Endocrine Abstracts

Uncoupling protein 2 (UCP2) is a mitochondrial protein located in the mitochondrial inner membrane, and it uncouples substrate oxidation from ATP synthesis, thereby dissipating the membrane potential energy and consequently decreasing reactive oxygen species (ROS) formation by mitochondrial respiratory chain. ROS overproduction is related to diabetic retinopathy (DR), a chronic complication of diabetes mellitus (DM). Recently, our group reported that the -866A/55Val/Ins haplotype (-866G/A, Ala55Val and Ins/Del polymorphisms) of the UCP2 was associated with risk for proliferative DR in both type 1 and type 2 diabetic patients. Additionally, this haplotype influenced UCP2 expression in human retina samples. Indeed, some studies suggest that the -866G/A polymorphism directly affect UCP2 expression; however, its effect on endothelial cells under different glucose concentrations is not well defined.

Objective: To investigate the effect of -866G/A polymorphism on UCP2 expression in an endothelial cell line under different glucose concentrations.

Methods: HUVECs cells were transfected with pGL3 plasmids containing the promoter region of UCP2 gene and the coding sequence of firefly luciferase, using LTX Lipofectamine (Life Technologies). The test conditions were: 1) transfection with the wild-type allele-containing plasmid (pGL3-UCP2-G); and 2) transfection with the mutated allele (pGL3-UCP2-A), under conditions of normoglycemia (4 mM) or hyperglycemia (25 mM) after 24 h and 48 h. Plasmid pCMV encoding renin luciferase and pEGFP were co-transfected as internal control and transfection control, respectively. Luciferase levels were measured with Luminescent Dual-luciferase Assay (Promega).

Results: HUVECs cells transfected for 24 h and 48 h with the plasmid containing the -866A allele under normal glucose conditions had 47% and 37% decrease in UCP2 expression than cells transfected with the plasmid containing the G allele (P=0.011 and P=0.0001; n=3). Interestingly, under high glucose conditions, cells containing the A allele had a more drastic decrease in UCP2 expression (70% and 54%) compared to cells with the G allele (P=0.0001 and P=0.028; n=3) after 24 and 48 h.

Conclusion: Our preliminary results demonstrate that the UCP2 -866A allele decreases UCP2 expression in HUVEC cells, which is exacerbated under high glucose conditions. The -866G/A polymorphism of the UCP2 gene is associated with increased UCP2 protein concentrations in human retina, which may explain the previous reported association between the -866A/55Val/Ins haplotype and risk for DR.