Insulin signalling and TBC1D1 in skeletal muscle metabolism

Type 2 diabetes mellitus (T2DM) has taken the form of a pandemic disease globally as people adopt a more western lifestyle. High circulating glucose levels indicates that an individual is in the risk zone for developing T2DM. One of the hallmarks for T2DM is insulin resistance in skeletal muscle. Skeletal muscle is the primary target for insulin- stimulated glucose uptake. Thus, it is of clinical importance to understand of how the key hormone, insulin, affects the sensitivity of the tissues to maintain the rate of glucose uptake, and also how glucose uptake could be altered on a cellular level through metabolic switches. The aim for thesis is to investigate whether functional insulin signalling, and hence glucose uptake, occurs in incubated skeletal muscle specimens. Another aim of this thesis is to understand how the molecular switches TBC1D1 and NT5C1A are involved in the regulation of glucose uptake in skeletal muscle. In paper I, the canonical insulin signalling cascade is investigated in homogenates and cross-sections of skeletal muscle. Muscle specimens were incubated in vitro, which is a commonly used experimental technique, to measure glucose uptake and utilisation in skeletal muscle. We show that the canonical pathway of insulin signalling is activated throughout the muscle specimen due to insulin diffusion. We also validate the preparation for the study of insulin signalling. We provide evidence that the experimental approach is valid to assess insulin signalling. In paper II the effect of silencing the NT5C1A enzyme in skeletal muscle and NT5C2 in cultured myotubes and intact muscle was investigated. We hypothesised that AMPK could be increased if less AMP was hydrolysed as a result of a reduction in the expression of the NT5-enzyme. We demonstrate that in myotubes grown from human biopsies, as well as in mouse tibialis anterior muscle, NT5C silencing increases phosphorylation of AMPK and ACC through changes in the AMP:ATP ratio to thereby increase gluco