DOWN-REGULATION OF HEPATIC MBOAT7 BY HYPERINSULINEMIA FAVORS STEATOSIS DEVELOPMENT

Background and aims: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver diseases in Western Countries. NAFLD could evolve into nonalcoholic steatohepatitis (NASH), which is associated with activation of fibrogenesis, possibly leading to cirrhosis and hepatocellular carcinoma (HCC). The genetic background plays a pivotal role in predisposing to development and progression of liver disease. Recently, our group have shown that the rs641738 C>T variant, in the Membrane bound o-acyltransferase domain-containing 7 locus (MBOAT7), increases the risk of NAFLD, NASH and fibrosis, by decreasing the hepatic expression of MBOAT7, a protein involved in phosphatidylinositol acyl-chain remodeling. Aim of this study was to evaluate the regulation of hepatic MBOAT7 and the impact on hepatic fat accumulation. Methods: We examined hepatic and adipose MBOAT7 expression in 119 obese patients and in experimental models. We silenced hepatic Mboat7 by i.v. administration of antisense oligonucleotides modified by morpholinos (MPO) for 4 consecutive days in C57Bl/6 male mice (n=6). Results: In obese patients, hepatic mRNA levels of MBOAT7 progressively decreased from normal liver to simple steatosis and NASH (p<0.05). At multivariate analysis, type 2 diabetes (p<0.05), necroinflammation (p<0.01) and MBOAT7 genotype (p<0.01) were independently associated with MBOAT7 down-regulation. This suggests that down-regulation of hepatic MBOAT7 is involved in NAFLD pathogenesis independently of MBOAT7 genotype. In line with this hypothesis, we found that mRNA and protein levels of Mboat7 were reduced in experimental models of NAFLD: in the methionine-choline deficient diet (MCD), but more so in genetically obese Lepob/ob mice and in insulin resistant mice with Insulin receptor haplo-insufficiency (InsR+/-), characterized by hyperinsulinemia (p<0.05). Furthermore, in wild-type male mice Mboat7 was physiologically down-regulated by refeeding concomitantly with the rise of insulin levels and activation of hepatic insulin signaling through Phosphatidyl Inositol 3-Kinase (PI3K) and the Serine/Threonine Kinase AKT. In keeping with these results, activation of insulin-receptor dependent signaling down-regulated Mboat7 protein expression in primary mouse hepatocytes in a PI3Kinase-depedent manner. Consistently, in InsR+/- hepatocytes insulin-mediated suppression of Mboat7 was lost (p<0.01). Moreover, the effect of insulin on Mboat7 expression was also abrogated in wild-type hepatocytes treated with antisense oligonucleotide stabilized by morpholination directed against FoxO1 (MPO) (p<0.05). These data suggest that FoxO1 is required to mediate the Mboat7 regulation by insulin. Consistently with this notion, the hampered Mboat7 expression observed in InsR+/- hepatocytes was rescued by FoxO1-silencing by antisense-MPO (p<0.05 vs scramble). Finally, in vivo administration of antisense-MPO against Mboat7 for 4 consecutive days, induced a 45% silencing of hepatic Mboat7 in wild-type male mice, which is comparable to that associated with the genetic risk variant, resulting in 80% increase in hepatic TG content (p<0.05 vs scramble) and in microvesicular steatosis development. In contrast, it did not affect circulating glucose, insulin and lipid levels. Steatosis development was not explained by altered de novo lipogenesis or β-oxidation or VLDL export, but was associated with increased expression of fatty acids transporters (such as Fatp1, Fabp1). Conversely, Mboat7 silencing was not associated with alterations in the hepatic expression of inflammatory cytokines and chemokines (such as Tnf-α and Cxcl10). These data are consistent with human genetic data suggesting that MBOAT7 down-regulation plays a causal role in NAFLD development. Conclusion: In summary, we found that hyperinsulinemia, a typical feature of metabolic syndrome and post-prandial conditions, reduces hepatic MBOAT7 expression via increased insulin signaling activation through the Insulin receptor - PI3K – FoxO1 pathway. Reduced MBOAT7 then favors hepatic fat accumulation. These data suggest that MBOAT7 down-regulation may be implicated in the pathogenesis of progressive NAFLD.