Uncoupling Protein 1 Decreases Superoxide Production in Brown Adipose Tissue Mitochondria

In thermogenic brown adipose tissue, uncoupling protein 1 (UCP1) catalyzes the dissipation of mitochondrial proton motive force as heat. In a cellular environment of high oxidative capacity such as brown adipose tissue (BAT), mitochondrial uncoupling could also reduce deleterious reactive oxygen species, but the specific involvement of UCP1 in this process is disputed. By comparing brown adipose tissue mitochondria of wild type mice and UCP1-ablated litter mates, we show that UCP1 potently reduces mitochondrial superoxide production after cold acclimation and during fatty acid oxidation. We address the sites of superoxide production and suggest diminished probability of “reverse electron transport” facilitated by uncoupled respiration as the underlying mechanism of reactive oxygen species suppression in BAT. Furthermore, ablation of UCP1 represses the cold-stimulated increase of substrate oxidation normally seen in active BAT, resulting in lower superoxide production, presumably avoiding deleterious oxidative damage. We conclude that UCP1 allows high oxidative capacity without promoting oxidative damage by simultaneously lowering superoxide production.     

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.     

Cold tolerance of UCP1-ablated mice: A skeletal muscle mitochondria switch toward lipid oxidation with marked UCP3 up-regulation not associated with increased basal,fatty acid- or ROS-induced uncoupling or enhanced GDP effects

Mice lacking the thermogenic mitochondrial membrane protein UCP1 (uncoupling protein 1) - and thus all heat production from brown adipose tissue - can still adapt to a cold environment (4 °C) if successively transferred to the cold. The mechanism behind this adaptation has not been clarified. To examine possible adaptive processes in the skeletal muscle, we isolated mitochondria from the hind limb muscles of cold-acclimated wild-type and UCP1(–/–) mice and examined their bioenergetic chracteristics. We observed a switch in metabolism, from carbohydrate towards lipid catabolism, and an increased total mitochondrial complement, with an increased total ATP production capacity. The UCP1(–/–) muscle mitochondria did not display a changed state-4 respiration rate (no uncoupling) and were less sensitive to the uncoupling effect of fatty acids than the wild-type mitochondria. The content of UCP3 was increased 3-4 fold, but despite this, endogenous superoxide could not invoke a higher proton leak, and the small inhibitory effect of GDP was unaltered, indicating that it was not mediated by UCP3. Double mutant mice (UCP1(–/–) plus superoxide dismutase 2-overexpression) were not more cold sensitive than UCP1(–/–), bringing into question an involvement of reactive oxygen species (ROS) in activation of any alternative thermogenic mechanism. We conclude that there is no evidence for an involvement of UCP3 in basal, fatty-acid- or superoxide-stimulated oxygen consumption or in GDP sensitivity. The adaptations observed did not imply any direct alternative process for nonshivering thermogenesis but the adaptations observed would be congruent with adaptation to chronically enhanced muscle activity caused by incessant shivering in these mice.     

Is the "mammalian" brown fat-specific mitochondrial uncoupling protein present in adipose tissues of birds?

1. Mitochondria were isolated from the furcular, subcutaneous, abdominal, nape and lateral adipose tissue depots of five species of bird (pheasant, Japanese quail, pigeon, house sparrow and great tit) acclimatized to the Northern winter. 2. Mitochondrial proteins were separated by SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes, and probed for the presence of the 32,000-33,000 Mr uncoupling protein characteristic of "mammalian" brown adipose tissue, using an anti-(ground squirrel uncoupling protein)serum. 3. Immunoreactivity consistent with uncoupling protein was not detected in mitochondria from any of the avian adipose tissues. Immunoreactivity was, however, evident in mitochondria from perirenal or interscapular adipose tissue from a range of mammals--rats, mice, golden hamsters, Orkney voles, wood mice, pipistrelle bats, wood lemmings, and newborn lambs, cattle, reindeer and red deer. 4. These results provide biochemical evidence that "mammalian-like" thermogenic brown adipose tissue is absent from avian species; adipose tissues in birds appear to be functionally "white".     

Cold-induced expression of the VEGF gene in brown adipose tissue is independent of thermogenic oxygen consumption

To examine whether cold-induced vascular endothelial growth factor (VEGF) gene expression in brown adipose tissue involved generation of hypoxic oxygen levels by thermogenic processes, we cold-exposed wild-type mice, as well as uncoupling protein-1 (UCP1)-ablated mice in which no thermogenesis in brown adipocytes can be induced. Cold exposure stimulated VEGF expression in both wild-type and UCP1-ablated mice. Unexpectedly, the effect was 3-fold higher in UCP1-ablated animals, whereas cultured brown adipocytes from both genotypes responded identically to norepinephrine stimulation. These results demonstrate that generation of low oxygen levels does not contribute to cold-induced VEGF expression in brown adipose tissue, but the results are consistent with an adrenergic regulation of expression.     

SIRT3, a mitochondrial sirtuin deacetylase, regulates mitochondrial function and thermogenesis in brown adipocytes

SIRT3 is one of the seven mammalian sirtuin homologs of the yeast Sir2 gene, which mediates the effect of caloric restriction on life span extension in yeast and Caenorhabditis elegans. Because adipose tissue is essential in energy homeostasis and also plays a role in life span determination, we decided to investigate the function of sirtuin members in fat. We report here that murine SIRT3 is expressed in brown adipose tissue and is localized on the mitochondria inner membrane. Caloric restriction activates SIRT3 expression in both white and brown adipose. Additionally, cold exposure up-regulates SIRT3 expression in brown fat, whereas elevated climate temperature reduces the expression. Enforced expression of SIRT3 in the HIB1B brown adipocytes enhances the expression of the uncoupling protein PGC-1alpha, UCP1, and a series of mitochondria-related genes. Both ADP-ribosyltransferase and deacetylase activities of SIRT3 are required for this action. Furthermore, the SIRT3 deacetylase mutant exhibits a dominant negative effect by inhibiting UCP1 expression. This inhibitive effect can be abolished by the coexpression of PGC-1alpha, indicating a major role of PGC-1alpha in the SIRT3 action. In addition, SIRT3 stimulates CREB phosphorylation, which reportedly activates PGC-1alpha promoter directly. Functionally, sustained expression of SIRT3 decreases membrane potential and reactive oxygen species production while increasing cellular respiration. Finally, SIRT3, along with genes related to mitochondrial function, is down-regulated in the brown adipose tissue of several genetically obese mice. In summary, our results demonstrate that SIRT3 activates mitochondria functions and plays an important role in adaptive thermogenesis in brown adipose.     

Evidence that fasting can induce a selective loss of uncoupling protein from brown adipose tissue mitochondria of mice

The effects of fasting and refeeding on the concentration of uncoupling protein in brown adipose tissue mitochondria have been investigated in mice. Fasting mice for 48 h led to a large decrease in the total cytochrome oxidase activity of the interscapular brown fat pad. Mitochondrial GDP binding and the specific mitochondrial concentration of uncoupling protein also fell on fasting. After 24 h refeeding both GDP binding and the mitochondrial concentration of uncoupling protein were normalized, but there was no alteration in the total tissue cytochrome oxidase activity. Fasting appears to induce a selective loss of uncoupling protein from brown adipose tissue mitochondria, which is rapidly reversible on refeeding.     

Lack of activation of UCP1 in isolated brown adipose tissue mitochondria by glucose-O-ω-modified saturated fatty acids of various chain lengths

We previously demonstrated that uncoupling protein 1 activity, as measured in isolated brown adipose tissue mitochondria (and as a native protein reconstituted into liposome membranes), was not activated by the non-flippable modified saturated fatty acid, glucose-O-ω-palmitate, whereas activity was stimulated by palmitate alone (40 nM free final concentration). In this study, we investigated whether fatty acid chain length had any bearing on the ability of glucose-O-ω-fatty acids to activate uncoupling protein 1. Glucose-O-ω-saturated fatty acids of various chain lengths were synthesized and tested for their potential to activate GDP-inhibited uncoupling protein 1-dependent oxygen consumption in brown adipose tissue mitochondria, and the results were compared with equivalent non-modified fatty acid controls. Here we demonstrate that laurate (12C), palmitate (16C) and stearate (18C) could activate GDP-inhibited uncoupling protein 1-dependent oxygen consumption in brown adipose tissue mitochondria, whereas there was no activation with glucose-O-ω-laurate (12C), glucose-O-ω-palmitate (16C), glucose-O-ω-stearate (18C), glucose-O-ω-arachidate (20C) or arachidate alone. We conclude that non-flippable fatty acids cannot activate uncoupling protein 1 irrespective of chain length. Our data further undermine the cofactor activation model of uncoupling protein 1 function but are compatible with the model that uncoupling protein 1 functions by flipping long-chain fatty acid anions.     

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.     

Thyroxine 5'-deiodinase in brown adipose tissue of the cynomolgus monkey Macaca fascicularis

Brown adipose tissue was identified in axillary, interscapular, subscapular, and cervical fat deposits of male and female cynomolgus monkeys (Macaca fascicularis) by histological and immunological techniques. Histology included staining of mitochondria with a Novelli stain and identification of mitochondria-rich multilocular cells. Immunological detection involved separation of homogenate proteins by sodium dodecyl sulphate--polyacrylamide gel chromatography, blotting on to nitrocellulose membranes, and identification of the specific uncoupling protein, unique to brown adipose tissue, with an antiserum to purified hamster uncoupling protein followed by detection with 125I-labelled protein A. The activity of thyroxine 5'-deiodinase in monkey brown adipose tissue homogenates was much higher than that seen previously in brown adipose tissue of rats, mice, and hamsters. This is the first demonstration of the presence of this enzyme in brown adipose tissue of a primate species.     

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.     

Antioxidant properties of UCP1 are evolutionarily conserved in mammals and buffer mitochondrial reactive oxygen species

Mitochondrial uncoupling reduces reactive oxygen species (ROS) production and appears to be important for cellular signaling/protection, making it a focus for the treatment of metabolic and age-related diseases. Whereas the physiological role of uncoupling protein 1 (UCP1) of brown adipose tissue is established for thermogenesis, the function of UCP1 in the reduction of ROS in cold-exposed animals is currently under debate. Here, we investigated the role of UCP1 in mitochondrial ROS handling in the Lesser hedgehog tenrec (Echinops telfairi), a unique protoendothermic Malagasy mammal with recently identified brown adipose tissue (BAT). We show that the reduction of ROS by UCP1 activity also occurs in BAT mitochondria of the tenrec, suggesting that the antioxidative role of UCP1 is an ancient mammalian trait. Our analysis shows that the quantity of UCP1 displays strong control over mitochondrial hydrogen peroxide release, whereas other factors, such as mild cold, nonshivering thermogenesis, oxidative capacity, and mitochondrial respiration, do not correlate. Furthermore, hydrogen peroxide release from recoupled BAT mitochondria was positively associated with mitochondrial membrane potential. These findings led to a model of UCP1 controlling mitochondrial ROS release and, presumably, being controlled by high membrane potential, as proposed in the canonical model of “mild uncoupling”. Our study further promotes a conserved role for UCP1 in the prevention of oxidative stress, which was presumably established during evolution before UCP1 was physiologically integrated into nonshivering thermogenesis.     

Carboxyatractyloside effects on brown-fat mitochondria imply that the adenine nucleotide translocator isoforms ANT1 and ANT2 may be responsible for basal and fatty-acid-induced uncoupling respectively

In brown-fat mitochondria, fatty acids induce thermogenic uncoupling through activation of UCP1 (uncoupling protein 1). However, even in brown-fat mitochondria from UCP1-/- mice, fatty-acid-induced uncoupling exists. In the present investigation, we used the inhibitor CAtr (carboxyatractyloside) to examine the involvement of the ANT (adenine nucleotide translocator) in the mediation of this UCP1-independent fatty-acid-induced uncoupling in brown-fat mitochondria. We found that the contribution of ANT to fatty-acid-induced uncoupling in UCP1-/- brown-fat mitochondria was minimal (whereas it was responsible for nearly half the fatty-acid-induced uncoupling in liver mitochondria). As compared with liver mitochondria, brown-fat mitochondria exhibit a relatively high (UCP1-independent) basal respiration ('proton leak'). Unexpectedly, a large fraction of this high basal respiration was sensitive to CAtr, whereas in liver mitochondria, basal respiration was CAtr-insensitive. Total ANT protein levels were similar in brown-fat mitochondria from wild-type mice and in liver mitochondria, but the level was increased in brown-fat mitochondria from UCP1-/- mice. However, in liver, only Ant2 mRNA was found, whereas in brown adipose tissue, Ant1 and Ant2 mRNA levels were equal. The data are therefore compatible with a tentative model in which the ANT2 isoform mediates fatty-acid-induced uncoupling, whereas the ANT1 isoform may mediate a significant part of the high basal proton leak in brown-fat mitochondria.     

Uncoupling protein in human brown adipose tissue mitochondria. Isolation and detection by specific antiserum

A protein of Mr 32 000 has been isolated from human infant brown adipose tissue mitochondria following the procedure used to purify the uncoupling protein from rat brown adipose tissue mitochondria. A specific antiserum has been raised against the human 32 kDa protein, and used to detect it by probing mitochondrial proteins separated by SDS-PAGE. The protein is present in large amounts in brown adipose tissue but is undetectable in human liver, heart or white adipose tissue. It has strong immunological cross-reactivity with rat brown adipose tissue uncoupling protein.     

Brown adipose tissue specific lack of uncoupling protein 3 is associated with impaired cold tolerance and reduced transcript levels of metabolic genes

Uncoupling protein 3 (Ucp3) is located within the mitochondrial inner membrane of brown adipose tissue and skeletal muscle. It is thought to be implicated in lipid metabolism and defense against reactive oxygen species. We previously reported on a mutation in our breeding colony of Djungarian hamsters (Phodopus sungorus) that leads to brown adipose tissue specific lack of Ucp3 expression. In this study we compared wildtype with mutant hamsters on a broad genetic background. Hamsters lacking Ucp3 in brown adipose tissue displayed a reduced cold tolerance due to impaired nonshivering thermogenesis. This phenotype is associated with a global decrease in expression of metabolic genes but not of uncoupling protein 1. These data implicate that Ucp3 is necessary to sustain high metabolic rates in brown adipose tissue.     

Number of mice per cage influences uncoupling protein content of brown adipose tissue.

The effect of housing density of mice on the thermogenic state and capacity of their brown adipose tissue was studied. Mice were housed one, two, or six per cage at 28 degrees C for 15 days. Increased housing density suppressed the thermogenic capacity of brown adipose tissue (decreased the total amount of uncoupling protein) and decreased the thermogenic state of brown adipose tissue mitochondria (decreased GDP binding). A density of six mice per cage had a greater effect than a density of two mice per cage. The size of brown adipose tissue (wet weight and protein content), the content of mitochondria in it (cytochrome oxidase content), and the total activity of thyroxine 5'-deiodinase were not altered by housing density. We conclude that even at a temperature close to thermoneutrality (29-33 degrees C for the mouse), the occurrence of social thermoregulation (huddling) reduces the requirement for brown adipose tissue thermogenesis and results in a reduction in its thermogenic capacity. It is clearly of importance that the design of studies of mouse brown adipose tissue take into account not only the temperature at which the mice are housed, but also the number of mice housed per cage.     

Brown adipose tissue mitochondria: modulation by GDP and fatty acids depends on the respiratory substrates

The UCP1 [first UCP (uncoupling protein)] that is found in the mitochondria of brown adipocytes [BAT (brown adipose tissue)] regulates the heat production, a process linked to non-shivering thermogenesis. The activity of UCP1 is modulated by GDP and fatty acids. In this report, we demonstrate that respiration and heat released by BAT mitochondria vary depending on the respiratory substrate utilized and the coupling state of the mitochondria. It has already been established that, in the presence of pyruvate/malate, BAT mitochondria are coupled by faf-BSA (fatty-acid-free BSA) and GDP, leading to an increase in ATP synthesis and mitochondrial membrane potential along with simultaneous decreases in both the rates of respiration and heat production. Oleate restores the uncoupled state, inhibiting ATP synthesis and increasing the rates of both respiration and heat production. We now show that in the presence of succinate: (i) the rates of uncoupled mitochondria respiration and heat production are five times slower than in the presence of pyruvate/malate; (ii) faf-BSA and GDP accelerate heat and respiration as a result and, in coupled mitochondria, these two rates are accelerated compared with pyruvate/malate; (iii) in spite of the differences in respiration and heat production noted with the two substrates, the membrane potential and the ATP synthesized were the same; and (iv) oleate promoted a decrease in heat production and respiration in coupled mitochondria, an effect different from that observed using pyruvate/malate. These effects are not related to the production of ROS (reactive oxygen species). We suggest that succinate could stimulate a new route to heat production in BAT mitochondria.     

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.