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Mitochondrial UCP2 in the central regulation of metabolism
Best Practice & Research Clinical Endocrinology & Metabolism xxx (2014) 1–8
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Mitochondrial UCP2 in the central regulation of metabolism Chitoku Toda, DVM, PhD, (Postdoctoral Fellow) a, b, Sabrina Diano, PhD, (Professor) a, b, c, d, * a
Departments of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT 06520, USA Program in Cell Signaling and Neurobiology of Metabolism, Yale University School of Medicine, New Haven, CT 06520, USA c Department of Neurobiology, Yale University School of Medicine, New Haven, CT 06520, USA d Section of Comparative Medicine, Yale University School of Medicine, New Haven, CT 06520, USA b
Keywords: UCP2 ghrelin AgRP POMC hypothalamus reward system dopamine synaptic plasticity mitochondrial dynamics
Uncoupling protein 2 (UCP2) is a mitochondrial anion carrier protein, which uncouples the oxidative phosphorylation from ATP production by dissipating the proton gradient generated across the mitochondrial inner membrane. UCP2 regulates not only mitochondrial ATP production, but also the generation of reactive oxygen species (ROS), considered important second-messenger signals within the cell. The importance of UCP2 was firstly reported in macrophages and pancreatic beta cells. However, several studies have revealed the important role of UCP2 in the Central Nervous System (CNS) in the regulation of homeostatic mechanisms including food intake, energy expenditure, glucose homeostasis and reward behaviors. The mechanisms by which central UCP2 affect these processes seem to be associated with synaptic and mitochondrial plasticity. In this review, we will describe recent findings on central UCP2 and discuss its role in CNS regulation of homeostasis. Ó 2014 Published by Elsevier Ltd.
* Corresponding author. Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT 06520, USA. Tel.: þ1 203 737 1216; Fax: þ1 203 785 4713. E-mail address: [email protected] (S. Diano).
http://dx.doi.org/10.1016/j.beem.2014.02.006 1521-690X/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Toda C, Diano S, Mitochondrial UCP2 in the central regulation of metabolism, Best Practice & Research Clinical Endocrinology & Metabolism (2014), http://dx.doi.org/ 10.1016/j.beem.2014.02.006
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Introduction The primary function of mitochondria is to produce ATP by using the electro-proton gradient across the inner mitochondrial membrane made by the electron transport chain (ETC). Uncoupling proteins (UCP1-5) are mitochondrial inner-membrane proteins which function to uncouple the oxidative phosphorylation from ATP production. UCP1 is the first uncoupler identified in the brown adipose tissue that dissipates the electron-proton gradient in the form of heat [1]. The other uncouplers, including UCP2, have been identified by sequence homology and their functions are still under investigation. Specifically, UCP2 has been involved in physiological/pathological mechanisms of inflammation [2], neurodegenerative disorder [3], cancer [4] and metabolic syndrome [5]. For example, polymorphisms of UCP2, such as -866G>A, Ala55Val or -5331G>A, have been reported to be associated with obesity and type II diabetes in human [6,7]. Regulation of UCP2 expression and activity The transcription of UCP2 gene is regulated by several factors. Fatty acids, for example, have been shown to enhance UCP2 gene expression through peroxisomal proliferators-activated receptors (PPARs) [8]. PPARg agonist enhances UCP2 expression in adipocytes [9] and in the hippocampus [10] and a PPARg and retinoid X receptor (RXR) complex, by binding to the intron 1 of UCP3 gene, located just before UCP2 gene, has been shown to facilitate the translational activation of UCP2 [11]. Furthermore, PPARg coactivator1-a (PGC1a) has also been reported to increase UCP2 gene expression in animal models of type II diabetes [12] by interacting with thyroid hormone response elements (TRE) in the UCP2 promoter region in pancreatic beta cell [12]. In addition to PGC1a, PGC1b and sterol regulatory element binding protein (SREBP) have also been shown to enhance UCP2 gene expression [13]. In contrast, Sirt1 [14], Foxa1 [15], SMAD [16], microRNA-133a [17] and -15a [18] have been shown to repress UCP2 gene expression. Once translated, UCP2 activity can also be regulated. It has been established that fatty acid (FA), especially polyunsaturated FA [19], and reactive oxygen species (ROS), generated during oxidative phosphorylation, can affect and specifically increase the activity of UCP2. For example, ROS-derived lipid peroxidation product, 4-hydroxynonenal, has been found to promote proton leak through UCP2 [20]. Furthermore, Nègre-Salvayre and collaborators showed that UCP2 is a negative regulator of mitochondrial hydrogen peroxide production [21]. The mechanism(s) by which ROS regulates UCP2 activity is unclear. However, a recent study [22] has proposed that when mitochondrial ROS levels are maintained at tolerable levels and cell redox state is normal, UCP2 is conjugated with glutathione (GSH). This glutathionylated form of UCP2 keeps it in an inactive state. When small increases in ROS levels occur, UCP2 is deglutathionylated, resulting in an increased proton leak and state 4 respiration [22]. Furthermore, besides allowing protons to pass from the matrix to the inter-membrane space, it has been proposed that UCP2 allows also the transport of C4 metabolites (malate, oxaloacetate, and aspartate) [23]. By doing that, Vozza and collaborators hypothesized that UCP2 reduces the substrates of Krebs cycle and therefore attenuates the activity of ETC, ATP production and ROS production [23]. Role of UCP2 in the pancreatic beta and alpha cells UCP2 is involved in glucose-stimulated insulin secretion (GSIS) in pancreatic beta cells [24–26]. Increased levels of glucose induce an increase in ATP/ADP ratio in the mitochondria. The increase in ATP levels will then inhibit ATP-sensitive potassium (KATP) channels, leading to plasma membrane depolarization and increase in insulin secretion. Simultaneously, increased ROS levels induced by high glucose will also increase GSIS. A genetic mouse model of UCP2 knockout (KO) on a mixed mouse background (129/SVJ and C57BL/6) was reported to display higher ATP levels in isolated beta cells resulting in increased insulin secretion, and thus suggesting a role for UCP2 in suppressing GSIS [24]. However, a different result has been reported in an UCP2KO mouse model with a different genetic background, suggesting the importance of the genetic background when analyzing the role of UCP2 [25]. Nevertheless, the hypothesis that UCP2 is an endogenous suppressor of GSIS has been supported Please cite this article in press as: Toda C, Diano S, Mitochondrial UCP2 in the central regulation of metabolism, Best Practice & Research Clinical Endocrinology & Metabolism (2014), http://dx.doi.org/ 10.1016/j.beem.2014.02.006
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by other experimental models including the overexpression of UCP2 in isolated pancreatic beta cells, which inhibits GSIS, and the short-term UCP2 knockdown or acute UCP2 inhibition, both increasing GSIS [26]. In addition to insulin, UCP2 has also been reported to regulate glucagon secretion from pancreatic alpha cells [27]. Alpha cell-specific KO of UCP2 or application of UCP2 inhibitor in human alpha cells has been shown to impair glucagon secretion during fasting. UCP2 ablation in alpha cells induces higher levels of intracellular ROS, due to enhanced mitochondrial coupling, which, in turn, will suppress glucagon secretion [27]. Considering the fundamental role of these 2 hormones in glucose homeostasis and the role of UCP2 in their secretion, this mitochondrial protein represents an important pancreatic glucose sensor in the regulation of blood glucose level. Role of UCP2 in the hypothalamus UCP2 in POMC and NPY/AgRP neurons The hypothalamus is a key center in the homeostatic control of energy metabolism. Within the hypothalamus, the melanocortin system represents the primum movens of energy homeostasis. Within the melanocortin system, the proopiomelanocortin (POMC)- and neuropeptide Y (NPY)/Agoutirelated peptide (AgRP)-expressing neurons of the arcuate nucleus (ARC) are well-characterized anorexigenic and orexigenic neurons, respectively (Fig. 1) [5,28]. POMC and NPY/AgRP neurons project to melanocortin receptor 4-expressing neurons (MC4R), where, by secreting a-melanocyte-
Fig. 1. Schematic illustration of brain circuits involved in the regulation of food intake. In response to high glucose levels, UCP2 affects POMC neuronal activity by lowering ATP levels. Decreased ATP levels will, in turn, induce the closing of KATP channels and thus the depolarization of the neurons. However, in response to low glucose levels such as in fasting, UCP2 increases neuronal activity in NPY/AgRP neurons. By lowering ROS production, byproduct of ghrelin-induced FA oxidation, UCP2 enables sustained NPY/AgRP neuronal activity during negative energy balance such as fasting condition. MC4R: melanocortin 4 receptor, CGRP: Calcitonin gene-related peptide, PBN: parabrachial nucleus.
Please cite this article in press as: Toda C, Diano S, Mitochondrial UCP2 in the central regulation of metabolism, Best Practice & Research Clinical Endocrinology & Metabolism (2014), http://dx.doi.org/ 10.1016/j.beem.2014.02.006
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stimulating hormone (a-MSH) and AgRP, respectively, they compete for the binding of this receptor. While a-MSH activates MC4R, AgRP competitively inhibits this receptor and NPY functionally antagonizes MC4R signaling. Activation of MC4R in the paraventricular nucleus of the hypothalamus (PVH) and in other brain regions, including brainstem, inhibits food intake and increases energy expenditure. Besides their role on MC4R neurons, recent studies have revealed the importance of GABA release from NPY/AgRP neurons in other brain areas, including the parabrachial nucleus (PBN), in regulating appetite [29,30]. Here, activation of a neuronal population expressing Calcitonin gene-related peptide (CGRP) has been recently shown to suppress food intake by projecting to the central nucleus of the amygdala forming a functional important circuit for suppressing appetite (Fig. 1) [30]. UCP2 is highly expressed in the hypothalamus, specifically in the arcuate and ventromedial nuclei [31,32]. Within the arcuate, UCP2 is expressed in both POMC and NPY/AgRP neurons where it plays an important role in regulating neuronal function. UCP2 ablation attenuates fasting- as well as ghrelininduced food intake and this is mediated by a reduction in NPY/AgRP neuronal activation due to an uncontrolled rise in ROS levels [33,34]. It has been hypothesized that fasting- (and ghrelin) induced UCP2 activation is mediated by increased fatty acid oxidation-induced elevation of ROS levels. By preventing and controlling ROS levels, UCP2 enables these neurons to sustain high activity levels at a time of negative energy balance such as fasting (Fig. 1) [33,34]. A different scenario occurs in POMC neurons. Intracerebroventricular (ICV) injection of ROS scavenger decreases, while ROS administration increases POMC neuronal activation [35]. It has been proposed that activation of UCP2 during dietinduced obesity by preventing an increase in ROS levels in POMC neurons impairs the activity of these neurons during elevated glucose and leptin levels, thus inducing the so-called leptin resistance, an enigmatic biological entity in which elevated levels of circulating leptin do not promote a reduction in feeding and an increase in energy expenditure. In both cases, UCP2 by regulating ROS production in POMC and NPY/AgRP neuron is an important regulator of neuronal function and plays a critical role in the control of energy and glucose homeostasis. Role of UCP2 and ROS in glucose sensing neurons Glucose is considered the main source of energy substrate for the brain. While hypoglycemia induces seizures, unconsciousness and brain injury, sustained hyperglycemia causes diabetes-associated diseases, such as diabetic retinopathy, nephropathy and stroke. Therefore, our body needs to keep blood glucose levels in a tight controlled range. To accomplish this, the brain has to sense changes in glucose levels and trigger mechanisms for restoring its normal levels. Changes in glucose levels are sensed in the brain by the so-called glucose excited (GE) and glucose inhibited (GI) neurons. These neurons are either activated (GE) or inhibited (GI) by increased glucose concentrations. Arcuate POMC neurons represent an example of GE neurons, while NPY/AgRP neurons are GI neurons [36,37]. Similar to what occurs in the pancreatic beta cells, high glucose levels in POMC neurons are metabolized and converted to high ATP levels. Increased ATP levels will then induce the closing of KATP channels causing the depolarization of these neurons (Fig. 1) [38]. Inhibition of UCP2 by genipin, a UCP2 inhibitor, has been shown to induce excitation of POMC neurons, and this effect was abolished in KATP channeldeficient POMC neurons [38]. Therefore, UCP2 decreases glucose sensing through the regulation of intracellular ATP level and subsequent modulation of KATP channel. Diet-induced obese (DIO) and genetic obese (Ob/Ob) mice display increased UCP2 expression in the hypothalamus, which is believed to be responsible for the decreased glucose sensing in these animal models. Interestingly, genipin or genetic deletion of UCP2 prevent the decreased glucose sensing in POMC neurons in DIO mice [38], suggesting that the increase in UCP2 during high fat feeding is in part responsible for the impairment of glucose sensing in POMC neurons. A similar mechanism in glucose sensing has been also reported in melanin-concentrating hormone (MCH)-expressing neurons of the lateral hypothalamus that regulate whole body glucose metabolism. MCH-specific UCP2 knockout mice show enhanced glucose-induced depolarization of MCH neurons and increased glucose tolerance [39], suggesting that MCH neurons regulates glucose metabolism to prevent hyperglycemia and UCP2 is a negative regulator in the glucose sensing. As for POMC neurons, it has been reported that KATP channels are necessary for the alteration in glucose tolerance by UCP2 in MCH neurons [39]. However, the role of MCH neurons in glucose metabolism is controversial since ICV Please cite this article in press as: Toda C, Diano S, Mitochondrial UCP2 in the central regulation of metabolism, Best Practice & Research Clinical Endocrinology & Metabolism (2014), http://dx.doi.org/ 10.1016/j.beem.2014.02.006
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injection of MCH promotes glucose intolerance [40] and deletion of MCH neurons increases glucose tolerance [41], suggesting that MCH neurons have a function to decrease glucose metabolism. Further studies are necessary to investigate this discrepancy. In addition to the arcuate and the lateral hypothalamus, UCP2 is highly expressed in ventromedial nucleus of the hypothalamus (VMH) [31]. VMH GE and GI neurons have been characterized and found to play an important role in the counter regulatory response to hypoglycemia [42]. Although this system plays a fundamental role in the hypoglycemia-associated autonomic failure (HAAF) and UCP2 is highly expressed in this nucleus, the role of this mitochondrial protein still remains to be elucidated. UCP2 in food motivation Animals need to learn where and how to find food in their environment, and they are better at this task when they are hungry. After they find food, their reward system strengthens the memory and motivation. Dopamine neurons are thought to regulate the hunger-promoted motivation and reward system. Dopamine neurons located in ventral tegmental area, and substantia pars compacta, project to striatal (nucleus accumbens and the dorsal striatum), limbic (amygdala and hippocampus) and cortical regions (prefrontal cortex, cingulate gyrus, temporal pole) and modulate the motivation and sustainability of effort necessary to accomplish tasks needed for survival. Ghrelin has been shown to play a role for the incentive value of food cues [43]. Intravenous ghrelin administration to healthy volunteers during functional magnetic resonance imaging showed an increase in neural response to food pictures in several regions of the brain, including the amygdala, orbitofrontal cortex, anterior insula, and striatum, implicated in encoding the incentive value of food cues, suggesting that ghrelin favors not only the homeostatic component of feeding but also the hedonic component by enhancing the incentive responses to food-related cues [43]. The mechanism by which ghrelin modulate hedonic feeding involves its control on the activity of dopamine neurons [44]. Similar to its effect on NPY/AgRP neurons, ghrelin directly activates dopamine neurons via an UCP2-dependent increase in mitochondrial respiration and proliferation and a decrease in ROS production [45]. In support of this, UCP2KO mice show reduced striatal dopamine turnover [46]. Besides its direct effect on the reward system, ghrelin has been shown to regulate dopamine neurons indirectly via AgRP neuronal activation. Optogenetic activation of AgRP neuron and subsequent inactivation of PVH neurons has been shown to increase motivational food seeking [47] and pharmacogenetic activation of AgRP neurons increases food seeking behavior [48]. Therefore, UCP2 seems to play a critical role in food motivation and reward system by altering the activity of dopamine neurons both directly and indirectly through the AgRP pathway. UCP2 in synaptic plasticity and mitochondrial dynamics Mitochondrial mechanisms related to UCP2 function have been shown to be essential for appropriate bioenergetic adaptation of neurons to increase not only neuronal activity but also synaptic plasticity. We have previously shown that ghrelin increases miniature inhibitory postsynaptic currents (mIPSC), which mainly represents GABA input without presynaptic activation, on POMC neurons [34]. However, ghrelin failed to increase mIPSC on POMC in UCP2KO mice [34]. Furthermore, we have also reported that in NPY/AgRP neurons Sirt1-induced synaptic plasticity onto POMC neurons is UCP2dependent [49]. A part the hypothalamus [34,49], these UCP2-dependent synaptic changes have been reported in other brain areas including the hippocampal formation [50]. Voluntary exercise, for example, has been shown to induce UCP2 mRNA expression and mitochondrial oxygen consumption in coupled as well as uncoupled respiratory states in the hippocampus. These changes in mitochondrial metabolism coincided with an increase in mitochondrial number and dendritic spine synapses in granule cells of the dentate gyrus and the stratum radiatum of the CA1 region. All of these changes were dependent on UCP2 expression, since no alterations in mitochondrial and synaptic plasticity were observed in UCP2 knockout mice [50]. We have shown that under different metabolic states such as fasting and feeding conditions, UCP2dependent mitochondrial plasticity occurs in the hypothalamus [33–35]: in NPY/AgRP neurons, for example, the mitochondrial number is significantly lower in fed versus overnight fasted mice [33–35]. Please cite this article in press as: Toda C, Diano S, Mitochondrial UCP2 in the central regulation of metabolism, Best Practice & Research Clinical Endocrinology & Metabolism (2014), http://dx.doi.org/ 10.1016/j.beem.2014.02.006
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Similar to the hypothalamus, this same UCP2-dependent change in mitochondrial density occurs in other brain areas including the hippocampus and the midbrain [44–46]. Furthermore, a recent study [51] found that in NPY/AgRP neurons the decrease in mitochondria number during the transition from fasted to fed to overfed states was associated with an increase in mitochondrial size, suggesting that mitochondrial plasticity through the combined actions of fission and fusion occurs in these neurons according to the metabolic state [51]. Indeed when Dietrich and collaborators [51] interfered with the mitochondrial fusion mechanism in AgRP neurons by cell-specific deletion of mitofusin 1 (Mfn1) or mitofusin 2 (Mfn2), an alteration in mitochondria size and density and impairment in the electric activity of AgRP neurons during high-fat diet (HFD) was observed. AgRP-specific Mfn1 or Mfn2 knockout mice gained less weight when fed an HFD due to decreased fat mass, unmasking the important role of mitochondrial dynamics in AgRP neurons in central regulation of whole-body energy metabolism. Interestingly, these fusion-like dynamic changes are cell-type specific, as they occurred in the opposite direction in anorexigenic POMC neurons [52]. POMC-specific deletion of Mfn2, resulting in altered mitochondrial number and morphology, induced hyperphagia, reduced energy expenditure, and obesity during HF feeding [52]. Although the role of UCP2 in the regulation of mitochondrial fission and fusion is unknown, it is conceivable that changes in mitochondrial morphology may be associated to changes in mitochondrial function. For example, the increased uncoupling activity in NPY/AgRP neurons during fasting state may induce mitochondrial fission and thus increased mitochondrial density. In support of this, it has been reported that dissipation of the mitochondrial inner membrane potential by a chemical uncoupler induces mitochondrial fragmentation by inhibiting Mitofusindependent mitochondrial fusion or activating dynamin-related protein (DRP)-dependent mitochondrial fission in vitro [53]. Furthermore, increased ROS production by docosahexaenoic acid has been found to be associated with mitochondrial fusion in L6 myocytes [54]. Thus, UCP2 and its effect on ROS levels may be an important component of mitochondrial dynamics. Future studies are needed to clarify the role of UCP2 in mitochondrial fission/fusion mechanisms in the regulation of energy metabolism.
Practice points UCP2 regulates ghrelin’s effect to increase food intake by decreasing ROS production in AgRP neuron. UCP2 attenuates glucose sensing in POMC neuron in obesity UCP2 in dopamine and AgRP neurons may regulate food motivation and reward system UCP2 regulates synaptic plasticity
Research agenda Whether UCP2-ROS pathway regulates glucose sensing in other brain regions Further studies are needed to investigate the role of brain UCP2-ROS pathway in glucose metabolism in peripheral tissues Role of UCP2-ROS pathway in food motivation and reward system Role of UCP2 in mitochondrial fission and fusion Relationship between UCP2 in the brain and pathological mechanism in obesity and diabetes
Summary UCP2 is a key mitochondrial protein in the central regulation of energy metabolism by modulating ROS production and neuronal activity. UCP2 is involved in the regulation of homeostatic and hedonic food intake, in addition to insulin and glucagon secretion from pancreas. Considering the role of UCP2 in regulating energy and glucose homeostasis via mitochondrial morphology and synaptic plasticity, it Please cite this article in press as: Toda C, Diano S, Mitochondrial UCP2 in the central regulation of metabolism, Best Practice & Research Clinical Endocrinology & Metabolism (2014), http://dx.doi.org/ 10.1016/j.beem.2014.02.006
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represents an important target for new strategies to combat metabolic disorders such as obesity and type 2 diabetes.
Acknowledgments This work was supported by NIH DK097566 (to S.D.), ADA 7-11-BS-33 (to S.D.), and Manpei Suzuki Diabetes Foundation (to C.T.).
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Mitochondrial UCP2 in the central regulation of metabolism Chitoku Toda, DVM, PhD, (Postdoctoral Fellow) a, b, Sabrina Diano, PhD, (Professor) a, b, c, d, * a
Departments of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT 06520, USA Program in Cell Signaling and Neurobiology of Metabolism, Yale University School of Medicine, New Haven, CT 06520, USA c Department of Neurobiology, Yale University School of Medicine, New Haven, CT 06520, USA d Section of Comparative Medicine, Yale University School of Medicine, New Haven, CT 06520, USA b
Keywords: UCP2 ghrelin AgRP POMC hypothalamus reward system dopamine synaptic plasticity mitochondrial dynamics
Uncoupling protein 2 (UCP2) is a mitochondrial anion carrier protein, which uncouples the oxidative phosphorylation from ATP production by dissipating the proton gradient generated across the mitochondrial inner membrane. UCP2 regulates not only mitochondrial ATP production, but also the generation of reactive oxygen species (ROS), considered important second-messenger signals within the cell. The importance of UCP2 was firstly reported in macrophages and pancreatic beta cells. However, several studies have revealed the important role of UCP2 in the Central Nervous System (CNS) in the regulation of homeostatic mechanisms including food intake, energy expenditure, glucose homeostasis and reward behaviors. The mechanisms by which central UCP2 affect these processes seem to be associated with synaptic and mitochondrial plasticity. In this review, we will describe recent findings on central UCP2 and discuss its role in CNS regulation of homeostasis. Ó 2014 Published by Elsevier Ltd.
* Corresponding author. Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT 06520, USA. Tel.: þ1 203 737 1216; Fax: þ1 203 785 4713. E-mail address: [email protected] (S. Diano).
http://dx.doi.org/10.1016/j.beem.2014.02.006 1521-690X/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Toda C, Diano S, Mitochondrial UCP2 in the central regulation of metabolism, Best Practice & Research Clinical Endocrinology & Metabolism (2014), http://dx.doi.org/ 10.1016/j.beem.2014.02.006
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Introduction The primary function of mitochondria is to produce ATP by using the electro-proton gradient across the inner mitochondrial membrane made by the electron transport chain (ETC). Uncoupling proteins (UCP1-5) are mitochondrial inner-membrane proteins which function to uncouple the oxidative phosphorylation from ATP production. UCP1 is the first uncoupler identified in the brown adipose tissue that dissipates the electron-proton gradient in the form of heat [1]. The other uncouplers, including UCP2, have been identified by sequence homology and their functions are still under investigation. Specifically, UCP2 has been involved in physiological/pathological mechanisms of inflammation [2], neurodegenerative disorder [3], cancer [4] and metabolic syndrome [5]. For example, polymorphisms of UCP2, such as -866G>A, Ala55Val or -5331G>A, have been reported to be associated with obesity and type II diabetes in human [6,7]. Regulation of UCP2 expression and activity The transcription of UCP2 gene is regulated by several factors. Fatty acids, for example, have been shown to enhance UCP2 gene expression through peroxisomal proliferators-activated receptors (PPARs) [8]. PPARg agonist enhances UCP2 expression in adipocytes [9] and in the hippocampus [10] and a PPARg and retinoid X receptor (RXR) complex, by binding to the intron 1 of UCP3 gene, located just before UCP2 gene, has been shown to facilitate the translational activation of UCP2 [11]. Furthermore, PPARg coactivator1-a (PGC1a) has also been reported to increase UCP2 gene expression in animal models of type II diabetes [12] by interacting with thyroid hormone response elements (TRE) in the UCP2 promoter region in pancreatic beta cell [12]. In addition to PGC1a, PGC1b and sterol regulatory element binding protein (SREBP) have also been shown to enhance UCP2 gene expression [13]. In contrast, Sirt1 [14], Foxa1 [15], SMAD [16], microRNA-133a [17] and -15a [18] have been shown to repress UCP2 gene expression. Once translated, UCP2 activity can also be regulated. It has been established that fatty acid (FA), especially polyunsaturated FA [19], and reactive oxygen species (ROS), generated during oxidative phosphorylation, can affect and specifically increase the activity of UCP2. For example, ROS-derived lipid peroxidation product, 4-hydroxynonenal, has been found to promote proton leak through UCP2 [20]. Furthermore, Nègre-Salvayre and collaborators showed that UCP2 is a negative regulator of mitochondrial hydrogen peroxide production [21]. The mechanism(s) by which ROS regulates UCP2 activity is unclear. However, a recent study [22] has proposed that when mitochondrial ROS levels are maintained at tolerable levels and cell redox state is normal, UCP2 is conjugated with glutathione (GSH). This glutathionylated form of UCP2 keeps it in an inactive state. When small increases in ROS levels occur, UCP2 is deglutathionylated, resulting in an increased proton leak and state 4 respiration [22]. Furthermore, besides allowing protons to pass from the matrix to the inter-membrane space, it has been proposed that UCP2 allows also the transport of C4 metabolites (malate, oxaloacetate, and aspartate) [23]. By doing that, Vozza and collaborators hypothesized that UCP2 reduces the substrates of Krebs cycle and therefore attenuates the activity of ETC, ATP production and ROS production [23]. Role of UCP2 in the pancreatic beta and alpha cells UCP2 is involved in glucose-stimulated insulin secretion (GSIS) in pancreatic beta cells [24–26]. Increased levels of glucose induce an increase in ATP/ADP ratio in the mitochondria. The increase in ATP levels will then inhibit ATP-sensitive potassium (KATP) channels, leading to plasma membrane depolarization and increase in insulin secretion. Simultaneously, increased ROS levels induced by high glucose will also increase GSIS. A genetic mouse model of UCP2 knockout (KO) on a mixed mouse background (129/SVJ and C57BL/6) was reported to display higher ATP levels in isolated beta cells resulting in increased insulin secretion, and thus suggesting a role for UCP2 in suppressing GSIS [24]. However, a different result has been reported in an UCP2KO mouse model with a different genetic background, suggesting the importance of the genetic background when analyzing the role of UCP2 [25]. Nevertheless, the hypothesis that UCP2 is an endogenous suppressor of GSIS has been supported Please cite this article in press as: Toda C, Diano S, Mitochondrial UCP2 in the central regulation of metabolism, Best Practice & Research Clinical Endocrinology & Metabolism (2014), http://dx.doi.org/ 10.1016/j.beem.2014.02.006
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by other experimental models including the overexpression of UCP2 in isolated pancreatic beta cells, which inhibits GSIS, and the short-term UCP2 knockdown or acute UCP2 inhibition, both increasing GSIS [26]. In addition to insulin, UCP2 has also been reported to regulate glucagon secretion from pancreatic alpha cells [27]. Alpha cell-specific KO of UCP2 or application of UCP2 inhibitor in human alpha cells has been shown to impair glucagon secretion during fasting. UCP2 ablation in alpha cells induces higher levels of intracellular ROS, due to enhanced mitochondrial coupling, which, in turn, will suppress glucagon secretion [27]. Considering the fundamental role of these 2 hormones in glucose homeostasis and the role of UCP2 in their secretion, this mitochondrial protein represents an important pancreatic glucose sensor in the regulation of blood glucose level. Role of UCP2 in the hypothalamus UCP2 in POMC and NPY/AgRP neurons The hypothalamus is a key center in the homeostatic control of energy metabolism. Within the hypothalamus, the melanocortin system represents the primum movens of energy homeostasis. Within the melanocortin system, the proopiomelanocortin (POMC)- and neuropeptide Y (NPY)/Agoutirelated peptide (AgRP)-expressing neurons of the arcuate nucleus (ARC) are well-characterized anorexigenic and orexigenic neurons, respectively (Fig. 1) [5,28]. POMC and NPY/AgRP neurons project to melanocortin receptor 4-expressing neurons (MC4R), where, by secreting a-melanocyte-
Fig. 1. Schematic illustration of brain circuits involved in the regulation of food intake. In response to high glucose levels, UCP2 affects POMC neuronal activity by lowering ATP levels. Decreased ATP levels will, in turn, induce the closing of KATP channels and thus the depolarization of the neurons. However, in response to low glucose levels such as in fasting, UCP2 increases neuronal activity in NPY/AgRP neurons. By lowering ROS production, byproduct of ghrelin-induced FA oxidation, UCP2 enables sustained NPY/AgRP neuronal activity during negative energy balance such as fasting condition. MC4R: melanocortin 4 receptor, CGRP: Calcitonin gene-related peptide, PBN: parabrachial nucleus.
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stimulating hormone (a-MSH) and AgRP, respectively, they compete for the binding of this receptor. While a-MSH activates MC4R, AgRP competitively inhibits this receptor and NPY functionally antagonizes MC4R signaling. Activation of MC4R in the paraventricular nucleus of the hypothalamus (PVH) and in other brain regions, including brainstem, inhibits food intake and increases energy expenditure. Besides their role on MC4R neurons, recent studies have revealed the importance of GABA release from NPY/AgRP neurons in other brain areas, including the parabrachial nucleus (PBN), in regulating appetite [29,30]. Here, activation of a neuronal population expressing Calcitonin gene-related peptide (CGRP) has been recently shown to suppress food intake by projecting to the central nucleus of the amygdala forming a functional important circuit for suppressing appetite (Fig. 1) [30]. UCP2 is highly expressed in the hypothalamus, specifically in the arcuate and ventromedial nuclei [31,32]. Within the arcuate, UCP2 is expressed in both POMC and NPY/AgRP neurons where it plays an important role in regulating neuronal function. UCP2 ablation attenuates fasting- as well as ghrelininduced food intake and this is mediated by a reduction in NPY/AgRP neuronal activation due to an uncontrolled rise in ROS levels [33,34]. It has been hypothesized that fasting- (and ghrelin) induced UCP2 activation is mediated by increased fatty acid oxidation-induced elevation of ROS levels. By preventing and controlling ROS levels, UCP2 enables these neurons to sustain high activity levels at a time of negative energy balance such as fasting (Fig. 1) [33,34]. A different scenario occurs in POMC neurons. Intracerebroventricular (ICV) injection of ROS scavenger decreases, while ROS administration increases POMC neuronal activation [35]. It has been proposed that activation of UCP2 during dietinduced obesity by preventing an increase in ROS levels in POMC neurons impairs the activity of these neurons during elevated glucose and leptin levels, thus inducing the so-called leptin resistance, an enigmatic biological entity in which elevated levels of circulating leptin do not promote a reduction in feeding and an increase in energy expenditure. In both cases, UCP2 by regulating ROS production in POMC and NPY/AgRP neuron is an important regulator of neuronal function and plays a critical role in the control of energy and glucose homeostasis. Role of UCP2 and ROS in glucose sensing neurons Glucose is considered the main source of energy substrate for the brain. While hypoglycemia induces seizures, unconsciousness and brain injury, sustained hyperglycemia causes diabetes-associated diseases, such as diabetic retinopathy, nephropathy and stroke. Therefore, our body needs to keep blood glucose levels in a tight controlled range. To accomplish this, the brain has to sense changes in glucose levels and trigger mechanisms for restoring its normal levels. Changes in glucose levels are sensed in the brain by the so-called glucose excited (GE) and glucose inhibited (GI) neurons. These neurons are either activated (GE) or inhibited (GI) by increased glucose concentrations. Arcuate POMC neurons represent an example of GE neurons, while NPY/AgRP neurons are GI neurons [36,37]. Similar to what occurs in the pancreatic beta cells, high glucose levels in POMC neurons are metabolized and converted to high ATP levels. Increased ATP levels will then induce the closing of KATP channels causing the depolarization of these neurons (Fig. 1) [38]. Inhibition of UCP2 by genipin, a UCP2 inhibitor, has been shown to induce excitation of POMC neurons, and this effect was abolished in KATP channeldeficient POMC neurons [38]. Therefore, UCP2 decreases glucose sensing through the regulation of intracellular ATP level and subsequent modulation of KATP channel. Diet-induced obese (DIO) and genetic obese (Ob/Ob) mice display increased UCP2 expression in the hypothalamus, which is believed to be responsible for the decreased glucose sensing in these animal models. Interestingly, genipin or genetic deletion of UCP2 prevent the decreased glucose sensing in POMC neurons in DIO mice [38], suggesting that the increase in UCP2 during high fat feeding is in part responsible for the impairment of glucose sensing in POMC neurons. A similar mechanism in glucose sensing has been also reported in melanin-concentrating hormone (MCH)-expressing neurons of the lateral hypothalamus that regulate whole body glucose metabolism. MCH-specific UCP2 knockout mice show enhanced glucose-induced depolarization of MCH neurons and increased glucose tolerance [39], suggesting that MCH neurons regulates glucose metabolism to prevent hyperglycemia and UCP2 is a negative regulator in the glucose sensing. As for POMC neurons, it has been reported that KATP channels are necessary for the alteration in glucose tolerance by UCP2 in MCH neurons [39]. However, the role of MCH neurons in glucose metabolism is controversial since ICV Please cite this article in press as: Toda C, Diano S, Mitochondrial UCP2 in the central regulation of metabolism, Best Practice & Research Clinical Endocrinology & Metabolism (2014), http://dx.doi.org/ 10.1016/j.beem.2014.02.006
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injection of MCH promotes glucose intolerance [40] and deletion of MCH neurons increases glucose tolerance [41], suggesting that MCH neurons have a function to decrease glucose metabolism. Further studies are necessary to investigate this discrepancy. In addition to the arcuate and the lateral hypothalamus, UCP2 is highly expressed in ventromedial nucleus of the hypothalamus (VMH) [31]. VMH GE and GI neurons have been characterized and found to play an important role in the counter regulatory response to hypoglycemia [42]. Although this system plays a fundamental role in the hypoglycemia-associated autonomic failure (HAAF) and UCP2 is highly expressed in this nucleus, the role of this mitochondrial protein still remains to be elucidated. UCP2 in food motivation Animals need to learn where and how to find food in their environment, and they are better at this task when they are hungry. After they find food, their reward system strengthens the memory and motivation. Dopamine neurons are thought to regulate the hunger-promoted motivation and reward system. Dopamine neurons located in ventral tegmental area, and substantia pars compacta, project to striatal (nucleus accumbens and the dorsal striatum), limbic (amygdala and hippocampus) and cortical regions (prefrontal cortex, cingulate gyrus, temporal pole) and modulate the motivation and sustainability of effort necessary to accomplish tasks needed for survival. Ghrelin has been shown to play a role for the incentive value of food cues [43]. Intravenous ghrelin administration to healthy volunteers during functional magnetic resonance imaging showed an increase in neural response to food pictures in several regions of the brain, including the amygdala, orbitofrontal cortex, anterior insula, and striatum, implicated in encoding the incentive value of food cues, suggesting that ghrelin favors not only the homeostatic component of feeding but also the hedonic component by enhancing the incentive responses to food-related cues [43]. The mechanism by which ghrelin modulate hedonic feeding involves its control on the activity of dopamine neurons [44]. Similar to its effect on NPY/AgRP neurons, ghrelin directly activates dopamine neurons via an UCP2-dependent increase in mitochondrial respiration and proliferation and a decrease in ROS production [45]. In support of this, UCP2KO mice show reduced striatal dopamine turnover [46]. Besides its direct effect on the reward system, ghrelin has been shown to regulate dopamine neurons indirectly via AgRP neuronal activation. Optogenetic activation of AgRP neuron and subsequent inactivation of PVH neurons has been shown to increase motivational food seeking [47] and pharmacogenetic activation of AgRP neurons increases food seeking behavior [48]. Therefore, UCP2 seems to play a critical role in food motivation and reward system by altering the activity of dopamine neurons both directly and indirectly through the AgRP pathway. UCP2 in synaptic plasticity and mitochondrial dynamics Mitochondrial mechanisms related to UCP2 function have been shown to be essential for appropriate bioenergetic adaptation of neurons to increase not only neuronal activity but also synaptic plasticity. We have previously shown that ghrelin increases miniature inhibitory postsynaptic currents (mIPSC), which mainly represents GABA input without presynaptic activation, on POMC neurons [34]. However, ghrelin failed to increase mIPSC on POMC in UCP2KO mice [34]. Furthermore, we have also reported that in NPY/AgRP neurons Sirt1-induced synaptic plasticity onto POMC neurons is UCP2dependent [49]. A part the hypothalamus [34,49], these UCP2-dependent synaptic changes have been reported in other brain areas including the hippocampal formation [50]. Voluntary exercise, for example, has been shown to induce UCP2 mRNA expression and mitochondrial oxygen consumption in coupled as well as uncoupled respiratory states in the hippocampus. These changes in mitochondrial metabolism coincided with an increase in mitochondrial number and dendritic spine synapses in granule cells of the dentate gyrus and the stratum radiatum of the CA1 region. All of these changes were dependent on UCP2 expression, since no alterations in mitochondrial and synaptic plasticity were observed in UCP2 knockout mice [50]. We have shown that under different metabolic states such as fasting and feeding conditions, UCP2dependent mitochondrial plasticity occurs in the hypothalamus [33–35]: in NPY/AgRP neurons, for example, the mitochondrial number is significantly lower in fed versus overnight fasted mice [33–35]. Please cite this article in press as: Toda C, Diano S, Mitochondrial UCP2 in the central regulation of metabolism, Best Practice & Research Clinical Endocrinology & Metabolism (2014), http://dx.doi.org/ 10.1016/j.beem.2014.02.006
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Similar to the hypothalamus, this same UCP2-dependent change in mitochondrial density occurs in other brain areas including the hippocampus and the midbrain [44–46]. Furthermore, a recent study [51] found that in NPY/AgRP neurons the decrease in mitochondria number during the transition from fasted to fed to overfed states was associated with an increase in mitochondrial size, suggesting that mitochondrial plasticity through the combined actions of fission and fusion occurs in these neurons according to the metabolic state [51]. Indeed when Dietrich and collaborators [51] interfered with the mitochondrial fusion mechanism in AgRP neurons by cell-specific deletion of mitofusin 1 (Mfn1) or mitofusin 2 (Mfn2), an alteration in mitochondria size and density and impairment in the electric activity of AgRP neurons during high-fat diet (HFD) was observed. AgRP-specific Mfn1 or Mfn2 knockout mice gained less weight when fed an HFD due to decreased fat mass, unmasking the important role of mitochondrial dynamics in AgRP neurons in central regulation of whole-body energy metabolism. Interestingly, these fusion-like dynamic changes are cell-type specific, as they occurred in the opposite direction in anorexigenic POMC neurons [52]. POMC-specific deletion of Mfn2, resulting in altered mitochondrial number and morphology, induced hyperphagia, reduced energy expenditure, and obesity during HF feeding [52]. Although the role of UCP2 in the regulation of mitochondrial fission and fusion is unknown, it is conceivable that changes in mitochondrial morphology may be associated to changes in mitochondrial function. For example, the increased uncoupling activity in NPY/AgRP neurons during fasting state may induce mitochondrial fission and thus increased mitochondrial density. In support of this, it has been reported that dissipation of the mitochondrial inner membrane potential by a chemical uncoupler induces mitochondrial fragmentation by inhibiting Mitofusindependent mitochondrial fusion or activating dynamin-related protein (DRP)-dependent mitochondrial fission in vitro [53]. Furthermore, increased ROS production by docosahexaenoic acid has been found to be associated with mitochondrial fusion in L6 myocytes [54]. Thus, UCP2 and its effect on ROS levels may be an important component of mitochondrial dynamics. Future studies are needed to clarify the role of UCP2 in mitochondrial fission/fusion mechanisms in the regulation of energy metabolism.
Practice points UCP2 regulates ghrelin’s effect to increase food intake by decreasing ROS production in AgRP neuron. UCP2 attenuates glucose sensing in POMC neuron in obesity UCP2 in dopamine and AgRP neurons may regulate food motivation and reward system UCP2 regulates synaptic plasticity
Research agenda Whether UCP2-ROS pathway regulates glucose sensing in other brain regions Further studies are needed to investigate the role of brain UCP2-ROS pathway in glucose metabolism in peripheral tissues Role of UCP2-ROS pathway in food motivation and reward system Role of UCP2 in mitochondrial fission and fusion Relationship between UCP2 in the brain and pathological mechanism in obesity and diabetes
Summary UCP2 is a key mitochondrial protein in the central regulation of energy metabolism by modulating ROS production and neuronal activity. UCP2 is involved in the regulation of homeostatic and hedonic food intake, in addition to insulin and glucagon secretion from pancreas. Considering the role of UCP2 in regulating energy and glucose homeostasis via mitochondrial morphology and synaptic plasticity, it Please cite this article in press as: Toda C, Diano S, Mitochondrial UCP2 in the central regulation of metabolism, Best Practice & Research Clinical Endocrinology & Metabolism (2014), http://dx.doi.org/ 10.1016/j.beem.2014.02.006
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represents an important target for new strategies to combat metabolic disorders such as obesity and type 2 diabetes.
Acknowledgments This work was supported by NIH DK097566 (to S.D.), ADA 7-11-BS-33 (to S.D.), and Manpei Suzuki Diabetes Foundation (to C.T.).
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Please cite this article in press as: Toda C, Diano S, Mitochondrial UCP2 in the central regulation of metabolism, Best Practice & Research Clinical Endocrinology & Metabolism (2014), http://dx.doi.org/ 10.1016/j.beem.2014.02.006