Non-alcoholic fatty liver disease (NAFLD), a hepatopathy syndrome affecting both adults and children, is characterized by a range of liver conditions affecting people who consume little to no alcohol, including simple fatty liver, steatohepatitis, fatty liver fibrosis, and cirrhosis. NAFLD can progress from simple fatty liver through nonalcoholic steatohepatitis (NASH) to liver fibrosis, and even lead to end-stage liver diseases, such as, liver cirrhosis, hepatocellular carcinoma (HCC), or liver failure.
According to previous research, NAFLD is one of the most significant causes of liver disease worldwide and will probably emerge as the leading cause of end-stage liver disease in the coming decades.
Liver biopsy is the gold standard for NASH diagnosis, which a very invasive method. Patients with NAFLD may sometimes have normal level of liver enzymes. Ultrasound can detect fatty liver but cannot detect fibrosis or inflammation. Even with a successful diagnosis, there is no approved treatment for NAFLD. Current research is not only aimed at the development of NASH therapeutic drugs, but new diagnostic methods - many pharmaceutical and biotechnology companies are investing heavily in NAFLD and NASH research.
Genome-wide association studies have been used to determine genetic modifiers of disease severity and progression. This has led to the identification of I148M polymorphism in patatin-like phosholipase domain-containing 3 (PNPLA3) gene as a strong modifier of NASH and progressive hepatic injury. Additional genetic variants have been implicated in the progression of NAFLD towards fibrosing NASH, through mechanisms such as insulin signaling, oxidative stress, and fibrogenesis.
Figure 1. Worldwide estimated prevalence of NAFLD and distribution of PNPLA3 genotypes.3
Animal models play an important role in elucidating the pathophysiological mechanisms of non-alcoholic fatty liver disease (NAFLD) and the development of new drugs. Preclinical studies require the application of different animal models based on the specific NAFLD phenotype as studied. The preclinical animal models of NAFLD and NASH can be divided into four categories: diet-induced models, chemically induced models, genetically modified models, and composite (combined) models.
1. Diet-Induced Models
As the most common NAFLD animal model, the diet-induced model is pathologically similar to development of human NAFLD. The diet-induced fatty liver model is established by feeding animals with high-fat and high-sugar diets, leading to overnutrition – from excessive amounts of lipids, cholesterol, and/or carbohydrates in the food - which cannot be fully absorbed and utilized. This overnutrition results in lipids accumulation, further inflammatory changes, and fibrosis. Characteristics of different diet-induced models vary, but the long construction time before starting the study remains a primary challenge for using this type of model.
Table 1. Characteristics of Different Diet-induced Models1
| Model | Mouse Type | Obesity | Insulin Resistance (IR) | Dyslipidemia | Elevated Transaminase | Fatty Degeneration of Liver | Hepatic Fibrosis |
| High-fat Diet Model | Mouse | Y | Y | Y | Y | Y | - |
| Rat | Y | Y | Y | Y | Y | - | |
| high-sugar Diet Model | Mouse | - | Y | Y | N | Y | - |
| Rat | Y | Y | Y | N | Y | - | |
| High-fat & High-sugar Diet Model | Mouse | Y | Y | Y | Y | Y | Y |
| Rat | Y | Y | Y | Y | Y | Y | |
| Methionine & Choline Deficient Diet Model | Mouse | N | N | N | Y | Y | Y |
| Rat | N | N | N | Y | Y | Y |
Note: Y: Yes, N: No, (-): without report
2. Chemical-Induced Models
The combination of low-dose streptozotocin with a high-fat diet has been used to model NAFLD in mice, resulting in steatosis, inflammation, fibrosis, and even hepatocellular carcinoma. Alternatively, carbon tetrachloride (CCl4) can cause liver damage and be administered alone or with a high-fat diet to induce fatty liver or liver fibrosis. The main mechanism is that CCI4 induces an oxidative stress reaction in the liver, resulting in the continuous production and accumulation of harmful lipid and protein peroxidation products, a severe necrosis reaction, and the destruction of liver cell structure and function. This method has a relatively short induction period, but the pathogenesis, course changes, and histological morphology are quite different from those of human fatty liver, and these drugs may be highly toxic and may cause animal death.
3. Genetically Modified Models
The production and clearance of fat in liver cells is regulated by a variety of genes, of which, mutation, deletion, overexpression, or modification may affect fat metabolism - thereby forming fatty liver. Human genes associated with particular pathologies can be replicated across species by targeting analogous genes to develop this specific NAFLD animal model.
Table 2. Characteristics of Different Genetic NAFLD Models1
| Model | Obesity | Insulin Resistance (IR) | Dyslipidemia | Elevated Transaminase | Fatty Degeneration of Liver | Hepatic Fibrosis |
| Inherited Leptin Deficiency & Resistance Model | ||||||
| ob/ob mice | Y | Y | Y | Y | Y | N |
| db/db mice | Y | Y | Y | Y | Y | N |
| Zucker obese rat | Y | Y | Y | Y | Y | N |
| Mouse Models with Inherited Defects of Fatty Acid β-Oxidation | ||||||
| JVS mice | N | N | - | Y | Y | N |
| PPAR-α Knockout Model | Y | N | Y | N | Y | N |
| MTP Knockout Model | - | Y | - | Y | Y | N |
| AOX Knockout Model | N | N | - | - | Y | N |
| Other Genetic Models | ||||||
| ADK Knockout Model | N | - | - | - | Y | N |
| ArKO Mouse Model | Y | N | - | - | Y | N |
| CD36 Deficient Model | N | N | Y | - | Y | N |
| MC4R Knockout Model | Y | - | - | - | Y | - |
| SREBP-1a Overexpression Transgenic Model | N | Y | Y | - | Y | N |
| FLS Mouse Model | N | N | Y | Y | Y | - |
Note: Y: Yes, N: No, (-): without report
For the pathogenesis of NAFLD, the “two-hit hypothesis" theory is widely accepted. Among them, the "first hit" refers to the liver fatty degeneration caused by insulin resistance, while the "second hit" refers to factors such as oxidative stress, inflammatory factors, and endotoxins which drive progression to steatohepatitis.
Obese (ob/ob) mice have homozygous mutations of Ob(Lep), resulting in obesity and steatosis. Zucker (fa/fa) rats and db/db mice have mutations in the fa or db genes, respectively, which encode the leptin receptor. These mutations induce the dysfunction of leptin receptor, causing leptin resistance to develop a genetic phenotype similar to that of ob/ob mice. However, these models cannot transition spontaneously from hepatic steatosis to steatohepatitis – they must be combined with either diet or chemical substances to induce factors associated with the "second hit" and progression to the NASH phenotype.
Phosphatase and tensin homolog (PTEN) is a lipid phosphatase involved in the fatty acidβ-oxidation of hepatic cells and triacylglycerol synthesis, which plays the role as a negative regulator in signaling pathways such as apoptosis, cell proliferation and differentiation, and tumor formation. One study found that liver damage discovered in liver tissue-specific PTEN gene knockout mice is similar to that of human NASH. Such mice can successively develop steatohepatitis, liver fibrosis, and liver adenoma within 10 weeks of age, with up to 66% of the cohort pathology complicated with HCC. Peroxisome proliferator-activated receptor alpha (PPARA) is a transcription factor involved in the regulation of mitochondrial and peroxisomal β-oxidation gene transcription in the liver and can regulate ATP production. Microsomal triglyceride transfer protein (MTP, MTTP) is a key enzyme for β-oxidation of mitochondrial fatty acids. Alternative oxidase (AOX) is the rate-limiting enzyme for β-oxidation of long-chain fatty acids in peroxisomes and can produce hydrogen peroxide. The modification of these genes can affect the β-oxidation of fatty acids and be used to develop NAFLD models.
Methionine adenosyltransferase 1A (MAT1A), a liver-specific rate-limiting enzyme for methionine metabolism, which can catalyze the synthesis of S-Adenosyl-l-methionine - a major methyl donor in the liver. MAT1A knockout (KO) mice exhibit both reduced levels of antioxidants (such as glutathione) and lowered expression of genes that participate in the liver lipid oxidation process.
Apoe (apolipoprotein E) gene knockout mice are commonly used in atherosclerosis (AS) studies. Interestingly, Apoe KO mice which are fed a high-fat, high-cholesterol diet develop NASH phenotypes, such as steatosis, inflammation, and fibrosis.
Methods of gene editing and chemical induction expedite model development, but the resulting models may be independent of disease induction mechanisms.
4. Composite (Combined) Models
None of the previous three models can completely simulate the pathogenesis of human NAFLD, and there are also phenotypic differences between the respective models and the human traits presented. Many researchers have combined the genetic modification model with diet or drug induction to produce a composite model, which makes the phenotype and pathogenesis of the model closer to that of human NAFLD. Composite models can more accurately reflect the progression of the disease development - from simple fatty liver to NASH, and progression to liver fibrosis.
Models with typical NAFLD histological changes: ob/ob mice + methionine-choline deficient (MCD) diet; db/db mice + methionine choline deficient (MCD) diet, Zucker rats + methionine choline deficient (MCD) diet / high fat diet.
The db/db mouse + MCD diet model is more severe than the ob/ob mouse + MCD diet model in both inflammation and fibrosis around the cells, and also demonstrates a significantly shortened induction period - which has led to predominate use of the db/db mouse + MCD diet model.
Despite the relatively complicated modeling process, a composite model can maximize simulation of human NAFLD, to develop more remarkable pathological changes.
The above fatty liver animal models prepared by different methods varied in their phenotypes and formation pathologies. In order to optimize the application of models, factors like research targets and corresponding complications should be taken into serious consideration. Experimental models that use simple methods to accomplish high similarity to human NAFLD, are highly recommended to demonstrate high success rate, low animal mortality, short development time, and good repeatability. However, the NAFLD/NASH models mentioned in the article are not exhaustive.
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Alongside the aging of society and the prevalence of obesity, the incidence of metabolic diseases is increasing yearly - which is a serious threat to human health. The most common metabolic diseases include obesity, type 2 diabetes caused by insulin resistance, and non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH).
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