Kitzman H H, McMahon R J, Aslanian A M, Fadia P M, Frost S C
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville 32610, USA.
Mol Cell Biochem. 1996 Sep 6;162(1):51-8. doi: 10.1007/BF00250995.
We tested the hypothesis that the constitutive glucose transporter (GLUT1) in 3T3-L1 adipocytes belongs to the family of glucose-regulated proteins which are transcriptionally regulated by glucose deprivation. Using cDNA probes for both GRP78 (BiP) and GLUT1, we show that the level of GRP78 mRNA increased by 15-fold within 24 h of glucose deprivation with little change in GLUT1 mRNA. The elevated GRP78 mRNA in turn led to a time-dependent increase in GRP78 protein. While glucose deprivation did not alter the expression of the normal glycoform of GLUT1, a lower molecular weight glycoform accumulated with extended deprivation. Mannose and fructose, but not galactose, prevented the induction of GRP78 and accumulation of the abnormal GLUT1. Because GRP78 acts as a chaperone in other cell systems, we also sought evidence to support this activity in 3T3-L1 adipocytes. Using the technique of co-immunoprecipitation, we demonstrate that GRP78 bound several proteins unique to the glucose-deprived state. No deprivation-specific proteins could be detected in association with GLUT1. These data lead us to conclude that GLUT1 does not display characteristics of the glucose-regulated proteins, at least in 3T3-L1 adipocytes, a widely used model for differentiation, hormone action, and nutrient control. However, the mechanisms for activating traditional members of this family appear intact.
我们验证了一个假说，即3T3-L1脂肪细胞中的组成型葡萄糖转运蛋白（GLUT1）属于葡萄糖调节蛋白家族，该家族受葡萄糖剥夺的转录调控。使用针对GRP78（BiP）和GLUT1的cDNA探针，我们发现，在葡萄糖剥夺24小时内，GRP78 mRNA水平增加了15倍，而GLUT1 mRNA变化不大。GRP78 mRNA升高进而导致GRP78蛋白随时间依赖性增加。虽然葡萄糖剥夺并未改变GLUT1正常糖型的表达，但随着剥夺时间延长，一种分子量较低的糖型会积累。甘露糖和果糖可阻止GRP78的诱导及异常GLUT1的积累，但半乳糖无此作用。由于GRP78在其他细胞系统中起伴侣蛋白作用，我们也寻找证据支持其在3T3-L1脂肪细胞中的这种活性。通过免疫共沉淀技术，我们证明GRP78与几种葡萄糖剥夺状态特有的蛋白结合。未检测到与GLUT1相关的剥夺特异性蛋白。这些数据使我们得出结论，至少在3T3-L1脂肪细胞（一种广泛用于分化、激素作用和营养控制研究的模型）中，GLUT1不具备葡萄糖调节蛋白的特征。然而，激活该家族传统成员的机制似乎是完整的。
