Lipid and insulin infusion-induced skeletal muscle insulin resistance is likely due to metabolic feedback and not changes in IRS-1, Akt, or AS160 phosphorylation

Abstract

Type 2 diabetes is characterized by hyperlipidemia, hyperinsulinemia, and insulin resistance. The aim of this study was to investigate whether acute hyperlipidemia-induced insulin resistance in the presence of hyperinsulinemia was due to defective insulin signaling. Hyperinsulinemia (approximately 300 mU/l) with hyperlipidemia or glycerol (control) was produced in cannulated male Wistar rats for 0.5, 1 h, 3 h, or 5 h. The glucose infusion rate required to maintain euglycemia was significantly reduced by 3 h with lipid infusion and was further reduced after 5 h of infusion, with no difference in plasma insulin levels, indicating development of insulin resistance. Consistent with this finding, in vivo skeletal muscle glucose uptake (31%, P < 0.05) and glycogen synthesis rate (38%, P < 0.02) were significantly reduced after 5 h compared with 3 h of lipid infusion. Despite the development of insulin resistance, there was no difference in the phosphorylation state of multiple insulin-signaling intermediates or muscle diacylglyceride and ceramide content over the same time course. However, there was an increase in cumulative exposure to long-chain acyl-CoA (70%) with lipid infusion. Interestingly, although muscle pyruvate dehydrogenase kinase 4 protein content was decreased in hyperinsulinemic glycerol-infused rats, this decrease was blunted in muscle from hyperinsulinemic lipid-infused rats. Decreased pyruvate dehydrogenase complex activity was also observed in lipid- and insulin-infused animals (43%). Overall, these results suggest that acute reductions in muscle glucose metabolism in rats with hyperlipidemia and hyperinsulinemia are more likely a result of substrate competition than a significant early defect in insulin action or signaling.

Fig. 1.

Blood glucose (A), plasma insulin (B), and plasma nonesterified fatty acids (NEFA; C) in rats infused with lipid or glycerol (control) for 5 h. Values are means ± SE (n = 10–12 rats per group).

Fig. 2.

Effect of lipid infusion on whole body indexes of insulin sensitivity. A: glucose infusion rate (GIR) required to maintain euglycemia in rats infused with lipid or glycerol and insulin for 5 h. *P < 0.05 vs. 60 min of lipid infusion (by ANOVA). #P < 0.05 vs. control infusion (by ANOVA). B–D: rate of glucose disappearance (R_d), red quadriceps glucose uptake, and glycogen synthesis rate in animals after 3 and 5 h of glycerol or lipid infusion in the presence of hyperinsulinemia. *P < 0.05 vs. 3-h lipid infusion (by ANOVA). E: glycogen content in animals infused with lipid or glycerol and insulin for 5 h. Values are means ± SE (n = 5–7 rats per group). *P < 0.05 vs. Basal.

Fig. 3.

Effect of lipid infusion on skeletal muscle insulin signaling intermediates, expressed as ratio of phosphorylated (p) to total (t) intermediate. A: insulin receptor (IR) activation (Tyr^1162/63). B: insulin receptor substrate 1 (IRS-1) activation (Tyr⁶¹²). C: Akt (Ser⁴⁷³). D: AS160 (Thr⁶⁴²). E: glycogen synthase kinase (GSK)-3β (Ser²¹). F: GSK3α (Ser⁹). Intermediates were measured in red quadriceps in the basal state and after 3 and 5 h of glycerol or lipid infusion in the presence of hyperinsulinemia. Values are means ± SE (n = 5–7 rats per group). *P < 0.05 vs. basal (by ANOVA).

Fig. 4.

Effects of lipid infusion on skeletal muscle inflammatory signaling. IκBα and JNK (Thr¹⁸³/Tyr¹⁸⁵) were measured in red quadriceps from control and lipid-infused animals in the presence of insulin.

Fig. 5.

Effects of lipid infusion on skeletal muscle lipid intermediates: triglyceride (A), diacylglyceride (B), ceramide (C), and long chain acyl-CoA (LCACoA, D) content and accumulated exposure (insets). Lipid intermediates were measured in red quadriceps from control and lipid-infused animals in the presence of insulin. Values are means ± SE (n = 5–7 rats per group). #P < 0.05 vs. Basal; *P < 0.05 vs. 5-h control infusion.

Fig. 6.

Effect of lipid infusion on skeletal muscle pyruvate dehydrogenase kinase (PDK4) content (A) and pyruvate dehydrogenase complex (PDHC) activity (B). Activities were measured in the basal state and after 3 and 5 h of glycerol or lipid infusion in the presence of hyperinsulinemia in red quadriceps. Values are means ± SE (n = 5–7 rats per group). *P < 0.05 vs. basal; #P < 0.05 vs. 3 h control; ‡P < 0.05 vs. 5 h control (by 1-way ANOVA).