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

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

Evidence from immunoblotting studies on uncoupling protein that brown adipose tissue is not present in the domestic pig

Adipose tissues and other tissues of the pig have been examined for the presence of the mitochondrial "uncoupling protein," characteristic of brown adipose tissue, in order to assess whether brown fat is present in this species. Mitochondria were prepared from various tissues and the proteins separated on the basis of molecular weight by sodium dodecyl sulphate--polyacrylamide gel electrophoresis. Immunoblotting procedures were then used to probe for uncoupling protein, employing a rabbit anti-(rat uncoupling protein) serum. Pigs were examined at 4 days, 4 weeks, and 8 weeks of age. No evidence for the presence of uncoupling protein was found at any of these ages. The protein was, however, readily detected in brown adipose tissue from rats, mice, golden hamsters, guinea pigs, Richardson's ground squirrel, and lambs. An additional group of pigs was acclimated to the cold (10 degrees C) for a period of 10 days prior to the examination of tissues, but again uncoupling protein was not detected in any tissue. These results indicate that uncoupling protein is either absent from adipose tissues of the pig or is present at such a low concentration that it is unlikely to support thermogenesis. It is concluded that the pig does not contain adipose tissue that is functionally "brown;" adipose tissues in this species appear to be exclusively "white."     

Measurement by radioimmunoassay of the mitochondrial uncoupling protein from brown adipose tissue of obese (ob/ob) mice and Zucker (fa/fa) rats at different ages

The concentration of the 'uncoupling protein' in brown adipose tissue mitochondria has been measured in lean and obese (ob/ob) mice and Zucker (fa/fa) rats at different ages using a specific radioimmunoassay. During the suckling period the concentration of the protein was similar in normal and mutant animals of both types, despite the decrease in mitochondrial GDP binding observed in the obese. The concentration of uncoupling protein was, however, decreased in adult ob/ob mice and adult Zucker rats compared with their respective lean siblings, in parallel with the decrease in GDP binding. It is concluded that there is a 'masked', or inactive, form of uncoupling protein in young ob/ob mice and fa/fa rats.     

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

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

Rapid quantitation of uncoupling protein in brown adipose tissue mitochondria by a dot immunobinding ("dot blot") procedure: application to the measurement of uncoupling protein in Richardson's ground squirrel, rats, and mice

A dot immunobinding ("dot blot") method for measuring uncoupling protein in brown adipose tissue mitochondria is described. Mitochondrial proteins were solubilized in sodium dodecyl sulfate and applied directly to a nitrocellulose membrane housed in a 96-well microfiltration manifold. Spare binding sites on the nitrocellulose membrane were blocked with bovine serum albumin and then anti-(uncoupling protein) serum was applied. The antigen-antibody complex was detected by the addition of 125I-labelled protein A. Each nitrocellulose "dot" was cut out and its radioactivity was counted. A calibration curve was constructed from purified uncoupling protein standards, taken through the entire procedure. The dot immunobinding method is sensitive (nanogram quantities of uncoupling protein), and in contrast to conventional radioimmunoassay and enzyme-linked immunosorbent assay procedures, it is also rapid and appears to be very robust. The method has been successfully applied to the measurement of uncoupling protein in brown adipose tissue mitochondria of Richardson's ground squirrel, rats, and mice.     

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

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

Mitochondria of adult human brown adipose tissue contain a 32 000-Mr uncoupling protein

Using both immunohistological study and photoaffinity labelling with radioactive azido-ATP, evidence is presented that the mitochondrial membranes of the brown adipose tissue of the human adult contain a 32 000-Mr uncoupling protein, which is probably similar to the uncoupling protein of BAT of rodents.     

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     

Immunochemical detection and quantitation of brown adipose tissue uncoupling protein

A polyclonal antisera against rat brown adipose tissue mitochondrial uncoupling protein was used to examine mitochondrial samples from liver and white and brown adipose tissue from several mammalian species. A sodium dodecyl sulfate--polyacrylamide gel electrophoretic separation of proteins combined with an immunochemical method allowed for visualization of antigen--antibody complexes on nitrocellulose blots. Hamster, cavy, monkey, and mouse brown adipose tissue mitochondrial samples cross-reacted with the antisera. Mitochondria prepared from white fat obtained from young swine and sheep contained two closely migrating, antigenically active proteins. Hepatic mitochondria samples did not contain antigenically active protein. Reflectance densitometry was used for quantitation of the uncoupling protein in various mitochondrial samples. In rats fed diets low in protein, there appears to be a dissociation between the concentration of uncoupling protein and the number of nucleotide binding sites as given by the [3H]GDP binding assay. These results are indicative of a physiological activation of the uncoupling protein.     

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.     

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.     

Differentiation of ovine brown adipocyte precursor cells in a chemically defined serum-free medium. Importance of glucocorticoids and age of animals

The rapid apparent conversion of brown adipose tissue into white adipose tissue in newborn offspring of large mammals, such as sheep and cattle is not explained at the cellular level. To study the differentiation of lamb brown adipocyte, a genomic fragment corresponding to the uncoupling protein was cloned from an ovine DNA library. Stromal vascular fibroblasts isolated from the perirenal adipose tissue of newborn lambs completely differentiated into brown adipocytes expressing the uncoupling protein gene, in a chemically defined serum-free medium. Dexamethasone was necessary for the expression of the uncoupling protein gene. When stromal vascular fibroblasts were isolated from 3-week-old lambs, the glucocorticoid analog still promoted in vitro differentiation of adipocytes. However those adipocytes were unable to express uncoupling mRNA and could be considered as white adipocytes. The data indicate that dexamethasone is necessary but not sufficient clone for the complete differentiation of brown adipocytes, and that the preadipocytes are committed to differentiation into brown or white adipocytes before culture.     

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

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

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.     

Postnatal appearance of uncoupling protein and formation of thermogenic mitochondria in hamster brown adipose tissue

Brown adipose tissue of developing hamster was characterized by western blotting, enzyme activity measurements and immunoelectron microscopy. During the first postnatal week the tissue contained significant amounts of differentiating mitochondria and comparable quantities of active cytochrome oxidase and ATP synthase. The uncoupling protein appeared on the 7/8th day and its specific content increased 80-times between day 8 and day 17. In parallel, the specific content and activity of cytochrome oxidase increased 3-times but ATP synthase decreased 2-times. The total content of uncoupling protein and of cytochrome oxidase in interscapular brown adipose tissue increased 360- and 11-times, respectively. Analysis of isolated mitochondria showed that the observed differences result mainly from changes of the enzymic equipment of the mitochondrial membrane. During the same interval, propylthiouracil-insensitive "type II' thyroxine 5'-deiodinase activity in brown adipose tissue increased 10-times. It was concluded that the thermogenic function of the hamster brown adipose tissue develops after the first postnatal week due to highly differentiated synthesis of mitochondrial proteins leading to replacement of preexisting, uncoupling protein-lacking nonthermogenic mitochondria by thermogenic ones, similarly as shown in brown adipose tissue of the embryonic mouse and rat (Houst?k, J., et al. (1988) Biochim. Biophys. Acta 935, 19-25).     

The role of UCP 1 in production of reactive oxygen species by mitochondria isolated from brown adipose tissue

We provide evidence that ablation or inhibition of, uncoupling protein 1 increases the rate of reactive oxygen containing species production by mitochondria from brown adipose tissue, no matter what electron transport chain substrate is used (succinate, glycerol-3-phosphate or pyruvate/malate). Consistent with these data are our observations that (a) the mitochondrial membrane potential is maximal when uncoupling protein 1 is ablated or inhibited and (b) oxygen consumption rates in mitochondria from uncoupling protein 1 knock-out mice, are significantly lower than those from wild-type mice, but equivalent to those from wild-type mice in the presence of GDP. In summary, we show that uncoupling protein 1 can affect reactive oxygen containing species production by isolated mitochondria from brown adipose tissue.     

An immunological study of the uncoupling protein of brown adipose tissue mitochondria

1. Ewes were injected with purified 32,000-Mr uncoupling protein from mitochondria of brown adipose tissue of cold-adapted rats in order to raise antibodies. 2. The existence of antibodies in the plasma of ewes and the cross-reactivity of plasmas were demonstrated and studied by 125I-labelled antigen-antibody reaction, double immunodiffusion, the inhibition of GDP binding to the 32,000 Mr protein and by immunohistochemistry. 3. The antibodies raised against the homogeneous protein yielded a single immunoprecipitation band with detergent-solubilized mitochondrial membranes of brown adipose tissue from rat, hamster, guinea-pig, rabbit and with the purified uncoupling protein of these animals. No immunoprecipitation was obtained with the protein purified from brown adipose tissue of term lamb foetus. 4. The GDP-binding activity of the uncoupling protein (isolated or in solubilized membranes) was largely inhibited by the antiserum. 5. The anti-(rat uncoupling protein) could not cross-react with solubilized membranes from liver or muscle, nor with the purified beef heart or rat liver ADP/ATP translocator.     

Immunohistochemical identification of the uncoupling protein in rat brown adipose tissue

Brown adipose tissue mitochondria are characterized by the presence of an uncoupling protein that gives them an exceptional capacity for substrate-controlled respiration and thermogenesis. The specific localization of this protein in rat brown adipocytes was demonstrated using an immunohistochemical technique, the peroxidase-antiperoxidase (PAP) method. Light microscopy observations showed that serum antibodies raised against the uncoupling protein selectively reacted with multilocular brown adipocytes. No labeling could be detected in either unilocular adipocytes, capillaries, or muscle fibers (striated and vascular smooth muscle). Staining was more intensive in certain adipocytes than in others, suggesting the presence of cellular heterogeneity. The specificity of the staining technique was demonstrated by showing that treatment of the preparations with antiserum saturated with an excess of uncoupling protein almost entirely inhibited brown adipocyte labeling. The specificity and selectivity of the PAP method allow the clear differentiation of uncoupling protein-containing adipocytes from other cellular types, suggesting that this immunohistochemical technique will represent an extremely useful tool for studying adipocyte function and differentiation.     

Molecular approach to thermogenesis in brown adipose tissue. Cell-free translation of mRNA and characterization of the mitochondrial uncoupling protein

In order to develop a molecular approach of the thermogenesis mechanism in brown adipose tissue, cell-free translation was performed with mRNA obtained from control or thermoactive brown adipose tissue. Alterations were observed on analysis of the newly synthesized proteins and in particular at the 32,000 dalton level. Experiments using antibodies against the purified characteristic 32,000-dalton uncoupling protein of brown fat mitochondria were carried out. They indicated that the uncoupling protein was synthesized in the reticulocyte lysate with the same apparent molecular weight as the mature form. It is suggested that the development of the thermogenic capacity of brown fat cells is accompanied by an increase in specific mRNA coding for the uncoupling mitochondrial protein and that such a system could be an interesting one for study of mitochondrial membrane biogenesis.