Nonalcoholic fatty liver disease is strongly associated with hepatic insulin resistance (HIR); however, the key lipid species and molecular mechanisms linking these conditions are widely debated. We developed a subcellular fractionation method to quantify diacylglycerol (DAG) stereoisomers and ceramides in the endoplasmic reticulum (ER), mitochondria, plasma membrane (PM), lipid droplets, and cytosol. Acute knockdown (KD) of diacylglycerol acyltransferase-2 in liver induced HIR in rats. This was due to PM sn-1,2-DAG accumulation, which promoted PKCϵ activation and insulin receptor kinase (IRK)-T1160 phosphorylation, resulting in decreased IRK-Y1162 phosphorylation. Liver PM sn-1,2-DAG content and IRK-T1160 phosphorylation were also higher in humans with HIR. In rats, liver-specific PKCϵ KD ameliorated high-fat diet-induced HIR by lowering IRK-T1160 phosphorylation, while liver-specific overexpression of constitutively active PKCϵ-induced HIR by promoting IRK-T1160 phosphorylation. These data identify PM sn-1,2-DAGs as the key pool of lipids that activate PKCϵ and that hepatic PKCϵ is both necessary and sufficient in mediating HIR.

非酒精性脂肪肝与肝胰岛素抵抗 (HIR) 密切相关；然而，连接这些条件的关键脂质种类和分子机制存在广泛争议。我们开发了一种亚细胞分馏方法来量化内质网 (ER)、线粒体、质膜 (PM)、脂滴和细胞质中的二酰基甘油 (DAG) 立体异构体和神经酰胺。肝脏中二酰基甘油酰基转移酶-2 的急性敲低 (KD) 诱导大鼠 HIR。这是由于 PM sn -1,2-DAG 积累，促进 PKCϵ 活化和胰岛素受体激酶 (IRK)-T1160 磷酸化，导致 IRK-Y1162 磷酸化降低。肝PM SN-1,2-DAG 含量和 IRK-T1160 磷酸化在患有 HIR 的人中也更高。在大鼠中，肝脏特异性 PKCϵ KD 通过降低 IRK-T1160 磷酸化改善高脂肪饮食诱导的 HIR，而肝脏特异性过表达组成型活性 PKCϵ 诱导的 HIR 通过促进 IRK-T1160 磷酸化。这些数据表明 PM sn -1,2-DAGs 是激活 PKCϵ 的关键脂质池，肝脏 PKCϵ 在介导 HIR 中既是必要的又是充分的。