Changes in Ucp1, D2 (Dio2) and Glut4 (Slc2a4) mRNA expression in response to short-term cold exposure in the house musk shrew (Suncus murinus).

The house musk shrew (Suncus murinus), or so-called suncus, is a cold-intolerant mammal, but it is unclear why it is susceptible to low temperatures. Cold-intolerance may be the result of lower thermogenic activity in brown adipose tissue (BAT). The early phase of severe cold exposure is a critical period for suncus. Therefore, we exposed suncus to mildly cold temperatures (10-12 degrees C) for 1 to 48 h to increase non-shivering thermogenesis without causing stress and measured changes in the expression of uncoupling protein 1 (Ucp1), type II iodothyronine 5'-deiodinase (Dio2=D2), and glucose transporter 4 (Slc2a4=Glut4) in BAT. These mRNAs play a major role in non-shivering thermogenesis and are mainly regulated by the sympathetic nervous system via direct beta-noradrenergic innervation of BAT. During cold exposure, Ucp1 expression in BAT increased steadily over time, albeit only slightly. Neither D2 nor Glut4 expression in BAT increased immediately; however, they had increased significantly after 24 h and 48 h of cold exposure. These findings suggest that the responsiveness of mRNA regulation is weak and thus may be involved in cold-intolerance in suncus.     

Uncoupling protein1 mRNA, mitochondrial GTP-binding, and T4 5'-deiodinase of brown adipose tissue in euthermic Daurian ground squirrel during cold exposure

Regulation of thermogenic activity and uncoupling protein1 (UCP1) expression in brown adipose tissue (BAT) were studied in euthermic Daurian ground squirrel after acute and chronic cold exposure at 4 degrees C. The UCP1 concentration was indirectly determined by titration with its specific ligand [3H]-labeled GTP, and Ucp1 mRNA was detected by using a [32P]-labeled antisense oligonucleotide probe. Both acute and chronic cold exposure stimulated up-regulation of Ucp1 mRNA. Although UCP1 concentration is not significantly increased after 24 h of cold exposure, it is markedly elevated by 75% in squirrels after 4-week cold adaptation compared with controls raised at 22 degrees C. Changes in T4 5'-deiodinase activity were closely associated with variations of Ucp1 mRNA level. Ucp1 gene expression is significantly affected by cold exposure in BAT from euthermic Daurian ground squirrels. In addition, the activation of T4 5'-deiodinase may be an important regulatory factor in cold-induced Ucp1 expression.     

The molecular and biochemical basis of nonshivering thermogenesis in an African endemic mammal, Elephantulus myurus

Uncoupling protein 1 (UCP1) mediated nonshivering thermogenesis (NST) in brown adipose tissue (BAT) is an important avenue of thermoregulatory heat production in many mammalian species. Until recently, UCP1 was thought to occur exclusively in eutherians. In the light of the recent finding that UCP1 is already present in fish, it is of interest to investigate when UCP1 gained a thermogenic function in the vertebrate lineage. We elucidated the basis of NST in the rock elephant shrew, Elephantulus myurus (Afrotheria: Macroscelidea). We sequenced Ucp1 and detected Ucp1 mRNA and protein restricted to brown fat deposits. We found that cytochrome c oxidase activity was highest in these deposits when compared with liver and skeletal muscle. Consistent with a thermogenic function of UCP1 isolated BAT mitochondria showed increased state 4 respiration in the cold, as well as palmitate-induced, GDP-sensitive proton conductance, which was absent in liver mitochondria. On the whole animal level, evidence of thermogenic function was further corroborated by an increased metabolic response to norepinephrine (NE) injection. Cold acclimation (18 degrees C) led to an increased basal metabolic rate relative to warm acclimation (28 degrees C) in E. myurus, but there was no evidence of additional recruitment of NE-induced NST capacity in response to cold acclimation. In summary, we showed that BAT and functional UCP1 are already present in a member of the Afrotheria, but the seasonal regulation and adaptive value of NST in Afrotherians remain to be elucidated.     

Regulation of UCP1 and UCP3 in arctic ground squirrels and relation with mitochondrial proton leak.

Uncoupling protein (UCP) 1 (UCP1) catalyzes a proton leak in brown adipose tissue (BAT) mitochondria that results in nonshivering thermogenesis (NST), but the extent to which UCP homologs mediate NST in other tissues is controversial. To clarify the role of UCP3 in mediating NST in a hibernating species, we measured Ucp3 expression in skeletal muscle of arctic ground squirrels in one of three activity states (not hibernating, not hibernating and fasted for 48 h, or hibernating) and housed at 5 degrees C or -10 degrees C. We then compared Ucp3 mRNA levels in skeletal muscle with Ucp1 mRNA and UCP1 protein levels in BAT in the same animals. Ucp1 mRNA and UCP1 protein levels were increased on cold exposure and decreased with fasting, with the highest UCP1 levels in thermogenic hibernators. In contrast, Ucp3 mRNA levels were not affected by temperature but were increased 10-fold during fasting and >3-fold during hibernation. UCP3 protein levels were increased nearly fivefold in skeletal muscle mitochondria isolated from fasted squirrels compared with nonhibernators, but proton leak kinetics in the presence of BSA were unchanged. Proton leak in BAT mitochondria also did not differ between fed and fasted animals but did show classical inhibition by the purine nucleotide GDP. Levels of nonesterified fatty acids were highest during hibernation, and tissue temperatures during hibernation were related to Ucp1, but not Ucp3, expression. Taken together, these results do not support a role for UCP3 as a physiologically relevant mediator of NST in muscle.     

Uncoupling protein1 mRNA, mitochondrial GTP-binding, and T4 5′-deiodinase of brown adipose tissue in euthermic Daurian ground squirrel during cold exposure

Regulation of thermogenic activity and uncoupling protein1 (UCP1) expression in brown adipose tissue (BAT) were studied in euthermic Daurian ground squirrel after acute and chronic cold exposure at 4°C. The UCP1 concentration was indirectly determined by titration with its specific ligand [³H]-labeled GTP, and Ucp1 mRNA was detected by using a [³²P]-labeled antisense oligonucleotide probe. Both acute and chronic cold exposure stimulated up-regulation of Ucp1 mRNA. Although UCP1 concentration is not significantly increased after 24 h of cold exposure, it is markedly elevated by 75% in squirrels after 4-week cold adaptation compared with controls raised at 22°C. Changes in T₄ 5′-deiodinase activity were closely associated with variations of Ucp1 mRNA level. Ucp1 gene expression is significantly affected by cold exposure in BAT from euthermic Daurian ground squirrels. In addition, the activation of T₄ 5′-deiodinase may be an important regulatory factor in cold-induced Ucp1 expression.     

Mfn2 deletion in brown adipose tissue protects from insulin resistance and impairs thermogenesis

BAT‐controlled thermogenic activity is thought to be required for its capacity to prevent the development of insulin resistance. This hypothesis predicts that mediators of thermogenesis may help prevent diet‐induced insulin resistance. We report that the mitochondrial fusion protein Mitofusin 2 (Mfn2) in BAT is essential for cold‐stimulated thermogenesis, but promotes insulin resistance in obese mice. Mfn2 deletion in mice through Ucp1‐cre (BAT‐Mfn2‐KO) causes BAT lipohypertrophy and cold intolerance. Surprisingly however, deletion of Mfn2 in mice fed a high fat diet (HFD) results in improved insulin sensitivity and resistance to obesity, while impaired cold‐stimulated thermogenesis is maintained. Improvement in insulin sensitivity is associated with a gender‐specific remodeling of BAT mitochondrial function. In females, BAT mitochondria increase their efficiency for ATP‐synthesizing fat oxidation, whereas in BAT from males, complex I‐driven respiration is decreased and glycolytic capacity is increased. Thus, BAT adaptation to obesity is regulated by Mfn2 and with BAT‐Mfn2 absent, BAT contribution to prevention of insulin resistance is independent and inversely correlated to whole‐body cold‐stimulated thermogenesis.     

UCP1-independent thermogenesis in white adipose tissue of cold-acclimated Ucp1-/- mice

Apart from UCP1-based nonshivering thermogenesis in brown adipocytes, the identity of thermogenic mechanisms that can be activated to reduce a positive energy balance is largely unknown. To identify potentially useful mechanisms, we have analyzed physiological and molecular mechanisms that enable mice, genetically deficient in UCP1 and sensitive to acute exposure to the cold at 4 degrees C, to adapt to long term exposure at 4 degrees C. UCP1-deficient mice that can adapt to the cold have increased oxygen consumption and show increased oxidation of both fat and glucose as indicated from serum metabolite levels and liver glycogen content. Enhanced energy metabolism in inguinal fat was also indicated by increased oxygen consumption and fat oxidation in tissue suspensions and increased AMP kinase activity in dissected tissues. Analysis of gene expression in skeletal muscle showed surprisingly little change between cold-adapted Ucp1+/+ and Ucp1-/- mice, whereas in inguinal fat a robust induction occurred for type 2 deiodinase, sarcoendoplasmic reticulum Ca2+-ATPase, mitochondrial glycerol 3-phosphate dehydrogenase, PGC1alpha, CoxII, and mitochondrial DNA content. Western blot analysis showed an induction of total phospholamban and its phosphorylated form in inguinal fat and other white fat depots, but no induction was apparent in muscle. We conclude that alternative thermogenic mechanisms, based in part upon the enhanced capacity for ion and substrate cycling associated with brown adipocytes in white fat depots, are induced in UCP1-deficient mice by gradual cold adaptation.     

Thermogenic responses in brown fat cells are fully UCP1-dependent. UCP2 or UCP3 do not substitute for UCP1 in adrenergically or fatty scid-induced thermogenesis

To examine the thermogenic significance of the classical uncoupling protein-1 (UCP1), the thermogenic potential of brown adipocytes isolated from UCP1-ablated mice was investigated. Ucp1(-/-) cells had a basal metabolic rate identical to wild-type; the mitochondria within them were coupled to the same degree. The response to norepinephrine in wild-type cells was robust ( approximately 10-fold increase in thermogenesis); Ucp1(-/-) cells only responded approximately 3% of this. Ucp1(-/-) cells were as potent as wild-type in norepinephrine-induced cAMP accumulation and lipolysis and had a similar mitochondrial respiratory complement. In wild-type cells, fatty acids induced a thermogenic response similar to norepinephrine, but fatty acids (and retinoate) were practically without effect in Ucp1(-/-) cells. It is concluded that no other adrenergically induced thermogenic mechanism exists in brown adipocytes except that mediated by UCP1 and that entopic expression of UCP1 does not lead to overt innate uncoupling, and it is suggested that fatty acids are transformed to an intracellular physiological activator of UCP1. High expression of UCP2 and UCP3 in the tissue was not associated with an overt innate highly uncoupled state of mitochondria within the cells, nor with an ability of norepinephrine or endo- or exogenous fatty acids to induce uncoupled respiration in the cells. Thus, UCP1 remains the only physiologically potent thermogenic uncoupling protein in these cells.     

Let's burn whatever you have: mitofusin 2 metabolically re‐wires brown adipose tissue

Brown adipose tissue (BAT) has received enormous scientific and lay attention in the recent past as its thermogenic, energy‐consuming capacities represent prime candidates for therapeutic interventions toward obesity, glucose intolerance, and diabetes even in humans. The overall positive effects of BAT activation and recruitment on systemic energy homeostasis have been largely attributed to the inherent ability of brown adipocytes to combust fatty acid and glucose energy substrates through mitochondrial uncoupling, driven by the unique expression of uncoupling protein 1 (UCP1). Two recent reports by Boutant et al and Mahdaviani et al now identify the GTPase mitofusin (Mfn) 2 as a key determinant of BAT thermogenic function that is largely independent of its previously described role in mitochondrial fusion [1,2].     

长爪沙鼠褐色脂肪组织和肝脏产热特征的季节性变化

长爪沙鼠（Meriones unguiculatus）是一种栖息于典型草原和荒漠草原非冬眠群居性的小型哺乳动物。为研究其产热功能的季节变化,我们分别在2003年秋季（9月下旬）、冬季（11月下旬）、2004年春季（3月底-4月下旬）和夏季（7月下旬）,分别测定了其体重、褐色脂肪组织和肝脏的重量、线粒体总蛋白含量和细胞色素c氧化酶活力,以及褐色脂肪组织中解偶联蛋白1（Uncoupling protein1,UCPl）的含量等。结果显示：除雄鼠的体重显著高于雌鼠外,其它各项指标均无性别差异。体重和褐色脂肪组织的重量都在冬季较高,显著高于夏季,而肝脏的重量在夏季显著高于其它季节。褐色脂肪组织和肝脏的线粒体蛋白含量和细胞色素c氧化酶活力以及UCP1含量,都在冬季较高,夏季较低。这些结果表明：在野外条件下,褐色脂肪组织和肝脏在细胞水平上产热能力的提高和UCP1含量的增加,是长爪沙鼠抵御寒冷的重要方式。     

Adaptive character of metabolism in Eothenomys miletus in Hengduan Mountains region during cold acclimation

Environmental factors play an important role in the seasonal adaptation of body mass and thermogenesis in small, wild mammals. The purpose of the present study was to test the hypothesis that ambient temperature was a cue to trigger the seasonal adjustments in body mass, energy intake, uncoupling protein 1 (UCP1) in brown adipose tissue (BAT), and other biochemical characteristics of Eothenomys miletus during 49 days of cold exposure. Our data demonstrated that cold acclimation induced a remarkable decrease in body mass, a significant increase in energy intake and metabolic rate, and high expression of UCP1 in BAT of E. miletus. Biochemical characteristics of BAT and liver respiration were also increased following cold acclimation. These data suggest that E. miletus reduced the body mass and increased energy intake and expenditure under cold acclimation. Increased expression of UCP1 was potentially involved in the regulation of energy metabolism and thermogenic capacity following cold acclimation.     

Effects of the presence, absence, and overexpression of uncoupling protein-3 on adiposity and fuel metabolism in congenic mice

Uncoupling protein-3 (UCP3) is a poorly understood mitochondrial inner membrane protein expressed predominantly in skeletal muscle. The aim of this study was to examine the effects of the absence or constitutive physiological overexpression of UCP3 on whole body energy metabolism, glucose tolerance, and muscle triglyceride content. Congenic male UCP3 knockout mice (Ucp3-/-), wild-type, and transgenic UCP3 overexpressing (UCP3Tg) mice were fed a 10% fat diet for 4 or 8 mo after they were weaned. UCP3Tg mice had lower body weights and were less metabolically efficient than wild-type or Ucp3-/- mice, but they were not hyperphagic. UCP3Tg mice had smaller epididymal white adipose tissue and brown adipose tissue (BAT) depots; however, there were no differences in muscle weights. Glucose and insulin tolerance tests revealed that both UCP3Tg and Ucp3-/- mice were protected from development of impaired glucose tolerance and were more sensitive to insulin. 2-Deoxy-D-[1-3H]glucose tracer studies showed increased uptake of glucose into BAT and increased storage of liver glycogen in Ucp3-/- mice. Assessments of intramuscular triglyceride (IMTG) revealed decreases in quadriceps of UCP3Tg mice compared with wild-type and Ucp3-/- mice. When challenged with a 45% fat diet, Ucp3-/- mice showed increased accumulation of IMTG compared with wild-type mice, which in turn had greater IMTG than UCP3Tg mice. Results are consistent with a role for UCP3 in preventing accumulation of triglyceride in both adipose tissue and muscle.     

Increased mitochondrial proton leak in skeletal muscle mitochondria of UCP1-deficient mice

Mice having targeted inactivation of uncoupling protein 1 (UCP1) are cold sensitive but not obese (Enerb?ck S, Jacobsson A, Simpson EM, Guerra C, Yamashita H, Harper M-E, and Kozak LP. Nature 387: 90-94, 1997). Recently, we have shown that proton leak in brown adipose tissue (BAT) mitochondria from UCP1-deficient mice is insensitive to guanosine diphosphate (GDP), a well known inhibitor of UCP1 activity (Monemdjou S, Kozak LP, and Harper M-E. Am J Physiol Endocrinol Metab 276: E1073-E1082, 1999). Moreover, despite a fivefold increase of UCP2 mRNA in BAT of UCP1-deficient mice, we found no differences in the overall kinetics of this GDP-insensitive proton leak between UCP1-deficient mice and controls. Based on these findings, which show no adaptive increase in UCP1-independent leak in BAT, we hypothesized that adaptive thermogenesis may be occurring in other tissues of the UCP1-deficient mouse (e.g., skeletal muscle), thus allowing them to maintain their normal resting metabolic rate, feed efficiency, and adiposity. Here, we report on the overall kinetics of the mitochondrial proton leak, respiratory chain, and ATP turnover in skeletal muscle mitochondria from UCP1-deficient and heterozygous control mice. Over a range of mitochondrial protonmotive force (Deltap) values, leak-dependent oxygen consumption is higher in UCP1-deficient mice compared with controls. State 4 (maximal leak-dependent) respiration rates are also significantly higher in the mitochondria of mice deficient in UCP1, whereas state 4 Deltap is significantly lower. No significant differences in state 3 respiration rates or Deltap values were detected between the two groups. Thus the altered kinetics of the mitochondrial proton leak in skeletal muscle of UCP1-deficient mice indicate a thermogenic mechanism favoring the lean phenotype of the UCP1-deficient mouse.     

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.     

Inactivation of UCP1 and the glycerol phosphate cycle synergistically increases energy expenditure to resist diet-induced obesity

Our current paradigm for obesity assumes that reduced thermogenic capacity increases susceptibility to obesity, whereas enhanced thermogenic capacity protects against obesity. Here we report that elimination of two major thermogenic pathways encoded by the mitochondrial uncoupling protein (Ucp1) and mitochondrial glycerol-3-phosphate dehydrogenase (Gdm) result in mice with increased resistance to diet-induced obesity when housed at 28 degrees C, provided prior adaptation occurred at 20 degrees C. Obesity resistant Gdm(-/-).Ucp1(-/-) mice maintained at 28 degrees C have increased energy expenditure, in part through conversion of white to brown adipocytes in inguinal fat. Increased oxygen consumption in inguinal fat cell suspensions and the up-regulation of genes of mitochondrial function and fat metabolism indicated increased thermogenic activity, despite the absence of UCP1, whereas liver and skeletal muscle showed no changes in gene expression. Additionally, comparisons of energy expenditure in UCP1-deficient and wild type mice fed an obesogenic diet indicates that UCP1-based brown fat-based thermogenesis plays no role in so-called diet-induced thermogenesis. Accordingly, a new paradigm for obesity emerges in which the inactivation of major thermogenic pathways force the induction of alternative pathways that increase metabolic inefficiency.     

Nonshivering thermogenesis in a marsupial (the tasmanian bettong Bettongia gaimardi) is not attributable to brown adipose tissue

The Tasmanian bettong (Bettongia gaimardi, a marsupial) is a rat-kangaroo that increases nonshivering thermogenesis (NST) in response to norepinephrine (NE). This study attempted to assess whether brown adipose tissue (BAT), a specialized thermogenic effector, is involved in NST in the bettong. Regulatory NST, indicated by resting oxygen consumption (Vo2) of the whole body, was measured under conscious conditions at 20 degrees C with various stimuli: cold (4 degrees -5 degrees C) or warm (25 degrees C) acclimation, NE injection, and the beta3-adrenoceptor agonist (BRL) 37344. In line with the functional studies in vivo, the presence of BAT was evaluated by examining the expression of the uncoupling protein 1 (UCP1) with both rat cDNA and oligonucleotide probes. Both NE and BRL 37344 significantly stimulated NST in the bettong. After cold acclimation of the animals (at 4 degrees -5 degrees C for 2 wk), the resting Vo2 was increased by 15% and the thermogenic effect of NE was enhanced; warm-acclimated animals showed a slightly depressed response. However, no expression of UCP1 was detected in bettongs either before or after cold exposure (2 wk). These data suggest that the observed NST in the marsupial bettong is not attributable to BAT.     

大绒鼠解偶联蛋白1基因部分序列扩增与分析

解偶联蛋白1（Uncoupling protein 1,UCP1）是位于褐色脂肪组织线粒体内膜上的一种解偶联蛋白,该蛋白可以诱导质子漏从而产热。通过设计简并引物进行RT-PCR从大绒鼠BAT中获得UCP1基因cDNA核心序列,RTPCR所得产物长约458 bp,包含的开放阅读框（open reading frame,ORF）为456 bp,编码151个氨基酸。通过BLAST搜索,所得大绒鼠UCP1基因cDNA氨基酸序列与黑线仓鼠、橙腹草原田鼠、金黄仓鼠、小家鼠和褐家鼠等哺乳动物的UCP1氨基酸序列同源性均在80%以上,而与鱼类和两栖类的氨基酸序列同源性在61%以下。研究结果表明UCP1在哺乳类中高度保守。同时,通过NJ方法以UCP1序列构建系统进化树表明大绒鼠与橙腹草原田鼠聚成一支,构成田鼠类分支。