Overexpression Of Uncoupling Protein Research Articles

Mitochondrial Uncoupling Protein-2 Ameliorates Ischemic Stroke by Inhibiting Ferroptosis-Induced Brain Injury and Neuroinflammation.

Ischemic stroke is a devastating disease in which mitochondrial damage or dysfunction substantially contributes to brain injury. Mitochondrial uncoupling protein-2 (UCP2) is a member of the UCP family, which regulates production of mitochondrial superoxide anion. UCP2 is reported to be neuroprotective for ischemic stroke-induced brain injury. However, the molecular mechanisms of UCP2 in ischemic stroke remain incompletely understood. In this study, we investigated whether and how UCP2 modulates neuroinflammation and regulates neuronal ferroptosis following ischemic stroke in vitro and in vivo. Wild-type (WT) and UCP2 knockout (Ucp2-/-) mice were subjected to middle cerebral artery occlusion (MCAO). BV2 cells (mouse microglial cell line) and HT-22 cells (mouse hippocampal neuronal cell line) were transfected with small interfering (si)-RNA or overexpression plasmids to knockdown or overexpress UCP2 levels. Cells were then exposed to oxygen-glucose deprivation and reoxygenation (OGD/RX) to simulate hypoxic injury in vitro. We found that UCP2 expression was markedly reduced in a time-dependent manner in both in vitro and in vivo ischemic stroke models. In addition, UCP2 was mainly expressed in neurons. UCP2 deficiency significantly enlarged infarct volumes, aggravated neurological deficit scores, and exacerbated cerebral edema in mice after MCAO. In vitro knockdown of Ucp2 and in vivo genetic depletion of Ucp2 (Ucp2-/- mice) increased neuronal ferroptosis-related indicators, including Fe2+, malondialdehyde, glutathione, and lipid peroxidation. Overexpression of UCP2 in neuronal cells resulted in reduced ferroptosis. Moreover, knockdown of UCP2 exacerbated neuroinflammation in BV2 microglia and mouse ischemic stroke models, suggesting that endogenous UCP2 inhibits neuroinflammation following ischemic stroke. Upregulation of UCP2 expression in microglia appeared to decrease the release of pro-inflammatory factors and increase the levels of anti-inflammatory factors. Further investigation showed that UCP2 deletion inhibited expression of AMPKα/NRF1 pathway-related proteins, including p-AMPKα, t-AMPKα, NRF1, and TFAM. Thus, UCP2 protects the brain from ischemia-induced ferroptosis by activating AMPKα/NRF1 signaling. Activation of UCP2 represents an attractive strategy for the prevention and treatment of ischemic stroke.

UCP2 overexpression activates SIRT3 to regulate oxidative stress and mitochondrial dynamics induced by myocardial injury

ObjectiveOur previous study found that overexpression of uncoupling protein-2 (UCP2) had a protective effect on lipopolysaccharide (LPS)-induced sepsis cardiomyocytes. The aim of this study was to explore the effect and mechanism of uncoupling protein-2 (UCP2) on myocardial ischemia-reperfusion injury. MethodsIn this study, we established hypoxia-reoxygenation (HR) injury model in rats and isolated cardiomyocytes of newborn rats. We also carried out following methods which include virus transfection technology, cell counting Kit-8 (CCK8), flow cytometry, enzyme linked immunosorbent assay (ELISA), Western blot (WB), quantitative reverse transcription PCR (RT qPCR), transmission electron microscopy, fluorescence colocalization and immunoprecipitation. Main resultsThe results of this study showed that hypoxia-reoxygenation treatment in cardiomyocytes increased UCP2, myocardial enzyme and myocardial apoptosis and weakened cardiomyocyte viability. We observed increased cardiomyocyte viability and mitochondrial membrane potential, decreased myocardial enzyme and myocardial apoptosis, Inhibition of oxidative stress when UCP2 was overexpressed in cardiomyocytes. It also can Increase ATP and stabilize mitochondrial dynamics. Further studies founded that Sirtuin-3(SIRT3) changed with the expression of UCP2, which was confirmed by fluorescence co-localization and immunoprecipitation. ConclusionsOur findings revealed that UCP2 and SIRT3 were important targets of anti-myocardial injury by inhibiting cellular oxidative stress and stabilizing mitochondrial dynamics.

UCP2-SIRT3 Signaling Relieved Hyperglycemia-Induced Oxidative Stress and Senescence in Diabetic Retinopathy.

Diabetic retinopathy (DR) is one of the most common reasons for blindness. uncoupling protein 2 (UCP2), an uncoupling protein located in mitochondria, has been reported to be related to metabolic and vascular diseases. This research aimed to illustrate the function and mechanism of UCP2 in the pathogenesis of DR. Human epiretinal membranes were collected to investigate the expression of UCP2 by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence. Primary human retinal microvascular endothelial cells (HRECs) were cultured in high glucose (HG) to establish an in vitro cell model for DR. Flow cytometry analysis was used to measure intracellular reactive oxygen species (ROS). Senescence levels were evaluated by the senescence-associated beta-galactosidase (SA-β-gal) assay, the expression of senescence marker P21, and cell-cycle analysis. Adenovirus-mediated UCP2 overexpression or knockdown and specific inhibitors were administered to investigate the underlying regulatory mechanism. Proliferative fibrovascular membranes from patients with DR illustrated the downregulation of UCP2 and sirtuin 3 (SIRT3) by qRT-PCR and immunofluorescence. Persistent hyperglycemia-induced UCP2 downregulation in the progress of DR and adenovirus-mediated UCP2 overexpression protected endothelial cells from hyperglycemia-induced oxidative stress and senescence. Under hyperglycemic conditions, UCP2 overexpression attenuated NAD+ downregulation; hence, it promoted the expression and activity of SIRT3, an NAD+-dependent deacetylase regulating mitochondrial function. 3-TYP, a selective SIRT3 inhibitor, abolished the UCP2-mediated protective effect against oxidative stress and senescence. UCP2 overexpression relieved oxidative stress and senescence based on a novel mechanism whereby UCP2 can regulate the NAD+-SIRT3 axis. Targeting oxidative stress and senescence amelioration, UCP2-SIRT3 signaling may serve as a method for the prevention and treatment of DR and other diabetic vascular diseases.

Early Adipogenesis and Upregulation of UCP1 in Mesenchymal Stromal Cells Stimulated by Devitalized Microfragmented Fat (MiFAT).

Adipose tissue is mainly composed by adipocytes. Moreover, mesenchymal stromal/stem cells (MSCs), macrophages, endothelial cells, and extracellular matrix components are present. The variety of molecules as cytokines and growth factors of its structure very rich in blood vessel makes it also similar to a true endocrine organ that however needs still to be fully investigated. In our study, we used human lipoaspirate to obtain mechanically microfragmented fat (MiFAT) which was washed and then devitalized by freezing-thawing cycles. In our experiments, thawed MiFAT was used to stimulate cultures of MSCs from two different sources (adipose tissue and gingiva papilla) in comparison with a traditional stimulation in vitro obtained by culturing MSCs with adipogenic medium. MSCs stimulated with MiFAT showed a very early production of lipid droplets, after only 3 days, that correlated with an increased expression of adipokines. Furthermore, a significant upregulation of PPAR gamma 1 alpha coactivator (PPARGC1A) was observed with an overexpression of uncoupling protein 1 (UCP1) that suggest a pattern of differentiation compatible with the beige-brown fat.

Abstract 17359: UCP2 Induced Glycolysis Promotes Histone Acetylation to Regulate Cardiomyocyte Cell Cycle After Injury

Developmental cardiac tissue is proliferative capable of robust regenerative response after myocardial injury, but this ability is lost by P7 when cardiomyocytes (CMs) exit the cell cycle and become terminally differentiated. Postnatal CMs exhibit a specialized metabolic state that supports rapid proliferation and is altered with loss of regeneration in the heart. Studies have shown that reliance on glycolysis for energy generation is preferred by proliferating CMs and activation of glycolysis promotes cardiac repair. Nevertheless, the role of glycolysis in regulating CM gene program linked to cell cycle and repair in the heart remains poorly studied. Here, we identify a novel role for mitochondrial uncoupling protein 2 (UCP2) in the heart regulating CM cell cycle dynamics. UCP2 was found to be primarily active during cardiac development, rapidly decreases after birth and is expressed at low levels in the adult heart. UCP2 overexpression in human IPS derived CMs increases cell cycle progression as assessed by immunostaining and flow cytometry-based DNA content analysis. In parallel, neonatal rat ventricular myocytes (NRVMs) overexpressing UCP2 showed increased glycolysis and glycolytic metabolites, reduced mitochondrial membrane potential and mitochondrial activity compared to controls. Next, we generated αMHC-UCPKO mice that showed reduction in total number of CMs and pHH3 levels, increased CM size and ploidy during postnatal development. Adult αMHC-UCPKO subjected to myocardial infarction injury showed significant cardiac dysfunction and fibrosis at 4 weeks compared to controls. Mechanistically, UCP2 induced glycolysis increases ATP-citrate lyase (ACLY) mediated acetyl-CoA generation in the CMs. Additionally, CMs isolated from P1 αMHC-UCPKO showed several histone modifications including decrease in histone acetylation marks. Similarly, NRVMs overexpressing UCP2 showed increased histone H3 acetylation and expression of histone acetyltransferases (HATs) and corresponding decrease in histone deacetylases (HDACs). In conclusion, UCP2 is a novel developmental metabolic regulator of CM cell cycle in the neonatal and adult heart. Moreover, UCP2 induced glycolysis increases cell cycle via altering CM histone acetylation.

Uncoupling protein 2 modulates polarization and metabolism of human primary macrophages via glycolysis and the NF‑κB pathway.

Metabolic abnormalities, particularly the M1/M2 macrophage imbalance, play a critical role in the development of various diseases, leading to severe inflammatory responses. The present study aimed to investigate the role of uncoupling protein 2 (UCP2) in regulating macrophage polarization, glycolysis, metabolic reprogramming, reactive oxygen species (ROS) and inflammation. Primary human macrophages were first polarized into M1 and M2 subtypes, and these two subtypes were infected by lentivirus-mediated UCP2 overexpression or knockdown, followed by enzyme-linked immunosorbent assay, reverse transcription-quantitative PCR, western blotting and flow cytometry to analyze the effects of UCP2 on glycolysis, oxidative phosphorylation (OXPHOS), ROS production and cytokine secretion, respectively. The results demonstrated that UCP2 expression was suppressed in M1 macrophages and increased in M2 macrophages, suggesting its regulatory role in macrophage polarization. UCP2 overexpression decreased macrophage glycolysis, increased OXPHOS, decreased ROS production, and led to the conversion of M1 polarization to M2 polarization. This process involved NF-κB signaling to regulate the secretion profile of cytokines and chemokines and affected the expression of key enzymes of glycolysis and a key factor for maintaining mitochondrial homeostasis (nuclear respiratory factor 1). UCP2 knockdown in M2 macrophages exacerbated inflammation and oxidative stress by promoting glycolysis, which was attenuated by the glycolysis inhibitor 2-deoxyglucose. These findings highlight the critical role of UCP2 in regulating macrophage polarization, metabolism, inflammation and oxidative stress through its effects on glycolysis, providing valuable insights into potential therapeutic strategies for macrophage-driven inflammatory and metabolic diseases.

UCP2 Upregulation Increases Fra-1 Expression and S-phase Cell Population without Decreasing Apoptosis during Skin Cell Transformation

Upregulation of uncoupling protein 2 (UCP2) is considered a prosurvival mechanism for cancer cells. This prosurvival function is thought to be mediated by UCP2’s uncoupling activity which reduces the production of superoxide in the mitochondria. However, exactly how highly expressed UCP2 regulates cell proliferation, cell cycle, and cell death during the early stage of tumorigenesis has not been studied thoroughly. For this purpose, we generated UCP2 stably overexpressed JB6 Cl-41 cells (a skin cell transformation model) and performed studies to answer the above questions. Our results demonstrated that UCP2 overexpression enhanced cell proliferation, activation of the oncoprotein Fra-1, anchorage-independent growth, 3D spheroids growth, and glucose uptake during skin cell transformation. Next, our results demonstrated that UCP2 overexpression resulted in marked decreases in the proportion of the cells in the G1 phase and an increase of cells in the S phase of the cell cycle, which was accompanied by increased expression of Cyclin E and Cdk2. Lastly, UCP2 overexpression did not enhance or suppress apoptosis during skin cell transformation, as indicated by Annexin V and active caspase 3/7 staining. Taken together, these data suggest that UCP2 upregulation mainly enhances the Fra-1 oncogenic pathway which drives cell proliferation, without inhibiting apoptosis during skin cell transformation.

Overexpression of UCP3 decreases mitochondrial efficiency in mouse skeletal muscle in vivo.

Uncoupling protein-3 (UCP3) is a mitochondrial transmembrane protein highly expressed in the muscle that has been implicated in regulating the efficiency of mitochondrial oxidative phosphorylation. Increasing UCP3 expression in skeletal muscle enhances proton leak across the inner mitochondrial membrane and increases oxygen consumption in isolated mitochondria, but its precise function in vivo has yet to be fully elucidated. To examine whether muscle-specific overexpression of UCP3 modulates muscle mitochondrial oxidation in vivo, rates of ATP synthesis were assessed by 31 P magnetic resonance spectroscopy (MRS), and rates of mitochondrial oxidative metabolism were measured by assessing the rate of [2-13 C]acetate incorporation into muscle [4-13 C]-, [3-13 C]-glutamate, and [4-13 C]-glutamine by high-resolution 13 C/1 H MRS. Using this approach, we found that the overexpression of UCP3 in skeletal muscle was accompanied by increased muscle mitochondrial inefficiency in vivo as reflected by a 42% reduction in the ratio of ATP synthesis to mitochondrial oxidation.

Downregulation of Uncoupling Protein 2(UCP2) Mediated by MicroRNA-762 Confers Cardioprotection and Participates in the Regulation of Dynamic Mitochondrial Homeostasis of Dynamin Related Protein1 (DRP1) After Myocardial Infarction in Mice.

Acute myocardial infarction (MI) is one of the leading causes of death in the world, and its pathophysiological mechanisms have not been fully elucidated. The purpose of this study was to investigate the role and mechanism of uncoupling protein 2 (UCP2) after MI in mouse heart. Here, we examined the expression and role of UCP2 in mouse heart 4 weeks after MI. The expression of UCP2 was detected by RT-PCR and western blotting. Cardiac function, myocardial fibrosis, and cardiomyocyte apoptosis were assessed by echocardiography and immunohistochemistry. Phosphatase dynamin-related protein1 (P-DRP1) and myocardial fibrosis-related proteins were measured. Cardiomyocytes were exposed to hypoxia for 6 h to mimic the model of MI. Mdivi, an inhibitor of P-DRP1, was used to inhibit DRP1-dependent mitochondrial fission. Mitochondrial superoxide, membrane potential, oxygen consumption rate, and cardiomyocyte apoptosis were detected after hypoxia. It is shown mitochondrial superoxide, membrane potential, oxygen consumption rate, and cardiomyocyte apoptosis were dependent on the level of P-DRP1. UCP2 overexpression reduced cardiomyocyte apoptosis (fibrosis), improved cardiac function and inhibit the phosphorylation of DRP1 and the ratio of P-DRP1/DRP1. However, inhibition of DRP1 by mdivi did not further reduce cell apoptosis rate and cardiac function in UCP2 overexpression group. In addition, bioinformatics analysis, luciferase activity, and western blot assay proved UCP2 was a direct target gene of microRNA-762, a up-regulated microRNA after MI. In conclusion, UCP2 plays a protective role after MI and the mechanism is involved in microRNA-762 upstream and DRP1-dependent mitochondrial fission downstream.

An interplay between UCP2 and ROS protects cells from high-salt-induced injury through autophagy stimulation

The mitochondrial uncoupling protein 2 (UCP2) plays a protective function in the vascular disease of both animal models and humans. UCP2 downregulation upon high-salt feeding favors vascular dysfunction in knock-out mice, and accelerates cerebrovascular and renal damage in the stroke-prone spontaneously hypertensive rat. Overexpression of UCP2 counteracts the negative effects of high-salt feeding in both animal models. We tested in vitro the ability of UCP2 to stimulate autophagy and mitophagy as a mechanism mediating its protective effects upon high-salt exposure in endothelial and renal tubular cells. UCP2 silencing reduced autophagy and mitophagy, whereas the opposite was true upon UCP2 overexpression. High-salt exposure increased level of reactive oxygen species (ROS), UCP2, autophagy and autophagic flux in both endothelial and renal tubular cells. In contrast, high-salt was unable to induce autophagy and autophagic flux in UCP2-silenced cells, concomitantly with excessive ROS accumulation. The addition of an autophagy inducer, Tat-Beclin 1, rescued the viability of UCP2-silenced cells even when exposed to high-salt. In summary, UCP2 mediated the interaction between high-salt-induced oxidative stress and autophagy to preserve viability of both endothelial and renal tubular cells. In the presence of excessive ROS accumulation (achieved upon UCP2 silencing and high-salt exposure of silenced cells) autophagy was turned off. In this condition, an exogenous autophagy inducer rescued the cellular damage induced by excess ROS level. Our data confirm the protective role of UCP2 toward high-salt-induced vascular and renal injury, and they underscore the role of autophagy/mitophagy as a mechanism counteracting the high-salt-induced oxidative stress damage.

The effects of UCP2 on autophagy through the AMPK signaling pathway in septic cardiomyopathy and the underlying mechanism.

BackgroundMitochondrial dysfunction plays an important role in the development of septic cardiomyopathy. This study aimed to reveal the protective role of uncoupling protein 2 (UCP2) in mitochondria through AMP-activated protein kinase (AMPK) on autophagy during septic cardiomyopathy.MethodsUCP2 knockout mice via a cecal ligation and puncture (CLP) model and the H9C2 cardiomyocyte cell line in response to lipopolysaccharide (LPS) in vitro were used to study the effect. The myocardial morphological alterations, indicators of mitochondrial injury and levels of autophagy-associated proteins (pAMPK, pmTOR, pULK1, pTSC2, Beclin-1, and LC3-I/II) were assessed. In addition, the mechanism of the interaction between UCP2 and AMPK was further studied through gain- and loss-of-function studies.ResultsCompared with the wild-type mice, the UCP2 knockout mice exhibited more severe cardiomyocyte injury after CLP, and the AMPK agonist AICAR protected against such injury. Consistent with this result, silencing UCP2 augmented the LPS-induced pathological damage and mitochondrial injury in the H9C2 cells, limited the upregulation of autophagy proteins and reduced AMPK phosphorylation. AICAR protected the cells from morphological changes and mitochondrial membrane potential loss and promoted autophagy. The silencing and overexpression of UCP2 led to correlated changes in the AMPK upstream kinases pLKB1 and CAMKK2.ConclusionsUCP2 exerts cardioprotective effects on mitochondrial dysfunction during sepsis via the action of AMPK on autophagy.

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.

JNK inhibition alleviates oxidative DNA damage, germ cell apoptosis, and mitochondrial dysfunction in testicular ischemia reperfusion injury.

The aim of this study is to determine whether the c-Jun N-terminal kinase (JNK) signaling is a regulator of oxidative DNA damage, germ cell apoptosis (GCA), and mitochondrial dysfunction during testicular ischemia reperfusion injury (tIRI) using the JNK inhibitor SP600125. Male Sprague Dawley rats (n = 36) were equally divided into three groups: sham, tIRI only, and tIRI + SP600125 (15mg/kg). Testicular ischemia was induced for 1h followed by 4h of reperfusion prior to animal sacrifice. Spermatogenesis was evaluated by light microscopy, while expression of oxidative stress and GCA-related mRNAs and proteins were evaluated by real-time polymerase chain reaction and colorimetric assays, respectively. Expressions of JNK, p53, and survivin were detected by immunofluorescence (IF) staining. Indicators of mitochondrial dysfunction were examined by western blot analysis and colorimetric assay. In comparison to sham, the tIRI testes showed a significant increase in lipid and protein oxidation products. Oxidative DNA damage was reflected by a significant increase in the number of DNA strand breaks, increased concentration of 8-OHdG, and elevated poly (ADP-ribose) polymerase activity. Spermatogenic damage was associated with the activation of caspase 3 and elevated Bax to Bcl2 ratio. This was also accompanied by a significantly heightened IF expression of the phosphorylated forms of JNK and p53 paralled with the suppression of survivin. Mitochondrial dysfunction was reflected by NAD+ depletion, overexpression of uncoupling protein 2, and increased level of cytochrome c. Such tIRI-induced modulations were all attenuated by SP600125 treatment prior to reperfusion. In conclusion, JNK signaling regulates oxidative DNA damage, GCA, and mitochondrial dysfunction through activation of p53 and suppression of survivin during tIRI.

Brain Overexpression of Uncoupling Protein-2 (UCP2) Delays Renal Damage and Stroke Occurrence in Stroke-Prone Spontaneously Hypertensive Rats.

The downregulation of uncoupling protein-2 (UCP2) is associated with increased brain and kidney injury in stroke-prone spontaneously hypertensive rats (SHRSP) fed with a Japanese style hypersodic diet (JD). Systemic overexpression of UCP2 reduces organ damage in JD-fed SHRSP. We examined the effect of brain-specific UCP2 overexpression on blood pressure (BP), stroke occurrence and kidney damage in JD-fed SHRSP. Rats received a single i.c.v. injection of a lentiviral vector encoding UCP2 (LV-UCP2), or an empty vector. The brain delivery of LV-UCP2 significantly delayed the occurrence of stroke and kidney damage. The large reduction of proteinuria observed after LV-UCP2 injection was unexpected, because BP levels were unchanged. At the time of stroke, rats treated with LV-UCP2 still showed a large UCP2 upregulation in the striatum, associated with increases in OPA1 and FIS1 protein levels, and reductions in PGC1-α, SOD2, TNFα mRNA levels and NRF2 protein levels. This suggested UCP2 overexpression enhanced mitochondrial fusion and fission and reduced oxidative damage and inflammation in the striatum of JD-fed SHRSP rats. Our data suggest the existence of central mechanisms that may protect against hypertension-induced organ damage independently of BP, and strengthen the suitability of strategies aimed at enhancing UCP2 expression for the treatment of hypertensive damage.

UCP2 silencing in glioblastoma reduces cell proliferation and invasiveness by inhibiting p38 MAPK pathway

Uncoupling protein-2 (UCP2) is a mitochondrial inner membrane anion carrier and is emerging as a negative regulator of ROS production. Overexpression of UCP2 has been detected in various tumors, but its role in glioblastoma remains unclear. Using tissue microarrays and interrogations of public databases, we explored that the expression of UCP2 is upregulated in glioma, especially in GBM, and overexpression of UCP2 correlates with poor prognosis in glioma patients. To further reveal the role of UCP2 in glioma, UCP2-slienced cell lines (U251, U87MG and A172) by lentivirus were constructed to study how silenced UCP2 expression affects cellular functions in vitro, and tumorigenicity in vivo. RNA-Seq based genome and pathway analysis were performed to elucidate the underlying mechanisms of action of UCP2. Our results revealed that UCP2 silenced glioma cells show inhibited migration, invasiveness, clonogenicity, proliferation, promoted cell apoptosis in vitro, and weaker tumorigenicity in nude mice. Transcriptome analysis suggested a UCP2-dependent regulation of p38 MAPK (Mitogen-activated protein kinase) signaling networks, which was further validated by qRT-PCR and Western blot. Our research provides a new insight into the biological significance of UCP2 in glioma and its potential application in treatment and diagnosis.

Mitochondrial Dysfunction Contributes to Germ Cell Apoptosis via the JNK/p53/ survivin Pathway

The aim is to show whether the c‐Jun N‐terminal kinases (JNK) signaling is a regulator of oxidative DNA damage, germ cell apoptosis (GCA) and mitochondrial dysfunction during testicular ischemia reperfusion injury (tIRI) using the JNK inhibitor SP600125. Male Sprague‐Dawley rats (n=36) were equally divided into 3 groups: sham, tIRI only and tIRI + SP600125 (15 mg/Kg, i.p.). Testicular ischemia was induced for 1 hour followed by 4 hours reperfusion prior to animal sacrifice. Spermatogenesis was evaluated by light microscopy using the Johnsen’s scoring system. Expression of oxidative stress and GCA genes and proteins were evaluated by real‐time PCR and colorimetric assays, respectively. Activation of the JNK/p53/survivin signaling pathway was detected by immunofluorescence (IF) staining. Indicators of mitochondrial dysfunction were examined by Western blot and colorimetric assays. In comparison to sham group, the tIRI testes showed a significant decrease in the antioxidant activity of superoxide dismutase, which was associated with increased lipid and protein oxidation products. Oxidative DNA damage was reflected by a significant increase in the number of single and double DNA strand breaks and increased concentration of 8‐OHdG DNA adducts. Spermatogenic damage was associated with the activation of caspases 9 and 3 and an elevated Bax to Bcl2 ratio. This was also accompanied by a significantly heightened IF expression of the phosphorylated forms of JNK and p53 paralleled with the down‐regulation of survivin. Mitochondrial dysfunction was reflected by NADH depletion, overexpression of uncoupling protein 2 and increased levels of cytochrome c protein. Such tIRI‐induced modulations were all attenuated by SP600125 treatment prior to reperfusion. In conclusion, mitochondrial dysfunction could contribute to GCA during tIRI under the control of the JNK/p53/survivin signaling pathway.Support or Funding InformationThis study was supported by Kuwait University Grants YM 15/17 (College of Graduate Studies ) and SRU02/13 (Research Administration).

PGC-1andalpha; and p38 MAPK are involved in uncoupling protein 2-dependent reactive oxygen species production in renal tubular epithelial cells

Object: The objective was to investigate Uncoupling protein 2 (UCP2)-dependent Reactive oxygen species (ROS) production in renal tubular epithelial cells treated with high glucose. Methods: In this study, we investigated the associations among PGC-1α, p38 MAPK and UCP2 in HK-2 cells treated with high glucose. Target genes overexpression and silence were obtained by plasmid transfection, and SB203580 was applied as a p38 MAPK inhibitor. The mutual effect of these three proteins was evaluated by RTPCR and western blot. ROS production was detected by a fluorescent probe. Results: We found that UCP2 overexpression enhanced PGC-1α expression, as well as inhibited p38 MAPK activation and ROS generation. These changes were reversed by UCP2 siRNA. However, neither PGC-1α nor p38 MAPK could exert significant influence on the expressions of the other two proteins, although their capacity to regulate ROS was demonstrated. Conclusion: These results indicate UCP2 plays a pivotal and relatively upstream role in oxidative stress induced by high glucose in renal tubular epithelial cells.

Effects of uncoupling protein 2 overexpression on myocardial mitochondrial dynamics in sepsis rats

To investigate the effects of uncoupling protein 2 (UCP2) overexpression on mitochondrial dynamics (mitochondrial division and fusion) of sepsis myocardial injury in rats. Forty male Sprague-Dawley (SD) rats were randomly divided into four groups (n = 10): sham operation group (Sham group) using normal saline instead of transfection and simulating cecal ligation and perforation (CLP); CLP group using normal saline instead of transfection, performing CLP to induce sepsis; adeno-associated virus (AAV) group using CLP after myocardial transfection with empty virus; UCP2 overexpression group (UCP2 group) CLP was performed 3 weeks after AAV-UCP2 (1×1015 vg/L, a total of 60 μL) myocardial transfection. The rats in each group were examined by echocardiography at 24 hours after the CLP, and then the rats were sacrificed immediately to harvest myocardial tissue. Myocardial ultrastructural changes were observed under the electron microscope, the expression of regulatory proteins related to myocardial mitochondrial dynamics [optic atrophy 1 (Opa1), dynamin-related protein 1 (Drp1) and fission 1 (Fis1)] were detected by Western Blot, and the level of mitochondrial adenosine triphosphate (ATP) production was detected by chemiluminescence. (1) The echocardiographic results showed that there was no significant difference in left ventricular mass (LVM) and stroke volume (SV). Compared with Sham group, left ventricular diastolic anterior wall thickness (LVAWd), left ventricular systolic anterior wall thickness (LVAWs), left ventricular diastolic posterior wall thickness (LVPWd), left ventricular systolic posterior wall thickness (LVPWs), left ventricular ejection fraction (LVEF) and left ventricular short axis shortening rate (LVFS) were significantly increased in CLP group and AAV group, while left ventricular systolic diameter (LVEDs), left ventricular diastolic diameter (LVEDd), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV) were significantly decreased. Compared with CLP group and AAV group, LVAWs, LVEF, LVFS were significantly decreased in UCP2 group, and LVEDs, LVEDV and LVESV were significantly increased [LVAWs (mm): 3.82±0.42 vs. 4.34±0.30, 4.44±0.12; LVEF: 0.921±0.038 vs. 0.979±0.019, 0.991±0.010; LVFS: (65.33±6.56)% vs. (80.11±8.23)%, (85.31±6.11)%; LVEDs (mm): 1.81±0.36 vs. 0.89±0.54, 0.60±0.17; LVEDV (μL): 137.09±50.05 vs. 89.72±53.04, 85.42±40.99; LVESV (μL): 10.48±4.59 vs. 2.48±3.52, 2.58±2.50, all P < 0.05]. (2) Electron microscope showed that the structure of myocardial fibers in the Sham group was clear and aligned with complete intervertebral disc and mitochondrial structure, no damage to mitochondrial membranes, and tight arrangement of cristae. In CLP group and AAV group, muscle fiber breakage, sarcoplasmic reticulum expansion, severe mitochondrial swelling and even cristage structure disorder were observed. In the UCP2 group, only myocardial fiber edema was observed, and the muscle fiber structure was more complete than that of Sham group and AAV group. The mitochondria were slightly swollen and the cristae were intact. (3) Western Blot showed that there was no significant difference in the expression of Opa1 and Fis1 in the four groups. The expression of Drp1 in CLP group and AAV group were significantly higher than that in Sham group. The expression of Drp1 in UCP2 group was significantly lower than that in CLP group and AAV group (Drp1/β-actin: 1.01±0.03 vs. 1.39±0.03, 1.49±0.03, both P < 0.05). (4) The results of immunofluorescence showed that the ATP content of CLP group and AAV group were significantly lower than that of Sham group; the ATP content of UCP2 group was significantly higher than that of CLP group and AAV group (μmol/L: 1.99±0.15 vs. 1.10±0.17, 1.13±0.19, both P < 0.05). UCP2 overexpression can significantly improve the systemic systolic function of myocardium in sepsis rats, protect myocardial mitochondrial ultrastructure, inhibit mitochondrial division, and improve mitochondrial ATP synthesis.

MicroRNA-152 Promotes Slow-Twitch Myofiber Formation via Targeting Uncoupling Protein-3 Gene.

Simple SummaryDifferent pig breeds exhibit evident diversities in pork quality characteristics, which largely resulted from the differences in myofiber type compositions. To explore the decisive role of microRNAs (miRNAs) in myofiber specification, myofiber type compositions, miR-152 expression patterns in various tissues and miR-152 expression level in the longissimus dorsi (LD) muscles from either Rongchang (RC) or large white (LW) pigs were determined. We found that the longissimus dorsi (LD) muscles of the RC pigs have higher proportion of slow-twitch myofibers and more abundant level of miR-152 than those of the LW pigs, which indicated that miR-152 might be involved in myofiber type compositions and myogenesis. Then, the gain- and loss-of-function trials and luciferase activity assays confirmed that miR-152 could promote slow-twitch myofiber formation and skeletal myogenesis via targeting uncoupling protein 3 (UCP3) gene. The knockdown of UCP3 and rescue experiment further verified that UCP3 mediates miR-152 action in slow-twitch myofiber formation. Our findings suggested that miR-152 and its target gene UCP3 might be feasible markers for interposition intended to promoting meat quality in domesticated animals.The differences of pork quality characteristics among different pig breeds mainly came from the differences in myofiber type compositions. Growing evidence indicated the key role of miRNAs in myofiber specification. In the present study, we found that miR-152 is more abundant in the slow-twitch myofiber-enriched muscles. However, its role in myofiber type transformation and myogenesis is largely unknown. Overexpression of miR-152 in porcine myotubes promoted the formation of slow-twitch myofibers and myogenesis. While, inhibition of miR-152 expression showed the opposite effect to miR-152 mimics transfection. The luciferase reporter analysis confirmed that miR-152 straightly targets the 3′-untranslated region (3’-UTR) of uncoupling protein 3 (UCP3) to cause its post-transcriptional inhibition in the protein level. The knockdown of UCP3 by siRNA showed the similar effect of miR-152 on myofiber type transition. Furthermore, the rescue experiment in the porcine myotube transfected with miR-152 mimics or/and UCP3 overexpression plasmid with or without the 3’UTR revealed that UCP3 mediates the action of miR-152 in slow-twitch myofiber formation. Taken together, our findings proposed a novel molecular mechanism through which miR-152 epigenetically regulates meat quality via promoting slow-twitch myofiber formation and skeletal myogenesis.

2161-P: Overexpression of UCP2 in Pancreatic Beta Cells Caused Impaired Insulin Secretion through Mitochondrial Dysfunction and Aldolase B Expression

Uncoupling protein 2 (UCP2), a mitochondrial protein, is known to be upregulated in pancreatic islets of patients with type 2 diabetes (T2DM); however, the pathological significance of the increase in UCP2 expression remained unclear. In this study, we investigated the regulatory mechanisms of UCP2 expression and its effects on β-cell functions. The mRNA expression of UCP2 in mouse islets was upregulated by the treatment with glucose, glucokinase activator, or insulin (p&amp;lt;0.05), while metformin downregulated it (p&amp;lt;0.01). Islets from db/db mice and insulin receptor substrate-2 knockout (IRS-2KO) mice showed the increment in the UCP2 expression. Transgenic mouse with specific UCP2 overexpression in β-cells (βUCP2Tg) demonstrated impaired glucose tolerance due to reduced insulin secretion. Adenoviral expression of UCP2 in MIN6-M9 β-cell line also attenuated glucose-induced insulin secretion. The ATP production in response to glucose and mitochondrial Complex I protein level were reduced (p&amp;lt;0.01) in βUCP2Tg islets. Dilatation (1.8-fold, p&amp;lt;0.05) and morphological alteration of mitochondria were observed in βUCP2Tg β-cells. Gene expression microarray analysis revealed 18 upregulated genes (1.5-fold, p&amp;lt;0.05) and 2 downregulated genes (&amp;lt;67%, p&amp;lt;0.05) in βUCP2Tg islets. The most upregulated mRNA by UCP2 expression encoded the aldolase B (Aldob) (77-fold), whose expression was reported to be negatively associated with insulin secretion in human islets (J Clin Endocrinol Metab. 103(12):4373-4383, 2018). We validated increased Aldob expression in islets from βUCP2Tg mice, db/db mice, and IRS-2KO mice. Taken together, UCP2 expression seemed to be increased under T2DM conditions including hyperglycemia or hyperinsulinemia. Our data also suggested that the increase in UCP2 expression in T2DM β-cells cell-autonomously attenuated glucose-induced insulin secretion, possibly via mitochondrial abnormality and Aldob expression. Disclosure R. Inoue: None. J. Shirakawa: None. Y. Togashi: None. Y. Terauchi: Advisory Panel; Self; AstraZeneca, Daiichi Sankyo Company, Limited, Eli Lilly and Company, Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk A/S, Sanofi. Research Support; Self; Daiichi Sankyo Company, Limited, Eli Lilly and Company, Merck Sharp &amp; Dohme Corp., Novartis Pharmaceuticals Corporation, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Sanofi, Shionogi &amp; Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd. Speaker's Bureau; Self; Astellas Pharma Inc., AstraZeneca, Bayer Yakuhin, Ltd., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho, Shionogi &amp; Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd.