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The underlining mechanisms of dietary cholesterol and nonalcoholic steatohepatitis (NASH) in contributing to hepatocellular carcinoma (HCC) remain undefined. Here we demonstrated that high-fat-non-cholesterol-fed mice developed simple steatosis, whilst high-fat-high-cholesterol-fed mice developed NASH. Moreover, dietary cholesterol induced larger and more numerous NASH-HCCs than non-cholesterol-induced steatosis-HCCs in diethylnitrosamine-treated mice. NASH-HCCs displayed significantly more aberrant gene expression-enriched signaling pathways and more non-synonymous somatic mutations than steatosis-HCCs (335 ± 84/sample vs 43 ± 13/sample). Integrated genetic and expressional alterations in NASH-HCCs affected distinct genes pertinent to five pathways: calcium, insulin, cell adhesion, axon guidance and metabolism. Some of the novel aberrant gene expression, mutations and core oncogenic pathways identified in cholesterol-associated NASH-HCCs in mice were confirmed in human NASH-HCCs, which included metabolism-related genes (ALDH18A1, CAD, CHKA, POLD4, PSPH and SQLE) and recurrently mutated genes (RYR1, MTOR, SDK1, CACNA1H and RYR2). These findings add insights into the link of cholesterol to NASH and NASH-HCC and provide potential therapeutic targets.

Unlabelled: The majority of patients with nonalcoholic fatty liver disease (NAFLD) have "simple steatosis," which is defined by hepatic steatosis in the absence of substantial inflammation or fibrosis and is considered to be benign. However, 10%-30% of patients with NAFLD progress to fibrosing nonalcoholic steatohepatitis (NASH), which is characterized by varying degrees of hepatic inflammation and fibrosis, in addition to hepatic steatosis, and can lead to cirrhosis. The cause(s) of progression to fibrosing steatohepatitis are unclear. We aimed to test the relative contributions of dietary fat and dietary cholesterol and their interaction on the development of NASH. We assigned C57BL/6J mice to four diets for 30 weeks: control (4% fat and 0% cholesterol); high cholesterol (HC; 4% fat and 1% cholesterol); high fat (HF; 15% fat and 0% cholesterol); and high fat, high cholesterol (HFHC; 15% fat and 1% cholesterol). The HF and HC diets led to increased hepatic fat deposition with little inflammation and no fibrosis (i.e., simple hepatic steatosis). However, the HFHC diet led to significantly more profound hepatic steatosis, substantial inflammation, and perisinusoidal fibrosis (i.e., steatohepatitis), associated with adipose tissue inflammation and a reduction in plasma adiponectin levels. In addition, the HFHC diet led to other features of human NASH, including hypercholesterolemia and obesity. Hepatic and metabolic effects induced by dietary fat and cholesterol together were more than twice as great as the sum of the separate effects of each dietary component alone, demonstrating significant positive interaction., Conclusion: Dietary fat and dietary cholesterol interact synergistically to induce the metabolic and hepatic features of NASH, whereas neither factor alone is sufficient to cause NASH in mice., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Background & Aims: Type 2 diabetes and nonalcoholic steatohepatitis (NASH) are associated with insulin resistance and disordered cholesterol homeostasis. We investigated the basis for hepatic cholesterol accumulation with insulin resistance and its relevance to the pathogenesis of NASH., Methods: Alms1 mutant (foz/foz) and wild-type NOD.B10 mice were fed high-fat diets that contained varying percentages of cholesterol; hepatic lipid pools and pathways of cholesterol turnover were determined. Hepatocytes were exposed to insulin concentrations that circulate in diabetic foz/foz mice., Results: Hepatic cholesterol accumulation was attributed to up-regulation of low-density lipoprotein receptor via activation of sterol regulatory element binding protein 2 (SREBP-2), reduced biotransformation to bile acids, and suppression of canalicular pathways for cholesterol and bile acid excretion in bile. Exposing primary hepatocytes to concentrations of insulin that circulate in diabetic Alms1 mice replicated the increases in SREBP-2 and low-density lipoprotein receptor and suppression of bile salt export pump. Removing cholesterol from diet prevented hepatic accumulation of free cholesterol and NASH; increasing dietary cholesterol levels exacerbated hepatic accumulation of free cholesterol, hepatocyte injury or apoptosis, macrophage recruitment, and liver fibrosis., Conclusions: In obese, diabetic mice, hyperinsulinemia alters nuclear transcriptional regulators of cholesterol homeostasis, leading to hepatic accumulation of free cholesterol; the resulting cytotoxicity mediates transition of steatosis to NASH., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, can progress to steatohepatitis (NASH) and advanced liver disease. Mechanisms that underlie this progression remain poorly understood, partly due to lack of good animal models that resemble human NASH. We previously showed that several metabolic syndrome features that develop in LDL receptor-deficient (LDLR-/-) mice fed a diabetogenic diet are worsened by dietary cholesterol. To test whether dietary cholesterol can alter the hepatic phenotype in the metabolic syndrome, we fed LDLR-/- mice a high-fat, high-carbohydrate diabetogenic diet (DD) without or with added cholesterol (DDC). Both groups of mice developed obesity and insulin resistance. Hyperinsulinemia, dyslipidemia, hepatic triglyceride, and alanine aminotransferase (ALT) elevations were greater with DDC. Livers of DD-fed mice showed histological changes resembling NAFLD, including steatosis and modest fibrotic changes; however, DDC-fed animals developed micro- and macrovesicular steatosis, inflammatory cell foci, and fibrosis resembling human NASH. Dietary cholesterol also exacerbated hepatic macrophage infiltration, apoptosis, and oxidative stress. Thus, LDLR-/- mice fed diabetogenic diets may be useful models for studying human NASH. Dietary cholesterol appears to confer a second "hit" that results in a distinct hepatic phenotype characterized by increased inflammation and oxidative stress.