The complementary and divergent roles of uncoupling proteins 1 and 3 in thermoregulation.

KEY POINTS: Both uncoupling protein 1 (UCP1) and UCP3 are important for mammalian thermoregulation. UCP1 and UCP3 in brown adipose tissue mediate early and late phases of sympathomimetic thermogenesis, respectively. Lipopolysaccharide thermogenesis requires skeletal muscle UCP3 but not UCP1. Acute noradrenaline-induced hyperthermia requires UCP1 but not UCP3. Loss of both UCP1 and UCP3 accelerate the loss of body temperature compared to UCP1KO alone during acute cold exposure. ABSTRACT: Uncoupling protein 1 (UCP1) is the established mediator of brown adipose tissue-dependent thermogenesis. In contrast, the role of UCP3, expressed in both skeletal muscle and brown adipose tissue, in thermoregulatory physiology is less well understood. Here, we show that mice lacking UCP3 (UCP3KO) have impaired sympathomimetic (methamphetamine) and completely abrogated lipopolysaccharide (LPS) thermogenesis, but a normal response to noradrenaline. By comparison, UCP1 knockout (UCP1KO) mice exhibit blunted methamphetamine and fully inhibited noradrenaline thermogenesis, but an increased febrile response to LPS. We further establish that mice lacking both UCP1 and 3 (UCPDK) fail to show methamphetamine-induced hyperthermia, and have a markedly accelerated loss of body temperature and survival after cold exposure compared to UCP1KO mice. Finally, we show that skeletal muscle-specific human UCP3 expression is able to significantly rescue LPS, but not sympathomimetic thermogenesis blunted in UCP3KO mice. These studies identify UCP3 as an important mediator of physiological thermogenesis and support a renewed focus on targeting UCP3 in metabolic physiology.

Caenorhabditis elegans UCP4 protein controls complex II-mediated oxidative phosphorylation through succinate transport.

The novel uncoupling proteins (UCP2-5) are implicated in the mitochondrial control of oxidant production, insulin signaling, and aging. Attempts to understand their functions have been complicated by overlapping expression patterns in most organisms. Caenorhabditis elegans nematodes are unique because they express only one UCP ortholog, ceUCP4 (ucp4). Here, we performed detailed metabolic analyzes in genetically modified nematodes to define the function of the ceUCP4. The knock-out mutant ucp4 (ok195) exhibited sharply decreased mitochondrial succinate-driven (complex II) respiration. However, respiratory coupling and electron transport chain function were normal in ucp4 mitochondria. Surprisingly, isolated ucp4 mitochondria showed markedly decreased succinate uptake. Similarly, ceUCP4 inhibition blocked succinate respiration and import in wild type mitochondria. Genetic and pharmacologic inhibition of complex I function was selectively lethal to ucp4 worms, arguing that ceUCP4-regulated succinate transport is required for optimal complex II function in vivo. Additionally, ceUCP4 deficiency prolonged lifespan in the short-lived mev1 mutant that exhibits complex II-generated oxidant production. These results identify a novel function for ceUCP4 in the regulation of complex II-based metabolism through an unexpected mechanism involving succinate transport.

Identification of a redox-modulatory interaction between uncoupling protein 3 and thioredoxin 2 in the mitochondrial intermembrane space.

UNLABELLED: Uncoupling protein 3 (UCP3) is a member of the mitochondrial solute carrier superfamily that is enriched in skeletal muscle and controls mitochondrial reactive oxygen species (ROS) production, but the mechanisms underlying this function are unclear. AIMS: The goal of this work focused on the identification of mechanisms underlying UCP3 functions. RESULTS: Here we report that the N-terminal, intermembrane space (IMS)-localized hydrophilic domain of mouse UCP3 interacts with the N-terminal mitochondrial targeting signal of thioredoxin 2 (Trx2), a mitochondrial thiol reductase. Cellular immunoprecipitation and in vitro pull-down assays show that the UCP3-Trx2 complex forms directly, and that the Trx2 N-terminus is both necessary and sufficient to confer UCP3 binding. Mutation studies show that neither a catalytically inactivated Trx2 mutant, nor a mutant Trx2 bearing the N-terminal targeting sequence of cytochrome c oxidase (COXMTS-Trx2) bind UCP3. Biochemical analyses using permeabilized mitochondria, and live cell experiments using bimolecular fluorescence complementation show that the UCP3-Trx2 complex forms specifically in the IMS. Finally, studies in C2C12 myocytes stably overexpressing UCP3 (2.5-fold) and subjected to Trx2 knockdown show that Trx2 is required for the UCP3-dependent mitigation of complex III-driven mitochondrial ROS generation. UCP3 expression was increased in mice fed a high fat diet, leading to increased localization of Trx2 to the IMS. UCP3 overexpression also increased expression of the glucose transporter GLUT4 in a Trx2-dependent fashion. INNOVATION: This is the first report of a mitochondrial protein-protein interaction with UCP3 and the first demonstration that UCP3 binds directly, and in cells and tissues with mitochondrial thioredoxin 2. CONCLUSION: These studies identify a novel UCP3-Trx2 complex, a novel submitochondrial localization of Trx2, and a mechanism underlying UCP3-regulated mitochondrial ROS production.

Pharmacodynamic characterization of insulin on MDMA-induced thermogenesis.

Sympathomimetic drugs (MDMA; ecstasy) induce a potentially catastrophic hyperthermia that involves free fatty acid (FFA) activation of mitochondrial uncoupling proteins (UCP). Insulin is an important regulator of plasma FFA levels, although its role in thermogenesis is unclear. The aims of the present study were 1) to characterize the pharmacodynamic effects of MDMA on plasma insulin and glucose, 2) to examine the effects of insulin on MDMA-induced thermogenesis and 3) to examine MDMA-induced thermogenesis in an animal model of insulin resistance, the obese Zucker rat. Insulin levels peaked 15 min after MDMA (40 mg/kg, s.c.), which preceded the peak temperature change at 60 min. Plasma glucose levels also peaked 15 min. after MDMA and remained elevated throughout the 90-min. monitoring period. Insulin pretreatment (10 units/kg, s.c.) 30 min. before a low dose of MDMA (5 mg/kg, s.c.) potentiated the thermogenic response. Insulin resistant, fa/fa (obese) Zucker rats demonstrated an attenuated thermogenic response to MDMA (40 mg/kg, s.c.). Consistent with the role for FFA in UCP3 expression, immunoblot analysis showed significantly increased levels of UCP3 protein obese compared to lean Zucker skeletal muscle. In conclusion, the results of the present study suggest a potential role of insulin signaling in sympathomimetic-induced thermogenesis.

The hyperthermia mediated by 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) is sensitive to sex differences.

Female subjects have been reported to be less sensitive to the hyperthermic effects of 3,4-methylenedioxymethamine (MDMA) than males. Studies were designed to examine the cellular mechanisms involved in these sex sensitive differences. Gonadectomized female and male rats were treated with a 200 microg 100 microL(-1) of estrogen or 100 microg 100 microL(-1) of testosterone respectively every 5 days for a total of three doses. Rats were then challenged with either saline or MDMA (20 mg kg(-1), sc). Rats were then euthanized and aortas were constricted, in vitro, by serial phenylephrine (Phe) addition with or without the inhibitor of nitric oxide (NO) synthase, g-nitro-L-Arginine-Methyl Ester (L-NAME). Skeletal muscle uncoupling protein-3 (UCP3) expression was measured as well as plasma norepinephrine (NE) levels. All males but no females developed hyperthermia following MDMA treatment. The EC(50) for Phe dose response curves increased only in the females treated with MDMA and T(max) for Phe increased following L-NAME only in the females. Both males and females demonstrated an increase in plasma NE following MDMA treatment; however, males displayed a significantly greater NE concentration. Skeletal muscle UCP3 expression was 80% less in females than in males. These results suggest that the inability of MDMA to induce a thermogenic response in the female subjects may be due to four sex-specific mechanisms: 1) Female subjects have reduced sympathetic activation following MDMA challenge; 2) Female vasculature is less sensitive to alpha(1)-AR stimulation following MDMA challenge; 3) Female vasculature has an increased sensitivity to NO; 4) UCP3 expression in skeletal muscle is less in females.