Nagamatsu S, Nakamichi Y, Sawa H
Department of Biochemistry, Kyorin University School of Medicine, Tokyo, Japan.
Am J Physiol. 1995 Aug;269(2 Pt 1):C480-6. doi: 10.1152/ajpcell.1995.269.2.C480.
It was previously reported that insulin biosynthesis in mouse beta TC3 cells was regulated by glucose (Nagamatsu, S., and D. F. Steiner. Endocrinology 130: 748-754, 1992). In the present study, we examined the effect of glucose on the glucose transporter expression and hexokinase activities and determined the relationship between them and glucose-stimulated insulin biosynthesis in beta TC3 cells. Reverse transcriptase-polymerase chain reaction and Northern blot analysis revealed that beta TC3 cells expressed GLUT-1 and GLUT-3 glucose transporter mRNAs, but not GLUT-2. The levels of GLUT-1 and GLUT-3 mRNAs were not affected by glucose (0 or 11 mM glucose) over a period of 48 h. Immunoprecipitation of metabolically labeled beta TC3 cells with specific antibodies against GLUT-1 or GLUT-3 proteins revealed no effect of glucose on the biosynthesis of glucose transporters. Hexokinase [low Michaelis constant (Km) hexokinase] activity from cells incubated in 11 mM glucose for 48 h increased nearly twofold compared with cells maintained in 0 mM glucose, although the amount of cellular hexokinase protein detected by immunoblot analysis was unchanged between 0 and 11 mM glucose conditions. Glucokinase (high Km hexokinase) activity, in contrast, was not affected by glucose. Preincubation of beta TC3 cells with 2-deoxyglucose to inhibit hexokinase, thereby inhibiting all glycolysis, resulted in the decrease of glucose-stimulated insulin biosynthesis. Thus, in mouse beta TC3 cells that do not express GLUT-2, there is a close relationship between hexokinase activity and glucose-stimulated insulin biosynthesis, but not between the glucose transporter and glucose-stimulated insulin biosynthesis.
先前有报道称，小鼠β TC3细胞中的胰岛素生物合成受葡萄糖调节（永松，S.，和D. F. 施泰纳。《内分泌学》130: 748 - 754，1992）。在本研究中，我们检测了葡萄糖对葡萄糖转运蛋白表达和己糖激酶活性的影响，并确定了它们与β TC3细胞中葡萄糖刺激的胰岛素生物合成之间的关系。逆转录聚合酶链反应和Northern印迹分析显示，β TC3细胞表达GLUT - 1和GLUT - 3葡萄糖转运蛋白mRNA，但不表达GLUT - 2。在48小时内，GLUT - 1和GLUT - 3 mRNA的水平不受葡萄糖（0或11 mM葡萄糖）的影响。用针对GLUT - 1或GLUT - 3蛋白的特异性抗体对代谢标记的β TC3细胞进行免疫沉淀，结果显示葡萄糖对葡萄糖转运蛋白的生物合成没有影响。与在0 mM葡萄糖中培养的细胞相比，在11 mM葡萄糖中培养48小时的细胞的己糖激酶[低米氏常数（Km）己糖激酶]活性增加了近两倍，尽管通过免疫印迹分析检测到的细胞己糖激酶蛋白量在0和11 mM葡萄糖条件下没有变化。相比之下，葡萄糖激酶（高Km己糖激酶）活性不受葡萄糖影响。用2 - 脱氧葡萄糖预孵育β TC3细胞以抑制己糖激酶，从而抑制所有糖酵解，导致葡萄糖刺激的胰岛素生物合成减少。因此，在不表达GLUT - 2的小鼠β TC3细胞中，己糖激酶活性与葡萄糖刺激的胰岛素生物合成之间存在密切关系，但葡萄糖转运蛋白与葡萄糖刺激的胰岛素生物合成之间不存在这种关系。
