During the previous grant period a model of glucose induced insulin secretion was developed suggesting that oscillations in metabolism generate high and low signals in the ATP/ADP ratio that lead to an increase and oscillatory changes in cytosolic free Ca2+. Additionally, further metabolism of glucose leads to production of malonyl CoA which inhibits fatty acid (FFA) oxidation and elevates cytosolic long chain acyl CoA (LC-CoA). This model has been supported in numerous ways and proven to be useful in enhancing our understanding of secretion. Although many aspects of the model are now widely accepted, some need to be investigated more thoroughly and further advances must be made, such as the relationship of the model to physiologically relevant concentrations of glucose in conjunction with other modulators of secretion and the efflux of 40-50 percent of cellular phosphate. Elucidation is also needed on whether recruitment occurs with graded responses to glucose or all or none responses; and how oscillatory metabolism communicates with the exocytotic pathway. Challenges have arisen from evidence against a role for LC-CoA in glucose-induced insulin secretion and from mitochondrial studies implying a major role for mitochondrial Ca2+ in stimulating ATP production.
Aim 1 investigates how beta-cells are recruited to respond to graded increases in glucose. A microscope-based digital imaging system and self-referencing probes will be used to evaluate single beta-cells to determine whether parallel metabolic and ionic changes occur in the same cells. Small step-jumps in glucose will be performed and O2 consumption, membrane potential, insulin secretion and Ca2+ movements measured.
Aim 2 evaluates the role of LC-CoA in glucose-induced insulin secretion. We will test the effect of mutations on KATP channel activity. Studies will also attempt to reconcile apparent discrepancies in the role of LC-CoA in secretion.
Aim 3 will assess oscillations in translocation or phosphorylation of exocytotic proteins using a multiwell system to determine whether LC-CoA, phosphorylation or acylation oscillate. Translocation will be monitored using GFP- or BFP- tagged proteins.
Aim 4 will study the role of mitochondria and the phosphate flush in fuel-induced insulin secretion. In addition, the presence of the uncoupling protein, UCP-2, in beta-cells suggests a possible role for FFA in mitochondrial energetics. Experiments will measure the effect of FFA and the Ca2+ dependence of the changes in O2 consumption, membrane potential and pyridine and flavin nucleotide fluorescence. 31P-NMR analysis of samples taken at different times after stimulation of beta-cells by glucose will be used to identify the source of Pi.
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