Effects of the presence, absence, and overexpression of uncoupling protein-3 on adiposity and fuel metabolism in congenic mice

Uncoupling protein-3 (UCP3) is a poorly understood mitochondrial inner membrane protein expressed predominantly in skeletal muscle. The aim of this study was to examine the effects of the absence or constitutive physiological overexpression of UCP3 on whole body energy metabolism, glucose tolerance, and muscle triglyceride content. Congenic male UCP3 knockout mice (Ucp3-/-), wild-type, and transgenic UCP3 overexpressing (UCP3Tg) mice were fed a 10% fat diet for 4 or 8 mo after they were weaned. UCP3Tg mice had lower body weights and were less metabolically efficient than wild-type or Ucp3-/- mice, but they were not hyperphagic. UCP3Tg mice had smaller epididymal white adipose tissue and brown adipose tissue (BAT) depots; however, there were no differences in muscle weights. Glucose and insulin tolerance tests revealed that both UCP3Tg and Ucp3-/- mice were protected from development of impaired glucose tolerance and were more sensitive to insulin. 2-Deoxy-D-[1-3H]glucose tracer studies showed increased uptake of glucose into BAT and increased storage of liver glycogen in Ucp3-/- mice. Assessments of intramuscular triglyceride (IMTG) revealed decreases in quadriceps of UCP3Tg mice compared with wild-type and Ucp3-/- mice. When challenged with a 45% fat diet, Ucp3-/- mice showed increased accumulation of IMTG compared with wild-type mice, which in turn had greater IMTG than UCP3Tg mice. Results are consistent with a role for UCP3 in preventing accumulation of triglyceride in both adipose tissue and muscle.     

Effects of fasting on muscle mitochondrial energetics and fatty acid metabolism in Ucp3(-/-) and wild-type mice

Uncoupling protein-3 (UCP3) is a mitochondrial carrier protein of as yet undefined physiological function. To elucidate characteristics of its function, we studied the effects of fasting on resting metabolic rate, respiratory quotient, muscle Ucp3 expression, and mitochondrial proton leak in wild-type and Ucp3(-/-) mice. Also analyzed were the fatty acid compositions of skeletal muscle mitochondria in fed and fasted Ucp3(-/-) and wild-type mice. In wild-type mice, fasting caused significant increases in Ucp3 (4-fold) and Ucp2 (2-fold) mRNA but did not significantly affect mitochondrial proton leak. State 4 oxygen consumption was not affected by fasting in either of the two groups. However, protonmotive force was consistently higher in mitochondria of Ucp3(-/-) animals (P = 0.03), and fasting further augmented protonmotive force in Ucp3(-/-) mice; there was no effect in wild-type mitochondria. Resting metabolic rates decreased with fasting in both groups. Ucp3(-/-) mice had higher respiratory quotients than wild-type mice in fed resting states, indicating impaired fatty acid oxidation. Altogether, results show that the fasting-induced increases in Ucp2 and Ucp3 do not correlate with increased mitochondrial proton leak but support a role for UCP3 in fatty acid metabolism.     

UCP1 deficiency increases susceptibility to diet-induced obesity with age

Loss of nonshivering thermogenesis in mice by inactivation of the mitochondrial uncoupling protein gene (Ucp1-/- mice) causes increased sensitivity to cold and unexpected resistance to diet-induced obesity at a young age. To clarify the role of UCP1 in body weight regulation throughout life and influence of UCP1 deficiency on longevity, we longitudinally analyzed the phenotypes of Ucp1-/- mice maintained in a room at 23 degrees C. There was no difference in body weight and lifespan between genotypes under the standard chow diet condition, whereas the mutant mice developed obesity with age under the high-fat (HF) diet condition. Compared with Ucp1+/+ mice, Ucp1-/- mice showed increased expression of genes related to thermogenesis and fatty acid metabolism, such as beta3-adrenergic receptor, in adipose tissues of the 3-month-old mutants; however, the augmented expression was reduced in Ucp1+/+ mice in 11-month-old Ucp1-/- mice fed the HF diet. Likewise, the increased levels of UCP3 and cAMP-dependent protein kinase in the brown adipose tissue of Ucp1-/- mice given the standard diet were decreased significantly in that of Ucp1-/- mice fed the HF diet, which animals showed impaired norepinephrine-induced lipolysis in their adipose tissues. These results suggest profound attenuation of beta-adrenergic responsiveness and fatty acid utilization in Ucp1-/- mice fed the HF diet, bringing them to late-onset obesity. Our findings provide evidence that UCP1 is neither essential for body weight regulation nor for longevity under conditions of standard diet and normal housing temperature, but deficiency increases susceptibility to obesity with age in combination with HF diet.     

The bioenergetics of brown fat mitochondria from UCP1-ablated mice. Ucp1 is not involved in fatty acid-induced de-energization ("uncoupling").

The bioenergetics of brown fat mitochondria isolated from UCP1-ablated mice were investigated. The mitochondria had lost the high GDP-binding capacity normally found in brown fat mitochondria, and they were innately in an energized state, in contrast to wild-type mitochondria. GDP, which led to energization of wild-type mitochondria, was without effect on the brown fat mitochondria from UCP1-ablated mice. The absence of thermogenic function did not result in reintroduction of high ATP synthase activity. Remarkably and unexpectedly, the mitochondria from UCP1-ablated mice were as sensitive to the de-energizing ("uncoupling") effect of free fatty acids as were UCP1-containing mitochondria. Therefore, the de-energizing effect of free fatty acids does not appear to be mediated via UCP1, and free fatty acids would not seem to be the intracellular physiological activator involved in mediation of the thermogenic signal from the adrenergic receptor to UCP1. In the UCP1-ablated mice, Ucp2 mRNA levels in brown adipose tissue were 14-fold higher and Ucp3 mRNA levels were marginally lower than in wild-type. The Ucp2 and Ucp3 mRNA levels were therefore among the highest found in any tissue. These high mRNA levels did not confer on the isolated mitochondria any properties associated with de-energization. Thus, the mere observation of a high level of Ucp2 or Ucp3 mRNA in a tissue cannot be taken as an indication that mitochondria isolated from that tissue will display innate de-energization or thermogenesis.     

Lack of obesity and normal response to fasting and thyroid hormone in mice lacking uncoupling protein-3

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 beta3-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.     

Thermogenic responses in brown fat cells are fully UCP1-dependent. UCP2 or UCP3 do not substitute for UCP1 in adrenergically or fatty scid-induced thermogenesis

To examine the thermogenic significance of the classical uncoupling protein-1 (UCP1), the thermogenic potential of brown adipocytes isolated from UCP1-ablated mice was investigated. Ucp1(-/-) cells had a basal metabolic rate identical to wild-type; the mitochondria within them were coupled to the same degree. The response to norepinephrine in wild-type cells was robust ( approximately 10-fold increase in thermogenesis); Ucp1(-/-) cells only responded approximately 3% of this. Ucp1(-/-) cells were as potent as wild-type in norepinephrine-induced cAMP accumulation and lipolysis and had a similar mitochondrial respiratory complement. In wild-type cells, fatty acids induced a thermogenic response similar to norepinephrine, but fatty acids (and retinoate) were practically without effect in Ucp1(-/-) cells. It is concluded that no other adrenergically induced thermogenic mechanism exists in brown adipocytes except that mediated by UCP1 and that entopic expression of UCP1 does not lead to overt innate uncoupling, and it is suggested that fatty acids are transformed to an intracellular physiological activator of UCP1. High expression of UCP2 and UCP3 in the tissue was not associated with an overt innate highly uncoupled state of mitochondria within the cells, nor with an ability of norepinephrine or endo- or exogenous fatty acids to induce uncoupled respiration in the cells. Thus, UCP1 remains the only physiologically potent thermogenic uncoupling protein in these cells.     

GDP and carboxyatractylate inhibit 4-hydroxynonenal-activated proton conductance to differing degrees in mitochondria from skeletal muscle and heart

The lipid peroxidation product 4-hydroxynonenal (HNE) increases the proton conductance of the inner mitochondrial membrane through effects on uncoupling proteins (UCPs) and the adenine nucleotide translocase (ANT); however, the relative contribution of the two carriers to these effects is unclear. To clarify this we isolated mitochondria from skeletal muscle and heart of wild-type and Ucp3 knockout (Ucp3KO) mice. To increase UCP3 expression, some mice were i.p. injected with LPS (12 mg/kg body weight). In spite of the increased UCP3 expression levels, basal proton conductance did not change. HNE increased the proton conductance of skeletal muscle and heart mitochondria. In skeletal muscle, this increase was lower in Ucp3KO mice and higher in LPS-treated wild-type mice, and was partially abolished by GDP (UCPs inhibitor) and completely abolished by carboxyatractylate (ANT inhibitor) or addition of both inhibitors. GDP had no effect on HNE-induced conductance in heart mitochondria, but carboxyatractylate or administration of both inhibitors had a partial effect. GDP-mediated inhibition of HNE-activated proton conductance in skeletal muscle mitochondria was not observed in Ucp3KO mice, indicating that GDP is specific for UCP3, at least in muscle. Carboxyatractylate was able to inhibit UCP3, probably through an indirect mechanism. Our results are consistent with the conclusion that, in skeletal muscle, HNE-induced increase in proton conductance is mediated by UCP3 (30%) and ANT, whereas in the heart the increase is mediated by ANT and other carriers, possibly including UCP3.     

The role of uncoupling protein 2 in macrophages and its impact on obesity-induced adipose tissue inflammation and insulin resistance

The development of a chronic, low-grade inflammation originating from adipose tissue in obese subjects is widely recognized to induce insulin resistance, leading to the development of type 2 diabetes. The adipose tissue microenvironment drives specific metabolic reprogramming of adipose tissue macrophages, contributing to the induction of tissue inflammation. Uncoupling protein 2 (UCP2), a mitochondrial anion carrier, is thought to separately modulate inflammatory and metabolic processes in macrophages and is up-regulated in macrophages in the context of obesity and diabetes. Here, we investigate the role of UCP2 in macrophage activation in the context of obesity-induced adipose tissue inflammation and insulin resistance. Using a myeloid-specific knockout of UCP2 (Ucp2^ΔLysM), we found that UCP2 deficiency significantly increases glycolysis and oxidative respiration, both unstimulated and after inflammatory conditions. Strikingly, fatty acid loading abolished the metabolic differences between Ucp2^ΔLysM macrophages and their floxed controls. Furthermore, Ucp2^ΔLysM macrophages show attenuated pro-inflammatory responses toward Toll-like receptor-2 and -4 stimulation. To test the relevance of macrophage-specific Ucp2 deletion in vivo, Ucp2^ΔLysM and Ucp2^fl/fl mice were rendered obese and insulin resistant through high-fat feeding. Although no differences in adipose tissue inflammation or insulin resistance was found between the two genotypes, adipose tissue macrophages isolated from diet-induced obese Ucp2^ΔLysM mice showed decreased TNFα secretion after ex vivo lipopolysaccharide stimulation compared with their Ucp2^fl/fl littermates. Together, these results demonstrate that although UCP2 regulates both metabolism and the inflammatory response of macrophages, its activity is not crucial in shaping macrophage activation in the adipose tissue during obesity-induced insulin resistance.     

Fatty acid transport protein 4 is dispensable for intestinal lipid absorption in mice

FA transport protein 4 (FATP4), one member of a multigene family of FA transporters, was proposed as a major FA transporter in intestinal lipid absorption. Due to the fact that Fatp4(-/-) mice die because of a perinatal skin defect, we rescued the skin phenotype using an FATP4 transgene driven by a keratinocyte-specific promoter (Fatp4(-/-);Ivl-Fatp4(tg/+) mice) to elucidate the role of intestinal FATP4 in dietary lipid absorption. Fatp4(-/-);Ivl-Fatp4(tg/+) mice and wild-type littermates displayed indistinguishable food consumption, growth, and weight gain on either low or high fat (Western) diets, with no differences in intestinal triglyceride (TG) absorption or fecal fat losses. Cholesterol absorption and intestinal TG absorption kinetics were indistinguishable between the genotypes, although Western diet fed Fatp4(-/-);Ivl-Fatp4(tg/+) mice showed a significant increase in enterocyte TG and FA content. There was no compensatory upregulation of other FATP family members or any other FA or cholesterol transporters in Fatp4(-/-);Ivl-Fatp4(tg/+) mice. Furthermore, although serum cholesterol levels were lower in Fatp4(-/-);Ivl-Fatp4(tg/+) mice, there was no difference in hepatic VLDL secretion in-vivo or in hepatic lipid content on either a chow or Western diet. Taken together, our studies find no evidence for a physiological role of intestinal FATP4 in dietary lipid absorption in mice.     

Globular adiponectin resistance develops independently of impaired insulin-stimulated glucose transport in soleus muscle from high-fat-fed rats

High-fat (HF) diets induce insulin resistance and alter lipid metabolism, although controversy exists regarding the impact of saturated vs. polyunsaturated fats. Adiponectin (Ad) stimulates fatty acid (FA) oxidation and improves insulin sensitivity in humans and rodents, due in part to the activation of AMP-activated protein kinase (AMPK) and subsequent deactivation of acetyl coenzyme A carboxylase (ACC). In genetically obese, diabetic mice, this acute stimulatory effect on AMPK in muscle is lost. The ability of a HF diet to induce skeletal muscle Ad resistance has not been examined. The purpose of this study was to determine whether Ad's effects on FA oxidation and AMPK/ACC would be reduced following different HF diets, and if this coincided with the development of impaired maximal insulin-stimulated glucose transport. Rats were fed a control (10% kcal fat, CON), high unsaturated fat (60% kcal safflower oil, SAFF), or high saturated fat diet (60% kcal lard, LARD) for 4 wk. Following the dietary intervention, glucose transport, lipid metabolism, and AMPK/ACC phosphorylation were measured in the presence and absence of globular Ad (gAd, 2.5 microg/ml) in isolated soleus muscle. LARD rats showed reduced rates of maximal insulin-stimulated glucose transport compared with CON and SAFF (+68 vs. +172 and +184%, P < or = 0.001). gAd increased pACC (+25%, P < or = 0.01) and FA oxidation (+28%, P < or = 0.05) in CON rats, but not in either HF group. Thus 4 wk of HF feeding results in the loss of gAd stimulatory effect on ACC phosphorylation and muscle FA oxidation, and this can occur independently of impaired maximal insulin-stimulated glucose transport.     

No evidence for a basal,retinoic, or superoxide-induced uncoupling activity of the uncoupling protein 2 present in spleen or lung mitochondria

The phenotypes observed in mice whose uncoupling protein (Ucp2) gene had been invalidated by homologous recombination (Ucp2(-/-) mice) are consistent with an increase in mitochondrial membrane potential in macrophages and pancreatic beta cells. This could support an uncoupling (proton transport) activity of UCP2 in the inner mitochondrial membrane in vivo. We used mitochondria from lung or spleen, the two organs expressing the highest level of UCP2, to compare the proton leak of the mitochondrial inner membrane of wild-type and Ucp2(-/-) mice. No difference was observed under basal conditions. Previous reports have concluded that retinoic acid and superoxide activate proton transport by UCP2. Spleen mitochondria showed a higher sensitivity to retinoic acid than liver mitochondria, but this was not caused by UCP2. In contrast with a previous report, superoxide failed to increase the proton leak rate in kidney mitochondria, where no UCP2 expression was detected, and also in spleen mitochondria, which does not support stimulation of UCP2 uncoupling activity by superoxide. Finally, no increase in the ATP/ADP ratio was observed in spleen or lung of Ucp2(-/-) mice. Therefore, no evidence could be gathered for the uncoupling activity of the UCP2 present in spleen or lung mitochondria. Although this may be explained by difficulties with isolated mitochondria, it may also indicate that UCP2 has another physiological significance in spleen and lung.     

Reduced antioxidant capacity and diet-induced atherosclerosis in uncoupling protein-2-deficient mice

Vascular dysfunction in response to reactive oxygen species (ROS) plays an important role in the development and progression of atherosclerotic lesions. In most cells, mitochondria are the major source of cellular ROS during aerobic respiration. Under most conditions the rates of ROS formation and elimination are balanced through mechanisms that sense relative ROS levels. However, a chronic imbalance in redox homeostasis is believed to contribute to various chronic diseases, including atherosclerosis. Uncoupling protein-2 (UCP2) is a mitochondrial inner membrane protein shown to be a negative regulator of macrophage ROS production. In response to a cholesterol-containing atherogenic diet, C57BL/6J mice significantly increased expression of UCP2 in the aorta, while mice lacking UCP2, in the absence of any other genetic modification, displayed significant endothelial dysfunction following the atherogenic diet. Compared with wild-type mice, Ucp2(-/-) mice had decreased endothelial nitric oxide synthase, an increase in vascular cell adhesion molecule-1 expression, increased ROS production, and an impaired ability to increase total antioxidant capacity. These changes in Ucp2(-/-) mice were associated with increased aortic macrophage infiltration and more numerous and larger atherosclerotic lesions. These data establish that in the vasculature UCP2 functions as an adaptive antioxidant defense to protect against the development of atherosclerosis in response to a fat and cholesterol diet.     

Uncoupling protein 2, but not uncoupling protein 1, is expressed in the female mouse reproductive tract

Uncoupling proteins (UCPs) are transporters of the inner mitochondrial membrane. Whereas UCP1 is uniquely present in brown adipose tissue where it uncouples respiration from ATP synthesis and activates respiration and heat production, UCP2 is present in numerous tissues, and its exact function remains to be clarified. Two sets of data provided the rationale for this study: (i) the intriguing report that UCP1 is present in uterus of mice (Nibbelink, M., Moulin, K., Arnaud, E., Duval, C., Penicaud, L., and Casteilla, L. (2001) J. Biol. Chem. 276, 47291-47295); and (ii) an observation that Ucp2(-/-) female mice (homozygous matings) have smaller litters compared with Ucp2(+/+) animals (S. Rousset and A.-M. Cassard-Doulcier, unpublished observations). These data prompted us to examine the expression of UCP1 and UCP2 in the reproductive tract of female mice. Using wild type, Ucp1(-/-) mice, and Ucp2(-/-) mice, we were unable to detect UCP1 in uterus of mice with appropriate antibodies, and we conclude that the signal assigned to UCP1 by others was neither UCP1 nor UCP2. Using a polyclonal antibody against UCP2 and tissues from Ucp2(-/-) mice as controls, UCP2 was detected in ovary, oviduct, and uterus. Expression of Ucp2 mRNA was also observed in ovary and uterus using in situ hybridization analysis. Bone marrow transplantation experiments revealed that the UCP2 signal of the ovary was restricted to ovarian cells. UCP2 level in ovary decreased during follicular growth and increased during the pre-ovulatory period, during which aspects of an inflammatory process are known to exist. Because UCP2 down-regulates reactive oxygen species, a role in the regulation of inflammatory events linked to the preparation of ovulation is suggested.     

The effect of high-fat feeding on intramuscular lipid and lipid peroxidation levels in UCP3-ablated mice

Uncoupling protein-3 (UCP3) has been suggested to protect against lipid-induced oxidative damage. Therefore, we studied intramuscular lipid peroxide levels and high-fat diet induced alterations in muscle lipid metabolism of UCP3-ablated mice. UCP3-/- mice showed approximately 3-fold higher levels of intramuscular lipid peroxides upon standard chow feeding, compared to wild-type littermates. Remarkably, this difference was no longer apparent on the high-fat diet. However, upon high-fat feeding, intramuscular triacylglycerol levels were approximately 50% lower in UCP3-/- mice, in comparison to UCP3+/+ animals. Succinate dehydrogenase activity, and total protein content of the muscle fatty acid transporter FAT/CD36 were however similar between UCP3-/- and UCP3+/+ mice.     

Inactivation of UCP1 and the glycerol phosphate cycle synergistically increases energy expenditure to resist diet-induced obesity

Our current paradigm for obesity assumes that reduced thermogenic capacity increases susceptibility to obesity, whereas enhanced thermogenic capacity protects against obesity. Here we report that elimination of two major thermogenic pathways encoded by the mitochondrial uncoupling protein (Ucp1) and mitochondrial glycerol-3-phosphate dehydrogenase (Gdm) result in mice with increased resistance to diet-induced obesity when housed at 28 degrees C, provided prior adaptation occurred at 20 degrees C. Obesity resistant Gdm(-/-).Ucp1(-/-) mice maintained at 28 degrees C have increased energy expenditure, in part through conversion of white to brown adipocytes in inguinal fat. Increased oxygen consumption in inguinal fat cell suspensions and the up-regulation of genes of mitochondrial function and fat metabolism indicated increased thermogenic activity, despite the absence of UCP1, whereas liver and skeletal muscle showed no changes in gene expression. Additionally, comparisons of energy expenditure in UCP1-deficient and wild type mice fed an obesogenic diet indicates that UCP1-based brown fat-based thermogenesis plays no role in so-called diet-induced thermogenesis. Accordingly, a new paradigm for obesity emerges in which the inactivation of major thermogenic pathways force the induction of alternative pathways that increase metabolic inefficiency.     

Effects of 2-G exposure on temperature regulation, circadian rhythms, and adiposity in UCP2/3 transgenic mice.

Altered ambient force environments affect energy expenditure via changes in thermoregulation, metabolism, and body composition. Uncoupling proteins (UCPs) have been implicated as potential enhancers of energy expenditure and may participate in some of the adaptations to a hyperdynamic environment. To test this hypothesis, this study examined the homeostatic and circadian profiles of body temperature (T(b)) and activity and adiposity in wild-type and UCP2/3 transgenic mice exposed to 1 and 2 G. There were no significant differences between the groups in the means, amplitudes, or phases of T(b) and activity rhythms at either the 1- or 2-G level. Percent body fat was significantly lower in transgenic (5.2 +/- 0. 2%) relative to the wild-type mice (6.2 +/- 0.1%) after 2-G exposure; mass-adjusted mesenteric and epididymal fat pads in transgenic mice were also significantly lower (P < 0.05). The data suggest that 1) the actions of two UCPs (UCP2 and UCP3) do not contribute to an altered energy balance at 2 G, although 2) UCP2 and UCP3 do contribute to the utilization of lipids as a fuel substrate at 2 G.     

Pten positively regulates brown adipose function, energy expenditure, and longevity

Aging in worms and flies is regulated by the PI3K/Akt/Foxo pathway. Here we extend this paradigm to mammals. Pten(tg) mice carrying additional genomic copies of Pten are protected from cancer and present a significant extension of life span that is independent of their lower cancer incidence. Interestingly, Pten(tg) mice have an increased energy expenditure and protection from metabolic pathologies. The brown adipose tissue (BAT) of Pten(tg) mice is hyperactive and presents high levels of the uncoupling protein Ucp1, which we show is a target of Foxo1. Importantly, a synthetic PI3K inhibitor also increases energy expenditure and hyperactivates the BAT in mice. These effects can be recapitulated in isolated brown adipocytes and, moreover, implants of Pten(tg) fibroblasts programmed with Prdm16 and Cebpβ form subcutaneous brown adipose pads more efficiently than wild-type fibroblasts. These observations uncover a role of Pten in promoting energy expenditure, thus decreasing nutrient storage and its associated damage.     

Acylation-stimulating protein (ASP)/complement C3adesArg deficiency results in increased energy expenditure in mice

Acylation-stimulating protein (ASP) acts as a paracrine signal to increase triglyceride synthesis in adipocytes. In mice, C3 (the precursor to ASP) knock-out (KO) results in ASP deficiency and leads to reduced body fat and leptin levels yet they are hyperphagic. In the present study, we investigated the mechanism for this energy repartitioning. Compared with wild-type (WT) mice, male and female C3(-/-) ASP-deficient mice had elevated oxygen consumption (VO2) in both the active (dark) and resting (light) phases of the diurnal cycle: +8.9% males (p < 0.05) +9.4% females (p < 0.05). Increased physical activity (movement) was observed during the dark phase in female but not in male KO animals. Female WT mice moved 16.9 +/- 2.4 m whereas KO mice moved 30.1 +/- 5.4 m, over 12 h, +78.4%, p < 0.05). In contrast, there was no difference in physical activity in male mice, but a repartitioning of dietary fat following intragastric fat administration was noted. This was reflected by increased fatty acid oxidation in liver and muscle in KO mice, with increased UCP2 (inguinal fat) and UCP3 (muscle) mRNA expression (p = 0.005 and 0.036, respectively). Fatty acid uptake into brown adipose tissue (BAT) and white adipose tissue (WAT) was reduced as reflected by a decrease in the fatty acid incorporation into lipids (BAT -68%, WAT -29%. The decrease of FA incorporation was normalized by intraperitoneal administration of ASP at the time of oral fat administration. These results suggest that ASP deficiency results in energy repartitioning through different mechanisms in male and female mice.     

Mitochondrial proton leak in obesity-resistant and obesity-prone mice

We quantified uncoupling proteins (UCPs) in molar amounts and assessed proton conductance in mitochondria isolated from interscapular brown adipose tissue (IBAT) and hindlimb muscle [known from prior work to contain ectopic brown adipose tissue (BAT) interspersed between muscle fibers] of obesity-resistant 129S6/SvEvTac (129) and obesity-prone C57BL/6 (B6) mice under conditions of low (LF) and high-fat (HF) feeding. With usual feeding, IBAT mitochondrial UCP1 content and proton conductance were greater in 129 mice than B6. However, with HF feeding, UCP1 and proton conductance increased more in B6 mice. Moreover, with HF feeding GDP-inhibitable proton conductance, specific for UCP1, equaled that seen in the 129 strain. UCP1 expression was substantial in mitochondria from hindlimb muscle tissue (ectopic BAT) of 129 mice as opposed to B6 but did not increase with HF feeding in either strain. As expected, muscle UCP3 expression increased with HF feeding in both strains but did not differ by strain. Moreover, the proton conductance of mitochondria isolated from hindlimb muscle tissue did not differ by strain or diet. Our data uncover a response to weight gain in obesity-prone (compared to resistant) mice unrecognized in prior studies that examined only UCP1 mRNA. Obesity-prone mice have the capacity to increase both IBAT UCP1 protein and mitochondrial proton conductance as much or more than obesity-resistant mice. But, this is only achieved only at a higher body mass and, therefore, may be adaptive rather than preventative. Neither obesity-prone nor resistant mice respond to HF feeding by expressing more UCP1 in ectopic BAT within muscle tissue.     

The role of uncoupling protein 1 in the metabolism and adiposity of RII beta-protein kinase A-deficient mice

Mice lacking the RII beta regulatory subunit of protein kinase A exhibit a 50% reduction in white adipose tissue stores compared with wild-type littermates and are resistant to diet-induced obesity. RII beta(-/-) mice also have an increase in resting oxygen consumption along with a 4-fold increase in the brown adipose-specific mitochondrial uncoupling protein 1 (UCP1). In this study, we examined the basis for UCP1 induction and tested the hypothesis that the induced levels of UCP1 in RII beta null mice are essential for the lean phenotype. The induction of UCP1 occurred at the protein but not the mRNA level and correlated with an increase in mitochondria in brown adipose tissue. Mice lacking both RII beta and UCP1 (RII beta(-/-)/Ucp1(-/-)) were created, and the key parameters of metabolism and body composition were studied. We discovered that RII beta(-/-) mice exhibit nocturnal hyperactivity in addition to the increased oxygen consumption at rest. Disruption of UCP1 in RII beta(-/-) mice reduced basal oxygen consumption but did not prevent the nocturnal hyperactivity. The double knockout animals also retained the lean phenotype of the RII beta null mice, demonstrating that induction of UCP1 and increased resting oxygen consumption is not the cause of leanness in the RII beta mutant mice.