GLUCOSE-TRANSPORT AND PHOSPHORYLATION IN SINGLE CARDIAC MYOCYTES - RATE-LIMITING STEPS IN GLUCOSE-METABOLISM
GLUCOSE-TRANSPORT AND PHOSPHORYLATION IN SINGLE CARDIAC MYOCYTES - RATE-LIMITING STEPS IN GLUCOSE-METABOLISM
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DOI：
10.1152/ajpendo.1994.266.3.e326
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发表时间：
1994-03-01
影响因子：
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通讯作者：
LAWRENCE, JC
中科院分区：
文献类型：
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作者：
MANCHESTER, J;KONG, XM;LAWRENCE, JC
采用微量分析方法研究了胰岛素对成年大鼠心室单个肌细胞糖代谢的调节作用。培养的肌细胞用胰岛素或不加胰岛素孵育，用葡萄糖或8-脱氧葡萄糖（2-DG）孵育，冲洗，冷冻干燥。称量单个细胞，酶促扩增后测定2- dg -6-磷酸（2-DG-6-P）或葡萄糖和葡萄糖-6-磷酸（G-6-P）的水平。在用2-DG孵育的细胞中，胰岛素使2-DG-6- p的水平增加了30倍，这表明葡萄糖转运被显著激活。在与葡萄糖孵育的细胞中，胰岛素使G-6-P的水平增加了大约三倍。在不使用胰岛素的情况下增加细胞外葡萄糖也会增加G-6-P；然而，细胞内葡萄糖浓度没有增加，表明葡萄糖转运在未受刺激的肌细胞中受到速率限制。相反，胰岛素使细胞内葡萄糖浓度增加了一个数量级以上，达到细胞外葡萄糖浓度的60%。葡萄糖和G-6-P的测量显示，当细胞外葡萄糖增加0.2 mM时，G-6-P的积累达到平台。此时，估计细胞内葡萄糖浓度为300 μ M，约为葡萄糖己糖激酶Michaelis常数的10倍。这些结果表明，在胰岛素和葡萄糖的生理浓度存在下，己糖激酶饱和葡萄糖。因此，胰岛素刺激的葡萄糖利用的限速步骤是葡萄糖磷酸化而不是葡萄糖转运。
Microanalytic methods were used to investigate the regulation of glucose metabolism by insulin in single myocytes isolated from adult rat ventricles. Cultured myocytes were incubated with or without insulin and, with either glucose or 8-deoxyglucose (2-DG), rinsed, and freeze-dried. Individual cells were weighed and levels of 2-DG-6-phosphate (2-DG-6-P) or glucose and glucose 6-phosphate (G-6-P) were determined after enzymatic amplification. In cells incubated with 2-DG, insulin increased the level of 2-DG-6-P by as much as 30-fold, indicative of dramatic activation of glucose transport. In cells incubated with glucose, insulin increased the levels of G-6-P by approximately threefold. Increasing extracellular glucose without insulin also increased G-6-P; however, intracellular glucose concentrations were not increased, indicating that glucose transport is rate limiting in nonstimulated myocytes. In contrast, intracellular glucose concentrations were increased by over an order of magnitude by insulin, reaching 60% of the extracellular glucose concentration. Measurements of glucose and G-6-P in the same insulin-treated cells revealed that accumulation of G-6-P reached a plateau when extracellular glucose was increased >2 mM. At this point the estimated intracellular glucose concentration was 300 mu M, or approximately 10 times the Michaelis constant of hexokinase for glucose. These results indicate that in the presence of insulin and physiological concentrations of glucose, hexokinase is saturated with glucose. Consequently, the rate-limiting step for insulin-stimulated glucose utilization is glucose phosphorylation rather than glucose transport.