Long-term profile of serological biomarkers, hepatic inflammation, and fibrosis in a mouse model of non-alcoholic fatty liver disease

Highlights

• Mouse models of NAFLD are useful tools for understanding the pathogenesis and progression of NAFLD.

• Time-dependent changes in serum lipids and biochemical markers hepatic inflammation, and fibrosis were investigated.

• The changes in AST, ALT, LDH, and TB showed an inverse U-shaped curve in the CDAHFD-fed mice for 12 weeks.

• Inflammatory foci and hepatic fibrosis markedly increased after 6 weeks of CDAHFD feeding.

Abstract

Non-alcoholic fatty liver disease (NAFLD) can be typically classified into two subgroups: non-alcoholic fatty liver and non-alcoholic steatohepatitis. Mouse models of NAFLD are useful tools for understanding the pathogenesis and progression of NAFLD and for developing drugs for its treatment. Here, we investigated the time-dependent changes in serum lipids and biochemical markers of hepatic function, hepatic inflammation, and fibrosis in mice fed a normal diet (ND) or a NAFLD diet (choline deficient, L-amino acid-defined, high-fat diet; CDAHFD) for 12 weeks. CDAHFD-fed mice showed significantly reduced serum levels of total cholesterol, triglyceride, and high-density lipoprotein cholesterol throughout the treatment period compared with ND-fed mice. The changes in aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and total bilirubin showed an inverse U-shaped curve in the CDAHFD-fed mice. The serum alkaline phosphatase levels decreased in both ND- and CDAHFD-fed mice in a time-dependent manner. Furthermore, CDAHFD-fed mice showed a significant increase in the number of inflammatory foci and hepatic fibrosis at 6–12 weeks, although inflammatory foci and hepatic fibrogenesis were observable at relatively early stages as well (1–4 weeks). In conclusion, the long-term profile of serological biomarkers, hepatic inflammation, and fibrosis in CDAHFD-fed mice identified in this study may provide a better understanding of NAFLD pathogenesis.

Introduction

Non-alcoholic fatty liver disease (NAFLD) can be typically classified into two subgroups: non-alcoholic fatty liver (NAFL; simple steatosis) and non-alcoholic steatohepatitis (NASH). NAFL is characterized by hepatic steatosis with no hepatocellular damage and inflammation, while NASH is characterized by the presence of hepatocellular damage, hepatic steatosis, inflammation, and fibrosis. NASH, especially with fibrosis, may lead to liver cirrhosis, which is associated with increased risk of developing hepatocellular carcinoma (HCC) (Starley et al., 2010; Vernon et al., 2011).

NAFLD mouse models are useful tools for understanding the pathogenesis and progression of NAFLD and for developing drugs for its treatment (Farrell et al., 2019; Hansen et al., 2017; Lau et al., 2017). Several NAFLD mouse models have been reported: 1) diet-induced models developed mainly through feeding the mice a high-fat diet; 2) nutrient-deficient diet-induced models developed through feeding mice a choline and/or methionine-deficient diet (with or without high-fat diet); 3) chemical-induced models developed through the injection of liver-targeted chemotoxins (with or without high-fat diet), such as carbon tetrachloride (CCl₄) or streptozotocin; and 4) genetic models developed through use of high-fat or nutrient-deficient diet-fed genetic mice, such as ob/ob, db/db, and foz/foz mice (Farrell et al., 2019; Hansen et al., 2017; Lau et al., 2017).

Among these animal models, mice fed methionine and choline deficient (MCD) diets are broadly used as NAFLD mouse models. MCD diet-fed mice exhibit hepatic damage and inflammation at a relatively early stage (1–3 weeks), compared to the other animal models (Hansen et al., 2017; Lau et al., 2017). Choline deficiency disrupts liver functions, causing altered lipid metabolism, phosphatidylcholine synthesis, mitochondrial dysfunction, and reactive oxygen species-mediated DNA damage, which are closely associated with NAFLD development (Corbin and Zeisel, 2012; Sherriff et al., 2016). Furthermore, impaired S-adenosylmethionine synthesis caused by methionine deficiency leads to reduced liver metabolism, enhanced liver injury, and HCC (Anstee and Day, 2012; Mato et al., 2013).

MCD diet-fed mice show sustained reduction in body weight (BW) while on the diet. To overcome BW reduction in the MCD diet-fed mice, proteins are replaced with equivalent amounts of L-amino acids and/or high-fat, or high-sucrose diets are added to MCD diets (Farrell et al., 2019; Hansen et al., 2017; Lau et al., 2017). However, limited data are available on the time-dependent profiles of serum lipids and biochemical markers of hepatic function, hepatic inflammation, and fibrosis in the long-term in mice fed MCD diets containing L-amino acids and high-amounts of fat.

Here, we investigated the time-dependent change in serum lipids and biochemical markers of hepatic function, hepatic inflammation, and fibrosis in mice fed a NAFLD diet (choline deficient, L-amino acid-defined, high-fat diet; CDAHFD) for 12 weeks. Our data provides a better understanding of NAFLD pathogenesis.