Gene regulation, intracellular signaling and membrane traffic : Studies in primary human skeletal muscle cultures

Skeletal muscle is an insulin sensitive organ and plays a major role in whole body electrolyte and substrate homeostasis in the post prandial state. Impaired insulin action in skeletal muscle may lead to the pathological condition of insulin resistance. In this thesis primary human skeletal muscle cell culture (HSMC) has been used to investigate gene regulation, cellular signaling and membrane traffic. The expression of several myogenic and insulin responsive proteins, as well as insulin action, was followed in HSMC cultures as they differentiate from single cell myoblasts to mutlicellular myotubes. Insulin signaling to phosphatidyl inositol (PI) 3-kinase, extracellular regulated kinase (ERK) 1/2 mitogen activated protein kinase (MAPK) and protein kinase B (PKB) increased with differentiation. At the same time protein expression of PKBbeta, and ERK1/2 MAPK, and the insulin regulated glucose transported GLUT4, increased. In contrast, expression of GLUT1 decreased with differentiation. Compared to mature muscle, HSMC cultures express high levels of GLUT1. To differentiate between insulin effects on GLUT1 and GLUT4 membrane traffic we studied appearance of glucose transporter proteins at the plasma membrane using a specific photolabelling reagent. Using this technique we show that insulin increases plasma membrane content of GLUT4 but not GLUT1. On the other hand, GLUT1 content increases at the cell surface in response to serum stimulation. GLUT1 content in HSMC cells was specifically reduced using RNAi methodology. GLUT1 RNAi reduced GLUT1 content by 70%, and reduced serum-induced glucose uptake. The ability to specifically knock down proteins in primary human muscle cultures increases the utility of HSMC in the study of cellular signaling. Insulin also increases plasma membrane appearance of Na+, K+ATPase, as well as Na+, K+ATPase activity (determined by ouabain-suppressible 86Rb+ uptake) and specific [3H] ouabain binding in HSMC. We compared two different methods to study insulinmediated plasma membrane appearance of Na+, K+-ATPase alpha 1 and alpha 2 subunits. Using discontinuous sucrose gradients, insulin stimulation increased Na+, K+-ATPase alpha2 but not alpha 1 subunit in plasma membrane fraction. In contrast, when cell surface proteins were biotin labeled, we detected an insulinstimulated increase in both Na+, K+-ATPase a I a nd alpha2 s ubunits a t the c ell s urface. T hus insulin regulates membrane appearance of Na+, K+-ATPase alpha1 subunit, an effect which may previously have been overlooked due to technological limitations. Insulin-mediated regulation of Na+, K+-ATPase a subunits was determined in HSMC. In rodents a key regulatory role is played by protein kinase C phosphorylation of Ser23, however, this site is not present in human Na+, K+ATPase. We hypothesized insulin-mediated phosphorylation of Na+, K+-ATPase is dependent on ERK 1/2 signaling. Insulin-stimulated phosphorylation and translocation of Na+, K+-ATPase a was inhibited by the MEK1 inhibitor PD98059. Furthermore, purified ERK is able to directly phosphorylate Na+, K+-ATPase a subunits in vitro. Thus we conclude that insulin regulates Na+, K+ATPase via ERK dependent pathways. Insulin, AICAR, contraction and cellular stress increased the transcription factor myocyte enhancer factor 2 (MEF2) DNA binding activity in HSMC. Insulin-induced MEF2 DNA binding activity could be blocked using inhibitors against PI 3-kinase, PKC, p38- or MEK1. In contrast, AICAR mediated activation was only sensitive to the AMPK inhibitor compound C. Cellular stress-mediated activation of MEF2 DNA binding was sensitive to inhibition of p38 and MEK1, and partially sensitive to compound C. In isolated rat muscle, contraction-mediated activation of MEF2 DNA binding was sensitive to inhibition of p38 and MEK 1. In conclusion, GLUT4 mediates insulin-stimulated glucose uptake in primary cultures of human skeletal muscle, while GLUT1 primarily mediates serum induced glucose uptake. Differentiation of muscle cultures increases insulin-mediate activation of PI3-kinase, PKB, and ERK. We show that insulin activation of ERK is likely to be required for insulin induced activation of Na+, K+-ATPase and MEF2 DNA-binding. Thus although primary human skeletal muscle cell cultures differs in some aspects from mature skeletal muscle, it is a valuable and useful tool in the study of human cellular signaling and skeletal muscle research