Experimental study of mechanisms of hypoxia-induced expression of glucose transporter 1 in rat liver cells

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-βgalactosidase 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 (P0.05). When construct N was cotransfected with p(HA)HIF-1, the expression of the reporter gene increased by 16 folds (P0.05), but when construct M was cotransfected with p(HA)HIF-1, no difference in the expression was found as compared with transfection with construct M alone (P0.05). Conclusion HIF-1 binding site of HRE may play an important role in the hypoxia-induced GLUT-1 mRNA expression in rat liver cells.