The first step in resolving abnormalities in insulin secretion is to understand how the beta-cell is normally stimulated by glucose to secrete insulin. Data obtained during the preceding grant period led us to propose a model linking oscillatory release of insulin from the pancreatic beta- cell with glucose metabolism. According to the model an increase in the ambient glucose concentration causes oscillatory changes in glucose phosphorylation and glycolysis leading to oscillations in the ATP/ADP ratio and O2 consumption. Peaks in ATP/ADP close ATP-sensitive K+-channels depolarizing the cell and causing opening of voltage regulated Ca2+- channels. As a consequence of the latter, oscillatory increases in cytosolic free Ca2+ occur in the beta-cell. A second change caused by enhanced glucose metabolism is an increase in malonyl CoA which leads to increases in diacylglycerol and cytosolic long chain acyl CoA. According to our model these may activate protein kinase C or cause the acylation of regulatory proteins. Finally, we have found that increases in glucose concentration increase the amplitude of oscillations in metabolism, cytosolic free Ca2+ and insulin release. This appears to be due to a synchronous recruitment of responding cells suggesting communication among cells and islets. The proposed studies will use methods and collaborative arrangements that have been established during the current grant period to test and extend the metabolic model and examine the phenomena of recruitment and synchronization. We will attempt to 1, substantiate that changes in the ATP/ADP ratio and O2 consumption precede those in cytosolic Ca2+; 2, identify the physiological changes in oscillating metabolites that regulate beta-cell ion channel activity; 3, establish whether isolated beta-cells are recruited to respond to glucose through cell contact, intracellular metabolites, or extracellular ions; 4, document how cells and islets are synchronized to respond to glucose in an oscillatory manner; 5, determine whether protein kinase C and GTP binding protein activity is regulated by acyl CoA esters in beta-cells; and 6, discover how fuels such as alpha-keto-isocaproic acid and glyceraldehyde cause ionic and metabolic changes in the beta-cell similar to those caused by glucose. These studies will address fundamental questions concerning metabolic events, electrical activity and Ca2+ levels in the framework of a complex model that includes both first and second phase insulin secretion.
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