Cloning of putative uncoupling protein 1 cDNA in a cold-intolerant mammal, the house musk shrew (Suncus murinus)

The house musk shrew (Suncus murinus), or suncus, is a unique experimental animal. We recently showed that this mammal is cold intolerant and hypothesized that its sensitivity to cold is caused by low thermogenic activity in brown adipose tissue (BAT). Thermogenesis in BAT is performed by a unique mitochondrial protein, uncoupling protein 1 (UCP1). Interestingly, only eutherians possess UCP1, and some traits in the suncus resemble those in the Ucp1-ablated mouse, including cold intolerance, histology of BAT, and obesity resistance. In a previous study, we hypothesized that UCP1 may not be present in BAT of the suncus or may be dysfunctional. Therefore, we performed cDNA cloning of suncus Ucp1 and compared it to homologs from other species. The deduced amino acid sequence showed high similarity to other mammalian UCP1. Northern blot analysis revealed mRNA in BAT, as in other mammals. However, a difference in an amino acid residue was observed in an important residue for thermogenesis. Genomic sequence analysis showed that this difference existed in our two genetically distant laboratory colonies. These results suggest that cold intolerance in the suncus is derived from low thermogenic activity of UCP1 and may exist in wild house musk shrews.     

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 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.     

Isoform and tissue dependent impact of apolipoprotein E on adipose tissue metabolic activation: The role of apolipoprotein A1

Adipose organ is made of white (WAT) and brown (BAT) adipose tissue which are primarily responsible for lipid storage and energy production (heat and ATP) respectively. Metabolic activation of WAT may ascribe to this tissue characteristics of BAT, namely non-shivering thermogenesis and ATP production. Recent data indicate that apolipoproteins E (APOE) and A1 (APOA1) regulate WAT mitochondrial metabolic activation. Here, we investigated the functional cross-talk between natural human APOE2 and APOE4 isoforms with APOA1 in this process, using Apoe2^knock-in and Apoe4^knock-in mice. At baseline when Apoe2^knock-in and Apoe4^knock-in mice express both APOE and Apoa1, the Apoe2^knock-in strain appears to have higher mitochondrial oxidative phosphorylation levels and non-shivering thermogenesis in WAT compared to Apoe4^knock-in mice. When mice were switched to a high-fat diet for 18 weeks, circulating levels of endogenous Apoa1 in Apoe2^knock-in mice became barely detectable though significant levels of APOE2 were still present. This change was accompanied by a significant reduction in WAT mitochondrial Ucp1 expression while BAT Ucp1 was unaffected. Ectopic APOA1 expression in Apoe2^knock-in animals potently stimulated WAT but not BAT mitochondrial Ucp1 expression providing further evidence that APOA1 potently stimulates WAT non-shivering thermogenesis in the presence of APOE2. Ectopic expression of APOA1 in Apoe4^knock-in mice stimulated BAT but no WAT mitochondrial Ucp1 levels, suggesting that in the presence of APOE4, APOA1 is a trigger of BAT non-shivering thermogenesis. Overall, our data identified a tissue-specific role of the natural human APOE2 and APOE4 isoforms in WAT- and BAT-metabolic activation respectively, that appears dependent on circulating APOA1 levels.     

Cold exposure increases glucose utilization and glucose transporter expression in brown adipose tissue.

When rats were exposed to a cold environment (4 degrees C) for 10 days, tissue glucose utilization was increased in brown adipose tissue (BAT), a tissue specified for non-shivering thermogenesis, but not in skeletal muscle. Cold exposure also caused an increase in the amount of GLUT4, an isoform of glucose transporters expressed in insulin-sensitive tissues, in parallel with an increased cellular level of GLUT4 mRNA. In contrast to BAT, no significant effect of cold exposure was found in skeletal muscle. The results suggest the cold-induced increase in glucose utilization by BAT is attributable, at least in part, to the increased expression of GLUT4.     

Tissue-specific functional interaction between apolipoproteins A1 and E in cold-induced adipose organ mitochondrial energy metabolism

White (WAT) and brown (BAT) adipose tissue, the two main types of adipose organ, are responsible for lipid storage and non-shivering thermogenesis, respectively. Thermogenesis is a process mediated by mitochondrial uncoupling protein 1 (UCP1) which uncouples oxidative phosphorylation from ATP production, leading to the conversion of free fatty acids to heat. This process can be triggered by exposure to low ambient temperatures, caloric excess, and the immune system. Recently mitochondrial thermogenesis has also been associated with plasma lipoprotein transport system. Specifically, apolipoprotein (APO) E3 is shown to have a bimodal effect on WAT thermogenesis that is highly dependent on its site of expression. Similarly, APOE2 and APOE4 differentially affect BAT and WAT mitochondrial metabolic activity in processes highly modulated by APOA1. Furthermore, the absence of classical APOA1 containing HDL (APOA1-HDL), is associated with no measurable non-shivering thermogenesis in WAT of mice fed high fat diet. Based on these previous observations which indicate important regulatory roles for both APOA1 and APOE in adipose tissue mitochondrial metabolic activity, here we sought to investigate the potential roles of these apolipoproteins in BAT and WAT metabolic activation in mice, following stimulation by cold exposure (7 °C). Our data indicate that APOA1-HDL promotes metabolic activation of BAT only in the presence of very low levels (virtually undetectable) of APOE3-containing HDL (APOE3-HDL), which acts as an inhibitor in this process. In contrast, induction of WAT thermogenesis is subjected to a more complicated regulation which requires the combined presence of both APOA1-HDL and APOE3-HDL.     

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.     

Liver and brown fat mitochondrial response to cold in the garden dormouse (Eliomys quercinus)

The involvement of two organs, i.e. the liver and the brown adipose tissue (BAT) in response to cold in a hibernating species such as the garden dormouse has been studied. 2. In animals living in the cold, mitochondrial respiratory rates significantly increased (with respect to those living at 28 degrees C) in both organs with a larger increase in the BAT (+152% in the BAT and 67% in the liver). 3. The increase in BAT activity was obtained by a concomitant increase in: (a) the BAT mass (+30%), (b) the total mitochondrial mass (+20%), and (c) the mitochondrial respiratory rate (+64%). In the liver the increase was due only to an augmentation in mitochondrial mass and activity. 4. These results indicate that: (a) the BAT exerts a pre-eminent role in the physiological response to cold of garden dormouse, (b) a certain non-shivering thermogenesis (NST) is present in the liver of such species. In addition we suggest that a local thermoregulatory response would take place in a metabolically important organ such as the liver.     

Homeostatic non-shivering thermogenesis in humans facts and hypotheses

This review considers current research of different forms of non-shivering thermogenesis related to thermoregulatory and substrate homeostasis. The term “homeostatic non-shivering thermogenesis (HNST)” is proposed for explanation of facultative heat production stimulated by exposure to cold, food intake and accumulation of lactate during intensive muscle loading. Similarities and differences in physiological activity are displayed in three HNST types. Existence of a number of common points makes it possible to propose common physiological mechanisms of HNST realization. Among other candidates for HNST location, the brown adipose tissue (BAT) fits best as its function is specifi between thermogenic function in cold environment and diet-induced thermogenesis that makes it possible to link these two HNST types with BAT activity. Here we present the data indirectly confirming BAT functioning in processes of homeostatic normalization not related to cold acclimation or food intake. We also consider new data about BAT functional activity, its topographic body location, mechanisms of uncoupled respiration in different tissues in adult humans and about methods of BAT diagnostics which include the use of molecular markers. We list a number of facts confirming our suggestion about BAT activity being related to homeostatic normalization after physical loading. In conclusion, we propose an experimental research program for the testing of our hypothesis regarding BAT universal homeostatic function in humans.     

Homeostatic non-shivering thermogenesis in man: facts and speculations

In this review it is considered up-to date researches of different forms of non-shivering thermogenesis that related to thermoregulatory and substrate homeostasis. Term "homeostatic non-shivering thermogenesis (HNST)" is proposed for explanation of facultative heat production stimulated by cold exposure, food intake and accumulation of lactate during intensive muscle load. There are common and different features of physiological activity displayed in three HNST types. Existence of these common points gets a probability to propose general physiological mechanisms of HNST realization. Between other candidates for HNST location brown adipose tissue (BAT) has real unquestionable advantage for this specific function. There is close relationship between thermogenic function in cold environment and diet-induced thermogenesis that allows to link two HNST types and BAT activity together. Here we present data indirectly confirming BAT functioning in processes of homeostatic normalization not due to cold acclimation or food intake. Also we give consideration to new data about BAT functional activity, its topographic body location, mechanisms of uncoupled respiration in different tissues in adult humans and methods of BAT diagnostics which include molecular marker using. We adduce a number of facts confirming our suggestion about BAT activity can be related to homeostatic normalization after physical load. At last, we bring forward experimental research program for examination of our hypothesis about BAT universal homeostatic function in humans.     

Ontogeny of cardiac rate regulation and brown fat thermogenesis in golden hamsters (Mesocricetus auratus)

It was shown previously in infant rats (Rattus norvegicus) that the ability to produce heat in the cold using brown adipose tissue (BAT) is closely related to the ability to maintain cardiac rate. When the limits of BAT thermogenesis were exceeded, interscapular temperature (which reflects the temperature of the interscapular BAT depot) and cardiac rate fell together. As an extension of this earlier study, the relation between BAT thermogenesis and cardiac rate was examined here in the golden hamster (Mesocricetus auratus), a species whose young do not exhibit BAT thermogenesis until the end of the 2nd week postpartum. It was found that 3 to 12-day-old hamsters were unable to increase shivering or nonshivering thermogenesis in the cold and exhibited decreases in cardiac rate that proceeded in lock-step with decreases in interscapular temperature. In contrast, as the thermogenic capability of hamsters increased after 12 days of age, cardiac rate was maintained within narrow limits across a wide range of air temperatures. These results support the hypothesis that heat produced by BAT helps to warm the heart and thus aids in the maintenance of cardiac rate during cold exposure. Accepted: 16 August 1997     

Brown adipose tissue in morbidly obese subjects

Background

Cold-stimulated adaptive thermogenesis in brown adipose tissue (BAT) to increase energy expenditure is suggested as a possible therapeutic target for the treatment of obesity. We have recently shown high prevalence of BAT in adult humans, which was inversely related to body mass index (BMI) and body fat percentage (BF%), suggesting that obesity is associated with lower BAT activity. Here, we examined BAT activity in morbidly obese subjects and its role in cold-induced thermogenesis (CIT) after applying a personalized cooling protocol. We hypothesize that morbidly obese subjects show reduced BAT activity upon cold exposure.

Methods and Findings

After applying a personalized cooling protocol for maximal non-shivering conditions, BAT activity was determined using positron-emission tomography and computed tomography (PET-CT). Cold-induced BAT activity was detected in three out of 15 morbidly obese subjects. Combined with results from lean to morbidly obese subjects (n = 39) from previous study, the collective data show a highly significant correlation between BAT activity and body composition (P<0.001), respectively explaining 64% and 60% of the variance in BMI (r = 0.8; P<0.001) and BF% (r = 0.75; P<0.001). Obese individuals demonstrate a blunted CIT combined with low BAT activity. Only in BAT-positive subjects (n = 26) mean energy expenditure was increased significantly upon cold exposure (51.5±6.7 J/s versus 44.0±5.1 J/s, P = 0.001), and the increase was significantly higher compared to BAT-negative subjects (+15.5±8.9% versus +3.6±8.9%, P = 0.001), indicating a role for BAT in CIT in humans.

Conclusions

This study shows that in an extremely large range of body compositions, BAT activity is highly correlated with BMI and BF%. BAT-positive subjects showed higher CIT, indicating that BAT is also in humans involved in adaptive thermogenesis. Increasing BAT activity could be a therapeutic target in (morbid) obesity.     

Chronic high-fat feeding impairs adaptive induction of mitochondrial fatty acid combustion-associated proteins in brown adipose tissue of mice

Since brown adipose tissue (BAT) is involved in thermogenesis using fatty acids as a fuel, BAT activation is a potential strategy for treating obesity and diabetes. However, whether BAT fatty acid combusting capacity is preserved in these conditions has remained unclear. We therefore evaluated expression levels of fatty acid oxidation-associated enzymes and uncoupling protein 1 (Ucp1) in BAT by western blot using a diet-induced obesity C57BL/6J mouse model. In C57BL/6J mice fed a high-fat diet (HFD) over 2–4 weeks, carnitine palmitoyltransferase 2 (Cpt2), acyl-CoA thioesterase (Acot) 2, Acot11 and Ucp1 levels were significantly increased compared with baseline and control low-fat diet (LFD)-fed mice. Similar results were obtained in other mouse strains, including ddY, ICR and KK-Ay, but the magnitudes of the increase in Ucp1 level were much smaller than in C57BL/6J mice, with decreased Acot11 levels after HFD-feeding. In C57BL/6J mice, increased levels of these mitochondrial proteins declined to near baseline levels after a longer-term HFD-feeding (20 weeks), concurrent with the accumulation of unilocular, large lipid droplets in brown adipocytes. Extramitochondrial Acot11 and acyl-CoA oxidase remained elevated. Treatment of mice with Wy-14,643 also increased these proteins, but was less effective than 4 week-HFD, suggesting that mechanisms other than peroxisome proliferator-activated receptor α were also involved in the upregulation. These results suggest that BAT enhances its fatty acid combusting capacity in response to fat overload, however profound obesity deprives BAT of the responsiveness to fat, possibly via mitochondrial alteration.     

Norepinephrine release in brown adipose tissue remains robust in cold-exposed senescent Fischer 344 rats

Near the end of life, old F344 rats undergo a transition, marked by spontaneous and rapidly declining function. Food intake and body weight decrease, and these rats, which we call senescent, develop severe hypothermia in the cold due in part to blunted brown fat [brown adipose tissue (BAT)] thermogenesis. We tested the hypothesis that this attenuation may involve diminished sympathetic signaling by measuring cold-induced BAT norepinephrine release in freely moving rats using linear microdialysis probes surgically implanted into interscapular BAT 24 and 48 h previously. In response to 2 h at 15 degrees C, senescent rats increased BAT norepinephrine release 6- to 10-fold but did not maintain homeothermy. This increase was comparable to that of old presenescent (weight stable) rats that did maintain homeothermy during even greater cold exposure (2 h at 15 degrees C followed by 1.5 h at 8 degrees C). Tail temperatures, an index of vasoconstrictor responsiveness to cold, exhibited similar cooling curves in presenescent and senescent rats. Thus cold-induced sympathetic signaling to BAT and tail vasoconstrictor responsiveness remain robust in senescent rats and cannot explain their cold-induced hypothermia.     

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.     

Leptin-programmed rats respond to cold exposure changing hypothalamic leptin receptor and thyroid function differently from cold-exposed controls

We showed that neonatal leptin treatment programmes for hyperleptinemia and central leptin resistance both at 30days-old and adulthood, while programmes for lower serum T3 at 30days-old, but higher thyroid hormones (TH) at adulthood. As in these animals, acute cold at 30days-old normalized leptinemia and restored the expression of hypothalamic leptin receptor (OBR), here we evaluate the effect of cold exposure on the thyroid function and OBR in adult rats programmed by neonatal hyperleptinemia. Pups were divided into 2 groups: Lep-injected with leptin (8μg/100g/BW, sc) for the first 10days of lactation, and C-injected with saline. At 150days, both groups were subdivided into: LepC and CC, which were exposed to 8°C for 12h. Serum leptin, TH, TSH, liver type I and brown adipose tissue (BAT) type II deiodinases (D1 and D2) activities, liver mitochondrial alpha-glycerol-3-phosphate dehydrogenase (mGPD) activity and adrenal catecholamine content were measured. Hypothalamic and thyroid OBR protein contents were evaluated. Differences were significant when p<0.05. Lep group had hyperleptinemia (+19%), higher T4 (+20%) and T3 (+30%) with lower TSH (-55%), higher liver D1 (1.4 fold-increase), lower BAT D2 (-44%) and liver mGPD activities (-55%), higher adrenal catecholamines (+44%), lower hypothalamic OBR (-51%) and normal thyroid OBR. Cold exposure normalized leptinemia, D1, mGPD, catecholamine and hypothalamic OBR. However, cold exposure further increased TH and decreased D2. Thus, cold exposure normalizes most of the changes programmed by neonatal hyperleptinemia, at the expense of worsening the hyperthyroidism and BAT thermogenesis.     

Alterations of glucose-dependent insulinotropic polypeptide (GIP) during cold acclimation

Cold acclimation is initially associated with shivering thermogenesis in skeletal muscle followed by adaptive non-shivering thermogenesis, particularly in brown adipose tissue (BAT). In response, hyperphagia occurs to meet increased metabolic demand and thermoregulation. The present study investigates the effects of cold (4 ± 1 °C) acclimation and hyperphagia on circulating and intestinal levels of gastric inhibitory polypeptide (GIP) in rats. Pair fed animals were used as additional controls in some experiments. Cold acclimation for 42 days significantly (p<0.01) increased daily food intake. There was no corresponding change in body weight. However, body weights of pair fed cold exposed rats were significantly (p<0.01) reduced compared to controls and ad libitum fed cold exposed rats. By day 42, non-fasting plasma glucose was increased (p<0.05) by chronic cold exposure regardless of food intake. Corresponding plasma insulin concentrations were significantly (p<0.01) lower in pair fed cold exposed rats. Circulating GIP levels were elevated (p<0.05) in ad libitum fed cold acclimated rats on days 18 and 24, but returned to normal levels by the end of the study. The glycaemic response to oral glucose was improved (p<0.01) in all cold exposed rats, with significantly (p<0.05) elevated GIP responses in ad libitum fed rats and significantly (p<0.05) reduced insulin responses in pair fed rats. In keeping with this, insulin sensitivity was enhanced (p<0.05) in cold exposed rats compared to controls. By the end of the study, cold acclimated rats had significantly (p<0.01) increased BAT mass and intestinal concentrations of GIP and GLP-1 compared to controls, independent of food intake. These data indicate that changes in the secretion and actions of GIP may be involved in the metabolic adaptations to cold acclimation in rats.