Attenuated cold-induced increase in mRNA for uncoupling protein in brown adipose tissue of obese (ob/ob) mice

The level of mRNA for uncoupling protein was measured in brown adipose tissue of young (8-10 weeks) and old (11 months) lean and ob/ob mice using a cDNA clone constructed previously. The level of poly(A)+ RNA was also measured using an oligo(dT)18 probe. Mice were kept at 28 degrees C or exposed to 14 degrees C for 12 h. The level of mRNA for uncoupling protein was normal in brown adipose tissue of younger obese mice but reduced in brown adipose tissue of old obese mice. The cold-induced absolute increase in uncoupling protein mRNA was smaller in obese mice, regardless of age. It is concluded that the known attenuation of the acute thermogenic response of brown adipose tissue of the ob/ob mouse to cold is accompanied by a similar attenuation of the initiation of the trophic response. It is likely, however, that these defects are secondary to the chronic reduction in sympathetic nervous system activity in brown adipose tissue of the ob/ob mouse, which results in a functional atrophy of the tissue.     

Loss of brown adipose tissue uncoupling protein mRNA on deacclimation of cold-exposed rats

The effect of environmental temperature on the level of uncoupling protein mRNA from rat brown adipose tissue was examined using a cDNA probe. A 4.4 fold increase in the mRNA level was observed after 1 day exposure of rats to 6 degrees C, which was followed by a slow loss with longer times of exposure. When rats were returned to a thermoneutral environment, there was a dramatic loss of uncoupling protein mRNA within 1 day. Comparison wih poly(A)+ RNA levels suggest that the response to temperature is specific for uncoupling protein mRNA.     

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.     

Effect of unilateral surgical denervation of brown adipose tissue on uncoupling protein mRNA level and cytochrom-c-oxidase activity in the Djungarian hamster

The bilateral lobe of interscapular brown adipose tissue of the Djungarian hamster was unilaterally denervated in order to study the role of the sympathetic innervation for maintenance and cold-induced increase of non-shivering thermogenesis. Denervation decreased the noradrenaline content of brown adipose tissue to less than 9% of the intact contralateral pad. This low noradrenaline level was maintained for 1–14 days after denervation. First, to study the role of the sympathetic innervation of brown adipose tissue in the maintenance of the high thermogenic capacity characteristic of the cold acclimated state, brown adipose tissue was denervated in hamsters either kept at thermoneutrality or acclimated to 5°C ambient temperature for 4 weeks. Cold-acclimated hamsters had elevated levels of uncoupling protein messenger ribonucleic acid (8.1-fold) and cytochrom-c oxidase-activity (3-fold). Denervation of brown adipose tissue decreased uncoupling protein-messenger ribonucleic acid level and cytochrom-c-oxidase-activity as compared to the intact pad in thermoneutral and in cold-acclimated hamsters. However, in cold-acclimated hamsters uncoupling protein-messenger ribonucleic acid level and cytochrom-c-oxidase-activity in denervated brown adipose tissue both were maintained on an elevated 6-fold higher levels as compared to thermoneutral controls. Second, to study the role of the sympathetic innervation of brown adipose tissue in the cold-induced increase in thermogenic capacity, hamsters were denervated prior to cold acclimation and responses were measured after 3 and 14 days of cold exposure. Uncoupling protein-messenger ribonucleic acid level and cytochrom-c-oxidase-activity of intact brown adipose tissue increased after 14 days cold acclimation. Denervation did not completely prevent a cold-induced 1.5-fold increase of cytochrom-c-oxidase-activity and a 3.2-fold increase of the uncoupling protein-messenger ribonucleic acid level in denervated brown adipose tissue after 14 days of cold acclimation. In conclusion, high levels of uncoupling protein-messenger ribonucleic acid and cytochrom-c-oxidase activity in brown adipose tissue of cold-acclimated hamsters can partially be maintained without intact sympathetic innervation, suggesting a considerable contribution of trophic factors not requiring sympathetic innervation for maintenance. The cold-induced increase of cytochrom-c-oxidase activity and expression of uncoupling protein-messenger ribonucleic acid largely depends upon sympathetic innervation of brown adipose tissue.Abbreviations ANOVA analysis of variance - BAT brown adipose tissue - COX cytochrom-c-oxidase - HPLC high performance liquid chromatography - mRNA messenger ribonucleie acid - NA noradrenaline - T _a ambient temperature - UCP uncoupling protein     

Thyroid-stimulating hormone receptor in brown adipose tissue is involved in the regulation of thermogenesis

C.RF- Tshr(hyt/hyt) mice have a mutated thyroid-stimulating hormone receptor (TSHR), and, without thyroid hormone supplementation, these mice develop severe hypothyroidism. When hypothyroid Tshr(hyt/hyt) mice were exposed to cold (4 degrees C), rectal temperature rapidly dropped to 23.9 +/- 0.40 degrees C at 90 min, whereas the wild-type mice temperatures were 37.0 +/- 0.15 degrees C. When we carried out functional rat TSHR gene transfer in the brown adipose tissues by plasmid injection combined with electroporation, there was no effect on the serum levels of thyroxine, although rectal temperature of the mice transfected with pcDNA3.1/Zeo-rat TSHR 90 min after cold exposure remained at 34.6 +/- 0.34 degrees C, which was significantly higher than that of Tshr(hyt/hyt) mice. Transfection of TSHR cDNA increased mRNA and protein levels of uncoupling protein-1 (UCP-1) in brown adipose tissues, and the weight ratio of brown adipose tissue to overall body weight also increased. Exogenous thyroid hormone supplementation to Tshr(hyt/hyt) mice restored rectal temperature 90 min after exposure to cold (36.8 +/- 0.10 degrees C). These results indicate that not only thyroid hormone but also thyroid-stimulating hormone (TSH)/TSHR are involved in the expression mechanism of UCP-1 in mouse brown adipose tissue. TSH stimulates thermogenesis and functions to protect a further decrease in body temperature in the hypothyroid state.     

Postnatal recruitment of brown adipose tissue is induced by the cold stress experienced by the pups. An analysis of mRNA levels for thermogenin and lipoprotein lipase.

In order to investigate the postnatal recruitment process, gene expression in the brown adipose tissue of rat pups was followed during the first 20 h of life. In normal pups, the level of mRNA coding for the uncoupling protein thermogenin increased markedly but gradually within the first 24 h. Lipoprotein lipase and actin mRNA levels were relatively low and remained constant. In pups exposed to thermoneutral temperature (35 degrees C) for the first 12 h after birth, no increase in thermogenin mRNA or lipoprotein lipase mRNA was observed, whereas in pups exposed to 28 degrees C a clear increase in both thermogenin and lipoprotein lipase mRNA levels was found. Actin mRNA levels were not affected by the environmental temperature under these circumstances. It was concluded that the postnatal recruitment in brown adipose tissue is a consequence of the cold stress experienced by the newborn pups. Thus, postnatal recruitment is not ontogenically predetermined.     

Brown fat thermogenesis in cold-acclimated rats is not abolished by the suppression of thyroid function

The effects of long-term cold exposure on brown adipose tissue (BAT) thermogenesis in hypothyroid rats have been examined. Thyroid ablation was performed in normal rats after 2 mo of exposure to 4 degrees C, when BAT hypertrophy and thermogenic activity were maximal. After ablation, hypothyroid and normal controls remained in the cold for 2 additional months. At the end of the 4-mo cold exposure, all untreated hypothyroid rats were alive, had normal body temperature, and had gained an average 12.8% more weight than normal controls. Long-term cold exposure of hypothyroid rats markedly increased BAT weight, mitochondrial proteins, uncoupling protein (UCP)-1, mRNA for UCP-1, and oxygen consumption to levels similar to those seen in cold-exposed normal rats. The results indicate that thyroid hormones are required for increased thermogenic capacity to occur as an adaptation to long-term cold exposure. However, cold adaptation can be maintained in the absence of thyroid hormone.     

The concentration of uncoupling protein correlates with Mg2+ activated mitochondrial binding of GDP

Rats were housed at 4 degrees C for periods of up to 26 days. As little as 2 h of cold exposure caused an increase in the binding of [3H]GDP to mitochondria from brown adipose tissue. Incubation of mitochondria in vitro with 10 mM Mg2+ caused a marked increase in the subsequent binding of GDP to mitochondria from rats housed at 28 degrees C and a smaller increase in that from rats exposed to 4 degrees C for 2 h. Chronic exposure to cold led to an even greater increase in the amount of GDP bound to mitochondria incubated with Mg2+. The time course for the increase in the concentration of uncoupling protein was compared with that for GDP binding to mitochondria with and without Mg2+ treatment. The concentration of uncoupling protein appears to be correlated with the GDP-binding values for mitochondria treated with Mg2+ (r = 0.70) but not with the GDP binding to untreated mitochondria (r = 0.36). Therefore, the binding of GDP to untreated mitochondria may represent thermogenic activity at the time of death, whereas that after treatment with Mg2+ may more closely reflect total thermogenic capacity of the mitochondrion.     

Uncoupling protein is expressed in liver mitochondria of cold-exposed and newborn rats

Uncoupling protein has been thought to be expressed only in the brown adipose tissue mitochondria of mammals. However, mRNA encoding mitochondrial uncoupling protein was detected in the liver of newborn rats and adult rats after cold exposure, although not in the liver of untreated adult rats.     

Effect of acclimation temperature on the concentration of the mitochondrial 'uncoupling' protein measured by radioimmunoassay in mouse brown adipose tissue

The effect of acclimation temperature on the concentration of the mitochondrial 'uncoupling' protein (Mr 32000) from brown adipose tissue of mice has been investigated. The uncoupling protein was measured by a specific radioimmunoassay. Between 33 degrees C (thermoneutrality) and -2 degrees C there was a progressive increase with decreasing environmental temperature in the amount of uncoupling protein. For mice at -2 degrees C the mitochondrial concentration of the protein was 9-times higher than at 33 degrees C, while the total amount of the protein in interscapular brown adipose tissue was estimated to be nearly 80-times greater at -2 degrees C compared to 33 degrees C.     

Opposite regulation of uncoupling protein 1 and uncoupling protein 3 in vivo in brown adipose tissue of cold-exposed rats

Earlier we reported a 14-fold increase of glycogen in the brown adipose tissue (BAT) in rats when the animals were placed back from cold to neutral temperature. To elucidate the mechanism, here we compared the level of glucose transporter 4 (GLUT4) protein, uncoupling protein (UCP) 1 and UCP3 mRNA and protein expressions in the BAT under the same conditions. We found that the increased GLUT4 level in cold was maintained during the reacclimation. After 1 week cold exposure the mRNA and protein content of UCP1 increased parallel, while the protein level of UCP3 decreased, contrary to its own mRNA level.     

Mitochondrial uncoupling protein from mouse brown fat. Molecular cloning, genetic mapping, and mRNA expression

We have identified cDNAs clones for several cold-inducible mRNAs from the brown adipose tissue of mice. pCIN-1, a plasmid with a 900-base pair insert, encoded the mitochondrial uncoupling protein (UCP) as determined by the ability of the cDNA insert to select, by hybridization, an mRNA that could be translated into a 32,000-Da protein immunoprecipitable with anti-UCP antibodies. Nine tissues were analyzed; however, UCP cDNA hybridized to an mRNA species of 1.6 and 2.0 kilobase pairs only in brown adipose tissue. A maximum induction of 10-fold occurred within 6 h of exposure to cold (5 degrees C). A BamHI restriction fragment polymorphism detected by Southern blot analysis of genomic DNA in recombinant inbred mouse strains allowed us to map the UCP gene to Chromosome 8. The analysis of the UCP gene expression in diabetic (db) and obese (ob) mice maintained at 27 degrees C for 3 days followed by cold exposure for 4 h at 5 degrees C indicated that UCP mRNA levels in mutant mice were unaffected at 27 degrees C and only slightly reduced at 5 degrees C. Accordingly, the inability of diabetic and obese mice to thermoregulate is not associated with a lack of UCP mRNA induction.     

Physiological activation of brown adipose tissue destabilizes thermogenin mRNA

The amount of mRNA coding for the brown fat specific uncoupling protein thermogenin was followed in the brown adipose tissue of adult mice. As expected, cold exposure or norepinephrine injection caused an increase in the amount of thermogenin mRNA. However, contrary to expectation, the half-life of thermogenin mRNA was dramatically reduced, from about 18 h to about 3 h, when the mice were cold exposed. This destabilization of thermogenin mRNA was not related to the activity of protein synthesis. It was concluded that in brown adipose tissue an unusual mechanism operates which leads to a destabilization of thermogenin mRNA under the same physiological conditions which increase thermogenin gene expression.     

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.     

Impaired response of UCP family to cold exposure in diabetic (db/db) mice

Impaired activity of the uncoupling protein (UCP) family has been proposed to promote obesity development. The present study examined differences in UCP responses to cold exposure between leptin-resistance obese (db/db) mice and their lean (C57Ksj) littermates. Basal UCP1 and UCP3 mRNA expression in brown adipose tissue was lower in obese mice compared with lean mice, but UCP2 expression in white adipose tissue (WAT) was higher. Basal skeletal muscle UCP3 did not change remarkably. The UCP family mRNAs, which were upregulated 12 and 24 h after cold exposure (4 degrees C), were returned to prior levels 12 h after rewarming exposure (21 degrees C) in lean mice. The accelerating effects of cold exposure on the UCP family were impaired in db/db obese mice. Together with these changes, WAT lipoprotein lipase mRNA was downregulated, and the concentration of serum free fatty acid was increased in response to cold exposure in the lean mice but not in db/db obese littermates. The impaired function of the UCP family and diminished lipolysis in response to cold exposure indicate that the reduced lipolytic activity may contribute to the inactivation of the UCP family in db/db obese mice.     

Morpholigic and physiologic repercussions of partial removal of brown adipose tissue in rats acclimated to different temperatures]

Rats were chronically acclimated to 28 degrees C or 5 degrees C or submitted to daily variations of ambiant temperature. Ten or thirty days after removal of about 40% of the total brown adipose tissue (whole interscapular and 25% of abdominal tissues), the weight of the different pads of brown adipose tissue, thyroid and adrenals were determined. In all the groups, there was a large decrease of brown adipose tissue weight for the first ten days due to the shock following the operation. Then, the brown adipose tissue weight was restaured and, only in constant cold accliclimated rats, compensative hypertrophies of axillary and thoracic brown adipose tissue were found. Adrenals weight was significantly increased after the operation; in the two groups of cold acclimated rats, that increase was still significant one month later. However, the corticosterone production rate was not increased. These results are discussed in relation to the physiolocical role of brown adipose tissue in cold acclimated animals.     

Convertible adipose tissue in mice

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

Full expression of uncoupling protein gene requires the concurrence of norepinephrine and triiodothyronine

We have examined the uncoupling (UCP) protein gene expression in euthyroid and hypothyroid rats. UCP mRNA levels were estimated by northern blot analysis of total RNA from brown adipose tissue (BAT). Stimuli were endogenous (cold) and exogenous norepinephrine (NE), isoproterenol, T3, and T4. While the euthyroid rats UCP mRNA levels increase 2- to 3-fold by 2 h after NE or overnight cold exposure, these stimuli and isoproterenol are ineffective in hypothyroid rats. One single dose of T4, equal to the daily production rate, brings about a normal response in hypothyroid rats exposed to cold overnight. Hypothyroid rats recover their responsiveness to NE approximately 4 h after a receptor saturating dose of T3. On the other hand, such a dose of T3 induces a 3- to 4-fold increase in UCP mRNA levels in hypothyroid rats without the need of exogenous NE, and this response is not reduced by raising ambient temperature to thermoneutrality (28 C). However, the following evidence indicates that T3 requires adrenergic input to stimulate the accumulation of UCP mRNA: first, euthyroid animals maintained at 28 C do not respond to such a treatment. Second, when T3 was injected to hypothyroid rats with unilaterally denervated BAT, only the intact side responded to T3 with an elevation of the UCP mRNA levels, but both sides remained responsive to T3 + NE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of breeding temperature on the growth and composition of brown fat in the newborn rat]

The weight and the lipid, water and norepinephrine contents of the interscapular brown adipose tissue from 1, 3, 7, 11, 14 and 21 day aged rats were measured. The animals were maintained at an ambiant temperature of 16 degrees, 23 degrees or 28 degrees C from birth. It is concluded that nonshivering thermogenesis is not necessary after 3 days of age in animals kept at 28 degrees C and after 11 days of age in the ones kept at 23 degrees C. However that thermogenesis persists for all the suckling period in those kept at 16 degrees C.