Acute effects of a beta-adrenoceptor agonist (BRL 26830A) on rat brown-adipose-tissue mitochondria. Increased GDP binding and GDP-sensitive proton conductance without changes in the concentration of uncoupling protein.

GDP binding, proton conductance and the specific concentration of uncoupling protein were measured in brown-adipose-tissue mitochondria of rats treated acutely with the novel beta-agonist, BRL 26830A. At 1 h after dosing with BRL 26830A, mitochondrial GDP binding was increased more than 2-fold. The increase in binding resulted from an increase in the number of binding sites. An iterative analysis of Scatchard binding data suggested that there is only one high-affinity GDP-binding site (Kd 0.3 microM) in brown-adipose-tissue mitochondria. The acute increase in GDP binding produced by treatment with BRL 26830A occurred without any alteration in the specific mitochondrial concentration of uncoupling protein, as determined by radioimmunoassay. Treatment with the beta-agonist did, however, lead to a small increase in the GDP-sensitive component of mitochondrial proton conductance. These results indicate that GDP-binding sites on uncoupling protein can be rapidly unmasked after treatment with a brown-fat-specific beta-agonist, and that the increase in binding reflects an increase in the activity of the mitochondrial proton-conductance pathway.     

The influence of GDP on Ca2+ uptake by mitochondria of brown adipose tissue from lean and genetically obese (ob/ob) mice.

The specific binding capacity for purine nucleotides in brown-adipose-tissue mitochondria is thought to indicate the capacity of the proton-conductance pathway which leads to uncoupled respiration. This functional relationship was investigated in studies measuring initial Ca2+-uptake rates and membrane potential in the presence or absence of GDP in brown-adipose-tissue mitochondria with different GDP-binding capacities. The mitochondria from pre-obese and obese ob/ob mice were less able than those from lean control mice to dissipate membrane potential in the absence of GDP. Mitochondria from the obese animals also maintained a higher Ca2+-uptake rate without GDP in comparison with the rate found with mitochondria from the lean mice. The GDP-dependence of Ca2+ uptake was greater in brown-adipose-tissue mitochondria from cold-adapted animals than in those from animals kept at 22 degrees C or at thermoneutrality (33 degrees C). It is concluded that Ca2+-uptake rate and membrane-potential values are depressed in the absence of GDP and indicate indirectly the influence of purine nucleotides on maintaining the proton electrochemical gradient in brown-adipose-tissue mitochondria. It is also apparent that the lower GDP-binding capacity in mitochondria from ob/ob mice is related to a decreased ability to dissipate the proton electrochemical gradient.     

Evidence that the acute unmasking of GDP-binding sites in brown adipose tissue mitochondria is not dependent on mitochondrial swelling

The role of mitochondrial swelling in the unmasking of GDP-binding sites on brown adipose tissue mitochondria has been examined in mice. Acute cold exposure (6 degrees C for 1 h) led to increases in GDP binding without changes in the concentration of uncoupling protein, indicating that an unmasking of binding sites had occurred. Measurements of mitochondrial matrix volume suggested that an acute unmasking of GDP-binding sites took place without swelling of the mitochondria. In addition, the induction of a rapid preswelling of the mitochondria by incubation in KCl in the presence of valinomycin did not affect the cold-induced unmasking of GDP-binding sites. It is concluded that the acute unmasking of GDP-binding sites on uncoupling protein in brown adipose tissue is not due simply to mitochondrial swelling.     

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.     

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

Scatchard analysis of³H-guanosine diphosphate (GDP) binding to rat brown-adipose-tissue mitochondria demonstrated that binding to the high- and low-affinity sites (K_d=0.05 and 2.0 M) 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.     

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.     

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

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.     

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

Scatchard analysis of specific guanosine-diphosphate-([³H]GDP-) binding to rat brown-adipose-tissue mitochondria revealed two distinct binding sites with apparent dissociation constants (K_d) 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 K_d 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 (B_max, 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.     

The effects of corticosterone, cold exposure and overfeeding with sucrose on brown adipose tissue of obese Zucker rats (fa/fa).

GDP binding to brown-adipose-tissue mitochondria was decreased in obese Zucker rats. Adrenalectomy restored both GDP binding and serum tri-iodothyronine of obese rats to values observed in lean rats. The effects of adrenalectomy on GDP binding and serum tri-iodothyronine were reversed by corticosterone. Decreasing food intake had no effect on brown-adipose-tissue GDP binding in obese rats. Young (5-week-old) obese rats showed a normal increase in brown-adipose-tissue mitochondrial GDP binding after housing at 4 degrees C for 7 days, but this response was attenuated in 10-week-old obese rats. Overfeeding with sucrose increased brown-adipose-tissue thermogenesis in lean, but not in obese, rats. After adrenalectomy, overfeeding with sucrose enhanced brown-adipose-tissue mitochondrial GDP binding in obese rats.     

Brown fat activity in fasted and refed rats

Four days of fasting in the rat reduced brown-adipose-tissue (BAT) mass, mitochondrial protein, and tissue protein content. Specific binding of guanosine diposphate (GDP) to BAT mitochondria was depressed by 55% in 4d-fasted rats. Rats fasted for 3 d, and then refed a single carbohydrate meal (40 kJ), showed a significant increase in specific GDP-binding (27% above fasted) 24 h later, and a large increase in total binding. Specific activities of cytochrome oxidase and -glycerophosphate dehydrogenase in BAT mitochondria were not significantly affected by fasting or refeeding. These results suggest that BAT may be partly responsible for the fall in metabolic rate associated with fasting and the delayed increase after carbo-hydrate refeeding. These effects may be due to changes in the mitochondrial proton-conductance pathway in brown fat.     

Apparent unmasking of [3H]GDP binding in rat brown-fat mitochondria is due to mitochondrial swelling

The presence of and biochemical background for the so-called 'unmasking' phenomenon in rat brown-fat mitochondria was investigated (i.e. the apparent increase in [3H]GDP binding to the 'uncoupling' protein thermogenin, without a concomitant increase in the amount of the protein). It was found that an unmasking could be observed both 1 h after norepinephrine injection and after 1 h cold stress, provided that the rats were preacclimated to 28 degrees C. The unmasking could be observed both when a filtration method and when a centrifugation method for determination of [3H]GDP-binding capacity were used; however, the absolute values were higher with the filtration method. Based on observations of slower cytochrome-c oxidase sedimentation during centrifugation, the possibility that the matrix volume of brown-fat mitochondria isolated from warm-acclimated animals was smaller than that of cold-stressed animals was investigated with 3H2O. The cold stress increased the matrix volume from being nearly non-existent to about 1 microliter/mg. A preswelling procedure in an ionic medium could similarly increase the matrix volume in mitochondria from warm-acclimated animals but was without significant effect in the already swollen mitochondria from cold-stressed animals or from animals adapted to a lower temperature. In mitochondria from warm-acclimated animals, the ionic preswelling procedure was fully able to increase the apparent amount of GDP binding to that observed in mitochondria from cold-stressed animals, but it was practically without effect on GDP binding in mitochondria from cold-stressed animals or from animals acclimated to a lower temperature. It is concluded that the apparent 'unmasking' phenomenon, observed when the tissue is less activated than in normal control situations, is not (as hitherto anticipated) due to a specific change in thermogenin as such, but is a reflection of a general mitochondrial phenomenon.     

The mitochondrial uncoupling protein from guinea-pig brown adipose tissue. Synchronous increase in structural and functional parameters during cold-adaptation.

The time-course for the induction of the uncoupling pathway in the inner membrane of brown-fat mitochondria from cold-adapting guinea pigs was studied. The amount of the protein was quantified from the capacity for high-affinity binding of GDP to the intact mitochondria, and was compared with the functional parameters diagnostic of the protein, namely the nucleotide-sensitive proton conductance and the sensitivity to uncoupling by low concentrations of fatty acids. A monophasic increase in nucleotide titre was observed, with no evidence of an early 'unmasking' of preexisting nucleotide-binding sites. The nucleotide-sensitive conductance increased in precise synchrony with the nucleotide-binding capacity. Mitochondria from newborn animals, and those from acutely cold-adapted animals, showed anomalously low sensitivities to uncoupling by fatty acids.     

Infrared spectroscopic studies of detergent-solubilized uncoupling protein from brown-adipose-tissue mitochondria

The uncoupling protein of brown-adipose-tissue mitochondria has been purified in the form of mixed micelles with lipid and reduced Triton X-100. This surfactant has the advantage over conventional Triton X-100, that it does not interfere with amide bands in infrared spectra. The structure of the uncoupling protein in micellar form has been examined by Fourier-transform infrared spectroscopy (FTIR). In order to decompose the amide I contour into its components, band-narrowing (Fourier derivation and deconvolution) and band-decomposition techniques have been used. Combining data from spectra taken in H2O and 2H2O media, the following percentage distribution of secondary structure patterns has been obtained: 50% alpha-helix, 28-30% beta-structure; 13-15% beta-turns and 7% unordered. Thermal denaturation of the uncoupling protein has also been monitored by FTIR. In accordance with previous observations of different proteins, thermal denaturation is marked by a shift in the amide I maximum and the appearance of two new peaks in 2H2O, at around 1620 cm-1 and 1685 cm-1. Denaturation occurs in the 40-50 degrees C temperature range, in agreement with studies of GDP-binding capacity. Cooling down the thermally denatured protein produces a new change in its secondary structure; however, the original conformation is not restored. The uncoupling protein possesses a nucleotide-binding site. On addition of GDP, small changes in protein conformation occur, attributable to changes in tertiary structure. However, no detectable effects are seen in the presence or absence of the other physiological regulators, the free fatty acids. The uncoupling protein shares important similarities in its primary structure with other anion carriers of the mitochondrial membrane; one of these, the adenine-nucleotide translocator, has been used in a comparative study, applying the same FTIR techniques described above for the uncoupling protein. Both proteins have a similar proportion of alpha-helix, probably corresponding to the segments spanning the membrane, but the conformation of the polar domains appears to differ.     

Parallel increases in amount of (3H)GDP binding and thermogenin antigen in brown-adipose-tissue mitochondria of cafeteria-fed rats

In order to investigate the possible existence of a ‘masked’ (i.e. non-GDP-binding) form of thermogenin (the brown-adipose-tissue specific, 32 000 Da so-called “uncoupling” protein), rats were fed a routine pellet diet or, in addition to this, a cafeteria diet. Brown-adipose-tissue mitochondria isolated from the cafeteria-fed animals showed as expected an increased (³H)GDP binding capacity (from 0.26 to 0.41 nmol/mg protein; an increase of 57%). However, when analysed by a quantitative enzyme-linked immuno-assay system for thermogenin, the mitochondria also showed an increased content of thermogenin (from 14.9 to 20.5 μg per mg; an increase of 38%). The ratio between thermogenin and GDP binding was 61 000 and 53 000 g/mol in the two cases; these values were not significantly different and were in good agreement with suggestions that thermogenin binds 1 GDP per thermogenin dimer. It was concluded that under the conditions investigated, there was no reason to assume the existence of a masked form of thermogenin.     

Cold-induced changes in the energy coupling and the UCP3 level in rodent skeletal muscles

The mechanism of thermoregulatory uncoupling of respiration and phosphorylation in skeletal muscles has been studied. It is found that 24 h cold exposure results in (i) a 3-fold increase in the amount of UCP3 protein in rat skeletal muscle mitochondria, and (ii) pronounced lowering of the membrane potential in isolated rat or mouse skeletal muscle mitochondria. The decrease in membrane potential is reversed by adding bovine serum albumin. Cold exposure is also found to sensitize the membrane potential to the uncoupling action of added fatty acid (laurate). After laurate addition, the recoupling effects of GDP and carboxyatractylate decrease whereas that of albumin increases in mitochondria from cold-treated rats or mice. Changes similar to those induced by cold can be initiated by the in vivo addition of thyroxine. Cold exposure does not affect energy coupling in liver mitochondria. The possible involvement of UCP3 isoforms in nucleotide-sensitive and -insensitive uncoupling is discussed.     

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

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.     

Increase in mitochondrial content of long-chain acyl-CoA in brown adipose tissue during cold-acclimation

The mitochondrial content of long-chain acyl-CoA esters in the brown adipose tissue of guinea pigs increased 3.5-fold from a level of 92 +/- 17 pmol per mg protein (+/- S.E.; n = 7) in the control animals adapted at 22 degrees C to a new steady-state level of 328 +/- 20 pmol per mg protein (+/- S.E.; n = 46) after 10 days of cold-acclimation (5 degrees C). These low values of long-chain acyl-CoA species and the slow adaptive response for their increase do not support the proposal (Cannon, B., Sindin, U. and Romert, L. (1977) FEBS Lett. 4, 43-46) that the fatty acid CoA-esters have a physiological function in the regulation of the H+ (or OH-) permeability of the mitochondrial inner membrane. Experimental evidence is presented supporting the proposal that the long-chain acyl-CoA species are largely confined to the cytosolic side of the inner membrane. The activity of the adenine nucleotide translocase, as estimated at 25 degrees C in the reverse direction, was found to increase 5-fold upon depletion of the mitochondria of fatty acids (free and esterified) by preincubation with bovine serum albumin. The presence of potent inhibitors, i.e., long-chain acyl-CoA species, of adenine nucleotide translocation in brown adipose tissue of thermogenically active animals further supports the conclusion that ATP hydrolyzing mechanisms contribute insignificantly to long-term thermogenesis. The low values of long-chain acyl-CoA hydrolase (EC 3.1.2.1) activity, as measured in intact mitochondria and on a mitochondrial matrix fraction (i.e., 1.6 nmol X min-1 per mg protein), do not support the proposal that the hydrolase activity plays a significant role in the loose-coupling of brown adipose tissue mitochondria, either by a futile cycle mechanism or promoted by free fatty acid-induced uncoupling.     

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.     

Protein--non-ionic detergent interaction. Interaction of bovine serum albumin with alkyl glucosides studied by equilibrium dialysis and infrared spectroscopy.

The binding isotherms of bovine serum albumin with octylglucoside and decyl glucoside were determined at 7 degrees C and 25 degrees C at pH 7.4 and ionic strength 0.1 M. The average number of detergent molecules bound was found to increase with increasing hydrocarbon chain length. Competitive binding indicates that alkylglycosides combine with the same sites as alkyl sulphates. Native bovine serum albumin has about 12 and 10 sites for non-ionic ligands at 7 degrees C and about 15 and 13 sites at 25 degrees C for octyl and decyl glucosides respectively. The values for standard free energy changes--delta G0, were calculated from the intrinsic association constants. Fourier-transformed infrared spectroscopy was used to study the effects of alkyl glucosides on the conformation of albumin. The results obtained indicate that there are no significant changes in protein structure.     

Induction of functional uncoupling protein in guinea pigs infused with noradrenaline. Studies with isolated brown adipocytes.

Continuous infusion of noradrenaline over the interscapular brown fat of guinea pigs maintained at thermoneutrality (28-32 degrees C) induces changes similar to those after cold-adaptation. (1) Multilocular fat droplets appear within the brown adipocytes. (2) The number of mitochondria per adipocyte and the total number of adipocytes both increase. (3) Noradrenaline addition to isolated adipocytes causes near maximal uncontrolled respiration. (4) The cells become more sensitive to fatty acid-induced uncoupling. (5) The tissue-specific uncoupling protein per mg of mitochondrial protein is increased 5-fold. Specific alpha- and beta-agonists were also chronically infused. (6) Separate infusion of phenylephrine or isoprenaline was not able to stimulate mitochondriogenesis or hyperplasia. (7) Adipocytes from these animals could not be uncoupled by acute noradrenaline. (8) Simultaneous chronic infusion of phenylephrine and isoprenaline reproduced the effects of chronic noradrenaline infusion.