Unmasking of GDP binding sites on hamster brown adipose tissue mitochondria and uncoupling protein.

1. A rapid unmasking of GDP binding sites on brown adipose tissue (BAT) mitochondria was observed when hamsters acclimatized to 28 degrees C were exposed to a temperature of 4 degrees C for 2 hr. 2. No rapid unmasking of GDP binding sites was observed when hamsters housed at 22 degrees C were briefly exposed to 4 degrees C. 3. The amount of GDP bound to BAT mitochondria from hamsters increased during 2 weeks of exposure to 4 degrees C, but did not change between 2 weeks and 30 days of cold exposure. 4. Incubation of mitochondria with 10 mM Mg2+ prior to the GDP binding assay increased the subsequent GDP binding to BAT mitochondria from hamsters housed at 28, 22 or 4 degrees C, albeit to different degrees. 5. The amount of GDP bound to uncoupling proteins isolated from untreated and Mg(2+)-treated mitochondria of hamsters and rats was measured. Scatchard analyses of the binding of GDP to purified uncoupling protein indicate that increases in the number of binding sites due to Mg2+ treatment of mitochondria do not change the affinity of the protein for GDP.     

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.     

Cold acclimation and oxygen consumption in the thymus

Mitochondrial uncoupling protein 1 is usually associated with brown adipose tissue but has recently been discovered in rat and mouse thymus. We wished to establish whether there was a thermogenic role for UCP 1 in thymus and thus examined the effect of 5 weeks cold-acclimation on rat thymus tissue abundance, thymocyte oxygen consumption, thymus mitochondrial abundance, uncoupling protein 1 expression and function. We found that thymocytes from cold-acclimated rats had oxygen consumption rates 8 times less than those from rats held at room temperature and that thymocytes from cold-acclimated rats or rats kept at room temperature were noradrenaline insensitive. In addition, we found that thymus tissue or mitochondrial abundance was not increased after cold-acclimation. However uncoupling protein 1 expression per unit mass of mitochondria was increased after cold-acclimation, as determined by immunoblotting (approximately 1.7-fold) and GDP binding (approximately 1.5-fold). Consistent with our protein expression data, we also observed an increased, state 4 (approximately 1.5-fold), GDP-inhibitable (approximately 1.3-fold) and palmitate activatable (approximately 1.6-fold) oxygen consumption rates in isolated thymus mitochondria. However, extrapolation of our data showed that cold-acclimation only increased the amount of UCP 1 per gram of thymus tissue approximately 1.2-fold. Taken together, we conclude that UCP 1 does not have a thermogenic role in thymus.     

GDP在体外对大鼠脑线粒体脱耦联蛋白活性和表达的影响

本研究通过GDP体外处理大鼠脑组织块,观察GDP对脑线粒体脱耦联蛋白(uncoupling proteins,UCPs)活性、UCP4和UCP5表达的影响,以探讨嘌呤核苷酸对大鼠脑UCPs的调节作用.取Sprague-Dawley大鼠双侧大脑半球,将脑组织切成约8-10 mm3的脑组织块,与含1 mmol/L GDP的孵育介质共孵育30 min后,匀浆并差速离心分离提取大鼠脑组织线粒体,采用[3H]-GTP结合法测定UCPs活性,并以Scatehard作图法计算两者结合的解离常数(Kd)和最大结合量(Bmax);RT-PCR和Western blot分别检测UCP4和UCP5的mRNA和蛋白表达.结果显示,1 mmol/L GDP可降低体外大鼠脑组织线粒体中UCPs与[3H]-GTP结合的Bmax,提高Kd,但对脑纰织中UCP4和UCP5 mRNA和蛋白表达量的改变无统计学意义.上述结果提示,GDP可直接抑制体外大鼠脑组织中UCPs的活性,但并不影响UCP4和UCP5的表达.     

Are the short-photoperiod-induced decreases in serum prolactin responsible for the seasonal changes in energy balance in Syrian and Siberian hamsters?

Serum prolactin (PRL) decreases in Syrian (Mesocricetus auratus) and Siberian (Phodopus sungorus sungorus) hamsters following short-photoperiod exposure. Both species also exhibit short-photoperiod-induced changes in body and lipid mass, but in opposite directions; Syrian hamsters increase and Siberian hamsters decrease their body weight, changes reflected nearly exclusively in their carcass lipid content. The purpose of these experiments was to determine whether the photoperiod-induced changes in PRL were responsible for the seasonal changes in energy balance in Syrian and Siberian hamsters by using the strategy of experimentally producing serum PRL levels opposite to those normally associated with the photoperiod in which the animals were housed. In long photoperiods serum PRL was reduced to short-day levels by subcutaneous (s.c.) CB-154 (bromoergocryptine, a dopamine agonist) injections. In short photoperiods, serum PRL was elevated to long-day levels in Syrian hamsters by ectopic pituitary explants, and in Siberian hamsters, serum PRL was elevated by chronic s.c. infusions of ovine PRL (oPRL). In both species, manipulations of serum PRL did not affect food intake, carcass composition, or the wet weight of various white and brown adipose tissue pads (WAT and BAT, respectively). Body weight increased in CB-154-treated Syrian hamsters and decreased in Siberian hamsters, an effect partially reversed by coadministration of oPRL in Syrian, but not Siberian, hamsters. Thus, lowering serum PRL to short-day levels in long-day-housed hamsters of both species mimicked the directional change in body weight appropriate for each species when they are exposed to short days. This effect of CB-154 on body weight may be a result of some as yet unidentified effect of dopaminergic stimulation on overall growth since 1) these changes in body weight were not reflected as changes in lipid mass, as occurs naturally following short-day exposure for each species, and 2) neither species exhibited a reciprocal change in body weight when serum PRL was experimentally elevated in short days. Alternatively, it may be that once the energetic responses to short-day exposure have been fully expressed, the ability of PRL to stimulate the target sites of action for PRL for these responses may be decreased. BAT protein content, cytochrome oxidase activity (measures of metabolic growth of this tissue), and retroperitoneal total and specific lipoprotein lipase (LPL) activities were increased by CB-154 treatment in Syrian hamsters.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased GDP binding and thermogenic activity in brown adipose tissue mitochondria during arousal of the hibernating garden dormouse (Eliomys quercinus L.).

1. The thermogenic activity of brown adipose tissue in hibernating garden dormice during hypothermic torpor and at different states of arousal were studied. High levels of GDP binding were observed on isolated brown fat mitochondria, indicating that the thermogenic proton conductance pathway is very active in brown fat during arousal. 2. In order to investigate this phenomenon, the uncoupling protein was assessed by immunological assay and the mRNA for UCP was analysed. 3. Animals during arousal exhibited neither increase in UCPmRNA nor an increase in UCP. 4. Our results suggest that during the rewarming of garden dormice there is an acute unmasking of GDP binding sites on the protein.     

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.     

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 approximately 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 degrees C and that UCP 3 expression is decreased (1.4-fold) in rats acclimated to 30 degrees 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.     

Developmental changes in uncoupling protein 1 and F1-ATPase subunit levels in the golden hamster brown adipose tissue mitochondria as determined by electron microscopy in situ immunocytochemistry

The postnatal developmental changes in mitochondrial uncoupling protein 1 (UCP 1) and F1-ATP synthase (ATPase) subunit levels in the interscapular brown adipose tissue (BAT) were studied in golden Syrian hamsters (Mesocricetus auratus) using electron microscopy in situ immunocytochemistry. The relatively low initial density of 5 nm gold conjugated anti-UCP 1 immunocomplexes gradually increased from 7- to 21-day-old animals and numerous immunocomplexes were found on the mitochondrial membranes of adult hamsters. At the age of 7-9 days, a positive reaction was also detected in the cytoplasm of BAT adipocytes. Immunolocalization of F1-ATPase subunit indicated its presence in BAT mitochondria and cytoplasm of 7- to 9-day-old animals. However, contrary to UCP 1, intensity of the immunostaining of F1-ATPase subunit rapidly decreased both in mitochondria and cytoplasm between the 10th and 21st postnatal day and it became stabilized in adult animals at a very low level restricted to mitochondria. These results confirm that profound changes in the enzymatic apparatus of BAT mitochondrial membranes, leading to formation of thermogenic mitochondria, occur not until the early postnatal period of hamster ontogenetic development.     

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.     

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

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

Cold acclimation and oxygen consumption in the thymus

Mitochondrial uncoupling protein 1 is usually associated with brown adipose tissue but has recently been discovered in rat and mouse thymus. We wished to establish whether there was a thermogenic role for UCP 1 in thymus and thus examined the effect of 5 weeks cold-acclimation on rat thymus tissue abundance, thymocyte oxygen consumption, thymus mitochondrial abundance, uncoupling protein 1 expression and function. We found that thymocytes from cold-acclimated rats had oxygen consumption rates 8 times less than those from rats held at room temperature and that thymocytes from cold-acclimated rats or rats kept at room temperature were noradrenaline insensitive. In addition, we found that thymus tissue or mitochondrial abundance was not increased after cold-acclimation. However uncoupling protein 1 expression per unit mass of mitochondria was increased after cold-acclimation, as determined by immunoblotting (∼ 1.7-fold) and GDP binding (∼ 1.5-fold). Consistent with our protein expression data, we also observed an increased, state 4 (∼ 1.5-fold), GDP-inhibitable (∼ 1.3-fold) and palmitate activatable (∼ 1.6-fold) oxygen consumption rates in isolated thymus mitochondria. However, extrapolation of our data showed that cold-acclimation only increased the amount of UCP 1 per gram of thymus tissue ∼ 1.2-fold. Taken together, we conclude that UCP 1 does not have a thermogenic role in thymus.     

Synthesis of mitochondrial uncoupling protein in brown adipocytes differentiated in cell culture

In order to characterize the biogenesis of unique thermogenic mitochondria of brown adipose tissue, differentiation of precursor cells isolated from mouse brown adipose tissue was studied in cell culture. Synthesis of mitochondrial uncoupling protein (UCP), F1-ATPase, and cytochrome oxidase was examined by L-[35S]methionine labeling and immunoblotting. For the first time, synthesis of physiological amounts of the UCP, a key and tissue-specific component of thermogenic mitochondria, was observed in cultures at about confluence (day 6), indicating that a complete differentiation of brown adipocytes was achieved in vitro. In postconfluent cells (day 8) the content of UCP decreased rapidly, in contrast to some other mitochondrial proteins (beta subunit of F1-ATPase, cytochrome oxidase). In these cells, it was possible, by using norepinephrine, to induce specifically the synthesis of the UCP but not of F1-ATPase or cytochrome oxidase. The maximal response was observed at 0.1 microM norepinephrine and the synthesis of UCP remained activated for at least 24 h. Detailed analysis revealed a major role of the beta-adrenergic receptors and elevated intracellular concentration of cAMP in stimulation of UCP synthesis. A quantitative recovery of the newly synthesized UCP in the mitochondrial fraction indicated completed biogenesis of functionally competent thermogenic mitochondria.     

Effects of fasting and food restriction on brown adipose tissue composition in normal and dystrophic hamsters

Fasting for 36-48 h or food restriction (30% reduction of daily food intake for 6 weeks) caused brown adipose tissue (BAT) atrophy in hamsters. Fasting-induced atrophy was characterized by reductions in tissue mass, DNA, protein, and thermogenin. By contrast, food restriction had no effect on tissue cellularity (DNA) but markedly reduced the tissue protein and thermogenin contents. The concentration of thermogenin in isolated mitochondria was unchanged by fasting or food restriction. Dystrophic hamsters had a reduced BAT mass when compared with weight-matched control hamsters. This resulted from a reduction in tissue cellularity since BAT DNA, protein and thermogenin contents were all reduced. The extent of binding of [3H]guanosine diphosphate to isolated mitochondria and their content of thermogenin were similar in normal and dystrophic hamsters. In response to cold exposure, as in normal hamsters, BAT of dystrophic hamsters grew and the tissue thermogenin increased, but the mitochondrial concentration of thermogenin did not change. In response to fasting, in contrast with normal hamsters, there was no significant reduction in BAT DNA in dystrophic animals and the loss of tissue protein was reduced. However, the relative changes in BAT composition during chronic food restriction were similar in normal and dystrophic animals. Thus, reduction in hamster BAT thermogenic capacity during food deprivation may occur by loss of cells and (or)reduction in the tissue protein and thermogenin contents. The extent of protein and (or) DNA loss may be dependent upon the original tissue mass and the severity of food deprivation.     

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.     

Sequential changes in brown adipose tissue composition, cytochrome oxidase activity and GDP binding throughout pregnancy and lactation in the rat

The sequential appearance of changes in interscapular brown adipose tissue composition, cytochrome oxidase activity and GDP binding was studied throughout pregnancy and lactation in the rat. Brown adipose tissue was hypertrophied during pregnancy because of progressive lipid accumulation, whereas its mitochondrial component and GDP binding to brown fat mitochondria were unchanged. In early lactation (day 5) there was a decrease in the overall GDP binding to brown fat only because of the lower mitochondrial protein content. In late stages of lactation (days 10 and 15), the amount of tissue and its mitochondrial protein content were minimal and the GDP binding per mitochondrial protein decreased substantially. Scatchard analysis in day-15-lactating rats indicated a large decrease in GDP binding sites without any changes in affinity. It is concluded that the diminished thermogenic activity of brown fat in lactation is attained through changes at different structural levels of the tissue occurring in a characteristic sequential trend; first a reduction in its mitochondrial component, and only later, at mid-lactation, a decrease in the specific mitochondrial proton conductance pathway activity.     

Development of Phodopus sungorus brown preadipocytes in primary cell culture: effect of an atypical beta-adrenergic agonist, insulin, and triiodothyronine on differentiation, mitochondrial development, and expression of the uncoupling protein UCP

A new cellular model for the study of brown adipocyte development and differentiation in vitro is presented. Preadipocytes isolated from brown adipose tissue (BAT) of the djungarian dwarf hamster Phodopus sungorus are able to proliferate and differentiate in vitro into true brown adipocytes able to express the BAT marker protein the uncoupling protein (UCP). Whereas basal UCP expression is very low, its mRNA levels as well as the UCP detected by immunoblotting are highly increased by beta-adrenergic stimulation. The novel, atypical beta-adrenergic compound D7114 (ICI Pharmaceuticals, Macclesfield, Cheshire, England) was found to increase the number of adipocytes as well as UCP mRNA and UCP content of mitochondria, indicating the involvement of an atypical or beta 3 receptor. Insulin was found to play an important role in brown adipocyte differentiation and mitochondrial development, whereas T3 seemed to be implicated more directly in UCP expression. In a defined, serum-free medium a synergistic stimulatory action of insulin and T3 on UCP expression was found, which seems to involve a pathway different from that of beta-adrenergic UCP stimulation.     

Functional characterization of UCP1 in mammalian HEK293 cells excludes mitochondrial uncoupling artefacts and reveals no contribution to basal proton leak

Mechanistic studies on uncoupling proteins (UCPs) not only are important to identify their cellular function but also are pivotal to identify potential drug targets to manipulate mitochondrial energy transduction. So far, functional and comparative studies of uncoupling proteins in their native environment are hampered by different mitochondrial, cellular and genetic backgrounds. Artificial systems such as yeast ectopically expressing UCPs or liposomes with reconstituted UCPs were employed to address crucial mechanistic questions but these systems also produced inconsistencies with results from native mitochondria. We here introduce a novel mammalian cell culture system (Human Embryonic Kidney 293 - HEK293) to study UCP1 function. Stably transfected HEK293 cell lines were derived that contain mouse UCP1 at concentrations comparable to tissue mitochondria. In this cell-based test system UCP1 displays native functional behaviour as it can be activated with fatty acids (palmitate) and inhibited with purine nucleotides guanosine-diphosphate (GDP). The catalytic centre activity of the UCP1 homodimer in HEK293 is comparable to activities in brown adipose tissue supporting functionality of UCP1. Importantly, at higher protein levels than in yeast mitochondria, UCP1 in HEK293 cell mitochondria is fully inhibitable and does not contribute to basal proton conductance, thereby emphasizing the requirement of UCP1 activation for therapeutic purposes. These findings and resulting analysis on UCP1 characteristics demonstrate that the mammalian HEK293 cell system is suitable for mechanistic and comparative functional studies on UCPs and provides a non-confounding mitochondrial, cellular and genetic background.     

Effects of neonatal sympathectomy on brown fat development and susceptibility to high fat diet induced obesity in mice.

Injections of 6-hydroxydopamine in mouse neonates caused extensive and long lasting damage to the sympathetic nervous system and impaired brown fat development. Brown adipose tissue (BAT) thermogenic capacity of sympathectomized mice (up to 120 days old) was reduced because of marked reductions in the tissue mitochondrial protein content and the mitochondrial concentration of uncoupling protein, as assessed by [3H]GDP binding and immunoassay. Neonatal sympathectomy did not affect BAT DNA content. Sympathectomized mice also had reduced epinephrine-stimulated rates of oxygen consumption. BAT of sympathectomized mice failed to respond by increases in [3H]GDP binding to isolated mitochondria and uncoupling protein concentration when animals were offered a palatable high-fat dietary supplement that increased calorie intake of both normal and sympathectomized mice. The high-fat diet caused increases in body weight, carcass fat, and gonadal white fat pad weights in sympathectomized animals that were similar to those of control mice. These results show that inactivation of BAT metabolism did not accentuate the development of obesity caused by a dietary supplement rich in fat and suggest that stimulation of BAT metabolism was not very effective in counteracting the obesity-inducing effect of this diet.