A Membrane-Bound Diacylglycerol Species Induces PKCϵ-Mediated Hepatic Insulin Resistance

Abstract

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.

Keywords: ceramides; dicylglycerols; hepatic glucose production; hepatic glycogen synthesis; hepatic insulin resistance; insulin receptor phosphorylation; liquid chromatography-tandem mass spectrometry; nonalcoholic fatty liver disease; protein kinase C-epsilon; type 2 diabetes.

Figure 1.. Acute Hepatic DGAT2 KD Induces HIR

(A) Hepatic and WAT DGAT2 protein content measured by western blot (top) and its quantification (bottom). (B) EGP and its suppression by insulin during a hyperinsulinemic-euglycemic clamp in Ctrl vs acute hepatic DGAT2 KD rats. (C) Levels of insulin-stimulated liver pIRK-Y1162, pAkt-S473, pGSK3β-S9 and pFOXO1-S256 as measured by western blot (top) and with its quantification (bottom). (D) Liver PKCε translocation from cytosol to membrane as measured by western blot (top) and with its quantification (bottom). (E) Levels of liver pIRK-T1160 in Ctrl vs acute hepatic DGAT2 KD rats as measured by western blot (top) and with its quantification (bottom). In all panels, data are the mean±S.E.M. In (A) and (C), n = 6 per group. In (B), n = 8 per group. In (D), n = 5 or 6 per group. In (E), n = 5 per group. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 2.. Liver PM sn-1,2-DAG Content Tracks with HIR in Rats and Humans

(A) Separation of five subcellular compartments in liver measured by western blot. (B) Representative chromatogram (n = 13) of DAG stereoisomer separation on LC/MS-MS. (C), (D) and (E) Liver DAG stereoisomer content in five subcellular compartments in Ctrl vs DGAT2 KD rats. (F), (G) and (H) Liver DAG stereoisomer content in five subcellular compartments in human individuals who were insulin-sensitive (black) or insulin-resistant (red). In all panels, data are the mean±S.E.M. In (C), (D) and (E), n = 8 per group. In (F), (G) and (H), n = 4 per group. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 3.. Liver-Specific PKCε KD Ameliorates HFD- and Acute DGAT2 KD-Induced HIR

(A) Hepatic and WAT PKCε protein content measured by western blot (top) and its quantification (bottom). (B) EGP and its suppression by insulin during a hyperinsulinemic-hyperglycemic clamp in Ctrl vs hepatic PKCε KD rats. (C) Hepatic glycogen synthesis rate during a hyperinsulinemic-hyperglycemic clamp and post-clamp hepatic glycogen content. (D) and (E) Levels of insulin-stimulated liver pIRK-Y1162, pAkt-S473, pGSK3β-S9 and pFOXO1-S256 as measured by western blot (top) and with its quantification (bottom). (F) Levels of liver pIRK-T1160 as measured by western blot (top) and with its quantification (bottom). (G) Fasted body weight, plasma glucose and insulin levels during an oGTT in Ctrl vs hepatic PKCε KD rats. (H) EGP, EGP’s suppression by insulin and hepatic glycogen synthesis rate during a hyperinsulinemic-hyperglycemic clamp and post-clamp hepatic glycogen content in Ctrl vs hepatic PKCε KD rats. In all panels, data are the mean±S.E.M. In (A), (D), (E) and (F), n = 6 per group. In (B), (C) and (H), n = 7 per group. In (G), n = 9 per group. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Figure 4.. Liver-Specific OE of Constitutively Active PKCε Induces HIR

(A) Hepatic PKCε protein content, hepatic PKCε translocation from cytosol to membrane and WAT PKCε content measured by western blot (top) and its quantification (bottom). (B) Hepatic glycogen synthesis rate during a hyperinsulinemic-hyperglycemic clamp and post-clamp liver glycogen content in Ctrl vs hepatic PKCε OE rats. (C) and (D) Levels of insulin-stimulated liver pIRK-Y1162, pAkt-S473, pGSK3β-S9 and pFOXO1-S256 as measured by western blot (top) and with its quantification (bottom). (E) Levels of liver pIRK-T1160 as measured by western blot (top) and with its quantification (bottom). (F) Fasted body weight, plasma glucose and insulin levels during an oGTT in Ctrl vs hepatic PKCε OE rats. In all panels, data are the mean±S.E.M. In (A), n = 5 or 7 per group. In (B), n = 8 per group. In (C), (D) and (E), n = 6 per group. In (F), n = 9 per group. *P < 0.05, **P < 0.01 and ***P < 0.001.