Chronic Reduction of the Cytosolic or Mitochondrial NAD(P)-malic Enzyme Does Not Affect Insulin Secretion in a Rat Insulinoma Cell Line
Chronic Reduction of the Cytosolic or Mitochondrial NAD(P)-malic Enzyme Does Not Affect Insulin Secretion in a Rat Insulinoma Cell Line
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DOI：
10.1074/jbc.m109.040394
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发表时间：
2009-12-18
影响因子：
4.8
通讯作者：
MacDonald, Michael J.
中科院分区：
文献类型：
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作者：
Brown, Laura J.;Longacre, Melissa J.;MacDonald, Michael J.
细胞质苹果酸酶（ME1）已被认为通过苹果酸-丙酮酸和/或柠檬酸-丙酮酸穿梭体，通过产生NADPH或其他代谢物来增加胰岛素分泌。我们使用表达短发夹RNA （short hairpin RNA, shRNA）的可选载体，在ins - 1832 /13胰岛素瘤细胞系中稳定地降低了Me1 mRNA水平80-86%，Me1酶活性78-86%。与已发表的短期ME1敲除实验相反，我们的长期靶细胞在葡萄糖或谷氨酰胺加2-氨基双环[2,2,1]庚烷-2-羧酸的作用下显示出正常的胰岛素分泌。我们发现胞质内异柠檬酸脱氢酶或葡萄糖-6-磷酸脱氢酶的mrna和酶活性没有增加，这两种酶也会产生胞质内NADPH。线粒体苹果酸酶Me2或Me3的mrna没有代偿性诱导。在早期小干扰RNA实验中诱导的干扰素通路基因在长期shRNA实验中未被诱导。我们用含有Tol2转位序列的改进载体重复了我们的研究，以产生更高的稳定转移率和缩短测试时间，但这并没有改变结果。同样，我们使用稳定表达的shRNA将线粒体NAD(P)-苹果酶（Me2） mRNA降低了95%，Me2蛋白严重降低，酶活性降低了90%。胰岛素对葡萄糖或谷氨酰胺加2-氨基双环[2,2,1]庚烷-2-羧酸的释放保持正常。胰岛素瘤细胞中丙酮酸循环和/或胞质内NADPH生成通路的冗余性与降低其中任何一种苹果酸酶的表达后维持强劲的胰岛素分泌是一致的。
The cytosolic malic enzyme (ME1) has been suggested to augment insulin secretion via the malate-pyruvate and/or citrate-pyruvate shuttles, through the production of NADPH or other metabolites. We used selectable vectors expressing short hairpin RNA (shRNA) to stably decrease Me1 mRNA levels by 80-86% and ME1 enzyme activity by 78-86% with either of two shRNAs in the INS-1 832/13 insulinoma cell line. Contrary to published short term ME1 knockdown experiments, our long term targeted cells showed normal insulin secretion in response to glucose or to glutamine plus 2-aminobicyclo[2,2,1]heptane-2- carboxylic acid. We found no increase in the mRNAs and enzyme activities of the cytosolic isocitrate dehydrogenase or glucose-6-phosphate dehydrogenase, which also produce cytosolic NADPH. There was no compensatory induction of the mRNAs for the mitochondrial malic enzymes Me2 or Me3. Interferon pathway genes induced in preliminary small interfering RNA experiments were not induced in the long term shRNA experiments. We repeated our study with an improved vector containing Tol2 transposition sequences to produce a higher rate of stable transferents and shortened time to testing, but this did not alter the results. We similarly used stably expressed shRNA to reduce mitochondrial NAD(P)-malic enzyme (Me2) mRNA by up to 95%, with severely decreased ME2 protein and a 90% decrease in enzyme activity. Insulin release to glucose or glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid remained normal. The maintenance of robust insulin secretion after lowering expression of either one of these malic enzymes is consistent with the redundancy of pathways of pyruvate cycling and/ or cytosolic NADPH production in insulinoma cells.