NNT reverse mode of operation mediates glucose control of mitochondrial NADPH and glutathione redox state in mouse pancreatic β-cells.
NNT reverse mode of operation mediates glucose control of mitochondrial NADPH and glutathione redox state in mouse pancreatic β-cells.
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DOI：
10.1016/j.molmet.2017.04.004
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发表时间：
2017-06
影响因子：
8.1
通讯作者：
Jonas JC
中科院分区：
文献类型：
--
作者：
Santos LRB;Muller C;de Souza AH;Takahashi HK;Spégel P;Sweet IR;Chae H;Mulder H;Jonas JC
葡萄糖刺激胰腺β细胞的胰岛素分泌（GSIS）主要依赖于代谢偶联因子（包括NADPH）的增加。烟酰胺核苷酸转氢酶（NNT）通常以NADH和ΔpH为代价在激活的线粒体中产生NADPH。在C57BL/6J小鼠中，其自发失活被证明会改变ATP的产生、Ca2+内流和GSIS，从而导致葡萄糖耐受不良。在这里，我们测试了NNT在线粒体NADPH和谷胱甘肽氧化还原状态的葡萄糖调节中的作用，并重新研究了其在小鼠胰岛GSIS偶联事件中的作用。从缺乏功能性NNT的雌性C57BL/6J小鼠（J-islets）和基因相近的C57BL/6N小鼠（N-islets）中分离出胰岛。野生型小鼠通过腺病毒感染在j型胰岛中表达NNT。用glutaredoxin - 1融合的roGFP2探针检测线粒体和细胞质谷胱甘肽氧化，这些探针靶向或不靶向线粒体基质。采用生化法测定NADPH和NADH氧化还原状态。通过标准程序在动态或静态条件下测量胰岛素分泌和上游耦合事件。NNT主要负责急性葡萄糖诱导的胰岛NADPH/NADP+比值升高和线粒体谷胱甘肽氧化降低，对胞质谷胱甘肽影响较小。然而，与目前关于β-细胞中NNT的观点相反，这些影响是由于NNT反向运作模式导致NADPH消耗的葡萄糖依赖性减少，而不是刺激其正向运作模式。因此，j型胰岛缺乏NNT降低了它们在非刺激性葡萄糖下对外源性H2O2的敏感性。令人惊讶的是，缺乏NNT并没有改变Ca2+内流和上游线粒体事件的葡萄糖刺激，但它通过改变Ca2+诱导的胞吐及其代谢扩增显着减少了GSIS的两个阶段。这些结果极大地改变了目前对胰腺β细胞NNT操作和线粒体功能的看法。
The glucose stimulation of insulin secretion (GSIS) by pancreatic β-cells critically depends on increased production of metabolic coupling factors, including NADPH. Nicotinamide nucleotide transhydrogenase (NNT) typically produces NADPH at the expense of NADH and ΔpH in energized mitochondria. Its spontaneous inactivation in C57BL/6J mice was previously shown to alter ATP production, Ca2+ influx, and GSIS, thereby leading to glucose intolerance. Here, we tested the role of NNT in the glucose regulation of mitochondrial NADPH and glutathione redox state and reinvestigated its role in GSIS coupling events in mouse pancreatic islets. Islets were isolated from female C57BL/6J mice (J-islets), which lack functional NNT, and genetically close C57BL/6N mice (N-islets). Wild-type mouse NNT was expressed in J-islets by adenoviral infection. Mitochondrial and cytosolic glutathione oxidation was measured with glutaredoxin 1-fused roGFP2 probes targeted or not to the mitochondrial matrix. NADPH and NADH redox state was measured biochemically. Insulin secretion and upstream coupling events were measured under dynamic or static conditions by standard procedures. NNT is largely responsible for the acute glucose-induced rise in islet NADPH/NADP+ ratio and decrease in mitochondrial glutathione oxidation, with a small impact on cytosolic glutathione. However, contrary to current views on NNT in β-cells, these effects resulted from a glucose-dependent reduction in NADPH consumption by NNT reverse mode of operation, rather than from a stimulation of its forward mode of operation. Accordingly, the lack of NNT in J-islets decreased their sensitivity to exogenous H2O2 at non-stimulating glucose. Surprisingly, the lack of NNT did not alter the glucose-stimulation of Ca2+ influx and upstream mitochondrial events, but it markedly reduced both phases of GSIS by altering Ca2+-induced exocytosis and its metabolic amplification. These results drastically modify current views on NNT operation and mitochondrial function in pancreatic β-cells.