Karnieli E, Garvey W T, Olefsky J M, Hueckstead T P, Harel C, Maianu L, Armoni M
Metabolic Unit, Rambam Medical Center, Technion, Haifa, Israel.
Endocrinology. 1993 Dec;133(6):2943-50. doi: 10.1210/endo.133.6.8243322.
We examined the hypothesis that insulin stimulation of cellular glucose transport may involve a protein synthesis-dependent regulation of glucose transporter (GTer) activity independent of GTer translocation to the cell surface. Rat adipocytes were isolated, incubated with or without 10 micrograms/ml (36 microM) cycloheximide (CHX) for 60 min, and then with or without 7 nM insulin for 30 min. Glucose transport rates were assessed in intact cells, and both glucose transport rates and GTer levels were assessed in subcellular fractions of membrane vesicles before and after reconstitution into artificial liposomes. GTer functional and intrinsic activities were calculated as the ratio between these transport rates and GTer levels in native and reconstituted membranes, respectively. Insulin increased functional activity by 340% in native plasma membrane (PM) vesicles and intrinsic activity by 60% in reconstituted membranes (from 54 +/- 4 to 86 +/- 4 molecules transported per GTer/sec, P < 0.02). CHX preincubation of cells did not interfere with the insulin effect to stimulate glucose transport rate in either intact cells or in native PMs; it did, however, reduce PM GTer levels by 27-30%, but not affecting those in the intracellular pool. However, CHX additively increased the insulin-stimulated intrinsic activity of PM GTers by 67%. Relative reconstitution efficiencies, assessed by immunoblotting both native and reconstituted membranes against specific antibodies, were similar for GLUT 1 and GLUT 4. Although insulin did not alter this efficiency, CHX slightly decreased it for GLUT 4. Our data suggest that insulin stimulation of glucose transport may involve, as part of its mechanism, modulation of the GTer intrinsic activity. We further hypothesize that CHX effects on increasing this activity state of GTer may involve as yet unknown protein synthesis-dependent regulator(s).
胰岛素对细胞葡萄糖转运的刺激作用可能涉及一种依赖蛋白质合成的葡萄糖转运体（GTer）活性调节，而与GTer转位至细胞表面无关。分离大鼠脂肪细胞，分别在有无10微克/毫升（36微摩尔）环己酰亚胺（CHX）的情况下孵育60分钟，然后再分别在有无7纳摩尔胰岛素的情况下孵育30分钟。评估完整细胞中的葡萄糖转运速率，并在将膜囊泡亚细胞组分重构成人工脂质体之前和之后评估葡萄糖转运速率和GTer水平。GTer的功能活性和内在活性分别计算为这些转运速率与天然膜和重构膜中GTer水平之间的比率。胰岛素使天然质膜（PM）囊泡中的功能活性增加340%，使重构膜中的内在活性增加60%（从每GTer/秒转运54±4个分子增加到86±4个分子，P<0.02）。细胞预先用CHX孵育并不干扰胰岛素对完整细胞或天然PM中葡萄糖转运速率的刺激作用；然而，它确实使PM GTer水平降低了27 - 30%，但不影响细胞内池中的水平。然而，CHX使胰岛素刺激的PM GTer内在活性额外增加了67%。通过针对特异性抗体对天然膜和重构膜进行免疫印迹评估的相对重构效率，对于GLUT 1和GLUT 4来说是相似的。虽然胰岛素没有改变这种效率，但CHX使GLUT 4的效率略有降低。我们的数据表明，胰岛素对葡萄糖转运的刺激作用可能在其机制中涉及对GTer内在活性的调节。我们进一步假设，CHX对增加GTer这种活性状态的作用可能涉及尚未知晓的依赖蛋白质合成的调节因子。
