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

1990 ◽  
Vol 68 (6) ◽  
pp. 973-979 ◽  
Author(s):  
Rachel E. Milner ◽  
Paul Trayhurn
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Ground Squirrel ◽  
Uncoupling Protein ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Nitrocellulose Membrane ◽  
Dot Blot ◽  
Brown Adipose

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.Key words: brown adipose tissue, uncoupling protein, dot immunobinding, immunoassay, thermogenesis.

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

1990 ◽  
Vol 68 (1) ◽  
pp. 231-237 ◽  
Author(s):  
Anna-Lisa Kates ◽  
Ian R. A. Park ◽  
Jean Himms-Hagen ◽  
Rudolf W. Mueller
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Macaca Fascicularis ◽  
Uncoupling Protein ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Fat Deposits ◽  
Brown Adipose ◽  
Tissue Homogenates

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.Key words: uncoupling protein, primate, 3,5,3′-triiodothyronine, thermogenesis; white adipose tissue.

Size analysis of uncoupling protein and its precursor from brown adipose tissue of different species

1985 ◽  
Vol 63 (9) ◽  
pp. 988-991 ◽  
Author(s):  
Karl B. Freeman ◽  
Karen Meyrick ◽  
Hasmukh V. Patel ◽  
Robert G. Ridley
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Uncoupling Protein ◽  
Mammalian Species ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Size ◽  
Size Analysis ◽  
Brown Adipose ◽  
Different Sources

The apparent size of the monomeric form of uncoupling protein from brown adipose tissue of several mammalian species was compared by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Including earlier results the apparent molecular mass of the protein from rat was about 32 000 daltons and varied about 1500–2000 daltons from the different sources with the size increasing in the order human < rat ≤ mouse ≤ hamster ≤ rabbit. The size of newly synthesized uncoupling protein was also found to vary among species. However in the two cases examined, rat and rabbit, the precursor and its respective mature monomeric protein had the same apparent size as shown by coelectrophoresis.

Immunochemical detection and quantitation of brown adipose tissue uncoupling protein

1987 ◽  
Vol 65 (3) ◽  
pp. 245-251 ◽  
Author(s):  
Mary F. Henningfield ◽  
Robert W. Swick
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Uncoupling Protein ◽  
Mammalian Species ◽  
Sodium Dodecyl ◽  
Mitochondrial Uncoupling ◽  
Immunochemical Method ◽  
Nucleotide Binding Sites ◽  
Mitochondrial Uncoupling Protein ◽  
Brown Adipose

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.

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

1989 ◽  
Vol 67 (12) ◽  
pp. 1480-1485 ◽  
Author(s):  
Paul Trayhurn ◽  
Norman J. Temple ◽  
Johny Van Aerde
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Uncoupling Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mitochondrial Uncoupling ◽  
Adipose Tissues ◽  
Mitochondrial Uncoupling Protein ◽  
Additional Group ◽  
Brown Adipose

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 °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."Key words: brown adipose tissue, white adipose tissue, uncoupling protein, thermogenesis, immunoblotting.

Brown fat thermogenesis during hibernation and arousal in Richardson's ground squirrel

1989 ◽  
Vol 256 (1) ◽  
pp. R42-R48 ◽  
Author(s):  
R. E. Milner ◽  
L. C. Wang ◽  
P. Trayhurn
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Cold Acclimation ◽  
Binding Sites ◽  
Ground Squirrel ◽  
Protein Concentration ◽  
Uncoupling Protein ◽  
Ground Squirrels ◽  
Mitochondrial Content ◽  
Brown Adipose

The thermogenic activity [mitochondrial guanosine 5'-diphosphate (GDP) binding] and capacity (uncoupling protein concentration, cytochrome oxidase activity) of brown adipose tissue have been investigated at different phases of the seasonally linked hibernation cycle in Richardson's ground squirrel. The amount of axillary brown adipose tissue and the total mitochondrial content of the tissue were substantially greater in hibernating squirrels than in squirrels caught posthibernation in April or May; cold acclimation induced qualitatively similar differences. The specific mitochondrial concentration of uncoupling protein was high under all conditions (compared with other species), differing little between hibernating, posthibernating, and cold-acclimated squirrels. The thermogenic capacity of brown adipose tissue in Richardson's ground squirrels is therefore modulated almost exclusively by changes in the mitochondrial content of the tissue. Mitochondrial GDP binding was increased on cold acclimation, but similar binding levels were observed in hibernating and posthibernation (May) animals. GDP binding and the GDP-sensitive component of acetate-induced mitochondrial swelling were increased during the early stages of arousal from hibernation. These changes, which indicate an activation of the thermogenic proton conductance pathway in arousal, occurred without an alteration in the specific mitochondrial concentration of uncoupling protein. Increased GDP binding during arousal is clearly due to the unmasking of binding sites, reflecting an acute activation of preexisting uncoupling protein.

Role of prolactin in lactation-induced changes in brown adipose tissue

1990 ◽  
Vol 258 (1) ◽  
pp. R51-R56 ◽  
Author(s):  
E. Chan ◽  
R. Swaminathan
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Uncoupling Protein ◽  
Energy Utilization ◽  
Serum Prolactin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Simultaneous Administration ◽  
Brown Adipose ◽  
Induced Changes

During lactation the efficiency of energy utilization is increased and brown adipose tissue (BAT) thermogenesis is suppressed. We have examined the role of prolactin in the suppression of BAT during lactation in the rat. Changes in BAT were studied using an enzyme-linked immunosorbent assay for the uncoupling protein (UCP). In the first experiment BAT from pregnant and lactating rats was examined at various times. Total UPC content of BAT decreased from 364 +/- 34 to 32 +/- 9 micrograms during late lactation (20 days). Serum prolactin (PRL) concentration was elevated during lactation and was highest at 10 days. When endogenous PRL was stimulated by injection of metoclopramide (1.67 mg/kg) for 5 days, UCP content of BAT decreased by 50%. This action of metoclopramide could be blocked by simultaneous administration of bromocriptine, an inhibitor of PRL secretion. Serum PRL concentration was elevated during metoclopramide administration but not when metoclopramide and bromocriptine were given together. We conclude that PRL is a possible mediator of the suppression of BAT during lactation.

scholarly journals A single mutation in uncoupling protein of rat brown adipose tissue mitochondria abolishes GDP sensitivity of H+ transport.

1994 ◽  
Vol 269 (10) ◽  
pp. 7435-7438
Author(s):  
D.L. Murdza-Inglis ◽  
M. Modriansky ◽  
H.V. Patel ◽  
G. Woldegiorgis ◽  
K.B. Freeman ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Uncoupling Protein ◽  
Single Mutation ◽  
Brown Adipose

scholarly journals TAF7L modulates brown adipose tissue formation

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Haiying Zhou ◽  
Bo Wan ◽  
Ivan Grubisic ◽  
Tommy Kaplan ◽  
Robert Tjian
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Core Promoter ◽  
Uncoupling Protein ◽  
Dna Looping ◽  
Chromatin Interactions ◽  
Brown Adipose ◽  
Important Regulator

Brown adipose tissue (BAT) plays an essential role in metabolic homeostasis by dissipating energy via thermogenesis through uncoupling protein 1 (UCP1). Previously, we reported that the TATA-binding protein associated factor 7L (TAF7L) is an important regulator of white adipose tissue (WAT) differentiation. In this study, we show that TAF7L also serves as a molecular switch between brown fat and muscle lineages in vivo and in vitro. In adipose tissue, TAF7L-containing TFIID complexes associate with PPARγ to mediate DNA looping between distal enhancers and core promoter elements. Our findings suggest that the presence of the tissue-specific TAF7L subunit in TFIID functions to promote long-range chromatin interactions during BAT lineage specification.

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