Clancy B M, Harrison S A, Buxton J M, Czech M P
Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605.
J Biol Chem. 1991 Jun 5;266(16):10122-30.
In this study, we tested the hypothesis that hexose transport regulation may involve proteins with relatively rapid turnover rates. 3T3-L1 adipocytes, which exhibit 10-fold increases in hexose transport rates within 30 min of the addition of 100 nM insulin, were utilized. Exposure of these cells to 300 microM anisomycin or 500 microM cycloheximide caused a maximal, 7-fold increase in 2-deoxyglucose transport rate after 4-8 h. The effects due to either insulin (0.5 h) or anisomycin (5 h) on the kinetics of zero-trans 3-O-methyl[14C]glucose transport were similar, resulting in 2.5-3-fold increases in apparent Vmax values (control Vmax = 1.6 +/- 0.3 x 10(-7) mmol/s/10(6) cells) coupled with approximately 2-fold decreases in apparent Km values (control Km = 23 +/- 3.3 mM). Insulin elicited the expected increases in plasma membrane levels of HepG2/erythrocyte (GLUT1) and muscle/adipocyte (GLUT4) transporters (1.6- and 2.8-fold, respectively) as determined by protein immunoblotting. In contrast, neither total cellular contents nor plasma membrane levels of these two transporter isoforms were increased when 3T3-L1 adipocytes were treated with either anisomycin or cycloheximide. 3-[125I]Iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n labeling of glucose transporters in plasma membrane fractions of similarly treated cells was also unaffected by these agents. Thus, a striking discrepancy was observed between the marked increase in cellular hexose transport rates due to these protein synthesis inhibitors and the unaltered amounts of glucose transporter proteins in the plasma membrane fraction. These data indicate that short-term protein synthesis inhibition in 3T3-L1 adipocytes leads to large increases in the intrinsic catalytic activity of one or both of the GLUT1 and GLUT4 transporter isoforms.
在本研究中，我们检验了己糖转运调节可能涉及周转率相对较快的蛋白质这一假说。我们使用了3T3-L1脂肪细胞，在添加100 nM胰岛素后30分钟内，其己糖转运速率会增加10倍。将这些细胞暴露于300 μM茴香霉素或500 μM环己酰亚胺中4 - 8小时后，2-脱氧葡萄糖转运速率出现了最大7倍的增加。胰岛素（0.5小时）或茴香霉素（5小时）对零转运3-O-甲基[14C]葡萄糖转运动力学的影响相似，导致表观Vmax值增加2.5 - 3倍（对照Vmax = 1.6 ± 0.3×10⁻⁷ mmol/s/10⁶细胞），同时表观Km值下降约2倍（对照Km = 23 ± 3.3 mM）。通过蛋白质免疫印迹法测定，胰岛素引起了HepG2/红细胞（GLUT1）和肌肉/脂肪细胞（GLUT4）转运蛋白的质膜水平预期增加（分别为1.6倍和2.8倍）。相比之下，当用茴香霉素或环己酰亚胺处理3T3-L1脂肪细胞时，这两种转运蛋白亚型的总细胞含量和质膜水平均未增加。同样处理的细胞的质膜部分中葡萄糖转运蛋白的3-[¹²⁵I]碘-4-叠氮苯乙酰胺-7-O-琥珀酰去乙酰佛司可林标记也不受这些试剂影响。因此，在这些蛋白质合成抑制剂导致细胞己糖转运速率显著增加与质膜部分葡萄糖转运蛋白量未改变之间观察到了明显差异。这些数据表明，3T3-L1脂肪细胞中的短期蛋白质合成抑制导致GLUT1和GLUT4转运蛋白亚型中的一种或两种的内在催化活性大幅增加。
