AbstractsBiology & Animal Science

This thesis focuses on the control of glucose uptake and protein synthesis in electrically stimulated rat skeletal muscle incubated ex vivo. The study is divided into two parts: the first part aims to investigate the role of phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) in the regulation of contraction-stimulated glucose uptake, while the second part focuses on the regulation of translation factors and signaling components that control protein synthesis during muscle contraction. Skeletal muscle represents the major site for plasma glucose disposal. Both insulin action and muscle contraction lead to an increase in glucose uptake in muscle. In Type 2 diabetic patients, insulin-stimulated glucose uptake in skeletal muscle is impaired, while contraction-stimulated glucose uptake is unaffected. Thus, a better understanding of the signaling pathways involved in contraction-induced glucose uptake will be of great value to find potential drug targets for treating Type 2 diabetes. PIKfyve is a lipid kinase that phosphorylates PtdIns3P to PtdIns(3,5)P2, implicated in insulin-stimulated GLUT4 translocation. In this study, we found that inhibition of PIKfyve by a selective inhibitor or by siRNA knockdown decreased contraction- or 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside (AICA riboside, an AMPK activator)-stimulated glucose uptake. Moreover, contraction increased muscle PtdIns(3,5)P2 levels along with PIKfyve phosphorylation. PIKfyve was phosphorylated by AMPK at Ser307 both in vitro and in intact cells. By subcellular fractionation and vital confocal imaging experiments, we demonstrated that PIKfyve phosphorylation was associated with its recruitment to PtdIns3P-enriched endosomes where it can catalyze PtdIns(3,5)P2 synthesis. We conclude that PIKfyve activity is important for contraction/AMPK activation -stimulated glucose uptake in skeletal muscle. PIKfyve phosphorylation by AMPK could favor its recruitment to endosomal membrane and thus increase PtdIns(3,5)P2 to facilitate GLUT4 translocation. Maintenance of skeletal muscle mass is of prime importance for good health. Exercise or muscle contraction is an effective way to maintain and increase muscle mass. The build-up of muscle mass depends on protein synthesis. Indeed, rates of protein synthesis significantly increase following exercise, likely due to the activation of mTORC1 (mammalian target of rapamycin complex 1) signaling. By contrast, protein synthesis is blocked during contraction, mainly due to the inhibition of translation elongation. Previous studies primarily focused on signaling pathways that control protein synthesis during post-exercise recovery, via mTORC1 and its downstream effectors S6K1 and 4E-BP1. Less attention has been paid to these signaling pathways during exercise or contraction. In the second part of the thesis, the changes in phosphorylation state of S6K1 and 4E-BP1 and other signaling components that control protein synthesis during high frequency contraction in skeletal muscle were studied. We found that S6K1 Thr389…