Université catholique de Louvain, Institute of Experimental and Clinical Research, Pole of Endocrinology, Diabetes and Nutrition, Brussels, B-1200, Belgium.
Lund University, Department of Clinical Sciences in Malmö, Unit of Molecular Metabolism, Malmö, 205 02, Sweden.
Mol Metab. 2017 Apr 21;6(6):535-547. doi: 10.1016/j.molmet.2017.04.004. eCollection 2017 Jun.
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, Ca 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 HO at non-stimulating glucose. Surprisingly, the lack of NNT did not alter the glucose-stimulation of Ca influx and upstream mitochondrial events, but it markedly reduced both phases of GSIS by altering Ca-induced exocytosis and its metabolic amplification.
These results drastically modify current views on NNT operation and mitochondrial function in pancreatic β-cells.
胰腺 β 细胞的葡萄糖刺激胰岛素分泌（GSIS）严重依赖于代谢偶联因子的增加产生，包括 NADPH。烟酰胺核苷酸转氢酶（NNT）通常以 NADH 和能量化线粒体中的 ΔpH 为代价产生 NADPH。之前的研究表明，C57BL/6J 小鼠中 NNT 的自发失活会改变 ATP 产生、Ca2+内流和 GSIS，从而导致葡萄糖不耐受。在这里，我们测试了 NNT 在葡萄糖调节胰腺胰岛中线粒体 NADPH 和谷胱甘肽氧化还原状态中的作用，并重新研究了其在 GSIS 偶联事件中的作用。
从缺乏功能性 NNT 的雌性 C57BL/6J 小鼠（J-胰岛）和遗传上相近的 C57BL/6N 小鼠（N-胰岛）中分离胰岛。通过腺病毒感染表达野生型小鼠 NNT。用靶向或不靶向线粒体基质的谷氧还蛋白 1 融合 roGFP2 探针测量线粒体和胞质谷胱甘肽氧化。用生化方法测量 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偶联事件。
NNT 主要负责急性葡萄糖诱导的胰岛 NADPH/NADP 比升高和线粒体谷胱甘肽氧化降低，对胞质谷胱甘肽的影响较小。然而，与目前对 β 细胞中 NNT 的观点相反，这些效应是由于 NNT 反向模式操作中 NADPH 消耗的葡萄糖依赖性降低，而不是其正向模式操作的刺激。因此，J-胰岛中缺乏 NNT 降低了它们对非刺激葡萄糖下外源性 HO 的敏感性。令人惊讶的是，缺乏 NNT 并没有改变葡萄糖对 Ca2+内流和上游线粒体事件的刺激，但它通过改变 Ca2+诱导的胞吐作用及其代谢放大作用，显著减少了 GSIS 的两个阶段。
这些结果极大地改变了目前对 NNT 操作和胰腺 β 细胞中线粒体功能的看法。
