Calcium Sensitivy of β-cell Transcription Factor Binding to an Insulin Enhancer

Gary Frank Scott, Calcium Sensitivity of β-cell Transcription Factor Binding to an Insulin Enhancer. Master of Science (Biochemistry and Molecular Biology), June 1998, 104 pp., 16 illustrations, bibliography, 94 titles. Insulin is an essential hormone and is produced exclusively in endocrine pancreas β-cells for the control of glucose homeostasis in mammals. The hypothesis tested in this thesis is that increased intracellular Ca2+ ([Ca2+]i) contributes to activation of glucose-induced insulin gene transcription. Glucose-induced insulin transcription has been mapped to binding of transcription factors by β-cell sequence motifs from -197 to -247, a glucose-response-enchancer (GRE), in the rat insulin1 gene (rINS1) promoter. Using oligonucleotide probes representing this glucose-response-enhancer (GRE) in electrophorectic mobility shift assays (EMSA), we have examined the Ca2+-sensitivity of transcription factor binding to nuclear extracts from cultured rat insulinoma β-cells (INS-1). In the presence or absence of kinase inhibitors, Ca2+ chelators, and Ca2+ channel blockers, binding was assayed for the following cell conditions: 1) in situ permeabilized cells exposed to Ca2+; 2) in vitro 32p-phosphorylated nuclear extracts; and 3) in situ glucose-stimulated and K+-depolarized intact cells. Binding was Ca2+-sensitive due to activation by K+depolarization as well as inhibition by a Ca2+-chelator, a Ca2+-channel blocker, and KN-93, specific for Ca2+/calmodulin kinases, suggesting a phosphorylation-dependent mechanism. Taken together, these findings identify a role for the Ca2+ second messenger in the glucose regulation of the insulin gene which points to novel treatments for type II diabetes.