2179-P: Overexpression Screening Identifies Mitochondrial Carbonic Anhydrase 5b as an Important Regulator of Glucose-Stimulated Insulin Secretion in Beta Cells

Abstract

Despite extensive research efforts, a precise picture of how glucose itself and its metabolites initiates insulin secretion in a concentration-dependent manner has unveiled. To identify candidate genes important for glucose-stimulated insulin secretion (GSIS), we have conducted screening of differentially expressed genes identified by microarray analyses between highly glucose-responsive and less responsive MIN6 subclones. For this purpose, a platform containing zeocin resistant gene flanked with a wild type and mutated flippase recognition target sites was integrated in the genome of MIN6 cells expressing Tet3G. The modified MIN6 cells have been used to generate stable transfectants expressing mRNA or shRNA of genes of interest by means of the recombinease-mediated cassette exchange method. Using this system, more than ten stable MIN6 cell transformants can be gnenerated at a time. By creating approximately 100 overexpressing transfectants, we have identified mitochondrial carbonic anhydrase 5b (Car5b) as an important regulator of GSIS. Car5b is a one of 15 members of carbonic anhydrases, which catalyze the interconversion of carbon dioxide and water to bicarbonate and protons, and modulate various intracellular processes, such as urea formation, HCO3- supply for carboxylation and regulation of intracellular acid-base balance. Car5b overexpression in MIN6 cells increased 14CO2 formation from H14CO3- (37 ± 14% increases, p < 0.05) in isolated mitochondria and resulted in 32 ± 12% increases (P < 0.05) in GSIS at 20 mM, while shCar5b expression reduced Car5b protein levels to 31.4 ± 4.4% (p < 0.05), 14CO2 formation to 68.7 ± 8.1% (p < 0.05), and GSIS at 20 mM to 77 ± 5% (p < 0.05). In addition, we found that Car5b expressions in ob/ob mice were 3.7 ± 0.4-fold higher (p < 0.01) and tended to be higher (p = 0.073) in HFD-mice. These data indicated that mitochondrial carbonic anhydrase Car5b plays a role in GSIS in pancreatic β-cells. Disclosure M. Kosuda: None. S. Yamaguchi: None. A. Nagasawa: None. M. Yamana: None. M. Koike: None. H. Nishioka: None. H. Ishihara: Research Support; Self; Daiichi Sankyo Company, Limited, Lilly Diabetes. Speaker's Bureau; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Kyowa Hakko Kirin Co., Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited.