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.     

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

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.     

Immunological and enzymological localization of carbonyl reductase in ovary and liver of various species

The tissue distribution of carbonyl reductase in ovary and liver of various animal species was investigated by measuring the reduction of 13,14-dihydro-15-keto-prostaglandin F2a, a specific substrate for rat ovarian carbonyl reductases, and by means of Western blotting analysis using anti-rat ovarian carbonyl reductase antibody. The highest ovarian carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was found in rat among ten animal species tested, followed by hamster and monkey. The immunoreactive protein was detected in hamster and monkey ovaries. Although carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was not detectable in non-pregnant rabbit ovary, pregnant rabbit ovary showed not only moderate activity but also immunoreactivity with anti-rat ovarian carbonyl reductase antibody. On the other hand, carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was detected in hepatic tissue of all the species tested, except for rat and left-eye flounder. Immunoreactive proteins were present in hepatic tissue of various species that exhibited measurable carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a.     

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.     

Evidence for sterol carrier protein2-like activity in hepatic, adrenal and ovarian cytosol

Purified sterol carrier protein2 (SCP2) from rat liver stimulated utilization of endogenous cholesterol for pregnenolone synthesis by adrenal mitochondria. Cytosolic preparations of rat liver, adrenal and luteinized ovary were also stimulatory in mitochondrial pregnenolone synthesis to different extents. Treatment of all preparations with rabbit anti-rat SCP2 IgG neutralized the stimulatory effects, and immunoprecipitated proteins gave similar patterns on SDS-gradient polyacrylamide gel electrophoresis. Treatment with rabbit pre-immune IgG had no effect on these parameters. Thus, proteins which are immunochemically compatible with hepatic SCP2 appear to be present in steroidogenic tissues and may play a role in control of mitochondrial cholesterol side chain cleavage activity.     

A plasmid region encoding the active fragment and the inhibitor protein of colicin E3--CA38

Thermogenin is the purine-nucleotide binding polypeptide in brown adipose tissue mitochondria (M_r 32 000) which confers upon these mitochondria the ability to produce heat. An enzyme-linked immunosorbent assay (ELISA) has been developed to demonstrate and quantitate the occurrence of thermogenin antigen in small amounts of tissue, and thus to characterize different depots of fat tissue as white or brown. The extreme sensitivity of the method allows determination of thermogenin in samples equivalent to <1 mg tissue. The results indicate that thermogenin seems to be exclusively localised in brown fat mitochondria (as compared to white fat, liver or heart muscle mitochondria), and thermogenin antigen could only be found in brown adipocytes (as compared to white adipocytes). Thus, brown and white adipose tissue are probably ontogenetically different     

Species differences in lung mitochondrial monoamine oxidase activities

Pulmonary mitochondrial monoamine oxidase (MAO) activity was examined in preparations from rat, rabbit and guinea-pig with 12 different amines as substrates: serotonin, norepinephrine, and octopamine (type A specific); tryptamine, benzylamine, 5-methoxytryptamine, 5-methyltryptamine, p-methoxyphenylethylamine, and 3,4-dimethoxyphenethylamine (type B specific); and tyramine, dopamine and 3-methoxytyramine (type A + B specific). The oxidation of type A and type A + B substrates was greater in guinea-pig lung mitochondria than in rat or rabbit preparations. Except for benzylamine, the oxidation of type B substrates was similar in all three species. Benzylamine was not oxidized by guinea-pig lung mitochondria but was actively metabolized by rat and rabbit preparations.     

Cytosolic and microsomal epoxide hydrolases are immunologically distinguishable from each other in the rat and mouse

Antibodies raised to homogeneous rat liver microsomal epoxide hydrolase were used to distinguish microsomal epoxide hydrolase from epoxide hydrolase of cytosolic origin in mice and rats. Using double diffusion analysis in agarose gels, we show that anti-rat liver microsomal epoxide hydrolase forms a single precipitin line with solubilized microsomes from rat and mouse liver, but no reaction is seen with the corresponding cytosolic fractions. Rat or mouse microsomal epoxide hydrolase activity (using benzo[a]pyrene 4,5-oxide as substrate) can be completely precipitated out of solubilized preparations by the antibody, which is equipotent against rat and mouse microsomal epoxide hydrolase. No precipitation of cytosolic hydrolase activity (using trans-beta-ethyl styrene oxide as substrate) is seen with any concentration of the antibody tested. Thus, in the case of microsomal epoxide hydrolase, extensive immunological cross-reactivity exists between the two species, rat and mouse. In contrast, no cross-reactivity is detectable between cytosolic and microsomal epoxide hydrolase, even when enzymes from the same species are compared. We conclude that microsomal and cytosolic epoxide hydrolase activities represent distinct and immunologically non-cross-reactive protein species.     

Brown adipocytes differentiated in vitro can express the gene for the uncoupling protein thermogenin: effects of hypothyroidism and norepinephrine

Expression of the gene for the brown-fat specific uncoupling protein thermogenin was investigated in cell cultures by hybridization of isolated RNA with a cDNA clone corresponding to mouse thermogenin. The RNA was isolated 3-4 days after confluence from cells differentiated in culture from precursors isolated from the interscapular brown adipose tissue of 5-week-old mice. Very low thermogenin mRNA levels were found in cells derived from untreated mice, and there was only little effect of added norepinephrine on thermogenin gene expression in these cells. However, in cells derived from hypothyroid (methimazole-treated) mice there was a higher expression of thermogenin, and norepinephrine had a marked augmenting effect on the thermogenin mRNA level in these cells. These effects of thermogenin mRNA levels were specific, in that they contrasted with the effects of hypothyroidism and norepinephrine on the level of other mRNA species in these cells (coding for beta-actin, lipoprotein lipase, cytochrome-c oxidase, and glycerol-3-phosphate dehydrogenase). It was concluded that brown-fat cells in culture can reach a differentiated state, sufficiently advanced that the unique properties of these cells can be expressed, and that thermogenin gene expression (i.e., the level of thermogenin mRNA) is under direct control of norepinephrine.     

Differentiation of brown adipose tissue and biogenesis of thermogenic mitochondria in situ and in cell culture

Differentiation and biogenesis of mitochondria in brown adipose tissue (BAT) was studied in situ and in cell culture by Western blotting, enzyme activity measurements, [35S]methionine incorporation and immunofluorescence microscopy. In different rodent species the perinatal development of BAT thermogenic function resulted from the formation of thermogenic mitochondria which replaced the preexisting nonthermogenic mitochondria. Their biogenesis was characterized by the sudden appearance and rapid increase of the uncoupling protein (UCP), increase of cytochrome oxidase (COX) and decrease of H(+)-ATPase. In primary cell culture, differentiation of precursor cells from mouse BAT to typical multilocular adipocytes was accompanied by increasing content of COX and H(+)-ATPase. A selective synthesis of UCP was induced by activation of beta-adrenergic receptors or by elevated levels of cellular cAMP. UCP was quantitatively incorporated into mitochondria and within 24 h after stimulation reached near physiological concentration. Both in situ and in cell culture, the conditions enabling the expression of UCP gene were accompanied by activation of intracellular thyroxine 5'-deiodinase.     

Brown adipose tissue mitochondria: modulation by GDP and fatty acids depends on the respiratory substrates

The UCP1 [first UCP (uncoupling protein)] that is found in the mitochondria of brown adipocytes [BAT (brown adipose tissue)] regulates the heat production, a process linked to non-shivering thermogenesis. The activity of UCP1 is modulated by GDP and fatty acids. In this report, we demonstrate that respiration and heat released by BAT mitochondria vary depending on the respiratory substrate utilized and the coupling state of the mitochondria. It has already been established that, in the presence of pyruvate/malate, BAT mitochondria are coupled by faf-BSA (fatty-acid-free BSA) and GDP, leading to an increase in ATP synthesis and mitochondrial membrane potential along with simultaneous decreases in both the rates of respiration and heat production. Oleate restores the uncoupled state, inhibiting ATP synthesis and increasing the rates of both respiration and heat production. We now show that in the presence of succinate: (i) the rates of uncoupled mitochondria respiration and heat production are five times slower than in the presence of pyruvate/malate; (ii) faf-BSA and GDP accelerate heat and respiration as a result and, in coupled mitochondria, these two rates are accelerated compared with pyruvate/malate; (iii) in spite of the differences in respiration and heat production noted with the two substrates, the membrane potential and the ATP synthesized were the same; and (iv) oleate promoted a decrease in heat production and respiration in coupled mitochondria, an effect different from that observed using pyruvate/malate. These effects are not related to the production of ROS (reactive oxygen species). We suggest that succinate could stimulate a new route to heat production in BAT mitochondria.     

Monoclonal antibodies to thromboxane synthase from porcine lung. Production and application to development of a tandem immunoradiometric assay

Two hybridoma cell lines secreting antibodies against thromboxane synthase of porcine lung were produced. Clone TS1 secretes IgG2a antibody of lower affinity, while clone TS2 secretes IgG1 antibody of higher affinity. Both antibodies (when bound to rabbit anti-mouse IgG-Staphylococcus aureus complex) can immunoprecipitate thromboxane synthase from crude enzyme preparations in an active form suggesting that binding was not directed at the active site. Each antibody showed a distinctive pattern of cross-reactivity with thromboxane synthase from different porcine tissues. Neither of the antibodies cross-reacted with the enzyme from tissues of other species tested, indicating the heterogeneous nature of the enzyme among species. Competitive binding assay revealed that TS1 and TS2 recognized different determinants on the enzyme. The fact that two antibodies bind to separate epitopes on the same enzyme allows the development of a sensitive tandem immunoradiometric assay. The assay, based on binding of 125I-TS2 to thromboxane synthase immobilized on TS1-S. aureus complex, was linear with 7.5 approximately 75 ng of purified lung thromboxane synthase as standards and applicable to enzyme preparations regardless of their purity. The concentration of immunoreactive thromboxane synthase in porcine tissues as determined by this assay followed the order of platelet greater than colon greater than duodenum greater than lung greater than kidney greater than stomach. The fact that gastrointestinal tract is enriched with thromboxane synthase suggests that thromboxane may have significant physiological roles to be recognized in these organs.     

cDNA sequence and bacterial expression of mouse liver sterol carrier protein-2.

Sterol carrier protein-2 (SCP-2) is an intracellular protein of Mr 13,096. In vitro studies have shown that it is involved in the transport and metabolism of cholesterol. This protein is believed to participate in these activities by forming a stoichiometric complex with the sterol. Because these activities occur in different intracellular locations, i.e. mitochondria, peroxisomes, and cytosol, it can be predicted that SCP-2 targets to these sites. In this report we show that a mouse cDNA (785 base pairs) encodes a precursor form of SCP-2 containing a N-terminal presequence and an additional C-terminal residue. These additional amino acid residues are found in proteins targeted to the mitochondria and peroxisomes, respectively. These signals are not found in SCP-2 purified from rat liver cytosol which is believed to be a cytosolic form. Northern analysis shows that there are four species of mRNA which hybridize to a SCP-2-specific probe at 1.0, 1.7, 2.2, and 2.9 kilobases. Southern analysis shows that the gene is distributed over a large amount of DNA or that there are multiple genes. We have cloned the cytosolic/peroxisomal form of mouse SCP-2 into the Escherichia coli expression vector pKK233-2 and have expressed and purified recombinant mouse SCP-2, Mr 13,034. The purified recombinant SCP-2 is immunoreactive to rabbit anti-rat SCP-2 antibody. It also has biological activity equivalent to homogeneous rat liver SCP-2 in stimulating the microsomal conversion of 7-dehydrocholesterol to cholesterol and in the esterification of cholesterol by acyl-CoA cholesterol acyltransferase by rat liver microsomes.     

The relationship between extramitochondrial Ca2+ concentration, respiratory rate, and membrane potential in mitochondria from brown adipose tissue of the rat

The ability of isolated mitochondria from rat brown-adipose tissue to regulate extramitochondrial Ca2+ (measured by arsenazo) was studied in relation to their ability to produce heat (measured polarographically). The energetic state of the mitochondria was expressed as a membrane potential, delta psi (estimated with safranine), and was varied semi-physiologically by the use of different GDP concentrations. In these mitochondria GDP binds to the 32-kDa polypeptide, thermogenin, which regulates coupling. Ca2+ uptake (at 5 microM extramitochondrial Ca2+) was maximal at delta psi greater than 150 mV. Basal Ca2+ release increased from 1 to 2 nmol x min-1 x mg-1 below 150 mV. Na+ -stimulated rate of Ca2+ release was stable within the investigated delta psi span (100-160 mV). Initial Ca2+ levels were maintained below 0.2 microM for 100 mV less than delta psi less than 160 mV. Ca2+ levels maintained after Ca2+ challenge (20 nmol Ca2+ x mg-1) were below 0.4 microM for delta psi greater than 135 mM. Respiration was unstimulated for delta psi greater than 150 mV and was maximal at delta psi less than or equal to 135 mV. In the presence of well-oxidised substrates, the respiration at maximally activated thermogenin was markedly below fully uncoupled respiration and was probably limited by thermogenin activity--i.e. by a limited H+ reentry (OH- exit) and therefore by a membrane potential maintained at about 135 mV. It is concluded that at membrane potentials of 135 mV and above the mitochondria exhibit full Ca2+ control and are able to regulate thermogenic output up to maximum without interfering with this Ca2+ control. Membrane potential probably does not decrease below 135 mV in vivo. Therefore, Ca2+ homeostasis and thermogenesis are non-interfering and can be hormonally independently regulated, e.g. by alpha-adrenergic and beta-adrenergic stimuli, respectively.     

Human adipose tissue hormone-sensitive lipase: identification and comparison with other species

The mRNA for human hormone-sensitive lipase (HSL) was identified using Northern blot analysis and a cDNA-probe for rat HSL. As in the rat, human adipose tissue expresses a single mRNA species of 3.3 kb. Using Western blotting with a polyclonal rabbit antibody towards rat adipose tissue HSL, the corresponding enzyme in human adipose tissue was identified with an apparent 88 kDa polypeptide, thus slightly larger than the rat and bovine 84 kDa, and the mouse and guinea-pig 82 kDa species. Additional evidence for the identification was provided by the inhibition of HSL diacylglycerol lipase activity by the anti-rat HSL antibody, and by NaF, DFP and Hg2+, known inhibitors of HSL. The concentration of the enzyme, as reflected by its activity per g tissue and the specific activity was about two thirds of that in the rat adipose tissue (200 g rats). The identification of the human enzyme protein made it possible to directly demonstrate its phosphorylation by cAMP-dependent protein kinase, thus extending the previous report regarding activation of the lipase with this kinase and ATP-Mg2+ in human adipose tissue extracts (Khoo, J.C., Aquino, A.A. and Steinberg, D. (1974) J. Clin. Invest. 53, 1124-1131).     

Improved protection against lung colonization by Actinobacillus pleuropneumoniae ghosts: characterization of a genetically inactivated vaccine

Pigs immunized with Actinobacillus pleuropneumoniae ghosts or a formalin-inactivated bacterin were found to be protected against clinical disease in both vaccination groups, whereas colonization of the lungs with A. pleuropneumoniae was only prevented in ghost-vaccinated pigs. Bacterial ghosts are empty cell envelopes created by the expression of a cloned bacteriophage lysis gene and, unlike formalin-inactivated bacteria, suffer no denaturing steps during their production. This quality may lead to a superior presentation of surface antigens to the immune system. Analysis by SDS-PAGE and immunoblotting of the two vaccine preparations revealed different contents of antigenic proteins. In order to better understand the immunogenic properties of A. pleuropneumoniae ghosts and formalin-inactivated bacteria, we compared the serum antibody response induced in both treatment groups. Immune sera were tested on whole cell antigen or purified virulence factors including outer membrane protein preparations (OMPs), outer membrane lipoprotein OmlA1, transferrin binding proteins (TfbA1, TfbA7 and TfbB) and Apx toxins (ApxI, II and III). SDS-PAGE and immunoblots revealed no specific antibody response against the single virulence factors tested in any vaccinated animal. The two vaccination groups showed different recognition patterns of whole cell antigen and OMP-enriched preparations. A 100 kDa protein was recognized significantly stronger by ghost-vaccinated pigs than convalescent pigs. This unique antibody population induced by ghosts could play a determining role in the prevention of lung colonization. The same 100 kDa antigen was recognized by ghost-sera in homologous as well as heterologous serotype A. pleuropneumoniae protein preparations. Indications for a crossprotective potential in the ghost vaccine were supported by studies on rabbit hyperimmune sera.     

Ribosomal Ribonucleic Acids of Chloroplastic and Mitochondrial Preparations

RNA prepared from fractions of chloroplasts and mitochondria sedimented at rates characteristic of ribosomal RNA. A predominance of the 18S species was frequently observed in preparations from chloroplasts from romaine lettuce and endive. The usual distribution, a preponderance of the 28S species, was observed in studies on tomato and spinach chloroplasts and mitochondria from mushroom and cauliflower. Comparisons of the base composition of RNA from organelles with their cytoplasmic ribosomal counterparts revealed that the 18S component from romaine lettuce chloroplast was different. A marginally significant difference was observed in the 28S particle from mushroom mitochondria preparations whereas distinct differences, reflected in all the bases, were seen when the 18S component of cauliflower mitochondria preparations was compared with cytoplasmic RNA.     

Isolation of mouse N-CAM-related cDNA: detection and cloning using monoclonal antibodies.

Clones coding for the mouse neural cell adhesion molecule (N-CAM) were isolated from a cDNA library prepared in the expression vector lambda gt 11 from mRNA extracted from a mouse neuroblastoma cell line. This library was screened with two anti-N-CAM monoclonal antibodies directed against different sites on the molecule and with rabbit anti-N-CAM serum. Two clones were identified with the first monoclonal antibody, three with the second one, none reacted with both. The relevance of these cDNA clones to N-CAM was confirmed by several observations. First, cDNA sequences detected with one monoclonal antibody cross-hybridized with those identified by the other antibody. Second, the different fusion proteins all bound the rabbit serum in addition to one monoclonal antibody. Finally, the probes hybridized to discrete mRNA species of sufficient lengths to code for the very large N-CAM polypeptides in RNA preparations from N-CAM-expressing, but not from N-CAM-negative cells. An additional mRNA species not seen in embryonic brain was expressed in adult mouse brain. Genomic blot experiments indicated that sequences corresponding to one of our probes are present only a few times in the mouse genome.     

Mammalian skeletal muscle myosin light chain kinases. A comparison by antiserum cross-reactivity

Purified myosin light chain kinases from skeletal muscle are reported to be significantly smaller (Mr = 75,000-90,000) than the kinases purified from smooth muscle (Mr = 130,000-155,000). It has been suggested that the smaller kinases from striated muscle are proteolytic fragments of a larger enzyme which is homologous, if not identical, to myosin light chain kinase from smooth muscle. Therefore, we have used an antiserum to rabbit skeletal muscle myosin light chain kinase and Western blot analysis to compare the subunit molecular weight of the kinase in skeletal muscle extracts of several mammalian species. In rabbit skeletal muscle, the antiserum only recognized a polypeptide of Mr = 87,000, with no indication that this polypeptide was a proteolyzed fragment of a larger protein. The apparent molecular weights observed in different animal species were 75,000 (mouse), 83,000 (guinea pig), 82,000 (rat), 87,000 (rabbit), 100,000 (dog), and 108,000 (steer). The molecular weight of myosin light chain kinase was constant within an animal species, regardless of skeletal muscle fiber type. The antiserum inhibited the catalytic activity of skeletal muscle myosin light chain kinase. Similar antibody dilution curves for inhibition of myosin light chain kinase activity in extracts were observed for all animal species (rabbit, rat, mouse, guinea pig, dog, cat, steer, and chicken) and different fibers (slow twitch oxidative, fast twitch oxidative glycolytic, and fast twitch glycolytic) tested. The antiserum did not inhibit the activity of rabbit smooth muscle myosin light chain kinase. These results suggest that there may be at least two classes of muscle myosin light chain kinase represented in skeletal and smooth muscles, respectively.