Cloning and expression of rat liver type glucose transporter and translocation by insulin in Chinese hamster ovary cells.
The 5'- and 3'-side half of liver type glucose transporter (GLUT2) cDNA was amplified from total RNA or mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). The amplified 5'-side fragment of GLUT2 cDNA was inserted into pGEM4Z and named pGLGT1, and the 3'-side fragment of GLUT2 cDNA was inserted into the HindIII site of pGLGT1 to construct pGLGT2 which contains an entire open reading frame of GLUT2 cDNA. The GLUT2 cDNA in pGLGT2 was transferred to an eukaryotic expression vector (pMAM) to construct pMLGT, which was expressed in the insulin-sensitive Chinese hamster ovary (CHO) cells. Western blot analysis showed that the GLUT2 gene in pMLGT was expressed in the transfected CHO cells successfully. The GLUT2 content in the plasma membrane fraction of insulin-treated CHO cells expressing GLUT2 increased 3.8-fold compared to that of the control group. This result suggests that GLUT2, which is not subjected to translocation by insulin in the cells of its major distribution, can be translocated if it is expressed in the suitable cells sensitive to insulin action.
Ahn
Y H
YH
Department of Biochemistry, Yonsei University College of Medicine, Seoul, Korea.
Yoon
D J
DJ
Han
G S
GS
Lee
B G
BG
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D051273:Glucose Transporter Type 2; D007328:Insulin / Q000494:pharmacology*; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D015345:Oligonucleotide Probes / Q000235:genetics; D014178:Translocation, Genetic
D051273:Glucose Transporter Type 2; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D015345:Oligonucleotide Probes