Germinario R J, Manuel S, Chang Z, Leckett B
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, Canada.
J Cell Physiol. 1992 Apr;151(1):156-63. doi: 10.1002/jcp.1041510120.
We have investigated the effect of various inhibitors of protein synthesis on hexose transport in human skin fibroblasts using 2-deoxy-D-glucose (2-DG) and 3-0-methyl-D-glucose (3-OMG) to measure hexose transport. Exposure of glucose-fed, serum-free cultures to cycloheximide (CHX) (50 micrograms/ml) for 6 h resulted in increased 2-DG transport (3.81 +/- .53 vs. 6.62 +/- .88 nmoles/mg protein/2 min; n = 9) and 3-OMG transport (1.36 +/- .66 vs. 3.18 +/- .83 nmoles/mg protein/30 sec; n = 4) in the CHX exposed group. Under these conditions inhibition of protein synthesis was greater than 90%. This CHX induced transport increase was time dependent (approaching maximum within 1 h of exposure to CHX) and related to an increase in the Vmax of hexose transport in the CHX exposed group (18.4 +/- 2.4 vs. 4.8 +/- 1.1 nmoles 2-DG/mg protein/min) with no difference in the transport Km (1.55 +/- .63 vs. 2.92 +/- .59 mM). Further, the CHX induced increase in hexose transport was reversible. Exposure of human fibroblasts to inhibitors of protein synthesis with different mechanisms of action (e.g., puromycin, pactamycin, or CHX) all generated hexose transport increases in a concentration-dependent fashion correlating with their increasing inhibitory effects on protein synthesis. Nucleotidase enriched (i.e., plasma membrane) fractions of control and CHX-exposed cells showed no differences in D-glucose inhibitable cytochalasin B binding activity. Further, quantitative Western analysis of nucleotidase enriched fractions indicated CHX exposure resulted in no significant increase in glucose transporter mass compared with control plasma membrane fractions. Glucose deprived cells, however, which exhibited increased sugar transport comparable to the CHX-exposed group, did show increased glucose transporter mass in the plasma membrane fraction. The data indicate that inhibitors of protein synthesis can cause a significant elevation in hexose transport and that the hexose transporter mass in the isolated plasma membrane fractions did not reflect the whole cell transport change. It is suggested that a mechanism other than glucose transporter translocation to the plasma membrane may be involved in causing this sugar transport increase.
我们使用2-脱氧-D-葡萄糖（2-DG）和3-O-甲基-D-葡萄糖（3-OMG）来测量己糖转运，研究了各种蛋白质合成抑制剂对人皮肤成纤维细胞中己糖转运的影响。将用葡萄糖喂养的无血清培养物暴露于环己酰亚胺（CHX）（50微克/毫升）6小时，导致CHX暴露组中2-DG转运增加（3.81±0.53对6.62±0.88纳摩尔/毫克蛋白质/2分钟；n = 9）和3-OMG转运增加（1.36±0.66对3.18±0.83纳摩尔/毫克蛋白质/30秒；n = 4）。在这些条件下，蛋白质合成的抑制率大于90%。这种CHX诱导的转运增加是时间依赖性的（在暴露于CHX后1小时内接近最大值），并且与CHX暴露组中己糖转运的Vmax增加有关（18.4±2.4对4.8±1.1纳摩尔2-DG/毫克蛋白质/分钟），而转运Km没有差异（1.55±0.63对2.92±0.59毫摩尔）。此外，CHX诱导的己糖转运增加是可逆的。将人成纤维细胞暴露于具有不同作用机制的蛋白质合成抑制剂（例如嘌呤霉素、密旋霉素或CHX）均以浓度依赖性方式导致己糖转运增加，这与其对蛋白质合成的抑制作用增强相关。对照细胞和CHX暴露细胞的富含核苷酸酶（即质膜）的组分在D-葡萄糖可抑制的细胞松弛素B结合活性方面没有差异。此外，对富含核苷酸酶的组分进行的定量蛋白质免疫印迹分析表明，与对照质膜组分相比，CHX暴露导致葡萄糖转运体质量没有显著增加。然而，葡萄糖剥夺的细胞，其表现出与CHX暴露组相当的糖转运增加，确实在质膜组分中显示出葡萄糖转运体质量增加。数据表明，蛋白质合成抑制剂可导致己糖转运显著升高，并且分离的质膜组分中的己糖转运体质量并未反映全细胞转运变化。有人提出，除了葡萄糖转运体易位到质膜之外的一种机制可能参与导致这种糖转运增加。
