Glucose produces, through glycolysis, pyruvate, a substrate for mitochondrial metabolism. Mitochondrial metabolism generates signals for insulin exocytosis, which include ATP and other as yet unidentified molecules. Although important roles of mitochondrial metabolism of glucose for insulin secretion is established, roles of glycolysis are not completely understood. Does glycolysis merely produce pyruvate for mitochondrial metabolism or generate its own signal? To study this question, we have created mitochondrial pyruvate carrier 2 (Mpc2) knockout MIN6 cells by the Crispr technology. We first generated MIN6 cells expressing MPC2-Flag under the control of Tet3G transcriptional activator, to assure mitochondrial metabolism of glucose in a condition of Mpc2 alleles being knocked out. Then, stop codons followed by the zeocin or puromycin-resistant gene units were introduced in the second exons of the gene. We have confirmed a complete loss of endogenous MPC2 expression by Western blot analyses and these cells were maintained in the presence of doxycycline, expressing MPC2-Flag. Upon withdrawal of doxycycline, glycolytic flux estimated by 5-[3H]glucose utilization was unaltered at 5 mM glucose but reduced by 37.2% ± 1.2% (Mean ± SE, n = 3, p < 0.05) at 12.5 mM and 55.3% ± 9.6% (p < 0.05) at 20 mM. In MPC2-deficient MIN6 cells, glucose-stimulated insulin secretion was completely abolished at 12.5 and 20 mM glucose, while glutamine (5 mM) plus leucine (5 mM) induced insulin secretion was comparable to that by doxycycline-treated MPC2-Flag expressing cells. Interestingly, when MPC2-deficient MIN6 cells were activated with glutamine plus leucine, 5-20 mM glucose was able to cause further dose-dependent increases in insulin secretion with 1.9 ± 0.3-fold (n = 4, p < 0.05) augmentation at 20 mM. These data suggested that glycolysis generates signals for insulin secretion distinct from those produced by the mitochondria in glucose-stimulated insulin secreting cells.
H. Ishihara: Advisory Panel; Self; Astellas Pharma Inc. Research Support; Self; Daiichi Sankyo, Eli Lilly Japan K.K., Kowa Company, Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi. H. Nishioka: None. M. Yamana: None. A. Nagasawa: None. M. Kosuda: None. M. Koike: None. G. Kohno: None. H. Saito: None.