Recent Advances in Nutritional Sciences Uncoupling Protein ...

Recent Advances in Nutritional Sciences Uncoupling Protein Homologs: Emerging Views of Physiological Function1

port chain drives outward proton pumping, thus forming a protonmotive force (⌬p) with concomitant O2 consumption; the ⌬p then drives proton flux inward through F1F0 ATP synthase during ATP formation (Fig. 1). Proton leak or “uncoupling” occurs when the proton flow arising from this well-coupled system is short-circuited by inward proton flow that is independent of F1F0 ATP synthase, resulting in a “drain” on ⌬p. In this case, fuel combustion, electron transport and O2 consumption increase in an effort to reestablish some steady-state ⌬p. An important role for proton leak in overall O2 consumption has been established; it is estimated that leak accounts for between ⬃20 and ⬃40% of the metabolic rate (2,3). The molecular underpinnings of bodywide mitochondrial proton leak are yet to be firmly established, and the assertion that UCPs regulate such leak has sparked a considerable number of studies over the last few years. Nevertheless, it is well accepted that the archetypal uncoupling protein UCP1 drives adaptational thermogenesis in the brown adipose tissue (BAT) of rodents through facilitation of proton leak. An overview of UCP1 biology and mechanism of action is beyond the scope of this review, and the reader may find more comprehensive information elsewhere (4 –7).

Sean H. Adams Department of Endocrinology, Genentech, Incorporated, South San Francisco, CA 94080

KEY WORDS: ● metabolic rate ● oxidative phosphorylation

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ABSTRACT The widespread occurrence of excess weight and related diseases demands that efforts be made to understand energy expenditure from the gene to the whole animal. For some time, it has been understood that mitochondrial oxidation of fuels generates an electrochemical gradient via outward pumping of protons by the electron transport chain. ATP production via F1F0 ATP synthase is then facilitated by the inward flux of protons down the gradient. There is a growing appreciation that a significant portion of the metabolic rate of endotherms is attributable to counteracting “proton leak” (uncoupling), wherein a flux of protons down the electrochemical gradient generates heat independently of ATP production. Proton leak is especially apparent in thermogenic brown adipose tissue, which expresses a tissue-specific uncoupling protein (UCP1). The recent discovery of widely expressed putative UCP1 homologs [UCP2, UCP3, UCP4, UCP5/brain mitochondrial carrier protein-1 (BMCP1)] raised the possibility that innate proton leak and metabolic rate are regulated by UCP1-like proteins. On the basis of current published data, one may not exclude the possibility that UCP homologs influence metabolic rate. J. Nutr. 130: 711–714, 2000.

Discovery of Four New Members of the UCP Family.

The demonstration of body-wide proton leak (2,3,8) despite confinement of UCP1 expression to BAT raised the possibility that additional UCP family members are involved in regulating mitochondrial proton flux in different tissues. Advancements in molecular biology and bioinformatics have allowed researchers to discover genes encoding proteins with varying degrees of sequence and domain homology to UCP1. To date, four UCP homologs have been characterized, with each protein displaying six transmembrane spans and three mitochondrial carrier domains. The first such gene emerged in 1997 with the characterization of UCP2 (9,10), whose amino acid sequence is 59% identical to that of UCP1 in humans. Discovery of UCP3 (57– 73% identical to UCP1 and UCP2, respectively in humans) soon followed (11–13). Homologs with abundant expression in brain have been described recently. UCP4 shares 29, 33 and 34% homology with UCP1, UCP2 and UCP3, respectively, in humans (14). UCP5, termed brain mitochondrial carrier protein-1 (BMCP1) in the report by Sanchis et al. (15) is a protein with 34, 38 and 39% identity to UCP1, UCP2, and UCP3, respectively (15,16). Remarkable strides in the identification of interesting mitochondrial carrier proteins based on sequence and domain homologies have been augmented by biochemical and physiologic studies further addressing function. The following sections shall attempt to clarify the current state of knowledge regarding the putative thermogenic function of published UCP homologs. Additional details and perspectives, including more in-depth aspects of UCP homolog gene regulation, may be gathered elsewhere (4,17). UCP2. Ectopic expression of UCP2 cDNA in mammalian cells in culture or in transformed yeast elicits a drop in ⌬p (9,10) and increased heat production (18), consistent with UCP2-induced uncoupling of mitochondrial respiration. Reconstitution of UCP2 protein in liposomes increases proton flux reminiscent of the effect of UCP1 (19). The widespread expression of UCP2 (9,10) raised the possibility that UCP2 underlies a portion of global proton leak. Expression in the immune system and rodent

proton leak

A cursory view of body weight control suggests simplicity, i.e., an imbalance of metabolic energy (energy absorbed and available for metabolism) compared with energy expenditure yields a loss or gain of body mass. Although true, this level of thinking belies the biological complexity of energy balance. Food intake and metabolic rate regulation involve interplay among many factors in humans, including social influences of behavior, appetite regulators generated centrally or peripherally, ambient and core body temperature signals affecting metabolic rate, autonomic regulation and activity level (1). This brief review emphasizes the energy expenditure portion of the energy balance equation, with a specific focus on recently described proteins, the uncoupling proteins or UCPs,2 hypothesized to drive a portion of mitochondrial respiration and hence metabolic rate. Which mitochondrial factors, if any, drive differences in cellular energy consumption? One candidate process to help explain flexibility in the metabolic rate is “proton leak.” As fuels are combusted in mitochondria, electron flow in the electron trans1

Manuscript received 17 January 2000. 2 Abbreviations: BAT, brown adipose tissue; BMCP1, brain mitochondrial carrier protein-1; ⌬p, protonmotive force; PPAR, peroxisome proliferator-activated receptor; UCP, uncoupling protein; WAT, white adipose tissue.

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BAT led to suggestions of a role in the immune response and adaptational thermogenesis, respectively (9,10,20). UCP2 is expressed in the brain, prompting the suggestion of its involvement in neuronal function or perhaps localized thermogenesis (21). Its genomic localization maps to human or mouse regions thought to contain genes involved with energy expenditure and hyperinsulinemia (9,22–24). Interestingly, ectopic expression of the newly described PPAR␥ co-activator 1 (PGC-1) triggers the UCP2 gene along with UCP1 and numerous genes known to be central to oxidative phosphorylation and mitochondrial replication (25). Thus, UCP2 is an interesting candidate for involvement with thermogenesis. However, expression data yield conflicting evidence for the role of UCP2 in situ. A number of findings are consistent with an uncoupling function of UCP2 in vivo. First, thyroid status affects metabolic rate and has been reported to correlate positively with mitochondrial proton leak in liver (see Ref. 26) and skeletal muscle (27). In rodents, UCP2 expression in a variety of tissues rises and falls in the hyperthyroid and hypothyroid states, respectively (28,29). Second, hepatocyte UCP2 expression is nominal in rodents (20,30), but is induced in the leptin-deficient ob/ob mouse (30) in which liver mitochondrial proton leak is clearly elevated (30,31). Interestingly, leptin administration to ob/ob mice normalized liver proton leak (31), but unfortunately leptin-induced changes in hepatocyte UCP2 expression were not presented. Third, UCP2

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FIGURE 1 Simplified overview of the mitochondrial proton (H⫹) circuit. Mitochondrial oxidation of fuels yields reducing equivalents (i.e., NADH and FADH2), which may then donate e- to the electron transport chain. Shown here is the flow of fatty acids into mitochondria via the carnitine palmitoyl-transferase/carnitine-acylcarnitine translocase apparatus (in lower box) and their combustion in the matrix to yield reducing equivalents. Flow of e- toward Complex IV, and ultimately to oxygen, occurs due to favorable differences in reduction potentials, providing sufficient energy to pump H⫹ outward. The resulting proton electrochemical gradient provides a protonmotive force (⌬p), which is the driving force behind ATP synthesis; energy is released as H⫹ flow down their electrochemical gradient through ATP synthase. The entire process is coupled in that fuel combustion, e- flow and outward H⫹ pumping increase to maintain a steady-state ⌬p when inward H⫹ flow rises concomitantly with ATP production. ⌬p may also be dissipated when the system is “short-circuited” by inward H⫹ flow independent of ATP synthase (“proton leak” or “uncoupling”). This event increases heat production as fuel oxidation and e- flow again increase in response to a dissipated ⌬p in an effort to maintain some new steady-state ⌬p. Such uncoupling may be facilitated by specific uncoupling proteins (UCPs).

expression in BAT rises in response to cold concurrently with UCP1 expression and BAT thermogenesis (32,33), and there are reports that a muscle group–specific and time-dependent increase of UCP2 mRNA occurs in rodent skeletal muscle with short-term cold exposure (32,34). Fourth, recovery of body temperature after endotoxin-mediated hypothermia in mice was preceded by upregulation of UCP2 expression in liver and skeletal muscle (unpublished data). Fifth, UCP2 expression in white adipose tissue (WAT) or BAT of obesity-resistant A/J mice was greater compared with that of obesity-prone C57BL6/J mice fed a high fat diet for ⬃2–3 wk (9,35; but also see Ref. 23). Despite the positive correlations just outlined, numerous data have emerged which raise the question whether UCP2 acts as an uncoupler in situ. For instance, skeletal muscle expression of UCP2 is generally reported to rise with fasting in rodents (32,36 –39) and is sometimes reported to increase in WAT or muscle of obese humans fed a hypocaloric diet (40,41), despite the decline in metabolic rate expected under such conditions. Furthermore, UCP2 transcript in WAT or liver is increased in obese ob/ob or db/db mice (10,30), although in this case the notion that UCP2-driven uncoupling is increased to counteract obesity (10) cannot be excluded. With respect to the brain, UCP2 expression in this organ was reported to remain unchanged despite metabolic challenges such as cold exposure (21). Finally, proton leak measured in isolated mitochondria often does not correlate with UCP2 gene expression. In UCP1 knockouts, for instance, GDPinsensitive proton leak kinetics in isolated BAT mitochondria are similar to those of controls (42) despite a marked upregulation of BAT UCP2 mRNA in the knockouts (43). Thyroid hormone increases hepatocyte proton leak (see Ref. 26) but may not alter liver UCP2 expression (28), whereas in endotoxin-treated mice, no alterations of liver or muscle proton leak were observed despite great fluctuations in UCP2 expression (unpublished data). Fasting elicits an increase in skeletal muscle UCP2 abundance with no alteration of proton leak in isolated mitochondria (36). UCP3. Transfection studies overexpressing UCP3 cDNA in mammalian cells (14,16,44) and construction of UCP3-transformed yeast (12,45,46) have established that under these conditions, UCP3 elicits a drop in ⌬p, consistent with an uncoupling activity. Introduction of purified UCP3 protein into artificial liposomes increased proton flux in one study (19) but not in another (47). It is notable that in humans, UCP3 transcripts exist in short (UCP3S, lacking the carboxy 37 amino acids and sixth transmembrane span) and long (UCP3L) forms (11) due to alternative splicing events (48). UCP3S retains the mitochondrial carrier motifs and the ability to lower ⌬p and increase cellular oxygen consumption, albeit not as strongly as UCP3L for the latter (45). The existence of both forms has unknown physiologic relevance. As reviewed by Chung et al. (49), mutations in the human UCP3 gene exist, leading to diminished UCP3L at the expense of UCP3S; either no metabolic effect or decreased fat oxidation with increased respiratory quotient was described. The UCP3 gene is located near UCP2, in a region linked to metabolic rate and hyperinsulinemia (12,23,24,48). Relatively abundant expression of UCP3 in skeletal muscle and rodent BAT (11–13,50) appears consistent with the characterization of UCP3 as a thermogenic protein. Indeed, UCP3 gene expression increases in skeletal muscle in response to thyroid hormone administration (12,27,51), a treatment that increases metabolic rate and proton leak (27; but also see Ref. 51). On balance, however, studies correlating UCP3 expression with metabolic status do not yield compelling evidence to confirm an important contribution of this homolog’s activity toward driving metabolic rate in vivo. First, skeletal muscle

EMERGING VIEWS OF UCP HOMOLOG FUNCTION

(16). First, after a 3-wk high fat dietary regimen, liver UCP5 expression in obesity-resistant A/J mice was significantly elevated compared with obesity-prone C57BL6/J mice. Second, cold exposure in mice sparked an induction of UCP5 mRNA in the liver and brain, potentially signaling increased thermogenesis in response to this challenge. Third, UCP5 expression in mouse liver was decreased significantly after a 24-h fast, but restored by refeeding. Finally, UCP5 expression in the skeletal muscle and liver of endotoxin-treated mice displayed a delayed (greater than threefold) induction, which preceded recovery from the hypothermia resulting from such treatment (unpublished data). Such relationships are not inconsistent with a hypothesized uncoupling function for UCP5. However, large fluctuations in hepatic UCP5 expression did not result in altered proton leak in isolated mitochondria after an endotoxin challenge (unpublished data), illustrating that additional work remains to further clarify UCP5 function in vivo.

Future Analysis of UCP Homolog Function and Regulation. Most analyses of putative UCP homologs rely on indirect indices of function, and challenges remain to optimize such assessments further. Such efforts may include development of satisfactory antibodies to define the correlation between UCP homolog mRNA and protein levels, exploration of post-translational modifiers of UCP activity [on the basis of the results suggesting that such factors exist (19,55)], and integrative analysis of the relationship among UCP homolog protein abundance, ⌬p, and other components that influence the proton circuit (ATP production, electron transport). On the basis of published information, one may not exclude the possibility that UCP homologs have thermogenic behavior, although additional metabolic roles have been postulated (37,38,56,57). Our understanding of UCP biology will benefit greatly from the following: 1) studies identifying possible cellular regulators of UCP homolog activity, 2) development of methods that facilitate whole-animal assessments of proton leak and mitochondrial function and 3) titration of UCP homolog abundance in vivo through transgenesis, gene therapy and knockout technologies. ACKNOWLEDGMENTS

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UCP3 expression is generally reported to rise significantly with fasting or food restriction (12,36 – 40,44,52,53), a period in which metabolic rate drops. The fasting-induced increase in muscle UCP3 expression is not matched with changes in proton leak in isolated mitochondria (36), and changes in rodent muscle UCP3 mRNA via alterations in dietary fat did not correlate with metabolic rate (54). Second, although it has been reported that muscle UCP3 mRNA rises with short-term (3-h) cold exposure in mice (34), there is no strong evidence of an increase with longer-term exposure in rodents (11,44). Third, UCP3 expression was not different among mouse strains displaying large differences in metabolic efficiency (35). Fourth, muscle UCP3 gene expression was stimulated acutely as hypothermia and depressed metabolic rate developed in endotoxin-treated mice (unpublished data). Fifth, stimulation of rodent BAT UCP3 expression by cold exposure remains an open question (33,44). UCP4. Experimental overexpression of UCP4 in mammalian cells results in lowered ⌬p, consistent with uncoupling under these conditions (14). The demonstration of its exclusive expression in the brain (14) points to intriguing possibilities with respect to physiologic function. It has been hypothesized (14) that UCP4 drives a portion of the innate proton leak observed in brain mitochondria (8) and may participate in localized thermogenesis. Supportive of this idea is our recent finding that acute cold exposure induces whole-brain UCP4 mRNA abundance (16). These cold-challenge studies using whole brain did not pinpoint the regions of the brain that displayed increased expression; thus, the possibility that cold exposure stimulated preferential expression in specific central nervous system areas is an interesting subject for additional study. The possibility that this mitochondrial carrier influences nervous system signaling or reactive oxygen species generation (14) is fertile ground for future experiments. Due to its negligible expression outside of the brain, the extent to which UCP4 activity influences whole-animal physiology remains to be clarified further. UCP5/BMCP1. Ectopic expression of UCP5 in mammalian cells or transformed yeast lowers ⌬p (15,16), and mitochondria derived from UCP5-transformed yeast displayed increased proton leak (15). These results indicate that under specific conditions, UCP5 may facilitate uncoupling. Initial results (15) indicated almost exclusive expression of UCP5 in human brain (hence the name “BMCP1”). Although UCP5 mRNA is particularly abundant in the brain (i.e., ⬎6- to ⬎40-fold higher vs. liver in the human and mouse, respectively; Ref. 16), widespread expression has been observed in rodents (15,16) and more recently in humans (16). At least two forms of UCP5 exist (short-form/UCP5S and long-form/ UCP5L; UCP5S lacks an amino acid insert Val-Ser-Gly beginning at position 23 of UCP5L)(15,16) and have potentially different biochemical potencies (16). It is interesting to consider that in the mouse, UCP5S is the sole isoform detected in all tissues except the brain and WAT (where UCP5L comprises only 2 and 0.1% of total UCP5 mRNA, respectively)(16). In contrast, humans express only the UCP5L isoform in the brain, with the UCP5S transcript comprising ⬃50 –90% of total UCP5 mRNA in other tissues. Unlike UCP2 (see above), much of whole-liver UCP5 expression takes place in hepatocytes of mice (unpublished data). The physiologic ramifications of these findings are not fully known. Body-wide expression of this homolog may point to a physiologically relevant role in contributing to global proton leak, and further studies linking expression of UCP5 to whole-animal metabolism are warranted. Nevertheless, interesting correlations of UCP5 expression and metabolic status have emerged from our initial studies, a number of which are consistent with such a role

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The author thanks M.D. Brand, G. Pan, T. Stewart, and X.X. Yu for thoughtful consideration of the manuscript and for spirited discussions of UCP biology.

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