The A allele of the UCP2 -866G/A polymorphism changes UCP2 promoter activity in HUVECs treated with high glucose.

The mitochondrial uncoupling protein 2 (UCP2) decreases reactive oxygen species (ROS) formation by mitochondria. Our group previously showed that the UCP2 -866A allele was associated with risk of diabetic retinopathy (DR), which is caused by hyperglycemia-induced oxidative stress. To date, it is still unclear if the -866A allele directly affects UCP2 expression in endothelial cells. Thus, we investigated the effect of the A allele on UCP2 promoter activity in HUVECs treated with high glucose (HG) or hydrogen peroxide (H2O2). To quantify UCP2 promoter activity, HUVECs were transfected with pGL3 plasmids containing the UCP2 promoter and the firefly luciferase coding sequence. Experimental groups were: (1) pGL3-866G-transfected cells and (2) pGL3-866A cells, both under normal (4 mM) or HG (25 mM) concentrations for 24 h and 48 h or incubated with H2O2 (0.1 mM) for 1 h. UCP2 promoter activity was monitored by Luminescent Dual-luciferase Assay. HG induced an upregulation of UCP2 promoter activity in PGL3-866G cells after 24 h of treatment (P = 0.027), but not after 48 h. Compared to pGL3-866G cells, pGL3-866A cells seems to have reduced UCP2 promoter activity following 24 h and 48 h of normal glucose treatment (P = 0.087 and P = 0.022). After HG treatment, pGL3-866A cells had more marked UCP2 downregulation (24 h: - 3.2-folds, P < 0.001; and 48 h: - 2.5-folds, P < 0.001 vs. G cells). Both pGL3-866G and pGL3-866A cells treated with H2O2 showed a ≅ 4-fold increase in UCP2 promoter activity (both P < 0.001). The -866A allele modifies UCP2 promoter activity in HUVECs under HG treatment but not in the H2O2 condition.

Association of the UCP polymorphisms with susceptibility to obesity: case-control study and meta-analysis.

This paper describes a case-control study and a meta-analysis performed to evaluate if the following polymorphisms are associated with presence of obesity: -3826A/G (UCP1); -866G/A, Ala55Val and Ins/Del (UCP2) and -55C/T (UCP3). The case-control study enrolled 282 obese and 483 non-obese patients with type 2 diabetes. A literature search was made to identify all studies that evaluated associations between UCP1-3 polymorphisms and obesity. In the case-control study the distributions of the UCP variants did not differ between obese and non-obese groups (P > 0.05). Forty-seven studies were eligible for the meta-analysis and the results showed that the UCP2 -866G/A and UCP3 -55C/T polymorphisms were associated with protection to obesity in Europeans (OR = 0.89, 95% CI 0.82-0.97 and OR = 0.88, 95% CI 0.80-0.97, respectively). The UCP2 Ala55 val polymorphism was associated with obesity in Asians (OR = 1.61, 95% CI 1.13-2.30). The UCP2 Ins/Del polymorphism was associated with obesity mainly in Europeans (OR = 1.19, 95% CI 1.00-1.42). There was no significant association of the UCP1 -3826A/G polymorphism with obesity. In our case-control study we were not able to demonstrate any association between UCP polymorphisms and obesity in T2DM patients; however, in the meta-analysis we detected a significant association of UCP2 -866G/A, Ins/Del, Ala55Val and UCP3 -55C/T polymorphisms with obesity.

Blueberry Consumption and Changes in Obesity and Diabetes Mellitus Outcomes: A Systematic Review.

Low-grade inflammation and oxidative stress are key mechanisms involved in obesity and related disorders. Polyphenols from blueberry (BB) and bilberries (BiB) might protect against oxidative damage and inflammation. To summarize the effects of BiB or BB consumption in parameters related to obesity and its comorbidities, a search of the literature was performed in PubMed, Embase, and Cochrane Library repositories to identify all studies that evaluated associations of whole BB or BiB with obesity and associated disorders. Thirty-one studies were eligible for inclusion in this review: eight clinical trials and 23 animal studies. In humans, BB consumption only consistently decreased oxidative stress and improved endothelial function. In rodents, BB or BiB consumption caused positive effects on glucose tolerance, nuclear factor-kappa B (Nf-κb) activity, oxidative stress, and triglyceride (TG) content in the liver and hepatic steatosis. The high content of anthocyanins present in BB and BiB seems to attenuate oxidative stress. The decrease in oxidative stress may have a positive impact on glucose tolerance and endothelial function. Moreover, in rodents, these berries seem to protect against hepatic steatosis, through the decreased accumulation of hepatic TGs. BB and BiB might also attenuate inflammation by decreasing Nf-κb activity and immune cell recruitment into the adipose tissue.

The rs2304256 Polymorphism in TYK2 Gene Is Associated with Protection for Type 1 Diabetes Mellitus.

BACKGROUND: Tyrosine kinase 2 (TYK2) is a candidate gene for type 1 diabetes mellitus (T1DM) since it plays an important role in regulating apoptotic and pro-inflammatory pathways in pancreatic ß-cells through modulation of the type I interferon signaling pathway. The rs2304256 single nucleotide polymorphism (SNP) in TYK2 gene has been associated with protection for different autoimmune diseases. However, to date, only two studies have evaluated the association between this SNP and T1DM, with discordant results. This study thus aimed to investigate the association between the TYK2 rs2304256 SNP and T1DM in a Southern Brazilian population. METHODS: This case-control study comprised 478 patients with T1DM and 518 non-diabetic subjects. The rs2304256 (C/A) SNP was genotyped by real-time polymerase chain reaction technique using TaqMan minor groove binder (MGB) probes. RESULTS: Genotype and allele frequencies of the rs2304256 SNP differed between T1DM patients and non-diabetic subjects (P<0.0001 and P=0.001, respectively). Furthermore, the A allele was associated with protection against T1DM under recessive (odds ratio [OR], 0.482; 95% confidence interval [CI], 0.288 to 0.806) and additive (OR, 0.470; 95% CI, 0.278 to 0.794) inheritance models, adjusting for human leukocyte antigen (HLA) DR/DQ genotypes, gender, and ethnicity. CONCLUSION: The A/A genotype of TYK2 rs2304256 SNP is associated with protection against T1DM in a Southern Brazilian population.

A functional genomic approach to identify reference genes for human pancreatic beta cell real-time quantitative RT-PCR analysis.

Exposure of human pancreatic beta cells to pro-inflammatory cytokines or metabolic stressors is used to model events related to type 1 and type 2 diabetes, respectively. Quantitative real-time PCR is commonly used to quantify changes in gene expression. The selection of the most adequate reference gene(s) for gene expression normalization is an important pre-requisite to obtain accurate and reliable results. There are no universally applicable reference genes, and the human beta cell expression of commonly used reference genes can be altered by different stressors. Here we aimed to identify the most stably expressed genes in human beta cells to normalize quantitative real-time PCR gene expression.We used comprehensive RNA-sequencing data from the human pancreatic beta cell line EndoC-ßH1, human islets exposed to cytokines or the free fatty acid palmitate in order to identify the most stably expressed genes. Genes were filtered based on their level of significance (adjusted P-value >0.05), fold-change (|fold-change| <1.5) and a coefficient of variation <10%. Candidate reference genes were validated by quantitative real-time PCR in independent samples.We identified a total of 264 genes stably expressed in EndoC-ßH1 cells and human islets following cytokines - or palmitate-induced stress, displaying a low coefficient of variation. Validation by quantitative real-time PCR of the top five genes ARF1, CWC15, RAB7A, SIAH1 and VAPA corroborated their expression stability under most of the tested conditions. Further validation in independent samples indicated that the geometric mean of ACTB and VAPA expression can be used as a reliable normalizing factor in human beta cells.

MiR-30e-5p and MiR-15a-5p Expressions in Plasma and Urine of Type 1 Diabetic Patients With Diabetic Kidney Disease.

INTRODUCTION: Diabetic kidney disease (DKD) is a common microvascular complication that affects 40% of patients with diabetes mellitus (DM). Emerging evidence suggests a role for several microRNAs (miRNAs) in the development of DKD. In this context, miR-15a-5p and miR-30e-5p have been shown to regulate the expression of the uncoupling protein 2 (UCP2), a mitochondrial protein that decreases reactive oxygen species (ROS) formation by the mitochondria. Since ROS overproduction is a key contributor to the pathogenesis of DKD, dysregulation of these two miRNAs could be involved in DKD pathogenesis. Thus, the aim of this study was to compare the expressions of miR-15a-5p and miR-30e-5p in type 1 DM (T1DM) patients with DKD (cases) and without this complication (controls), and to perform bioinformatics analyses to investigate their putative targets and biological pathways under their regulation. METHODS: MiR-15a-5p and miR-30e-5p expressions were analyzed in plasma and urine of 17 T1DM controls and 23 DKD cases (12 with moderate DKD and 11 with severe DKD) using qPCR. Bioinformatics analyses were performed in Cytoscape software. RESULTS: MiR-30e-5p expression was downregulated in plasma of patients with moderate and severe DKD compared to T1DM controls. Moreover, this miRNA was also downregulated in urine of patients with severe DKD compared to the other groups. No difference was found in miR-15a-5p expression between groups. Bioinformatics analyses indicated that miR-30e-5p and miR-15a-5p regulate various genes that participate in pathways related to angiogenesis, apoptosis, cell differentiation, oxidative stress, and hypoxia. CONCLUSION: MiR-30e-5p seems to be downregulated in plasma and urine of patients with DKD.

Effect of co-culture of mesenchymal stem/stromal cells with pancreatic islets on viability and function outcomes: a systematic review and meta-analysis.

The maintenance of viable and functional pancreatic islets is crucial for successful islet transplantation from brain-dead donors. To overcome islet quality loss during culture, some studies have co-cultured islets with mesenchymal stem/stromal cells (MSC). However, it is still uncertain if MSC-secreted factors are enough to improve islet quality or if a physical contact between MSCs and islets is needed. Therefore, we performed a systematic review and meta-analysis to clarify the effect of different culture contact systems of islets with MSCs on viability and insulin secretion outcomes. Pubmed and Embase were searched. Twenty studies fulfilled the eligibility criteria and were included in the qualitative synthesis and/or meta-analysis. For both outcomes, pooled weighted mean differences (WMD) between islet cultured alone (control group) and the co-culture condition were calculated. Viability mean was higher in islets co-cultured with MSCs compared with islet cultured alone [WMD = 18.08 (95% CI 12.59-23.57)]. The improvement in viability was higher in islets co-cultured in indirect or mixed contact with MSCs than in direct physical contact (P <0.001). Moreover, the mean of insulin stimulation index (ISI) was higher in islets from co-culture condition compared with islet cultured alone [WMD = 0.83 (95% CI 0.54-1.13)], independently of contact system. Results from the studies that were analyzed only qualitatively are in accordance with meta-analysis data. Co-culture of islets with MSCs has the potential for protecting islets from injury during culture period. Moreover, culture time appears to influence the beneficial effect of different methods of co-culture on viability and function of islets.

Polymorphisms of the UCP2 Gene Are Associated with Glomerular Filtration Rate in Type 2 Diabetic Patients and with Decreased UCP2 Gene Expression in Human Kidney.

INTRODUCTION: Uncoupling protein 2 (UCP2) reduces production of reactive oxygen species (ROS) by mitochondria. ROS overproduction is one of the major contributors to the pathogenesis of chronic diabetic complications, such as diabetic kidney disease (DKD). Thus, deleterious polymorphisms in the UCP2 gene are candidate risk factors for DKD. In this study, we investigated whether UCP2 -866G/A, Ala55Val and Ins/Del polymorphisms were associated with DKD in patients with type 2 diabetes mellitus (T2DM), and whether they had an effect on UCP2 gene expression in human kidney tissue biopsies. MATERIALS AND METHODS: In a case-control study, frequencies of the UCP2 -866G/A, Ala55Val and Ins/Del polymorphisms as well as frequencies of the haplotypes constituted by them were analyzed in 287 T2DM patients with DKD and 281 T2DM patients without this complication. In a cross-sectional study, UCP2 gene expression was evaluated in 42 kidney biopsy samples stratified according to the presence of the UCP2 mutated -866A/55Val/Ins haplotype. RESULTS: In the T2DM group, multivariate logistic regression analysis showed that the -866A/55Val/Ins haplotype was an independent risk factor for DKD (OR = 2.136, 95% CI 1.036-4.404), although neither genotype nor allele frequencies of the individual polymorphisms differed between case and control groups. Interestingly, T2DM patients carrying the mutated haplotype showed decreased estimated glomerular filtration rate (eGFR) when compared to subjects with the reference haplotype (adjusted P= 0.035). In kidney biopsy samples, UCP2 expression was significantly decreased in UCP2 mutated haplotype carriers when compared to kidneys from patients with the reference haplotype (0.32 ± 1.20 vs. 1.85 ± 1.16 n fold change; adjusted P< 0.000001). DISCUSSION: Data reported here suggest that the UCP2 -866A/55Val/Ins haplotype is associated with an increased risk for DKD and with a lower eGFR in T2DM patients. Furthermore, this mutated haplotype was associated with decreased UCP2 gene expression in human kidneys.