Investigation of the effects of thrombin and electrical pulse stimulation on metabolic function in cultured human skeletal muscle cells

Abstract

Hyper-coagulability (elevated thrombin) is a feature of T2D. There is an emerging evidence of a correlation between the genetic basis of hypercoagulation and T2D. We hypothesized that thrombin affects insulin activity and/or exercise responses in human skeletal muscle cells which could link the hypercoagulability and insulin resistance in T2D. Furthermore, we hypothesized that the metabolic benefits of exercise are decreased in cultured muscle cells from T2D patients. Cultured human myotubes were used aiming to explore the effects of thrombin on insulin signalling and glucose uptake as well as the effect of thrombin on metabolic function in the presence and absence of EPS as an in vitro model of exercise. Furthermore, to explore the effects of EPS on metabolic function in muscle cell cultures derived from T2D and non-diabetic control subjects. The findings of the first three chapters of this thesis demonstrated that thrombin was shown to have multiple metabolic effects represented by a decrease in insulin stimulated IRS1 and Akt activation which was mediated through PKCα, but thrombin had no effect on the parallel insulin-stimulated aPKC and AS160 pathway. Thrombin directly increased glucose uptake through an AMPK mediated mechanism. Furthermore, the increase in AMPK activity, elevation of glucose uptake and the rise in cytokine release in response to EPS (above basal values) that were noted with non-thrombin treated myotubes was lost upon thrombin treatment. The key findings of the last chapter there were, AMPK activation and glucose uptake increased in response to EPS in control myotubes, and EPS enhanced the effect of insulin on glucose uptake and distal insulin signalling pathway (AS160). In diabetic myotubes, EPS did not increase AMPK activation and glucose uptake, nor enhanced the action of insulin. Thus, hypercoagulation associated with diabetes could be involved in multiple metabolic effects in skeletal muscle including insulin signalling, exercise signalling, proinflammatory pathways, and glucose uptake. There is an intrinsic defect in diabetic myotubes represented by defective AMPK, glucose uptake and distal insulin signalling in response to EPS that are consistent with the changes observed in vivo.