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.     

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.     

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.     

Undecanesulfonate does not allosterically activate H+ uniport mediated by uncoupling protein-1 in brown adipose tissue mitochondria

Undecanesulfonate is transported by uncoupling protein-1. Its inability to induce H+ uniport with reconstituted uncoupling protein-1 supports fatty acid cycling hypothesis. Rial et al. [Rial, E., Aguirregoitia, E., Jimenez-Jimenez, J., & Ledesma, A. (2004). Alkylsulfonates activate the uncoupling protein UCP1: Implications for the transport mechanism. Biochimica et Biophysica Acta, 1608, 122-130], have challenged the fatty acid cycling by observing uncoupling of brown adipose tissue mitochondria due to undecanesulfonate, interpreted as allosteric activation of uncoupling protein-1. We have estimated undecanesulfonate effects after elimination of endogenous fatty acids by carnitine cycle in the presence or absence of bovine serum albumin. We show that the undecanesulfonate effect is partly due to fatty acid release from albumin when undecanesulfonate releases bound fatty acid and partly represents a non-specific uncoupling protein-independent acceleration of respiration, since it proceeds also in rat heart mitochondria lacking uncoupling protein-1 and membrane potential is not decreased upon addition of undecanesulfonate without albumin. When the net fatty acid-induced uncoupling was assayed, the addition of undecanesulfonate even slightly inhibited the uncoupled respiration. We conclude that undecanesulfonate does not allosterically activate uncoupling protein-1 and that fatty acid cycling cannot be excluded on a basis of its non-specific effects.     

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.     

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.     

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.     

The possible proton translocating activity of the mitochondrial uncoupling protein of brown adipose tissue. Reconstitution studies in liposomes

Loose coupling of thermogenic mitochondria of brown adipose tissue is related to a high proton (or hydroxyl) conductance of the inner membrane and to the presence of a unique 32 kDa uncoupling protein. Reconstitution experiments of the purified protein in liposomes are reported which suggest that this component could form proton channels in the membrane.     

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).     

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.     

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.     

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.     

Sulfhydryl groups are involved in H+ translocation via the uncoupling protein of brown adipose tissue mitochondria

Mersalyl inhibits H+ transport via the uncoupling protein (UP) in brown adipose tissue (BAT) mitochondria estimated as swelling in potassium acetate (Ki 67 microM) or as valinomycin-induced H+ extrusion in K2SO4 (Ki 55 microM) and KCl. The swelling in KCl is depressed only slightly. Some other SH-reagents (p-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoate) and thiolyte DB), but not hydrophobic reagents (N-ethylmaleimide and eosin-5-maleimide), exhibit analogous inhibition. Thus an essential SH-group localized at the water-accessible cytosolic surface of UP was found to be involved in H+ transport via UP but not in Cl- transport.     

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.     

New substrates and competitive inhibitors of the Cl- translocating pathway of the uncoupling protein of brown adipose tissue mitochondria

A large number of new substrates for anion uniport by the uncoupling protein of brown adipose tissue mitochondria have been found. These include alkylsulfonates, alkylsulfates and their derivatives, benzenesulfonate, oxohalogenides, hypophosphate, hexafluorophosphate, and pyruvate. Although the spectrum of anion selectivity is far wider than had previously been suspected, there are strong structural requirements for transport. The anion must be monovalent, and polar groups must not be attached to alkyl or aryl chains. The most striking finding is that transport increases dramatically with anion hydrophobicity. Anions that are transported are shown to compete with Cl- for transport by the reconstituted uncoupling protein. For each anion, the Ki for GDP inhibition of transport increases with its rate of transport and correlates inversely with its Ki for competitive inhibition of Cl- transport. For alkylsulfonates, transport rate, Ki for GDP inhibition, and Ki for inhibition of Cl- transport each depend monotonically on alkyl chain length. These findings suggest several new hypotheses relating to the molecular mechanism of transport through uncoupling protein and suggest explanations for observed functional differences among porters belonging to the same gene family.     

Number of mice per cage influences uncoupling protein content of brown adipose tissue.

The effect of housing density of mice on the thermogenic state and capacity of their brown adipose tissue was studied. Mice were housed one, two, or six per cage at 28 degrees C for 15 days. Increased housing density suppressed the thermogenic capacity of brown adipose tissue (decreased the total amount of uncoupling protein) and decreased the thermogenic state of brown adipose tissue mitochondria (decreased GDP binding). A density of six mice per cage had a greater effect than a density of two mice per cage. The size of brown adipose tissue (wet weight and protein content), the content of mitochondria in it (cytochrome oxidase content), and the total activity of thyroxine 5'-deiodinase were not altered by housing density. We conclude that even at a temperature close to thermoneutrality (29-33 degrees C for the mouse), the occurrence of social thermoregulation (huddling) reduces the requirement for brown adipose tissue thermogenesis and results in a reduction in its thermogenic capacity. It is clearly of importance that the design of studies of mouse brown adipose tissue take into account not only the temperature at which the mice are housed, but also the number of mice housed per cage.     

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.     

Limited proteolysis reveals conformational changes in uncoupling protein-1 from brown adipose tissue mitochondria

Limited proteolytic digestion of uncoupling protein-1 (UCP1) from hamster brown adipose tissue mitochondria was studied. Under optimal conditions, trypsin and chymotrypsin cleave at Lys-292 and at Phe-102, yielding major products 31-kDa T1 and 22-kDa Ch1. Both T1 and Ch1 remained dimers, as in UCP1. Using fluorescent nucleotide derivative 2'-O-dansyl GTP, it is shown that T1 retains the nucleotide binding affinity (K(D)=1 microM for dansyl GTP) while Ch1 does not bind nucleotide. Previously kinetic binding and H(+) transport studies [Biochemistry 35 (1996) 7846] have shown that UCP1 forms tight complexes to varying degrees with nucleotides and their derivatives. Nucleotides strongly protect against tryptic digestion but less against chymotryptic digestion, because the chymotryptic product Ch1 does not bind nucleotide. The nucleotides and derivatives show the same potency profile in protecting against both trypsinolysis and chymotryptic digestion, suggesting that UCP1 undergoes a major conformational change upon nucleotide binding from an initial loose complex into a tight complex, in which the cleavage sites become masked from proteolysis.