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.

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.