin resistance [16, 17].

The IRS-1 is critical in its signaling, since it is an important target in the inflammatory process and, following serine phosphorylation of IRS-1, there is a reduction in the activation of phosphotidylinositol-3-kinase (PI3k), as well as other proteins involved in the normal insulin signaling process, such as protein kinase B (Akt). These deleterious events are shown to be mediated by proteins that activate inflammatory pathways, such as the c-Jun NH2-terminal kinase (JNK), the kinase IκB (Iκκβ) and PKCθ [18–21]. NFκB is a gene transcription factor that alters insulin signaling and after stimulation, Iκκβ is phosphorylated, leading to the translocation of NFκB to the cell nucleus and ̶ subsequent activation of proinflammatory cytokine genes, such as TNF-α, IL- 6 and IL-1β [22–24].

The mechanism of resistance to peripheral insulin point to the fat-induced induction of several inflammatory signaling kinases (PKCθ, IKK-β and JNK1), which may in turn increase serine phosphorylation in IRS-1, thus preventing their participation in the insulin signaling cascade. As a result, the insulin capacity to increase GLUT4 translocation is impaired. The mechanisms of fat-induced hepatic insulin resistance are similar to peripheral resistance. Thus, accumulation of fat within the liver results in a blockage in insulin signaling in IRS-2. This proximal blockade in the insulin signaling cascade may limit the ability of insulin to activate hepatic glycogen synthesis and suppress hepatic glucose production, involving the role of PKC, IKK-β and/or JNK1 in the pathogenesis of hepatic insulin resistance induced by fat [17]. Based on a study of patients with diabetes, accumulation of fat in the liver may also be associated with increased hepatic gluconeogenesis [25].

and chronic lipid overload, ROS lead to peroxidation of fatty acids, further interfering in mitochondrial function through oxidative damage to mitochondrial DNA and proteins [12]. The reason some obese individuals are able to regulate mitochondrial function and compensate lipid overload, while others are not, are still unclear. Multiple pathways involving a complex interaction between excess lipids, systemic inflammation and cellular stress probably

Obesity is an important risk factor for non-alcoholic fatty liver disease (NAFLD). Although it is not a risk factor present in all obese individuals, a minority of patients with NAFLD are lean. In the survey conducted by the National Health and Nutrition Examination Survey III, 7.4% of lean adults and 27.8% of overweight/obese adults had hepatic steatosis that could be detected by ultrasound, which highlights the higher prevalence in overweight or obessed individuals. [14]. The pathophysiology of NAFLD is complex, since it is a multifactorial disease, whose disorders contribute to the metabolic syndrome, involving obesity, diabetes mellitus, hypertension and dyslipidemia [15]. Patients with NAFLD and metabolic syndrome (MS) have a higher prevalence and severity of fibrosis and necroinflammatory activity, compared to individuals of NAFLD without MS. In addition, the presence of MS is associated with a high risk of NASH

among NAFLD individuals, after correction for gender, age and body mass [15].

to focus on the mechanism of hepatic insulin resistance [16, 17].

Insulin resistance, an important feature of MS and type 2 diabetes mellitus (DM 2), is classified as peripheral insulin resistance and/or hepatic insulin resistance [16, 17]. Peripheral insulin resistance refers primarily to decreased insulin-mediated glucose uptake in skeletal muscle and adipocytes, whereas hepatic insulin resistance relates to the inability of insulin to decrease hepatic glucose production. The accumulation of fat in skeletal muscle has been considered the main pathogenic event leading to resistance to peripheral insulin. Briefly, the accumulation of arachidonic acid fatty acid metabolites in the muscle initiates a series of signaling reactions that increase the phosphorylation of specific serine residues (e.g, S307) on the insulin-1 receptor substrate (IRS-1) in the muscle. However, several researchers have begun

The IRS-1 is critical in its signaling, since it is an important target in the inflammatory process and, following serine phosphorylation of IRS-1, there is a reduction in the activation of phosphotidylinositol-3-kinase (PI3k), as well as other proteins involved in the normal insulin signaling process, such as protein kinase B (Akt). These deleterious events are shown to be mediated by proteins that activate inflammatory pathways, such as the c-Jun NH2-terminal kinase (JNK), the kinase IκB (Iκκβ) and PKCθ [18–21]. NFκB is a gene transcription factor that alters insulin signaling and after stimulation, Iκκβ is phosphorylated, leading to the translocation of NFκB to the cell nucleus and ̶ subsequent activation of proinflammatory cytokine genes, such as TNF-α, IL- 6 and IL-1β [22–24]. The mechanism of resistance to peripheral insulin point to the fat-induced induction of several inflammatory signaling kinases (PKCθ, IKK-β and JNK1), which may in turn increase serine phosphorylation in IRS-1, thus preventing their participation in the insulin signaling cascade. As a result, the insulin capacity to increase GLUT4 translocation is impaired. The mechanisms of fat-induced hepatic insulin resistance are similar to peripheral resistance.

contribute to the development and progression of NAFLD [13].

90 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

**2.1. Crosstalk between obesity and NAFLD**

In addition, the hormonal and biological activity of adipose tissue through the secretion of adipokines by white adipose tissue (WAT) contributes to the insulin and metabolic resistance of the disease, including NASH. This is due to the release of inflammatory cytokines by macrophages in the adipose tissue and the increased release of free fatty acids from adipocytes [12].

In the context of chronic overfeeding, white adipose tissue becomes expanded with lipids, leading to adipocyte hyperplasia or hypertrophy. The remodeling of white adipose tissue helps to accommodate adipocyte hypertrophy, but, eventually, impaired innervation and vascularization result in hypoxia and adipocyte dysfunction [26]. This dysfunction would increase adipocyte secretion mediated by JNK1 - inflammatory cytokines [27], resulting in increases and changes of immune cells in adipose tissue and eventual systemic metabolic stress with mitochondrial dysfunction, lipolysis, decreased lipid storage capacity and signaling rupture of insulin [28].

In addition, the Western dietary pattern with excessive intake of high-calorie, rich in fat, sugar and cholesterol and sedentary lifestyle are risk factors for hepatic steatosis. Such factors have an impact on lipid metabolism related to liver diseases, which include decreased conversion of cholesterol to bile acids; increased hydrolysis of cholesterol esters to free cholesterol [29]; increased endogenous cholesterol synthesis [30]; increased absorption of cholesterol-rich lipoproteins [29]; and decreased cholesterol excretion [31]. Excess cholesterol influences the fluidity of the membrane, affecting the function of its proteins [32]. It is also worth mentioning that this excessive consumption activates Kupffer cells and hepatic stellate cells, exacerbating liver inflammation, increasing extracellular matrix synthesis and eventually accelerating progress for NASH [33, 34].
