Uncoupling protein-3 (UCP3) is a mitochondrial protein that can diminish the mitochondrial membrane potential. Levels of muscle Ucp3 mRNA are increased by thyroid hormone and fasting. Ucp3 has been proposed to influence metabolic efficiency and is a candidate obesity gene. We have produced a Ucp3 knockout mouse to test these hypotheses. The Ucp3 (؊/؊) mice had no detectable immunoreactive UCP3 by Western blotting. In mitochondria from the knockout mice, proton leak was greatly reduced in muscle, minimally reduced in brown fat, and not reduced at all in liver. These data suggest that UCP3 accounts for much of the proton leak in skeletal muscle. Despite the lack of UCP3, no consistent phenotypic abnormality was observed. The knockout mice were not obese and had normal serum insulin, triglyceride, and leptin levels, with a tendency toward reduced free fatty acids and glucose. Knockout mice showed a normal circadian rhythm in body temperature and motor activity and had normal body temperature responses to fasting, stress, thyroid hormone, and cold exposure. The base-line metabolic rate and respiratory exchange ratio were the same in knockout and control mice, as were the effects of fasting, a ␤3-adrenergic agonist (CL316243), and thyroid hormone on these parameters. The phenotype of Ucp1/Ucp3 double knockout mice was indistinguishable from Ucp1 single knockout mice. These data suggest that Ucp3 is not a major determinant of metabolic rate but, rather, has other functions.Human obesity is the result of energy intake greater than metabolic expenditure and is increasing in incidence (1). On an evolutionary time scale, obesity is a recent development, attributed to the interaction of predisposing genetic backgrounds with a sedentary lifestyle and an abundance of food (2, 3). Little is known about the molecular mechanisms and genes that contribute to the regulation of metabolic rate. For example, metabolic efficiency decreases with increased food intake, and it increases with lowered food intake (4), but the mechanistic details are unknown.The discovery of uncoupling protein (UCP, 1 now named UCP1) illustrated one way to regulate metabolic efficiency. UCP1 uncouples oxidative phosphorylation by allowing leakage of protons into the mitochondrial matrix without the phosphorylation of ADP (5). Heat is released because UCP1 degrades the proton gradient energy without storing it chemically or using it to perform physical work. At the whole-body level, this shows up as metabolic inefficiency. Ucp1 is expressed only in brown adipose tissue (BAT), which is a major heat-producing tissue in small mammals. In addition to cold-induced thermogenesis, BAT and UCP1 have been implicated in diet-induced thermogenesis, the increased energy expenditure that accompanies increased food intake (6). Activation of BAT and increased expression of Ucp1 cause reduced adiposity (7-9). However, BAT is present in only small amounts in large mammals, so its role in regulating energy homeostasis in adult humans is problematic (10).Interest in UCPs incr...

Weight loss in response to caloric restriction is variable. Because skeletal muscle mitochondrial proton leak may account for a large proportion of resting metabolic rate, we compared proton leak in diet-resistant and dietresponsive overweight women and compared the expression and gene characteristics of uncoupling protein (UCP)2 and UCP3. Of 1,129 overweight women who completed the University of Ottawa Weight Management Clinic program, 353 met compliance criteria and were free of medical conditions that could affect weight loss. Subjects were ranked according to percent body weight loss during the first 6 weeks of a 900-kcal meal replacement protocol. The highest and lowest quintiles of weight loss were defined as diet responsive and diet resistant, respectively. After body weight had been stable for at least 10 weeks, 12 of 70 subjects from each group consented to muscle biopsy and blood sampling for determinations of proton leak, UCP mRNA expression, and genetic studies. Despite similar baseline weight and age, weight loss was 43% greater, mitochondrial proton leak-dependent (state 4) respiration was 51% higher (P ‫؍‬ 0.0062), and expression of UCP3 mRNA abundance was 25% greater (P < 0.001) in diet-responsive than in diet-resistant subjects. There were no differences in UCP2 mRNA abundance. None of the known polymorphisms in UCP3 or its 5 flanking sequence were associated with weight loss or UCP3 mRNA abundance. Thus, proton leak and the expression of UCP3 correlate with weight loss success and may be candidates for pharmacological regulation of fat oxidation in obese diet-resistant subjects. Diabetes 51: 2459 -2466, 2002 A t the Weight Management Program at the University of Ottawa, we have documented a 10-fold variation in the rate of weight loss in 353 highly compliant women on a standard exercise program and standard 900-kcal meal replacement protocol. These women were ranked according to percent body weight loss, and highest and lowest quintiles were defined as diet responsive and diet resistant, respectively. Regression analyses demonstrated that the known variables regulating energy requirements, including initial weight, age, and plasma free triiodothyronine (T3) concentrations, accounted for only half of this variability (1), leading us to search for novel molecular determinants of weight loss success.Variable responses to overfeeding have been reported. Rodent studies have demonstrated that genetic factors not only regulate weight gain in response to high-fat highcalorie diets but also determine the susceptibility to obesity when energy intake is controlled (2). In response to the ingestion of hypercaloric diets, the majority of subjects gain less weight than anticipated, and a process of adaptive thermogenesis appears to play a role in the defense against obesity (3). Several studies have demonstrated marked interindividual variability in the susceptibility to weight gain in response to overfeeding (4), and identical twins show marked similarity in this regard, suggesting an important genetic cont...

Age-related changes in mitochondria, including decreased respiratory control ratios and altered mitochondrial inner membrane lipid composition, led us to study oxidative phosphorylation in hepatocytes from old (30 mo) and young (3 mo) male C57BL/J mice. Top-down metabolic control analysis and its extension, elasticity analysis, were used to identify changes in the control and regulation of the three blocks of reactions constituting the oxidative phosphorylation system: substrate oxidation, mitochondrial proton leak, and the ATP turnover reactions. Resting oxygen consumption of cells from old mice was 15% lower ( P < 0.05) than in young cells. This is explained entirely by a decrease in oxygen consumption supporting ATP turnover reactions. At all values of mitochondrial membrane potential assessed, the proportion of total oxygen consumption used to balance the leak was greater in the old cells than in the young cells. Metabolic control coefficients indicate a shift in control over respiration and phosphorylation away from substrate oxidation toward increased control by leak and by ATP turnover reactions. Control of the actual number of ATP molecules synthesized by mitochondria for each oxygen atom consumed by the ATP turnover and leak reactions was greater in old than in young cells, showing that efficiency in older cells is more sensitive to changes in these two blocks of reactions than in young cells.

Long-term caloric restriction (CR) retards aging processes and increases maximum life span. We investigated the influence of CR on mitochondrial proton leaks in rat skeletal muscle. Because CR lowers oxidative damage to mitochondrial membrane lipids and proteins, we hypothesized that leak would be lower in mitochondria from old CR rats than in age-matched controls. Three groups (n = 12) were studied: 4-month-old "young" control rats (body weight: 404 g +/- 7 SEM), 33-month-old CR rats (body weight: 262 g +/- 3), and 33-month-old control rats (body weight: 446 g +/- 5). CR rats received 67% of the energy intake of old control rats, with adequate intakes of all essential nutrients. Maximum leak-dependent O2 consumption (State 4) was 23% lower in CR rats than in age-matched controls, whereas protonmotive force values were similar, supporting our hypothesis. The overall kinetics of leak were similar between the two groups of old rats; in the young, kinetics indicated higher protonmotive force values. The latter indication is consistent with aging-induced alterations in proton leak kinetics that are independent of dietary intervention. There was no influence of age or diet on serum T4 level, whereas T3 was lower in young than in old control rats. These results support and extend the oxidative stress hypothesis of aging.

Mice deficient in mitochondrial uncoupling protein (UCP) 1 are cold sensitive, despite abundant expression of the homologues, Ucp2 and Ucp3 (S. Enerbäck, A. Jacobsson, E. M. Simpson, C. Guerra, H. Yamashita, M.-E. Harper, and L. P. Kozak. Nature 387: 90–94, 1997). We have analyzed characteristics of mitochondrial proton leak from brown adipose tissue (BAT) of Ucp1-deficient mice and normal controls and conducted the first top-down metabolic control analysis of oxidative phosphorylation in BAT mitochondria. Because purine nucleotides inhibit UCP1 and because UCP2 and the long form of UCP3 have putative purine nucleotide-binding regions, we predicted that proton leak in BAT mitochondria from Ucp1-deficient mice would be sensitive to GDP. On the contrary, although control over mitochondrial oxygen consumption and proton leak reactions at state 4 are strongly affected by 1 mM GDP in mitochondria from normal mice, there is no effect in UCP1-deficient mitochondria. In the presence of GDP, the overall kinetics of proton leak were not significantly different between Ucp1-deficient mice and controls. In its absence, state 4 respiration in normal BAT mitochondria was double that in its presence. Leak-dependent oxygen consumption was higher over a range of membrane potentials in its absence than in its presence. Thus proton leak, potentially including that through UCP2 and UCP3, is GDP insensitive. However, our measurements were made in the presence of albumin and may not allow for the detection of any fatty acid-induced UCP-mediated leak; this possibility requires investigation.