Membrane-bound sn-1,2-diacylglycerols explain the dissociation of hepatic insulin resistance from hepatic steatosis in MTTP knockout mice

Abstract

Microsomal triglyceride transfer protein (MTTP) deficiency results in a syndrome of hypolipidemia and accelerated NAFLD. Animal models of decreased hepatic MTTP activity have revealed an unexplained dissociation between hepatic steatosis and hepatic insulin resistance. Here, we performed comprehensive metabolic phenotyping of liver-specific MTTP knockout (L-Mttp^-/-) mice and age-weight matched wild-type control mice. Young (10-12-week-old) L-Mttp^-/- mice exhibited hepatic steatosis and increased DAG content; however, the increase in hepatic DAG content was partitioned to the lipid droplet and was not increased in the plasma membrane. Young L-Mttp^-/- mice also manifested normal hepatic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps, no PKCε activation, and normal hepatic insulin signaling from the insulin receptor through AKT Ser/Thr kinase. In contrast, aged (10-month-old) L-Mttp^-/- mice exhibited glucose intolerance and hepatic insulin resistance along with an increase in hepatic plasma membrane sn-1,2-DAG content and PKCε activation. Treatment with a functionally liver-targeted mitochondrial uncoupler protected the aged L-Mttp^-/- mice against the development of hepatic steatosis, increased plasma membrane sn-1,2-DAG content, PKCε activation, and hepatic insulin resistance. Furthermore, increased hepatic insulin sensitivity in the aged controlled-release mitochondrial protonophore-treated L-Mttp^-/- mice was not associated with any reductions in hepatic ceramide content. Taken together, these data demonstrate that differences in the intracellular compartmentation of sn-1,2-DAGs in the lipid droplet versus plasma membrane explains the dissociation of NAFLD/lipid-induced hepatic insulin resistance in young L-Mttp^-/- mice as well as the development of lipid-induced hepatic insulin resistance in aged L-Mttp^-/- mice.

Keywords: diabetes; drug therapy; lipids; liver; liver microsomal triglyceride transfer protein; liver-targeted mitochondrial uncoupler; metabolic disease; nonalcoholic fatty liver disease.

Fig. 1.

Young L-Mttp^−/− mice develop hepatic steatosis without the accumulation of plasma membrane sn-1,2-DAGs. A: Liver TAG content. B: Liver ceramide content. C: Total DAG. D–F: sn-1,2-DAG (D), sn-1,3-DAG (E), and sn-2,3-DAG (F) content in five compartments of the liver from young WT and L-Mttp^−/− mice: plasma membrane (PM), mitochondrial (Mito), ER, lipid droplet (LD), and cytosol (Cyto). Individual statistical comparisons were evaluated by Student’s two-tailed t-test. Data are means ± SEMs of n = 6–8 per group.

Fig. 2.

Normal whole-body insulin sensitivity in young L-Mttp^−/− mice. A, B: Plasma glucose concentrations (A) and glucose infusion rate (B) during the hyperinsulinemic portion of the clamp study. C: Whole-body insulin-stimulated peripheral glucose metabolism. D: Endogenous glucose production. E: Insulin-mediated suppression of endogenous glucose production. F: PKCε translocation in the liver. G: Liver AKT phosphorylation comparing basal and insulin-stimulated liver. Statistical comparisons were made by unpaired two-way Student’s t-test. Data are means ± SEMs of n = 7–8 per group.

Fig. 3.

Aged L-Mttp^−/− mice demonstrated glucose intolerance that improved with CRMP. A, B: Plasma glucose concentration time course (A) and area under the glucose versus time curve (B) during intraperitoneal glucose tolerance tests. C, D: Plasma insulin concentration time course (C) and area under the insulin versus time curve (D) during intraperitoneal glucose tolerance tests. Mice were fasted overnight before the glucose tolerance tests. Data are represented as means ± SEMs. Statistical comparisons were made by two-way ANOVA. Data are means ± SEMs of n = 7–8 per group.

Fig. 4.

Hepatic steatosis, hepatic plasma membrane sn-1,2 DAG accumulation, and increased PKCε membrane translocation were all observed in aged L-Mttp^−/− mice. A: Plasma TAG concentration. B: Liver TAG content. C: Liver ceramide content. D: Liver total DAG. E–G: sn-1,2-DAG (E), sn-1,3-DAG (F), and sn-2,3-DAG (G) content in five hepatic subcellular compartments from aged WT mice, aged L-Mttp^−/− mice, and aged L-Mttp^−/− mice treated with CRMP: plasma membrane (PM), mitochondrial (Mito), ER, lipid droplet (LD), and cytosol (Cyto). H: Hepatic PKCε translocation. Statistical comparisons were made by two-way ANOVA. Data are means ± SEMs of n = 6–8 per group.

Fig. 5.

Whole-body energy balance was not different between aged WT mice, aged L-Mttp^−/− mice, and aged L-Mttp^−/− mice treated with CRMP. A: Body weight. B: Oxygen consumption (V_O2). C: Carbon dioxide production (V_CO2). D: Respiratory exchange ratio. E: Energy expenditure throughout the day. F: Food intake. G: Daily activity. Statistical comparisons were made by two-way ANOVA. Data are means ± SEMs of n = 6 per group.

Fig. 6.

Liver inflammatory markers and activation of the unfolded protein response were unaltered by genotype or drug treatment in aged L-Mttp^−/− mice and L-Mttp^−/− mice treated with CRMP. A: Hepatic cytokine concentrations. B: ER stress/unfolded protein response markers assessed by immunoblot. C: GRP78. D: GRP94. E: Phosphorylation of eIF2. F: Phosphorylation of IRE1. Statistical comparisons were made by two-way ANOVA. Data are means ± SEMs of n = 3 per group.

Fig. 7.

CRMP treatment improved hepatic insulin sensitivity of aged L-Mttp^−/− mice as assessed by hyperinsulinemic-euglycemic clamps. A: Plasma glucose during the clamp. B: Glucose infusion rate during the clamp. C: Insulin-stimulated peripheral glucose metabolism. D: EGP in both the basal and hyperinsulinemic clamped state. E: Insulin-mediated suppression of EGP represented as percentage suppression. F: Liver AKT phosphorylation under basal and insulin-stimulated conditions. G: Liver insulin receptor β (insulin receptor kinase; IRK) phosphorylation under basal and insulin-stimulated conditions. Statistical comparisons made by Student’s t-test. Data are means ± SEMs of n = 6–7 per group.