Sterol O-acyltransferase (SOAT/ACAT) activity is required to form cholesterol crystals in hepatocyte lipid droplets.

OBJECTIVE: Excess cholesterol storage can induce the formation of cholesterol crystals in hepatocyte lipid droplets. Such crystals distinguish metabolic dysfunction associated steatohepatitis (MASH) from simple steatosis and may underlie its pathogenesis by causing cell damage that triggers liver inflammation. The mechanism linking cholesterol excess to its crystallization in lipid droplets is unclear. As cholesteryl esters localize to and accumulate in lipid droplets more readily than unesterified free cholesterol, we investigated whether cholesterol esterification by sterol O-acyltransferase (SOAT), also known as acyl co-A cholesterol acyltransferase (ACAT), is required for hepatocyte lipid droplet crystal formation. METHOD: Cholesterol crystals were measured in cholesterol loaded Hep3B hepatocytes, RAW264.7 macrophages, and mouse liver using polarizing light microscopy. We examined the effect of blocking SOAT activity on crystal formation and compared these results to features of cholesterol metabolism and the progression to intracellular crystal deposits. RESULTS: Cholesterol loading of Hep3B cells caused robust levels of lipid droplet localized crystal formation in a dose- and time-dependent manner. Co-treatment with SOAT inhibitors and genetic ablation of SOAT1 blocked crystal formation. SOAT inhibitor also blocked crystal formation in low density lipoprotein (LDL) treated Hep3B cells, acetylated LDL treated RAW 264.7 macrophages, and in the liver of mice genetically predisposed to hepatic cholesterol overload and in mice with cholesterol enriched diet-induced MASH. CONCLUSION: SOAT1-mediated esterification may underlie cholesterol crystals associated with MASH by concentrating it in lipid droplets. These findings imply that inhibiting hepatocyte SOAT1 may be able to alleviate cholesterol associated MASH. Moreover, that either a lipid droplet localized cholesteryl ester hydrolase is required for cholesterol crystal formation, or the crystals are composed of cholesteryl ester.

HDL functionality is dependent on hepatocyte stress defense factors Nrf1 and Nrf2.

High density lipoproteins (HDL) promote homeostasis and counteract stressful tissue damage that underlie cardiovascular and other diseases by mediating reverse cholesterol transport, reducing inflammation, and abrogating oxidative damage. However, metabolically stressful conditions associated with atherosclerosis can impair these effects. Hepatocytes play a major role in the genesis and maturation of circulating HDL, and liver stress elicits marked regulatory changes to circulating HDL abundance and composition, which affect its functionality. The mechanisms linking liver stress to HDL function are incompletely understood. In this study, we sought to determine whether stress defending transcription factors nuclear factor erythroid 2 related factor-1 (Nrf1) and -2 (Nrf2) promote hepatocyte production of functional HDL. Using genetically engineered mice briefly fed a mild metabolically stressful diet, we investigated the effect of hepatocyte-specific deletion of Nrf1, Nrf2, or both on circulating HDL cholesterol, protein composition, and function. Combined deletion, but not single gene deletion, reduced HDL cholesterol and apolipoprotein A1 levels as well as the capacity of HDL to accept cholesterol undergoing efflux from cultured macrophages and to counteract tumor necrosis factor α-induced inflammatory effect on cultured endothelial cells. This coincided with substantial alteration to the HDL proteome, which correlated with liver gene expression profiles of corresponding proteins. Thus, our findings show complementary actions by hepatocyte Nrf1 and Nrf2 play a role in shaping HDL abundance and composition to promote production of functionally viable HDL. Consequently, our study illuminates the possibility that enhancing stress defense programming in the liver may improve atheroprotective and perhaps other health promoting actions of HDL.

Euglycemia is affected by stress defense factor hepatocyte NRF1, but not NRF2.

Hepatocyte stress signaling has been established to alter glucose metabolism and impair systemic glucose homeostasis. In contrast, the role of stress defenses in the control of glucose homeostasis is less understood. Nuclear factor erythroid 2 related factor-1 (NRF1) and -2 (NRF2) are transcription factors that promote stress defense and can exert hepatocyte stress defense programming via complementary gene regulation. To identify whether there are independent or complementary roles of these factors in hepatocytes on glucose homeostasis, we investigated the effect of adult-onset, hepatocyte-specific deletion of NRF1, NRF2, or both on glycemia in mice fed 1-3 weeks with a mildly stressful diet enriched with fat, fructose, and cholesterol. Compared to respective control, NRF1 deficiency and combined deficiency reduced glycemia, in some cases resulting in hypoglycemia, whereas there was no effect of NRF2 deficiency. However, reduced glycemia in NRF1 deficiency did not occur in the leptin-deficient mouse model of obesity and diabetes, suggesting hepatocyte NRF1 support defenses that counteract hypoglycemia but does not promote hyperglycemia. Consistent with this, NRF1 deficiency was associated with reduced liver glycogen and glycogen synthase expression as well as marked alteration to circulating level of glycemia-influencing hormones, growth hormone and insulin-like growth factor-1 (IGF1). Overall, we identify a role for hepatocyte NRF1 in modulating glucose homeostasis, which may be linked to liver glycogen storage and the growth hormone/IGF1 axis.

Complementary gene regulation by NRF1 and NRF2 protects against hepatic cholesterol overload.

Hepatic cholesterol overload promotes steatohepatitis. Insufficient understanding of liver stress defense impedes therapy development. Here, we elucidate the role of stress defense transcription factors, nuclear factor erythroid 2 related factor-1 (NRF1) and -2 (NRF2), in counteracting cholesterol-linked liver stress. Using a diet that increases liver cholesterol storage, expression profiles and phenotypes of liver from mice with hepatocyte deficiency of NRF1, NRF2, or both are compared with controls, and chromatin immunoprecipitation sequencing is undertaken to identify target genes. Results show NRF1 and NRF2 co-regulate genes that eliminate cholesterol and mitigate inflammation and oxidative damage. Combined deficiency, but not deficiency of either alone, results in severe steatohepatitis, hepatic cholesterol overload and crystallization, altered bile acid metabolism, and decreased biliary cholesterol. Moreover, therapeutic effects of NRF2-activating drug bardoxolone require NRF1 and are supplemented by NRF1 overexpression. Thus, we discover complementary gene programming by NRF1 and NRF2 that counteract cholesterol-associated fatty liver disease progression.

Immunometabolic factors contributing to obesity-linked hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major public health concern that is promoted by obesity and associated liver complications. Onset and progression of HCC in obesity is a multifactorial process involving complex interactions between the metabolic and immune system, in which chronic liver damage resulting from metabolic and inflammatory insults trigger carcinogenesis-promoting gene mutations and tumor metabolism. Moreover, cell growth and proliferation of the cancerous cell, after initiation, requires interactions between various immunological and metabolic pathways that provide stress defense of the cancer cell as well as strategic cell death escape mechanisms. The heterogenic nature of HCC in addition to the various metabolic risk factors underlying HCC development have led researchers to focus on examining metabolic pathways that may contribute to HCC development. In obesity-linked HCC, oncogene-induced modifications and metabolic pathways have been identified to support anabolic demands of the growing HCC cells and combat the concomitant cell stress, coinciding with altered utilization of signaling pathways and metabolic fuels involved in glucose metabolism, macromolecule synthesis, stress defense, and redox homeostasis. In this review, we discuss metabolic insults that can underlie the transition from steatosis to steatohepatitis and from steatohepatitis to HCC as well as aberrantly regulated immunometabolic pathways that enable cancer cells to survive and proliferate in the tumor microenvironment. We also discuss therapeutic modalities targeted at HCC prevention and regression. A full understanding of HCC-associated immunometabolic changes in obesity may contribute to clinical treatments that effectively target cancer metabolism.

Perturbed adipose tissue hydrogen peroxide metabolism in centrally obese men: Association with insulin resistance.

OBJECTIVE: Although adipose tissue hydrogen peroxide (H2O2) and its metabolizing enzymes have been linked to obesity and insulin resistance in animal studies, this relation remains to be evaluated in humans. METHODS: Non-diabetic men (N = 43, median age, 49 (37, 54 y)) undergoing abdominal surgeries were studied. Participants were classified by body mass index (BMI) into normal-weight (N = 19), or overweight/obese (Ow/Ob; BMI ≥25; N = 24). Centrally obese men were identified by waist-height ratio ≥0.5. H2O2 and activities of superoxide dismutase, catalase and glutathione peroxidase enzymes were assayed in subcutaneous fat samples, and visceral fat (available from N = 33), and their associations with anthropometric parameters, fasting serum lipids, and the homeostasis model of insulin resistance (HOMA-IR) were tested using correlations and multivariate linear regression. RESULTS: H2O2 concentrations and catalase activity were increased in visceral fat from Ow/Ob men, compared to normal-weight subjects (+32%, P = 0.038 and +51%, P = 0.043 respectively). Centrally obese subjects had >2-fold higher superoxide dismutase activity (P = 0.005), 46% higher H2O2 (P = 0.028), and 89% higher catalase activity (P = 0.009) in visceral fat, compared to lean subjects. Central obesity did not alter these markers in subcutaneous fat, apart from a 50% increase in catalase, and did not affect glutathione peroxidase in either fat depot. H2O2 in visceral fat positively correlated with insulin resistance (r = 0.40, P = 0.032). Catalase activity in visceral fat was an independent determinant of HOMA-IR, explaining ~18% of the variance (ß = 0.42, P = 0.016), after adjustment for age and BMI. CONCLUSION: These findings suggest that adipose tissue catalase shows compensatory up-regulation in response to obesity-induced H2O2 accumulation, and that perturbed H2O2 metabolism in visceral fat is linked to insulin resistance in obese humans.