Insulin signaling and glucose transport in insulin resistant human skeletal muscle

Abstract

Insulin resistance in skeletal muscle is a hallmark feature of Type 2 diabetes mellitus. The overall aim of this thesis was to investigate downstream intermediates in the insulin signaling pathway in an attempt to characterize the molecular mechanism of skeletal muscle insulin resistance in Type 2 diabetes. Skeletal muscle biopsies were obtained from healthy and Type 2 diabetic subjects before and after an in vivo hyperinsulinemic infusion. Insulin infusion increased the phosphorylation of several proteins reacting with a phospho-Akt substrate antibody. We focused on AS160, as this Akt substrate has been linked to glucose transport. A phosphorylated protein of 160 kDa was identified as AS160 using an AS160-specific antibody. Insulin-stimulated AS160 phosphorylation was reduced in Type 2 diabetic patients, whereas AS160 protein expression was similar between Type 2 diabetic and control subjects. Impaired AS160 phosphorylation was related to a reduction in Akt Thr308-phosphorylation.

To investigate whether pharmaceutical treatment improves glucose uptake due to enhanced insulin signaling in skeletal muscle, biopsies were obtained during a euglycemic hyperinsulinemic clamp from newly diagnosed Type 2 diabetic subjects before and after 26 weeks of metformin, rosiglitazone or placebo treatment. Insulin-mediated whole body and leg muscle glucose uptake was enhanced 36% and 32%, respectively, after rosiglitazone, but not after metformin or placebo treatment. Insulin-stimulated IRS-1 Tyr-phosphorylation, IRS-1 associated PI 3-kinse activity, Akt Ser473-phosphorylation, and AS160 phosphorylation were assessed. Gene expression of several targets involved in lipid and glucose metabolism was also determined. Insulin signaling parameters and gene expression were unaltered after metformin or rosiglitazone treatment, despite the improvement in glucose uptake.

Since an altered metabolic milieu may secondarily cause insulin resistance in Type 2 diabetic subjects, we studied a cohort of healthy glucose tolerant first-degree relatives of Type 2 diabetic patients to determine whether early metabolic and molecular defects contribute to insulin resistance in skeletal muscle. Skeletal muscle from first-degree relatives and control subjects were incubated in vitro in the absence or presence of increasing concentrations of insulin. Glucose transport, AS160 phosphorylation, and GLUT4 expression were assessed. Insulin-stimulated glucose transport rate at a maximal insulin concentration tended to be reduced in skeletal muscle from first-degree relatives. Insulin increased AS160 phosphorylation in a dose-dependent manner, with no difference between first-degree relatives and control subjects. A tight correlation was observed between insulin action on AS160 and glucose transport in control subjects, whereas a weak correlation was observed in first-degree relatives.

Tanis, a recently described putative receptor for serum amyloid A, has been implicated in inflammatory responses. We determined mRNA expression of the human homologue of Tanis, SelS/AD-015 in skeletal muscle and adipose tissue biopsies obtained from Type 2 diabetic patients and healthy subjects. Expression of Tanis/SelS mRNA in skeletal muscle and adipose tissue biopsies was similar between Type 2 diabetic and control subjects. Adipose tissue Tanis/SelS mRNA expression was unchanged after insulin infusion in control subjects, whereas Tanis/SelS mRNA increased following insulin stimulation in Type 2 diabetic subjects. Skeletal muscle and adipose tissue Tanis/SelS mRNA expression were positively correlated with plasma serum amyloid A.

In summary, defects in insulin action on AS160 may impair GLUT4 trafficking in Type 2 diabetes. Further, insulin-sensitizing effects of rosiglitazone are independent of enhanced insulin signaling of the IRS-1/PI 3-kinase/Akt/AS160 pathway in Type 2 diabetes, suggesting that improvements might be achieved at distal events at the level of GLUT4 translocation. In addition, healthy first-degree relatives of Type 2 diabetic subjects have a tendency of impaired glucose transport at a super-physiological insulin concentration, despite a normal insulin action on AS160, implicating early insulin signaling defects are likely to be a consequence of an altered metabolic milieu. Moreover, the positive correlation of Tanis/SelS mRNA expression and serum amyloid A suggests a potential interaction between immune system responses and Tanis/SelS expression in muscle and adipose tissue. In conclusion, future strategies designed to treat insulin resistance in human skeletal muscle may target multiple sites along insulin signaling and GLUT4 pathways depending on the progression of the disease.