Uncoupling Protein 2 Protein Expression Research Articles

The role of uncoupling protein 2 in experimental periodontitis-associated renal injury in rats.

This study aims to explore changes in uncoupling protein 2 (UCP2) in experimental periodontitis-associated renal injury induced by ligation and investigate the effect of UCP2 on renal injury induced by periodontitis. Twelve Wistar male rats were randomly divided into two groups: control and periodontitis groups. A periodontal model was built by ligating the maxillary first molars area with 0.2 mm orthodontic ligature wire. After 8 weeks, the intraoral condition of the rats was observed and periodontal clinical indices such as gingival bleeding index (BI), periodontal probing depth (PD), and tooth mobility (TM) were detected. The maxillary bone was scanned by Micro CT to observe the alveolar bone resorption. The tissue mineral density (TMD), bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular bone separation (Tb.Sp) were recorded, and the distance from the enamel bone boundary to the alveolar crest (CEJ-ABC) of the maxillary first molar was measured. The oxidative stress indexes such as malondialdehyde, glutathione (GSH), and superoxide dismutase (SOD) were detected using frozen rat kidney tissue. The gene expression of UCP2, nuclear factor erythroid 2-related factor 2 (Nrf2), and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) was observed by quantitative real-time polymerase chain reaction (qRT-PCR) test. The gingival tissue of the rats was used for immunohistochemical staining to observe the expression of the UCP2 protein. The fixed rat kidney tissue was used for hematoxylin-eosin (HE), periodic acid-schiff (PAS), MitoSOX Red, JC-1, and immunohistochemical staining to observe the renal histopathology, the level of reactive oxygen species (ROS), the level of mitochondrial membrane potential, and the expression of UCP2, Nrf2, and PGC-1α protein. Rat serum was collected to detect renal function indices, namely, blood urea nitrogen (BUN), creatinine (Cre), and albumin (Alb). Compared with the control group, the periodontitis group showed red, swollen, and soft gingival tissue, with gingival probing bleeding, periodontal PD increased, tooth loosening, alveolar bone resorption, decreased TMD, BMD, BV/TV, and Tb.Th indices, and increased Tb.Sp index, CEJ-ABC, and gingival UCP2 protein expression. Compared with the control group, the levels of MDA and ROS in the kidney tissue of periodontitis rats and the gene and protein expression of UCP2 increased, and the levels of MMP, GSH, and SOD and the gene and protein expression of Nrf2 and PGC-1α decreased. Renal functional indices, namely, BUN, Cre, and Alb, were not significantly different between the two groups. UCP2 may play a role in renal injury induced by periodontitis through oxidative stress.

Imbalances in mitochondrial metabolism and uncoupling protein 2 in policystic kidney disease cells.

Polycystic kidney disease (PKD) is proposed as a cancer-type disease. We demonstrated that PKD cells are more proliferative and glycolytic than tubular epithelial cells. Uncoupling protein 2 (UCP2), highly expressed in glycolytic cancer cells, enhances fatty acid and glutamine oxidation. Our aim was to characterize a potential role of UCP2 in PKD mitochondrial metabolism. We assessed respiration using a Seahorse XF Analyzer; NADH, TMRM and reactive oxygen species (ROS, cm-DCF) fluorescence by confocal microscopy; ATP by HPLC; and protein expression by western blotting. We studied PKD cells in comparison with normal kidney tubular epithelial cells. PKD cells displayed higher respiration, NADH/NAD+, and ROS levels (∼59%, ∼36%, and 58%, respectively), and lower mitochondrial membrane potential (ΔΨ) and ATP (∼50% and ∼32%, respectively), compared to controls. The antioxidant Trolox decreased ROS formation, basal respiration (∼60%), and glycolysis in PKD cells suggesting a regulatory effect of ROS on mitochondrial metabolism and glycolysis. N-acetyl-cystein (NAC), structurally unrelated to Trolox, decreased basal respiration in both control and PKD cells (10% and 30% vs vehicle, respectively). NAC also decreased maximum respiratory capacity in PKD cells (50% vs vehicle). UCP2 protein expression was ∼70% higher in PKD cells compared to control. The UCP2 inhibitor genipin (50 μM) increased ΔΨ and decreased respiration in PKD cells to the level of control cells suggesting that UCP2 may be involved in a metabolic switch in PKD. A futile cycle of substrate oxidation with minimal production of ATP, possibly mediated by UCP2, may trigger enhanced glycolysis as a compensatory mechanism favoring proliferation in PKD.

Thioredoxin Prevents Loss of UCP2 in Hyperoxia via MKK4-p38 MAPK-PGC1α Signaling and Limits Oxygen Toxicity.

Administration of high concentrations of oxygen (hyperoxia) is one of few available options to treat acute hypoxemia-related respiratory failure, as seen in the current coronavirus disease (COVID-19) pandemic. Although hyperoxia can cause acute lung injury through increased production of superoxide anion (O2•-), the choice of high-concentration oxygen administration has become a necessity in critical care. The objective of this study was to test the hypothesis that UCP2 (uncoupling protein 2) has a major function of reducing O2•- generation in the lung in ambient air or in hyperoxia. Lung epithelial cells and wild-type; UCP2-/-; or transgenic, hTrx overexpression-bearing mice (Trx-Tg) were exposed to hyperoxia and O2•- generation was measured by using electron paramagnetic resonance, and lung injury was measured by using histopathologic analysis. UCP2 expression was analyzed by using RT-PCR analysis, Western blotting analysis, and RNA interference. The signal transduction pathways leading to loss of UCP2 expression were analyzed by using IP, phosphoprotein analysis, and specific inhibitors. UCP2 mRNA and protein expression were acutely decreased in hyperoxia, and these decreases were associated with a significant increase in O2•- production in the lung. Treatment of cells with rhTrx (recombinant human thioredoxin) or exposure of Trx-Tg mice prevented the loss of UCP2 protein and decreased O2•- generation in the lung. Trx is also required to maintain UCP2 expression in normoxia. Loss of UCP2 in UCP2-/- mice accentuated lung injury in hyperoxia. Trx activates the MKK4-p38MAPK (p38 mitogen-activated protein kinase)-PGC1α (PPARγ [peroxisome proliferator-activated receptor γ] coactivator 1α) pathway, leading to rescue of UCP2 and decreased O2•- generation in hyperoxia. Loss of UCP2 in hyperoxia is a major mechanism of O2•- production in the lung in hyperoxia. rhTrx can protect against lung injury in hyperoxia due to rescue of the loss of UCP2.

Uncoupling protein 2 overexpression alleviates sepsis-induced myocardial injury via inhibiting reactive oxygen species production and inflammation

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. 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). 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/β-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/β-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. 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.

Down regulation of UCP2 expression in retinal pigment epithelium cells under oxidative stress: an in vitro study.

To evaluate the expression of uncoupling protein 2 (UCP2) in a retinal pigment epithelium cell line (ARPE-19), under oxidative stress (OS). ARPE-19 cells were divided into groups treated with various concentrations of hydrogen peroxide (H2O2; 0, 150, 300, 500, 700, and 900 µmol/L) for 24h, to induce oxidative damage and cell viability was assessed by MTT assay. UCP2 mRNA expression in cells treated with H2O2 was investigated by reverse transcription-polymerase chain reaction (RT-PCR). UCP2 protein expression was assessed by Western blotting and ROS levels analyzed by flow cytometry (FCM). Further, UCP2-siRNA treated cultures were exposed to H2O2 (0, 75, 150, and 300 µmol/L) for 2h and cell viability determined by MTT assay. Cells treated with higher concentrations of H2O2 appeared shrunken; their adhesion to adjacent cells was disrupted, and the number of dead cells increased. The results of cell viability assays demonstrated that the numbers of cells were decreased in a dose-dependent manner following treatment with H2O2. Compared with untreated controls, cell viability was significantly reduced after treatment with >300 µmol/L H2O2 (P<0.05). Cell metabolic activity was decreased with increased concentrations of H2O2 as detected by MTT assay. Levels of OS were further decreased in cells treated with UCP2-siRNA compared with those treated with H2O2 alone (P<0.05). The results of RT-PCR and Western blotting demonstrated that UCP2 expression was reduced in H2O2-treated groups compared with controls (P<0.05). FCM analysis showed that cell reactive oxygen species (ROS) levels were increased in H2O2-treated groups and further upregulated by UCP2-siRNA treatment (P<0.05). Expression levels of UCP2 are decreased in ARPE-19 cells treated with H2O2. ROS levels are further increased in cells treated with UCP2-siRNA relative to those treated with H2O2 alone. UCP2 may have a protective role in ARPE-19 cells during oxidative injury.

MRNA m6A plays opposite role in regulating UCP2 and PNPLA2 protein expression in adipocytes.

N6-methyladenosine (m6A) modification of mRNA plays an important role in regulating adipogenesis. However, its underlying mechanism remains largely unknown. Using Jinhua and Landrace pigs as fat and lean models, we presented a comprehensive transcriptome-wide m6A profiling in adipose tissues from these two pig breeds. Two differentially methylated genes were selected to explore the mechanisms of m6A-mediated regulation of gene function. The ratio of m6A/A in the layer of backfat (LB) was significantly higher in Landrace than that in Jinhua. Transcriptome-wide m6A profiling revealed that m6A modification on mRNA occurs in the conserved sequence motif of RRACH and that the pig transcriptome contains 0.53-0.91 peak per actively expressed transcript. The relative density of m6A peaks in the 3'UTR were higher than in 5'UTR. Genes with common m6A peaks from both Landrace (L-LB) and Jinhua (J-LB) were enriched in RNA splicing and cellular lipid metabolic process. The unique m6A peak genes (UMGs) from L-LB were mainly enriched in the extracellular matrix (ECM) and collagen catabolic process, whereas the UMGs from J-LB are mainly involved in RNA splicing, etc. Lipid metabolism processes were not significantly enriched in the UMGs from L-LB or J-LB. Uncoupling protein-2 (UCP2) and patatin-like phospholipase domain containing 2 (PNPLA2) were two of the UMGs in L-LB. Synonymous mutations (MUT) were conducted to reduce m6A level of UCP2 and PNPLA2 mRNAs. Adipogenesis test showed that UCP2-MUT further inhibited adipogenesis, while PNPLA2-MUT promoted lipid accumulation compared with UCP2-WT and PNPLA2-WT, respectively. Further study showed m6A negatively mediates UCP2 protein expression and positively mediates PNPLA2 protein expression. m6A modification affects the translation of PNPLA2 most likely through YTHDF1, whereas UCP2 is likely neither the target of YTHDF2 nor the target of YTHDF1. Our data demonstrated a conserved and yet dynamically regulated m6A methylome in pig transcriptomes and provided an important resource for studying the function of m6A epitranscriptomic modification in obesity development.

Atorvastatin Attenuates Metabolic Remodeling in Ischemic Myocardium through the Downregulation of UCP2 Expression.

Uncoupling protein 2 (UCP2) is primarily expressed in the myocardium and is closely related to myocardial ischemia/reperfusion injury and myocardial metabolism. To explore the effects and the mechanisms of UCP2 on atorvastatin-mediated myocardium protection, the rat model of myocardial ischemia was established by ligation of the left anterior descending coronary arteries (LADs). The rats were divided into the sham operation (SO) group, myocardial infarction (MI) group and MI-atorvastatin group. The study that atorvastatin reduced myocardial remodeling and improved the disturbed myocardial energy metabolism after MI. Furthermore, the mechanisms of myocardial metabolic remodeling affected by atorvastatin were explored. The atorvastatin group showed a significantly decreased expression of UCP2 mRNA and protein. Furthermore, the primary rat cardiomyocytes were cultured and treated with angiotensin II (Ang II) to induce cardiomyocyte hypertrophy. The results showed that in the atorvastatin group, the surface area of the cardiomyocytes, the total protein content per unit of cells, and the expression of the UCP2 protein were significantly decreased. These data suggested that atorvastatin significantly attenuated the myocardial remodeling by downregulating the expression of UCP2 that was found to improve the myocardial energy metabolism, inhibit myocardial hypertrophy, and eventually reduce myocardial remodeling

A differential expression of uncoupling protein-2 associates with renal damage in stroke-resistant spontaneously hypertensive rat/stroke-prone spontaneously hypertensive rat-derived stroke congenic lines.

Uncoupling protein-2 (UCP2), a mitochondrial anion transporter involved in mitochondrial uncoupling, limiting reactive oxygen species formation, is significantly downregulated in kidneys of high-salt-fed stroke-prone spontaneously hypertensive rat (SHRSP), where it associates with increased renal damage occurrence. We aimed at establishing whether UCP2 differential expression associates with renal damage in two stroke-resistant spontaneously hypertensive rat (SHRSR)/SHRSP-derived stroke congenic lines. For this purpose, SHRSR, SHRSP, and two reciprocal stroke congenic lines carrying the (D1Rat134-Mt1pa) segment of chromosome 1 were fed with Japanese style diet for 8 weeks. At 4, 6, and 8 weeks of Japanese diet, kidneys were removed and analyzed for UCP2 gene and protein expression [UCP2 maps within (D1Rat134-Mt1pa)]; nuclear factor kappa-light-chain-enhancer of activated B cells protein expression; oxidized total protein levels; mitochondrial function; gene expression of cubulin, megalin, and nephrin. At 6 and 8 weeks of Japanese diet, histological damage and percentage of high molecular weight urinary proteins excretion were assessed. Introgression of UCP2 in the SHRSP configuration within the SHRSR genome led to UCP2 downregulation upon Japanese diet, as compared with the SHRSR, with significantly reduced ATP levels, increased rate of inflammation, oxidative stress, renal damage, and excretion of high molecular weight proteins. The opposite phenomena were observed in the reciprocal congenic line, compared with the SHRSP. In vitro, high-NaCl medium led to UCP2 downregulation, increased apoptosis/necrosis, and reduced viability in primary renal proximal tubular epithelial cells isolated from SHRSP. Exposure of the SHRSP/proximal tubular epithelial cells to recombinant UCP2 rescued the high-salt-dependent deleterious effects. A differential UCP2 expression associates with different degree of renal damage upon Japanese diet in two SHRSR/SHRSP-derived stroke congenic lines through modulation of mitochondrial function, inflammation, and oxidative stress.

Protective effects and mechanism of insulin on brain in septic rats

Objective To investigate the protective effects and mechanism of insulin(INS) on brain in septic rats, and explore the possible role of uncoupling protein 2 (UCP2) in these effects. Methods Fifty male specific pathogen free(SPF) Sprague-Dawley rats were randomly divided into normal control (CN) group(n=10), lipopolysaccharide(LPS) group(n=20) and INS group (n=20) according to random number table.The septic rat model was established through an intraperitoneal injection of 15 mg/kg LPS of gram- negative bacteria.The rats in the INS group received a 1 U/kg INS injection subcutaneously 30 minutes before the injection of LPS, and the rats in the CN group were given equivalent 9 g/L saline in the same way.Eight rats in each group were killed, and their cerebral cortex were collected after the injection of LPS for 24 h. Pathological change of cerebral cortex was detected by Hematoxylin-Eosin(HE) staining.The cerebral cortex mitochondia were extracted for detecting the levels of reactive oxygen species(ROS), malondialdehyde (MDA) and the activity of superoxide dismutase(SOD). Neuronal apoptosis was detected by terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling staining.UCP2 mRNA expression was detected by quantitative real-time(RT)-PCR.Apoptosis-associated protein B lymphocyte tumor-2(Bcl-2), Bcl-2 associated X protein(Bax), cleaved cysteinyl aspartate specific protease(cleaved Caspase-9) and UCP2 protein expression were determined by Western blot. Results (1)Compared with the CN group, obvious abnormal pathological change was revealed by HE staining in cerebral cortex of rats in the LPS group and the INS group, but the pathological change was attenuated in the INS group compared with the LPS group.(2)Compared with the CN group, the levels of mitochondrial ROS[(210.01±14.09) RFU vs.(49.06±7.28) RFU] and MDA[(2.19±0.18) nmol/mg pro vs.(1.25±0.11)nmol/mg pro]in the LPS group significantly increased, whereas SOD activity significantly decreased [(238.49±35.60) U/g pro vs.(446.66±24.90)U/g pro], and the differences were significant(all P<0.05). Compared with the LPS group, the levels of ROS [(152.69±15.83) RFU vs.(210.01±14.09) RFU] and MDA[(1.55±0.14) nmol/mg pro vs.(2.19±0.18) nmol/mg pro] in the INS group decreased, while SOD activity increased[(327.8±23.26) U/g pro vs.(238.49± 35.60) U/g pro], and the differences were significant(all P<0.05). (3)Compared with the CN group, the neuronal apoptosis index of cortex in the LPS group was elevated[(54.16±6.84)% vs.(5.45±1.43)%], while the expression of Bcl-2 decreased (627±0.018 vs.0.739±0.020), but the expressions of Bax(0.768±0.019 vs.0.520±0.010) and cleaved Caspase-9(0.739±0.016 vs.0.467±0.030) increased, and the differences were significant(all P<0.05). Compared with the LPS group, the neuronal apoptosis index of cortex in the INS group decreased [(33.30±3.07)% vs.(54.16±6.84)%], but the Bcl-2 expression increased (0.743±0.022 vs.0.627±0.018), and Bax (0.687±0.034 vs.0.768±0.019) and cleaved Caspase-9(0.551±0.013 vs.0.739±0.016) were reduced, and the differences were significant (all P<0.05). (4)Compared with the CN group, the mRNA (2.248±0.155 vs.1.000±0.100) and protein expression of UCP2 (0.659±0.016 vs.0.599±0.018) were elevated in the LPS group.Compared with the LPS group, the UCP2 mRNA (2.944±0.117 vs.2.248±0.155) and UCP2 protein (0.719±0.018 vs.0.659±0.016) increased, and the differences were significant(all P<0.05). Conclusions INS can protect the brain of septic rats through alleviating mitochondrial oxidative stress and inhibiting the mitochondrial-initiated apoptotic pathway to reduce neuronal apoptosis.INS upregulates UCP2 expression in the brain of septic rats, which may play a role in the protective effects mentioned above. Key words: Sepsis; Brain; Insulin; Reactive oxygen species; Oxidative stress; Apoptosis; Uncoupling protein 2

Abstract 1033: Cigarette smoke and bile acids induce metabolic reprogramming mediated by uncoupling protein-2 (UCP2) during esophageal carcinogenesis

Abstract Background: In cancer cells, glucose is processed not through mitochondrial respiration, but mainly through glycolysis to form lactate and minimal ATP (Warburg effect). Glutamine is an essential amino acid in cancer cells and the primary fuel for the mitochondria. Uncoupling protein 2 (UCP2) decreases mitochondrial proton membrane potential and reduces reactive oxygen species (ROS). Limited information is available regarding mechanisms that link environmental risk factors such as gastro-esophageal reflux and cigarette smoking to metabolomic alterations in esophageal adenocarcinoma (EAC). The present study was undertaken to examine the effects of cigarette smoke and bile acids on UCP2 expression, metabolic activity, and malignant phenotype of EAC cells. Methods: Ten EAC lines were screened for UCP2 expression using qRT-PCR and immunoblot techniques. Two EAC lines, Flo-1 and NCI-SB-ESC2 (Esc2), had low and high UCP2 expression, respectively. Flo-1 and Esc2 cells were exposed to normal media with or without cigarette smoke condensate (CSC) and/or the bile acid, deoxycholic acid (DCA) for 5 days. Cell growth, migration, invasion, and clonogenicity were examined using CellTiter 96® Aqueous Kit, scratch, and soft agar clonogenicity assays. Mitochondrial respiration and glycolytic capacity were analyzed using the Seahorse Extracellular Flux Analyzer, and ATP and Lactate Production Kits. Levels of ROS were quantitated using Cellular Reactive Oxygen Species Detection Kit. Results: Flo-1 cells had low UCP2 protein expression and large spare mitochondrial respiratory capacity, whereas Esc2 cells exhibited high UCP2 protein expression and no spare mitochondrial respiratory capacity. CSC and DCA mediated synergistic increases in lactate levels with decreases in ATP production (Warburg effect) without altering levels of UCP2 or ROS in Flo-1 cells. In contrast, similar treatment did not alter lactate or ATP production, but did synergistically deplete UCP2 and increase ROS levels in Esc2 cells. Whereas CSC and DCA exposure did not alter proliferation, migration, or invasion of Flo-1 cells, these treatments enhanced proliferation, migration, and invasion of Esc2 cells. Conclusions: Following exposure to CSC and DCA, EAC cells can shift toward glycolysis when spare mitochondrial respiration is present. In contrast, EAC cells with maximal utilization of glycolysis have no ability to increase glycolysis with CSC or DCA exposure. However, in these cells, CSC and DCA down-regulates UCP2 and increases ROS; these metabolic effects coincide with increased malignant phenotype of EAC cells. These differences suggest that low levels of UCP2 may function as a metabolic sensor whereas high levels may serve as a cellular redox trigger effecting ROS. These preliminary insights into mitochondrial function may help guide the development of novel therapeutic strategies for EAC. Citation Format: Yuan Xu, Kate Brown, Deborah R. Depew, Paul L. Feingold, Mahadev Rao, Jeremy Davis, David S. Schrump, R. Taylor Ripley. Cigarette smoke and bile acids induce metabolic reprogramming mediated by uncoupling protein-2 (UCP2) during esophageal carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1033.

Mechanism of uncoupling protein2‑mediated myocardial injury in hypothermic preserved rat hearts.

In the present study, the alterations in uncoupling protein2 (UCP2) expression following hypothermic preservation in rat hearts were investigated. Isolated rat hearts were preserved in Celsior solution for 3‑12h followed by 60min of reperfusion. The cardiac function was evaluated using the Langendorff perfusion system. UCP2 and silent mating type information regulation2 homolog1 (SIRT1) proteins were detected by western blot analysis. The ATP production and mitochondrial reactive oxygen species (ROS) levels were assessed. Subsequent to preservation in ice‑cold Celsior solution for 3‑12h, the UCP2 protein expression in rat hearts was observed to increase in a time‑dependent manner. The UCP2 inhibitor genipin inhibited the hypothermic preservation‑induced cardiac dysfunction, prevented a decline in ATP production induced by 9h of preservation, however had no effect on the hypothermic preservation‑induced increase in mitochondrial ROS levels. Compared with the control group, the SIRT1 protein expression in rat hearts reduced following hypothermic preservation. Compared with the 9‑h preservation group, Celsior solution supplemented with the SIRT1 activator resveratrol (20 or 40µmol/l) inhibited UCP2 protein overexpression, prevented the decline in ATP production and resulted in an improvement cardiac function. The SIRT1 inhibitor EX‑527 abolished the resveratrol‑induced inhibition of UCP2 overexpression and cardiac protection in the hypothermic preserved rat heart. These observations suggest that downregulation of UCP2 expression in the hypothermic preserved rat heart in part initiated the protective mechanism via the SIRT1 pathway.

Dynamic regulation of uncoupling protein 2 expression by microRNA-214 in hepatocellular carcinoma.

Gemcitabine (GEM), a commonly used chemotherapeutic agent in hepatocellular carcinoma (HCC) patients, uses oxidative stress induction as a common effector pathway. However, GEM alone or in combination with oxaliplatin hardly renders any survival benefits to HCC patients. We have recently shown that this is part due to the overexpression of the mitochondrial uncoupling protein 2 (UCP2) that in turn mediates resistance to GEM in HCC patients. However, not much is known about regulatory mechanisms underlying UCP2 overexpression in HCC. Differential protein expression in HCC cell lines did not show a concomitant change in UCP2 transcript level, indicating post-transcriptional or post-translational regulatory mechanism. In situ analysis revealed that UCP2 is a putative target of miR-214 miR-214 expression is significantly down-regulated in HCC patient samples as compared with normal adjacent tissues and in cell line, human hepatoblastoma cells (HuH6), with high UCP2 protein expression. We demonstrated using miR-214 mimic and antagomir that the miRNA targeted UCP2 expression by directly targeting the wild-type, but not a miR-214 seed mutant, 3' UTR of UCP2 Overexpression of miR-214 significantly attenuated cell proliferation. Finally, analysis in 20 HCC patients revealed an inverse correlation in expression of UCP2 and miR-214 (Pearson's correlation coefficient, r=-0.9792). Cumulatively, our data indicate that in the context of HCC, miR-214 acts as a putative tumour suppressor by targeting UCP2 and defines a novel mechanism of regulation of UCP2.

Expression of UCP2 in Wistar rats varies according to age and the severity of obesity.

Obesity, a complex metabolic disorder, is characterized by mitochondrial dysfunction and oxidative stress. Increased expression of uncoupling protein 2 (UCP2) during obesity is an adaptive response to suppress the production of reactive oxygen species. The aims of this study were to compare the expression of UCP2 in diet-induced obese Wistar rats that differed according to age and their severity of obesity, and to compare UCP2 expression in the liver and muscle of these rats. UCP2 messenger RNA and protein expression was increased 4.6-fold (p < 0.0001) and 3.0-fold (p < 0.05), respectively, in the liver of the older and heavier rats. In contrast, UCP2 expression was decreased twofold (p < 0.005) in the muscle of these rats, while UCP3 messenger RNA (mRNA) was increased twofold (p < 0.01). Peroxisome proliferator-activated receptor alpha (PPARα) was similarly increased (3.0-fold, p < 0.05) in the liver of the older and more severe obese rats. Total protein content was increased (2.3-fold, p < 0.0001), while 5' adenosine monophosphate-activated protein kinase (AMPK) activity was decreased (1.3-fold, p = 0.05) in the liver of the older, heavier rats. No difference in total protein content and AMPK expression was observed in the muscle of these rats. This study showed that the expression of UCP2 varies according to age and the severity of obesity and supports the widely held notion that increased UCP2 expression is an adaptive response to increased fatty acid β-oxidation and reactive oxygen species production that occurs during obesity. An understanding of metabolic adaptation is imperative to gain insight into the underlying causes of disease, thus facilitating intervention strategies to combat disease progression.

Abstract P160: Role of Uncoupling Protein 2 in Stroke Susceptibility of Stroke-prone Spontaneously Hypertensive Rat

Mitochondrial dysfunction causes severe cellular derangements potentially underlying tissue injury and consequent diseases. Evidence of a direct involvement of mitochondrial dysfunction in hypertensive target organ damage is still poor. The gene encoding Uncoupling Protein 2 (UCP2), a inner mitochondrial membrane protein, maps inside stroke QTL/STR1 in stroke prone spontaneously hypertensive rat (SHRSP). We explored the role of UCP2 in stroke pathogenesis of SHRSP. Male SHRSP, stroke resistant SHR (SHRSR) and reciprocal STR1/congenic rats were fed with stroke permissive Japanese style diet (JD). A group of SHRSP received JD plus fenofibrate (150 mg/kg/die). Rats were sacrificed at stroke occurrence. Additional SHRSR and SHRSP rats were sacrificed at 1, 3, 6, 12 months of age upon regular diet. SBP, BW, proteinuria, stroke signs were monitored. Brains were used for molecular analysis (UCP2 gene and protein expression, Nf-kB protein expression, oxidative stress quantification) and for histological analyses. As a result, brain UCP2 expression was reduced to 20% by JD only in SHRSP (showing 100% stroke occurrence by 7 weeks of JD). Fenofibrate protected SHRSP from stroke and upregulated brain UCP2 (+ 100%). Congenic rats carrying STR1/QTL showed increased (+100%) brain UCP2 expression, as compared to SHRSP, when resistant to stroke, and, viceversa, decreased (-50%) brain UCP2 levels, as compared to SHRSR, when susceptible to stroke. Brain UCP2 expression progressively decreased with aging only in SHRSP, down to 15% level at one year of age (when SHRSP showed spontaneous stroke). Both brain Nf-kB expression and oxidative stress levels increased when UCP2 expression was downregulated, and viceversa. Histological analysis showed both ischemic and haemorrhagic lesions at stroke occurrence. Our results highlight a role of UCP2 in stroke predisposition associated to hypertension in an animal model of complex human disease.

Uncoupling protein 2 mediates resistance to gemcitabine-induced apoptosis in hepatocellular carcinoma cell lines.

Oxidative stress induction is a common effector pathway for commonly used chemotherapeutic agents like gemcitabine (GEM) in hepatocellular carcinoma (HCC) patients. However, GEM alone or in combination with oxiplatin hardly renders any survival benefits to HCC patients. Mitochondrial uncoupling protein 2 (UCP2) is known to suppress mitochondrial reactive oxygen species (ROS) generation, thus mitigating oxidative stress-induced apoptosis. We demonstrate in the present study, using a panel of HCC cell lines that sensitivity to GEM in HCC well correlate with the endogenous level of UCP2 protein expression. Moreover, ectopic overexpression of UCP2 in a HCC cell line with low endogenous UCP2 expression, HLE, significantly decreased mitochondrial superoxide induction by the anti-cancer drug GEM. Conversely, UCP2 mRNA silencing by RNA interference in HCC cell lines with high endogenous UCP2 expression significantly enhanced GEM-induced mitochondrial superoxide generation and apoptosis. Cumulatively, our results suggest a critical role for mitochondrial uncoupling in GEM resistance in HCC cell lines. Hence, synergistic targeting of UCP2 in combination with other chemotherapeutic agents might be more potent in HCC patients.

Lopimune-induced mitochondrial toxicity is attenuated by increased uncoupling protein-2 level in treated mouse hepatocytes.

Although the protease inhibitor (PI) Lopimune has proven to be effective, no studies have examined the side effects of Lopimune on mitochondrial bioenergetics in hepatocytes. The objective of the present study is to evaluate mitochondrial respiration, production of reactive oxygen species (ROS) and expression of uncoupling protein-2 (UCP2) in mouse hepatocytes following Lopimune administration. Mitochondria were extracted from mouse liver using differential centrifugation and hepatocytes were isolated by the collagenase perfusion procedure. Mitochondrial respiration was measured using a Rank Brothers oxygen electrode. ROS production in hepatocytes was monitored by flow cytometry using a 2',7'-dichlorofluorescin diacetate probe and UCP2 protein expression was detected by Western blotting. We found that Lopimune induced a significant decrease of approximately 30% in the respiratory control ratio (RCR) starting from day 4 until day 9 of treatment. This decrease was due to an increase in state 4 respiration, reflecting an increase in mitochondrial proton leak. State 2 and state 3 respirations were not affected. Moreover, ROS production significantly increased by about 2-fold after day 1 of treatment and decreased after day 3, returning to the resting level on day 5. Interestingly, UCP2 which is absent from control hepatocytes, was expressed starting from day 4 of treatment. Our findings indicate that Lopimune-induced proton leak, mediated by UCP2, may represent a response to inhibit the production of ROS as a negative feedback regulatory mechanism. These results imply a potential involvement of UCP2in the regulation of oxidative stress and add new insights into the understanding of mitochondrial toxicity induced by PIs.

The Role of Uncoupling Protein 2 During Myocardial Dysfunction in a Canine Model of Endotoxin Shock.

To explore the role of uncoupling protein 2 (UCP2) during myocardial dysfunction in a canine model of endotoxin shock, 26 mongrel canines were randomly divided into the following four groups: A (control group; n = 6), B2 (shock after 2 h; n = 7), B4 (shock after 4 h; n = 7), and B6 (shock after 6 h; n = 6). Escherichia coli endotoxin was injected into the canines via the central vein, and hemodynamics were monitored. Energy metabolism, UCP2 mRNA and protein expression, and UCP2 localization were analyzed, and the correlation between energy metabolism changes, and UCP2 expression was determined. After the canine endotoxin shock model was successfully established, the expression of UCP2 mRNA and protein was found to increase, with later time points showing significant increases (P < 0.05). Immunofluorescence assays of UCP2 in heart tissue showed that UCP2 was localized in the cytoplasm, and its expression pattern was the same as that found in the mRNA and protein analyses. The energy metabolism results revealed that the ADP levels increased, but the ATP and phosphocreatine (PCr) levels and ATP/ADP and PCr/ATP ratios decreased in the model. In particular, the PCr/ATP ratio was significantly different from that of the control group 6 h after shock (P < 0.05). Furthermore, correlation analysis showed that the UCP2 protein and mRNA levels were negatively correlated with myocardial energy levels. In summary, decreased energy synthesis can occur in the myocardium during endotoxin shock, and UCP2 may play an important role in this process. The negative correlation between UCP2 expression and energy metabolism requires further study, as the results might contribute to the treatment of sepsis with heart failure.

Hippocampal UCP2 is essential for cognition and resistance to anxiety but not required for the benefits of exercise

Uncoupling protein-2 (UCP2) reduces oxidative stress by facilitating the influx of protons into mitochondrial matrix, thus dissociating mitochondrial oxidation from ATP synthesis. UCP2 is expressed abundantly in brain areas and plays a key role in neuroprotection. Here, we sought to determine if UCP2 deficiency produces cognitive impairment and anxiety in young mice, and to determine if hippocampal UCP2 is essential for the beneficial effects of voluntary exercise. Antisense oligonucleotide (ASO) was used to produce UCP2 knockdown in mice. Our results firstly showed that UCP2-targeted ASO significantly reduced UCP2 mRNA and protein expression in the hippocampus. ASO treatment impaired learning and memory of the mice in Y-maze, T-maze, and object recognition tests (ORT). ASO-treated mice exhibited more anxiously in OPT, light/dark box test, and elevated plus maze (EPM) than the control mice. We also found that wheel running ameliorated cognitive dysfunction and anxiety-like behaviors in ASO-treated mice. Furthermore, voluntary exercise reversed ASO-induced changes in hippocampal levels of serotonin (5-HT), dopamine (DA), and norepinephrine (NE). However, UCP2 protein in the hippocampus was not correlated with cognitive and anxiolytic benefits of exercise. These findings suggest that hippocampal UCP2 is essential for cognitive function and the resistance to anxiety of mice, but not required for the beneficial effects of exercise.

The RNA binding protein hnRNP-K mediates post-transcriptional regulation of uncoupling protein-2 by angiopoietin-1

Angiopoietin-1 (Ang1) is a ligand for the receptor tyrosine kinase Tie2 and has key roles in the development of the vascular system and vascular protection. In a screen to define signalling pathways regulated by Ang1 in endothelial cells we found the RNA-binding protein hnRNP-K to be phosphorylated in response to Ang1. The ligand stimulated both tyrosine phosphorylation of hnRNP-K and recruitment of the tyrosine kinase Src to the RNA-binding protein. In endothelial cells hnRNP-K was found bound to mRNA encoding the mitochondrial protein uncoupling protein-2 (UCP2). Ang1 stimulation of cells resulted in the release of UCP2 mRNA from hnRNP-K. Using in vitro assays we confirmed direct binding between hnRNP-K and UCP2 mRNA. Furthermore Src induced phosphorylation of purified hnRNP-K and prevented UCP2 mRNA binding. Tyrosine 458 in the RNA-binding protein was found to be required for suppression of UCP2 mRNA binding by Src phosphorylation. In addition to releasing UCP2 mRNA from hnRNP-K, Ang1 induced an increase in UCP2 protein expression in endothelial cells without affecting total UCP2 mRNA levels. Consistent with the known effects of UCP2 to suppress generation of reactive oxygen species, Ang1 limited ROS production in endothelium stimulated with tumour necrosis factor-α. Taken together these data suggest that UCP2 mRNA is present in endothelial cells bound to hnRNP-K, which holds it in a translationally inactive state, and that Ang1 stimulates Src interaction with hnRNP-K, phosphorylation of the RNA-binding protein, release of these transcripts and upregulation of UCP2 protein expression. This study demonstrates a new mechanism for post-transcriptional regulation of UCP2 by the vascular protective ligand Ang1. The ability to rapidly upregulate UCP2 protein expression may be important in protecting endothelial cells from excessive generation of potentially damaging reactive oxygen species.

Differential expression of cardiac uncoupling proteins 2 and 3 in response to myocardial ischemia-reperfusion in rats

AimsWe aimed to evaluate the transcription and translation of genes for uncoupling protein 2 (UCP2) & uncoupling protein 3 (UCP3) in rat heart mitochondria of both ventricles after myocardial ischemia followed by various periods of reperfusion. Main methodsSeven groups of 8 male Wistar rats were evaluated for the effects of ischemia and also reperfusion, using Western blot of isolated mitochondrial proteins in addition to RNA extraction followed by real-time RT-PCR analysis. Key findingsIn rats with 30min of reperfusion (R30) UCP2 protein was increased 213±33%, which is meaningfully more than the control group (P<0.001). Western blot showed increase in UCP2 protein level in groups receiving reperfusion for 60min (R60), 120min (R120) and 180min (R180) as much as 152±28% (P<0.001 vs. control), 123±19% (P<0.01 vs. control) and 131±30% (P<0.01 vs. control), respectively. There was no statistically important difference in UCP2 mRNA between either right or left ventricles of ischemic and ischemia-reperfusion (IR) groups vs. control group. In the groups R180 and R240, UCP3 protein levels showed 131±27% and 102±18% increase, respectively (both P<0.001 vs. control group). However, the change in UCP3 level in other groups was not significantly different from the control group. SignificanceUCP2 and UCP3 protein levels are considerably increased in the ischemic area early after acute myocardial IR. The right ventricular UCP2 protein expression does not change, that is, effect of IR on UCP2 protein is a local process. However, UCP3 protein level increased both in ischemic area of the left ventricle and in non-ischemic area of the right ventricle.