MOLECULAR BASIS OF ANTIDIABETOGENIC HORMONE ACTION

胰高血糖素样肽-1（GLP-1）是一种有效的降糖药物， 刺激胰腺分泌胰岛素的激素， 细胞，并且其正在临床研究中用于治疗 非胰岛素依赖型糖尿病（NIDDM）。 该提案寻求 资助支持新发现的细胞内Ca 2+的研究 GLP-1激活原代培养大鼠的信号系统 pancratic β-细胞。 初步研究表明， GLP-1刺激[Ca 2 +]i的大幅上升， 和持续的成分，并通过激活一个 cAMP依赖的第二信使系统。 值得注意的是， cAMP介导的[Ca 2 +]i升高是已知的一种强有力的刺激， 胰岛素的分泌。 本建议的第一个具体目标是 确定是否响应GLP-1测量的[Ca 2 +]i瞬时升高 由于蛋白质的作用， 激酶A介导的兰尼碱受体磷酸化 细胞内Ca 2+释放通道。 第二个具体目标是 确定[Ca 2 +]i的持续升高是否由Ca 2+的流入引起， 并且如果是，确定哪种类型的离子通道是起作用的。 本文认为Ca ~（2+）的内流是由于Ca ~（2+）通道开放所致。 NS非选择性阳离子通道，产生内向膜 目前的IcAMP。 令人惊讶的是，这些渠道的开放不仅是为了回应 GLP-1，但也响应口服降糖磺酰脲类药物， 如格列本脲，用于治疗NIDDM。 因此 第三个具体目标是确定磺酰脲类药物的这种作用是否 由高亲和力磺酰脲受体（SUR）介导， CA-NS通道的跨膜传导调节剂。 实现这些 具体目的，[Ca 2 +]i和膜电流的测量将 使用Fura-2荧光分光光度法从大鼠β细胞中获得， 结合膜片钳电生理学。 我们的近期目标是 描绘GLP-1影响的信号转导途径 β细胞内Ca 2+稳态。 我们的长期目标是 为了确定这种Ca 2+信号系统的激活是否解释了 GLP-1治疗NIDDM的疗效。

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.