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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK052166-03
Application #
2905958
Study Section
Metabolism Study Section (MET)
Program Officer
Laughlin, Maren R
Project Start
1997-05-01
Project End
2001-04-30
Budget Start
1999-05-01
Budget End
2000-04-30
Support Year
3
Fiscal Year
1999
Total Cost
Indirect Cost
Name
New York University
Department
Physiology
Type
Schools of Medicine
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10016
Leech, Colin A; Chepurny, Oleg G; Holz, George G (2010) Epac2-dependent rap1 activation and the control of islet insulin secretion by glucagon-like peptide-1. Vitam Horm 84:279-302
Holz, George G (2004) Epac: A new cAMP-binding protein in support of glucagon-like peptide-1 receptor-mediated signal transduction in the pancreatic beta-cell. Diabetes 53:5-13
Holz, George G; Chepurny, Oleg G (2003) Glucagon-like peptide-1 synthetic analogs: new therapeutic agents for use in the treatment of diabetes mellitus. Curr Med Chem 10:2471-83
Kang, Guoxin; Holz, George G (2003) Amplification of exocytosis by Ca2+-induced Ca2+ release in INS-1 pancreatic beta cells. J Physiol 546:175-89
Kang, Guoxin; Joseph, Jamie W; Chepurny, Oleg G et al. (2003) Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP as a stimulus for Ca2+-induced Ca2+ release and exocytosis in pancreatic beta-cells. J Biol Chem 278:8279-85
Chepurny, Oleg G; Hussain, Mehboob A; Holz, George G (2002) Exendin-4 as a stimulator of rat insulin I gene promoter activity via bZIP/CRE interactions sensitive to serine/threonine protein kinase inhibitor Ro 31-8220. Endocrinology 143:2303-13
Chepurny, Oleg G; Holz, George G (2002) Over-expression of the glucagon-like peptide-1 receptor on INS-1 cells confers autocrine stimulation of insulin gene promoter activity: a strategy for production of pancreatic beta-cell lines for use in transplantation. Cell Tissue Res 307:191-201
Kang, G; Chepurny, O G; Holz, G G (2001) cAMP-regulated guanine nucleotide exchange factor II (Epac2) mediates Ca2+-induced Ca2+ release in INS-1 pancreatic beta-cells. J Physiol 536:375-85
Skoglund, G; Hussain, M A; Holz, G G (2000) Glucagon-like peptide 1 stimulates insulin gene promoter activity by protein kinase A-independent activation of the rat insulin I gene cAMP response element. Diabetes 49:1156-64
Holz, G G; Leech, C A; Habener, J F (2000) Insulinotropic toxins as molecular probes for analysis of glucagon-likepeptide-1 receptor-mediated signal transduction in pancreatic beta-cells. Biochimie 82:915-26

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