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Challenges Encountered in NAFLD Animal Models: History
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Subjects: Medicine, Research & Experimental
Contributor: Mohammed Alshawsh

Several experimental in vivo animal models for NAFLD/NASH have been established. However, no reproducible experimental animal model displays the full spectrum of pathophysiological, histological, molecular, and clinical features associated with human NAFLD/NASH progression. Although methionine-choline-deficient (MCD) diet and high-fat diet (HFD) models can mimic histological and metabolic abnormalities of human disease, respectively, the molecular signaling pathways are extremely important for understanding the pathogenesis of the disease.

  • animal models
  • non-alcoholic fatty liver disease
  • liver fibrosis
  • signaling pathway
  • high-fat diet
  • methionine-choline-deficient diet

1. Introduction

As a term, non-alcoholic fatty liver disease (NAFLD) was proposed to describe the histopathological fatty changes in hepatocytes in which alcohol consumption is not involved as the etiological inducer [1]. In general, NAFLD embraces several forms of liver disorders involving fat disposition in hepatocytes ranging from the simple steatosis to the advanced stage, namely, non-alcoholic steatohepatitis (NASH) [2]. Moreover, NAFLD is known as the hepatic manifestation of metabolic syndrome since it is associated with metabolic disorders, such as obesity, type 2 diabetes mellitus, dyslipidemia, and insulin resistance [1]. NASH is characterized by the finding of hepatic steatosis with inflammation due to the excessive disposition of fats in hepatocytes, which makes hepatocytes predisposed to oxidative stress and the subsequent inflammatory and fibrosis cascades [3]. However, obesity or simple hepatic steatosis is normally not sufficient to develop inflammation and fibrosis, thus the “second-hit” hypothesis was proposed to further overstate the liver injury [4]. On the other hand, NAFLD is considered a multisystem disease as it is significantly related to extra-hepatic complications, including type 2 diabetes, chronic kidney disease [5], cardiovascular disease [6], and even neurological diseases [7]. These complications make it difficult to develop a comprehensive experimental model that fully mimics the underlying mechanisms of NAFLD. Depending on the study objectives, many models have been developed to elucidate the pathophysiological mechanisms of NAFLD, including genetically modified animals, such as leptin deficiency (ob/ob) mice [6], leptin receptor (LepR) deficiency (db/db) mice [8], KK-Ay mice [9], phosphatase and tensin homolog (PTEN)-deficient mice [10], CD36-deficient mice [11], peroxisome proliferator-activated receptor alpha (PPAR-α) knockout mice [12], acyl CoA oxidase (AOX)-null mice [13], methionine adenosyltransferase-1A (MAT1A)-null mice [14], nuclear factor erythroid 2-related factor 2 (Nrf2)-deficient mice [15], Zucker (fa/fa) rats [16], Otsuka Long-Evans Tokushima Fatty (OLETF) rats [17], and Koletsky f/f rats [18]. Furthermore, NAFLD can be induced by feeding mice different kinds of diets, including a high-fat diet (HFD) [19], high-carbohydrate diet (HCD) [20], methionine- and choline-deficient (MCD) diet [21], and fast food (FF) diet [22]. Furthermore, to reach the NASH and fibrosis stage, some models need to be triggered by a second stimulus or agent (“second hit”), such as tunicamycin [23], dexamethasone [24], and carbon tetrachloride (CCl4) [25]. It was found that male C57BL/6J mice fed a high-fat, -sucrose, and -cholesterol diet with high fructose or glucose water and injected with CCl4 showed the closest similarity to the human NAFLD pattern [26]. However, the most common dietary animal models used to elucidate the molecular and cellular progression of NAFLD either induce hepatotoxicity with MCD or over-nutrition disorders with HFD [4] as both cover most NAFLD manifestations. Although a significant number of studies regarding the cellular and molecular pathogenicity of NAFLD/NASH have been conducted on those two dietary animal models, gene expression could provide an extra essential approach to elucidate the complex pathogenesis of NAFLD. 

2. Challenges Encountered in NAFLD Animal Models

In humans, NAFLD is the outcome of a complex of genetic and environmental factors [9]. Since NAFLD is a polygenic disease, a full understanding of its pathogenicity has still been limited because of the genetic heterogeneity within populations, the suppression of a certain gene by another [27], and ethical issues regarding exposing humans to the whole experimental approaches and techniques during the study of NAFLD compared to what animals are exposed to [28]. For such reasons, several animal models were developed to achieve a better understanding of NAFLD. Firstly, relative similarities regarding physiological, metabolic, and anatomical features exist between humans and animals. Secondly, animal models provide a valuable opportunity to study the pathogenesis of NAFLD in a single homogeneous population since the effects of variations in age, genetic heterogeneity, gender, and diet are minimized in animal models [27]. In addition, the safer and easier collection of liver biopsies is an advantage of animal models over humans [28]. Nonetheless, an animal model that successfully reflects human NASH should feature the pathological profile of NASH, such as steatosis, inflammation, and fibrosis [3]. The latter fact directs us to another challenge: how to distinguish between bland steatosis and steatohepatitis. Simple steatosis and steatohepatitis share similar metabolic determinants, which supports the idea of looking for clear-cut metabolic boundaries to distinguish steatosis from steatohepatitis [28] and which animal model could solve such a challenge or achieve the transition from simple steatosis to steatohepatitis exactly as it develops in humans [28]. It has been reported that bland steatosis is well recognized by the presence of fatty infiltration while steatohepatitis involves the presence of both fatty infiltration and inflammation [29].
Regardless of the pathogenesis of NASH, over-nutrition is the critical factor responsible for the development of NAFLD since it leads to the most common metabolic disorders, namely, insulin resistance, glucose intolerance, obesity, and dyslipidemia, which are considered risk factors for the establishment of NASH [28]. Simple steatosis usually develops after insulin resistance, which results in a reversible cumulative disposition of fat in hepatocytes due to increases in lipid delivery to hepatocytes and the uptake of lipids by hepatocytes, an increase in the biosynthesis of triglycerides and fatty acids in the cytoplasm of hepatocytes, failure in the biosynthesis of very-low-density lipoprotein, and the export of triglycerides and the impairment in β-oxidation of hepatic mitochondria. To achieve the transition of steatosis to NASH, the combination of oxidative stress, lipid peroxidation, cell death, and proinflammatory cytokine-mediated liver injury constitutes the second hit [27]. Therefore, many etiological pathways are involved in the development of NASH, including mainly oxidative stress, lipid peroxidation, and inflammation [30]. Although the genetically altered ob/ob mice is the most common model used for studying obesity and NAFLD, it does not acquire NASH spontaneously and requires a second hit with lipopolysaccharide to trigger inflammatory events [31].
HFD- and MCD-fed animal models were found to be appropriate models for the study of NAFLD/NASH for humans since the HFD-fed animal model can mimic the metabolic abnormalities of NAFLD and other spectra of oxidative stress and inflammation but is unable to reach advanced stages, such as fibrosis and cirrhosis [4]. The MCD-fed animal model provides the histological hallmark of NASH because of its vulnerability to transition from simple steatosis to steatohepatitis and can reach fibrosis stages [4]. Furthermore, the HFD model develops insulin resistance [32] unlike the MCD-fed model, which shows lower insulin resistance levels [33]. Further, the HFD-fed model cannot mimic the whole features of histological changes that are associated with NASH as in humans while the MCD model fails to establish the transition from NAFLD (simple steatosis) to the advanced stage (NASH) [34]. Interestingly, the histological changes in the liver can be recovered among HFD-fed animals but not among MCD-fed animals after stopping the tested diet [35]. Table 1 shows a comparison between the diet models. However, the effects may differ depending on the mice strains, for example, feeding MCD to C57BL/6 mice [36], FVB/NJ mice [33], and db/db mice [37] resulted in noticeably lower serum insulin levels compared to chow diet-fed mice, but db/m mice showed a slight increase in serum insulin levels [37]. Moreover, to emphasize the important difference between intracellular oxidative stress caused by different types of diet, a recent study compared the mitochondrial (mt)DNA content between two mice models. A significantly lower mtDNA copy number was found in the MCD group compared to the HFD group, which could be attributed to the upregulation of both mitochondrial biogenesis and degradation-related genes. Interestingly, after stopping feeding, the MCD-fed group showed irreversible and consistently low levels of mtDNA, unlike the HFD group, which recovered to similar levels to those observed in the control chow-fed mice [38]. This finding justifies the increase in body weight with time and fat accumulation without fibrosis among the HFD group while a decrease in body weight and steatohepatitis with fibrosis was observed among the MCD group [35]. In general, high liver steatosis results in a higher average mtDNA copy number [39] while high liver steatohepatitis results in lower mtDNA copy numbers [40].
Table 1. A comparison between the NAFLD model mice fed MCD and HFD.
Comparison HFD * MCD ** HFD & MCD ***
Body weight Higher Lower No change
Liver-to-body weight ratio No change Slightly lower Higher
Serum biomarkers TC Higher Lower Lower
TG Slightly lower Lower Lower
AST Lower Higher NA
Glucose Higher Lower Slightly higher
Insulin Higher Lower Lower
Steatosis Higher Higher (but not as high as HFD) Higher
Fibrosis No change Higher No change
Inflammation lobular Slightly higher Higher Higher
Hepatocellular ballooning Slightly higher Slightly higher No change
* Compared (HFD 60% of energy as fat) to a standard chow diet as control ([22]); ** Compared to a standard chow diet as the control ([33][35][36]); *** Compared to an L-amino acid rodent diet as the control ([41]). TC: total cholesterols; TG: total triglycerides; AST: alanine aminotransferase; NA: not available.

This entry is adapted from the peer-reviewed paper 10.3390/molecules27030858

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