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

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

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 agents that can prevent or revert diseases [36].

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. In these models, the disease can be induced through specific diets or with the use of chemical substances and may result in non-alcoholic hepatic steatosis [37–39].

Despite the diversity of existing models, it is of utmost importance that they can replicate the histological patterns and pathophysiological mechanisms characteristic of each stage of NAFLD in humans, develop comorbidities associated with this disease such as increase of body weight, peripheral resistance to insulin, dyslipidemia, release of adipokines by adipose tissue, among others [40].

Mice and rats are mammals belonging to the Muridae family and the Murinae subfamily, order Rodentia and genus of the mouse *Mus*, whose scientific name *Mus musculus/domesticus* and the rat of the genus *Rattus* and the species *Rattus Norvegicus/rattus*. Both are heavily used in research due to their practical handling and playback performance in short period of time. Its lineage classification may be as genetically variable (heterozygous) termed outbreed or genetically defined inbreed. Outbreds are non-consanguineous and heterogeneous, which brings them closer to representing natural populations, larger litters and lower mortality rate, for example the Swiss colony (NIH, Webster, ICR and CD-1) [41]. Conversely, inbreds are consanguineous and isogenic, from crossbreeding between siblings which determine them to be identical, such as C57BL/ J6, BALB/ C, C3H, FVB, 129, DBA and CBA [42, 43].

Controlled expression of some genes results in the animal model called transgenic, they are susceptible to genome changes with specific DNA fragments, may have a mutated or increased gene, and result in a genetic modification that is transmissible to their offspring. "Knockout" animals, i.e. animals having a knockout gene, have a total or partial sequence of a withdrawn gene, contributing to determine a pathology. And "knock-in" animals acquire a total or partial sequence of a gene introduced in their genome predisposing it to a certain pathology by excess of the gene product [44, 45].

The ob/ob model behaves similarly to the methionine and choline deficiency models, since they affect oxidative stress, lipid peroxidation, and cell death. They are characterized by a chronic subclinical inflammatory condition with constant release of proinflammatory cytokines, monocyte infiltration of reactive oxygen species (ROS), oxidation of lipid molecules, cholesterol and proteins present in low density lipoprotein (LDL). This is in addition to mitochondrial DNA damage (measured by the mitochondrial levels of 8-hydroxy-2′-deoxyguanosine) and reduced expression of the DNA mismatch repair enzyme [51, 52]. Lipid peroxidation of polyunsaturated fatty acids generates by-products of toxic aldehydes, including malondaldehyde and hydroxynonenal, which are more persistent than ROS and damage more distant intracellular organelles which can cause cell death. These products activate fibrous hepatic

**Genetic alteration Characteristics Considerations "Second hit": NASH**

Obesity, IR, and NAFLD 3 months is confirmed

Accumulation of fat at liver 1 week presented

**model**

C57BL/J6 SD C57BL/J6

C57BL/J6 Wistar C57BL/J6 Wistar Sprague Dawley

30 weeks 48 weeks 3 months

4 months 5 weeks 8 weeks 4 weeks 4 months

**Follow-up Obesity NAFLD IR NASH**

Yes Yes Yes

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

Yes Yes Yes Yes Yes No Yes No

Yes No Yes No Yes

Hepatotoxic stimuli, specifics diets

Diet HFD or MCD

Diet MCD, a HFD or use of endotoxin

Not necessary

Yes Yes No 93

No Yes No No No

Yes Yes Yes

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

Yes Yes Yes Yes Yes

C57BL/J6 25 weeks Yes Yes Yes Yes

10–12 weeks is confirmed

NAFLD. Features similar to Nafld in humans.

Altered gene expression of lipid metabolism.

microvesicular steatosis. At 2 months, livers show extensive steatosis.

NAFLD.

stellate cells and are chemotactic for neutrophils [53, 54].

Hyperphagia, Obesity, diabetes, IR and NAFLD

Obesity, hyperphagia, hyperinsulinemia, hyperglycaemia and NAFLD

**Table 2.** Characteristics of mouse genetic models for the development of NAFLD.

**Diet Concentration/nutrient Animal** 

30% fructose/glicose/sacarose

**Table 1.** Characteristics of obesity/NAFLD diet composition in various animal models.

15% +1% 30% +2% 49%

70% fructose 65% sacarose 70% sacarose

FF 40% HCD + 2% cholesterol e fructose drink

HCD 30% fructose

HFD SFAs +Cholesterol

**Genetic models**

*ob ob* Absence of the leptin gene

*db db* Deficient in leptin receptor

ACOX knockout mice for the enzyme acylcoenzyme A oxidase

melanocortin receptor 4

KK-AY knockout for

Animals with genetic obesity or induced obesity (**Table 1**) present non-alcoholic hepatic steatosis, either by genetic alteration (**Table 2**) and increased lipid synthesis as in *ob/ob* mice, the *db/db* rat, agouti (obese yellow); and environmental factors such as diets deficient in methionine or choline, rich in carbohydrates or lipids and can also be combined with genetic alterations [46].

#### **3.1. Genetic models**

#### *3.1.1. ob/ob mice*

The *ob/ob* mice are spontaneous, obese mutants that do not have the Leptin gene (ob) which is autosomal recessive located on chromosome 6. From 4 weeks of age they are able to triple the normal weight when they are exposed to open offer of food due to lack of satiety caused by the absence of the hormone leptin. They are hypothermic and unable to stay warm. Its characteristics go beyond obesity, including hyperphagia, diabetes and non-alcoholic hepatic steatosis that presents with 10–12 weeks of life. The *ob/ob* is considered a good model of obesity linked hepatic steatosis, since metabolism of lipids and carbohydrates is related to the amount of white adipose tissue (WAT), which is increased in obesity. WAT in abundance reflects on increased expression of TNF-alpha and lipolysis, whose fatty acids are released to the liver for deposition [47, 48]. In this model, for hepatic steatoectomy, regarded as a second stimulus or agent ("second hit" such as ischemia induction) specific drugs or methionine and choline-deficient diets, is needed [46, 49, 50].


**Table 1.** Characteristics of obesity/NAFLD diet composition in various animal models.

In these models, the disease can be induced through specific diets or with the use of chemical

Despite the diversity of existing models, it is of utmost importance that they can replicate the histological patterns and pathophysiological mechanisms characteristic of each stage of NAFLD in humans, develop comorbidities associated with this disease such as increase of body weight, peripheral resistance to insulin, dyslipidemia, release of adipokines by adipose

Mice and rats are mammals belonging to the Muridae family and the Murinae subfamily, order Rodentia and genus of the mouse *Mus*, whose scientific name *Mus musculus/domesticus* and the rat of the genus *Rattus* and the species *Rattus Norvegicus/rattus*. Both are heavily used in research due to their practical handling and playback performance in short period of time. Its lineage classification may be as genetically variable (heterozygous) termed outbreed or genetically defined inbreed. Outbreds are non-consanguineous and heterogeneous, which brings them closer to representing natural populations, larger litters and lower mortality rate, for example the Swiss colony (NIH, Webster, ICR and CD-1) [41]. Conversely, inbreds are consanguineous and isogenic, from crossbreeding between siblings which determine them to

Controlled expression of some genes results in the animal model called transgenic, they are susceptible to genome changes with specific DNA fragments, may have a mutated or increased gene, and result in a genetic modification that is transmissible to their offspring. "Knockout" animals, i.e. animals having a knockout gene, have a total or partial sequence of a withdrawn gene, contributing to determine a pathology. And "knock-in" animals acquire a total or partial sequence of a gene introduced in their genome predisposing it to a certain

Animals with genetic obesity or induced obesity (**Table 1**) present non-alcoholic hepatic steatosis, either by genetic alteration (**Table 2**) and increased lipid synthesis as in *ob/ob* mice, the *db/db* rat, agouti (obese yellow); and environmental factors such as diets deficient in methionine or choline, rich in carbohydrates or lipids and can also be combined with genetic alterations [46].

The *ob/ob* mice are spontaneous, obese mutants that do not have the Leptin gene (ob) which is autosomal recessive located on chromosome 6. From 4 weeks of age they are able to triple the normal weight when they are exposed to open offer of food due to lack of satiety caused by the absence of the hormone leptin. They are hypothermic and unable to stay warm. Its characteristics go beyond obesity, including hyperphagia, diabetes and non-alcoholic hepatic steatosis that presents with 10–12 weeks of life. The *ob/ob* is considered a good model of obesity linked hepatic steatosis, since metabolism of lipids and carbohydrates is related to the amount of white adipose tissue (WAT), which is increased in obesity. WAT in abundance reflects on increased expression of TNF-alpha and lipolysis, whose fatty acids are released to the liver for deposition [47, 48]. In this model, for hepatic steatoectomy, regarded as a second stimulus or agent ("second hit" such as ischemia induction) specific drugs or methionine and choline-deficient diets, is needed [46, 49, 50].

be identical, such as C57BL/ J6, BALB/ C, C3H, FVB, 129, DBA and CBA [42, 43].

pathology by excess of the gene product [44, 45].

substances and may result in non-alcoholic hepatic steatosis [37–39].

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

tissue, among others [40].

**3.1. Genetic models**

*3.1.1. ob/ob mice*

The ob/ob model behaves similarly to the methionine and choline deficiency models, since they affect oxidative stress, lipid peroxidation, and cell death. They are characterized by a chronic subclinical inflammatory condition with constant release of proinflammatory cytokines, monocyte infiltration of reactive oxygen species (ROS), oxidation of lipid molecules, cholesterol and proteins present in low density lipoprotein (LDL). This is in addition to mitochondrial DNA damage (measured by the mitochondrial levels of 8-hydroxy-2′-deoxyguanosine) and reduced expression of the DNA mismatch repair enzyme [51, 52]. Lipid peroxidation of polyunsaturated fatty acids generates by-products of toxic aldehydes, including malondaldehyde and hydroxynonenal, which are more persistent than ROS and damage more distant intracellular organelles which can cause cell death. These products activate fibrous hepatic stellate cells and are chemotactic for neutrophils [53, 54].


**Table 2.** Characteristics of mouse genetic models for the development of NAFLD.

#### *3.1.2. db/db mice and Zucker fa/fa rats and Koletsky f/f*

Obese Zucker fa/fa rats and the *db/db* mouse are deficient in leptin receptor function, resulting from mutations in the leptin gene which occur on chromosome 4. Quite close phenotypically to these models is the obese Koletsky *f/f* rat which has the leptin deficiency with reduced energy expenditure and neuropeptidergic alterations of the hypothalamus [55].

A higher fat supply leads to increased adiposity in adipose tissue, frequent stimulation of proinflammatory cytokines secretion, increased free fatty acids, insulin resistance, and lipolysis, with consequent increase in the transport of free fatty acids to the liver via the portal vein and increased intake of fatty acids. In the liver, hyperinsulinemia inhibits beta-oxidation, reducing the output of fatty acids, leading to unanticipated the accumulation of triglycerides in the cytoplasm of hepatocytes which hallmarks NAFLD. Both actions favor the accumulation of fat in the hepatocytes, a condition that promotes hepatic resistance to the action of insulin. Loss of insulin's ability to suppress hepatic glucose production aggravates overall insulin resistance and exacerbates the manifestation of metabolic syndrome components [66–69].

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

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95

The addition of cholesterol along with saturated fatty acids has shown to have a good disease predisposition, with progression of NAFLD to NASH. Savard et al. [70] tested the effect of this diet on the respective proportions of saturated fatty acids and cholesterol on the C57BL/J6 model for 30 weeks: control (4% fat and 0% cholesterol); high cholesterol[HC] (4% fat and 1% cholesterol); high fat[HF] (15% fat and 0% cholesterol); and high fat, high cholesterol[HFHC] (15% fat and 1% cholesterol). The animals treated with HCHC showed the highest weight gain, hepatic lipid content, inflammation of adipose tissue and reduction in adiponectin plasma levels, leading to NAFLD in a profound way, as they developed macrovesicular steatosis (grade 3) associated with inflammatory spots (grade 2) and peripheral fibrosis, with these effects being twice as

Similarly, adult Sprague Dawley rats fed HFD made with 30% lipids (lard and 2% cholesterol) were evaluated at 4, 8, 12, 16, 24, 36, 48 weeks intervals. At week 8 body weight and epididymal fat weight began to increase, which was associated with increased serum levels of free fatty acids, cholesterol and TNF-α, as well as the development of NAFLD fatty liver. Steatohepatitis occurred between weeks 12 and 48. Apparent hepatic fibrosis did not occur until week 24, and went from week 36 to 48 with insulin resistance reproducing the pathologi-

Obesity models induced by HFD consumption have also been characterized by inflammation in peripheral tissues as well as in hypothalamic areas critical for energy homeostasis, in an attempt to interrupt body weight control and glucose homeostasis [72]. According to Thaler et al. [73] unlike inflammation in peripheral tissues, which develops as a consequence of obesity, inflammatory signaling of the hypothalamus is confirmed in rats and mice within 1 to 3 days after initiation of HFD treatment, ie before substantial weight gain. In addition, both reactive glucose and markers suggestive of neuronal injury were evident in the arched hypothalamic nuclei of rodents in the first week of dietary feeding, leading to the knowledge that obesity is associated with neuronal injury in an area of the brain, suggesting a crucial aspect for the control of body weight [73]. Induction of FHD obesity in AFasKO mice with death receptor Fas deficiency (also known as CD95), specifically in adipocyte cells, were protected from adipose tissue inflammation and also from hepatic steatosis (more sensitive to insulin,

both at the level of the body and in the liver) and hepatic insulin resistance [74].

In C57BL/6 mouse fed with HF diet (49% of lipids) during gestation and/or lactation, or both, the presence of non-alcoholic fatty liver steatosis was verified by expression of protein-1c binding to the sterol regulatory element. There was an exacerbation of NAFLD phenotype in utero and during lactation, demonstrating the development of hepatic steatosis already in fetal life [75].

large as in the HC and HF groups [70].

cal sequence of events typical of human NASH [71].

Obese fa/fa Zucker rats fed with high fat diet (HFD) made with 60% of saturated lipids for 8 weeks, confirmed the occurance of hyperglycemia and hepatic steatosis. NADPH oxidase activity increased 2.5-fold leading to hepatic liver injury of the animals thus contributing to the progression of NASH [56].

#### *3.1.3. KK-ay mice and knockout for melanocortin receptor 4*

The KK-Ay genotype results from the crossing of KK diabetic rats with yellow coat and agouti background (Ay) [57], they develop obesity due to the antagonistic action of the agouti protein in the central nervous system capable of promoting alimentary hyperphagia and consequent obesity [58]. Changes in the genes of the agouti rat and in the encoding gene of the melanocortin receptor 4 (MC4R) are related to the involvement of the melanocortins system in the pathogenesis of NAFLD [59]. They present indicators of resistance to insulin and leptin, conditions that favor the appearance of hepatic steatosis. Also the genetic depletion of MC4R is associated with a severe obesity phenotype such as hyperphagia, hyperinsulinemia, hyperglycemia and hyperleptinemia [60]. To progress to NASH, a "second hit" is needed again as an example: diet MCD, a HFD or use of endotoxin [61].

#### *3.1.4. ACOX and knockout mice for the enzyme acyl-coenzyme A oxidase*

The enzyme acyl-coenzyme A oxidase-1 (ACOX 1) is the first enzyme of peroxisomal β-oxidation of long chain fatty acids. Peroxisomal and mitochondrial fatty acid beta-oxidation occuring in mice nullizygous for both peroxisome proliferator-activated receptor alpha and peroxisomal fatty acyl-CoA oxidase (ACOX−/−) exhibits a wide range of microvesicular steatohepatitis. These animals develop NAFLD due to the accumulation of fat generated by impaired β-oxidation [62]. At 1 week of age, the liver of the AOX−/− mice already presented microvesicular steatosis, which intensifies at 2 months of age to inflammatory infiltration [63]. However, when they are 6 to 8 months old, the liver of these mice exhibits reversal of steatosis from hepatocyte regeneration and also exhibits growth retardation [64].

#### **3.2. Animal models induced by diets**

#### *3.2.1. High fat diet (HFD)*

High fat diet (HFD) treated animals acquire obesity and also increased epididymal fat, hyperglycemia and insulin resistance, because these associated comorbidities are also widely used as a method of inducing hepatic steatosis, as they cause liver changes similar to that of human disease. Usually fat sources such as lard and soybean oil are used, different concentrations of lipids are taken into account in the preparation, varying in proportion from 40 to 60%. The most commonly used rodents are C57BL/6, Swiss, Sprague Dawley, Wistar and SHR [62, 65].

A higher fat supply leads to increased adiposity in adipose tissue, frequent stimulation of proinflammatory cytokines secretion, increased free fatty acids, insulin resistance, and lipolysis, with consequent increase in the transport of free fatty acids to the liver via the portal vein and increased intake of fatty acids. In the liver, hyperinsulinemia inhibits beta-oxidation, reducing the output of fatty acids, leading to unanticipated the accumulation of triglycerides in the cytoplasm of hepatocytes which hallmarks NAFLD. Both actions favor the accumulation of fat in the hepatocytes, a condition that promotes hepatic resistance to the action of insulin. Loss of insulin's ability to suppress hepatic glucose production aggravates overall insulin resistance and exacerbates the manifestation of metabolic syndrome components [66–69].

*3.1.2. db/db mice and Zucker fa/fa rats and Koletsky f/f*

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

*3.1.3. KK-ay mice and knockout for melanocortin receptor 4*

an example: diet MCD, a HFD or use of endotoxin [61].

and also exhibits growth retardation [64].

**3.2. Animal models induced by diets**

*3.2.1. High fat diet (HFD)*

*3.1.4. ACOX and knockout mice for the enzyme acyl-coenzyme A oxidase*

the progression of NASH [56].

Obese Zucker fa/fa rats and the *db/db* mouse are deficient in leptin receptor function, resulting from mutations in the leptin gene which occur on chromosome 4. Quite close phenotypically to these models is the obese Koletsky *f/f* rat which has the leptin deficiency with reduced

Obese fa/fa Zucker rats fed with high fat diet (HFD) made with 60% of saturated lipids for 8 weeks, confirmed the occurance of hyperglycemia and hepatic steatosis. NADPH oxidase activity increased 2.5-fold leading to hepatic liver injury of the animals thus contributing to

The KK-Ay genotype results from the crossing of KK diabetic rats with yellow coat and agouti background (Ay) [57], they develop obesity due to the antagonistic action of the agouti protein in the central nervous system capable of promoting alimentary hyperphagia and consequent obesity [58]. Changes in the genes of the agouti rat and in the encoding gene of the melanocortin receptor 4 (MC4R) are related to the involvement of the melanocortins system in the pathogenesis of NAFLD [59]. They present indicators of resistance to insulin and leptin, conditions that favor the appearance of hepatic steatosis. Also the genetic depletion of MC4R is associated with a severe obesity phenotype such as hyperphagia, hyperinsulinemia, hyperglycemia and hyperleptinemia [60]. To progress to NASH, a "second hit" is needed again as

The enzyme acyl-coenzyme A oxidase-1 (ACOX 1) is the first enzyme of peroxisomal β-oxidation of long chain fatty acids. Peroxisomal and mitochondrial fatty acid beta-oxidation occuring in mice nullizygous for both peroxisome proliferator-activated receptor alpha and peroxisomal fatty acyl-CoA oxidase (ACOX−/−) exhibits a wide range of microvesicular steatohepatitis. These animals develop NAFLD due to the accumulation of fat generated by impaired β-oxidation [62]. At 1 week of age, the liver of the AOX−/− mice already presented microvesicular steatosis, which intensifies at 2 months of age to inflammatory infiltration [63]. However, when they are 6 to 8 months old, the liver of these mice exhibits reversal of steatosis from hepatocyte regeneration

High fat diet (HFD) treated animals acquire obesity and also increased epididymal fat, hyperglycemia and insulin resistance, because these associated comorbidities are also widely used as a method of inducing hepatic steatosis, as they cause liver changes similar to that of human disease. Usually fat sources such as lard and soybean oil are used, different concentrations of lipids are taken into account in the preparation, varying in proportion from 40 to 60%. The most commonly used rodents are C57BL/6, Swiss, Sprague Dawley, Wistar and SHR [62, 65].

energy expenditure and neuropeptidergic alterations of the hypothalamus [55].

The addition of cholesterol along with saturated fatty acids has shown to have a good disease predisposition, with progression of NAFLD to NASH. Savard et al. [70] tested the effect of this diet on the respective proportions of saturated fatty acids and cholesterol on the C57BL/J6 model for 30 weeks: control (4% fat and 0% cholesterol); high cholesterol[HC] (4% fat and 1% cholesterol); high fat[HF] (15% fat and 0% cholesterol); and high fat, high cholesterol[HFHC] (15% fat and 1% cholesterol). The animals treated with HCHC showed the highest weight gain, hepatic lipid content, inflammation of adipose tissue and reduction in adiponectin plasma levels, leading to NAFLD in a profound way, as they developed macrovesicular steatosis (grade 3) associated with inflammatory spots (grade 2) and peripheral fibrosis, with these effects being twice as large as in the HC and HF groups [70].

Similarly, adult Sprague Dawley rats fed HFD made with 30% lipids (lard and 2% cholesterol) were evaluated at 4, 8, 12, 16, 24, 36, 48 weeks intervals. At week 8 body weight and epididymal fat weight began to increase, which was associated with increased serum levels of free fatty acids, cholesterol and TNF-α, as well as the development of NAFLD fatty liver. Steatohepatitis occurred between weeks 12 and 48. Apparent hepatic fibrosis did not occur until week 24, and went from week 36 to 48 with insulin resistance reproducing the pathological sequence of events typical of human NASH [71].

Obesity models induced by HFD consumption have also been characterized by inflammation in peripheral tissues as well as in hypothalamic areas critical for energy homeostasis, in an attempt to interrupt body weight control and glucose homeostasis [72]. According to Thaler et al. [73] unlike inflammation in peripheral tissues, which develops as a consequence of obesity, inflammatory signaling of the hypothalamus is confirmed in rats and mice within 1 to 3 days after initiation of HFD treatment, ie before substantial weight gain. In addition, both reactive glucose and markers suggestive of neuronal injury were evident in the arched hypothalamic nuclei of rodents in the first week of dietary feeding, leading to the knowledge that obesity is associated with neuronal injury in an area of the brain, suggesting a crucial aspect for the control of body weight [73]. Induction of FHD obesity in AFasKO mice with death receptor Fas deficiency (also known as CD95), specifically in adipocyte cells, were protected from adipose tissue inflammation and also from hepatic steatosis (more sensitive to insulin, both at the level of the body and in the liver) and hepatic insulin resistance [74].

In C57BL/6 mouse fed with HF diet (49% of lipids) during gestation and/or lactation, or both, the presence of non-alcoholic fatty liver steatosis was verified by expression of protein-1c binding to the sterol regulatory element. There was an exacerbation of NAFLD phenotype in utero and during lactation, demonstrating the development of hepatic steatosis already in fetal life [75].

#### *3.2.2. High carbohydrate diet (HCD)*

Prolonged consumption of HCD causes obesity and non-alcoholic fatty liver disease (NAFLD), in addition to oxidative stress in the liver and insulin resistance. The most common sources of simple carbohydrates in the diet are fructose, glucose and sucrose, for example the addition of corn syrup (50–90% fructose) or refined sugar (50% fructose). Fructose is highly lipogenic and has been more widely used than sucrose and glucose, however, induction of chronic models in NAFLD requires longer treatment periods [76–78]. Fructose is primarily metabolized in the liver without the need for insulin, its phosphorylation consumes ATP and accumulates ADP thus stimulating the formation of uric acid and reactive oxygen species (ROS) which rapidly increases the synthesis and hepatic deposition of triglycerides leading to a fatty liver in rodents [79–81].

*3.2.3. Fast food diet (FF)*

and elevated levels of endotoxemia and glycemia [86].

**3.3. Combined models (genetic and environmental)**

exacerbation of inflammatory and profibrogenic responses [96].

is considered the second hit in NASH models [99].

when fed with the MCD diet [95].

Charlton et al. [93] proposed the mouse fast food diet model. C57BL/6 mice were fed for 25 weeks on a diet composed of 40% fat (12% saturated fatty acids, 2% cholesterol) and the water offered contained corn syrup, it was found that the animals developed obesity and resistance to insulin. I Other studies used the same model and also stated that the intake of FF diet in the form of emulsion for 6 weeks led to the onset of hepatic steatosis and inflammation,

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This diet model was very similar to the Western diet, since it presents high concentrations of fat, fructose and cholesterol, appearing as a good model related to human diets and with the

The use of the C57BL LDLr male mouse (low density lipoprotein receptor deficient) treated for 21 weeks with HFD, was associated with weight gain, macrovesicular steatosis and lobular inflammation. Inflammation in adipose tissue and liver provides a positive attenuation for studies of obesity and associated cardiometabolic diseases such as NAFLD and atherosclerosis [94]. *db/db* mice subjected to methionine and choline deficient diet (MCD) showed macrovesicular steatosis, and increased hepatic collagen type 1 mRNA levels in comparison to the control group. This outcome suggests an important model for the study of NASH, i.e. establishing obesity, diabetes, insulin resistance and dietary MCD results in steatohepatitis indicating leptin activity in liver fibrosis. In this way, an interesting comparison is made with the *ob/ob* model that is deficient in leptin which also develops steatohepatitis but not hepatic fibrosis

The verification in the obese and diabetic C57BI/6-A and KK-Ay models of diet treatment (MCD) for 8 weeks demonstrated that KK-Ay rats exhibited increased susceptibility to steatohepatitis and inflammatory infiltration as well as increased levels of TNFα mRNA and lipid peroxidation in the liver where hypoadiponectinemia probably played a key role in the

The treatment of the KK-Ay (MC4R) model with hyperlipidic diets at concentrations of 35 and 60% of lipids derived from soybean oil and lard, develop severe hepatic steatosis and show liver changes in the lipogenic gene profile [97]. Another study has shown that the exposure of these animals to 60% HFD for 1 year leads to the appearance of more severe forms of NAFLD such as NASH, fibrosis and hepatocellular cancer, as well as leading to the development of systemic metabolic alterations very similar to those observed in humans [98]. This same model was used for a study of hepatic lesions induced by D-galactosamine/lipopolysaccharide LPS (GalN/LPS) endotoxin, there were significant increases in the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the blood, apoptotic and necrotic changes in hepatocytes and/or showed a high degree of lethality. GalN/LPS-induced liver injury was more pronounced in KK-Ay obese than in the control group [61]. LPS is a key component of many bacteria present in the microbiota, plays a central role in innate immune responses and

capacity to induce obesity, NAFLD among other comorbidities in rodents.

Treatment with a 30% fructose, glucose and sucrose diet for 4 months in Sprague Dawley rats demonstrated that such treatment induced metabolic syndrome, intrahepatic accumulation of uric acid and triglycerides, increased MCP-1 and TNF-alpha, as well as hepatic steatosis [82]. This simple carbohydrate intake is associated with a greater translocation of the endotoxin from the intestine to the portal vein, ROS formation in the liver (due to the greater oxidation of fatty acids in the hepatocytes) and induction of the TNF-alpha factor. TNF-alpha has been associated with the development of NAFLD because it is involved in the dysregulation of hepatic lipid metabolism and in insulin signaling [83, 84].

The C57BL/6 mouse model fed a high calorie and sucrose diet (65%) for 8 weeks showed obesity, insulin resistance and macrovesicular steatosis [85]. In another study with C57BL/J6 mice with TNF-alpha receptor 1/− TNFR1/− (sterol regulatory element-binding protein 1) were protected against the onset of hepatic steatosis and also the insulin resistance induced by HCD with fructose (30%), this result was associated with increased phosphate levels of adenosine monophosphate-activated protein kinase (AMPK) and protein kinase (AKT), decreased expression of SREBP-1, fatty acid synthesis in the liver and decreased levels of retinol binding protein (RBP4) that behaved differently from the control group [86].

As with Wistar rats, the effect of the sucrose diet (70%) when compared to starch (70%) for 28 days resulted in a significant difference in the group that received sucrose, thus the amount of hepatic fat and serum fructosamine concentration was increased in sucrose diet group and in both hepatic steatosis confirmed in the two groups [87]. However, when using HCD with 70% fructose for 5 weeks, obesity, elevated levels of hepatic triglycerides, macrovesicular steatosis, lobular inflammation (as were observed [88]. Another report with the same animal model in (HCD) added 10% of fructose (corn syrup) in drinking water and evaluated at 7, 14 and 21 days. At the start of treatment, an increase in triglycerides,oxidative stress and hepatic sensitivity to hyperinsulinemia (β-cell reaction) were was observed in serum and liver, suggesting that this increase is related to metabolism that occurred in the liver and probably in the adipose tissue as well [89].

Epidemiological studies have shown that increasing the intake of fructose mono- or disaccharides by humans is a considerable risk factor for NALFD [90, 91], and it is estimated that patients with fructosemia present a fructose consumption about 2–3 times higher than healthy individuals or with other liver diseases [92]. These data stimulated the study of experimental models of NAFLD induced by fructose.

#### *3.2.3. Fast food diet (FF)*

*3.2.2. High carbohydrate diet (HCD)*

Prolonged consumption of HCD causes obesity and non-alcoholic fatty liver disease (NAFLD), in addition to oxidative stress in the liver and insulin resistance. The most common sources of simple carbohydrates in the diet are fructose, glucose and sucrose, for example the addition of corn syrup (50–90% fructose) or refined sugar (50% fructose). Fructose is highly lipogenic and has been more widely used than sucrose and glucose, however, induction of chronic models in NAFLD requires longer treatment periods [76–78]. Fructose is primarily metabolized in the liver without the need for insulin, its phosphorylation consumes ATP and accumulates ADP thus stimulating the formation of uric acid and reactive oxygen species (ROS) which rapidly increases the synthesis and hepatic deposition of triglycerides leading to a fatty liver in rodents [79–81].

Treatment with a 30% fructose, glucose and sucrose diet for 4 months in Sprague Dawley rats demonstrated that such treatment induced metabolic syndrome, intrahepatic accumulation of uric acid and triglycerides, increased MCP-1 and TNF-alpha, as well as hepatic steatosis [82]. This simple carbohydrate intake is associated with a greater translocation of the endotoxin from the intestine to the portal vein, ROS formation in the liver (due to the greater oxidation of fatty acids in the hepatocytes) and induction of the TNF-alpha factor. TNF-alpha has been associated with the development of NAFLD because it is involved in the dysregulation of

The C57BL/6 mouse model fed a high calorie and sucrose diet (65%) for 8 weeks showed obesity, insulin resistance and macrovesicular steatosis [85]. In another study with C57BL/J6 mice with TNF-alpha receptor 1/− TNFR1/− (sterol regulatory element-binding protein 1) were protected against the onset of hepatic steatosis and also the insulin resistance induced by HCD with fructose (30%), this result was associated with increased phosphate levels of adenosine monophosphate-activated protein kinase (AMPK) and protein kinase (AKT), decreased expression of SREBP-1, fatty acid synthesis in the liver and decreased levels of retinol binding

As with Wistar rats, the effect of the sucrose diet (70%) when compared to starch (70%) for 28 days resulted in a significant difference in the group that received sucrose, thus the amount of hepatic fat and serum fructosamine concentration was increased in sucrose diet group and in both hepatic steatosis confirmed in the two groups [87]. However, when using HCD with 70% fructose for 5 weeks, obesity, elevated levels of hepatic triglycerides, macrovesicular steatosis, lobular inflammation (as were observed [88]. Another report with the same animal model in (HCD) added 10% of fructose (corn syrup) in drinking water and evaluated at 7, 14 and 21 days. At the start of treatment, an increase in triglycerides,oxidative stress and hepatic sensitivity to hyperinsulinemia (β-cell reaction) were was observed in serum and liver, suggesting that this increase is related to

Epidemiological studies have shown that increasing the intake of fructose mono- or disaccharides by humans is a considerable risk factor for NALFD [90, 91], and it is estimated that patients with fructosemia present a fructose consumption about 2–3 times higher than healthy individuals or with other liver diseases [92]. These data stimulated the study of experimental

metabolism that occurred in the liver and probably in the adipose tissue as well [89].

models of NAFLD induced by fructose.

hepatic lipid metabolism and in insulin signaling [83, 84].

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

protein (RBP4) that behaved differently from the control group [86].

Charlton et al. [93] proposed the mouse fast food diet model. C57BL/6 mice were fed for 25 weeks on a diet composed of 40% fat (12% saturated fatty acids, 2% cholesterol) and the water offered contained corn syrup, it was found that the animals developed obesity and resistance to insulin. I Other studies used the same model and also stated that the intake of FF diet in the form of emulsion for 6 weeks led to the onset of hepatic steatosis and inflammation, and elevated levels of endotoxemia and glycemia [86].

This diet model was very similar to the Western diet, since it presents high concentrations of fat, fructose and cholesterol, appearing as a good model related to human diets and with the capacity to induce obesity, NAFLD among other comorbidities in rodents.

#### **3.3. Combined models (genetic and environmental)**

The use of the C57BL LDLr male mouse (low density lipoprotein receptor deficient) treated for 21 weeks with HFD, was associated with weight gain, macrovesicular steatosis and lobular inflammation. Inflammation in adipose tissue and liver provides a positive attenuation for studies of obesity and associated cardiometabolic diseases such as NAFLD and atherosclerosis [94].

*db/db* mice subjected to methionine and choline deficient diet (MCD) showed macrovesicular steatosis, and increased hepatic collagen type 1 mRNA levels in comparison to the control group. This outcome suggests an important model for the study of NASH, i.e. establishing obesity, diabetes, insulin resistance and dietary MCD results in steatohepatitis indicating leptin activity in liver fibrosis. In this way, an interesting comparison is made with the *ob/ob* model that is deficient in leptin which also develops steatohepatitis but not hepatic fibrosis when fed with the MCD diet [95].

The verification in the obese and diabetic C57BI/6-A and KK-Ay models of diet treatment (MCD) for 8 weeks demonstrated that KK-Ay rats exhibited increased susceptibility to steatohepatitis and inflammatory infiltration as well as increased levels of TNFα mRNA and lipid peroxidation in the liver where hypoadiponectinemia probably played a key role in the exacerbation of inflammatory and profibrogenic responses [96].

The treatment of the KK-Ay (MC4R) model with hyperlipidic diets at concentrations of 35 and 60% of lipids derived from soybean oil and lard, develop severe hepatic steatosis and show liver changes in the lipogenic gene profile [97]. Another study has shown that the exposure of these animals to 60% HFD for 1 year leads to the appearance of more severe forms of NAFLD such as NASH, fibrosis and hepatocellular cancer, as well as leading to the development of systemic metabolic alterations very similar to those observed in humans [98]. This same model was used for a study of hepatic lesions induced by D-galactosamine/lipopolysaccharide LPS (GalN/LPS) endotoxin, there were significant increases in the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the blood, apoptotic and necrotic changes in hepatocytes and/or showed a high degree of lethality. GalN/LPS-induced liver injury was more pronounced in KK-Ay obese than in the control group [61]. LPS is a key component of many bacteria present in the microbiota, plays a central role in innate immune responses and is considered the second hit in NASH models [99].
