Mitochondrial uncoupling proteins: redox-metabolic homeostasis and disease pathogenesis

Transient cardiac ischemia activates cell survival signaling, conferring subsequent ischemia tolerance to the heart. This biological phenomenon, termed ischemic preconditioning, results in improved clinical outcome and attenuated infarct size following myocardial infarction. To explore genomic modifications underpinning this ischemia tolerance, we delineated the regulation and function of the cardiac enriched mitochondrial uncoupling proteins 2 and 3 during delayed ischemic preconditioning in the rat. Cardiac transcripts of genes encoding uncoupling proteins 2 and 3 are up-regulated in parallel with infarct size reduction in preconditioned hearts. Mitochondria isolated from preconditioned hearts exhibit an augmented inducible proton leak. In parallel, following anoxia-reoxygenation these mitochondria generate less hydrogen peroxide compared with non-preconditioned mitochondria. Preconditioning in rat cardiac derived myoblasts is abolished following uncoupling protein-2 depletion by RNA-interference. RNAi of uncoupling protein-3 partially attenuates the capacity to precondition these cells. Functional characterization of anoxia and reoxygenation tolerance following uncoupling protein 2 or 3 and combined 2 and 3 RNAi shows the largest reduction in viability follows depletion of both homologues. Uncoupling protein-2 depletion alone significantly attenuates anoxia-reoxygenation tolerance but uncoupling protein-3 depletion does not reduce anoxia tolerance. In parallel combined uncoupling protein depletion and isolated uncoupling protein-2 depletion augments ROS production in viable cardiomyocytes following anoxia-reoxygenation. Concurrent anti-oxidant administration ameliorates the uncoupling protein-depleted anoxia-susceptible phenotype. In conclusion, mitochondrial uncoupling proteins are necessary components of ischemia tolerance and function as components of the cellular antioxidant defense program. In the cytoprotective hierarchy, uncoupling protein-2 appears to play a greater role than uncoupling protein-3 in modulating ischemia/anoxia tolerance in heart-derived cells.

Steatotic livers are not used for transplantation because they have a reduced tolerance for ischemic events with reduced ATP levels and greater levels of cellular necrosis, which ultimately result in total organ failure. Mitochondrial uncoupling protein-2 (UCP2) is highly expressed in steatotic livers and may be responsible for liver sensitivity to ischemia through mitochondrial and ATP regulation. To test this hypothesis, experiments were conducted in lean and steatotic (ob/ob), wild-type, and UCP2 knock-out mice subjected to total warm hepatic ischemi-a/reperfusion. Although ob/ob UCP2 knock-out mice and ob/ob mice have a similar initial phenotype, ob/ob UCP2 knock-out animal survival was 83% when compared with 30% in ob/ob mice 24 h after reperfusion. Serum alanine aminotransferase concentrations and hepatocellular necrosis were decreased in the ob/ob UCP2 knock-out mice when compared with ob/ob mice subjected to ischemia. Liver ATP levels were increased in the ob/ob UCP2 knock-out animals after reperfusion when compared with the ob/ob mice but remained below the concentrations from lean livers. Lipid peroxidation (thiobarbituric acid-reactive substances) increased after reperfusion most significantly in the steatotic groups, but the increase was not affected by UCP2 deficiency. These results reveal that UCP2 expression is a critical factor, which sensitizes steatotic livers to ischemic injury, regulating liver ATP levels after ischemia and reperfusion.

Mitochondrial abnormalities are long suspected to play important roles in tumor cell differentiation, proliferation and metabolism. Dysfunction of mitochondrial-dependent processes appear to be key features of cancerous cells. Among many such abnormal features of cancerous cells, a mitochondrial uncoupling protein 2 (UCP2) is shown to be up-regulated in various aggressive human cancers. Uncoupling proteins are a family of mitochondrial proteins present in the inner mitochondrial membrane whose physiological role is to decrease membrane potential and reactive oxygen species (ROS) production. UCP2 over-expression recently been proposed as a novel survival mechanism for cancer cells. However, till date, the exact role of UCP2 in cancer remains inconclusive. We recently reported that UCP2 appears to be a key regulator of cell proliferation, cell cycle and cell death during skin tumorigenesis. Using JB6P+ cells that overexpress UCP2, we showed that UCP2 expression correlated closely with cell proliferation and cell transformation. Moreover, inhibition of UCP2, decreased colony formation and 3D culture growth. Since UCP2 is the crucial player in the regulation of reactive oxygen species, and mitochondrial bioenergetics, we wanted to dissect out the role of UCP2 in redox regulation and tumor promotion. Our data demonstrated that UCP2 differentially regulated ROS. UCP2 upregulation, decreased superoxide production, whereas increased hydrogen peroxide production with a concomitant increase in manganese superoxide dismutase (MnSOD) expression and activity. Furthermore, hydrogen peroxide was responsible for induction of lipid peroxidation and PLCγ-1 activation in UCP2 overexpressed cells. Moreover, PLCγ-1 activation enhanced tumorigenecity. Strikingly, pharmacological and siRNA mediated inhibition of PLCγ-1 markedly reduced colony formation and 3D growth in vitro. Lastly, restricting hydrogen peroxide production with hydrogen peroxide scavenger catalase, suppressed lipid peroxidation and dampened PLCγ-1 activity. Taken together, our data suggest that hydrogen peroxide might be the mediator of UCP2’s tumor promoting role, and pharmacological disruption of PLCγ-1, and/or hydrogen peroxide may have clinical utility for UCP2-overexpressed cancers. This research was funded by NCI and Feist-Wellier Cancer Center of LSUHSC-S Citation Format: Annapoorna Sreedhar, Yunfeng Zhao. UCP2’s tumor-promoting role via regulating lipid signaling and PLCγ -1 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3561. doi:10.1158/1538-7445.AM2017-3561

Vascular dysfunction in response to reactive oxygen species (ROS) plays an important role in the development and progression of atherosclerotic lesions. In most cells, mitochondria are the major source of cellular ROS during aerobic respiration. Under most conditions the rates of ROS formation and elimination are balanced through mechanisms that sense relative ROS levels. However, a chronic imbalance in redox homeostasis is believed to contribute to various chronic diseases, including atherosclerosis. Uncoupling protein-2 (UCP2) is a mitochondrial inner membrane protein shown to be a negative regulator of macrophage ROS production. In response to a cholesterol-containing atherogenic diet, C57BL/6J mice significantly increased expression of UCP2 in the aorta, while mice lacking UCP2, in the absence of any other genetic modification, displayed significant endothelial dysfunction following the atherogenic diet. Compared with wild-type mice, Ucp2(-/-) mice had decreased endothelial nitric oxide synthase, an increase in vascular cell adhesion molecule-1 expression, increased ROS production, and an impaired ability to increase total antioxidant capacity. These changes in Ucp2(-/-) mice were associated with increased aortic macrophage infiltration and more numerous and larger atherosclerotic lesions. These data establish that in the vasculature UCP2 functions as an adaptive antioxidant defense to protect against the development of atherosclerosis in response to a fat and cholesterol diet.

Study question Are mitochondrial uncoupling proteins (UCPs) expressed in human spermatozoa and if yes, what is their role? Summary answer UCP1, UCP2, and UCP3 are expressed in human spermatozoa. The inhibition of UCPs irreversibly arrests human spermatozoa motility without compromising viability. What is known already UCPs are expressed in the mitochondrial inner membrane, where they act as channels between the intermembrane space and the matrix. Six UCP homologs had already been identified in mammals (UCP1-6). UCPs act as regulators of reactive oxygen species (ROS) production, general cellular redox state, and mitochondrial function. The altered expression or function of UCPs is positively linked with the onset of metabolic diseases, such as obesity and diabetes mellitus. Male infertility is closely related to metabolic diseases since the testis is susceptible to metabolic alterations and oxidative stress, however, the expression and function of UCPs in human spermatozoa are unknown. Study design, size, duration We performed a control-versus-treatment study. High motility spermatozoa were isolated through density gradient centrifugation from human normozoospermic seminal samples (n = 16) and incubated with genipin, a selective UCP inhibitor (0, 0.5, 5, and 50 µM), for 3 h at 37 °C. Cells and culture media were collected for analysis. Participants/materials, setting, methods UCP1-3 protein expression was detected by western blot and immunofluorescence. After UCPs inhibition, spermatozoa viability and motility were assessed. Mitochondrial membrane potential and ROS production were evaluated. Media were collected and the metabolic profile and antioxidant potential were analysed by 1H-NMR and FRAP, respectively. Main results and the role of chance We were able to identify the expression of UCP1, UCP2, and UCP3 in human spermatozoa. UCP1-3 are mainly located at the equatorial segment of the head, whereas UCP1 and UCP2 are also expressed at the spermatozoa midpiece, where mitochondria are located. The inhibition of UCPs by 50 µM genipin, resulting in the UCP3 inhibition, led to the complete and irreversible loss of motility that persisted despite washing or incubation with theophylline, a cAMP activator. These effects were associated with decreased mitochondrial membrane potential and lactate production. Interestingly, the loss of motility did not compromise spermatozoa viability. In addition, the inhibition of UCPs led to no alterations concerning ROS production, possibly due to the decreased mitochondrial activity and genipin antioxidant properties. Limitations, reasons for caution This is an in vitro study with a relatively small sample size. Genipin is considered a specific yet a general inhibitor of UCPs. The development and use of specific inhibitors for each homolog will further disclose their role in human spermatozoa motility and bioenergetics. Wider implications of the findings UCPs are expressed in human spermatozoa. UCPs are important regulators of human spermatozoa motility and metabolism. The discovery and characterization of UCPs’ role in human spermatozoa open the path for studies on ROS-related pathways and bioenergetics physiology of human spermatozoa. Trial registration number 'not applicable'

Over several years we have provided evidence that uncoupling protein 1 (UCP1) is present in thymus mitochondria. We have demonstrated the conclusive evidence for the presence of UCP1 in thymus mitochondria and we have been able to demonstrate a GDP-sensitive UCP1-dependent proton leak in non-phosphorylating thymus mitochondria. In this chapter, we show how to detect UCP1 in mitochondria isolated from whole thymus using immunoblotting. We show how to measure GDP-sensitive UCP1-dependent oxygen consumption in non-phosphorylating thymus mitochondria and we show that increased reactive oxygen species production occurs on addition of GDP to non-phosphorylating thymus mitochondria. We conclude that reactive oxygen species production rate can be used as a surrogate for detecting UCP1 catalyzed proton leak activity in thymus mitochondria.

See related article, pages 103–112 Winter hibernation carries the promise of rejuvenation in the spring. In a similar fashion, myocardial “hibernation” describes a clinical phenomenon in which patients with ischemic left ventricular dysfunction demonstrate improved cardiac function following bypass surgery.1 The signature of myocardial hibernation is decreased blood flow with preserved glucose uptake, as demonstrated by positron emission tomography imaging, and identifies individuals with ischemic cardiomyopathy who may benefit from revascularization.2 In experimental models of hibernating myocardium, oxygen consumption is reduced in the absence of active ischemia.3,4 This implies that hibernation is a coordinated response to balance myocardial energy utilization with energy production capacity.5 However, within hibernating myocardium, several morphological and functional changes have been observed that can identify regions in which complete revascularization may not result in normalization of contraction.6–10 In fact, those myocardial regions with the greatest metabolic abnormalities in the hibernating tissue demonstrate the longest delay in recovery.11 In the current issue, Page et al12 demonstrate that the process of hibernation is associated with altered expression of mitochondrial proteins. Using 2D differential-in-gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry in a swine model of hibernation, they have found that key mitochondrial proteins associated with the electron transport chain are reduced. The functional importance of the decreased protein expression is documented by reduced activity measurements of the pyruvate dehydrogenase complex, cytochrome c oxidase, and citrate synthase. The parallel reductions in mitochondrial proteins and contractile function 5 months after placement of the coronary artery constrictor suggest that the “downregulation” of electron transport proteins is related to the reduced oxygen consumption. In fact, the reductions in ATPase correlate with the reduction in subendocardial blood flows in the hibernating myocardium. In addition, “upregulation” of several cytosolic proteins has been observed, including …

176 - Mitochondrial UCP2 in Age-Related Lung Fibrosis

Uncoupling proteins as a therapeutic target for the development of new era drugs against neurodegenerative disorder

Mitochondrial uncoupling protein 2 (UCP2) deficiency exacerbates brain damage following cerebral ischemia/reperfusion (I/R). The Nod-like receptor protein-3 (NLRP3) inflammasome also plays a vital role in cerebral I/R damage. However, the effect of UCP2 on NLRP3 inflammasome-mediated hyperglycemia and I/R damage is not clear. In the present study, UCP2-knockout (UCP2−/−) and wild-type (WT) mice were used to establish a model of middle cerebral artery occlusion (MCAO) and reperfusion under normo- and hyperglycemic conditions. HT22 cells were established as a model of oxygen–glucose deprivation and reoxygenation (OGD/R) with high glucose to mimic hyperglycemia and I/R in vitro. HT22 cells were treated with/without different concentrations of the UCP2-specific inhibitor genipin for different periods of time. The results showed that UCP2 deficiency significantly increased histopathological changes and apoptosis after cerebral I/R damage in hyperglycemic mice. Moreover, UCP2 deficiency enhanced NLRP3 inflammasome activation in neurons when cerebral I/R damage was exacerbated by hyperglycemia. Furthermore, UCP2 deficiency enhanced NLRP3 inflammasome activation and reactive oxygen species (ROS) production in HT22 cells under OGD/R and high-glucose conditions. UCP2 deficiency aggravated hyperglycemia-induced exacerbation of cerebral I/R damage. UCP2 deficiency also enhanced NLRP3 inflammasome activation and ROS production in neurons in vitro and in vivo. These findings suggest that UCP2 deficiency enhances NLRP3 inflammasome activation following hyperglycemia-induced exacerbation of cerebral I/R damage in vitro and in vivo. UCP2 may be a potential therapeutic target for hyperglycemia-induced exacerbation of cerebral I/R damage.

Microglial activation is a dynamic process, central to neuroinflammation, which can have beneficial or pathogenic effects to human health. Mitochondria are key players in neuroinflammatory and neurodegenerative processes, common to most brain diseases. To the best of our knowledge on the role of mitochondria in the modulation of neuroinflammation, we focused on the mitochondrial uncoupling protein-2 (UCP2), known to control mitochondrial functions and to be implicated in a variety of physiological and pathological processes. In primary microglial cultures, the M1 stimulus lipopolysaccharide induced an early and transitory decrease in UCP2 levels. The initial UCP2 down-regulation was paralleled by mitochondrial inner membrane potential (mMP) depolarization and increased mitochondrial reactive oxygen species production. The key role of UCP2 in controlling mMP and reactive oxygen species production was confirmed by both pharmacological inhibition and down-regulation by RNA interference. Additionally, UCP2-silenced microglia stimulated with lipopolysaccharide showed an enhanced inflammatory response, characterized by a greater production of nitric oxide and interleukin-6. UCP2 was differently regulated by M2 stimuli, as indicated by its persistent up-regulation by interleukin-4. In UCP2-silenced microglia, interleukin-4 failed to induce M2 genes (mannose receptor 1 and interleukin-10) and to reduce M1 genes (inducible nitric oxide synthase and tumour necrosis factor-α). Our findings indicate that UCP2 is central to the process of microglial activation, with opposite regulation of M1 and M2 responses, and point to UCP2 manipulation as a potential strategy for redirecting microglial response towards protective phenotypes in several brain diseases where neuroinflammation is recognized to contribute to neurodegeneration. We show that the mitochondrial uncoupling protein-2 (UCP2) is central to the process of microglial activation, with opposite regulation of M1 and M2 responses. In UCP2-silenced microglia, lipopolysaccharide (LPS) triggers an enhanced inflammatory response characterized by a greater expression of M1 genes, whereas interleukin-4 (IL-4) fails in inducing M2 genes and reducing M1 genes. We propose UCP2 manipulation as a potential strategy for redirecting microglial response towards protective phenotypes.

Uncoupling protein 2 modulation of the NLRP3 inflammasome in astrocytes and its implications in depression

The uncoupling protein 1 homologue, uncoupling protein 3, is able to uncouple adenosine triphosphate production from mitochondrial respiration, thereby dissipating energy as heat and affecting the efficiency of energy metabolism. Uncoupling protein 3 is expressed predominantly in skeletal muscle, and has been associated with whole-body energy metabolism. However, on the basis of present evidence it has been concluded that the primary function of uncoupling protein 3 is not in the regulation of energy expenditure. For example, fasting, an energy expenditure attenuating condition, upregulates uncoupling protein 3 expression, and uncoupling protein 3 knockout mice have a normal metabolic rate. The exact function of uncoupling protein 3 remains to be elucidated, but at present putative roles for uncoupling protein 3 include involvement in the regulation of the production of reactive oxygen species, mitochondrial fatty acid transport and the regulation of glucose metabolism in skeletal muscle. Because all these putative functions assume that uncoupling protein 3 affects mitochondrial coupling, a secondary effect of the function of uncoupling protein 3 might still be that it influences (but not regulates) energy metabolism, consistent with observations in linkage and association studies. Therefore, uncoupling protein 3 remains an interesting target for pharmacological upregulation in the treatment of obesity and diabetes.

The expression status of mitochondrial uncoupling protein 2 (UCP2) was investigated in undifferentiated mouse myeloid leukemia (M1) and its differentiated macrophage-like cells (Mm1). Mm1 cells have a high ability of phagocytosis along with significantly high levels of reactive oxygen species (ROS) production, UCP2 protein and manganese superoxide dismutase (Mn-SOD), in contrast to undifferentiated leukemia cells (M1). Mm1 cells expressed 10-fold more UCP2 protein compared with undifferentiated M1 cells, although the UCP2 mRNA levels in both cell types were similar. The higher expression of UCP2 in the Mm1 cells suggests a regulatory role of UCP2 in the ROS production. Furthermore, the transfection of UCP2-GFP-expression vector in Mm1 cells dissipated the mitochondrial membrane potential and reduced ROS production, which was shown by their direct visualization using MitoTracker Red CM-H2Xros. The macrophage gp91phox protein, a membrane catalytic component of the NADPH oxidase complex, was at a similar level in both of UCP2-GFP expressed and non-expressed Mm1 cells. These results suggest that the UCP2 protein of the undifferentiated cell is regulated at a quite low level and the higher UCP2 protein of the differentiated macrophages involves with the regulation of ROS production.

Mitochondrial uncoupling proteins (UCPs) are central in the regulation of mitochondrial activity and reactive oxygen species (ROS) production. High oxidative stress is a major cause of male infertility; however, UCPs expression and function in human spermatozoa are still unknown. Herein, we aimed to assess the expression and function of the different homologs (UCP1-6) in human spermatozoa. For this purpose, we screened for the mRNA expression of all UCP homologs. Protein expression and immunolocalization of UCP1, UCP2, and UCP3 were also assessed. Highly motile spermatozoa were isolated from human normozoospermic seminal samples (n = 16) and incubated with genipin, an inhibitor of UCPs (0, 0.5, 5, and 50 µM) for 3 h at 37 °C. Viability and total motility were assessed. Mitochondrial membrane potential and ROS production were evaluated. Media were collected and the metabolic profile and antioxidant potential were analyzed by 1H-NMR and FRAP, respectively. The expression of all UCP homologs (UCP1-6) mRNA by human spermatozoa is herein reported for the first time. UCP1-3 are predominant at the head equatorial segment, whereas UCP1 and UCP2 are also expressed at the spermatozoa midpiece, where mitochondria are located. The inhibition of UCPs by 50 µM genipin, resulting in the UCP3 inhibition, did not compromise sperm cell viability but resulted in irreversible total motility loss that persisted despite washing or incubation with theophylline, a cAMP activator. These effects were associated with decreased mitochondrial membrane potential and lactate production. No differences concerning UCP3 expression, however, were observed in spermatozoa from normozoospermic versus asthenozoospermic men (n = 6). The inhibition of UCPs did not increase ROS production, possibly due to the decreased mitochondrial activity and genipin antioxidant properties. In sum, UCPs are major regulators of human spermatozoa motility and metabolism. The discovery and characterization of UCPs' role in human spermatozoa can shed new light on spermatozoa ROS-related pathways and bioenergetics physiology.