Impaired anaplerosis and insulin secretion in insulinoma cells caused by small interfering RNA-mediated suppression of pyruvate carboxylase
Impaired anaplerosis and insulin secretion in insulinoma cells caused by small interfering RNA-mediated suppression of pyruvate carboxylase
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DOI：
10.1074/jbc.m804170200
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发表时间：
2008-10-17
影响因子：
4.8
通讯作者：
MacDonald, Michael J.
中科院分区：
文献类型：
--
作者：
Hasan, Noaman M.;Longacre, Melissa J.;MacDonald, Michael J.
由胰腺β细胞线粒体合成的柠檬酸循环中间体，被认为对胰岛素分泌和线粒体能量产生同样重要。然而，旨在降低β细胞骨质疏松率的研究尚无定论。为了验证过敏性反应对胰岛素分泌有重要作用的假设，我们降低了β细胞中过敏性反应的主要酶丙酮酸羧化酶（PC）的活性。通过稳定转染短发夹RNA，我们获得了大量的ins - 1832 /13衍生细胞系，这些细胞系具有不同水平的PC酶活性，但保持了正常水平的对照酶、胰岛素含量和葡萄糖氧化。葡萄糖诱导的胰岛素释放与PC活性的降低成比例地减少。单独丙酮酸、2-氨基双环[2,2,1]庚烷-2-羧酸（BCH）加谷氨酰胺或琥珀酸甲酯加β -羟基丁酸的胰岛素释放也在PC敲低细胞中减少。与PC的阻滞一致，大多数缺乏PC的细胞在PC表现出代谢交叉点，增加基础和/或葡萄糖刺激的丙酮酸和乳酸，减少苹果酸和柠檬酸。此外，在BCH +谷氨酰胺刺激的PC敲低细胞中，丙酮酸和乳酸升高，柠檬酸严重降低，苹果酸和天冬氨酸轻微降低。在PC敲除的细胞中，[U-14C]葡萄糖的脂质中C-14的掺入减少。这些结果证实了PC和anaerosis对葡萄糖代谢产物的中心重要性，这些代谢产物支持胰岛素分泌，甚至表明PC胰岛素分泌受到非碳水化合物胰岛素分泌剂的刺激。
Anaplerosis, the synthesis of citric acid cycle intermediates, by pancreatic beta cell mitochondria has been proposed to be as important for insulin secretion as mitochondrial energy production. However, studies designed to lower the rate of anaplerosis in the beta cell have been inconclusive. To test the hypothesis that anaplerosis is important for insulin secretion, we lowered the activity of pyruvate carboxylase ( PC), the major enzyme of anaplerosis in the beta cell. Stable transfection of short hairpin RNA was used to generate a number of INS-1 832/13-derived cell lines with various levels of PC enzyme activity that retained normal levels of control enzymes, insulin content, and glucose oxidation. Glucose-induced insulin release was decreased in proportion to the decrease in PC activity. Insulin release in response to pyruvate alone, 2-aminobicyclo[ 2,2,1] heptane-2-carboxylic acid (BCH) plus glutamine, or methyl succinate plus beta-hydroxybutyrate was also decreased in the PC knockdown cells. Consistent with a block at PC, the most PC-deficient cells showed a metabolic crossover point at PC with increased basal and/or glucose-stimulated pyruvate plus lactate and decreased malate and citrate. In addition, in BCH plus glutamine-stimulated PC knockdown cells, pyruvate plus lactate was increased, whereas citrate was severely decreased, and malate and aspartate were slightly decreased. The incorporation of C-14 into lipid from [U-14C] glucose was decreased in the PC knockdown cells. The results confirm the central importance of PC and anaplerosis to generate metabolites from glucose that support insulin secretion and even suggest PC insulin secretion stimulated by noncarbohydrate insulin secretagogues.