Clinical and Investigative Medicine

 

Obesity research continues to spring leaks

Mary-Ellen Harper, PhD

Clin Invest Med 1997;20(4):239-44.

[résumé]

Dr. Harper is Assistant Professor of Biochemistry in the Faculty of Medicine, University of Ottawa, Ottawa, Ont.

(Original manuscript submitted Dec. 6, 1995; received in revised form Feb. 6, 1997; accepted Feb. 10, 1997)

Reprint requests to: Dr. Mary-Ellen Harper, Department of Biochemistry, Faculty of Medicine, University of Ottawa, 451 Smyth Rd., Ottawa ON K1H 8M5; fax 613 562-5440; mharper@uottawa.ca

Contents

Abstract

Recent discoveries about the roles of 2 uncoupling proteins are changing the way we view obesity and its treatment. The author is also a coauthor of a recent Nature report that mice deficient in uncoupling protein 1 (UCP1) did not become fat, as anticipated, but lean. She found that the other uncoupling protein (UCP2) was up-regulated in the brown adipose tissue (BAT) of these mice, compensating, at least in part, for the lack of UCP1 and preventing obesity. Researchers have known for 40 years that the function of BAT is heat production. In 1978, researchers discovered UCP1, the protein responsible for this function. Subsequent investigation focused on the role of this protein in staving off obesity in animal models. In the early 1990s, surprising evidence from tissues other than BAT show that 20% to 40% of resting cellular energy expenditure is used to counter a proton leak down the electrochemical gradient across the mitochondrial inner membrane. This leak was found to be related to metabolic rate; the search for the mechanism of the leak led to the discovery of UCP2. Both uncoupling proteins have been found to act as leaks in mitochondrial inner membranes, allowing the dissipation of proton motive force. These findings could lead to new treatments for obesity and non-insulin-dependent diabetes mellitus.

Résumé

Des découvertes récentes sur le rôle de deux protéines découplantes modifient notre façon d'envisager l'obésité et son traitement. L'auteur est aussi coauteur d'un compte rendu publié récemment dans Nature, selon lequel des souris qui avaient un déficit des protéines découplantes 1 (PDC1) ne sont pas devenues grasses comme prévu, mais sont restées maigres. Elle a constaté que l'autre protéine découplante (PDC2) était régularisée à la hausse dans les tissus adipeux bruns (TAB) de ces souris, ce qui compensait, du moins en partie, le manque de PDC1 et prévenait l'obésité. Les chercheurs savent depuis 40 ans que les TAB servent à la production de la chaleur. En 1978, des chercheurs ont découvert la PDC1, protéine responsable de cette fonction. Des études subséquentes ont porté avant tout sur le rôle de cette protéine dans le blocage de l'obésité chez des modèles animaux. Des données probantes recueillies au début des années 1990 démontrent que dans les tissus autres que le TAB, de 20 % à 40 % des dépenses d'énergie cellulaire au repos servent à contrer une fuite de protons dans le gradient électrochimique à travers la membrane interne des mitochondries. On a découvert que cette fuite était liée au taux métabolique. La recherche du mécanisme de la fuite a débouché sur la découverte de la PDC2. Les deux protéines découplantes provoquent des fuites dans les membranes internes des mitochondries, ce qui permet de dissiper la force motrice des protons. Ces découvertes pourraient déboucher sur de nouveaux traitements contre l'obésité et le diabète sucré non insulino dépendant.

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Introduction

 A report in a recent issue of Nature described mice deficient in uncoupling protein 1 (UCP1), which were anticipated to become fat but turned out to be thin.1 This exciting finding, in conjunction with the recent discovery of a new uncoupling protein (UCP2),2 is forcing us to re-evaluate the role of UCP1 in obesity. These discoveries are contributing to current views on the mechanism and treatment of this serious scourge of modern societies. This review traces the evolution of these ideas, including important contributions from Canadian scientists whose work has been funded by the Medical Research Council of Canada (MRC) and the Natural Sciences and Engineering Research Council of Canada (NSERC).

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Brown adipose tissue and energy balance

The presence of UCP1 has long been used as the diagnostic criterion to identify brown adipose tissue (BAT). Although much of our understanding of the role of this protein has recently changed, this hallmark feature has not. UCP1 is found only in BAT, where it performs the function of that tissue, heat production. The thermogenic function of BAT was identified almost 40 years ago by 2 California researchers, Robert Smith and Raymond Hock.3 However, it wasn't until 20 years ago that the physiologic significance of BAT was demonstrated quantitatively by a Canadian researcher, David Foster, at the National Research Council of Canada. With the use of the radioactive microsphere method to assess blood flow as well as arteriovenous differences in oxygen tension to measure oxygen uptake of BAT, he showed that BAT accounts for 40% of the twofold increase in the resting metabolic rate of a rat infused with norepinephrine or exposed to cold.4,5 During the same year (1978), University of Ottawa researcher Jean Himms-Hagen showed that the binding of guanosine-5´-diphosphate (GDP) to BAT mitochondria could act as an index of the response of BAT to stimulation by cold or norepinephrine.6 That was indeed an enlightening year, for it was also in 1978 that Himms-Hagen's group and 2 others, David Nicholls's group in Scotland and Daniel Ricquier's group in France, demonstrated that the thermogenic component of BAT mitochondria is a 32-kDa protein,7­9 dubbed "uncoupling protein."

Until this point, interest in BAT thermogenesis was limited to researchers studying mechanisms of adaptation to cold environments and hibernation. The role of BAT in obesity was not an issue until later in 1978 and during 1979, when 2 published reports showed that BAT metabolism played a role in obesity development. Because it was known that the genetically obese (ob/ob) mouse became hypothermic in cold environments, Himms-Hagen and doctoral student Michel Desautels decided to study BAT metabolism in these mice. They found that the obese mice had a defect in the mechanisms necessary for the activation of BAT thermogenesis and hypothesized that this defect might be the basis for obesity in the ob/ob mouse.10 (We now know that the specific genetic defect in the ob/ob mouse is the absence of the satiety factor, leptin, and that defects in BAT thermogenesis, although important in obesity development, are secondary.) This research was funded by the MRC, and the data supporting the idea that obesity was due to malfunctions in BAT were first reviewed by Himms-Hagen in an article published in the Canadian Medical Association Journal in 1979.11 The second key finding concerning the importance of BAT in obesity came from Nancy Rothwell and Michael Stock, 2 researchers in London, England. In an article published in Nature in 1979, they showed that diet-induced thermogenesis in BAT, like cold-induced nonshivering thermogenesis in BAT, was to some degree responsible for staving off obesity, which would otherwise develop as a result of overeating.12

During the ensuing 14 years, defective BAT thermogenesis was identified in many animal models of obesity, including genetic models such as the ob/ob mouse, the db/db mouse, the fa/fa rat and the cp/cp rat, as well as hypothalamic models of obesity such as the hypothalamic-lesioned rat and the gold-thioglucose obese mouse. However, 2 problems persisted doggedly. First, in all models studied, the animals were hyperphagic, and it was not possible to separate completely the effect of overeating from the proposed hypometabolism of BAT. The second and markedly more difficult issue was that it was impossible to quantify the deficit in energy expenditure that results from lower-than-normal amounts of BAT. Measuring this deficit was necessary to prove that BAT thermogenesis contributed significantly to energy balance in obesity.

This second problem was rectified to some extent in 1993 with the creation, by Brad Lowell and Jeff Flier (both at Harvard University) as well as Himms-Hagen and others, of a transgenic mouse with gene-targeted ablation of BAT.13 The method resulted in the destruction of the cells expressing UCP1 and involved "hooking up" the expression of diptheria toxin A chain to the promoter region of the UCP1 gene. However, the ablation achieved was not complete: about 30% of the normal amount of uncoupling protein in BAT remained. Nevertheless, at a young age the mice became obese, then they became hyperphagic, and yet later they developed non-insulin-dependent diabetes mellitus. These findings clearly showed the importance of BAT in energy balance and obesity.

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Mitochondrial proton leaks in other tissues in the body

In the early 1990s rapidly accumulating evidence supported the idea that a substantial amount of energy expended by all cells in the body, not just cells in BAT, is used to counter a leak of protons down the electrochemical gradient across the mitochondrial inner membrane. The amount of energy used to balance this leak was shown to be in the order of 20% to 40% of a cell's resting energy expenditure.14­16 That this proportion of resting energy metabolism was being "wasted" was unbelievable to some, who argued, among other things, that evolution would not allow such a wasteful process to persist.

The researcher who has championed the quantitative importance of the mitochondrial proton leak is Martin Brand of the University of Cambridge. In 1986 he and Guy Brown, then a doctoral student in the same Cambridge laboratory, began to scrutinize the relationship between the permeability of the mitochondrial inner membrane and mitochondrial proton motive force.17 Interestingly, they were following up the findings of David Nicholls, who later played an important role in the elucidation of thermogenic mechanisms in BAT. In 1974 Nicholls had published an article in which he identified a nonproportional relationship between mitochondrial respiration and proton motive force.18 In 1990 Brand published a review article in which he first advanced the hypothesis that the mitochondrial proton leak contributes significantly to metabolic rate.19 Previous results from his laboratory showed that thyroid hormones, the most important endocrine regulators of metabolic rate, significantly increased the leak in rat liver mitochondria.20

While doing postdoctoral research (funded by the NSERC) in Brand's laboratory, I studied the effects of hypo- and hyperthyroidism on the mitochondrial proton leak in intact liver cells and in muscle mitochondria. My colleagues and I confirmed the quantitative importance of the leak in intact cells (rather than in isolated mitochondria, since the leaks in mitochondria could be artifactual). We showed that the leak decreased during hypothyroidism and increased during hyperthyroidism in rats.15,21,22 Roughly half of the increase in cellular energy demand in hyperthyroidism was found to be due to increased leakage; the remainder was due to increases in cytoplasmic adenosine triphosphate (ATP) demands (e.g., sodium-potassium adenosine triphosphatase and calcium adenosine triphosphatase). These findings supported the hypothesis that the proton leak was related to metabolic rate. Brand and Richard Porter, also a postdoctoral researcher in the laboratory, then published a series of articles showing that proton leak decreases with increasing body mass in animals. They hypothesized that the differences in proton leak may explain in part the known differences in the standard metabolic rate among mammals of different mass.16,23

Many questions regarding the proton leak remained and certainly still remain. At the top of the list of questions was the leak's mechanism. Initial hypotheses involved the proportional relationship between leak and the total surface area of the mitochondrial inner membrane, which pointed toward the fatty-acid composition of the membrane. Striking correlations between proton permeability and fatty-acid composition have been observed. However, pursuit of the latter possibility by examination of liposomes made from phospholipids of mitochondrial inner membranes has led to the very recent finding that the phospholipid leak could account for only about 5% of the proton leak flux under comparable conditions in isolated mitochondria.24 Thus, the leak pathway through the bulk phospholipid bilayer represents only a small proportion of mitochondrial proton leak. These findings led various researchers to hypothesize that the mechanism involved the action of a protein.

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Discovery of a new leak protein, UCP2

Within months of the publication of the latter finding came a report, in Nature Genetics, of the discovery of a novel uncoupling protein, UCP2.2 Here, perhaps, was the answer to the question of the leak's mechanism. Considering the predicted importance of this protein in basal energy metabolism and the similarity of this protein to the original uncoupling protein, it is surprising that it has only now been identified.

Fleury and associates2 have shown that both UCP1 and UCP2 reside in the mitochondrial inner membrane in cells. Whereas UCP1 is expressed only in BAT, UCP2 is apparently expressed ubiquitously throughout tissues of the body. The highest levels of expression were found in white adipose tissue, BAT, heart, kidney, spleen, thymus, macrophage, bone marrow and stomach. UCP2 is 56% identical to UCP1 at the amino acid level. Both proteins can act as leaks in the membrane -- as "metabolic waste valves" if you like -- to allow the dissipation of proton motive force. Proton motive force is used mainly to fuel the synthesis of ATP by ATP synthase; however, in the presence of an active uncoupling protein, the gradient is dissipated as protons leak through this protein. It is readily apparent how such a leak translates to a loss of a potential source of fuel.

The fact that UCP2 is expressed in a wide range of tissues is consistent with the idea that it may play a very important role in establishing basal metabolic rate. Moreover, Fleury and associates also report that UCP2 messenger RNA levels in white adipose tissue were not increased by cold exposure (which induces UCP1 expression dramatically in BAT) but were increased by eating a high-fat diet. UCP mRNA levels were also higher in a strain of mouse that is susceptible to obesity development and lower in another strain that is resistant to obesity development. This indicates, first, that the expression of UCP2 is sensitive to dietary factors and, second, that the 2 uncoupling proteins are regulated quite distinctly in the body.

In BAT the function of the UCP1-mediated leak is clearly heat production. The specific function of UCP2 is less clear. Again, why should such a wasteful process in such a wide variety of tissue types be retained throughout evolution? Perhaps the function of UCP2 is to maintain an idling rate of oxygen consumption by tissues so that when there is a rapid demand for ATP during, for example, a "flight-or-fight" situation, the immediate increased requirement for oxygen is minimized. We know that when cells are challenged to produce ATP efficiently, the oxygen used to support proton leak is essentially shut off.15 This amount of idling oxygen consumption can then be used to support ATP synthesis.

Quantitative answers regarding the importance of the protein in cellular and whole body energy expenditure are not yet available; they await studies in human populations in which decreased expression of UCP2, or expression of mutated forms of UCP2, is associated with obesity. Further advances will certainly be gleaned from various studies involving transgenic and knockout mice with decreased expression of UCP2 or mutations in UCP2.

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A revolutionary finding: UCP1 knockout mice are lean

Given the extensive findings supporting the importance of BAT metabolism in the development of obesity in experimental animals, and given the obese phenotype seen in the mouse with transgenic ablation of BAT, we aimed to create and study a transgenic mouse in which UCP1 was completely absent. This work, published in Nature,1 was directed by Leslie Kozak and his colleagues at the Jackson Laboratory in Bar Harbor, Maine. The contributions from my laboratory, funded by the NSERC, focused on aspects of metabolic physiology, including analyses of resting metabolic rates. We expected that the mice with targeted inactivation of UCP1 would become obese and diabetic at a young age. It came as a great surprise to us that these mice had a lean phenotype, even when fed a high-fat diet (58% of energy as fat), and were not hyperphagic. The mice' only abnormal phenotypic characteristic was that they were extremely sensitive to cold. We also found that, although their resting metabolism (resting oxygen consumption per mouse) was normal, their response to the ß3-adrenergic agonist CL 316,243 was significantly blunted. This finding confirmed that there was a defect in thermoregulation.

Because we suspected that compensatory thermogenic mechanisms were protecting these mice from the development of obesity, and because we were interested in mitochondrial proton leaks in other tissues, we searched for increases in UCP2 expression. We examined various tissues and found that the only tissue showing a significant up-regulation of UCP2 mRNA was BAT, which had a fivefold up-regulation. This up-regulation is probably the essential compensatory mechanism that allows these mice to remain lean.

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Brown fat metabolism is of fundamental importance in energy balance and obesity

The finding that UCP2 mRNA levels were up-regulated only in BAT of UCP1 knockout mice points to the importance of BAT in energy balance and obesity development. This finding, in tandem with the previous finding that mice with transgenic ablation of intact cells in BAT are obese,13 conclusively shows that BAT metabolism is fundamentally important in energy balance and obesity. In the mice with transgenic ablation of cells in BAT, entire cells that presumably express both UCP1 and UCP2 are destroyed; hence, the mice become obese. Other compensatory mechanisms, if any, are insufficient.

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Hope for human obesity?

Most of the findings I have described here resulted from many years of research on rodent models of obesity. In humans, the amount of BAT in adults is known to be quite small. However, it is widely accepted that BAT thermogenesis plays an essential role in thermoregulation immediately after birth. Moreover, BAT thermogenesis is hypothesized to be important in controlling feeding in newborns.25

Recent research suggests that there may soon be interventions to "awaken" BAT thermogenesis in adults through the use of the ß3-adrenergic agonists. One of the reasons why ß3-adrenergic agonists hold such promise is that their receptors are found exclusively in the target tissues, white and brown adipose tissues. It has been argued that receptors are also found in the gastrointestinal tract and perhaps in the heart. However, recent results of agonist treatment in ß3-receptor knockout mice, in knockout mice with targeted re-expression only in white and brown adipose tissues and in controls militate against any significant physiologic role of the receptors in these tissues.26,27 This research has also been supported in part by funding from the MRC and NSERC.

Many recent reviews have described the potential of ß3-adrenergic agonists. These agents have been shown to be remarkably effective in treating rodents having various types of obesity and non-insulin-dependent diabetes mellitus, which often accompanies chronic obesity.28­30 In view of the limited scope of this review, I will not elaborate on these fascinating recent findings.

Given that UCP2 is expressed widely in tissues and given the data suggesting that the resulting energy expenditure is very significant, the many studies to come will advance our understanding of the control and regulation of basal energy expenditure in humans. Aberrations in these processes will likely be correlated with obesity development, and efforts to stem the tide of obesity development and its clinical sequelae will likely take a new direction.

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Postscript

Just before the publication of this review, 2 independent groups reported the cloning of a third uncoupling protein. Olivier Boss and colleagues31 in Geneva screened a human skeletal muscle cDNA library and isolated 3 clones, UCP2 and long and short forms of a new uncoupling protein, UCP3L and UCP3S, respectively. UCP3 is 57% and 73% identical to human UCP1 and UCP2, respectively; it is specific to skeletal muscle; and, in rats, its expression is unaffected by cold acclimation. The other research group was that of Antonio Vidal-Puig and colleagues in Brad Lowell's laboratory at Harvard University. They have also cloned UCP3, and their results show abundant and preferential expression in skeletal muscle in humans, and in BAT and skeletal muscle in rodents.32 It is quite likely that there are other, as yet unidentified, uncoupling protein homologues, given their seemingly fundamental importance in governing idling energy expenditure. The pace of research in this area is certainly fast, and rapid and extensive advances in our understanding of the efficiency of tissue respiration and its control can be expected.

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References
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  2. Fleury C, Neverova M, Collins S, Raimbault S, Champigny O, Levi-Meyrueis C, et al. Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nature Genet 1997;15:269-72.
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