Effect of hyperglycemia on glucose transport and intracellular signal transduction in skeletal muscle

Abstract: Recent studies revealing the components of insulin signal transduction have raised the question of whether the defects in insulin signaling account for disturbed glucose metabolism in people with NIDDM. The major focus of this thesis is to elucidate the role of insulin signal transduction in the development of peripheral insulin resistance. Furthermore, since hyperglycemia is a main clinical future of diabetes mellitus and a known confounding factor of insulin resistance, the effect of hyperglycemia on signal transduction and glucose transport in skeletal muscle was determined. Skeletal muscle was obtained from lean-to-moderately obese NIDDM and control subjects after in vivo insulin-stimulation. Tyrosine-phosphorylation of insulin receptor substrate (IRS)-1 and phosphatidylinositol (PI) 3 kinase activity were significantly increased in muscle from control subjects whereas no appreciable increase was noted in muscle from NIDDM subjects. IRS-1 protein expression was similar between control and NIDDM subjects. Insulin-stimulated 3-O-methylglucose transport assessed in vitro was reduced by 40% in NIDDM muscle. In the Goto-Kakizaki (GK) rat, a non-obese model of NIDDM, defects in insulin signaling and glucose transport were observed in a muscle fiber type-specific manner. In oxidative (soleus) muscle, insulinstimulated IRS-1 phosphorylation and PI 3-kinase activity and glucose transport activity was reduced compared to Wistar rats. In glycolytic (extensor digitrum longus: EDL) muscle, insulin-stimulated IRS-1 phosphorylation and PI 3-kinase activity were not impaired, whereas submaximal insulin-stimulated glucose transport was impaired. Regardless of muscle fiber type, insulin-stimulated Akt kinase activity was impaired in GK rats. Correction of glycemia of in GK rats by phlorizin-treatment improved whole body glucose tolerance. Furthermore, insulin-stimulated Akt kinase and glucose transport activity was normalized regardless of muscle fiber type. This improvement was independent of restorations in early events of insulin signaling namely IRS-1 phosphorylation and PI 3-kinase activation. Acute elevation of extracellular glucose increased glucose transport activity. This increase was abolished by the dantroline, an inhibitor of Cae release from sarcoplasmic reticulum or by PKC inhibitors (H-7, GF109203X), but not by a phosphatidylinositol-specific PLC inhibitor (U72133). Western blot analysis revealed that PKC02 is a candidate isoform that functions in this pathway. Activation of PKC02 does not require glucose metabolism, as 3-O-methylglucose also activated PKC[beta]2. Stimulation of skeletal muscle with high glucose did not increase the cell surface GLUT I and GLUT4 content as assessed by the exofacial photolabeling technique. Although glucose has been reported to stimulate the mitogen-activated protein kinase (MAPK) cascade in several cells, an acute elevation of glucose did not induce phosphorylation of either extracellular signal regulated protein kinase 1/2 or p38 MAPK in skeletal muscle. In conclusion, insulin signaling is impaired in skeletal muscles from both human NIDDM and an animal model of NIDDM, suggesting these defects contribute to impaired glucose metabolism in NIDDM. Furthermore, Akt kinase and glucose transport, are sensitive to change in glycemia, suggesting level of glycemia modulates insulin signal transduction in skeletal muscle. In addition glucose transport is activated in response to an acute elevation in extracellular glucose, presumably via Ca2+/PKC[beta]2-dependent mechanism. Thus, high glucose is able to directly interact with skeletal muscle and modulate glucose transport and signal-transduction.