The goal has been and remains elucidation of the mechanisms of hormones and other effectors in the regulation of hepatic glycogen metabolism and of the defects in glycogen metabolism associated with the diabetic state or severe insulin deficiency. The hypotheses to be tested are that insulin and some other hormones facilitate hepatic glycogen synthesis chronically by maintenance of glycogenic enzymes in a responsive state, and that hepatic glycogen synthase is acutely regulated by insulin and glucose through changes in the phosphorylation state of specific serine residues. The premise is that a complete understanding of hepatic defects in glycogen synthesis observed in the diabetic state will lead to a better understanding of the mechanics of insulin action.
Specific aims are to 1) Study diabetic defects and the acute and chronic regulation of glycogen synthetic enzymes using the primry adult rat hepatocyte culture system. 2) Produce polyclonal antibodies to rat liver glycogen synthase to be used to study the acute effects of insulin and glucose on the phosphorylation state and chronic control of synthase. 3) Determine specific amino acid sequences around phosphorylation sites in rat liver synthase, and to produce site specific antibodies to be used for determination of site specific phosphorylation/dephosporylation as effected by glucose and insulin. 4) Produce antibodies to PP-1 and PP-2C (phosphatase-2C), if necessary, to be used to determine if one of these is the physiologically meaningful form of glycogen synthase phosphatase. 5) Determine if glycogen synthase phosphatase is physically missing in the diabetic state or is in some way altered so that synthase is no longer a good substrate. 6) Determine if synthase phosphtase is regulated acutely, and if so, how. and 7) Identify a clonal hepatic cell line which closely mimics our normal cell line so that future mechanistic studies can be carried out in a manipulatable system not dependent upon animal sacrifice. Results of these studies should clarify the roles of hormonal interactions in diabetes-related abnormalities in hepatic glycogen metabolism, and should clarify the mechanistic roles of insulin and glucose in acute regulation of hepatic glycogen synthase. It is hoped that these studies will eventually help to improve the prognosis for human diabetics.
