Cold-induced alterations of phospholipid fatty acyl composition in brown adipose tissue mitochondria are independent of uncoupling protein-1

The recruitment process induced by acclimation of mammals to cold includes a marked alteration in the acyl composition of the phospholipids of mitochondria from brown adipose tissue: increases in 18:0, 18:2(n-6), and 20:4(n-6) and decreases in 16:0, 16:1, 18:1, and 22:6(n-3). A basic question is whether these alterations are caused by changes in the concentration of uncoupling protein-1 (UCP1) or the thermogenesis it mediates-implying that they are secondary effects-or whether they are an integrated, independent part of the recruitment process. This question was addressed here using wild-type and UCP1-ablated C57BL/6 mice acclimated to 24 degrees C or 4 degrees C. In wild-type mice, the phospholipid fatty acyl composition of mitochondria from brown adipose tissue showed the changes in response to cold that were expected from observations in other species and strains. The changes were specific, as different changes occurred in skeletal muscle mitochondria. In UCP1-ablated mice, cold acclimation induced acyl alterations in brown adipose tissue that were qualitatively identical and quantitatively similar to those in wild-type mice. Therefore, neither the increased content of UCP1 nor mitochondrial uncoupling altered the effect of cold on acyl composition. Cold acclimation in wild-type mice had little effect on phospholipid acyl composition in muscle mitochondria, but cold-acclimation in UCP1-ablated mice caused significant alterations, probably due to sustained shivering. Thus, the alterations in brown adipose tissue phospholipid acyl composition are revealed to be an independent part of the recruitment process, and their functional significance for thermogenesis should be elucidated.     

An ancient look at UCP1

Brown adipose tissue serves as a thermogenic organ in placental mammals to defend body temperature in the cold by nonshivering thermogenesis. The thermogenic function of brown adipose tissue is enabled by several specialised features on the organ as well as on the cellular level, including dense sympathetic innervation and vascularisation, high lipolytic capacity and mitochondrial density and the unique expression of uncoupling protein 1 (UCP1). This mitochondrial carrier protein is inserted into the inner mitochondrial membrane and stimulates maximum mitochondrial respiration by dissipating proton-motive force as heat. Studies in knockout mice have clearly demonstrated that UCP1 is essential for nonshivering thermogenesis in brown adipose tissue. For a long time it had been presumed that brown adipose tissue and UCP1 emerged in placental mammals providing them with a unique advantage to survive in the cold. Our subsequent discoveries of UCP1 orthologues in ectotherm vertebrates and marsupials clearly refute this presumption. We can now initiate comparative studies on the structure–function relationships in UCP1 orthologues from different vertebrates to elucidate when during vertebrate evolution UCP1 gained the biochemical properties required for nonshivering thermogenesis.     

An ancient look at UCP1

Brown adipose tissue serves as a thermogenic organ in placental mammals to defend body temperature in the cold by nonshivering thermogenesis. The thermogenic function of brown adipose tissue is enabled by several specialised features on the organ as well as on the cellular level, including dense sympathetic innervation and vascularisation, high lipolytic capacity and mitochondrial density and the unique expression of uncoupling protein 1 (UCP1). This mitochondrial carrier protein is inserted into the inner mitochondrial membrane and stimulates maximum mitochondrial respiration by dissipating proton-motive force as heat. Studies in knockout mice have clearly demonstrated that UCP1 is essential for nonshivering thermogenesis in brown adipose tissue. For a long time it had been presumed that brown adipose tissue and UCP1 emerged in placental mammals providing them with a unique advantage to survive in the cold. Our subsequent discoveries of UCP1 orthologues in ectotherm vertebrates and marsupials clearly refute this presumption. We can now initiate comparative studies on the structure-function relationships in UCP1 orthologues from different vertebrates to elucidate when during vertebrate evolution UCP1 gained the biochemical properties required for nonshivering thermogenesis.     

The role of skeletal‐muscle‐based thermogenic mechanisms in vertebrate endothermy

Thermogenesis is one of the most important homeostatic mechanisms that evolved during vertebrate evolution. Despite its importance for the survival of the organism, the mechanistic details behind various thermogenic processes remain incompletely understood. Although heat production from muscle has long been recognized as a thermogenic mechanism, whether muscle can produce heat independently of contraction remains controversial. Studies in birds and mammals suggest that skeletal muscle can be an important site of non‐shivering thermogenesis (NST) and can be recruited during cold adaptation, although unequivocal evidence is lacking. Much research on thermogenesis during the last two decades has been focused on brown adipose tissue (BAT). These studies clearly implicate BAT as an important site of NST in mammals, in particular in newborns and rodents. However, BAT is either absent, as in birds and pigs, or is only a minor component, as in adult large mammals including humans, bringing into question the BAT‐centric view of thermogenesis. This review focuses on the evolution and emergence of various thermogenic mechanisms in vertebrates from fish to man. A careful analysis of the existing data reveals that muscle was the earliest facultative thermogenic organ to emerge in vertebrates, long before the appearance of BAT in eutherian mammals. Additionally, these studies suggest that muscle‐based thermogenesis is the dominant mechanism of heat production in many species including birds, marsupials, and certain mammals where BAT‐mediated thermogenesis is absent or limited. We discuss the relevance of our recent findings showing that uncoupling of sarco(endo)plasmic reticulum Ca²⁺‐ATPase (SERCA) by sarcolipin (SLN), resulting in futile cycling and increased heat production, could be the basis for NST in skeletal muscle. The overall goal of this review is to highlight the role of skeletal muscle as a thermogenic organ and provide a balanced view of thermogenesis in vertebrates.     

Lecithin cholesterol acyltransferase null mice are protected from diet-induced obesity and insulin resistance in a gender-specific manner through multiple pathways

Complete lecithin cholesterol acyltransferase (LCAT) deficiency uniformly results in a profound HDL deficiency. We recently reported unexpected enhanced insulin sensitivity in LCAT knock-out mice in the LDL receptor knock-out background (Ldlr(-/-)×Lcat(-/-); double knock-out (DKO)), when compared with their Ldlr(-/-)×Lcat(+/+) (single knock-out (SKO)) controls. Here, we report that LCAT-deficient mice (DKO and Lcat(-/-)) are protected against high fat high sucrose (HFHS) diet-induced obesity without hypophagia in a gender-specific manner compared with their respective (SKO and WT) controls. The metabolic phenotypes are more pronounced in the females. Changes in endoplasmic reticulum stress were examined as a possible mechanism for the metabolic protection. The female DKO mice developed attenuated HFHS-induced endoplasmic reticulum stress as evidenced by a lack of increase in mRNA levels of the hepatic unfolded protein response (UPR) markers Grp78 and CHOP compared with SKO controls. The DKO female mice were also protected against diet-induced insulin resistance. In white adipose tissue of chow-fed DKO mice, we also observed a reduction in UPR, gene markers for adipogenesis, and markers for activation of Wnt signaling. In skeletal muscles of female DKO mice, we observed an unexpected increase in UCP1 in association with increase in phospho-AMPKα, PGC1α, and UCP3 expressions. This increase in UCP1 was associated with ectopic islands of brown adipocytes between skeletal muscle fibers. Our findings suggest that LCAT deficiency confers gender-specific protection against diet-induced obesity and insulin resistance at least in part through regulation in UPR, white adipose tissue adipogenesis, and brown adipocyte partitioning.     

Selective detection of UCP 3 expression in skeletal muscle: effect of thyroid status and temperature acclimation

A novel peptide antibody to UCP 3 is characterized which is sensitive and discriminatory for UCP 3 over UCP 2, UCP 1 and other mitochondrial transporters. The peptide antibody detects UCP 3 expression in E. coli, COS cells and yeast expression systems. The peptide antibody detects a single ∼33 kDa protein band in mitochondria from isolated rat skeletal muscle, mouse and rat brown adipose tissue, and in whole muscle groups (soleus and extensor digitorum longus) from mice. No 33 kDa band is detectable in isolated mitochondria from liver, heart, brain, kidney and lungs of rats, or gastrocnemius mitochondria from UCP 3 knock-out mice. From our data, we conclude that the peptide antibody is detecting UCP 3 in skeletal muscle, skeletal muscle mitochondria and brown adipose tissue mitochondria. It is also noteworthy that the peptide antibody can detect human, mouse and rat forms of UCP 3. Using the UCP 3 peptide antibody, we confirm and quantify the increased (2.8-fold) UCP 3 expression observed in skeletal muscle mitochondria isolated from 48-h-starved rats. We show that UCP 3 expression is increased (1.6-fold) in skeletal muscle of rats acclimated over 8 weeks to 8 °C and that UCP 3 expression is decreased (1.4-fold) in rats acclimated to 30 °C. Furthermore, UCP 3 expression is increased (2.3-fold) in skeletal muscle from hyperthyroid rats compared to euthyroid controls. In addition, we show that UCP 3 expression is only coincident with the mitochondrial fraction of skeletal muscle homogenates and not peroxisomal, nuclear or cytosolic and microsomal fractions.     

Inactivation of the mitochondrial carrier SLC25A25 (ATP-Mg2+/Pi transporter) reduces physical endurance and metabolic efficiency in mice

An ATP-Mg(2+/)P(i) inner mitochondrial membrane solute transporter (SLC25A25), which is induced during adaptation to cold stress in the skeletal muscle of mice with defective UCP1/brown adipose tissue thermogenesis, has been evaluated for its role in metabolic efficiency. SLC25A25 is thought to control ATP homeostasis by functioning as a Ca(2+)-regulated shuttle of ATP-Mg(2+) and P(i) across the inner mitochondrial membrane. Mice with an inactivated Slc25a25 gene have reduced metabolic efficiency as evidenced by enhanced resistance to diet-induced obesity and impaired exercise performance on a treadmill. Mouse embryo fibroblasts from Slc25a25(-/-) mice have reduced Ca(2+) flux across the endoplasmic reticulum, basal mitochondrial respiration, and ATP content. Although Slc25a25(-/-) mice are metabolically inefficient, the source of the inefficiency is not from a primary function in thermogenesis, because Slc25a25(-/-) mice maintain body temperature upon acute exposure to the cold (4 °C). Rather, the role of SLC25A25 in metabolic efficiency is most likely linked to muscle function as evidenced from the physical endurance test of mutant mice on a treadmill. Consequently, in the absence of SLC25A25 the efficiency of ATP production required for skeletal muscle function is diminished with secondary effects on adiposity. However, in the absence of UCP1-based thermogenesis, induction of Slc25a25 in mice with an intact gene may contribute to an alternative thermogenic pathway for the maintenance of body temperature during cold stress.     

Only UCP1 can mediate adaptive nonshivering thermogenesis in the cold.

Adaptive nonshivering thermogenesis may have profound effects on energy balance and is therefore therefore is a potential mechanism for counteracting the development of obesity. The molecular basis for adaptive nonshivering thermogenesis has remained a challenge that sparked acute interest with the identification of proteins (UCP2, UCP3, etc.) with high-sequence similarity to the original uncoupling protein-1 (UCP1), which is localized only in brown adipose tissue. Using UCP1-ablated mice, we examined whether any adaptive nonshivering thermogenesis could be recruited by acclimation to cold. Remarkably, by successive acclimation, the UCP1-ablated mice could be made to subsist for several weeks at 4C during which they had to constantly produce heat at four times their resting levels. Despite these extreme requirements for adaptive nonshivering thermogenesis, however, no substitution of shivering by any adaptive nonshivering thermogenic process occurred. Thus, although the existence of, for example, muscular mechanisms for adaptive nonshivering thermogenesis has recurrently been implied, we did not find any indication of such thermogenesis. Not even during prolonged and enhanced demand for extra heat production was any endogenous hormone or neurotransmitter able to recruit any UCP1-independent adaptive nonshivering thermogenic process in muscle or in any other organ, and no proteins other than UCP1-not even UCP2 or UCP3-therefore have the ability to mediate adaptive nonshivering thermogenesis in the cold.     

Convertible adipose tissue in mice

Summary Ability to express uncoupling protein (UCP) and establish UCP-dependent thermogenesis was analyzed in anatomical areas of mice that are generally considered to be white adipose tissue: mesenterial, perimetral, epididymal, inguinal, and superficial layer of interscapular white adipose tissue. The mice were acclimatized for 1 week to 4° C; the following week they were exposed to cold stress (1 h at-20° C, 2–3 times daily). In such conditions in inguinal adipose tissue, slot-blot analysis detected significant amount of UCP mRNA and lipoprotein lipase mRNA. Immuno-electron-microscopic localization of UCP showed that developed mitochondria of cold-stressed inguinal adipocytes contained UCP in the same amount as uncoupled (UC)-mitochondria of brown adipocytes. Morphological and morphometrical analysis showed that such inguinal adipose tissue appeared as brown adipose tissue. Since in control mice, inguinal adipose tissue was UCP-negative and tissue appeared as white adipose tissue, the duration of this white-to-brown adipose tissue conversion was analyzed. Mice, cold stressed for 1 week, were rewarmed at 28° C and their inguinal adipose tissue was analyzed in comparison with interscapular brown adipose tissue and epididymal white adipose tissue for another 37 days. During that time inguinal adipocytes ceased expressing UCP mRNA; UC-mitochondria in inguinal adipocytes were destroyed and replaced with common, C-mitochondria; and UCP was undetectable immunohistochemically. Adipocytes accumulated lipids, and the tissue morphologically once again resembled white adipose tissue. Described changes showed that besides typical brown and white adipose tissue in mice, there existed a third type of adipose tissue described as convertible adipose tissue.     

β₁-Adrenergic receptors increase UCP1 in human MADS brown adipocytes and rescue cold-acclimated β₃-adrenergic receptor-knockout mice via nonshivering thermogenesis

With the finding that brown adipose tissue is present and negatively correlated to obesity in adult man, finding the mechanism(s) of how to activate brown adipose tissue in humans could be important in combating obesity, type 2 diabetes, and their complications. In mice, the main regulator of nonshivering thermogenesis in brown adipose tissue is norepinephrine acting predominantly via β(3)-adrenergic receptors. However, vast majorities of β(3)-adrenergic agonists have so far not been able to stimulate human β(3)-adrenergic receptors or brown adipose tissue activity, and it was postulated that human brown adipose tissue could be regulated instead by β(1)-adrenergic receptors. Therefore, we have investigated the signaling pathways, specifically pathways to nonshivering thermogenesis, in mice lacking β(3)-adrenergic receptors. Wild-type and β(3)-knockout mice were either exposed to acute cold (up to 12 h) or acclimated for 7 wk to cold, and parameters related to metabolism and brown adipose tissue function were investigated. β(3)-knockout mice were able to survive both acute and prolonged cold exposure due to activation of β(1)-adrenergic receptors. Thus, in the absence of β(3)-adrenergic receptors, β(1)-adrenergic receptors are effectively able to signal via cAMP to elicit cAMP-mediated responses and to recruit and activate brown adipose tissue. In addition, we found that in human multipotent adipose-derived stem cells differentiated into functional brown adipocytes, activation of either β(1)-adrenergic receptors or β(3)-adrenergic receptors was able to increase UCP1 mRNA and protein levels. Thus, in humans, β(1)-adrenergic receptors could play an important role in regulating nonshivering thermogenesis.     

Brown adipose tissue dynamics in wild-type and UCP1-knockout mice: in vivo insights with magnetic resonance

We used noninvasive magnetic resonance imaging (MRI) and magnetic resonance spectroscopy to compare interscapular brown adipose tissue (iBAT) of wild-type (WT) and uncoupling protein 1 (UCP1)-knockout mice lacking UCP1-mediated nonshivering thermogenesis (NST). Mice were sequentially acclimated to an ambient temperature of 30°C, 18°C, and 5°C. We detected a remodeling of iBAT and a decrease in its lipid content in all mice during cold exposure. Ratios of energy-rich phosphates (ATP/ADP, phosphocreatine/ATP) in iBAT were maintained stable during noradrenergic stimulation of thermogenesis in cold- and warm-adapted mice and no difference between the genotypes was observed. As free fatty acids (FFAs) serve as fuel for thermogenesis and activate UCP1 for uncoupling of oxidative phosphorylation, brown adipose tissue is considered to be a main acceptor and consumer of FFAs. We measured a major loss of FFAs from iBAT during noradrenergic stimulation of thermogenesis. This mobilization of FFAs was observed in iBAT of WT mice as well as in mice lacking UCP1. The high turnover and the release of FFAs from iBAT suggests an enhancement of lipid metabolism, which in itself contributes to the sympathetically activated NST and which is independent from uncoupled respiration mediated by UCP1. Our study demonstrates that MRI, besides its potential for visualizing and quantification of fat tissue, is a valuable tool for monitoring functional in vivo processes like lipid and phosphate metabolism during NST.     

Absence of adaptive nonshivering thermogenesis in a marsupial,the fat-tailed dunnart (<Emphasis Type="Italic">Sminthopsis crassicaudata</Emphasis>)

The presence of nonshivering thermogenesis in marsupials is controversially debated. Survival of small eutherian species in cold environments is crucially dependent on uncoupling protein 1 (UCP1)-mediated, adaptive nonshivering thermogenesis that is executed in brown adipose tissue. In a small dasyurid marsupial species, the fat-tailed dunnart (Sminthopsis crassicaudata), an orthologue of UCP1 has been recently identified which is upregulated during cold exposure resembling adaptive molecular adjustments of eutherian brown adipose tissue. Here, we tested for a thermogenic function of marsupial brown adipose tissue and UCP1 by evaluating the capacity of nonshivering thermogenesis in cold-acclimated dunnarts. In response to an optimal dosage of noradrenaline, cold-acclimated dunnarts (12°C) showed no additional recruitment of noradrenaline-induced maximal thermogenic capacity in comparison to warm-acclimated dunnarts (24°C). While no differences in body temperature were observed between the acclimation groups, basal metabolic rate was significantly elevated after cold acclimation. Therefore, we suggest that adaptive nonshivering thermogenesis does not occur in this marsupial species despite the cold recruitment of oxidative capacity and UCP1 in the interscapular fat deposit. In conclusion, the ancient UCP orthologue in marsupials does not contribute to the classical nonshivering thermogenesis, and may exhibit a different physiological role.     

Absence of UCP3 in brown adipose tissue does not impair nonshivering thermogenesis

We report on a novel Djungarian hamster mutant lineage that exhibits a loss of uncoupling protein (UCP) 3 mRNA and protein in brown adipose tissue (BAT), whereas UCP3 expression in skeletal muscle is only mildly diminished. In response to 2 d of cold exposure, UCP3 mRNA was 4.5-fold elevated in BAT of wild-type hamsters but remained undetectable in mutant hamsters. Notably, in BAT of warm- and cold-exposed mutant hamsters, UCP1 and UCP2 mRNA levels were increased. The tissue specificity of UCP3 deficiency suggests that the underlying unknown mutation impairs a factor controlling UCP3 gene expression selectively in brown adipocytes. In wild-type but not mutant primary brown adipocytes, UCP3 gene expression was stimulated by treatment with peroxisome proliferator activated receptor (PPAR) ligands. This implies that the underlying mutation causing UCP3 deficiency is expressed within brown adipocytes and disrupts PPAR-dependent transactivation of the UCP3 gene. On the functional level, we found no direct phenotypic consequences of altered UCP expression in BAT. The absence of UCP3 in BAT of cold-acclimated mutant hamsters affected neither maximal nonshivering thermogenesis elicited by noradrenaline nor the uncoupled respiration of isolated mitochondria in the presence of oligomycin and in response to palmitate.     

Uncoupling Protein 3: Its Possible Biological Role and Mode of Regulation in Rodents and Humans

The recently discovered uncoupling protein 3 (UCP3) is highly homologous to the mitochondrialinner membrane protein UCP1, which generates heat by uncoupling the respiratory chainfrom oxidative phosphorylation. The thermogenic function of UCP1 protects against cold andregulates the energy balance in rodents. We review in vitro studies investigating the uncouplingactivity of UCP3 and in vivo studies, which address UCP3 gene expression in brown adiposetissue and skeletal muscle under various metabolic conditions. The data presented are, for themost, consistent with an uncoupling role for UCP3 in regulatory thermogenesis. We alsodiscuss mediators of UCP3 regulation and propose a potential role for intracellular fatty acidsin the mechanism of UCP3 modulation. Finally, we hypothesize a role for UCP3 in themetabolic adaptation of the mitochondria to the degradation of fatty acids.     

UCP1 is essential for adaptive adrenergic nonshivering thermogenesis

Participation of brown adipose tissue [through the action of the uncoupling protein-1 (UCP1)] in adaptive adrenergic nonshivering thermogenesis is recognized, but the existence of a response to adrenergic stimulation in UCP1-ablated mice implies that a mechanism for an alternative adaptive adrenergic thermogenesis may exist. Here, we have used UCP1-ablated mice to examine the existence of an alternative adaptive adrenergic nonshivering thermogenesis, examined as the oxygen consumption response to systemically injected norepinephrine into anesthetized or conscious mice acclimated to different temperatures. We confirm that UCP1-dependent adrenergic nonshivering thermogenesis is adaptive, but we demonstrate that the adrenergic UCP1-independent thermogenesis is not recruitable by cold acclimation. Thus, at least in the mouse, no other proteins or enzymatic pathways exist that can participate in or with time take over the UCP1 mediation of adaptive adrenergic nonshivering thermogenesis, even in the total absence of UCP1. UCP1 is thus the only protein capable of mediating cold acclimation-recruited adaptive adrenergic nonshivering thermogenesis.     

White adipose tissue contributes to UCP1-independent thermogenesis

Beta3-adrenergic receptors (AR) are nearly exclusively expressed in brown and white adipose tissues, and chronic activation of these receptors by selective agonists has profound anti-diabetes and anti-obesity effects. This study examined metabolic responses to acute and chronic beta3-AR activation in wild-type C57Bl/6 mice and congenic mice lacking functional uncoupling protein (UCP)1, the molecular effector of brown adipose tissue (BAT) thermogenesis. Acute activation of beta3-AR doubled metabolic rate in wild-type mice and sharply elevated body temperature and BAT blood flow, as determined by laser Doppler flowmetry. In contrast, beta3-AR activation did not increase BAT blood flow in mice lacking UCP1 (UCP1 KO). Nonetheless, beta3-AR activation significantly increased metabolic rate and body temperature in UCP1 KO mice, demonstrating the presence of UCP1-independent thermogenesis. Daily treatment with the beta3-AR agonist CL-316243 (CL) for 6 days increased basal and CL-induced thermogenesis compared with naive mice. This expansion of basal and CL-induced metabolic rate did not require UCP1 expression. Chronic CL treatment of UCP1 KO mice increased basal and CL-stimulated metabolic rate of epididymal white adipose tissue (EWAT) fourfold but did not alter BAT thermogenesis. After chronic CL treatment, CL-stimulated thermogenesis of EWAT equaled that of interscapular BAT per tissue mass. The elevation of EWAT metabolism was accompanied by mitochondrial biogenesis and the induction of genes involved in lipid oxidation. These observations indicate that chronic beta3-AR activation induces metabolic adaptation in WAT that contributes to beta3-AR-mediated thermogenesis. This adaptation involves lipid oxidation in situ and does not require UCP1 expression.     

Blockade of the activin receptor IIb activates functional brown adipogenesis and thermogenesis by inducing mitochondrial oxidative metabolism

Brown adipose tissue (BAT) is a key tissue for energy expenditure via fat and glucose oxidation for thermogenesis. In this study, we demonstrate that the myostatin/activin receptor IIB (ActRIIB) pathway, which serves as an important negative regulator of muscle growth, is also a negative regulator of brown adipocyte differentiation. In parallel to the anticipated hypertrophy of skeletal muscle, the pharmacological inhibition of ActRIIB in mice, using a neutralizing antibody, increases the amount of BAT without directly affecting white adipose tissue. Mechanistically, inhibition of ActRIIB inhibits Smad3 signaling and activates the expression of myoglobin and PGC-1 coregulators in brown adipocytes. Consequently, ActRIIB blockade in brown adipose tissue enhances mitochondrial function and uncoupled respiration, translating into beneficial functional consequences, including enhanced cold tolerance and increased energy expenditure. Importantly, ActRIIB inhibition enhanced energy expenditure only at ambient temperature or in the cold and not at thermoneutrality, where nonshivering thermogenesis is minimal, strongly suggesting that brown fat activation plays a prominent role in the metabolic actions of ActRIIB inhibition.     

Cold-induced changes in brown adipose tissue thermogenic capacity of immunocompetent and immunodeficient hairless mice

Mild cold acclimation (22°C, 3 weeks) of hairless mice was shown to increase 5-fold the brown adipose tissue uncoupling protein content in immunodeficient BALB/c nu/nu mice, but by only 2.3-fold in immunocompetent BFU mice. The difference in activation of brown adipose tissue thermogenic capacity was due to a 2-fold increase in the content of brown adipose tissue in nu/nu mice only, which was paralleled by an increase in brown adipose tissue protein but not DNA content. Likewise, only in nu/nu mice the cold acclimation increased the reaction of natural killer cells in blood and peritoneal exudate with a shift from spleen to lymph nodes and increased the phagocytic index. The results indicate that the immune system may influence the defence against cold at the level of brown adipose tissue thermogenesis.Abbreviations AU arbitrary unit(s) - bw body weight - HEMA 2-hydromethyl-metacrylate copolymer - BAT brown adipose tissue - UCP uncoupling protein - ATPase mitochondrial F_oF₁-ATPsynthase - IL-1 interleukin 1 - TNF tumour necrosis factor - NK cells natural killer cells - T _a ambient temperature     

Cold-induced mitochondrial uncoupling and expression of chicken UCP and ANT mRNA in chicken skeletal muscle

Although bird species studied thus far have no distinct brown adipose tissue (BAT) or a related thermogenic tissue, there is now strong evidence that non-shivering mechanisms in birds may play an important role during cold exposure. Recently, increased expression of the duckling homolog of the avian uncoupling protein (avUCP) was demonstrated in cold-acclimated ducklings [Raimbault et al., Biochem. J. 353 (2001) 441-444]. Among the mitochondrial anion carriers, roles for the ATP/ADP antiporter (ANT) as well as UCP variants in thermogenesis are proposed. The present experiments were conducted (i) to examine the effects of cold acclimation on the fatty acid-induced uncoupling of oxidative phosphorylation in skeletal muscle mitochondria and (ii) to clone the cDNA of UCP and ANT homologs from chicken skeletal muscle and study differences compared to controls in expression levels of their mRNAs in the skeletal muscle of cold-acclimated chickens. The results obtained here show that suppression of palmitate-induced uncoupling by carboxyatractylate was greater in the subsarcolemmal skeletal muscle mitochondria from cold-acclimated chickens than that for control birds. An increase in mRNA levels of avANT and, to lesser degree, of avUCP in the skeletal muscle of cold-acclimated chickens was also found. Taken together, the present studies on cold-acclimated chickens suggest that the simultaneous increments in levels of avANT and avUCP mRNA expression may be involved in the regulation of thermogenesis in skeletal muscle.     

Energy metabolism in uncoupling protein 3 gene knockout mice

Uncoupling protein 3 (UCP3) is a member of the mitochondrial anion carrier superfamily. Based upon its high homology with UCP1 and its restricted tissue distribution to skeletal muscle and brown adipose tissue, UCP3 has been suggested to play important roles in regulating energy expenditure, body weight, and thermoregulation. Other postulated roles for UCP3 include regulation of fatty acid metabolism, adaptive responses to acute exercise and starvation, and prevention of reactive oxygen species (ROS) formation. To address these questions, we have generated mice lacking UCP3 (UCP3 knockout (KO) mice). Here, we provide evidence that skeletal muscle mitochondria lacking UCP3 are more coupled (i.e. increased state 3/state 4 ratio), indicating that UCP3 has uncoupling activity. In addition, production of ROS is increased in mitochondria lacking UCP3. This study demonstrates that UCP3 has uncoupling activity and that its absence may lead to increased production of ROS. Despite these effects on mitochondrial function, UCP3 does not seem to be required for body weight regulation, exercise tolerance, fatty acid oxidation, or cold-induced thermogenesis. The absence of such phenotypes in UCP3 KO mice could not be attributed to up-regulation of other UCP mRNAs. However, alternative compensatory mechanisms cannot be excluded. The consequence of increased mitochondrial coupling in UCP3 KO mice on metabolism and the possible role of yet unidentified compensatory mechanisms, remains to be determined.