Schuit F, De Vos A, Farfari S, Moens K, Pipeleers D, Brun T, Prentki M
Diabetes Research Center, Faculty of Medicine, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium.
J Biol Chem. 1997 Jul 25;272(30):18572-9. doi: 10.1074/jbc.272.30.18572.
Previous studies in rat islets have suggested that anaplerosis plays an important role in the regulation of pancreatic beta cell function and growth. However, the relative contribution of islet beta cells versus non-beta cells to glucose-regulated anaplerosis is not known. Furthermore, the fate of glucose carbon entering the Krebs cycle of islet cells remains to be determined. The present study has examined the anaplerosis of glucose carbon in purified rat beta cells using specific 14C-labeled glucose tracers. Between 5 and 20 mM glucose, the oxidative production of CO2 from [3,4-14C]glucose represented close to 100% of the total glucose utilization by the cells. Anaplerosis, quantified as the difference between 14CO2 production from [3,4-14C]glucose and [6-14C]glucose, was strongly influenced by glucose, particularly between 5 and 10 mM. The dose dependence of glucose-induced insulin secretion correlated with the accumulation of citrate and malate in beta(INS-1) cells. All glucose carbon that was not oxidized to CO2 was recovered from the cells after extraction in trichloroacetic acid. This indirectly indicates that lactate output is minimal in beta cells. From the effect of cycloheximide upon the incorporation of 14C-glucose into the acid-precipitable fraction, it could be calculated that 25% of glucose carbon entering the Krebs cycle via anaplerosis is channeled into protein synthesis. In contrast, non-beta cells (approximately 80% glucagon-producing alpha cells) exhibited rates of glucose oxidation that were (1)/(3) to (1)/(6) those of the total glucose utilization and no detectable anaplerosis from glucose carbon. This difference between the two cell types was associated with a 7-fold higher expression of the anaplerotic enzyme pyruvate carboxylase in beta cells, as well as a 4-fold lower ratio of lactate dehydrogenase to FAD-linked glycerol phosphate dehydrogenase in beta cells versus alpha cells. Finally, glucose caused a dose-dependent suppression of the activity of the pentose phosphate pathway in beta cells. In conclusion, rat beta cells metabolize glucose essentially via aerobic glycolysis, whereas glycolysis in alpha cells is largely anaerobic. The results support the view that anaplerosis is an essential pathway implicated in beta cell activation by glucose.
以往对大鼠胰岛的研究表明，回补反应在胰腺β细胞功能和生长的调节中起重要作用。然而，胰岛β细胞与非β细胞对葡萄糖调节的回补反应的相对贡献尚不清楚。此外，进入胰岛细胞三羧酸循环的葡萄糖碳的去向仍有待确定。本研究使用特定的14C标记葡萄糖示踪剂，检测了纯化的大鼠β细胞中葡萄糖碳的回补反应。在5至20 mM葡萄糖浓度范围内，[3,4-14C]葡萄糖氧化产生的CO2占细胞总葡萄糖利用率的近100%。以[3,4-14C]葡萄糖和[6-14C]葡萄糖产生的14CO2之间的差异来量化的回补反应，受到葡萄糖的强烈影响，尤其是在5至10 mM之间。葡萄糖诱导的胰岛素分泌的剂量依赖性与β（INS-1）细胞中柠檬酸和苹果酸的积累相关。所有未氧化为CO2的葡萄糖碳在经三氯乙酸提取后从细胞中回收。这间接表明β细胞中乳酸输出极少。根据环己酰亚胺对14C-葡萄糖掺入酸沉淀部分的影响，可以计算出通过回补反应进入三羧酸循环的葡萄糖碳中有25%进入蛋白质合成。相比之下，非β细胞（约80%产生胰高血糖素的α细胞）的葡萄糖氧化速率仅为总葡萄糖利用率的1/3至1/6，且未检测到葡萄糖碳的回补反应。这两种细胞类型之间的这种差异与β细胞中回补酶丙酮酸羧化酶的表达高7倍以及β细胞与α细胞相比乳酸脱氢酶与FAD连接的甘油磷酸脱氢酶的比例低4倍有关。最后，葡萄糖导致β细胞中磷酸戊糖途径的活性呈剂量依赖性抑制。总之，大鼠β细胞基本上通过有氧糖酵解代谢葡萄糖，而α细胞中的糖酵解在很大程度上是无氧的。这些结果支持了回补反应是葡萄糖激活β细胞所涉及的重要途径这一观点。
