Uncoupling Protein 1 Knockout Research Articles

Not all mitochondrial carrier proteins support permeability transition pore formation: no involvement of uncoupling protein 1

The mPTP (mitochondrial permeability transition pore) is a non-specific channel that is formed in the mitochondrial inner membrane in response to several stimuli, including elevated levels of matrix calcium. The pore is proposed to be composed of the ANT (adenine nucleotide translocase), voltage-dependent anion channel and cyclophilin D. Knockout studies, however, have demonstrated that ANT is not essential for permeability transition, which has led to the proposal that other members of the mitochondrial carrier protein family may be able to play a similar function to ANT in pore formation. To investigate this possibility, we have studied the permeability transition properties of BAT (brown adipose tissue) mitochondria in which levels of the mitochondrial carrier protein, UCP1 (uncoupling protein 1), can exceed those of ANT. Using an improved spectroscopic assay, we have quantified mPTP formation in de-energized mitochondria from wild-type and Ucp1KO (Ucp1-knockout) mice and assessed the dependence of pore formation on UCP1. When correctly normalized for differences in mitochondrial morphology, we find that calcium-induced mPTP activity is the same in both types of mitochondria, with similar sensitivity to GDP (~50% inhibited), although the portion sensitive to cyclosporin A is higher in mitochondria lacking UCP1 (~80% inhibited, compared with ~60% in mitochondria containing UCP1). We conclude that UCP1 is not a component of the cyclosporin A-sensitive mPTP in BAT and that playing a role in mPTP formation is not a general characteristic of the mitochondrial carrier protein family but is, more likely, restricted to specific members including ANT.

Uncoupling protein-1 (UCP1) contributes to the basal proton conductance of brown adipose tissue mitochondria

Proton leak pathways uncouple substrate oxidation from ATP synthesis in mitochondria. These pathways are classified as basal (not regulated) or inducible (activated and inhibited). Previously it was found that over half of the basal proton conductance of muscle mitochondria was catalyzed by the adenine nucleotide translocase (ANT), an abundant mitochondrial anion carrier protein. To determine whether ANT is the unique protein catalyst, or one of many proteins that catalyze basal proton conductance, we measured proton leak kinetics in mitochondria isolated from brown adipose tissue (BAT). BAT can express another mitochondrial anion carrier, UCP1, at concentrations similar to ANT. Basal proton conductance was measured under conditions where UCP1 and ANT were catalytically inactive and was found to be lower in mitochondria from UCP1 knockout mice compared to wild-type. Ablation of another abundant inner membrane protein, nicotinamide nucleotide transhydrogenase, had no effect on proton leak kinetics in mitochondria from liver, kidney or muscle, showing that basal proton conductance is not catalyzed by all membrane proteins. We identify UCP1 as a second protein propagating basal proton leak, lending support to the hypothesis that basal leak pathways are perpetrated by members of the mitochondrial anion carrier family but not by other mitochondrial inner membrane proteins.

Uncoupling protein 2 regulates reactive oxygen species formation in islets and influences susceptibility to diabetogenic action of streptozotocin

Currently, the physiological function of uncoupling protein-2 (UCP2) in pancreatic islets and its role in the development of diabetes is a matter of great debate. To further investigate the impact of UCP2 on diabetes development, we used streptozotocin (STZ) to experimentally generate diabetes in both wild-type (WT) and UCP2-knockout (UCP2KO) mice. While multiple low-dose STZ injections led to hyperglycemia development over a 14-day period in both WT and UCP2KO mice, we found the development of hyperglycemia to be significantly less severe in the UCP2KO mice. Measurement of insulin and glucagon secretion (in vitro), as well as their plasma concentrations (in vivo), indicated that UCP2-deficiency showed enhanced insulin secretion but impaired alpha-cell function. Glucagon secretion was attenuated, despite reduced insulin secretion after exposure to STZ, which together contributed to less severe hyperglycemia development in UCP2KO mice. Further experimentation revealed that UCP2-deficient alpha- and beta-cells had chronically higher cellular reactive oxygen species (ROS) levels than the WT prior to STZ application, which correlated with increased basal beta- and alpha-cell mass. Overall, we suggest that increased chronic ROS signaling as a result of UCP2-deficiency contributes to enhanced beta-cell function and impairment of alpha-cell function, leading to an attenuation of STZ-induced hyperglycemia development.

Uncoupling protein-3 as a molecular determinant of the action of 3,5,3′-triiodothyronine on energy metabolism

Thyroid hormones are known to stimulate thermogenesis in rodents by exerting a permissive effect on norepinephrine that affects uncoupling protein-1 (UCP1) expression in brown adipose tissue (BAT). The aim of this study was to identify new targets of the thermogenic effects of T3 in tissues other than the BAT, such as skeletal muscle. In beta(1)/beta(2)/beta(3)-adrenoceptor knockout (beta-less) mice, that are dramatically cold intolerant, a normal body temperature was maintained throughout 48 h of cold exposure by T3 administration. In these mice, BAT UCP1 protein expression was not modified either by cold exposure or by T3 administration. To test the possibility that T3 might act via muscle uncoupling protein-3 (UCP3), an UCP3 knockout (KO) model was used. This model exhibited a normal phenotype except that, upon T3 administration, stimulated oxygen consumption of the UCP3KO mice was significantly lower by 6% than that of the wild-type (WT) mice. This difference was observed only during the dark period (between 7.00 p.m. and 7.00 a.m.), i.e. when the mice are the most active at consuming food. Therefore, UCP3 might participate in the correction by T3 of the dramatic cold intolerance of the beta-less mice. These results reactivate the idea that UCP3 might play a role in the control of energy balance.

Dysregulation of glucose homeostasis in nicotinamide nucleotide transhydrogenase knockout mice is independent of uncoupling protein 2

Glucose intolerance in C57Bl/6 mice has been associated with mutations in the nicotinamide nucleotide transhydrogenase (Nnt) gene. It has been proposed that the absence of NNT from mitochondria leads to increased mitochondrial reactive oxygen species production and subsequent activation of uncoupling protein 2 (UCP2). Activation of UCP2 has been suggested to uncouple electron transport from ATP synthesis in pancreatic beta cell mitochondria thereby decreasing glucose tolerance due to decreased insulin secretion through lower ATP/ADP ratios. The hypothesis tested in this paper is that UCP2 function is required for the dysregulation of glucose homeostasis observed in NNT ablated mice. Single and double Nnt and Ucp2 knockout mouse lines were used to measure glucose tolerance, whole animal energy balance and biochemical characteristics of mitochondrial uncoupling. As expected, glucose tolerance was diminished in mice lacking NNT. This was independent of UCP2 as it was observed either in the presence or absence of UCP2. The range of metabolic parameters examined in the mice and the proton conductance of isolated mitochondria remained unaltered in this double NNT and UCP2 knockout model. Ablation of UCP2 did not itself affect glucose tolerance and therefore previous observations of increased glucose tolerance of mice lacking UCP2 were not confirmed. We conclude that the decreased glucose tolerance in Nnt knockout mice observed in our experiments does not require UCP2.

The absence of UCP3 leads to tighter coupling of oxidative phosphorylation in skeletal muscle at rest

The role of uncoupling protein 3 (UCP3) in mitochondrial function in skeletal muscle remains controversial. To gain insight into the role of this mitochondrial protein, we sought to determine whether UCP3 uncouples oxidative phosphorylation in vivo, as suggested by the putative function of the homologue, UCP1. We hypothesized that, under resting conditions, the presence of UCP3 would correspond with a reduction in coupling. To test this we compared wild type mice with UCP3 knockout mice using non‐invasive methods for assessing in vivo resting metabolism, 31P nuclear magnetic resonance spectroscopy and optical spectroscopy. During a bout of ischemia, the former provides a measure of the resting rate of ATP production while the latter provides a measure of the resting rate of oxygen consumption. Dividing the rate of ATP production by the rate of oxygen consumption provides a measure of coupling (P/O ratio). We found a significant increase in the P/O ratio in mice lacking UCP3 compared to wild type mice (2.81 +/‐ 0.34 for knockout and 2.00 +/‐ 0.18 for wild type). This difference appears to be due to an increase in resting mitochondrial ATP production in the absence of UCP3. Our results indicate that UCP3 plays a role in regulating coupling of oxidative phosphorylation in skeletal muscle under resting conditions. This work was supported by NIH grants AG022385 and AG028455 and the Ellison Medical Foundation. Sample Data

Mutations in UCP2 in Congenital Hyperinsulinism Reveal a Role for Regulation of Insulin Secretion

Although the most common mechanism underlying congenital hyperinsulinism is dysfunction of the pancreatic ATP-sensitive potassium channel, the pathogenesis and genetic origins of this disease remains largely unexplained in more than half of all patients. UCP2 knockout mice exhibit an hyperinsulinemic hypoglycemia, suggesting an involment of UCP2 in insulin secretion. However, a possible pathogenic role for UCP2 protein in the development of human congenital hyperinsulinism or of any human disease has not yet been investigated. We studied ten children exhibiting congenital hyperinsulinism, without detectable mutations in the known congenital hyperinsulinism-causing genes. Parental-inherited heterozygous UCP2 variants encoding amino-acid changes were found in two unrelated children with congenital hyperinsulinism. Functional assays in yeast and in insulin-secreting cells revealed an impaired activity of UCP2 mutants. Therefore, we report the finding of UCP2 coding variants in human congenital hyperinsulinism, which reveals a role for this gene in the regulation of insulin secretion and glucose metabolism in humans. Our results show for the first time a direct association between UCP2 amino acid alteration and human disease and highlight a role for mitochondria in hormone secretion.

Effect of aging, caloric restriction, and uncoupling protein 3 (UCP3) on mitochondrial proton leak in mice

Mitochondrial proton leak may modulate reactive oxygen species (ROS) production and play a role in aging. The purpose of this study was to determine proton leak across the life span in skeletal mitochondria from calorie-restricted and UCP2/3 overexpressing mice. Proton leak in isolated mitochondria and markers of oxidative stress in whole tissue were measured in female C57BL/6J mice fed ad-libitum (WT-Control) or a 30% calorie-restricted (WT-CR) diet, and in mice overexpressing UCP2 and UCP3 (Positive-TG), their non-overexpressing littermates (Negative-TG) and UCP3 knockout mice (UCP3KO). Proton leak in WT-CR mice was lower than that of control mice at 8 and 26 months of age. The Positive-TG mice had greater proton leak than the Negative-TG and UCP3KO mice at 8 months of age, but this difference disappeared by 19 and 26 months. Lipid peroxidation was generally lower in WT-CR vs. WT-Control mice and UCP3KO mice had greater concentrations of T-BARS (thiobarbituric acid reactive substances, a measure of lipid peroxidation) than did Positive-TG and Negative-TG. The results of this study indicate that sustained increases in muscle mitochondrial proton leak are not responsible for alterations in life span with calorie restriction or UCP3 overexpression in mice. However, UCP3 may contribute to the actions of CR through mechanisms distinct from increasing basal proton leak.

Mitochondrial dysfunction contributes to the increased vulnerabilities of adiponectin knockout mice to liver injury†

Adiponectin is an adipocyte-derived hormone with a wide range of beneficial effects on obesity-related medical complications. Numerous epidemiological investigations in diverse ethnic groups have identified a lower adiponectin level as an independent risk factor for nonalcoholic fatty liver diseases and liver dysfunctions. Animal studies have demonstrated that replenishment of adiponectin protects against various forms of hepatic injuries, suggesting it to be a potential drug candidate for the treatment of liver diseases. This study was designed to investigate the cellular and molecular mechanisms underlying the hepatoprotective effects of adiponectin. Our results demonstrated that in adiponectin knockout (ADN-KO) mice, there was a preexisting condition of hepatic steatosis and mitochondrial dysfunction that might contribute to the increased vulnerabilities of these mice to secondary liver injuries induced by obesity and other conditions. Adenovirus-mediated replenishment of adiponectin depleted lipid accumulation, restored the oxidative activities of mitochondrial respiratory chain (MRC) complexes, and prevented the accumulation of lipid peroxidation products in ADN-KO mice but had no obvious effects on mitochondrial biogenesis. The gene and protein levels of uncoupling protein 2 (UCP2), a mitochondrial membrane transporter, were decreased in ADN-KO mice and could be significantly up-regulated by adiponectin treatment. Moreover, the effects of adiponectin on mitochondrial activities and on protection against endotoxin-induced liver injuries were significantly attenuated in UCP2 knockout mice. These results suggest that the hepatoprotective properties of adiponectin are mediated at least in part by an enhancement of the activities of MRC complexes through a mechanism involving UCP2.

Mitochondrial Uncoupling Protein-2 Mediates Steatotic Liver Injury following Ischemia/Reperfusion

Steatotic livers are not used for transplantation because they have a reduced tolerance for ischemic events with reduced ATP levels and greater levels of cellular necrosis, which ultimately result in total organ failure. Mitochondrial uncoupling protein-2 (UCP2) is highly expressed in steatotic livers and may be responsible for liver sensitivity to ischemia through mitochondrial and ATP regulation. To test this hypothesis, experiments were conducted in lean and steatotic (ob/ob), wild-type, and UCP2 knock-out mice subjected to total warm hepatic ischemi-a/reperfusion. Although ob/ob UCP2 knock-out mice and ob/ob mice have a similar initial phenotype, ob/ob UCP2 knock-out animal survival was 83% when compared with 30% in ob/ob mice 24 h after reperfusion. Serum alanine aminotransferase concentrations and hepatocellular necrosis were decreased in the ob/ob UCP2 knock-out mice when compared with ob/ob mice subjected to ischemia. Liver ATP levels were increased in the ob/ob UCP2 knock-out animals after reperfusion when compared with the ob/ob mice but remained below the concentrations from lean livers. Lipid peroxidation (thiobarbituric acid-reactive substances) increased after reperfusion most significantly in the steatotic groups, but the increase was not affected by UCP2 deficiency. These results reveal that UCP2 expression is a critical factor, which sensitizes steatotic livers to ischemic injury, regulating liver ATP levels after ischemia and reperfusion.

Ablation of Uncoupling Protein‐3 Does Not Inhibit Palmitate Export from Mouse Heart Mitochondria

Uncoupling protein‐3 (UCP3) has been implicated in the export of long‐chain free fatty acid (FA) from mitochondria. However, there is no direct evidence of FA export from mitochondria. If UCP3 is involved in FA export from mitochondria then its ablation should inhibit the process. Employing a novel technique developed in our laboratory that directly measures FA export from isolated mitochondria, we sought to determine whether or not the rate of export of palmitate generated in the matrix from palmitoyl‐carnitine is inhibited in mitochondria isolated from UCP3 knockout (UCP3KO) mouse hearts. UCP3KO mouse heart mitochondria had no detectable UCP3 protein. However, the UCP2 level was similar to wild type mouse heart mitochondria. The mitochondrial thioesterase‐1 (MTE‐1) which generates FA in the matrix was increased by ~30% in UCP3 knockout compared to WT mice. The rate of generation of palmitate from palmitoyl‐carnitine in mitochondria was also increased by ~25%, but there was no inhibition in the rate of export of palmitate from mitochondria of UCP3KO mouse hearts. We conclude that UCP3 does not exclusively export FA from mitochondria.

Studies of UCP2 transgenic and knockout mice reveal that liver UCP2 is not essential for the antiobesity effects of fish oil

Uncoupling protein 2 (UCP2) is a possible target molecule for energy dissipation. Many dietary fats, including safflower oil and lard, induce obesity in C57BL/6 mice, whereas fish oil does not. Fish oil increases UCP2 expression in hepatocytes and may enhance UCP2 activity by activating the UCP2 molecule or altering the lipid bilayer environment. To examine the role of liver UCP2 in obesity, we created transgenic mice that overexpressed human UCP2 in hepatocytes and examined whether UCP2 transgenic mice showed less obesity when fed a high-fat diet (safflower oil or lard). In addition, we examined whether fish oil had antiobesity effects in UCP2 knockout mice. UCP2 transgenic and wild-type mice fed a high-fat diet (safflower oil or lard) developed obesity to a similar degree. UCP2 knockout and wild-type mice fed fish oil had lower rates of obesity than mice fed safflower oil. Remarkably, safflower oil did not induce obesity in female UCP2 knockout mice, an unexpected phenotype for which we presently have no explanation. However, this unexpected effect was not observed in male UCP2 knockout mice or in UCP2 knockout mice fed a high-lard diet. These data indicate that liver UCP2 is not essential for fish oil-induced decreases in body fat.

CALORIE RESTRICTION REDUCES SUPEROXIDE PRODUCTION OF AGING MOUSE HYPOTHALAMUS

Previous investigations suggested that calorie restriction in mice significantly reduces reactive oxygen species (ROS), decreasing oxidative damage and extending lifespan. Calorie-restricted mice also increase expression of mitochondrial uncoupling proteins (UCPs), which have been suggested to reduce ROS generation. Thus, we hypothesized that the age-related increase in ROS would be attenuated in transgenic mice that overexpress UCP2 (TG-UCP2). To test this, we measured superoxide production in hypothalami from 8-, 19- and 26-month old female mice in the following ad libitum fed groups: TG-UCP2 mice, UCP2 knockout mice, and C57BL/6 wild type (WT) mice. Measurements were also made in hypothalami from C57BL/6 mice calorie-restricted to 70% of WT animals. Superoxide production in the fixed hypothalami was determined by measuring oxidation of fluorescent-labeled dihydroethidium to ethidium. We found a significant age-related increase in superoxide between 8 and 19 month WT mice without further increases at 26 months. Calorie restriction significantly reduced superoxide concentration compared to all other groups; and preliminary data suggest that increased UCP2 expression may contribute to this attenuation. (NIH grant AG19984; Genentech Support for Undergraduate Research)

Regulation of insulin secretion by uncoupling protein

UCPs (uncoupling proteins) can regulate cellular ATP production by uncoupling oxidative phosphorylation. UCP2 is expressed in islet beta-cells and its induction reduces glucose-stimulated insulin secretion. Under physiological conditions, superoxide, formed as a by-product of respiration, activates UCP2. This leads to reduced ATP production, which impairs closure of the ATP-dependent K+ channels to prevent insulin secretion. It is suggested that the physiological role of UCP2 is to prevent excessive superoxide generation through a feedback loop. UCP2 induction may also alter fatty acid metabolism by altering NAD/NADH or by facilitating cycling of fatty acid anions. Recently, UCP2 has been proposed to keep insulin secretion low during starvation, a function under the control of the transcription co-repressor, surtuin-1, which has been shown to bind to the UCP2 promoter. Pathological UCP2 expression or activation may suppress glucose-stimulated insulin secretion to the extent that diabetes onset is hastened. In ob/ob mice, induction of UCP2 at age 5 weeks precedes development of insulin secretion defects and hyperglycaemia. Activating protein kinase A-dependent pathways can normalize insulin secretion in UCP2-overexpressing islets. Conversely, lowering UCP2 expression may promote increased insulin secretion. UCP2 knockout mice were protected from the diabetogenic effects of a high-fat diet and their islets exhibited increased sensitivity to glucose and elevated ATP/ADP. These results support a role for UCP2 as a gene contributing to the pathogenesis of Type 2 diabetes.

Uncoupling Proteins: Role in Insulin Resistance and Insulin Insufficiency

Uncoupling proteins (UCPs) are modulators of mitochondrial metabolism that have been implicated in the development of both insulin resistance and insulin insufficiency, the two major pathophysiological events associated with type 2 diabetes. UCP2 mRNA is expressed in a wide range of tissues; however UCP2 protein expression is restricted to fewer tissues, including the endocrine pancreas, spleen, stomach, brain and the lung. To date, its role in the pathophysiology of diabetes has been most strongly associated with impaired glucose-stimulated insulin secretion from the beta-cell, particularly after its induction by free fatty acids. The physiological role of UCP2 remains controversial, but it may act as a downstream signal transducer of superoxide. UCP3 mRNA and protein are expressed in relatively few tissues, predominantly skeletal muscle, brown adipose tissue and heart. Increased expression of UCP3 in skeletal muscle is associated with protection from diet-induced insulin resistance in mice. In patients with type 2 diabetes UCP3 protein in muscle is reduced by 50% compared to healthy controls. The primary physiological role of the novel UCPs does not appear to be protection against positive energy balance and obesity; this is based largely on findings from studies of UCP2 and UCP3 knockout mice and from observed increases in UCP3 expression with fasting. The mechanism(s) of action of UCP2 and UCP3 are poorly understood. However, findings support roles for UCP2 and UCP3 as modifiers of fatty acid metabolism and in mitigating damage from reactive oxygen species.

Tough love: left out in the cold, but not abandoned, by UCP3

In rodents, the challenge of maintaining core body temperature in a cold environment is partially met by increased activity of mitochondrial uncoupling protein (UCP) 1 in brown adipose tissue (BAT) ([1][1]). The energy that would normally be preserved by coupling ATP synthesis with the translocation

Uncoupling Protein 2 Has Protective Function during Experimental Autoimmune Encephalomyelitis

Uncoupling protein 2 (UCP2) is a member of the mitochondrial transporter superfamily that is expressed in many tissues, including immune cells. UCP2 prevents oxidative stress by reducing reactive oxygen species. Using UCP2-deficient mice, it was shown that UCP2 is involved in the regulation of insulin secretion, in the resistance to infection, and in atherosclerosis. Here, we investigated the role of UCP2 in experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis. Immunized C57BL/6J UCP2-deficient mice showed a slightly delayed onset during experimental autoimmune encephalomyelitis (13.0 +/- 0.6 versus 11.5 +/- 0.8 in wild-type controls) and developed significantly higher disease scores than littermate controls (maximum disease score of 2.9 +/- 0.2 versus 1.7 +/- 0.2, P = 0.001). Higher levels of infiltrating T cells into the spinal cord meninges and parenchyma were observed. The T-cell proliferative response to the specific antigen was increased in UCP2-deficient mice compared with littermate controls, and CD4 cells of UCP2 knockout mice produced significantly higher levels of pro-inflammatory cytokines, eg, tumor necrosis factor-alpha and interleukin-2, resulting from a Th1 response. Mice lacking UCP2 also developed a higher B-cell response. Concomitantly, CD4 and CD8 cells of the UCP2-deficient mice showed increased production of reactive oxygen species. These results suggest a protective function of UCP2 in chronic inflammatory diseases such as multiple sclerosis.

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.

Indispensable role of mitochondrial UCP1 for antiobesity effect of β3-adrenergic stimulation

Mitochondrial uncoupling protein-1 (UCP1) has been thought to be a key molecule for thermogenesis during cold exposure and spontaneous hyperphagia and thereby in the autonomic regulation of energy expenditure and adiposity. However, UCP1 knockout (KO) mice were reported to be cold intolerant but unexpectedly did not get obese even after hyperphagia, implying that UCP1 may not be involved in the regulation of adiposity. Treatment of obese animals with beta3-adrenergic agonists is known to increase lipid mobilization, induce UCP1, and, finally, reduce body fat content. To obtain direct evidence for the role of UCP1 in the anti-obesity effect of beta3-adrenergic stimulation, in the present study, UCP1-KO and wild-type (WT) mice were fed on cafeteria diets for 8 wk and then given a beta3-adrenergic agonist, CL-316,243 (CL), or saline for 2 wk. A single injection of CL increased whole body oxygen consumption and brown fat temperature in WT mice but not in KO mice, and it elicited almost the same plasma free fatty acid response in WT and KO mice. WT and KO mice increased similarly their body and white fat pad weights on cafeteria diets compared with those on laboratory chow. Daily treatment with CL resulted in a marked reduction of white fat pad weight and the size of adipocytes in WT mice, but not in KO mice. Compared with WT mice, KO mice expressed increased levels of UCP2 in brown fat but decreased levels in white fat and comparable levels of UCP3. It was concluded that the anti-obesity effect of beta3-adrenergic stimulation is largely attributable to UCP1, but less to UCP2 and UCP3, and thereby to UCP1-dependent degradation of fatty acids released from white adipose tissue.

Uncoupling Proteins 2 and 3 Function in Concert to Augment Tolerance to Cardiac Ischemia

Transient cardiac ischemia activates cell survival signaling, conferring subsequent ischemia tolerance to the heart. This biological phenomenon, termed ischemic preconditioning, results in improved clinical outcome and attenuated infarct size following myocardial infarction. To explore genomic modifications underpinning this ischemia tolerance, we delineated the regulation and function of the cardiac enriched mitochondrial uncoupling proteins 2 and 3 during delayed ischemic preconditioning in the rat. Cardiac transcripts of genes encoding uncoupling proteins 2 and 3 are up-regulated in parallel with infarct size reduction in preconditioned hearts. Mitochondria isolated from preconditioned hearts exhibit an augmented inducible proton leak. In parallel, following anoxia-reoxygenation these mitochondria generate less hydrogen peroxide compared with non-preconditioned mitochondria. Preconditioning in rat cardiac derived myoblasts is abolished following uncoupling protein-2 depletion by RNA-interference. RNAi of uncoupling protein-3 partially attenuates the capacity to precondition these cells. Functional characterization of anoxia and reoxygenation tolerance following uncoupling protein 2 or 3 and combined 2 and 3 RNAi shows the largest reduction in viability follows depletion of both homologues. Uncoupling protein-2 depletion alone significantly attenuates anoxia-reoxygenation tolerance but uncoupling protein-3 depletion does not reduce anoxia tolerance. In parallel combined uncoupling protein depletion and isolated uncoupling protein-2 depletion augments ROS production in viable cardiomyocytes following anoxia-reoxygenation. Concurrent anti-oxidant administration ameliorates the uncoupling protein-depleted anoxia-susceptible phenotype. In conclusion, mitochondrial uncoupling proteins are necessary components of ischemia tolerance and function as components of the cellular antioxidant defense program. In the cytoprotective hierarchy, uncoupling protein-2 appears to play a greater role than uncoupling protein-3 in modulating ischemia/anoxia tolerance in heart-derived cells.