UCPs, at the interface between bioenergetics and metabolism.

The first member of the uncoupling protein (UCP) family, brown adipose tissue uncoupling protein 1 (UCP1), was identified in 1976. Twenty years later, two closely related proteins, UCP2 and UCP3, were described in mammals. Homologs of these proteins exist in other organisms, including plants. Uncoupling refers to a deterioration of energy conservation between substrate oxidation and ADP phosphorylation. Complete energy conservation loss would be fatal but fine-tuning can be beneficial for processes such as thermogenesis, redox control, and prevention of mitochondrial ROS release. The coupled/uncoupled state of mitochondria is related to the permeability of the inner membrane and the proton transport mediated by activated UCPs underlies the uncoupling activity of these proteins. Proton transport by UCP1 is activated by fatty acids and this ensures thermogenesis. In vivo in absence of this activation UCP1 remains inhibited with no transport activity. A similar situation now seems unlikely for UCP2 and UCP3 and while activation of their proton transport has been described its physiological relevance remains uncertain and their influence can be envisaged as a result of another transport pathway that takes place in the absence of activation. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

UCP2 transports C4 metabolites out of mitochondria, regulating glucose and glutamine oxidation.

Uncoupling protein 2 (UCP2) is involved in various physiological and pathological processes such as insulin secretion, stem cell differentiation, cancer, and aging. However, its biochemical and physiological function is still under debate. Here we show that UCP2 is a metabolite transporter that regulates substrate oxidation in mitochondria. To shed light on its biochemical role, we first studied the effects of its silencing on the mitochondrial oxidation of glucose and glutamine. Compared with wild-type, UCP2-silenced human hepatocellular carcinoma (HepG2) cells, grown in the presence of glucose, showed a higher inner mitochondrial membrane potential and ATP:ADP ratio associated with a lower lactate release. Opposite results were obtained in the presence of glutamine instead of glucose. UCP2 reconstituted in lipid vesicles catalyzed the exchange of malate, oxaloacetate, and aspartate for phosphate plus a proton from opposite sides of the membrane. The higher levels of citric acid cycle intermediates found in the mitochondria of siUCP2-HepG2 cells compared with those found in wild-type cells in addition to the transport data indicate that, by exporting C4 compounds out of mitochondria, UCP2 limits the oxidation of acetyl-CoA-producing substrates such as glucose and enhances glutaminolysis, preventing the mitochondrial accumulation of C4 metabolites derived from glutamine. Our work reveals a unique regulatory mechanism in cell bioenergetics and provokes a substantial reconsideration of the physiological and pathological functions ascribed to UCP2 based on its purported uncoupling properties.

Loss of UCP2 attenuates mitochondrial dysfunction without altering ROS production and uncoupling activity.

Although mitochondrial dysfunction is often accompanied by excessive reactive oxygen species (ROS) production, we previously showed that an increase in random somatic mtDNA mutations does not result in increased oxidative stress. Normal levels of ROS and oxidative stress could also be a result of an active compensatory mechanism such as a mild increase in proton leak. Uncoupling protein 2 (UCP2) was proposed to play such a role in many physiological situations. However, we show that upregulation of UCP2 in mtDNA mutator mice is not associated with altered proton leak kinetics or ROS production, challenging the current view on the role of UCP2 in energy metabolism. Instead, our results argue that high UCP2 levels allow better utilization of fatty acid oxidation resulting in a beneficial effect on mitochondrial function in heart, postponing systemic lactic acidosis and resulting in longer lifespan in these mice. This study proposes a novel mechanism for an adaptive response to mitochondrial cardiomyopathy that links changes in metabolism to amelioration of respiratory chain deficiency and longer lifespan.

White-to-brown transdifferentiation of omental adipocytes in patients affected by pheochromocytoma.

In all mammals, white adipose tissue (WAT) and brown adipose tissue (BAT) are found together in several fat depots, forming a multi-depot organ. Adrenergic stimulation induces an increase in BAT usually referred to as "browning". This phenomenon is important because of its potential use in curbing obesity and related disorders; thus, understanding its cellular mechanisms in humans may be useful for the development of new therapeutic strategies. Data in rodents have supported the direct transformation of white into brown adipocytes. Biopsies of pure white omental fat were collected from 12 patients affected by the catecholamine-secreting tumor pheochromocytoma (pheo-patients) and compared with biopsies from controls. Half of the omental fat samples from pheo-patients contained uncoupling protein 1 (UCP1)-immunoreactive-(ir) multilocular cells that were often arranged in a BAT-like pattern endowed with noradrenergic fibers and dense capillary network. Many UCP1-ir adipocytes showed the characteristic morphology of paucilocular cells, which we have been described as cytological marker of transdifferentiation. Electron microscopy showed increased mitochondrial density in multi- and paucilocular cells and disclosed the presence of perivascular brown adipocyte precursors. Brown fat genes, such as UCP1, PR domain containing 16 (PRDM16) and ß3-adrenoreceptor, were highly expressed in the omentum of pheo-patients and in those cases without visible morphologic re-arrangement. Of note, the brown determinant PRDM16 was detected by immunohistochemistry only in nuclei of multi- and paucilocular adipocytes. Quantitative electron microscopy and immunohistochemistry for Ki67 suggest an unlikely contribution of proliferative events to the phenomenon. The data support the idea that, in adult humans, white adipocytes of pure white fat that are subjected to adrenergic stimulation are able to undergo a process of direct transformation into brown adipocytes. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.

Listeria monocytogenes transiently alters mitochondrial dynamics during infection.

Mitochondria are essential and highly dynamic organelles, constantly undergoing fusion and fission. We analyzed mitochondrial dynamics during infection with the human bacterial pathogen Listeria monocytogenes and show that this infection profoundly alters mitochondrial dynamics by causing transient mitochondrial network fragmentation. Mitochondrial fragmentation is specific to pathogenic Listeria monocytogenes, and it is not observed with the nonpathogenic Listeria innocua species or several other intracellular pathogens. Strikingly, the efficiency of Listeria infection is affected in cells where either mitochondrial fusion or fission has been altered by siRNA treatment, highlighting the relevance of mitochondrial dynamics for Listeria infection. We identified the secreted pore-forming toxin listeriolysin O as the bacterial factor mainly responsible for mitochondrial network disruption and mitochondrial function modulation. Together, our results suggest that the transient shutdown of mitochondrial function and dynamics represents a strategy used by Listeria at the onset of infection to interfere with cellular physiology.

A central thermogenic-like mechanism in feeding regulation: an interplay between arcuate nucleus T3 and UCP2.

The active thyroid hormone, triiodothyronine (T3), regulates mitochondrial uncoupling protein activity and related thermogenesis in peripheral tissues. Type 2 deiodinase (DII), an enzyme that catalyzes active thyroid hormone production, and mitochondrial uncoupling protein 2 (UCP2) are also present in the hypothalamic arcuate nucleus, where their interaction and physiological significance have not been explored. Here, we report that DII-producing glial cells are in direct apposition to neurons coexpressing neuropeptide Y (NPY), agouti-related protein (AgRP), and UCP2. Fasting increased DII activity and local thyroid hormone production in the arcuate nucleus in parallel with increased GDP-regulated UCP2-dependent mitochondrial uncoupling. Fasting-induced T3-mediated UCP2 activation resulted in mitochondrial proliferation in NPY/AgRP neurons, an event that was critical for increased excitability of these orexigenic neurons and consequent rebound feeding following food deprivation. These results reveal a physiological role for a thyroid-hormone-regulated mitochondrial uncoupling in hypothalamic neuronal networks.

Clinical and biochemical heterogeneity associated with fumarase deficiency.

Fumarase deficiency (FD), caused by biallelic alteration of the Fumarase Hydratase gene (FH), and a rare metabolic disorder that affects the Krebs cycle, causes severe neurological impairment and fumaric aciduria. Less than 30 unrelated cases are known to date. In addition, heterozygous mutations of the FH gene are responsible for hereditary leiomyomatosis and renal cell cancer (HLRCC). We report three additional patients with dramatically different clinical presentations of FD and novel missense mutations in the FH gene. One patient had severe neonatal encephalopathy, polymicrogyria, <1% enzyme activity, and mildly increased levels of urinary fumarate. The second patient had microcephaly, mental retardation, 20% of fumarase activity, and intermediate levels of urinary fumarate. The third patient had mild mental retardation, polymicrogyria, 42-61% enzyme activity in different cell types and massive amounts of urinary fumarate. In silico analysis predicted minor yet significant structural changes in the encoded proteins. The nuclear translocation of hypoxia-inducible factor (HIF)-1alpha (HIF1A) in cultured fibroblasts was similar to controls. These results extend the range of clinical and biochemical variation associated with FD, supporting the notion that patients with moderate increases in fumarate excretion should be investigated for this disease. The tumoral risk in the patients and their relatives requires adequate screening protocols.

Multiple sources of metabolic disturbance in ETHE1-related ethylmalonic encephalopathy.

Ethylmalonic encephalopathy (EE) is a rare metabolic disorder caused by dysfunction of ETHE1, a mitochondrial dioxygenase involved in hydrogen sulfide (H2S) detoxification. Patients present in infancy with psychomotor retardation, chronic diarrhea, orthostatic acrocyanosis and relapsing petechiae. High levels of lactic acid, ethymalonic acid (EMA) and methylsuccinic acid (MSA) are detected in body fluids. Several pathways may contribute to the pathophysiology, including isoleucine, methionine and fatty acid metabolism. We report on a 15-month-old male presenting with typical EE associated with a homozygous ETHE1 mutation. We investigated oral isoleucine (150 mg/kg), methionine (100 mg/kg), fatty acid loading tests and isoleucine-restricted diet (200 mg/day) for any effects on several metabolic parameters. Before loading tests or specific dietary interventions, EMA, C4-C5 acylcarnitines and most acylglycines were elevated, indicating functional deficiency of short chain acyl-CoA (SCAD) as well as all branched acyl-CoA dehydrogenases. Excretion of EMA and n-butyrylglycine increased following each of the loads, and isoleucine led to increased levels of derivative metabolites. An isoleucine-restricted diet for 8 days corrected some of the abnormalities but led to no obvious clinical improvement and only partial effects on EMA. A principal component analysis supports the inference that these dietary conditions have consistent effects on the global metabolic profile. Our results suggest that multiple pathways modulate EMA levels in EE. They might all interact with H2S toxicity. Prolonged dietary interventions involving the restriction for branched aminoacids, fatty acids and methionine could be discussed as auxiliary therapeutical strategies in EE.

Role of uncoupling protein UCP2 in cell-mediated immunity: how macrophage-mediated insulitis is accelerated in a model of autoimmune diabetes.

Infiltration of inflammatory cells into pancreatic islets of Langerhans and selective destruction of insulin-secreting beta-cells are characteristics of type 1 diabetes. Uncoupling protein 2 (UCP2) is a mitochondrial protein expressed in immune cells. UCP2 controls macrophage activation by modulating the production of mitochondrial reactive oxygen species (ROS) and MAPK signaling. We investigated the role of UCP2 on immune cell activity in type 1 diabetes in Ucp2-deficient mice. Using the model of multiple low-dose streptozotocin (STZ)-induced diabetes, we found that autoimmune diabetes was strongly accelerated in Ucp2-KO mice, compared with Ucp2-WT mice with increased intraislet lymphocytic infiltration. Macrophages from STZ-treated Ucp2-KO mice had increased IL-1beta and nitric oxide (NO) production, compared with WT macrophages. Moreover, more macrophages were recruited in islets of STZ-treated Ucp2-KO mice, compared with Ucp2-WT mice. This finding also was accompanied by increased NO/ROS-induced damage. Altogether, our data show that inflammation is stronger in Ucp2-KO mice and islets, leading to the exacerbated disease in these mice. Our results highlight the mitochondrial protein UCP2 as a new player in autoimmune diabetes.

Modified glutamine catabolism in macrophages of Ucp2 knock-out mice.

Uncoupling protein 2 (UCP2) belongs to a family of transporters of the mitochondrial inner membrane and is reported to uncouple respiration from ATP synthesis. Our observation that the amino acid glutamine specifically induces UCP2 protein expression prompted us to investigate metabolic consequences of a UCP2 knockdown (Ucp2-KO) when glutamine is offered as a substrate. We found that Ucp2-KO macrophages incubated in the presence of glutamine exhibit a lower ammonium release, a decreased respiratory rate, and an intracellular accumulation of aspartate. Therefore, we conclude that UCP2 expression is required for efficient oxidation of glutamine in macrophages. This role of UCP2 in glutamine metabolism appears independent from the uncoupling activity of UCP2.

UCP2, a metabolic sensor coupling glucose oxidation to mitochondrial metabolism?

Mitochondrial uncoupling of oxidative phosphorylation may serve a variety of purposes such as the regulation of substrate oxidation, free radical production (a major by-product of mitochondrial respiration) and ATP production and turnover. As regulators of energy expenditure and antioxidant defenses, uncoupling proteins would seem to offer an attractive mechanism by which to explain the control of body weight, resting metabolic rate and aging. As a result, the discovery of UCP1 homologues has led to an impressive number of publications. However, 10 years after their identification, no consensus has been found concerning the function of UCP homologues, and there are controversies as to whether or not they even have physiologically significant uncoupling activity. Here, we discuss a potential new function for UCP2, as a carrier involved in the coupling between glucose oxidation and mitochondrial metabolism.

Uncoupling protein-2 controls proliferation by promoting fatty acid oxidation and limiting glycolysis-derived pyruvate utilization.

Uncoupling protein-2 (UCP2) belongs to the mitochondrial carrier family and has been thought to be involved in suppressing mitochondrial ROS production through uncoupling mitochondrial respiration from ATP synthesis. However, we show here that loss of function of UCP2 does not result in a significant increase in ROS production or an increased propensity for cells to undergo senescence in culture. Instead, Ucp2-/- cells display enhanced proliferation associated with a metabolic switch from fatty acid oxidation to glucose metabolism. This metabolic switch requires the unrestricted availability of glucose, and Ucp2-/- cells more readily activate autophagy than wild-type cells when deprived of glucose. Altogether, these results suggest that UCP2 promotes mitochondrial fatty acid oxidation while limiting mitochondrial catabolism of pyruvate. The persistence of fatty acid catabolism in Ucp2+/+ cells during a proliferative response correlates with reduced cell proliferation and enhances resistance to glucose starvation-induced autophagy.

UCP2 Deficiency Increases Colon Tumorigenesis by Promoting Lipid Synthesis and Depleting NADPH for Antioxidant Defenses.

Colorectal cancer (CRC) is associated with metabolic and redox perturbation. The mitochondrial transporter uncoupling protein 2 (UCP2) controls cell proliferation in vitro through the modulation of cellular metabolism, but the underlying mechanism in tumors in vivo remains unexplored. Using murine intestinal cancer models and CRC patient samples, we find higher UCP2 protein levels in tumors compared to their non-tumoral counterparts. We reveal the tumor-suppressive role of UCP2 as its deletion enhances colon and small intestinal tumorigenesis in AOM/DSS-treated and ApcMin/+ mice, respectively, and correlates with poor survival in the latter model. Mechanistically, UCP2 loss increases levels of oxidized glutathione and proteins in tumors. UCP2 deficiency alters glycolytic pathways while promoting phospholipid synthesis, thereby limiting the availability of NADPH for buffering oxidative stress. We show that UCP2 loss renders colon cells more prone to malignant transformation through metabolic reprogramming and perturbation of redox homeostasis and could favor worse outcomes in CRC.

Uncoupling protein-2 controls adiponectin gene expression in adipose tissue through the modulation of reactive oxygen species production.

Uncoupling protein-2 (UCP2) is a mitochondrial membrane transporter expressed in white adipose tissue. We observed that circulating adiponectin levels and adiponectin gene expression in adipose tissue are reduced in UCP2-null mice. We studied whether mitochondrial activity and its control by UCP2 may regulate adiponectin gene expression. In 3T3-L1 cells, increasing UCP2 mitochondrial levels by adenoviral-mediated gene transfer induced adiponectin gene expression, whereas oligomycin and antimycin A, inhibitors of ATP synthesis and mitochondrial respiration, led to a downregulation. Reactive oxygen species (ROS) scavengers alleviated the repression of adiponectin gene expression caused by oligomycin or antimycin A. The action of ROS involves the transcription factor CHOP-10, the abundance of which was reduced in response to UCP2 and was induced by oligomycin. CHOP-10 inhibited adiponectin gene expression by interfering with the -117/-73 CCAAT/enhancer binding protein-binding region in the adiponectin gene promoter. Moreover, CHOP-10 levels were increased in adipose tissue from UCP2-null mice. Results indicate that the modulation of ROS levels by mitochondrial activity, and specifically as a consequence of the action of UCP2, controls adiponectin gene expression. This provides a physiological mechanism by which the adipose tissue energetic status may determine the extent of adiponectin release and influence systemic insulin sensitivity.

Mitochondria contribute to LPS-induced MAPK activation via uncoupling protein UCP2 in macrophages.

The mitochondrion is a major organelle contributing to energy metabolism but also a main site of ROS (reactive oxygen species) production. LPS (lipopolysaccharide)-induced ROS signalling is a critical event in macrophage activation. In the present paper we report that part of LPS-mediated ROS signalling comes from mitochondria inside a signal amplification loop that enhances MAPK (mitogen-activated protein kinase) activation. More precisely, we have identified the inner mitochondrial membrane UCP2 (uncoupling protein 2) as a physiological brake on ROS signalling. Stimulation of murine bone marrow-derived macrophages by LPS quickly down-regulated UCP2 through the JNK (c-Jun N-terminal kinase) and p38 pathways. UCP2 down-regulation was shown to be necessary to increase mitochondrial ROS production in order to potentiate MAPK activation. Consistent with this, UCP2-deficient macrophages exhibit an enhanced inflammatory state characterized by increased nitric oxide production and elevated migration ability. Additionally, we found that the absence of UCP2 renders macrophages more resistant to nitric oxide-induced apoptosis.

A large genomic deletion in the PDHX gene caused by the retrotranspositional insertion of a full-length LINE-1 element.

The long interspersed element-1 (LINE-1 or L1) retrotransposition has altered the human genome in many ways. In particular, recent in vitro studies have demonstrated that the retrotranspositional insertion of L1 elements has resulted in significant genomic deletions. Here we provide evidence for its operation in the human genome by identifying a approximately 46-kb pathological genomic deletion in the PDHX gene directly linked to the insertion of a full-length L1 element, in a patient with pyruvate dehydrogenase complex (PDHc) deficiency. Both the deduced bottom and top strand cleavage sites in the PDHX gene coincide with the consensus L1 endonuclease (EN) target sequence 5'-TTTT/A-3', while the full-length L1 element is followed by a 67-bp poly(A) tail. Interestingly, two hairpin structures, potentially formed by the inverted repeats present immediately 5' to the top strand nick site and 3' to the bottom strand nick site, may have facilitated the accessibility of L1 EN to the target sequences and also brought the two otherwise distantly located sequences into close proximity. Since the L1 element inserted in the PDHX gene is full-length, we favor the model of the template jumping as opposed to that of the microhomology-mediated end-joining for linking the 5' end of the nascent L1 copy to its genomic target. Our finding not only serves as an important complement to the in vitro approaches to studying L1 retrotransposition, but also reveals a novel mechanism causing human genetic disease.

UCP2 is a mitochondrial transporter with an unusual very short half-life.

This study focused on the stability of UCP2 (uncoupling protein 2), a mitochondrial carrier located in the inner membrane of mitochondrion. UCP2 is very unstable, with a half-life close to 30min, compared to 30h for its homologue UCP1, a difference that may highlight different physiological functions. Heat production by UCP1 in brown adipocytes is generally a long and adaptive phenomenon, whereas control of mitochondrial ROS by UCP2 needs more subtle regulation. We show that a mutation in UCP2 shown to modify its activity, actually decreases its stability.

Induction of Ucp2 expression in brain phagocytes and neurons following murine toxoplasmosis: an essential role of IFN-gamma and an association with negative energy balance.

A model of murine toxoplasmosis was used to study cellular and temporal expression of uncoupling protein-2 (Ucp2) in the brain. In situ hybridization indicated that Ucp2 was located in neurons. Nuclei structures involved in energy balance, in particular the nucleus of the solitary tract (NST), was shown to have a positive association between negative energy balance and Ucp2 levels. Infection-induced Ucp2 expression colocalized mainly with microglial cells, but also with infiltrating macrophages and neutrophils in the brain, which was evident from day 9 post-infection. Using cytokine knockout mice we demonstrate that microglial Ucp2 induction in the brain was largely dependant on interferon-gamma, but not interleukin-6 or tumour-necrosis-factor-alpha in response to infection. In summary, this study shows that Ucp2 is regulated in a different manner in neurons than in microglia/phagocytes following infection. Our study indicates that an association exists between negative energy balance and neuronal Ucp2 levels in the NST, in particular.

Thymus uncoupling protein 1 is exclusive to typical brown adipocytes and is not found in thymocytes.

A large number of studies have established the mitochondrial uncoupling protein UCP1 as a specific marker of brown adipocytes, where it controls energy dissipation of fatty acid oxidation as heat in response to physiological requirements. Following the recent report of the detection of UCP1 in thymocytes of rats and mice, we reinvestigated its presence in thymus. Light microscopy and immunohistochemical analysis demonstrated that the UCP1 signal in thymus is entirely explained by the presence of typical brown adipocytes around the gland. Staining for UCP1 was not observed in thymocytes. Similarly, UCP1 failed to be observed in rat spleen, skeletal muscle, stomach, intestine, or uterus, even after exposure of animals to the cold. These data confirm the specificity of UCP1 expression in the thermogenic brown adipocytes and argue against a direct role for this mitochondrial transporter in immune cells. Whether brown adipocytes adjacent to thymic lobes play a role in thymus physiology remains to be investigated.