RESUMO
BACKGROUND: Evidence implying that metabolism reprogramming plays an important role in the regulation of sepsis is increasing; however, whether it has a similar role in septic organ dysfunction remains unclear. Here, we provide evidence to support a new role of uncoupling protein-2 (UCP2)-regulated Warburg effect, i.e., aerobic glycolysis, in promoting mitochondrial injury in the kidney. METHODS: To imitate sepsis condition, male C57BL/6 mice were operated by the cecal ligation puncture in vivo, whereas a normal human kidney cell line (HK-2) was treated with lipopolysaccharide in vitro. UCP2 small interfering RNA pretreatment was performed to knock down UCP2 expression in vitro. The glycolysis metabolite was detected by liquid chromatography/tandem mass spectrometry in vivo and detected by commercial kits in vitro. Oxidative phosphorylation level and glycolysis level were monitored by measuring the oxygen consumption rate (indicative of respiration) and extracellular acidification rate (indicative of glycolysis) in vitro. Exogenous lactate was supplied to stimulate HK-2 cells and indicators of mitochondrial dysfunction were also assessed. RESULTS: Aerobic glycolysis is enhanced in septic tubular epithelial cells, and the glycolysis inhibitor 2-deoxyglucose can partially restore mitochondrial membrane potential and decrease the reactive oxygen species production. With the knockdown of UCP2, the aerobic glycolysis level upregulates, and mitochondrial injury increases. CONCLUSIONS: These results provide insights on a new mechanism of metabolic regulation of mitochondrial injury and the importance of targeting aerobic glycolysis for the treatment of septic acute kidney injury.
Assuntos
Injúria Renal Aguda/metabolismo , Mitocôndrias/metabolismo , Sepse/metabolismo , Proteína Desacopladora 2/fisiologia , Injúria Renal Aguda/etiologia , Animais , Linhagem Celular , Glicólise , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sepse/complicaçõesRESUMO
OBJECTIVE: To investigate whether the overexpression of uncoupling protein 2 (UCP2) can protect myocardium from sepsis by inhibiting the production of reactive oxygen species (ROS) and inflammatory response. METHODS: Forty Sprague-Dawley rats were divided into four groups according to random number table method (n = 10): sham transfection and sham surgery group (Sham group), sham transfection and cecal ligation and perforation (CLP) group (CLP group), simple adeno-associated virus (AAV) transfection surgery group (AAV group), and UCP2 overexpression surgery group (UCP2 group). In UCP2 group, UCP2 adeno-associated virus (AAV-UCP2; titer 1×1012 v.g/mL, 10 µL per site, 60 µL in total) was injected into myocardium, and CLP was performed 3 weeks later. In AAV group, the myocardium was transfected with AAV virus and CLP was performed 3 weeks later. Twenty-four hours after modeling, whether the model was successfully prepared was evaluated. The transfection effect of AAV virus on the frozen sections of myocardial tissue was observed under fluorescence microscope, the expression of UCP2 protein was detected by Western blotting, ROS production was detected by dihydroethidine (DHE) staining, and serum myocardial markers and inflammatory cytokines were detected by enzyme linked immunosorbent assay (ELISA). RESULTS: Twenty-four hours after CLP, the rats showed stiff hair, increased secretions from eyes, nose and mouth, and symptoms of pyuria, loose stools, and dyspnea. After laparotomy, the cecum showed purple and black, and there was purulent exudation around the intestinal cavity. The virus was successfully transfected on frozen section under the fluorescence microscope (the site of the transfection was green fluorescence), and further Western blotting revealed that the expression of UCP2 in the CLP group was higher than that in the Sham group (UCP2/ß-tubulin: 1.53±0.06 vs. 1, P < 0.01). Compared with the AAV group, UCP2 expression was further increased in the UCP2 group (UCP2/ß-tubulin: 1.96±0.22 vs. 1.59±0.07, P < 0.01). Under the fluorescence microscope, ROS production in the CLP and AAV groups were found significantly increased compared with that in the Sham group; when UCP2 was overexpressed, ROS production were significantly decreased compared with the CLP and AAV groups (A value: 1.03±0.10 vs. 1.81±0.13, 1.67±0.08, both P < 0.01). ELISA showed that compared with the Sham group, the levels of lactate dehydrogenase (LDH), creatine kinase (CK), cardiac troponin I (cTnI), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were significantly increased in the CLP and AAV groups; when UCP2 was overexpressed, the above myocardial enzymes and inflammatory cytokines secretion were significantly decreased compared with the CLP group and AAV group [LDH (ng/L): 48.97±1.04 vs. 56.85±1.36, 57.08±1.54; CK (ng/L): 235.23±20.33 vs. 306.34±25.93, 304.76±25.29; cTnI (ng/L): 199.79±18.27 vs. 241.88±14.32, 243.33±23.79; TNF-α (ng/L): 385.71±20.09 vs. 488.92±26.92, 489.03±33.37; IL-6 (ng/L): 121.12±7.61 vs. 159.07±17.65, 157.61±15.13; all P < 0.01]. Kaplan-Meier survival curve showed that the survival rate of rats 36 hours after CLP was only 30.0%. When UCP2 overexpressed, the survival rate was significantly higher than that of the CLP group and AAV group (60.0% vs. 30.0%, 30.0%, both P < 0.05). There was no significant difference between the AAV group and CLP group. CONCLUSIONS: UCP2 overexpression can reduce myocardial injury and improve the survival rate of septic rats by reducing ROS production and inhibiting inflammatory reaction in septic myocardium.
Assuntos
Sepse , Proteína Desacopladora 2 , Animais , Inflamação , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio , Fator de Necrose Tumoral alfa , Proteína Desacopladora 2/genética , Proteína Desacopladora 2/fisiologiaRESUMO
Acute kidney injury (AKI) is a public health concern, with high morbidity and mortality rates in hospitalized patients and because survivors have an increased risk of progression to chronic kidney disease. Mitochondrial damage is the critical driver of AKI-associated dysfunction and loss of tubular epithelial cells; however, the pathways that mediate these events are poorly defined. Here, in murine ischemia/reperfusion (I/R)-induced AKI, we determined that mitochondrial damage is associated with the level of renal uncoupling protein 2 (UCP2). In hypoxia-damaged proximal tubular cells, a disruption of mitochondrial dynamics demonstrated by mitochondrial fragmentation and disturbance between fusion and fission was clearly indicated. Ucp2-deficient mice (knockout mice) with I/R injury experienced more severe AKI and mitochondrial fragmentation than wild-type mice. Moreover, genetic or pharmacological treatment increased UCP2 expression, improved renal function, reduced tubular injury and limited mitochondrial fission. In cultured proximal tubular epithelial cells, hypoxia-induced mitochondrial fission was exacerbated in cells with UCP2 deletion, whereas an increase in UCP2 ameliorated the hypoxia-induced disturbance of the balance between mitochondrial fusion and fission. Furthermore, results following modulation of UCP2 suggested it has a role in preserving mitochondrial integrity by preventing loss of membrane potential and reducing subsequent mitophagy. Taken together, our results indicate that UCP2 is protective against AKI and suggest that enhancing UCP2 to improve mitochondrial dynamics has potential as a strategy for improving outcomes of renal injury. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Injúria Renal Aguda/patologia , Mitocôndrias/fisiologia , Proteína Desacopladora 2/fisiologia , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/prevenção & controle , Animais , Hipóxia Celular/fisiologia , Células Cultivadas , Túbulos Renais Proximais/ultraestrutura , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica , Dinâmica Mitocondrial/fisiologia , Mitofagia/fisiologia , Proteínas Quinases/fisiologia , Traumatismo por Reperfusão/complicações , Proteína Desacopladora 2/deficiência , Proteína Desacopladora 2/genética , Proteína Desacopladora 2/metabolismo , Regulação para Cima/fisiologiaRESUMO
BACKGROUND: The uncoupling protein 2 (UCP2) gene plays an important role in the complications of type 2 diabetes (T2D). However, the association between variants in the UCP2 gene and diabetic retinopathy (DR) in Han Chinese T2D patients remains unclear. METHODS: Two single-nucleotide polymorphisms (SNPs) [rs659366 (-866G/A) and a 45-bp insertion/deletion (I/D) in the 3'-UTR] in the UCP2 gene were genotyped in a study cohort of 209 T2D patients with DR and 199 T2D patients without DR by direct DNA sequencing. RESULTS: Logistic regression analysis showed that the AA and GA genotypes of rs659366 were significantly associated with an increased risk for nonproliferative DR (NPDR) in the codominant model (corrected p-value <0.01) and the dominant model (corrected p-value = 0.006). Patients harboring the II and DI genotypes had a higher risk for PDR in the codominant model (corrected p-value = 0.011) and the dominant model (corrected p-value = 0.006), and the DI genotype showed a higher risk for NPDR in the dominant model (corrected p-value = 0.007) or codominant model (corrected p-value = 0.006). Further, haplotype analyses verified that the A-I haplotype is a risk haplotype for NPDR and PDR. CONCLUSION: This study suggests that the UCP2 gene may be involved in the pathogenesis of NPDR and PDR in Han Chinese patients with T2D.
Assuntos
Retinopatia Diabética/genética , Proteína Desacopladora 2/genética , Adulto , Alelos , Povo Asiático/genética , Estudos de Casos e Controles , China , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Retinopatia Diabética/metabolismo , Etnicidade/genética , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença/genética , Genótipo , Haplótipos/genética , Humanos , Mutação INDEL/genética , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , Proteína Desacopladora 2/fisiologiaRESUMO
The mitochondrial aspartate/glutamate transporter Aralar/AGC1/Slc25a12 is critically involved in brain aspartate synthesis, and AGC1 deficiency results in a drastic fall of brain aspartate levels in humans and mice. It has recently been described that the uncoupling protein UCP2 transports four carbon metabolites including aspartate. Since UCP2 is expressed in several brain cell types and AGC1 is mainly neuronal, we set to test whether UCP2 could be a mitochondrial aspartate carrier in the brain glial compartment. The study of the cerebral metabolism of (1-13C)-glucose in vivo in wild type and UCP2-knockout mice showed no differences in C3 or C2 labeling of aspartate, suggesting that UCP2 does not function as a mitochondrial aspartate carrier in brain. However, surprisingly, a clear decrease (of about 30-35 %) in the fractional enrichment of glutamate, glutamine and GABA was observed in the brains of UCP2-KO mice which was not associated with differences in either glucose or lactate enrichments. The results suggest that the dilution in the labeling of glutamate and its downstream metabolites could originate from the uptake of an unlabeled substrate that could not leave the matrix via UCP2 becoming trapped in the matrix. Understanding the nature of the unlabeled substrate and its precursor(s) as alternative substrates to glucose is of interest in the context of neurological diseases associated with UCP2.
Assuntos
Córtex Cerebral/metabolismo , Glucose/metabolismo , Proteína Desacopladora 2/fisiologia , Animais , Isótopos de Carbono/metabolismo , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Desacopladora 2/deficiência , Ácido gama-Aminobutírico/metabolismoRESUMO
AIMS/INTRODUCTION: Uncoupling protein 2 (UCP2), which was an important mitochondrial inner membrane protein associated with glucose and lipid metabolism, widely expresses in all kinds of tissues including hepatocytes. The present study aimed to explore the impact of UCP2 deficiency on glucose and lipid metabolism, insulin sensitivity and its effect on the liver-associated signaling pathway by expression profiling analysis. MATERIALS AND METHODS: Four-week-old male UCP2-/- mice and UCP2+/+ mice were randomly assigned to four groups: UCP2-/- on a high-fat diet, UCP2-/- on a normal chow diet, UCP2+/+ on a high-fat diet and UCP2+/+ on a normal chow diet. The differentially expressed genes in the four groups on the 16th week were identified by Affymetrix gene array. RESULTS: The results of intraperitoneal glucose tolerance test and insulin tolerance showed that blood glucose and ß-cell function were improved in the UCP2-/- group on high-fat diet. Enhanced insulin sensitivity was observed in the UCP2-/- group. The differentially expressed genes were mapped to 23 pathways (P < 0.05). We concentrated on the 'peroxisome proliferator-activated receptor (PPAR) signaling pathway' (P = 3.19 × 10(-11)), because it is closely associated with the regulation of glucose and lipid profiles. In the PPAR signaling pathway, seven genes (PPARγ, Dbi, Acsl3, Lpl, Me1, Scd1, Fads2) in the UCP2-/- mice were significantly upregulated. CONCLUSIONS: The present study used gene arrays to show that activity of the PPAR signaling pathway involved in the improvement of glucose and lipid metabolism in the liver of UCP2-deficient mice on a long-term high-fat diet. The upregulation of genes in the PPAR signaling pathway could explain our finding that UCP2 deficiency ameliorated insulin sensitivity. The manipulation of UCP2 protein expression could represent a new strategy for the prevention and treatment of diabetes.