Identification of two mitochondrial GDP-binding sites in rat brown adipose tissue

1983 ◽  
Vol 3 (7) ◽  
pp. 589-598 ◽  
Author(s):  
K. R. Bryant ◽  
N. J. Rothwell ◽  
M. J. Stock ◽  
D. Stribling
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Mitochondrial Protein ◽  
Scatchard Analysis ◽  
Nucleotide Binding Sites ◽  
Guanosine Diphosphate ◽  
Brown Adipose ◽  
Number Of Binding Sites

Scatchard analysis of specific guanosine-diphosphate-([3H]GDP-) binding to rat brown-adipose-tissue mitochondria revealed two distinct binding sites with apparent dissociation constants (Kd) of approximately 0.05 and 2.0 μM. Binding to both sites was insensitive to atractyloside. Reducing the pH of the binding medium from 7.1 to 6.6 caused marked reductions in the Kd of both sites, but at pH 7.6, the dissociation constants were increased about 3-fold. Acute treatment of rats with noradrenaline, 1 h before sacrifice, increased the maximum number of binding sites (Bmax, pmol/rng mitochondrial protein) of both sites and also increased the dissociation constants. The Bmax of the lower-affinity site was elevated in rats exposed to 5°C or fed a palatable cafeteria diet for 10 d, compared to control animals, with the greater changes occurring in the cold-adapted group. The high-affinity site was unaltered by cold adaptation or cafeteria feeding. These results indicate the presence of two distinct nucleotide-binding sites in brown-fat mitochondria, both of which may be involved in thermogenesis.

Acute influences on the two GDP-binding sites in brown-adipose-tissue mitochondria

1984 ◽  
Vol 4 (6) ◽  
pp. 523-533 ◽  
Author(s):  
K. R. Bryant ◽  
N. J. Rothwell ◽  
M. J. Stock
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Conformational Changes ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Mitochondrial Protein ◽  
Scatchard Analysis ◽  
Slight Reduction ◽  
High Affinity ◽  
Brown Adipose

Scatchard analysis of3H-guanosine diphosphate (GDP) binding to rat brown-adipose-tissue mitochondria demonstrated that binding to the high- and low-affinity sites (Kd = 0.05 and 2.0 μM) was abolished by denaturation at 100°C but non-specific binding remained constant (0.2% of free-GDP). Prior incubation of mitochondria at 37°C reduced binding to the high-affinity site, but this could be reversed by incubating samples at 0°C. Addition of palmitic acid (5–40 nmole/mg of mitochondrial protein) did not affect GDP-binding, but similar concentrations of palmitoyl CoA caused a slight reduction in the number of high-affinity sites and a significant decrease in the number of lower-affinity sites. Acute treatments known to stimulate thermogenesis in vivo (a single meal, cold exposure, or noradrenaline injection 40–80 min before sacrifice) all increased binding to both binding sites, and tended to raise the dissociation constants, whereas injection of 2-deoxy-D-glucose, which depresses metabolic rate in the rat, decreased dissociation constants of both sites and the maximum number of high-affinity sites. These data indicate that both GDP-binding sites respond rapidly to acute thermogenic stimuli, possibly due to conformational changes in the mitochondrial inner membrane, and that palmitoyl CoA may influence mitochondrial proton conductance via an association with purine nucleotide binding sites.

scholarly journals Evidence for decreased GDP binding to brown-adipose-tissue mitochondria of obese Zucker (fa/fa) rats in the very first days of life

1984 ◽  
Vol 221 (1) ◽  
pp. 241-245 ◽  
Author(s):  
R Bazin ◽  
D Eteve ◽  
M Lavau
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Binding Sites ◽  
Scatchard Analysis ◽  
Zucker Rat ◽  
Large Decrease ◽  
Sequential Order ◽  
Lean Control ◽  
Brown Adipose ◽  
Obese Zucker Rat

GDP binding to brown-adipose-tissue mitochondria of obese Zucker-rat (fa/fa) pups aged 2-14 days was significantly less than in lean control rats. Scatchard analysis in 10-day-old pups suggests that there was a large decrease in GDP-binding sites. However, a significant increase in fat content in brown adipose tissue of 2-day-old pre-obese pups raised the question of the sequential order and causal relationship between these two derangements.

scholarly journals GDP binding to brown-adipose-tissue mitochondria of diabetic-obese (db/db) mice. Decreased binding in both the obese and pre-obese states

1981 ◽  
Vol 194 (3) ◽  
pp. 1019-1022 ◽  
Author(s):  
A E Goodbody ◽  
P Trayhurn
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Binding Sites ◽  
Mutant Mice ◽  
Brown Adipose ◽  
Number Of Binding Sites

GDP binding to brown-adipose-tissue mitochondria from adult diabetic–obese (db/db) mice was significantly less than with lean siblings. Binding was also decreased in the mutant mice before obesity had begun to develop. Decreased GDP binding was found to result from a decrease in the number of binding sites.

Beta-adrenoreceptors in rat brown adipose tissue: proportions of beta 1- and beta 2-subtypes

1985 ◽  
Vol 248 (4) ◽  
pp. E397-E402 ◽  
Author(s):  
N. J. Rothwell ◽  
M. J. Stock ◽  
D. K. Sudera
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Binding Sites ◽  
Scatchard Analysis ◽  
Single Class ◽  
Brown Adipose ◽  
Ici 118,551 ◽  
Microsomal Membranes ◽  
Beta 2 ◽  
Equilibrium Dissociation

Binding of (-)-[3H]dihydroalprenolol [( 3H]DHA) to isolated brown adipose tissue (BAT) microsomal membranes was used to estimate beta-adrenoreceptor density, and this was found to be saturable, reversible, and stereospecific. Scatchard analysis indicated a single class of binding sites of equilibrium dissociation constant 2.2 nM, with a maximum number of binding sites of 160-170 fmol/mg protein. These values were unaffected by the age or sex of the animals, and similar Kd values were obtained for binding to heart and lung membranes. The kinetically derived Kd from forward and reverse reactions was 1.4 nM for BAT. Hofstee analysis of displacement of [3H]DHA produced linear plots for propranolol but curvilinear plots for atenolol and ICI 118,551. Atenolol showed 50-fold selectivity for beta 1-receptors and ICI 118,551 50-fold selectivity for beta 2-receptors in heart, lung, and BAT membranes. These plots indicated beta 1:beta 2-receptor ratios of 59:41 for BAT and heart and 25:75 for lung. It is possible that both beta-adrenoreceptor subtypes in BAT may be involved in the activation of thermogenesis in the tissue.

Uncoupling protein and its mRNA in brown adipose tissue of newborn rabbits

1989 ◽  
Vol 67 (1) ◽  
pp. 54-58 ◽  
Author(s):  
D. K. Rozon ◽  
W. H. Harris ◽  
A. M. Verrinder Gibbins
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Messenger Rna ◽  
Uncoupling Protein ◽  
Mitochondrial Protein ◽  
Brown Fat ◽  
Guanosine Diphosphate ◽  
Mitochondrial Inner Membrane ◽  
Age Related ◽  
Brown Adipose

Guanosine diphosphate binding to the uncoupling protein of isolated mitochondria of brown adipose tissue in newborn rabbits was measured as an index of thermogenic activity. The binding was 0.281 ± 0.022 nmol GDP/mg mitochondrial protein at 1 day of age, 0.214 ± 0.017 at 3 days, 0.428 ± 0.038 at 5 days, and 0.208 ± 0.016 at 7 days. The increase in binding between 3 and 7 days of age suggests that the brown fat has an increased thermogenic capacity at that age. In addition, the potential for synthesis of the uncoupling protein was investigated in 1- to 5-day-old newborn rabbits by probing the total cellular ribonucleic acid for the messenger that codes for uncoupling protein. The amount of uncoupling protein messenger was highest at 1 day of age and declined at least until 5 days of age. Because the amount of uncoupling protein messenger decreased as the GDP binding increased, the results suggest that either the initially translated uncoupling protein was unmasked at about 5 days of age or there was a delay in the incorporation of uncoupling protein into the mitochondrial inner membrane, or both.Key words: brown fat, neonatal rabbit, thermogenin, messenger RNA, age-related changes.

scholarly journals Evidence for unmasking of rat brown-adipose-tissue mitochondrial GDP-binding sites in response to acute cold exposure. Effects of washing with albumin on GDP binding

1986 ◽  
Vol 233 (3) ◽  
pp. 743-747 ◽  
Author(s):  
C L Gribskov ◽  
M F Henningfield ◽  
A G Swick ◽  
R W Swick
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Cold Exposure ◽  
Binding Sites ◽  
Uncoupling Protein ◽  
Fold Increase ◽  
Scatchard Analysis ◽  
Mitochondrial Uncoupling ◽  
Mitochondrial Uncoupling Protein ◽  
Brown Adipose

Rats, previously acclimated to 29 degrees C, were moved into the cold (4 degrees C) for 2 h. Scatchard analysis of GDP binding to the brown-adipose-tissue mitochondria of these animals showed a 2.3-fold increase in the number of high-affinity sites and a 1.5-fold increase in the number of low-affinity sites compared with binding in animals maintained at 29 degrees C. Immunochemical determination showed no increase in the amount of mitochondrial uncoupling protein during this period. This strongly suggests an unmasking of existing GDP-binding sites before a detectable increase in synthesis of uncoupling protein can occur. Washing with albumin increased the number of GDP-binding sites of brown-adipose-tissue mitochondria from both warm-housed and cold-exposed animals to the same extent. This indicates that the effects of washing with albumin and cold exposure are independent and additive.

scholarly journals The effect of adrenalectomy on GDP binding to brown-adipose-tissue mitochondria of obese rats

1982 ◽  
Vol 208 (3) ◽  
pp. 819-822 ◽  
Author(s):  
Susan Holt ◽  
David A. York
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Binding Sites ◽  
Zucker Rats ◽  
Lean Control ◽  
Brown Adipose ◽  
Obese Rats ◽  
Number Of Binding Sites ◽  
Obese Zucker Rats

GDP binding to brown-adipose-tissue mitochondria of young obese Zucker rats (fa/fa) was significantly lower than in lean control rats, as a result of a decrease in the number of binding sites. Adrenalectomy of fa/fa rats restored GDP binding to control values. Corticosterone replacement suppressed GDP binding in adrenalectomized obese rats.

Effects of fasting and food restriction on brown adipose tissue composition in normal and dystrophic hamsters

1986 ◽  
Vol 64 (7) ◽  
pp. 970-975 ◽  
Author(s):  
M. Desautels ◽  
R. A. Dulos ◽  
H. M. Yuen
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Food Deprivation ◽  
Food Restriction ◽  
Tissue Mass ◽  
Tissue Protein ◽  
Guanosine Diphosphate ◽  
Isolated Mitochondria ◽  
Daily Food ◽  
Brown Adipose

Fasting for 36–48 h or food restriction (30% reduction of daily food intake for 6 weeks) caused brown adipose tissue (BAT) atrophy in hamsters. Fasting-induced atrophy was characterized by reductions in tissue mass, DNA, protein, and thermogenin. By contrast, food restriction had no effect on tissue cellularity (DNA) but markedly reduced the tissue protein and thermogenin contents. The concentration of thermogenin in isolated mitochondria was unchanged by fasting or food restriction. Dystrophic hamsters had a reduced BAT mass when compared with weight-matched control hamsters. This resulted from a reduction in tissue cellularity since BAT DNA, protein and thermogenin contents were all reduced. The extent of binding of [3H]guanosine diphosphate to isolated mitochondria and their content of thermogenin were similar in normal and dystrophic hamsters. In response to cold exposure, as in normal hamsters, BAT of dystrophic hamsters grew and the tissue thermogenin increased, but the mitochondrial concentration of thermogenin did not change. In response to fasting, in contrast with normal hamsters, there was no significant reduction in BAT DNA in dystrophic animals and the loss of tissue protein was reduced. However, the relative changes in BAT composition during chronic food restriction were similar in normal and dystrophic animals. Thus, reduction in hamster BAT thermogenic capacity during food deprivation may occur by loss of cells and (or) reduction in the tissue protein and thermogenin contents. The extent of protein and (or) DNA loss may be dependent upon the original tissue mass and the severity of food deprivation.

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