In vivo studies of stimulus-secretion coupling and glucose metabolism dynamics
The research in our lab focuses on molecular events involved in glucose metabolism and insulin secretion in beta cells within intact functioning pancreatic islets. We have developed unique, state-of-the-art fluorescence imaging methods to assay the response at various points along the glucose transduction pathway. The quantitative microscopy is combined with standard biochemical and molecular biological techniques to obtain unique information that bridges the gap between the known details of the glucose transduction pathway in individual beta cells and the overall glucose response of a whole islet. As glucose is metabolized by the beta cell, there is a rise in NAD(P)H that can be monitored by its autofluorescence. Imaging of the temporal and spatial pattern of NAD(P)H has revealed that individual beta cells within the islet form a much more uniform population than was expected from studies of dissociated cells. Glucokinase dominates the rate of glucose signal transduction by beta cells, and we are investigating its allosteric regulation by protein-protein interactions with nitric oxide synthase. This interaction has not been found in vitro, but is considerable in vivo. We are also developing methods to correlate metabolic behavior to beta cell function by imaging cytoplasmic calcium activity, membrane potential, intracellular pH, and secretion of fluorescent insulin. Recently proposed models suggest that gap junctions and/or K+ channels play a role in insulin secretion from whole islets. Our new methods allow us to test these models' predictions, and thus permit determination of the important cell-cell communication mechanisms in the islet.