Insulin resistance differentially affects the PI 3-kinase- and MAP kinase-mediated signaling in human muscle

Abstract

The broad nature of insulin resistant glucose metabolism in skeletal muscle of patients with type 2 diabetes suggests a defect in the proximal part of the insulin signaling network. We sought to identify the pathways compromised in insulin resistance and to test the effect of moderate exercise on whole-body and cellular insulin action. We conducted euglycemic clamps and muscle biopsies on type 2 diabetic patients, obese nondiabetics and lean controls, with and without a single bout of exercise. Insulin stimulation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway, as measured by phosphorylation of the insulin receptor and IRS-1 and by IRS protein association with p85 and with PI 3-kinase, was dramatically reduced in obese nondiabetics and virtually absent in type 2 diabetic patients. Insulin stimulation of the MAP kinase pathway was normal in obese and diabetic subjects. Insulin stimulation of glucose-disposal correlated with association of p85 with IRS-1. Exercise 24 hours before the euglycemic clamp increased phosphorylation of insulin receptor and IRS-1 in obese and diabetic subjects but did not increase glucose uptake or PI 3-kinase association with IRS-1 upon insulin stimulation. Thus, insulin resistance differentially affects the PI 3-kinase and MAP kinase signaling pathways, and insulin-stimulated IRS-1-association with PI 3-kinase defines a key step in insulin resistance.

Figure 1

Representative immunoblots. Top panel: Effect of insulin infusion in lean, obese nondiabetic, and type 2 diabetic subjects on insulin receptor (a) and IRS-1 (b) tyrosine phosphorylation, and the association of p85 with IRS-1 (c). Phosphotyrosine content of specific proteins was determined by immunoprecipitation with insulin receptor or IRS-1 antibodies followed by immunoblot analysis with antiphosphotyrosine antibodies. The association of p85 with IRS-1 was determined by immunoblot analysis of anti–IRS-1 immunoprecipitates. Bottom panel: Effect of insulin infusion in lean, obese nondiabetic, and type 2 diabetic subjects on PI 3-kinase activity associated with IRS-1 (d) or p110 (e). PI 3-kinase activity was determined as described in the text in anti–IRS-1 immunoprecipitates; effect of insulin infusion on phosphorylation of ERK, determined by immunoblot analysis with an antiphosphoERK antibody (f). pTyr, phosphotyrosine.

Figure 2

Effect of exercise on subsequent insulin stimulation of insulin receptor (a) and IRS-1 (b) tyrosine phosphorylation. Obese (n = 7) and diabetic (n = 10) subjects underwent euglycemic clamps with muscle biopsies twice: 1 time without prior exercise and again 24 hours after a single 1-hour bout of moderate exercise. Homogenates of muscle biopsy specimens were immunoprecipitated with antibodies directed against the insulin receptor β subunit or IRS-1. Immunoprecipitated proteins were resolved on polyacrylamide gels, transferred to nitrocellulose membranes, probed with an antiphosphotyrosine antibody, and detected by chemiluminescence, and the digitized images were quantified. Basal values are shown as open bars, and insulin-stimulated values are shown as filled bars. Data were normalized to insulin receptor β subunit or IRS-1 protein content (determined on separate immunoblots) and are expressed as percent of the mean insulin-stimulated value in the lean control subjects. Data are presented as means ± SEM. *P < 0.05 versus basal values.

Figure 3

Effect of exercise on subsequent insulin stimulation of the association of p85 protein (a) and PI 3-kinase activity (b) with IRS-1. Obese (n = 7) and diabetic (n = 10) subjects underwent euglycemic clamps with muscle biopsies twice: 1 time without prior exercise and again 24 hours after a single 1-hour bout of moderate exercise. Homogenates of muscle biopsy specimens were immunoprecipitated with an antibody directed against IRS-1. Immunoprecipitates were alternatively analyzed for p85 protein by subsequent immunoblot analysis or for PI 3-kinase activity (for details see the text). Basal values are shown as open bars, and insulin-stimulated values are shown as filled bars. Data were expressed relative to IRS-1 content and are given as percent of the mean insulin-stimulated value in the lean control subjects. Data are presented as means ± SEM. *P < 0.05 versus basal values; ^†P < 0.05 versus lean control values.

Figure 4

Effect of exercise on insulin stimulation of ERK phosphorylation (a), ERK activity (b), and MEK activity (c). Obese (n = 6) and diabetic (n = 6) subjects underwent euglycemic clamps with muscle biopsies twice: 1 time without prior exercise and again 24 hours after a single 1-hour bout of moderate exercise. Homogenates of muscle biopsy specimens were analyzed for ERK phosphorylation by immunoblot analysis using an antiphosphoERK antibody. ERK activity was determined as described in the text using anti-ERK immunoprecipitates, and MEK activity was assayed using anti-MEK immunoprecipitates. Basal values are shown as open bars, and insulin-stimulated values are shown as filled bars. Data are presented as means ± SEM. *P < 0.05 versus basal values. ^†P < 0.05 versus lean control values.