Studies of the amylin signalling pathway in skeletal muscle with respect to carbohydrate metabolism

Abstract

Skeletal muscle accounts for up to 85% of glucose disposal following ingestion or glucose infusion and is the most important tissue involved in maintaining euglycaemia. Resistance to the action of insulin on glucose metabolism in skeletal muscle has been proposed as an early defect in the aetiology of type 2 diabetes, and experiments have demonstrated that amylin can evoke peripheral insulin resistance by antagonizing insulin action in a noncompetitive manner in vitro and in vivo. This study was designed to better elucidate the mechanism by which amylin regulates the metabolism of carbohydrate in skeletal muscle. Since amylin-mediated suppression of insulin-stimulated glycogen synthesis in skeletal muscle is a key action of amylin, it was determined to focus this study around the pathway of glycogen synthesis. All experiments were performed on isolated skeletal muscle incubated in vitro, in preparations whose viability was confirmed. Treatment with amylin at a concentration of 10 nM did not significantly influence glucose uptake. However, this concentration of amylin evoked a half-maximal inhibition of glycogen synthesis as determined by incorporation of D-[U-14C]-glucose into muscle glycogen. Therefore, amylin mediated glucose transport is not of primary importance in causing insulin resistance. These results also indicate that amylin-mediated glycogenolysis, leading to increased skeletal muscle glucose-6-phosphate, is likely to be sufficient to suppress glucose transport by its inhibitory effects on hexokinase. Amylin and CGRP evoked comparable effects on glucose uptake, however the effects of amylin were more potent than those of CGRP, judged by its EC50 (0.6 and 17.2 nM respectively). The antagonistic effect of 8-37 CGRP on amylin mediated glucose uptake suggests that inhibition of insulin-stimulated glucose transport by amylin might be l) mediated via a specific amylin receptor as well as a CGRP receptor 2) that 8-37 CGRP is a more effective blocker than 8-37 amylin at this receptor. It was found that CGRP is more potent than amylin with respect to effects on total glycogen content and de novo glycogen synthesis. The order of potencies in decreasing total glycogen content was sCT > CGRP > amylin, in the presence and absence of insulin. With respect to the inhibition of de novo glycogen synthesis, the order of potencies in the absence of insulin was sCT > CGRP > amylin and in the presence of insulin CGRP = sCT > amylin. Studies of concentration-dependence of hormone-evoked cAMP content revealed amylin's ability to increase [cAMP] at a concentration of 100 nM. This effect was completely reversed by insulin. In contrast, CGRP- mediated effects on [cAMP] were detected at 0.1 nM and were independent of insulin. Treatment of skeletal muscle with a maximally effective amylin concentration evoked a 58% decrease in protein phosphatase-l activity, as well as translocation of its catalytic subunit from the soluble to the particulate fraction. These results could explain, at least in part, the increased glycogenolysis seen with amylin. However, the effect of amylin on glycogen synthesis de novo, analyzed indirectly by testing glycogen synthase kinase 3 activity, did not show any effect of amylin on total glycogen synthase kinase 3. Therefore, these results suggest that amylin inhibition of glycogen synthase (mediated via increased phosphorylation of the important sites 3 a,b,c) is not the result of increased kinase activity, but rather decreased protein phosphatase 1 activity, which is the only glycogen synthase phosphatase present in skeletal muscle.