Ishihara H, Nakazaki M, Kanegae Y, Inukai K, Asano T, Katagiri H, Yazaki Y, Kikuchi M, Miyazaki J, Saito I, Oka Y
Institute for Adult Diseases, Asahi Life Foundation, Shinjuku-ku, Japan.
Diabetes. 1996 Sep;45(9):1238-44. doi: 10.2337/diab.45.9.1238.
The glycerol phosphate shuttle consists of FAD-linked mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) and its cytosolic NAD-linked isoform (cGPDH). Impaired mGPDH activity has recently been suggested to be one of the primary causes of insulin secretory defects in beta-cells. We found that mGPDH and cGPDH activities in MIN6 cells are comparable to those of isolated islets and higher than those in HIT cells by eightfold and threefold, respectively. Therefore, we selected the MIN6 cell line as a beta-cell model with normally regulated insulin secretion and normal shuttle enzyme activities and the HIT cell line as a beta-cell model with impaired insulin secretion and lower activities of these enzymes. The role of these dehydrogenases in glucose-stimulated insulin secretion was addressed by examining the effects of overexpression of mGPDH and/or cGPDH via recombinant adenoviruses in these cells. Infection with recombinant adenovirus with a cDNA encoding the Escherichia coli beta-galactosidase gene resulted in expression of its gene in 90% of MIN6 and HIT cells. Infection with a recombinant adenovirus with mGPDH cDNA (Adex1CAmGPDH) caused 2.1-fold and 5.7-fold increases in dehydrogenase activity as compared with those of control MIN6 and HIT cells, respectively. Infection with a recombinant adenovirus with cGPDH cDNA (Adex1CAcGPDH) caused a more than 50-fold increase in activity in both cell lines. Glycerol phosphate shuttle flux, as estimated by [2-3H]glycerol conversion to [3H]H2O, was increased to 120-130% by infection with Adex1CAmGPDH, but not with Adex1CAcGPDH infection, in both MIN6 and HIT cells. No further increase in flux through the glycerol phosphate shuttle was detected when the cells were infected with Adex1CAmGPDH together with Adex1CAcGPDH. Furthermore, neither [U-14C]glucose oxidation nor the insulin secretory response to glucose was affected in either cell line. Thus, mGPDH abundance in MIN6 and HIT cells is not directly related to their insulin secretory capacity in response to glucose, and reduced expression of mGPDH is not the primary cause of abnormal insulin secretory responses in HIT cells. The present data indicate that the emerging hypothesis pointing to mGPDH deficiency as a possible cause of NIDDM needs to be carefully evaluated.
磷酸甘油穿梭系统由与黄素腺嘌呤二核苷酸（FAD）相连的线粒体3-磷酸甘油脱氢酶（mGPDH）及其胞质中与烟酰胺腺嘌呤二核苷酸（NAD）相连的同工型（cGPDH）组成。最近有人提出，mGPDH活性受损是β细胞胰岛素分泌缺陷的主要原因之一。我们发现，MIN6细胞中的mGPDH和cGPDH活性与分离的胰岛相当，分别比HIT细胞中的活性高8倍和3倍。因此，我们选择MIN6细胞系作为胰岛素分泌正常调节且穿梭酶活性正常的β细胞模型，选择HIT细胞系作为胰岛素分泌受损且这些酶活性较低的β细胞模型。通过检测重组腺病毒介导的mGPDH和/或cGPDH过表达对这些细胞葡萄糖刺激的胰岛素分泌的影响，来探讨这些脱氢酶的作用。用编码大肠杆菌β-半乳糖苷酶基因的cDNA重组腺病毒感染后，其基因在90%的MIN6和HIT细胞中表达。与对照MIN6和HIT细胞相比，用携带mGPDH cDNA的重组腺病毒（Adex1CAmGPDH）感染后，脱氢酶活性分别增加了2.1倍和5.7倍。用携带cGPDH cDNA的重组腺病毒（Adex1CAcGPDH）感染后，两种细胞系的活性均增加了50倍以上。在MIN6和HIT细胞中，通过[2-³H]甘油转化为[³H]H₂O估算的磷酸甘油穿梭通量，用Adex1CAmGPDH感染后增加到120%-130%，而用Adex1CAcGPDH感染则无此效果。当细胞同时用Adex1CAmGPDH和Adex1CAcGPDH感染时，未检测到磷酸甘油穿梭通量的进一步增加。此外，两种细胞系中[U-¹⁴C]葡萄糖氧化及对葡萄糖的胰岛素分泌反应均未受影响。因此，MIN6和HIT细胞中mGPDH的丰度与其对葡萄糖的胰岛素分泌能力无直接关系，mGPDH表达降低不是HIT细胞胰岛素分泌异常反应的主要原因。目前的数据表明，认为mGPDH缺乏可能是2型糖尿病病因的新假说需要仔细评估。
