Effects of thrombin on insulin signalling and glucose uptake in cultured human myotubes

Abstract

Background: Hyper-coagulability (elevated thrombin) is a feature of type 2 diabetes and contributes to an increased risk of thrombotic and vascular events. Skeletal muscle is the key peripheral tissue site of insulin resistance in type 2 diabetes. Cultured human skeletal muscle cells were used to explore the effects of thrombin on insulin signalling and glucose uptake. We hypothesized that thrombin affects insulin activity in human skeletal muscle cells which could link the hypercoagulability and insulin resistance in type 2 diabetes.

Methods: Human skeletal muscle cell cultures (myotubes) were treated with +/-5 units/ml thrombin for 6h. Insulin signalling pathway components and AMPK were examined by Western blotting. Real time PCR and glucose uptake assays were performed.

Results: There was a significant decrease (p<0.01) in insulin mediated IRS-1 and Akt phosphorylation in response to thrombin in cultured myotubes. Diminished Akt phosphorylation was alleviated by treatment with a PKC inhibitor. Thrombin directly increased basal glucose uptake (p<0.05) that involved AMPK phosphorylation (p<0.01) and this was partly repressed by compound C (AMPK inhibitor). Thrombin also significantly increased the gene expression level of both GLUT1 and GLUT4 in cultured human skeletal muscle cells.

Conclusion: Thrombin decreased insulin signalling in skeletal muscle cells through a PKC mediated mechanism, but did not affect the net action of insulin on glucose uptake. The direct stimulatory effect of thrombin on glucose uptake was mediated, at least in part, via an AMPK dependent mechanism. We conclude that thrombin activation results in multiple metabolic effects beyond increased thrombogenicity but does not include a decrease in insulin sensitivity (glucose uptake) in cultured human skeletal muscle cells.

Keywords: AMPK; Protein kinase C; Signal transduction; Skeletal muscle metabolism; Thrombin.