Non-alcoholic fatty liver disease (NAFLD) is attributable to over-nutrition, but non-alcoholic steatohepatitis (NASH) is linked to its complications: insulin resistance, glucose intolerance/type 2 diabetes (T2D), adipose inflammation/hypoadiponectinemia. The links between severity of metabolic dysfunction and fatty liver phenotype (injury, inflammation, fibrosis) form a framework to consider that NASH (not just simple steatosis [SS]) results from metabolic disorder. The concept that lipid molecules can incite tissue injury (lipotoxicity) is gaining currency for NASH pathogenesis. Outstanding questions include: (1) Which is/are the lipotoxic mediators; (2) How does lipotoxicity result in persistent liver inflammation (roles of mitochondrial injury and ER stress, innate immunity sensing TLRs, inflammasome); (3) gut microbiotome; (4) adipose inflammation/adiponectin secretion. Human lipidomic studies show hepatic free cholesterol (FC) accumulation is strongly related to hepatocellular injury, inflammatory recruitment and fibrosis inNASH; FC is not increased in simple steatosis (SS). We have developed murine models of genetic and diet-induced NASH vs SS and control liver, coupled to studies in primary hepatocytes to clarify the lipotoxic pathogenesis of NASH. Alms1 mutant (foz/foz) mice have severely disordered appetite dysregulation and develop obesity, dyslipidemia, T2D and hypertension (metabolic syndrome) with low serum adiponectin; changes are accelerated by a high fat, high sucrose, 0.19% cholesterol diet. In foz/foz mice, hyperinsulinemia promotes hepatic FC accumulation by pathways mediated via SREBP2, LRH1, SHP and possibly FXR. They include LDLR-mediated cholesterol uptake, impaired biotransformation to bile acids, and suppression of canalicular FC and bile acid secretion. All except the uptake pathways have been confirmed by others in human liver. Dietary modulation of hepatic FC content (up or down) produced parallel changes in injury, liver inflammation, and fibrosis. Cholesterol-lowering drugs reduced JNK1 activation (a feature of lipotoxicity), hepatocyte injury/apoptosis, inflammation and fibrosis in metabolic syndrome NASH. Ezetimibe (blocks hepatic reuptake of cholesterol from bile) was as effective as atorvastatin, and ezetimibe/atorvastatin combination normalized hepatic FC with minimal effects on other lipids. Pharmacological lowering of hepatic FC abolished JNK activation, improved ALT, apoptosis, liver inflammation/NAFLD activity score, pathology designation as “NASH”, MCP-1 expression and liver fibrosis. With George Ioannou from UW Seattle, we have now shown that cholesterol crystals form in hepatocytes in our NASH model (as well as in human NASH). We adopted an old strategy for cholesterol-loading of macrophages to study direct effects of FC loading on primary hepatocytes. FC loading activated JNK (independently of ER stress) and caused mitochondrial injury to increases apoptosis and necrosis; both forms of cell death were dependent on HMGB1 secretion, were blocked by potent JNK inhibitors, and failed to occur in Jnk1^-/- or Tlr4^-/- mouse hepatocytes. FC-induced lipotoxicity activated Kupffer cells via release of HMGB1; such activation was totally abrogated in Kupffer cells from Tlr4^-/- mice. Cholesterol crystals also activate Kupffer cells; IL-1b release is abrogated by the novel inflammasome inhibitor, MCC-950.

In conclusion, FC is at least one lipotoxic mediator of NASH. Its hepatic accumulation in prediabetes/metabolic syndrome is programmed by hyperinsulinemia, involves profound dysregulation of hepatic cholesterol homeostasis, and can result in cholesterol crystal formation. These processes have mechanistic, diagnostic and management implications for NASH, such as the need to correct insulin resistance and the potential efficacy of potent cholesterol-lowering agents (ezetimibe/statin combination, FXR agonists). Implication of the NLRP3 inflammasome, JNK1 and TLR4 signaling offer the most logical drugable targets for pharmacological intervention when obese patients are refractory to lifestyle interventions that reverse insulin resistance.