Mitochondria couple respiration to ATP synthesis through an electrochemical proton gradient. Proton leak across the inner membrane allows adjustment of the coupling efficiency. The aim of this review is threefold: 1) introduce the unfamiliar reader to proton leak and its physiological significance, 2) review the role and regulation of uncoupling proteins, and 3) outline the prospects of proton leak as an avenue to treat obesity, diabetes, and age-related disease.
The Regulation and Physiology of Mitochondral Proton Leak
Peter Mitchell's chemiosmotic hypothesis revolutionized the study of biological energy transduction (143). Mitchell claimed that electron transfer and ATP synthesis were indirectly coupled by a transmembrane electrochemical proton gradient. His now-substantiated chemiosmotic theory provides the conceptual framework for not only oxidative phosphorylation by mitochondria but also photophosphorylation, active metabolite transport, and flagellal motion of bacteria.
During oxidative phosphorylation, the electrons stripped from oxidizable substrates (glucose, fatty acids, etc.) pass down a series of electron carriers in the respiratory chain to reduce molecular oxygen to water (180). The energy harvested from electron transfer drives the endergonic proton pumping activities of respiratory complexes I, III, and IV. Protons are vectorially pumped against their electrochemical gradient into the mitochondrial intermembrane space, creating a protonmotive force (Δp) across the inner membrane with both electrical (ΔΨm) and chemical (ΔpH) components. Dissipation of Δp through the FO/F1 ATP synthase drives ADP phosphorylation by rotary catalysis (161, 208).
Mitochondrial Proton Leak
Oxidative phosphorylation is incompletely coupled, since protons can leak across the inner membrane and relieve Δp independently of ATP synthase (FIGURE 1). Isolated mitochondria rapidly consume oxygen when given oxidizable substrates and ADP to allow ATP production (state 3 respiration), but slow respiration persists in the absence of ADP (state 4 respiration) and in the presence of the ATP synthase inhibitor, oligomycin (52).
State 4 respiration increases disproportionately as Δp rises, which can be entirely explained by proton leak across the mitochondrial inner membrane (154, 160). By analogy to a simple electrical circuit, the inner membrane can be thought of as a non-ohmic proton conductor: the oligomycin-insensitive respiration used to drive proton leak (current) increases nonlinearly with respect to Δp (electrical potential). Respiration is often used as an indirect measurement of leak rate, and proton fluxes can subsequently be calculated using appropriate stoichiometry.
Proton leak, as defined by the mitochondrial respiration rate in the presence of an ATP synthase inhibitor (oligomycin), is demonstrable in both mitochondria and intact cells (154, 160). Its kinetics can be quantified by simultaneously measuring respiration (54) and mitochondrial membrane potential (28) as both are titrated using respiratory inhibitors. A common, but overly simplistic, assumption is that state 4 respiration rate is a surrogate for the proton leakiness of the inner membrane. Rather, a proper quantification of proton conductance requires measurement of the respiration used to drive proton leak at a defined Δp (FIGURE 2).
Although proton leak is the predominant mechanism responsible for the incomplete coupling of substrate oxidation and ATP synthesis, other mechanisms are formally possible. For example, “slip” in the respiratory chain (electron transfer without concomitant proton pumping) can, in principle, cause this phenomenon. Most evidence, however, suggests its contribution is insignificant (33, 110, 148, 172).
The coupling efficiency of a cell can be defined as the proportion of mitochondrial respiratory rate used to drive ATP synthesis (e.g., perfectly coupled oxidative phosphorylation has a coupling efficiency of 100%, whereas pure state 4 respiration has one of 0%). It is usually approximated by the proportion of mitochondrial respiration sensitive to oligomycin. Oligomycin, however, will slightly increase proton leak rate by inhibiting phosphorylation and subsequently increasing Δp. Therefore, such estimates yield artificially low coupling efficiencies, but the error is generally <10% (6).
The coupling efficiency of hepatocytes in a broad range of species, both ectothermic and endothermic, is roughly 80% (30). Similar coupling efficiencies are observed in other cell types (5, 9, 43, 104), although a remarkable exception is the INS-1E insulinoma cell line, where up to 70% of respiration occurs without ATP production.
Proton leak is relatively high in perfused rat muscle. It comprises 35% (185) and 50% (184) of the respiration rate, respectively, for contracting or resting preparations. In vivo spectroscopic measurements of coupling efficiency in mouse skeletal muscle, however, suggest much tighter coupling (139), although both methods can be subjected to technical criticisms (30).
“Futile” proton cycling comes at a tremendous energetic cost: up to 20–25% of a rat's basal metabolic rate (BMR) may be attributable to proton leak (185). Its persistence throughout evolution, therefore, implies a conferred advantage significant enough to necessitate mitochondrial inefficiency (29).
What, then, is the physiological benefit of proton leak? Incompletely coupled oxidative phosphorylation permits adjustments in energy metabolism to regulate metabolic homeostasis and maintain body function. In specific cell types, demonstrable roles for proton leak include thermogenesis, maintaining carbon flux despite low ATP demand (213), and modulating the nutrient response in glucose-sensing cells.
A general theory to explain the ubiquity of proton leak, with regard to both cell type and phylogeny, is the “uncoupling to survive” hypothesis (29). Mitochondria produce superoxide as a by-product of oxidative metabolism and are a major source of intracellular reactive oxygen species (ROS) production (11, 27, 31, 149). ROS production and subsequent oxidative damage are thought to cause numerous degenerative disorders and may contribute to the aging process itself (20, 87). Since mitochondrial superoxide production is steeply dependent on Δp (116, 124, 132), proton leak pathways may therefore generally exist to minimize oxidative damage by tempering Δp and mitochondrial superoxide production.
Regulation and Physiological Role of Proton Leak
It is useful to classify proton leak into two categories: 1) constitutive, basal proton conductance and 2) regulated, inducible proton conductance catalyzed by uncoupling proteins (UCPs).
Basal Proton Conductance
Basal proton conductance in liver mitochondria strongly correlates with thyroid status (85) and phylogeny (34), and is inversely proportional to body mass (171). As previously mentioned, however, the broad changes in liver mitochondrial conductance and hepatocyte respiration across species (38, 171) are not manifested in altered coupling efficiencies because of matched changes in substrate oxidation and ADP phosphorylation (30).
The machinery responsible for basal proton leak is not fully understood. Although proton conductance correlates strongly with the fatty acyl composition of inner membrane phospholipids (38, 40, 74, 173), detergent-free liposome studies show that only a small percentage of constitutive leak is mediated by the lipid bilayer (41). A majority of basal proton conductance, however, is attributable to the abundance, but not activity, of the adenine nucleotide translocase (ANT) (36). This is not a property of all mitochondrial inner membrane proteins (209), but it may be characteristic of the mitochondrial solute carrier family.
Responsible for metabolite transport across the inner membrane, mitochondrial carriers share a tripartite structure: three repeats of ∼100 amino acids form six transmembrane helices with conserved salt-bridge networks on both faces of the inner membrane (168, 182, 225). Most are in such low abundance that any attributable basal proton conductance is likely negligible (186). Uncoupling protein 1 (UCP1), however, is found in brown adipose tissue mitochondria at comparable levels to ANT (210) and contributes to basal proton leak in this tissue (165), although this conclusion has been disputed (199).
ANT-mediated basal leak could be an evolved function: species with an increased capacity for oxidative phosphorylation, and thus a concomitantly raised ANT concentration, are also susceptible to increased free-radical production. It may therefore provide such species with a greater degree of protection from ROS via increased “mild uncoupling,” slightly lowering the protonmotive force to prevent ROS production without deleteriously lowering ATP synthesis (36). Although the phospholipid composition of the membrane itself does not appear to determine proton conductance, the observed correlations between phospholipid fatty acyl composition and basal proton conductance can easily be explained by separate correlations between each of them and basal metabolic rate (95).
Inducible Proton Conductance
UCPs catalyze proton conductance that is controlled on multiple levels: molecular, transcriptional, translational, and proteolytic (17). They are increasingly implicated in a variety of pathophysiological processes including obesity, Type 2 diabetes mellitus, the immune response, cancer, cardiovascular disease, and age-related disease caused by oxidative stress.
Studies into the bioenergetics and inner membrane composition of brown adipose tissue mitochondria led to the discovery of UCP1 (91, 131, 153, 155, 156, 175, 176), which dissipates Δp to generate heat in mammals during non-shivering thermogenesis (47).
Acute Regulation of UCP1
UCP1 is subject to strict, acute regulation: it is activated by fatty acids and inhibited by purine nucleoside di- and triphosphates (134). Precisely how this molecular-level regulation is executed remains contentious as, broadly, three competing models exist (FIGURE 3).
In the first, fatty acids act as obligatory cofactors, embedding their carboxyl side-chain into the protein to provide a critical proton-buffering site to transport protons across the inner membrane (113, 114, 227). A second model is informed by the halide-translocating activity of UCP1 (159) and the fatty acid anion transport activity of ANT (202). In this “flip-flop” model (39, 76, 77), protonated fatty acids cross the inner membrane and deprotonate in the mitochondrial matrix as governed by the pH gradient. The dissociated fatty acid anion is subsequently transported across the inner membrane by UCP1, driven by the membrane potential, and leading to a net relief of Δp without UCP1 explicitly translocating protons.
A third model contends that fatty acids are not required for proton transport but induce an allosteric change to overcome the persistent nucleotide inhibition of an inherently active UCP1. Simple competitive kinetics can describe the functional interaction between fatty acids and nucleotides in BAT mitochondria (198), but the kinetics of nucleotide binding are unchanged with fatty acids present (179, 227), suggesting that this functional competition is not achieved by simple competitive binding.
Much of the controversy stems from the use of different experimental systems. The co-factor and flip-flop models are grounded in evidence from UCP1 reconstituted into liposomes, a system that produces inconsistencies with results from isolated mitochondria with regard to crucial mechanistic questions. These include whether UCP1 activation requires ubiquinone (66, 71), whether the concentrations of fatty acids are at the level required to stimulate proton conductance (77, 82), and whether fatty acids can overcome nucleotide inhibition (106, 198). Even within liposome studies, stark differences exist regarding the specific activity of UCP1 (64, 77) and the dependence of catalyzed proton flux on membrane potential (105, 114). Proponents of all three activation models can rely on favorable evidence, and thus knowledge of how UCP1 is acutely regulated remains unresolved.
Regulation of UCP1 Concentration
The Ucp1 gene is under extensive transcriptional control (47, 55, 115, 200). Briefly, sympathetic innervation in BAT releases catecholamines, such as noradrenaline, in response to cold stimulus or overfeeding. These activate β3-adrenergic receptors, which trigger cyclic AMP (cAMP)-mediated pathways that affect UCP1 at both the molecular and the transcriptional level. cAMP-dependent protein kinase A (PKA) stimulates lipolysis to release fatty acids that acutely activate UCP1 (181), and a cAMP-responsive enhancer element upstream of the Ucp1 gene controls expression (50, 118). This region contains binding sites for transcription factors of the nuclear receptor family, including the peroxisome proliferator-activated receptor γ (PPARγ), thyroid hormone receptor (TR), and retinoic acid receptor (RXR). Both mRNA and protein levels of UCP1 are dramatically upregulated on cold-acclimation or noradrenaline treatment (26, 210).
Chronic noradrenergic stimulation also extends the half-life of UCP1 (144, 174), perhaps by inhibiting whole mitochondrial turnover (145). The adrenergic response that stimulates non-shivering thermogenesis is therefore an extraordinarily concerted process, regulating UCP1 at the levels of molecular activation, protein expression, and protein turnover.
Physiological Role of UCP1
Studies using Ucp1-null mice are mixed regarding whether UCP1 is obligatory for defense of body temperature (69, 141, 218), since thermal acclimation is a crucial parameter. White adipose tissue may be thermogenic under certain conditions (83, 218), although no compensatory UCP1-independent pathways can be recruited in the mouse for adaptive adrenergic non-shivering thermogenesis (80).
UCP1 may also possess additional physiological roles in controlling body weight and tempering oxidative damage. Early reports studying mice with ablated Ucp1 showed no obesogenic phenotype (69, 133), but UCP1 has a pronounced role in body weight maintenance in mice kept at thermoneutrality (72), although it has been argued that this function is indirect (117). Evidence using isolated BAT mitochondria is also consistent with a role for UCP1 in lowering ROS production (63, 163).
The previous idea that UCP1 co-evolved with BAT in placental mammals is likely incomplete. A UCP1 ortholog is present in marsupial adipose tissue and upregulated on cold exposure, indicating that an ancestral form of BAT likely predated the divergence of marsupials and eutherians (102). The Ucp1 gene is also present in amphibians and ectothermic fish (94, 102, 103). Despite an unlikely thermogenic role in these organisms, proton leak in carp liver mitochondria exhibits a regulatory response to fatty acids and nucleotides that is similar to UCP1 expressed in BAT (101), although this effect may be attributable to ANT.
Even within mammals the tissue distribution of UCP1 is not limited to BAT; UCP1 is functionally expressed in the rodent thymus (1, 49), where it may regulate T-cell selection and mitochondrial ROS production (2).
UCP2 and UCP3
Despite the growing body of evidence highlighting their importance in physiology and disease, an explicit description of the regulation and physiological function of UCP2 and UCP3 is lacking. UCP2 mRNA expression is ubiquitous in vertebrates, and the protein can be detected in spleen, kidney, thymus, pancreas, central nervous system, and macrophages (15, 73, 169). Expression of UCP3 is confined to skeletal muscle, brown adipose tissue, and, perhaps, heart (7, 24, 222). Plants (105, 123, 213, 220) and birds (177, 221) also express uncoupling protein homologs.
Acute Regulation of UCP2 and UCP3
It is uncertain to what extent UCP2 and UCP3 are regulated by fatty acids and nucleotides. They exhibit a fatty acid-inducible, nucleotide-sensitive proton flux when reconstituted into liposomes (65, 229), but evidence from isolated mitochondria is mixed regarding whether purine nucleotides are functional inhibitors of these UCPs (7, 136, 164, 178, 215). UCP2 and UCP3 may lack the amino acid sequence necessary to confer the fatty acid-sensitive proton conductance characteristic of UCP1 (86, 108).
UCP2 and UCP3 do not affect basal proton conductance (44, 57, 58). However, they catalyze an inducible uncoupling in the presence of specific activators including reactive alkenals and retinoic acid analogs (7, 119, 142, 164, 178). The induced UCP2 activity is probably physiologically relevant, given that its ablation in intact thymocytes decreases proton conductance (119) and its acute knockdown in INS-1E cells increases coupling efficiency (5).
Unfortunately, certain methods used to delineate UCP1 function may not be appropriate for studying other uncoupling proteins. UCP2 and UCP3 are expressed at levels of orders of magnitude less than UCP1 or ANT (89, 211). This, coupled with ANT's ability to catalyze inducible, nucleotide-sensitive proton conductance (112, 164), makes ascribing functions explicitly to UCP2 or UCP3 complicated in isolated mitochondria.
Furthermore, numerous studies demonstrate that UCP2 and UCP3 are incompetently folded when expressed in yeast mitochondria (89, 92, 211, 226), since observed uncoupling is unregulated and likely an artifact caused by the misfolded protein disrupting the integrity of the inner membrane. Even UCP1, which displays canonical regulatory properties when moderately expressed, can exhibit such behavior when expressed too strongly (210). This is also true for expression in mammalian cell systems (84), so artificial expression studies of UCPs (reviewed in Ref. 70) should be treated with caution, unless regulated proton leak is demonstrated.
Studying UCP2 and UCP3 in cells where they are natively expressed may therefore circumvent two problems that have stalled progress: 1) the need to add exogenous, specific activators to induce uncoupling in isolated mitochondria and 2) improper folding of ectopically expressed protein, and some progress has been made for UCP2 in this regard (5, 119).
Regulation of UCP2 and UCP3 Concentration
Like UCP1, UCP2 and UCP3 are subject to extensive transcriptional control. Many studies, reviewed in Ref. 51, have linked Ucp2 mRNA expression to the hyperglycemia and hyperlipidaemia associated with Type 2 diabetes mellitus. This regulation occurs via transcription factors including the sterol regulatory element-binding protein-1c (SREBP-1c) (162, 214), the PPAR family (167, 216), and forkhead transcription factors (162). Transcriptional regulation has also been linked to fatty acid oxidation (130), oxidative stress (78, 129), and Sirt1 protein (22).
Critically, UCP2 expression is also translationally regulated, so increases in mRNA are not necessarily indicative of increased protein concentration. An upstream open reading frame (ORF) in the 5′ untranslated region (5′ UTR) of Ucp2 mRNA affords translational control by glutamine, an amino acid implicated in the insulin secretion pathway (15, 97, 98).
Ucp3 mRNA is upregulated in response to nutrient deprivation (23, 44), serum free fatty acids (192), cytokines (42), retinoic acid (204), and thyroid hormones (125). Sirt1 represses Ucp3 mRNA expression (8). Transcriptional upregulation by fatty acids is mediated by PPARs and the myogenic regulatory factor MyoD (205), and thyroid hormone sensitivity is conferred by a thyroid response element (203). Avian Ucp mRNA responds to starvation and high-fat diet, which, coupled with phylogenetic inference and skeletal muscle expression, suggests the protein is a UCP3 ortholog (177, 221).
Like UCP1, UCP2 and UCP3 are also regulated by proteolysis. Unlike the archetypal UCP1 and other mitochondrial carriers, however, these UCPs are rapidly degraded with half-lives on the order of hours [∼1 h for UCP2 (15, 189), 1–4 h for UCP3 (18)]. In further contrast to the concerted regulation of UCP1, regulation of UCP2 in INS-1E cells is dynamic. Nutrients that increase UCP2 expression, such as glucose, do not change the protein's half-life (15).
Uncoupling by UCP1 is entirely reversible, as isolated BAT mitochondria recouple when free fatty acids are removed, mimicking a thermogenic cycle of fatty acids being transiently released then oxidized (135). Appropriate skepticism of the function of UCP2 and UCP3 has been raised given no comparable demonstration of reversible UCP2- and UCP3-mediated leak (157). Instead, rapid degradation of UCP2 and UCP3 may represent the physiological means by which their proton conductance is switched off following activation (17). Studies into the mechanism of degradation revealed that UCP2 and UCP3 are degraded by the cytosolic ubiquitin-proteasome machinery, the first convincing demonstration for a mitochondrial inner membrane protein (16, 18).
Physiological Role of UCP2 and UCP3
There is no widely accepted physiological role for UCP2 and UCP3, and many critiques of the proposed models are available (35, 48, 70, 107, 152, 187). It is well established, however, that they do not catalyze adaptive, non-shivering thermogenesis. Upregulation of Ucp2 and Ucp3 mRNA increases upon starvation (23, 44, 193), where thermogenesis is decreased, and both Ucp2- and Ucp3-null mice have a normal cold response (13, 81, 223). Rodent UCP3 may, however, be sufficiently thermogenic under appropriate pharmacological stimulation (142). Additionally, expression of UCP3 in BAT may be involved in the machinery required for thermogenesis (150), even if uncoupling by UCP3 itself is not sufficient to maintain body temperature (80).
Crucially, an insufficient role in thermogenesis and basal proton conductance does not imply that these UCPs do not uncouple. It should be stressed that, in isolated mitochondria, UCP2 and UCP3 do not catalyze proton leak in the absence of specific activators. Furthermore, the presence of an inducible proton conductance by UCP1 in ectotherms and plants, which do not participate in adaptive thermogenesis, suggests they may possess a shared ancestral function apart from heat production.
What might this function be? Given the strong correlation between conditions favoring fatty acid oxidation and Ucp3 mRNA expression, UCP3 might export fatty acid anions from the mitochondrial matrix, either to maintain fatty acid oxidation (93) or to protect against lipotoxicity (195). Experiments using mice with ablated Ucp3, however, show that UCP3 does not function physiologically as a fatty acid anion transporter (197).
Caution should therefore be exercised when inferring physiological function from transcriptional correlations without biochemical evidence. Likewise, the bioinformatic prediction that UCPs are keto-acid transporters (122) and the hypothesis that UCP2 functions physiologically as an anionic pyruvate uniporter (25) should be regarded as speculative pending experimental confirmation.
There is, however, abundant support for UCP2 and UCP3 protecting against oxidative damage, although such results may be dependent on genetic background. Studies with Ucp2-null mice indicate that, by attenuating ROS production, the protein confers cytoprotection (62, 68, 170) and can regulate atherosclerotic plaque formation (21, 146), the immune response (13, 188), and colon tumorigenesis (61). Additionally, UCP3 lowers ROS production in isolated skeletal muscle mitochondria (215), mitigates ROS-induced damage (37, 223), and allows for less harmful fatty acid oxidation (197).
Broadly, two non-exclusive models are consistent with the evidence that UCP2 and UCP3 protect against oxidative damage and uncouple when suitably activated. The first is a catalyzed mild uncoupling function, the fullest elaboration of which proposes that UCPs are activated by the reactive alkenals generated by superoxide-induced lipid peroxidation (32). UCPs therefore provide local feedback in response to ROS damage, lowering Δp to reduce the original, causative superoxide production. Notably, matrix superoxide production may be high during fatty acid oxidation (207) and can explain correlations between fatty acids and mRNA expression. This model has been corroborated in intact cells (121) but has not been reproduced in all laboratories (57, 201), nor is it free from criticism (157).
An alternative, but less substantiated, model suggests that UCP2 and UCP3 translocate lipid hydroperoxides out of the mitochondrial matrix (79). This prevents the formation of reactive oxidants that cause oxidative damage and allows them to reside more safely in the outer leaflet. It also accommodates a mild uncoupling function in a cycle analogous to the “flip-flop” model proposed for UCP1: translocation of fatty acid hydroperoxide anions followed by flip back of the protonated acids. This idea has some support from experiments with mitochondria, using Ucp3-null mice (136), and liposomes, where UCP2 can translocate lipid hydroperoxides (100) and all UCPs can transport a variety of anions.
Importantly, UCP2 may also modulate the glucose-sensing nutrient response in brain and pancreas (4, 51, 120), a function expanded on later. Briefly, UCP2 can negatively regulate glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells (230) and glucose-excitation in pro-opiomelanocortin (POMC) neurons (166). This is likely executed by UCP2-mediated uncoupling, which lowers the cytoplasmic ATP-to-ADP ratio to inhibit closure of downstream ATP-sensitive potassium (KATP) channels (5, 230). UCP2-catalyzed uncoupling may exert further regulation in glucose-sensing systems by attenuating ROS production (121), which is thought to be a required signal in nutrient-sensing (126, 127).
Proton Leak as a Therapeutic Target
Given their central role in regulating cellular energy transduction, UCPs may provide an attractive therapeutic target for diseases rooted in metabolic imbalance and oxidative stress.
Obesity ensues when energy intake chronically exceeds energy expenditure (90, 217). Most available pharmacological treatments reduce intake (by either appetite suppression via satiety centers or reduced intestinal absorption). Tremendous interest has been recently generated in targeting energy expenditure, however, by the rediscovery of active human brown adipose tissue that correlates inversely with body mass index (BMI) (59, 151, 191, 219, 224).
Although attempts to target β3-adrenergic receptors as obesity treatments have proven difficult (reviewed in Ref. 12), other treatments may hold considerable promise. These include increasing recruitment of brown adipocyte progenitors to induce BAT differentiation (111, 196) and increasing UCP1-mediated uncoupling, reiterating the need for an explicit description of UCP1's mechanism. Increasing uncoupling in skeletal muscle may also prove to be an effective obesity treatment, since overexpression of UCP3 in mice causes fat-specific weight loss (45, 53) and ectopic expression of UCP1 in skeletal muscle confers resistance to diet induced obesity (128).
Generic mitochondrial uncoupling, such as that achieved by the chemical protonophore 2,4-dinitrophenol (DNP), has proven effective as an anti-obesity agent, although the narrow margin between its therapeutic and toxic dosage is clinically prohibitive (56, 88, 217). The recent discoveries of chemical uncouplers with vastly improved pharmacodynamics (137) and machinery that maintains homeostatic ATP levels (183), however, may provide footholds from which nonspecific uncoupling can be revisited as an obesogenic treatment.
Type 2 Diabetes Mellitus
Uncoupling proteins may also provide avenues to treat the hallmark insulin resistance and pancreatic β-cell dysfunction associated with Type 2 diabetes (4, 51, 138). In pancreatic β-cells, abundant glucose triggers insulin secretion by a signaling cascade linked to the mitochondrial protonmotive force (FIGURE 4) (190). The high Δp generated by glucose oxidation results in a high ATP-to-ADP ratio, which closes KATP channels and depolarizes the plasma membrane. This leads to calcium influx via voltage-dependent calcium channels that signals the exocytosis of insulin granules. Uncoupling by UCP2 blunts insulin secretion, dropping the ATP-to-ADP ratio by lowering Δp.
The ablation of Ucp2 in mice results in improved insulin secretion and lower blood glucose levels than controls (230), but this effect is dependent on genetic background (170). Nonetheless, the acute knockdown of the protein in both cultured cells (5) and two diabetic animal models (60) provides examples where UCP2 attenuates GSIS.
Pancreatic β-cells are remarkably uncoupled (∼30% coupling efficiency) (5), and the high proton conductance strongly controls the ATP-to-ADP ratio and GSIS (3). A significant proportion of leak is attributable to UCP2, making it an obvious candidate for treating Type 2 diabetes.
Indeed, acute pharmacological inhibition of UCP2 may reverse the diabetogenic phenotype in pancreatic islets (231) and the obesity-induced disruption of glucose sensing in POMC neurons (166). The viability of inhibiting UCP2 in β-cells as an effective clinical therapy, however, has been questioned in light of its cytoprotective role in attenuating ROS production outlined earlier (170).
Additionally, increasing uncoupling in skeletal muscle would protect against insulin resistance (90). Inefficient oxidative phosphorylation would result in a lower plasma glucose concentration from an increased glucose oxidation rate, an idea that is experimentally supported by mice transgenically overexpressing UCP3 (53), and reduced lipotoxicity from an increased fatty acid oxidation rate.
Aging and Age-Related Disease
Given their role in attenuating superoxide production, UCPs may be clinically relevant to mitigating the ROS production thought to cause age-related disease and perhaps aging itself. Such a role for UCPs would be in line with the free radical theory of aging, which proposes that oxygen radicals are responsible for oxidative damage that causes aging (87). This has been subject to extensive experimental testing, and a clear verdict has yet to emerge (19, 20, 147, 194).
Mouse models are slowly appearing to allow the study of whether uncoupling can mediate extension of lifespan. Positive correlations between increased lifespan and proton conductance in skeletal muscle mitochondria (206), along with evidence for lifespan extension on dinitrophenol administration (46), lend credence to the “uncoupling to survive” hypothesis. Experiments to directly test the effects of uncoupling proteins on lifespan in both flies (75, 96) and mice (10, 14, 140), however, often produce confounding or conflicting results.
Mild uncoupling also short-circuits the chemiosmotic process, reducing the capacity of a mitochondrion to produce ATP, possibly causing irreparable damage outweighing potential antioxidant effects. This has been demonstrated in cultured rat cerebellar granule neurons, where a <10% drop in membrane potential insignificantly changes already low matrix superoxide levels but causes a pronounced decrease in the spare respiratory capacity thought to be important for neuronal survival (109, 158). Apart from this context, however, inducing mild uncoupling could prove effective in treating pathologies caused by high ROS production.
The need to better quantify the effect of mild uncoupling at the cellular level and thoroughly describe the physiological mechanism of UCP activation necessitates studying UCPs within the context of whole cells. As such, further progress in the field is likely contingent on developing improved methods for studying cellular bioenergetics and physiologically relevant ROS production.
Work in this laboratory is supported by grants from the National Institute on Aging (P01 AG-025901, PL1 AG-032118, and R01 AG-033542), The Ellison Medical Foundation (AG-SS-2288-09), and a British Marshall Scholarship and National Science Foundation Graduate Research Fellowship to A. S. Divakaruni.
No conflicts of interest, financial or otherwise, are declared by the author(s).
- ©2011 Int. Union Physiol. Sci./Am. Physiol. Soc.