MacDonald M J, Kaysen J H, Moran S M, Pomije C E
Department of Pediatrics, University of Wisconsin Medical School, Madison 53706.
J Biol Chem. 1991 Nov 25;266(33):22392-7.
It has been shown previously that glucose-induced insulin release is completely absent in rat pancreatic islets that had been cultured for 1 day at low glucose (1 mM) and that it is restored by culturing islets for a 2nd day at high (20 mM) glucose (MacDonald, M. J., Fahien, L. A., McKenzie, D. I., and Moran, S. M. (1991) Am. J. Physiol. 259, E548-E554). It has been suggested that the incapacitation of glucose's insulinotropism is due to down-regulation of the synthesis of enzymes that process glucose's metabolic signal for insulin release. In the current study, results of metabolic, enzymic, and molecular biologic experiments were each consistent with (an) intramitochondrial site(s) of down-regulation in islets cultured at low glucose. Glucose metabolism was inhibited 80% in islets cultured at 1 mM glucose. The suppression of release of 14CO2 from [6-14C]glucose greater than from [U-14C]glucose greater than [3,4-14C]glucose greater than from [1-14C]glucose in islets cultured at low glucose indicated a mitochondrial site of down-regulation because C-6 of glucose can only be converted to CO2 in the citric acid cycle, whereas C-1 can be released as CO2 in the 6-phosphogluconate dehydrogenase [corrected] reaction, and C-6 of glucose dwells in the citric acid cycle longer than carbons 2-5 of glucose. Since carbons 3 and 4 of glucose can be decarboxylated in the pyruvate dehydrogenase reaction, incomplete suppression of CO2 formation from these carbons is consistent with suppression of pyruvate carboxylation as well as decarboxylation. Formation of 3HOH from [5-3H]glucose was equal in the two groups of islets, indicating that glycolysis as far as phosphoenolpyruvate was intact. This idea was supported by assays which showed that activities of enzymes of the glycolytic pathway between glucokinase/hexokinase and pyruvate kinase were equal in both types of islets. Additional studies indicated that regulation by glucose was at transcription of genes coding for some mitochondrial enzymes. Glucokinase, malic enzyme, and fumarase mRNAs were not affected by glucose, whereas the pyruvate dehydrogenase E1 alpha subunit and pyruvate carboxylase mRNAs were decreased 85-90% in islets cultured at 1 mM glucose. Pyruvate dehydrogenase enzyme activity was decreased to a similar extent in these islets. About 24 h was required for maximal (de)induction of pyruvate dehydrogenase E1 alpha and pyruvate carboxylase mRNAs, and the amounts of transcripts were proportional to the concentrations of glucose between 1 and 20 mM.(ABSTRACT TRUNCATED AT 400 WORDS)
先前的研究表明，在低葡萄糖（1 mM）条件下培养1天的大鼠胰岛中，葡萄糖诱导的胰岛素释放完全缺失，而在高葡萄糖（20 mM）条件下再培养1天，胰岛素释放可恢复（MacDonald, M. J., Fahien, L. A., McKenzie, D. I., and Moran, S. M. (1991) Am. J. Physiol. 259, E548 - E554）。有人提出，葡萄糖促胰岛素分泌作用丧失是由于处理葡萄糖代谢信号以释放胰岛素的酶合成下调。在当前研究中，代谢、酶学和分子生物学实验结果均与低葡萄糖培养的胰岛中线粒体内的下调位点一致。在1 mM葡萄糖培养的胰岛中，葡萄糖代谢被抑制了80%。低葡萄糖培养的胰岛中，[6 - 14C]葡萄糖释放14CO2的抑制程度大于[U - 14C]葡萄糖、大于[3,4 - 14C]葡萄糖、大于[1 - 14C]葡萄糖，这表明存在线粒体下调位点，因为葡萄糖的C - 6只能在柠檬酸循环中转化为CO2，而C - 1可在6 - 磷酸葡萄糖酸脱氢酶反应中释放为CO2，且葡萄糖的C - 6在柠檬酸循环中的停留时间比葡萄糖的C - 2 - 5长。由于葡萄糖的C - 3和C - 4可在丙酮酸脱氢酶反应中脱羧，这些碳的CO2形成未被完全抑制，这与丙酮酸羧化以及脱羧的抑制一致。两组胰岛中[5 - 3H]葡萄糖生成3H OH的量相等，表明糖酵解直至磷酸烯醇丙酮酸是完整的。这一观点得到了测定结果的支持，测定结果显示，在两种类型的胰岛中，葡萄糖激酶/己糖激酶和丙酮酸激酶之间糖酵解途径的酶活性相等。进一步的研究表明，葡萄糖的调节作用发生在编码某些线粒体酶的基因转录水平。葡萄糖激酶、苹果酸酶和延胡索酸酶的mRNA不受葡萄糖影响，而在1 mM葡萄糖培养的胰岛中，丙酮酸脱氢酶E1α亚基和丙酮酸羧化酶的mRNA减少了85 - 90%。在这些胰岛中，丙酮酸脱氢酶的酶活性也下降到了类似程度。丙酮酸脱氢酶E1α和丙酮酸羧化酶mRNA的最大（去）诱导需要约24小时，且转录本的量与1至20 mM之间的葡萄糖浓度成正比。（摘要截选至400字）
