Estudo da associação de polimorfismos nos genes das UCPS com suscetibilidade ao diabetes mellitus tipo 2 e doença renal do diabetes

Está bem estabelecido que fatores genéticos têm um papel importante no desenvolvimento do diabetes mellitus tipo 2 (DM2) bem como de suas complicações crônicas e que indivíduos geneticamente suscetíveis podem desenvolver essa doença após exposição a fatores de risco ambientais. Sendo assim, grandes esforços têm sido feitos para se identificar os genes associados com DM2 e suas complicações crônicas. As proteínas desacopladoras 1 a 3 (UCP1-3) são membros de uma superfamília de proteínas localizadas na membrana mitocondrial interna. A UCP1 é expressa principalmente no tecido adiposo marrom, a UCP2 é amplamente distribuída em diversos tecidos, enquanto a UCP3 é basicamente restrita ao músculo esquelético. Essas proteínas desacoplam a oxidação dos substratos da síntese de ATP pela ATP-sintase, dissipando o potencial de membrana e, consequentemente, diminuindo a produção de ATP pela cadeia respiratória mitocondrial. A dissipação de energia pelas UCPs está associada a diversas funções: produção de calor (UCP1), regulação do metabolismo e transporte de ácidos graxos livres (UCP2 e UCP3), diminuição da formação de espécies reativas de oxigênio (EROs) (UCP1-3) e regulação negativa da secreção de insulina (UCP2), mecanismos envolvidos na patogênese do DM2 e/ou de suas complicações crônicas. Dessa forma, polimorfismos nos genes UCP1-3 podem estar envolvidos no desenvolvimento destas doenças. A relação entre polimorfismos nos genes UCP1-3 e suscetibilidade ao DM2 tem sido investigada em diversas populações. No entanto, o impacto desses polimorfismos no DM2 ainda está em debate, com resultados contraditórios sendo relatados. Sendo assim, realizou-se um estudo de caso-controle na nossa população, seguido de uma revisão sistemática e metanálise dos estudos disponíveis na literatura para se avaliar se os seguintes polimorfismos estavam associados com suscetibilidade ao DM2: -3826A/G (UCP1); -866G/A, Ala55Val e Ins/Del (UCP2) e -55C/T (UCP3). Cabe ressaltar que os resultados do nosso estudo caso-controle foram incluídos na metanálise. No estudo de caso-controle, em indivíduos brancos, não encontramos nenhuma associação dos polimorfismos analisados com suscetibilidade ao DM2. Nossos resultados da meta-análise demonstraram que os polimorfismos -3826A/G (UCP1; rs1800592), -866G/A (UCP2; rs659366) e Ins/Del (UCP2), não estão associados com DM2. Por outro lado, o alelo Ala do polimorfismo Ala55Val (rs660339) no gene UCP2 e o alelo C do polimorfismo -55C/T (rs1800849) no gene UCP3 foram associados com risco para o DM2 em asiáticos, mas não em europeus. Está bem definido que as UCP1-3 diminuem a formação de EROs pela mitocôndria e que a superprodução de EROs é um dos principais fatores envolvidos na patogênese das complicações crônicas diabéticas. Entre as UCPs, a UCP2 é a mais expressa na retina e rins; portanto, polimorfismos neste gene podem estar envolvidos na patogênese dessas complicações. Recentemente, nós relatamos que o haplótipo mutado - 866A/55Val/Ins no gene UCP2 foi associado com risco aumentado para retinopatia diabética proliferativa em pacientes diabéticos tipo 1 e 2. Posteriormente, também demonstramos que portadores deste haplótipo mutado tinham uma expressão diminuída de UCP2 na retina humana de doadores de córnea em comparação aos homozigotos para o haplótipo de referência (-886G/55Ala/Del). No presente estudo, nós investigamos se os polimorfismos -866G/A, Ala55Val e Ins/Del (UCP2) estavam associados com doença renal do diabético (DRD) em pacientes com DM2 (delineamento caso-controle) e também se existia algum efeito destes polimorfismos sobre a expressão de UCP2 em biópsias de tecido renal humano (delineamento transversal). No estudo de caso-controle foram analisados 287 pacientes com DM2 e DRD (casos) e 281 pacientes com DM2 sem esta complicação e com mais de 10 anos de duração do DM2 (controle). No estudo transversal foram incluídas 42 amostras de biópsia de rim obtidas a partir de indivíduos que sofreram nefrectomia terapêutica. No grupo de pacientes com DM2, a homozigose para o haplótipo - 866A/55Val/Ins foi um fator de risco independente para DRD (RC = 2,136, IC 95% 1,036-4,404). Interessantemente, pacientes com DM2 portadores do haplótipo mutado apresentaram diminuição da taxa de filtração glomerular estimada (TFGe) quando comparados com indivíduos com o haplótipo de referência. Em amostras de biópsias renais, a expressão do gene UCP2 foi significativamente menor em portadores do haplótipo mutado quando comparados com o haplótipo de referência (0,32 ± 1,20 vs. 1,85 ± 1,16 n fold change; p ajustado < 0,000001). Em conclusão, os polimorfismos -3826A/G (UCP1), -866G/A, A55Val e Ins/Del (UCP2) e -55C/T (UCP3) não estão associados com DM2 em indivíduos brancos da nossa população. Entretanto, nossa meta-análise mostrou uma associação significativa entre os polimorfismos A55Val e -55C/T e risco para DM2 em populações asiáticas. Já a presença do haplótipo UCP2 -866A/55Val/Ins foi associada com aumento do risco para DRD e com diminuição da TFGe em pacientes com DM2. Além disso, este haplótipo foi associado com a diminuição da expressão do gene UCP2 em rins humanos.It is well established that genetic factors play an important role in the development of type 2 diabetes mellitus (T2DM) and its chronic complications, and that genetically susceptible subjects might develop the disease after being exposed to environmental risk factors. Therefore, great efforts have been made to identify genes associated with T2DM and its chronic complications. Uncoupling proteins 1, 2 and 3 (UCP1-3) are members of an anion-carrier protein family located in the mitochondrial inner membrane. UCP1 is mainly expressed in brown adipose tissue, UCP2 is widely distributed in several tissues, whereas UCP3 is mainly restricted to the skeletal muscle. These proteins act by uncoupling the oxidation of substrates from ATP synthesis by ATP-synthase, thereby dissipating the membrane potential and, consequently, decreasing the ATP production by the mitochondrial respiratory chain. This uncoupling effect then leads to different functions, such as thermogenesis (UCP1), regulation of free fatty acids metabolism and transport (UCP2 and UCP3), reduction in reactive oxygen species (ROS) formation (UCP1-3), and negative regulation of insulin secretion (UCP2), mechanisms involved in the pathogenesis of T2DM and/or its chronic complications. Therefore, polymorphisms in UCP1-3 genes might be associated with the development of these diseases. The relationship between UCP1-3 polymorphisms and susceptibility to T2DM has been investigated in several populations. Nevertheless, the impact of these polymorphisms on T2DM is still under debate, with contradictory results being reported. Thus, we performed a case-control study in our population followed by a systematic review and meta-analysis of published studies in order to evaluate whether the following polymorphisms were associated with T2DM susceptibility: -3826A/G (UCP1), -866G/A, Ala55Val and Ins/Del (UCP2) and -55C/T (UCP3). It is worth noting that results obtained in our case-control study were also included in the metaanalysis. In the case-control study, analyzing white subjects, we did not found any association between the analyzed polymorphisms and susceptibility for T2DM. Metaanalysis results showed that the -3826A/G (UCP1; rs1800592), -866G/A (UCP2; rs659366) and Ins/Del (UCP2) polymorphisms were not associated with T2DM. In contrast, the A allele of the Ala55Val polymorphism (UCP2; rs660339) and the C allele of the -55C/T polymorphism (UCP3; rs1800849) were significantly associated with risk to T2DM in Asians but not in Europeans. It is well stablished that UCP1-3 decrease ROS formation by mitochondria, and that ROS overproduction is one of the major contributors to the pathogenesis of chronic diabetic complications. Among UCPs, UCP2 has the higher expression in human retina and kidneys; therefore, polymorphisms in this gene could be involved in the pathogenesis of these complications. Recently, we reported that the -866A/55Val/Ins haplotype in the UCP2 gene was associated with increased risk for proliferative diabetic retinopathy in both type 1 and type 2 diabetic patients. Afterwards, we also demonstrated that mutated haplotype carriers had a decreased UCP2 gene expression in retina from cornea donors when compared to retina from subjects homozygous for the reference haplotype (-886G/55Ala/Del). In the present study, we investigated whether the -866G/A, Ala55Val and Ins/Del polymorphisms (UCP2) were associated with diabetic kidney disease (DKD) in T2DM patients (case-control design), and also if they had any effect on UCP2 gene expression in human kidney tissue biopsies (cross-sectional design). In the case-control study, we analyzed 287 T2DM patients with DKD (cases) and 281 T2DM patients without this complication and with more than 10 years of T2DM duration (controls). In the cross-sectional study, we included 42 kidney biopsy samples obtained from patients who undergone therapeutic nephrectomy. In the T2DM group, homozygosis for the mutated haplotype (-866A/55Val/Ins) was an independent risk factor for DKD (OR = 2.136, 95% CI 1.036-4.404). Interestingly, T2DM patients carrying the mutated haplotype showed decreased estimated glomerular filtration rate (eGFR) when compared to subjects with the reference haplotype. In kidney biopsy samples, UCP2 gene expression was significantly decreased in UCP2 mutated haplotype carriers when compared to reference haplotype (0.32 ± 1.20 vs. 1.85 ± 1.16 n fold; adjusted P < 0.000001). In conclusion, -3826A/G (UCP1), -866G/A, A55Val and Ins/Del (UCP2) and - 55C/T (UCP3) polymorphisms are not associated with T2DM in white subjects from our population. However, our meta-analysis showed a significant association between Ala55Val and -55C/T polymorphisms and risk for T2DM in Asians but not in Europeans. The presence of the UCP2 -866A/55Val/Ins haplotype seems to be associated with risk for DKD and with a decreased eGFR in T2DM patients. Moreover, this mutated haplotype was associated with decreased UCP2 gene expression in human kidneys

Comparison of two techniques for assessing pancreatic islet viability : flow cytometry and fluorescein diacetate/propidium iodide staining

Introduction: The success of islet transplantation for patients with unstable type 1 diabetes mellitus depends, in part, on the number of isolated islets and their quality, which is assessed by functional and viability tests. The test currently employed to evaluate islet viability, used by the Collaborative Islet Transplant Registry to release products for transplantation, is fluorescein diacetate/propidium iodide (FDA/PI) staining. However, the efficacy of this method relies on researcher experience; in this context, a quantitative method may be useful. The aim of this study was to compare islet viability as assessed by flow cytometry and the FDA/PI assay. Methods: Viability was analyzed in islets isolated from 10 male Wistar rats. Upon FDA/PI staining, 50 islets from each animal were analyzed under fluorescence microscopy by two well-trained researchers. For flow cytometry, islets were dispersed and 100 000 single cells were incubated with the 7-amino-actinomycin D (7AAD) fluorophore (dyes necrotic and late apoptotic cells) and the Annexin V-APC antibody (marks early apoptotic cells). Results: A moderate correlation was found between techniques (r = 0.6; p = 0.047). The mean islet viability measured by flow cytometry was higher than that estimated using FDA/PI staining (95.5 ± 1.4% vs 89.5 ± 5.0%; p = 0.002). Conclusions: Although flow cytometry is more expensive and time-consuming than FDA/PI staining, it is a quantitative technique with greater reproducibility that is less subject to inter-observer variability than FDA/PI. Therefore, flow cytometry appears to be the technique of choice when aiming for a more precise determination of islet viability

The A allele of the rs1990760 polymorphism in the IFIH1 gene is associated with protection for arterial hypertension in type 1 diabetic patients and with expression of this gene in human mononuclear cells

Background: The rs1990760 polymorphism of interferon induced with helicase C domain 1 (IFIH1) has been associated with type 1 diabetes mellitus (T1DM). Here, we investigated whether this polymorphism is associated with T1DM or its clinical characteristics in a Brazilian population, and if IFIH1 gene expression in mononuclear cells from T1DM patients differs according to the genotypes of this polymorphism. A meta-analysis was also conducted to evaluate if the rs1990760 polymorphism is associated with T1DM. Methods: Frequencies of the rs1990760 polymorphism were analyzed in 527 T1DM patients and in 517 healthy subjects. IFIH1 gene expressions according to genotypes were measured in a sub-sample of 26 T1DM patients by quantitative realtime PCR. Results: Our data show the association of the A allele with risk to T1DM under a dominant model of inheritance [odds ratio (OR) = 1.421, P = 0.037], adjusting for ethnicity. The meta-analysis revealed significant association between the rs199760A allele and risk for T1DM for all analyzed inheritance models. Surprisingly, T1DM patients carrying the A allele showed lower levels of systolic (P = 0.001) and diastolic (P = 1610210) blood pressures as compared to G/G carriers. Furthermore, the A/A genotype seems to be associated with protection to arterial hypertension (AH) after adjustment for covariates (OR = 0.339, P = 0.019). IFIH1 gene expression in mononuclear cells from 26 T1DM patients did not differ among genotypes (P = 0.274). Nevertheless, IFIH1 gene expression was increased in mononuclear cells from T1DM patients with AH as compared with T1DM patients without AH [6.7 (1.7–2.0) vs. 1.8 (1.3–7.1) arbitrary units; P = 0.036]. The association with blood pressures and AH was not observed in patients with type 2 diabetes mellitus. Conclusions: Our results indicate that the rs1990760 polymorphism is associated with T1DM. Interestingly, the rs1990760 A allele seems to be associated with protection for AH in T1DM patients. Further studies are needed to confirm the association with AH

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

Associations between UCP1 -3826A/G, UCP2 -866G/A, Ala55Val and Ins/Del, and UCP3 -55C/T polymorphisms and susceptibility to type 2 diabetes mellitus : case-control study and meta-analysis

Background: Some studies have reported associations between five uncoupling protein (UCP) 1–3 polymorphisms and type 2 diabetes mellitus (T2DM). However, other studies have failed to confirm the associations. This paper describes a casecontrol study and a meta-analysis conducted to attempt to determine whether the following polymorphisms are associated with T2DM: -3826A/G (UCP1); -866G/A, Ala55Val and Ins/Del (UCP2) and -55C/T (UCP3). Methods: The case-control study enrolled 981 T2DM patients and 534 nondiabetic subjects, all of European ancestry. A literature search was run to identify all studies that investigated associations between UCP1–3 polymorphisms and T2DM. Pooled odds ratios (OR) were calculated for allele contrast, additive, recessive, dominant and co-dominant inheritance models. Sensitivity analyses were performed after stratification by ethnicity. Results: In the case-control study the frequencies of the UCP polymorphisms did not differ significantly between T2DM and nondiabetic groups (P.0.05). Twenty-three studies were eligible for the meta-analysis. Meta-analysis results showed that the Ala55Val polymorphism was associated with T2DM under a dominant model (OR = 1.27, 95% CI 1.03–1.57); while the - 55C/T polymorphism was associated with this disease in almost all genetic models: allele contrast (OR = 1.17, 95% CI 1.02– 1.34), additive (OR = 1.32, 95% CI 1.01–1.72) and dominant (OR = 1.18, 95% CI 1.02–1.37). However, after stratification by ethnicity, the UCP2 55Val and UCP3 -55C/T alleles remained associated with T2DM only in Asians (OR = 1.25, 95% CI 1.02– 1.51 and OR = 1.22, 95% CI 1.04–1.44, respectively; allele contrast model). No significant association of the -3826A/G, -866G/ A and Ins/Del polymorphisms with T2DM was observed. Conclusions: In our case-control study of people with European ancestry we were not able to demonstrate any association between the UCP polymorphisms and T2DM; however, our meta-analysis detected a significant association between the UCP2 Ala55Val and UCP3 -55C/T polymorphisms and increased susceptibility for T2DM in Asian