The expanding role of fish models in understanding non-alcoholic fatty liver disease

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a condition in which excessive fat accumulates in the liver of an individual who has not consumed excessive alcohol. Non-alcoholic steatohepatitis (NASH), a severe form of NAFLD, can progress to hepatic cirrhosis and/or hepatocellular carcinoma (HCC). NAFLD is considered to be a hepatic manifestation of metabolic syndrome, and its incidence has risen worldwide in lockstep with the increased global prevalence of obesity. Over the last decade, rodent studies have yielded an impressive list of molecules associated with NAFLD and NASH pathogenesis. However, the identification of currently unknown metabolic factors using mammalian model organisms is inefficient and expensive compared with studies using fish models such as zebrafish (Danio rerio) and medaka (Oryzias latipes). Substantial advances in unraveling the molecular pathogenesis of NAFLD have recently been achieved through unbiased forward genetic screens using small fish models. Furthermore, these easily manipulated organisms have been used to great advantage to evaluate the therapeutic effectiveness of various chemical compounds for the treatment of NAFLD. In this Review, we summarize aspects of NAFLD (specifically focusing on NASH) pathogenesis that have been previously revealed by rodent models, and discuss how small fish are increasingly being used to uncover factors that contribute to normal hepatic lipid metabolism. We describe the various types of fish models in use for this purpose, including those generated by mutation, transgenesis, or dietary or chemical treatment, and contrast them with rodent models. The use of small fish in identifying novel potential therapeutic agents for the treatment of NAFLD and NASH is also addressed.

Fig. 1.

Mechanisms of NASH: the two-hit model. (A) In the two-hit hypothesis of liver disease development, steatosis (fatty liver causing NAFLD) represents the ‘first hit’ that sensitizes the liver to injury mediated by ‘second hits’, such as from endotoxin, inflammatory cytokines, adipokines, oxidative stress, mitochondrial dysfunction and/or ER stress. Receipt of both hits leads to the steatohepatitis and fibrosis of NASH, and eventually the development of hepatocellular carcinoma. (B) Obesity and insulin resistance lead to an increased release of FFA from adipose tissues and enhanced FFA flux to the liver. In the liver, hyperinsulinemia induces SREBP-1c expression, which increases de novo lipogenesis via activation of lipogenic gene transcription. At the same time, hyperglycemia activates ChREBP, which also activates the transcription of lipogenic genes, increasing de novo lipogenesis and FFA levels. These fatty acids can either be oxidized in the mitochondria to generate ATP, or esterified to produce triglycerides. These triglycerides are either incorporated into VLDL for export from hepatocytes, or are stored within hepatocytes, leading to steatosis.

Fig. 2.

Drug screening strategy using fish disease models. (A) An overview of the NAFLD, NASH and HCC drug screening strategy in fish models. WT or transgenic (HCP) fish are raised on a specific diet (HFD) or treated with a specific carcinogen (TAA), giving rise to NAFLD and NASH, or NASH and HCC, respectively. The disease models are exposed to candidate drugs to determine whether the development of NAFLD, NASH or HCC can be mitigated. (B) Small-molecule inhibitor screen. Chemical libraries can be aliquoted to multi-well plates that contain fish growth medium. Large numbers of mutant or transgenic fish can be mated to generate thousands of embryos, which are placed in the multi-well plates containing medium and test reagents. These multi-well plates easily allow the detailed observation of embryo morphology under a dissecting microscope. To investigate chemical-induced changes in specific markers, several techniques have been established to perform high-throughput analysis, such as whole-mount immunohistochemistry or whole-mount in situ hybridization on large numbers of embryos.