Diabetologia:功能性烟酰胺核苷酸转氢酶的缺乏仅对糖耐量和葡萄糖刺激的胰岛素分泌的损害有中等程度的作用

2021-10-15 从医路漫漫 MedSci原创

烟酰胺核苷酸转氢酶(NNT)参与线粒体NADPH的产生,其自发失活突变(NntTr[Tr,截断])通常被认为是C57BL/6J与C57BL/6N小鼠糖耐量降低的主要原因。

目的/假设:烟酰胺核苷酸转氢酶(NNT)参与线粒体NADPH的产生,其自发失活突变(NntTr[Tr,截断])通常被认为是C57BL/6J与C57BL/6N小鼠糖耐量降低的主要原因。然而,这种突变对葡萄糖耐量的影响仍然存在争议。在这里,我们从C57BL/6J和C57BL/6N小鼠之间的其他遗传变异中挑出NntTrn对线粒体谷胱甘肽氧化还原状态(EGSH)、葡萄糖刺激的胰岛素分泌(GSIS)和糖耐量的影响。

方法:将N5BL/6JNntWT/Tr杂合子小鼠与N5BL/6J小鼠杂交,获得在C57BL/6J遗传背景上表达野生型Nnt(NntWT)或rNntTr的雄性和雌性N5BL/6J小鼠(N5-WT和N5-Tr小鼠)。C57BL/6J和C57BL/6N小鼠均来自Janvier实验室。每种类型小鼠各1只,经PCR和全基因组测序确定Nnt基因型。糖耐量测定采用IPGTT、ITT和禁食/再喂养试验。在分离的胰岛上测量刺激-分泌耦合事件和GSIS。用GRX1-roGFP2(谷胱甘肽1融合到氧化还原敏感型增强型GFP)荧光探针检测胞浆和线粒体EGSH。

结果:确定了各型小鼠的Nnt基因型和遗传背景。在C57BL/6N和C57BL/6J胰岛中,N5-WT与N5-Tr胰岛相比,N5-WT和N5-Tr胰岛对线粒体(但不是胞浆)EGSH和NAD(P)H自发荧光的葡萄糖调节作用明显增强,证实了NNT在线粒体氧化还原调节中的作用。然而,在体外,N5-WT胰岛的GSIS仅是N5-Tr胰岛的1.2-1.4倍,而C57BL/6N胰岛的GSIS是N5-WT胰岛的2.4倍,这对NNT在GSIS中的作用提出了质疑。在体内,N5-WT和N5-Tr或C57BL/6N小鼠的ITT结果没有差异。然而,雌性N5-WT小鼠在IPGTT过程中的血糖漂移仅比N5-Tr和C57BL/6J小鼠低15-20%,并且仍然是雌性C57BL/6N小鼠的3.5倍。在禁食/再喂养试验中也进行了类似的观察。在男性中,NNT对糖耐量的影响略大(~30%)。

图1 葡萄糖和FCCP对雌性N5-WT和N5-Tr小鼠胰岛线粒体和胞质谷胱甘肽氧化的影响。感染Ad-mt-GRX1-roGFP2(a,b)或a d GRX1-roGFP2(c)两天后,将胰岛与含有各种葡萄糖浓度(Gn=nmmol/l葡萄糖),15μmol/l H2O2或10μmol/l FCCP。将痕量标准化为在10mmol/l二硫苏糖醇(设定为0%)存在下最大还原探针的比例和在100μmol/l三硫醇(设定为100%)存在下最大氧化探针的比例,如ESM图6a所示。数据是11个胰岛/6个制剂(a)的平均值±SD;5胰岛/3制剂(b);或7个胰岛/4制剂(c)**p<0.01***与之前的条件或vs G30相比,p<0.001;†††与N5-WT胰岛相比,p<0.001。电子补充材料[ESM]中显示了各个迹线。

图2 NNT对雌性小鼠糖耐量的影响。(a,b;c,d;和i,j)N5-Tr,N5-WT,BL/6N和BL/6J小鼠隔夜禁食后IPGTT期间的血糖水平和相应的AUC。(e,f)测定N5-WT和N5-Tr小鼠IPGTT期间禁食状态(0min)和注射葡萄糖后30min的血糖和血浆胰岛素水平。(g,h)N5-WT和N5Tr小鼠ITT期间的血糖水平,以及前30分钟内曲线的相应斜率。小鼠在3到6个月大的时候使用。每种类型的8只(a,b)、5只(c,d)、5只(e-h)和4只(i,j)小鼠的数据为均值±SD或均值±SD,并带有单独的数据点。(B,d,h,j)采用非配对双尾t-t t检验,N5-WT或BL/6N小鼠†p<0.0 5,†††p<0.001,组间差异有统计学意义(P<0.0 5)。(E)**p˂0.0 1和*p<0.001 vs时间0;†p˂0.0 5 vs N5-WT小鼠。(F)全局时间效应经双因素方差分析(后检验)*p<0.05。各个轨迹如图所示

表 在这项研究中测试的六种类型的小鼠在IPGTT期间的AUC

结论:虽然我们的结果证实了NNT在葡萄糖调节线粒体氧化还原状态中的重要性,但它们明显削弱了NNT在C57BL/6J与C57BL/6N小鼠GSIS和糖耐量改变中的作用。因此,记录C57BL/6小鼠的NntWT型并不能证明它们的糖耐量和C57BL/6N小鼠一样好。

原文出处:

Close AF,  Chae H,  Jonas JC,The lack of functional nicotinamide nucleotide transhydrogenase only moderately contributes to the impairment of glucose tolerance and glucose-stimulated insulin secretion in C57BL/6J vs C57BL/6N mice.Diabetologia 2021 Aug 27

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time=2021-10-17, status=1, ipAttribution=)]
  5. 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  6. 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time=2021-10-17, status=1, ipAttribution=)]
    2022-06-12 baoya
  7. 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    2021-10-17 fengxx

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