Stark Romana, Pasquel Francisco, Turcu Adina, Pongratz Rebecca L, Roden Michael, Cline Gary W, Shulman Gerald I, Kibbey Richard G
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
J Biol Chem. 2009 Sep 25;284(39):26578-90. doi: 10.1074/jbc.M109.011775. Epub 2009 Jul 27.
Pancreatic beta-cells couple the oxidation of glucose to the secretion of insulin. Apart from the canonical K(ATP)-dependent glucose-stimulated insulin secretion (GSIS), there are important K(ATP)-independent mechanisms involving both anaplerosis and mitochondrial GTP (mtGTP). How mtGTP that is trapped within the mitochondrial matrix regulates the cytosolic calcium increases that drive GSIS remains a mystery. Here we have investigated whether the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M) is the GTPase linking hydrolysis of mtGTP made by succinyl-CoA synthetase (SCS-GTP) to an anaplerotic pathway producing phosphoenolpyruvate (PEP). Although cytosolic PEPCK (PEPCK-C) is absent, PEPCK-M message and protein were detected in INS-1 832/13 cells, rat islets, and mouse islets. PEPCK enzymatic activity is half that of primary hepatocytes and is localized exclusively to the mitochondria. Novel (13)C-labeling strategies in INS-1 832/13 cells and islets measured substantial contribution of PEPCK-M to the synthesis of PEP. As high as 30% of PEP in INS-1 832/13 cells and 41% of PEP in rat islets came from PEPCK-M. The contribution of PEPCK-M to overall PEP synthesis more than tripled with glucose stimulation. Silencing the PEPCK-M gene completely inhibited GSIS underscoring its central role in mitochondrial metabolism-mediated insulin secretion. Given that mtGTP synthesized by SCS-GTP is an indicator of TCA flux that is crucial for GSIS, PEPCK-M is a strong candidate to link mtGTP synthesis with insulin release through anaplerotic PEP cycling.
胰腺β细胞将葡萄糖氧化与胰岛素分泌联系起来。除了经典的依赖ATP敏感性钾通道（K(ATP)）的葡萄糖刺激胰岛素分泌（GSIS）外，还存在重要的不依赖K(ATP)的机制，涉及回补反应和线粒体鸟苷三磷酸（mtGTP）。被困在线粒体基质中的mtGTP如何调节驱动GSIS的胞质钙增加仍是个谜。在这里，我们研究了磷酸烯醇式丙酮酸羧激酶的线粒体同工型（PEPCK-M）是否是一种GTP酶，它将琥珀酰辅酶A合成酶（SCS-GTP）产生的mtGTP水解与产生磷酸烯醇式丙酮酸（PEP）的回补途径联系起来。尽管不存在胞质PEPCK（PEPCK-C），但在INS-1 832/13细胞、大鼠胰岛和小鼠胰岛中检测到了PEPCK-M的信使核糖核酸和蛋白质。PEPCK的酶活性是原代肝细胞的一半，并且仅定位于线粒体。在INS-1 832/13细胞和胰岛中采用新的碳-13（(13)C）标记策略测量了PEPCK-M对PEP合成的重要贡献。在INS-1 832/13细胞中高达30%的PEP以及在大鼠胰岛中41%的PEP来自PEPCK-M。随着葡萄糖刺激，PEPCK-M对整体PEP合成的贡献增加了两倍多。沉默PEPCK-M基因完全抑制了GSIS，突出了其在线粒体代谢介导的胰岛素分泌中的核心作用。鉴于由SCS-GTP合成的mtGTP是对GSIS至关重要的三羧酸循环通量的指标，PEPCK-M是通过回补性PEP循环将mtGTP合成与胰岛素释放联系起来的有力候选者。
