Background & Aims Nonalcoholic fatty liver disease (NAFLD) is a leading cause of liver damage and is characterized by steatosis. Genetic factors increase risk for progressive NAFLD. A genome-wide association study showed that the rs641738 C>T variant in the locus that contains the membrane bound O-acyltransferase domain-containing 7 gene (MBOAT7, also called LPIAT1) and transmembrane channel-like 4 gene (TMC4) increased the risk for cirrhosis in alcohol abusers. We investigated whether the MBOAT7/TMC4 is a susceptibility locus for the development and progression of NAFLD. Methods We genotyped rs641738 in DNA collected from 3854 participants from the Dallas Heart Study (a multi-ethnic population-based probability sample of Dallas County residents) and 1149 European individuals from the Liver Biopsy Cross-sectional Cohort. Clinical and anthropometric data were collected, and biochemical and lipidomics were measured in plasma samples from participants. A total of 2736 participants from the Dallas Heart Study underwent also proton magnetic resonance spectroscopy to measure hepatic triglyceride content. In the Liver Biopsy Cross-sectional Cohort, a total of 1149 individuals underwent liver biopsy to diagnose liver disease and disease severity. Results The genotype rs641738 at the MBOAT7/TMC4 locus associated with increased hepatic fat content in the 2 cohorts, and with more severe liver damage and increased risk of fibrosis compared to subjects without the variant. MBOAT7, but not TMC4, was found to be highly expressed in the liver. The MBOAT7 rs641738 T allele was associated with lower protein expression in the liver and changes in plasma phosphatidylinositol species consistent with decreased MBOAT7 function. Conclusions We provide evidence for an association between the MBOAT7 rs641738 variant and the development and severity of NAFLD in individuals of European descent. This association seems to be mediated by changes in the hepatic phosphatidylinositol acyl-chain remodeling.

Retinoids are micronutrients that are stored as retinyl esters in the retina and hepatic stellate cells (HSCs). HSCs are key players in fibrogenesis in chronic liver diseases. The enzyme responsible for hydrolysis and release of retinyl esters from HSCs is unknown and the relationship between retinoid metabolism and liver disease remains unclear. We hypothesize that the patatin-like phospholipase domain-containing 3 (PNPLA3) protein is involved in retinol metabolism in HSCs. We tested our hypothesis both in primary human HSCs and in a human cohort of subjects with non-alcoholic fatty liver disease (N = 146). Here we show that PNPLA3 is highly expressed in human HSCs. Its expression is regulated by retinol availability and insulin, and increased PNPLA3 expression results in reduced lipid droplet content. PNPLA3 promotes extracellular release of retinol from HSCs in response to insulin. We also show that purified wild-type PNPLA3 hydrolyzes retinyl palmitate into retinol and palmitic acid. Conversely, this enzymatic activity is markedly reduced with purified PNPLA3 148M, a common mutation robustly associated with liver fibrosis and hepatocellular carcinoma development. We also find the PNPLA3 I148M genotype to be an independent (P = 0.009 in a multivariate analysis) determinant of circulating retinol-binding protein 4, a reliable proxy for retinol levels in humans. This study identifies PNPLA3 as a lipase responsible for retinyl-palmitate hydrolysis in HSCs in humans. Importantly, this indicates a potential novel link between HSCs, retinoid metabolism and PNPLA3 in determining the susceptibility to chronic liver disease.

Background and AimsNonalcoholic fatty liver disease is epidemiologically associated with hepatic and metabolic disorders. The aim of this study was to examine whether hepatic fat accumulation has a causal role in determining liver damage and insulin resistance.MethodsWe performed a Mendelian randomization analysis using risk alleles in PNPLA3, TM6SF2, GCKR and MBOAT7, and a polygenic risk score for hepatic fat, as instruments. We evaluated complementary cohorts of at‐risk individuals and individuals from the general population: 1515 from the liver biopsy cohort (LBC), 3329 from the Swedish Obese Subjects Study (SOS) and 4570 from the population‐based Dallas Heart Study (DHS).ResultsHepatic fat was epidemiologically associated with liver damage, insulin resistance, dyslipidemia and hypertension. The impact of genetic variants on liver damage was proportional to their effect on hepatic fat accumulation. Genetically determined hepatic fat was associated with aminotransferases, and with inflammation, ballooning and fibrosis in the LBC. Furthermore, in the LBC, the causal association between hepatic fat and fibrosis was independent of disease activity, suggesting that a causal effect of long‐term liver fat accumulation on liver disease is independent of inflammation. Genetically determined hepatic steatosis was associated with insulin resistance in the LBC and SOS. However, this association was dependent on liver damage severity. Genetically determined hepatic steatosis was associated with liver fibrosis/cirrhosis and with a small increase in risk of type 2 diabetes in publicly available databases.ConclusionThese data suggest that long‐term hepatic fat accumulation plays a causal role in the development of chronic liver disease.

The patatin-like phospholipase domain containing 3 (PNPLA3, also called adiponutrin, ADPN) is a membrane-bound protein highly expressed in the liver. The genetic variant I148M (rs738409) was found to be associated with progression of chronic liver disease. We aimed to establish a protein purification protocol in a yeast system (Pichia pastoris) and to examine the human PNPLA3 enzymatic activity, substrate specificity and the I148M mutation effect. hPNPLA3 148I wild type and 148M mutant cDNA were cloned into P. pastoris expression vectors. Yeast cells were grown in 3L fermentors. PNPLA3 protein was purified from membrane fractions by Ni-affinity chromatography. Enzymatic activity was assessed using radiolabeled substrates. Both 148I wild type and 148M mutant proteins are localized to the membrane. The wild type protein shows a predominant lipase activity with mild lysophosphatidic acid acyl transferase activity (LPAAT) and the I148M mutation results in a loss of function of both these activities. Our data show that PNPLA3 has a predominant lipase activity and I148M mutation results in a loss of function.