Experimental study of mechanisms of hypoxia-induced expression of glucose transporter 1 in rat liver cells / 第三军医大学学报

ABSTRACT

Objective To explore the mechanisms of hypoxia-induced expression of glucose transporter 1 (GLUT-1) in rat liver cells. Methods Wild type hypoxia response element (HRE) plasmid (construct N) containing the potential HIF-1 binding site was constructed by using construct A which contains the full length of the 5′-flanking region of the rat GLUT-1 gene as a template of PCR, and the PCR product was subcloned into the reporter plasmid pGL3-Promoter. Mutation type HRE plasmid (construct M) was made using a two-step overlapping PCR strategy. Then the liver cells were transfected with constructs N and M, respectively. At 24 h after transfection, the cells were subjected to hypoxia (1% O 2) to mimic the hypoxic environment caused by burn for 12 h. The samples were harvested and the determination of the reporter gene luciferase activity and pSV-?galactosidase activity was performed. Results Constructs N and M were successfully constructed and the liver cells were successfully transfected with construct N or construct M. Hypoxia induced more enhanced luciferase activity of constructs N and M as compared with the control (P