Role Of UCP2 Research Articles

AAV-ie-mediated UCP2 overexpression accelerates inner hair cell loss during aging in vivo

BackgroundUncoupling protein 2 (UCP2), activated by excessive reactive oxygen species (ROS) in vivo, has the dual effect of reducing ROS to protect against oxidative stress and reducing ATP production to regulate cellular metabolism. Both the UCP2 and ROS are increased in cochleae in age-related hearing loss (ARHL). However, the role of UCP2 in sensory hair cells in ARHL remains unclear.MethodsMale C57BL/6 J mice were randomly assigned to an 8-week-old group (Group 1), a 16-week-old group (Group 2), a 16-week-old + adeno-associated virus-inner ear (AAV-ie) group (Group 3), and a 16-week-old + AAV-ie-UCP2 group (Group 4). Mice aged 8 weeks were administrated with AAV-ie-GFP or AAV-ie-UCP2 via posterior semicircular canal injection. Eight weeks after this viral intervention, hearing thresholds and wave-I amplitudes were tested by auditory brainstem response (ABR). Subsequently, the cochlear basilar membrane was dissected for investigation. The number of hair cells and inner hair cell (IHC) synapses, the level of ROS, and the expression of AMP-activated protein kinase α (AMPKα), were assessed by immunofluorescence staining. In addition, mitochondrial function was determined, and the expression of AMPKα and UCP2 proteins was further evaluated using western blotting.ResultsMice with early-onset ARHL exhibited enhanced oxidative stress and loss of outer hair cells and IHC synapses, while UCP2 overexpression aggravated hearing loss and cochlear pathophysiological changes in mice. UCP2 overexpression resulted in a notable decrease in the number of IHCs and IHC synapses, caused ATP depletion and excessive ROS generation, increased AMPKα protein levels, and promoted IHC apoptosis, especially in the apical and middle turns of the cochlea.ConclusionCollectively, our data suggest that UCP2 overexpression may cause mitochondrial dysfunction via energy metabolism, which activates mitochondrion-dependent cellular apoptosis and leads to IHC loss, ultimately exacerbating ARHL.

Role of UCP2 in the metabolic flexibility of murine cancer cells

Role of UCP2 in the metabolic flexibility of murine cancer cells

UCP1 modulates immune infiltration level and survival outcome in ovarian cancer patients

BackgroundThe uncoupling proteins (UCPs) are critical genes associated with tumorigenesis and chemoresistance. However, little is known about the molecular mechanism of the UCPs in ovarian cancer (OV).Material and methodsUCPs expression analysis was conducted using Gene Expression Profiling Interactive Analysis (GEPIA), and its potential in clinical prognosis was analyzed using Kaplan- Meier analyses. The influence of UCPs on immune infiltration was analyzed by TIMER. In addition, the correlation between UCPs expression and molecular mechanisms was investigated by TIMER and Cancer Single-cell State Atlas (CancerSEA).ResultsUCP1, UCP2, UCP3 and UCP5 expression levels correlated with a favorable prognosis and tumor progression. Moreover, UCP1 expression correlated to several immune cell markers and regulated tumorigenesis, such as tumor invasion, EMT, metastasis and DNA repair. In addition, UCP1 potentially involved in genes expression of SNAI2, MMP2, BRCA1 and PARP1.ConclusionsThese results implied a critical role of UCP1 in the prognosis and immune infiltration of ovarian cancer. In addition, UCP1 expression participated in regulating multiple oncogenes and tumorigenesis.

Abstract 1527: Integrated computational and experimental analysis identifies the mitochondrial uncoupling protein 2 (Ucp2) as a key regulator of T cell anti-tumor function

Abstract T cells play a central role in cancer immunosurveillance and current cancer immunotherapies, including adoptive cell transfer (ACT), T cell receptor or chimeric antigen receptor (CAR) T cell therapies and immune checkpoint blockade. Understanding the factors regulating T cell function is hence critical for improving the success of these immunotherapies. It has been recognized that metabolism can greatly affect different aspects of T cell function, including differentiation, cytokine production, longevity and exhaustion. Here we integrate genome-scale metabolic modeling (GEM) with biological experiments to discover novel metabolic determinants of T cell function. Previously we developed the metabolic transformation algorithm (MTA), a GEM method that was successfully applied to identify driving factors and targets for different diseases. Here applying MTA to data on CAR-T cell gene expression and patient response to anti-CD19 CAR-T therapy, we predicted mitochondrial metabolite transport and specifically proton transport in mitochondrial uncoupling, as key determinants of CAR-T therapy response. Mitochondrial uncoupling is also important for the in vivo persistence of adoptively transferred tumor-infiltrating lymphocytes, as further confirmed by analyzing their gene expressions from a KRAS-targeting ACT dataset. Focusing on the mitochondrial uncoupling protein 2 (Ucp2), which is abundantly expressed in T cells, we experimentally validated that it is required for T cell longevity and anti-tumor function. Specifically, the loss of Ucp2 either via knock-out (KO) or treatment by genipin (a Ucp2 inhibitor) in mice T cells results in accelerated differentiation into “terminal effector cells”, as shown by increased levels of T cell cytotoxicity and exhaustion markers, and decreased levels of central memory and stemness markers. Adoptive transfer of Ucp2-KO Pmel-1 T cells to mice bearing B16 melanomas displayed poorer anti-tumor efficacy and worse survival than the transfer of Ucp2-wildtype T cells. We find that Ucp2 modulates oxidative stress and DNA damage by regulating the levels of mitochondrial superoxide. Reducing mitochondrial reactive oxygen species was sufficient to rescue the loss of Ucp2-mediated effector T cell differentiation, senescence, cytokine production and anti-tumor activity Ucp2-KO Pmel-1 T cells. Tumor-specific CD8+ T cells could be metabolically reprogrammed by Ucp2 overexpression, which improved T cell longevity and anti-tumor function in the Pmel-1/B16 ACT mice model. In sum, our study establishes a novel role of Ucp2 in regulating T cell longevity and anti-tumor activity by repressing increased ROS levels accompanying mitochondrial dysfunction in differentiated and exhausted cells, suggesting that manipulating Ucp2 levels in T cells can be exploited to enhance T cell-based cancer immunotherapies. Citation Format: Madhusudhanan Sukumar, Kuoyuan Cheng, Arunakumar Gangaplara, Yogin Patel, Suman K. Vodnala, Rafiqul Islam, Arash Eidizadeh, Carolyn Subramaniam, Ping Lee, Rigel Kishton, Amanda N. Henning, Michael J. Kruhlak, Zhiya Yu, Ethan M. Shevach, Toren Finkel, Eytan Ruppin, Nicholas P. Restifo. Integrated computational and experimental analysis identifies the mitochondrial uncoupling protein 2 (Ucp2) as a key regulator of T cell anti-tumor function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1527.

Temperature-Dependent Effects of Eicosapentaenoic Acid on Browning of Subcutaneous Adipose Tissue From UCP1 Knockout Mice

ObjectivesActivation and recruitment of thermogenic cells in white adipose tissue (WAT browning), in response to cold exposure, can combat obesity and associated metabolic disorders. We have previously reported that the beneficial effects of eicosapentaenoic acid (EPA), a long-chain omega 3 polyunsaturated fatty acids, in obesity and insulin resistance are independent of UCP1. In this study, we investigate the protective effects of EPA and the role of UCP1 in the browning of subcutaneous adipose tissue (SAT) at ambient and thermoneutral environments using UCP1 knockout (KO) mice. We hypothesized that EPA promotes SAT browning to prevent diet-induced obesity at both temperatures, independently of UCP1. MethodsMale and UCP1 KO and wild type (WT) B6 littermates were housed at room temperature (22°C) or thermoneutrality (28–30°C) and fed a high fat (HF) diet (45% kcal fat) supplemented with or without EPA (36g/kg) for 14 weeks. Body weight and glucose tolerance test (GTT) were measured, and browning-related markers were assessed in SAT. Data were statistically analyzed via three-way ANOVA using GraphPad to determine the individual and interactive effects of temperature, genotype, and diet. ResultsCompared to the WT, the body weight (BW) of KO mice increased at thermoneutrality (P < 0.01) but decreased at ambient temperature (P < 0.0001). Additionally, EPA attenuated weight and fat mass gain at thermoneutrality and improved glucose tolerance at both temperatures in both genotypes. mRNA levels for brown fat markers (Dio2 and Cidea), lipid metabolism (Elovl3, PGC1α, FASN, Cpt1b, and Gpd1), and batokines (Bmp8b and FGF21) were significantly up-regulated in KO mice, compared to WT, at ambient temperature (P < 0.01). Moreover, compared to HF-fed mice, EPA increased above markers in the KO mice at ambient temperature. Compared to HF, EPA-fed mice had significantly higher serum adiponectin levels (P < 0.01) in both genotypes and temperatures. ConclusionsUCP1 KO male mice were protected from diet-induced obesity and glucose intolerance and had increased SAT browning at ambient temperature. These results indicate that alternative thermoregulatory pathways mediate protective metabolic effects of EPA, in the absence of UCP1. These findings may be translated to human subjects with obesity, who exhibit low amounts of brown fat and UCP1. Funding SourcesNIH R15AT008879-01A1.

The metabolic effects of mirabegron are mediated primarily by β3 -adrenoceptors.

The β3‐adrenoceptor agonist mirabegron is approved for use for overactive bladder and has been purported to be useful in the treatment of obesity‐related metabolic diseases in humans, including those involving disturbances of glucose homeostasis. We investigated the effect of mirabegron on glucose homeostasis with in vitro and in vivo models, focusing on its selectivity at β‐adrenoceptors, ability to cause browning of white adipocytes, and the role of UCP1 in glucose homeostasis. In mouse brown, white, and brite adipocytes, mirabegron‐mediated effects were examined on cyclic AMP, UCP1 mRNA, [3H]‐2‐deoxyglucose uptake, cellular glycolysis, and O2 consumption. Mirabegron increased cyclic AMP levels, UCP1 mRNA content, glucose uptake, and cellular glycolysis in brown adipocytes, and these effects were either absent or reduced in white adipocytes. In brite adipocytes, mirabegron increased cyclic AMP levels and UCP1 mRNA content resulting in increased UCP1‐mediated oxygen consumption, glucose uptake, and cellular glycolysis. The metabolic effects of mirabegron in both brown and brite adipocytes were primarily due to actions at β3‐adrenoceptors as they were largely absent in adipocytes derived from β3‐adrenoceptor knockout mice. In vivo, mirabegron increased whole body oxygen consumption, glucose uptake into brown and inguinal white adipose tissue, and improved glucose tolerance, all effects that required the presence of the β3‐adrenoceptor. Furthermore, in UCP1 knockout mice, the effects of mirabegron on glucose tolerance were attenuated. Thus, mirabegron had effects on cellular metabolism in adipocytes that improved glucose handling in vivo, and were primarily due to actions at the β3‐adrenoceptor.

Proton Transport in Mitochondrial UCP2 is Regulated by a Matrix-Oriented Salt-Bridge Network

Uncoupling proteins (UCPs) are ion carrier proteins causing regulated proton leak across the mitochondrial inner membrane, thus reducing the rate of ATP synthesis. Despite this important impact, the mechanism of UCP-mediated proton transport has not been fully understood. It has been speculated that the mitochondrial carrier family members contain two interconnected salt-bridge networks facilitating the substrate transport. One of these networks is close to the matrix side (matrix network) and the other is close to the intermembrane space. This study focuses on the role of matrix network on the regulation of proton transport in UCP2. UCP2 and eleven mutants (in matrix network) are expressed recombinantly in the E. coli inner membrane. Six mutants disrupt a single salt-bridge link by maintaining either a single negative (K to Q mutation) or positive (D to N mutation) charge on one end of the salt-bridge (K38Q, K141Q, K239Q, D35N, D138N and D236N). Two mutants (K38Q/K239Q and D138N/D236N) disrupt two salt-bridges, while the other two, lacking three salt-bridges, disrupt the total matrix network (K38Q/K141Q/K239Q and D35N/D138N/D236N). In the last mutant, the matrix network is inversed by switching the salt-bridge forming couples (D35K/K38D/D236K/K239D/D138K/K141D). Results show that the matrix network has a regulatory role, and perturbation of the network increases the rate of proton transport. It seems that individual salt-bridges within the network are cooperatively and equally responsible for regulation of transport. Various alterations of the constituting elements of matrix salt-bridge network do not have a considerable effect on inhibitor-protein interactions. Conformational analysis of the protein (CD and fluorescence) and MD simulations provided molecular insights into the role of UCP2 in proton transport. This study characterizes the physicochemical role of the matrix-oriented salt-bridge network on proton transport in UCP2 and its inhibition by purine nucleotides.

Presence of UCP2 and Amylin in human Alzheimer's disease brains

Although the role of amyloid beta in Alzheimer's disease has been studied for decades, the link between amyloid beta and amylin is fairly new. Amylin is a protein that is co‐secreted from pancreatic beta cells alongside insulin and has been shown to cross the blood‐brain barrier and impact the formation of amyloid plaques. The normal function of amylin in the brain is directly correlated to metabolism because amylin is considered a satiety protein. The output involved in the metabolic process involves oxidative phosphorylation and includes a set of proteins known as uncoupling proteins (UCPs), which are proteins that directly impact oxidative phosphorylation. Traditionally, UCPs are known to impact the ATP levels that occur in oxidative phosphorylation. However, recently it has been postulated that UCPs may have an additional role that involves cell signaling. In fact, UCP2 has been shown to be present in pancreatic beta cells and to have diminished protein levels in Alzheimer's disease brains. The experiments conducted in this study analyzed the relationship between UCP2 and amylin. Specifically, immunohistochemistry experiments that examine both the presence of amylin and UCP2 in Alzheimer's disease brains were conducted. Western Blot analysis experiments were also conducted to compare the findings from the immunohistochemistry experiments. The initial studies suggest that the role of UCP2 may influence the role of amylin or the role of amylin may influence the role of UCP2.Support or Funding InformationNational Science Foundation Research Initiation Award #24430This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acetoacetate is a more efficient energy-yielding substrate for human mesenchymal stem cells than glucose and generates fewer reactive oxygen species

Stem cells have been assumed to demonstrate a reliance on anaerobic energy generation, suited to their hypoxic in vivo environment. However, we found that human mesenchymal stem cells (hMSCs) have an active oxidative metabolism with a range of substrates. More ATP was consistently produced from substrate oxidation than glycolysis by cultured hMSCs. Strong substrate preferences were shown with the ketone body, acetoacetate, being oxidised at up to 35 times the rate of glucose. ROS-generation was 45-fold lower during acetoacetate oxidation compared with glucose and substrate preference may be an adaptation to reduce oxidative stress. The UCP2 inhibitor, genipin, increased ROS production with either acetoacetate or glucose by 2-fold, indicating a role for UCP2 in suppressing ROS production. Addition of pyruvate stimulated acetoacetate oxidation and this combination increased ATP production 27-fold, compared with glucose alone, which has implications for growth medium composition. Oxygen tension during culture affected metabolism by hMSCs. Between passages 2 and 5, rates of both glycolysis and substrate-oxidation increased at least 2-fold for normoxic (20% O2)- but not hypoxic (5% O2)-cultured hMSCs, despite declining growth rates and no detectable signs of differentiation. Culture of the cells with 3-hydroxybutyrate abolished the increased rates of these pathways. These findings have implications for stem cell therapy, which necessarily involves in vitro culture of cells, since low passage number normoxic cultured stem cells show metabolic adaptations without detectable changes in stem-like status.

Role of UCP2 on mitochondrial dysfunction in the renal oxidative stress‐mediated hypertension associated with DJ‐1 depletion

DJ‐1 and uncoupling protein 2 (UCP2) exert a protective role against mitochondrial oxidative stress. DJ‐1−/− mice, relative to wild‐type (WT) littermates, have increased systolic BP (30±3%). This study determined the mechanisms involved in the oxidative stress‐mediated hypertension due to DJ‐1 germline deletion. There were no differences in the sodium excretion and renal renin expression between DJ‐1−/− and WT mice (n=5). H&amp;E and picro sirius red staining did not show any abnormalities in renal morphology, including fibrosis in the kidneys of DJ‐1−/− mice. Renal NADPH oxidase activity (n=12) and serum creatinine levels were also not different between the two genotypes. However, the renal expression of nitro‐tyrosine was increased in DJ‐1−/− mice relative to their WT littermates (+176.8±31% vs WT mice, n=5). Tempol, a membrane‐permeable radical scavenger, normalized the BP (tempol: 118±2% vs 100±1% vs WT, n=4) and renal malondialdehyde production (tempol:160±23% vs 109±15% vs WT, n=4) in DJ‐1−/− mice, but had no effect in WT. Tempol‐treated DJ‐1−/− mice had higher serum nitrite/nitrate levels than placebo‐treated DJ‐1−/− mice (172±30% vs WT, n=4) suggesting a role of nitrate radicals on the high blood pressure of this model. Excretion of the renal damage marker KIM‐1 which was increased in DJ‐1−/− mice versus WT (148±22% vs WT, n=4) was decreased by tempol (−58±3% vs WT, n=4). By contrast, heat shock protein mtHSP60, but not mtHSP40, was increased in DJ‐1−/− mice (2.9±0.1 fold changes of WT, n=4), indicating mitochondrial stress. However, there were no changes in the renal mRNA expression of mitophagy markers Nix/BNIP3L, BNIP3, and PINK, mitochondrial fusion markers FIS1, MFN1 and MFN2 and mitochondrial biogenesis markers PPRC1, NRF‐1 and PGC1, indicating that, despite the high mitochondrial ROS production, mitochondrial function was not altered. Renal expression of UCP2 was elevated in DJ‐1−/− mice (4.1±1.1 fold change of WT, n=4), and was partially normalized by tempol (1.8±0.2 fold change of WT, n=4) indicating UCP2 may have a protective role on mitochondrial function in this model. UCP2 expression was silenced in mouse kidney via renal sub‐capsular infusion of siRNA UCP2 in WT and DJ‐1−/− mice (WT: 63%±7 vs control: DJ‐1−/− :60%±6 vs control; n=4). Interestingly, mRNA expression of mitophagy makers BNIP3 (−0.65±6 fold change of WT) and PINK1 (1.55±0.3 fold change of WT of WT), mitochondrial fusion and fission makers FIS1 (−0.29±0.03 fold‐change of WT) NFN2 (1.42±0.06 fold‐change of WT) and mitochondrial biosynthesis maker PPRC1 (1.71±0.07‐fold change of WT; n=4) had significant changes after the UCP2 transfection in DJ‐1−/− mice but not in WT. However no differences were found on NRF1 expression. In conclusion, deletion of DJ‐1 leads to oxidative stress‐induced hypertension mediated by the down‐regulation of NO function, and UCP2 seems to have a protective role against development of mitochondrial dysfunction in mitochondrial oxidative stress, conditions which may have important implications in the prevention of renal disease.

The complementary and divergent roles of uncoupling proteins 1 and 3 in thermoregulation.

Both uncoupling protein 1 (UCP1) and UCP3 are important for mammalian thermoregulation. UCP1 and UCP3 in brown adipose tissue mediate early and late phases of sympathomimetic thermogenesis, respectively. Lipopolysaccharide thermogenesis requires skeletal muscle UCP3 but not UCP1. Acute noradrenaline-induced hyperthermia requires UCP1 but not UCP3. Loss of both UCP1 and UCP3 accelerate the loss of body temperature compared to UCP1KO alone during acute cold exposure. Uncoupling protein 1 (UCP1) is the established mediator of brown adipose tissue-dependent thermogenesis. In contrast, the role of UCP3, expressed in both skeletal muscle and brown adipose tissue, in thermoregulatory physiology is less well understood. Here, we show that mice lacking UCP3 (UCP3KO) have impaired sympathomimetic (methamphetamine) and completely abrogated lipopolysaccharide (LPS) thermogenesis, but a normal response to noradrenaline. By comparison, UCP1 knockout (UCP1KO) mice exhibit blunted methamphetamine and fully inhibited noradrenaline thermogenesis, but an increased febrile response to LPS. We further establish that mice lacking both UCP1 and 3 (UCPDK) fail to show methamphetamine-induced hyperthermia, and have a markedly accelerated loss of body temperature and survival after cold exposure compared to UCP1KO mice. Finally, we show that skeletal muscle-specific human UCP3 expression is able to significantly rescue LPS, but not sympathomimetic thermogenesis blunted in UCP3KO mice. These studies identify UCP3 as an important mediator of physiological thermogenesis and support a renewed focus on targeting UCP3 in metabolic physiology.

Abstract P185: Role of Ucp2 in the Oxidative/nitrosative Stress-mediated Hypertension Associated with Dj-1 Depletion

DJ-1 -/- mice, relative to wild-type (WT) littermates, have increased blood pressure (BP) ( DJ-1 -/- :130±4 vs WT:100±3 mmHg, n=6/8). and renal expressions of nitro-tyrosine (+76±31% of WT mice, n=5) and malondialdenyde (+63±23% of WT mice, n=4). Tempol, a superoxide dismutase mimetic, decreased the BP of DJ-1 -/- mice ( DJ-1 -/- : before tempol:119±3; after tempol:100±1 mmHg vs WT, n=4) and renal malondialdehyde production ( DJ-1 -/- : before tempol:+40±5%; after tempol:-24±5% vs WT, n=4) but increased serum nitrate/nitrite levels (+72±30%, n=4), indicating the presence of both oxidative and nitrosative stress. Lack of DJ-1 makes some cells vulnerable to endoplasmic reticulum (ER) stress. However, renal mRNA expression of ER stress markers, GRP94, ATF-4, ATF-6, sXBP-1, CHOP, caspase-12, and caspase-3 was not different between DJ-1 -/- and WT (n=7) mice. Markers of inflammation, IL-6, TNF α, MCP-1, NFκB, and T-cell and macrophage infiltration, were also not increased in the kidney of DJ-1 -/- mice. By contrast, renal mitochondrial (mt) HSP60, but not mtHSP40, was increased in DJ-1 -/- mice (2.9±0.1 fold, n=4) but there were no changes in the renal mRNA expressions of Nix/BNIP3L, BNIP3, PINK, FIS1, MFN1, MFN2, PPRC1, NRF-1, and PGC1, indicating that mt oxidative stress did not affect mt function. The renal expression of UCP2, which is involved in the control of mt-reactive oxygen species production, was elevated in DJ-1 -/- mice (4.1±1.1 fold of WT, n=4). Silencing UCP2 in mouse renal proximal tubule cells (-0.46.5±0.01 fold) increased the expression of ER stress and apoptosis markers CHOP (2±0.4 fold), ATF4 (2.6±0.6 fold), caspase-3 (2.3±0.4 fold), and caspase-12 (1.7±0.2 fold)(n=3). There were no differences in renal renin expression, sodium excretion, and serum creatinine between DJ-1 -/- and WT mice (n=5). There were no abnormalities in renal morphology, including fibrosis, in the kidneys of DJ-1 -/- mice. However, urinary KIM-1 was increased in DJ-1 -/- mice (148±22% of WT mice, n=4) and decreased by tempol (-58±3%, n=4); renal UCP2 expression was also partially normalized by tempol (1.8±0.2 fold of WT, n=4). UCP2 may protect from the development of renal ER stress and damage in the mt oxidative/nitrosative stress associated with DJ-1 depletion.

Abstract P241: Irisin Protects Mitochondria Function During Pulmonary Ischemia Reperfusion Injury

Background: Ischemia and reperfusion (I/R) induced lung injury is one of the most important and common causes of early and high morbidity and mortality. The mitochondrial damage of alveolar epithelium cells leads to further damage in lung I/R by augmented ROS activity and inflammation. Previous study show a humoral myokine, irisin, has been found stimulate mitochondrial biogenesis. We hypothesize irisin might protect lung from I/R injury. Methods: The lung I/R injury model was performed on C57BL/6J mice. The pulmonary protection of exogenous irisin was indicated by lung edema and function measurement, and the survival of mice. The localization of irisin was measured by immunohistochemistry and immunofluorescence staining. And the protective effect of irisin to mitochondrial was demonstrated by ATP production, ROS production, mitochondria-dependent apoptosis measurement, and so on. Results: There was no endogenous irisin expression in the alveolar wall in normal mice. However, it is interesting to find that, irisin was in the alveoli after lung I/R injury. The increased irisin in lung might be from plasma, because in the mean time, the plasma irisin levels were decreased. Exogenous intravenous injection of irisin protect the lung from I/R injury. The protective effect might be via the protection on the mitochondria in lung, exogenous irisin was found to localize in the mitochondria, the impaired mitochondrial function in I/R mice was reversed after irisin treatment. Moreover, there was colocalization between the irisin and UCP2, exogenous irisin decreased the I/R-induced UCP2 degradation. The role of UCP2 on the protection of mitochondria is further confirmed in the in in-vivo experiment, inhibition of UCP2 or knockout of UCP2 would make the protection of irisin on lung function and mitochondrial function lost. Conclusions: Administration with exogenous irisin would alleviate the I/R damage, reduced the inflammatory and superoxide factors, ameliorated the mitochondrial dysfunction, via the binding of irisin and UCP2 in lung.

Role of UCP2 in the metabolic flexibility of the cancer cell line A549

Role of UCP2 in the metabolic flexibility of the cancer cell line A549

By Regulating Mitochondrial Ca2+-Uptake UCP2 Modulates Intracellular Ca2+.

IntroductionThe possible role of UCP2 in modulating mitochondrial Ca2+-uptake (mCa2+-uptake) via the mitochondrial calcium uniporter (MCU) is highly controversial.MethodsThus, we analyzed mCa2+-uptake in isolated cardiac mitochondria, MCU single-channel activity in cardiac mitoplasts, dual Ca2+-transients from mitochondrial ((Ca2+)m) and intracellular compartment ((Ca2+)c) in the whole-cell configuration in cardiomyocytes of wild-type (WT) and UCP2-/- mice.ResultsIsolated mitochondria showed a Ru360 sensitive mCa2+-uptake, which was significantly decreased in UCP2-/- (229.4±30.8 FU vs. 146.3±23.4 FU, P<0.05). Single-channel registrations confirmed a Ru360 sensitive voltage-gated Ca2+-channel in mitoplasts, i.e. mCa1, showing a reduced single-channel activity in UCP2-/- (Po,total: 0.34±0.05% vs. 0.07±0.01%, P<0.05). In UCP2-/- cardiomyocytes (Ca2+)m was decreased (0.050±0.009 FU vs. 0.021±0.005 FU, P<0.05) while (Ca2+)c was unchanged (0.032±0.002 FU vs. 0.028±0.004 FU, P>0.05) and transsarcolemmal Ca2+-influx was inhibited suggesting a possible compensatory mechanism. Additionally, we observed an inhibitory effect of ATP on mCa2+-uptake in WT mitoplasts and (Ca2+)m of cardiomyocytes leading to an increase of (Ca2+)c while no ATP dependent effect was observed in UCP2-/-.ConclusionOur results indicate regulatory effects of UCP2 on mCa2+-uptake. Furthermore, we propose, that previously described inhibitory effects on MCU by ATP may be mediated via UCP2 resulting in changes of excitation contraction coupling.

In the Early Stages of Diabetes, Rat Retinal Mitochondria Undergo Mild Uncoupling due to UCP2 Activity.

In order to maintain high transmembrane ionic gradients, retinal tissues require a large amount of energy probably provided by a high rate of both, glycolysis and oxidative phosphorylation. However, little information exists on retinal mitochondrial efficiency. We analyzed the retinal mitochondrial activity in ex vivo retinas and in isolated mitochondria from normal rat retina and from short-term streptozotocin-diabetic rats. In normal ex vivo retinas, increasing glucose concentrations from 5.6mM to 30mM caused a four-fold increase in glucose accumulation and CO2 production. Retina from diabetic rats accumulated similar amounts of glucose. However, CO2 production was not as high. Isolated mitochondria from normal rat retina exhibited a resting rate of oxygen consumption of 14.6 ± 1.1 natgO (min.mg prot)-1 and a respiratory control of 4.0. Mitochondria from 7, 20 and 45 days diabetic rats increased the resting rate of oxygen consumption and the activity of the electron transport complexes; under these conditions the mitochondrial transmembrane potential decreased. In spite of this, the ATP synthesis was not modified. GDP, an UCP2 inhibitor, increased mitochondrial membrane potential and superoxide production in controls and at 45 days of diabetes. The role of UCP2 is discussed. The results suggest that at the early stage of diabetes we studied, retinal mitochondria undergo adaptations leading to maintain energetic requirements and prevent oxidative stress.

Effects of Mitochondrial Uncoupling Protein 2 Inhibition by Genipin in Human Cumulus Cells.

UCP2 plays a physiological role by regulating mitochondrial biogenesis, maintaining energy balance, ROS elimination, and regulating cellular autophagy in numerous tissues. But the exact roles of UCP2 in cumulus cells are still not clear. Genipin, a special UCP2 inhibitor, was added into the cultural medium to explore the roles of UCP2 in human cumulus cells. There were no significant differences in ATP and mitochondrial membrane potential levels in cumulus cells from UCP2 inhibiting groups as compared with the control. The levels of ROS and Mn-SOD were markedly elevated after UCP2 inhibited Genipin. However, the ratio of reduced GSH to GSSG significantly declined after treatment with Genipin. UCP2 inhibition by Genipin also resulted in obvious increase in the active caspase-3, which accompanied the decline of caspase-3 mRNA. The level of progesterone in culture medium declined obviously after Genipin treatment. But there was no significant difference in estradiol concentrations. This study indicated that UCP2 is expressed in human cumulus cells and plays important roles on mediate ROS production, apoptotic process, and steroidogenesis, suggesting UCP2 may be involved in regulation of follicle development and oocyte maturation and quality.

Abstract 439: Mitochondrial uncoupling protein 2 promotes skin carcinogenesis

Abstract Mitochondrial uncoupling uncouples mitochondrial respiration and ATP production. There are five uncoupling proteins (UCPs), UCP2 is ubiquitous expressed and plays an important role in the reprogramming of cancer cell metabolism. However, little is known about at what stage of cancer development mitochondrial uncoupling occurs and what the role of UCP2 in cancer etiology. We will try to address these puzzles in present study. The well-established chemically-induced skin carcinogenesis mouse model was used and UCP2 wide-type (WT) and homozygous knockout (KO) mice were added to this study as direct evidence for the role of UCP2 during skin carcinogenesis. After forty-week treatment with chemical carcinogens, the incidence of papillomas in WT and KO TPA mice was 92.3% and 77.8%, respectively; and tumor multiplicity was 2.6 and 1.3, respectively. The incidence of carcinomas in WT and KO TPA treatment was 76.9% and 22.2%, respectively, and tumor multiplicity was 0.9 and 0.2, respectively. We next detected how the mitochondrial function and ATP generation would be changed in this model. UCP2 knockout slightly increased oxygen consumption compared with WT, whereas tumor promoter treatment suppressed oxygen consumption in both UCP2 KO and WT group. As for ATP generation, there was no significant change between WT and KO, or between vehicle and tumor promoter treatment. We next examined the mechanisms of tumor promotion by mitochondrial uncoupling by overexpressing UCP2 in JB6 P+ cells, which are the only characterized skin cell model to study tumor promotion. Anchorage-independent growth assay was performed and the results showed that overexpression of UCP2 enhanced colony formation in response to tumor promoter treatment. In addition, overexpression of UCP2 induced the upregulation of the oncoprotein Fra-1 whereas inhibited the increase in p53 expression. In summary, this study will add new knowledge to the understanding of the role of mitochondrial uncoupling in cancer etiology, and many help design a mitochondrial uncoupling-targeted approach for the suppression of cancer development. Citation Format: Wenjuan Li, Chunjing Zhang, Runhua Shi, Xin Gu, Yunfeng Zhao. Mitochondrial uncoupling protein 2 promotes skin carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 439. doi:10.1158/1538-7445.AM2014-439

Abstract 4325: Overexpression of UCP2 is associated with tumor progression in Iranian breast cancer patients

Abstract Introduction: Uncoupling proteins (UCPs) are a family of inner mitochondrial membrane proteins whose function is to allow the re-entry of protons to the mitochondrial matrix, to decrease membrane potential and production of reactive oxygen species (ROS). Due to their pivotal role in the intersection between energy efficiency and oxidative stress, UCPs are being investigated for a potential role in cancer researches .UCP2 proteins functions in a variety of cell types as a sensor of mitochondrial oxidative stress and may be activated by superoxide or subsequently formed lipid peroxidation products. The ability of cancer cells to regulate ROS levels contributes greatly to autonomous growth, the evasion of apoptosis, and other hallmark characteristics of adaptation. Thus, UCP2 activity, a negative regulator of ROS, should be related to cancer development or progression. Over expression of UCP2 has been shown in different cancers. However, until now the role of UCP2 in breast cancers is not well understood. The aim of the current research study was to evaluate the association of UCP2 expression and clinic-pathological features in Iranian breast cancer patients. Patients and Methods: Thirty five breast cancer patients who referred to the National Cancer Institute (NCI) at Imam Khomeini Hospital Complex, Tehran, Iran from Oct. 2007 to Oct. 2009 were contributed in this study. The pathologic changes in tumor samples were determined by two expert pathologists. Each specimen was immediately frozen following resection and stored at -80°C until subjected to RNA extraction. UCP2 expression was analyzed by relative PCR method was performed in subjects. Results and Conclusion: The expression of UCP2 mRNA in 31 out of 35 breast cancer tissue samples and its adjacent tissue samples was determined. The expression level of UCP2 mRNA was about four-fold higher in 58% of breast cancer tissue samples than its adjacent tissue samples. The correlation between UCP2 expression and clinical pathological features of breast cancer including tumor stage, histological grade, estrogen receptor status, progesterone receptor status, Her-2/neu status, lymph node status, P53 status and metastasis status were analyzed. There was not a significant association between expression levels of UCP2 and clinical pathological features (P&amp;gt; 0.05). However, there was a significant association between the UCP2 expression and tumor size (P&amp;lt; 0.028). This finding may significantly play a role in the etiology of tumor development of breast cancer. Furthermore, our finding may be lead to determine whether UCP2 expression is an effective factor for tumor development in breast cancer cells so, targeted therapy drugs to impact this gene expression in the nucleus may be useful. Further studies are warranted to confirm these findings. Note: This abstract was not presented at the meeting. Citation Format: Neda Maraghechi, Massoud Ghaffarpour, Golna Asaadi Tehrani. Overexpression of UCP2 is associated with tumor progression in Iranian breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4325. doi:10.1158/1538-7445.AM2014-4325

The Role of Uncoupling Proteins in Diabetes Mellitus

Uncoupling proteins (UCPs) are anion carriers expressed in the mitochondrial inner membrane that uncouple oxygen consumption by the respiratory chain from ATP synthesis. The physiological functions of UCPs have long been debated since the new UCPs (UCP2 to 5) were discovered, and the role of UCPs in the pathogeneses of diabetes mellitus is one of the hottest topics. UCPs are thought to be activated by superoxide and then decrease mitochondrial free radicals generation; this may provide a protective effect on diabetes mellitus that is under the oxidative stress conditions. UCP1 is considered to be a candidate gene for diabetes because of its role in thermogenesis and energy expenditure. UCP2 is expressed in several tissues and acts in the negative regulation of insulin secretion by β-cells and in fatty acid metabolism. UCP3 plays a role in fatty acid metabolism and energy homeostasis and modulates insulin sensitivity. Several gene polymorphisms of UCP1, UCP2, and UCP3 were reported to be associated with diabetes. The progress in the role of UCP1, UCP2, and UCP3 on diabetes mellitus is summarized in this review.