MOLECULAR BASIS OF ANTIDIABETOGENIC HORMONE ACTION

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Glucagon-like peptide-1 (GLP-1) is a potent blood glucose-lowering hormone that stimulates the secretion of insulin from pancreatic beta- cells and which is under clinical investigation for use in treatment of non-insulin-dependent diabetes mellitus (NIDDM). This proposal seeks funding to support studies of a newly discovered intracellular Ca2+ signaling system that is activated by GLP-1 in primary culture of rat pancratic beta-cells. Preliminary studies are presented demonstrating that GLP-1 stimulates a large rise of [Ca2+]i that consists of transient and sustained components, and which is triggered by activation of a cAMP-dependent second messenger system. This is noteworthy because a cAMP-mediated rise of [Ca2+]i is known to be a powerful stimulus for secretion of insulin. A first Specific Aim of this proposal is to determine if the transient rise of [Ca2+]i measured in response to GLP-1 results from mobilization of Ca2+ stores as a consequence of protein kinase A-mediated phosphorylation of ryanodine receptor (RYR) intracellular Ca2+ release channels. A second Specific Aim is to determine if the sustained rise of [Ca2+]i results from influx of Ca2+, and if so, to determine what type(s) of ion channels are responsible. Here it is proposed that influx of Ca2+ results from the opening of Ca- NS nonselective cation channels that generate the inward membrane current IcAMP. Surprisingly, these channels open not only in response to GLP-1, but also in response to oral hypoglycemic sulfonylureas such as glyburide that are prescribed for treatment of NIDDM. Therefore, a third Specific Aim is to determine if this action of sulfonylureas is mediated by a high affinity sulfonylurea receptor (SUR) acting as a transmembrane conductance regulator of CA-NS channels. To achieve these Specific Aims, measurements of [Ca2+]i and membrane current will be obtained from rat beta-cells using fura-2 spectrofluorimetry in combination with patch clamp electrophysiology. Our immediate goal is to delineate the signal transduction pathways by which GLP-1 influence intracellular Ca2+ homeostasis in the beta-cell. Our long term goal is to determine if activation of this Ca2+ signaling system explains the therapeutic efficacy of GLP-1 for treatment of NIDDM.