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).     

pH-temperature interactions on protein function and hibernation: GDP binding to brown adipose tissue mitochondria

1. [3H]GDP binding to the uncoupling protein of brown adipose tissue was determined on mitochondria isolated from hibernating European hamsters, at two temperatures, 35 and 15 degrees C, and four values of 25pH (pH corrected to 25 degrees C): 6.4, 6.8, 7.2 and 7.6, encompassing the physiological range of pH. Buffer composition was adjusted to get the same pH-temperature relationship as for mammalian blood, in which this relationship is mainly determined by protein imidazole buffers. 2. The maximal binding capacity was independent both of temperature and pH. The dissociation constant KD was highly pH-dependent, but was independent of temperature when 25pH was held constant. Under these conditions, the uncoupling protein thus fully conserves its regulatory properties over the temperature range studied (eurythermal adaptation). 3. The temperature coefficient of the apparent pK' for the pH effect (-0.012 +/- 0.004) differed significantly from that of GDP terminal phosphoryl group, but not from that of blood protein imidazole buffer groups, in good agreement with the imidazole alphastat theory. 4. The results indicate that GDP reaction with the protein involves an electrostatic binding with a titratable group of the protein, probably a sulfhydryl. 5. pH modulation of the uncoupling of brown adipose tissue mitochondria probably permits a reversible control of thermogenesis in the hibernation cycle, heat dissipation being inhibited by respiratory acidosis in deep hibernation, but facilitated by the hyperventilation of arousal.     

Partial purification and functional reconstitution of GDP sensitive brown adipose tissue mitochondria uncoupling protein using octyl glucoside

A partial purification of the uncoupling protein of brown adipose tissue mitochondria (BATM) was achieved by extraction of BATM with 40 mM octyl glucoside, followed by affinity chromatography on ATP-agarose. The isolated protein was functionally reconstituted into liposomes using octyl glucoside dialysis. Proteoliposomes containing the uncoupling protein had an increased proton or chloride conductance when subjected to a valinomycin-induced potassium diffusion potential. The increased ion conductance was consistently found to be inhibited by 200 microM GDP.     

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.     

Immunoelectron microscopical identification of the uncoupling protein in brown adipose tissue mitochondria

The distribution of the uncoupling protein (UCP) in brown adipocyte mitochondria of the hibernant Muscardinus avellanarius was obtained by ultrastructural immunocytochemistry. In both cryosections and sections of Lowicryl-embedded material UCP was localized in the mitochondrial cristae of brown adipocytes, but not in liver mitochondria. It should now be possible to easily identify the morphology of cells committed to BAT differentiation in the tissue as well as in cell culture.     

Fatty acid activation of the reconstituted brown adipose tissue mitochondria uncoupling protein

The effect of fatty acids, palmitoyl-CoA, and N',N-dicyclohexylcarbodiimide on the ion conductance of the reconstituted brown adipose tissue mitochondria uncoupling protein was investigated. 1, 5, and 10 microM palmitic acid induced a specific, GDP inhibited, increase in proton conductance in proteoliposomes containing the uncoupling protein but not in proteoliposomes prepared with purified protein extracts of liver mitochondria. 10 microM oleic acid, like palmitic acid, increased proton conductance in proteoliposomes prepared with the uncoupling protein. Palmitoyl-CoA and caprylic acid had no effect on increasing proton conductance. Similar to the observation in mitochondria, there was no effect of palmitic acid on Cl-conductance, but unlike mitochondria its activation by palmitoyl-CoA or inhibition by N',N-dicyclohexylcarbodiimide was lost. The results, obtained in an isolated system, provide support for the contention that long chain fatty acids act as an acute physiological activator of the uncoupling protein.     

High affinity GDP binding sites on brown adipose tissue mitochondria of genetically obese ‘fa/fa’ rats

The number of high affinity [³H]GDP binding sites in brown adipose tissue mitochondria is normal in obese ( f a / f a ) rats in contrast to the reduced number of low affinity GDP binding sites. Adrenalectomy corrected the loss of low affinity binding sites in fa/fa rats but had no effect on the number of high affinity sites in either lean or obese rats. Equilibrium dialysis was used to show the presence of both high and low affinity binding sites on the purified 32 kdalton protein.     

Binding of fatty acids to the uncoupling protein from brown adipose tissue mitochondria

The uncoupling protein 1 (UCP1) is a H(+) carrier which plays a key role in heat generation in brown adipose tissue. The H(+) transport activity of UCP1 is activated by long-chain fatty acids and inhibited by purine nucleotides. While nucleotide binding has been well characterized, the interaction of fatty acid with UCP1 remains unknown. Here I demonstrate the binding of fatty acids by competition with a fluorescent nucleotide probe 2(')-O-dansyl guanosine 5(')-triphosphate (GTP), which has been shown previously to bind at the nucleotide binding site in UCP1. Fatty acids but not their esters competitively inhibit the binding of 2(')-O-dansyl GTP to UCP1. The fatty acid effect was enhanced at higher pH, suggesting the binding of fatty acid anion to UCP1. The inhibition constants K(i) were determined by fluorescence titrations for various fatty acids. Short-chain (C<8) fatty acids display no affinity, whereas medium-chain (C10-14) and unsaturated C18 fatty acids exhibit stronger affinity (K(i)=65 microM, for elaidic acid). This specificity profile agrees with previous functional data obtained in both proteoliposomes and mitochondria, suggesting a possible physiological role of this fatty acid binding site.     

Evaluation of the specific dicyclohexylcarbodiimide binding sites in brown adipose tissue mitochondria

1. The content of the membrane sector of the ATPase complex (Fo) in brown adipose tissue mitochondria was determined by means of specific [14C]-DCCD binding. 2. The specific DCCD binding to the F0 protein was distinguished from the nonspecific binding to the other membrane proteins and phospholipids by: (a) Scatchard plot analysis of the equilibrium binding data, (b) SDS-polyacrylamide gel electrophoresis of the 14C-labelled membrane proteins, (c) partial purification of the chloroform-methanol extractable DCCD-binding protein. It was found that the specific DCCD binding was present in three polypeptides of a relative molecular weight of 9000, 16 000 and 32 000. In brown adipose tissue mitochondria the specific binding was 10-times lower than in heart or liver mitochondria. The binding to the other membrane proteins and to phospholipids was quite similar in all mitochondrial preparations studied. 3. The decreased quantity of the specific binding sites in brown adipose tissue mitochondria demonstrated that the reduction of F0 parallels the reduction of the F1-ATPase and revealed that in these mitochondrial membranes the ratio between the respiratory chain enzymes and the ATPase complex is 10- to 20- times higher than in heart or liver mitochondria.     

Functional atrophy of brown adipose tissue during lactation in mice. Effects of lactation and weaning on mitochondrial GDP binding and uncoupling protein.

The thermogenic activity and capacity of brown adipose tissue were determined in mice during lactation and after weaning. There was a marked fall during lactation in the mitochondrial content of the tissue, and in GDP binding and the specific mitochondrial concentration of uncoupling protein. The lactation-induced functional atrophy of brown adipose tissue was fully reversible after weaning; mitochondrial content and the mitochondrial concentration of uncoupling protein were both restored, although GDP binding was not normalized.     

A study of GDP binding to purified thermogenin protein from brown adipose tissue.

1. The binding of GDP to purified thermogenin protein was studied by using fluorescence spectroscopy and equilibrium dialysis. 2. GDP binding to thermogenin diminished fluorescence emission in a concentration-dependent manner that exhibited saturation. 3. Kd values for binding of nucleoside di- and tri-phosphates were lower than those for nucleoside monophosphates. 4. The GDP-induced fluorescence quenching was decreased by increasing pH, but the apparent Kd was unaltered by pH changes. 5. Equilibrium dialysis showed a Kd change from 3 to 6 microM when the pH was increased from 6.6 to 8.5. 6. The apparent pK of the fluorescence changes induced by pH (8.3) was identical with the apparent pK of the GDP-binding response. 7. The data are consistent with the existence of protonated and non-protonated forms of thermogenin protein that both bind GDP.     

Photoaffinity labeling of the nucleotide-binding site of the uncoupling protein from hamster brown adipose tissue.

The nucleotide binding center of the uncoupling protein from brown adipose tissue (UCP) was probed by photoaffinity labeling with 8-azido-ATP. The isolated dimeric UCP in non-ionic detergent was used. 8-azido-ATP binds to UCP with a Kd = 3 microM, i.e. with an only threefold lower affinity than ATP and a maximum number of binding sites of about 12 mumol/g protein corresponding to about 1 mol/mol dimer UCP. UCP is rapidly degraded by ultraviolet radiation, and therefore only near ultraviolet and visible light can be used for photoaffinity labeling. The total covalent incorporation is shown to be dependent on the concentration of azido-ATP and on competing phospholipids. The specific, i.e. ATP-sensitive incorporation only to the binding site depends on the presence of cysteine. With CNBr cleavage the 8-azido-[gamma-32P]ATP insertion within the primary structure was located by identifying ATP-sensitive labeled peptides in SDS/PAGE. A major specific 8-azido-ATP incorporation was found by autoradiography in the smallest CNBr fragments. Identification of the radioactive peptides was difficult since 8-azido-ATP insertion causes a distinct shift in the gels from the stained peptides. Identification was possible by specific disulfide formation at the C-terminal within the UCP dimer which only removed the CB7 (CB, CNBr fragment) portion of the low-molecular-mass peptides but did not move the radioactive band. This excludes the C-terminal CB7 and identifies the labeled peptide as CB6. Also, limited tryptic cleavage of intact UCP at Lys293 did not remove the radioactivity. Cleavage of tryptophanes support localization of 8-azido-ATP between residues 173-280 which includes CB6. Solid-phase sequencing of the labeled CB6 both after serine lactone and carboxyl coupling suggest incorporation into Thr260. These results indicate that the adenine-binding site is within the third domain of the tripartite UCP structure at a putative hydrophilic channel which can be assessed both from the cytosol and matrix of mitochondria.     

Regulation of the uncoupling protein in brown adipose tissue

Presumptive evidence suggests that the brown fat mitochondrial uncoupling protein, thermogenin, is involved in the mechanism of stimulation of respiration by norepinephrine in the intact tissue. Conflicting data have been reported which suggest involvement of either adenine nucleotides, or fatty acids, or long chain acyl-CoA, or protons in the physiological regulation. We measured the electrical potential gradient across the mitochondrial membrane (delta psi m) in control cells and in cells stimulated with norepinephrine, using the accumulation of lipophilic cation, tetraphenylphosphonium, as an indicator of the potential gradient. The value of delta psi m in the cells in the control state is 116 mV, and in the hormonally stimulated state it is 56.6 mV. This supports the view that the protein is involved in the mechanism of hormone action. Other studies were designed to distinguish between the effects of fatty acids and ATP levels on the uncoupling protein in isolated mitochondria and in the adipocytes. ATP levels and fatty acid levels inside intact cells were independently varied using oligomycin or external fatty acids. Their effect on thermogenin was monitored as the capacity of the cells for reverse electron transport from durohydroquinone. The results suggest that ATP modulates the activity of thermogenin, while fatty acids can alter the relationship between ATP and thermogenin activity such that the protein appears to be activated at a higher cellular ATP level in the presence of fatty acids than in their absence.     

Reconstitution of the uncoupling protein of brown adipose tissue mitochondria. Demonstration of GDP-sensitive halide anion uniport

The fluorescent anion indicator 6-methoxy-N-(3-sulfopropyl)quinolinium was trapped in proteoliposomes reconstituted with purified 32-kDa uncoupling protein and used to detect GDP-sensitive uniports of Cl-, Br-, and I-. Transport of these halide anions was rapid and potential-dependent. F- and nitrate were found to inhibit Cl- uptake competitively, suggesting that these anions are also substrates for transport. This preparation also exhibited H+(OH-) transport, showing that the reconstituted uncoupling protein possesses both halide and H+ transport functions, as is observed in intact brown adipose tissue mitochondria. Cl- transport was inhibited to the residual level observed in liposomes without protein when GDP was present on both sides of the membrane. Cl- transport was inhibited by about 50% when GDP was present only on one side of the membrane. We infer that uncoupling protein reconstitutes into proteoliposomes with a 1:1 ratio of sidedness orientation. The Km values for Cl- uniport were 100 and 65 mM, respectively, in GDP-loaded and non-GDP-loaded vesicles. Participation of the inner membrane anion channel in the observed transport is rendered unlikely by the fact that this carrier is insensitive to GDP. A variety of additional experiments probing for inner membrane anion channel yielded uniformly negative results, confirming the absence of contamination by this protein. Our results therefore demonstrate that the uncoupling protein mediates anion translocation, a function previously reported as lacking in the reconstituted system.     

New insights into mechanisms of anion uniport through the uncoupling protein of brown adipose tissue mitochondria.

GDP-sensitive Cl- uniport is a widely studied property of the uncoupling protein of brown adipose tissue mitochondria; nevertheless, little is known about its mechanism and there is even controversy over whether this protein transports Cl-. Using a fluorescent probe assay, we have demonstrated non-ohmic, electrophoretic, GDP-sensitive Cl- uniport into proteoliposomes reconstituted with purified uncoupler protein. We have also identified a large number of new anionic substrates for this porter that also inhibit Cl- uniport competitively. Anion transport, its inhibition by GDP and anion inhibition of Cl- uniport are all strongly dependent on anion hydrophobicity. These surprising results are consequential for hypotheses of common transport mechanisms in the gene family of mitochondrial anion porters.     

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.     

Selective loss of uncoupling protein from mitochondria of surgically denervated brown adipose tissue of cold-acclimated mice

The effects of unilateral surgical denervation on brown adipose tissue (BAT) composition were evaluated to assess the importance of the sympathetic innervation in the maintenance of a high concentration of the uncoupling protein thermogenin in cold-acclimated (CA) mice and to assess whether suppression of neural activity could account for BAT atrophy observed during fasting or when CA mice are returned to a thermoneutral environment (33 degrees C). Denervation-induced BAT atrophy was characterized by protein and thermogenin losses in absence of changes in the tissue cellularity (DNA content). There was a marked reduction in the concentration of thermogenin in mitochondria isolated from denervated BAT, but the concentration of the adenine nucleotide translocator was unchanged. Fasting or exposure of CA mice to 33 degrees C induced a rapid and extensive loss of tissue protein from both innervated and denervated BAT. In CA mice exposed to 33 degrees C, there was also reduction in tissue cellularity and loss of thermogenin from BAT mitochondria. Since surgical denervation suppressed BAT hyperplasia and the increase in the mitochondrial concentration of thermogenin observed during cold exposure, these results indicate that an intact innervation is required for both synthesis and maintenance of a high mitochondrial content of thermogenin in CA mice. In addition, the lesser changes in tissue composition caused by denervation compared with those caused by fasting or exposure of CA mice to 33 degrees C question the importance of the suppression of neural activity as the exclusive cause of rapid BAT atrophy in mice.     

Isolation of the GDP binding protein from brown adipose tissue mitochondria of several animals and amino acid composition study in rat

The nucleotide binding protein (uncoupling protein, GDP binding protein) of brown adipose tissue mitochondria has been isolated from cold adapted rat, newborn guinea pig and newborn rabbit. The purification, using hydroxyapatite in sucrose gradient centrifugation, follows the procedures established previously for the isolation of this protein from cold adapted hamster. A similar degree of purification was obtained, reaching 60 μmol GDP bound/g protein. In SDS gel electrophoresis the purified protein gave a single band of M_r 32 000 from all species.     

Effect of corticotropin on brown adipose tissue mitochondrial GDP binding in obese rats.

Corticotropin stimulated brown adipose tissue mitochondrial GDP binding of young obese rats to the levels seen in lean rats. This effect was attenuated by chronic increases in corticosterone. The stimulatory response to corticotropin was absent from lean rats unless endogenous secretion of corticosterone was prevented.