Muscle-specific IRS-1 Ser->Ala transgenic mice are protected from fat-induced insulin resistance in skeletal muscle

Abstract

Objective: Insulin resistance in skeletal muscle plays a critical role in the pathogenesis of type 2 diabetes, yet the cellular mechanisms responsible for insulin resistance are poorly understood. In this study, we examine the role of serine phosphorylation of insulin receptor substrate (IRS)-1 in mediating fat-induced insulin resistance in skeletal muscle in vivo.

Research design and methods: To directly assess the role of serine phosphorylation in mediating fat-induced insulin resistance in skeletal muscle, we generated muscle-specific IRS-1 Ser(302), Ser(307), and Ser(612) mutated to alanine (Tg IRS-1 Ser-->Ala) and IRS-1 wild-type (Tg IRS-1 WT) transgenic mice and examined insulin signaling and insulin action in skeletal muscle in vivo.

Results: Tg IRS-1 Ser-->Ala mice were protected from fat-induced insulin resistance, as reflected by lower plasma glucose concentrations during a glucose tolerance test and increased insulin-stimulated muscle glucose uptake during a hyperinsulinemic-euglycemic clamp. In contrast, Tg IRS-1 WT mice exhibited no improvement in glucose tolerance after high-fat feeding. Furthermore, Tg IRS-1 Ser-->Ala mice displayed a significant increase in insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity and Akt phosphorylation in skeletal muscle in vivo compared with WT control littermates.

Conclusions: These data demonstrate that serine phosphorylation of IRS-1 plays an important role in mediating fat-induced insulin resistance in skeletal muscle in vivo.

FIG. 1.

Generation of Tg IRS-1 Ser→Ala mice. A: Serine phosphorylation of IRS-1 protein in skeletal muscle. Serine phosphorylation of IRS-1 and protein expression of IRS-1 were analyzed by Western blotting. All mice were killed at age 16 weeks. The high-fat diet group was fed for 8 weeks. B: Vector construct of the mutated IRS-1 gene (Ser³⁰², Ser³⁰⁷, and Ser⁶¹² to Ala mutant) for Tg IRS-1 Ser→Ala and WT IRS-1 gene for Tg IRS-1 WT. C: Comparison of IRS-1 expression in different insulin-targeting tissues. Protein expression of total IRS-1 and actin were analyzed by Western blotting. D: Comparison of IRS-1 expression between Tg IRS-1 Ser→Ala and Tg IRS-1 WT mice. Each graph was expressed as fold difference to their littermates. GAS, Musculus gastrocnemius; QD, Musclus quadriceps; TA, Musculus tibialis anterior; WAT, white adipose tissue.

FIG. 2.

Tg IRS-1 Ser→Ala, but not Tg IRS-1 WT, mice were protected from high-fat diet–induced glucose intolerance during IPGTT. A: Growth curves. Body weights were measured weekly under control diet and high-fat diet feeding regimens. At age 7–8 weeks, mice were started on a high-fat, safflower oil–based diet (27% safflower oil, 59% fat-derived calories) and maintained for 8 weeks. with solid line, WT control diet; • with solid line, Tg IRS-1 Ser→Ala control diet; with dotted line, WT high-fat diet; • with dotted line, Tg IRS-1 Ser→Ala high-fat diet. B: IPGTT. A total of 1 g/kg of 10% glucose was injected in control diet–fed Tg IRS-1 Ser→Ala mice (• with solid line) and littermate control mice ( with solid line). A total of 1 g/kg of 10% glucose was also injected in high-fat–fed Tg IRS-1 Ser→Ala (• with dotted line), Tg IRS-1 WT (♦ with dotted line), and littermate control ( with dotted line) mice. C: Area under the curve of insulin concentration during IPGTTs. Results are expressed as means ± SE (WT, n = 11; Tg IRS-1 Ser→Ala, n = 16; Tg IRS-1 WT, n = 8). *P < 0.05 vs. WT high-fat diet.

FIG. 3.

Tg IRS-1 Ser→Ala mice were protected from high-fat diet–induced muscle insulin resistance. A: After 8 weeks of high-fat feeding (16 ± 1 weeks), age-matched mice were food deprived for 16 h and hyperinsulinemic-euglycemic clamp experiments (2.5 mU · kg⁻¹ · min⁻¹) were performed using Tg IRS-1 Ser→Ala mice and their littermates. Plasma glucose concentration was maintained by an intravenous 20% glucose infusion and glucose infusion rates (GINF) at steady state were assessed. B: Muscle-specific glucose uptake was analyzed by enrichment of [¹⁴C]-2-deoxy-glucose in Musculus gastrocnemius (GAS) (left) and Musclus quadriceps (QD) (right). Results are expressed as means ± SE (WT, n = 12; Tg IRS-1 Ser→Ala, n = 16). C: Insulin-stimulated Akt phosphorylation (Ser⁴⁷³) in GAS was analyzed 15 min after intraperitoneal injection of 0.6 units/kg insulin in control diet–fed mice (WT, n = 6) and high-fat diet–fed mice (WT, n = 16; Tg IRS-1 Ser→Ala, n = 15; Tg IRS-1 WT, n = 3). D: IRS-1–associated PI3-kinase activity in GAS of high-fat–fed mice (WT, n = 12; Tg IRS-1 Ser→Ala, n = 8). E: IRS-1 tyrosine phosphorylation (left) and IRS-1–associated p85 subunit of PI3-kinase (right) were analyzed 15 min after intraperitoneal injection of 0.6 units/kg insulin by immunoprecipitation. Results are expressed as means relative to insulin-stimulated samples of WT high-fat fed mice ± SE.

FIG. 4.

Tg IRS-1 Ser→Ala mice were protected from age-related glucose intolerance. Plasma glucose (A) and insulin (B) concentrations in 1-year-old Tg IRS-1 Ser→Ala transgenic and WT littermate mice following an IPGTT (1 g/kg) (WT, n = 12; Tg IRS-1 Ser→Ala, n = 6). A: WT elder; ♦, Tg IRS-1 Ser→Ala elder. Results are expressed as means ± SE. C: Generation of Tg IRS-1 Ser→Ala/ob/ob. Growth curves: body weights were measured weekly under control diet until age 16 weeks (• with dotted line) and compared with WT/ob/ob ( with dotted line), Tg IRS-1 Ser→Ala/^+/+ (• with solid line), and WT/^+/+ ( with solid line); n = 7–10. *P < 0.05 vs. WT littermate mice.