OBJECTIVE-Glucagon-like peptide-1 (GLP-1) increases intracellular Ca 2ϩ concentrations ([Ca 2ϩ ] i ), resulting in insulin secretion from pancreatic ␤-cells. The molecular mechanism(s) of the GLP-1-mediated regulation of [Ca 2ϩ ] i was investigated. RESEARCH DESIGN AND METHODS- GLP-1-induced changes in [Ca 2ϩ ] i were measured in ␤-cells isolated from Cd38 ϩ/ϩ and Cd38 Ϫ/Ϫ mice. Calcium-mobilizing second messengers were identified by measuring levels of nicotinic acid adenine dinucleotide

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... ate (NAADP) and cyclic ADP-ribose (ADPR), using a cyclic enzymatic assay. To locate NAADP-and cyclic ADPRproducing enzyme(s), cellular organelles were separated using the sucrose gradient method. RESULTS-A GLP-1-induced [Ca 2ϩ ] i increase showed a cooperative Ca 2ϩ signal, i.e., an initial [Ca 2ϩ ] i rise mediated by the action of NAADP that was produced in acidic organelles and a subsequent long-lasting increase of [Ca 2ϩ ] i by the action of cyclic ADPR that was produced in plasma membranes and secretory granules. GLP-1 sequentially stimulated production of NAADP and cyclic ADPR in the organelles through protein kinase A and cAMP-regulated guanine nucleotide exchange factor II. Furthermore, the results showed that NAADP production from acidic organelles governed overall Ca 2ϩ signals, including insulin secretion by GLP-1, and that in addition to CD38, enzymes capable of synthesizing NAADP and/or cyclic ADPR were present in ␤-cells. These observations were supported by the study with Cd38 Ϫ/Ϫ ␤-cells, demonstrating production of NAADP, cyclic ADPR, and Ca 2ϩ signal with normal insulin secretion stimulated by GLP-1. CONCLUSIONS-Our findings demonstrate that the GLP-1mediated Ca 2ϩ signal for insulin secretion in pancreatic ␤-cells is a cooperative action of NAADP and cyclic ADPR spatiotemporally formed by multiple enzymes.