Glucagon plays a key role in blood glucose homeostasis and abnormalities in glucagon secretion are evident in diabetes mellitus. To resolve the abnormalities in glucagon secretion, it is first necessary to understand how alpha-cells are regulated by glucose. The candidate has initiated a new line of investigation to study the regulation of alpha-cell glucagon secretion. Preliminary experiments with a clonal alpha-cell line (alphaTC- 6) indicate that it is a good model cell type to study alpha-cell function. Furthermore, these studies have revealed that glucose and amino-acids regulate alpha-cell glucagon secretion and have led to the novel finding that KATP channels are present in alphaTC-6 cells. The planned research will extend these observations and test the following model: that at low glucose concentrations, alpha-cell catabolism via non-glycolytic pathways raises ATP levels, or ATP/ADP ratios, causing closure of KATP channels and voltage-dependent Ca2+ influx into alpha-cells. The resulting elevation in free cytosolic Ca2+ levels triggers glucagon secretion. It is postulated that a switch to carbohydrate metabolism as glucose levels rise, reduces steady-state ATP levels and glucagon secretion. To test these hypotheses, the aims of the project are to: l) further characterize the dynamics and modulation of glucose-regulated glucagon secretion, 2) determine if metabolically-regulated KATP channels are involved in glucose-signal transduction in alpha-cells, 3) determine if changes in glucose alter free cytosolic Ca2+ in alpha-cells, and 4) examine alpha-cell catabolism at different glucose levels. alphaTC-6 cell studies will be complemented using native islet alpha cells purified by cell sorting. Methods will include measurement of glucagon secretion using static incubations, perifusions, and radioimmunoassay; the patch-clamp technique for ion channel recordings; measurement of free cytosolic Ca2+ by fura-2 fluorescence; other standard biochemical methods to study alpha-cell metabolism.