DEVELOPMENT OF NOVEL PANCREATIC BETA CELL MODELS

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DESCRIPTION (taken from the application) Insulin production in the islets of Langerhans is regulated by Multiple hormonal, neuronal and paracrine factors acting through interconnected signal transduction pathways. The intracellular concentration of free Ca2+ in the cytoplasm is an important determinant of insulin secretion. We propose that the concentration of Ca2+ in the endoplasmic reticulum (ER) is also important in regulation of the pancreatic beta cell, and that the distinct patterns of Ca2+ responses evoked by glucose and other stimuli are translated differentially into down stream responses. We will develop novel methods to determine how regulators of insulin production affect Ca2+ concentrations in the ER and how changes in cellular Ca2+ levels are translated into a prototypic response, activation of calmodulin. Two classes of fluorescent indicators, both containing two spectral variants of green fluorescent protein, will be expressed in beta cells and fluorescence resonance energy transfer (FRET) will be measured by fluorescence microscopy. One set of reporters, the cameleons, shows changes in FRET upon Ca2+ calmodulin binding. We will transfect MIN6 cells with Ca2+ sensing cameleons targeted to the ER, YCer3 and Ycer4, and determine: (1) how the concentration of Ca2+ in the ER changes in response to activation of a G protein-coupled receptor linked to phospholipase C, (2) whether high glucose release Ca2+ from the ER when it is added alone, with carbachol or with vasoactive intestinal peptide, and (3) whether changes in ER Ca2+ are restricted to certain regions of the ER. We will also introduce the fluorescence target FIP-CBsm into MIN6 cells and use FRET to measure the extent of Ca2+-calmodulin binding. We will use this model to determine the spatial and temporal patterns of Ca2+-calmodulin activation when beta cells are: (1) exposed to stimuli that cause release of Ca2+ from intracellular stores and those that cause Ca2+ influx through voltage-operated Ca2+ channels, and 2) exposed to stimuli that cause sustained versus oscillatory increases in the cytoplasmic free Ca2+ concentration.