Endocrine Abstracts

Introduction: Uncoupling protein 2 (UCP2) is a mitochondrial transporter present in the inner membrane of mitochondria, and it uncouples substrate oxidation from ATP synthesis, thereby dissipating the membrane potential energy and consequently decreasing ATP production by mitochondrial respiratory chain. As a consequence of the uncoupling, UCP2 decreases reactive oxygen species (ROS) formation by mitochondria. 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 gene was associated with risk for DR in patients with type 1 or type 2 DM. Afterwards, we showed that this haplotype influences UCP2 mRNA expression in human retina samples.

Objective: To evaluate whether the UCP2 −866A/55Val/Ins haplotype influences the amounts of UCP2 protein in human retina.

Methods: The sample was constituted by 84 healthy cadaveric cornea donors from two hospitals from Porto Alegre, Brazil. Genotyping of the −866G/A, Ala55Val and Ins/Del polymorphisms were performed by Real-time PCR using TaqMan probes. UCP2 protein distributions and intensities were determined by immunohistochemistry in formalin-fixed, paraffin-embedded retina sections, using an anti-UCP2 rabbit polyclonal antibody. Ten fields of each slide were photographed, and the intensity of UCP2 immunostaining was analyzed by two independent researchers using the Image ProPlus version 4.5 program.

Results: UCP2 immunoreactivity was not exclusive to a specific retina cell layer. The concentration of UCP2 protein in retina did not differ significantly between 28 −866A/55Val/Ins haplotype carriers and 30 WT haplotype carriers (27.1±15.6 vs 20.2±13.1 pixels; P=0.064). However, when we analyzed each polymorphism individually, we observed that A allele carriers of the −866G/A polymorphism showed increased UCP2 levels as compared to the G/G genotype (27.9±20.9 vs 20.1±13.1 pixels; P=0.027).

Conclusion: The −866G/A polymorphism in the promoter region of the UCP2 gene seems to be associated with increased UCP2 protein concentrations in human retina, which may explain the reported association between the −866G/55Val/Ins haplotype and risk for DR. We hypothesized that in a glucotoxicity environment, as occurring in the diabetic milieu, the A allele would be a marker of excessive ROS production, which is the actual risk factor for DR.