The biochemistry of an inefficient tissue: brown adipose tissue.
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Acclimatization↗
Biomedical subjects
B Cannon
Publications and source records attributed to B Cannon.
A direct comparison between peroxisomal and mitochondrial preferences for fatty-acyl beta-oxidation predicts channelling of medium-chain and very-long-chain unsaturated fatty acids to peroxisomes.
A well-characterized crude peroxisomal fraction from brown adipose tissue was used to compare peroxisomal beta-oxidation with beta-oxidation in isolated mitochondria. The apparent Km and chain-length specificity for peroxisomal (acyl-CoA) and mitochondrial (acyl-carnitine) beta-oxidation were determined with saturated C4-C22 fatty acyls and some unsaturated fatty acyls. Peroxisomes showed the lowest Km for medium-chain (9:0-10:0) and mono-unsaturated long-chain (16:1-22:1) fatty acids, and highest oxidation rates with lauroyl-CoA (12:0). Mitochondria showed the lowest Km for long-chain fatty acids (16:0-18:0) and highest oxidation rates with 12:0-16:0 and with 18:2. These in vitro results offer an explanation of previous results obtained in situ by Foerster et al. (Foerster, E.-C., Fährenkemper, T., Rabo, U., Graf, P. and Sies, H. (1981) Biochem. J. 196, 705-712) and indicate a role for peroxisomes in degradation of medium-chain and mono-unsaturated long-chain fatty acids. It is concluded that no mechanism, other than relative preferences, needs to be suggested for channelling of fatty acids between the two subcellular organelles.
Adipose Tissue, Brown↗
Metabolic consequences of limited substrate anion permeability in brown fat mitochondria from a hibernator, the golden hamster.
Brown fat mitochondria obtained from a hibernator, the golden hamster, were investigated in order to elucidate the significance of membrane permeability for metabolic functioning at different temperatures. The mitochondria were shown to have active permeases for phosphate and pyruvate, but very poorly developed permeases for di- and tricarboxylate substrate anions. This was shown with both osmotic swelling techniques and respiration-driven uptake studies. It was shown that the very limited malate permeation observed was compatible with it being a non-carrier-mediated diffusion process. The role of malate transport in supporting fatty-acid oxidation in vitro as a function of temperature was studied in detail. The results support our earlier suggestion that physiologically pyruvate carboxylase probably functions to generate oxaloacetate when high concentrations of condensing partner are needed during thermogenesis. They may also explain earlier observations that acetate was produced from palmitoyl-carnitine at low temperatures even when malate was present; this is here shown to be due to the limited malate permeability at these low temperatures. Thus, even at the body temperature of the hibernating hamster (4-5 degrees C), brown fat is probably able to combust fatty acids totally.
Adipose Tissue, Brown↗
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 micrograms 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.
Adipose Tissue, Brown↗
Alpha-adrenergic effects on 86Rb+ (K+) potentials and fluxes in brown fat cells.
Net K+ fluxes in isolated hamster brown fat cells were studied by the use of the K+ analogue 86Rb+. In isolated cells, cold-stored overnight to diminish K+ gradients, an equilibrium 86Rb+ (K+) clearance value of 27 microliter/million cells was obtained after 30 min incubation at 37 degrees C. This corresponds to a 10-fold K+ gradient over the plasma membrane, and a K+ potential of about -60 mV. The attainment of this equilibrium was dependent upon the presence of Na+ in the extracellular medium, and the uptake was fully inhibited by the (Na+ + K+)-ATPase inhibitor ouabain. Ouabain had, however, no significant acute effect on the maximal rate of thermogenesis achieved after norepinephrine stimulation of the cells, but if the restoration of ionic equilibrium was inhibited by ouabain in prolonged incubations, a decreased thermogenesis was observed. This was probably due to the low cytosolic K+ content then encountered, and the resulting inhibition of lipolysis. The addition of norepinephrine to cells in which 86Rb+ (K+) equilibrium had been attained resulted in a rapid efflux of 86Rb+ and the establishment of a new equilibrium value, at about 65% of the unstimulated value. This corresponds to a decrease in K+ potential of about 15 mV. The effect of norepinephrine was stereospecific and reversible, and had an EC50 value of about 10 nM. As catecholamine effects were much more sensitive to phentolamine than to propranolol, the adrenergically-induced efflux was classified as predominantly alpha-adrenergic. It is suggested that the norepinephrine-induced K+ efflux is due to a (probably Ca2+-mediated) opening of K+ channels in the cell membrane, and that this effect occurs secondarily to the alpha-adrenergically induced membrane depolarization (and increase in cytosolic Ca2+). The increased PK over the cell membrane would counteract further depolarization, and the K+ gradient would then approach the Nernst equilibrium under the new steady-state conditions.
Adipose Tissue, Brown↗
Enzyme-linked immunosorbent assay (ELISA) studies of the interaction between mammalian and avian anti-thermogenin antibodies and brown-adipose-tissue mitochondria from different species.
Three different antibody preparations, rabbit anti-hamster and rabbit anti-rat thermogenin sera and chicken anti-rat thermogenin IgG, were tested for cross-reactivity towards isolated thermogenin and BAT mitochondria from different mammalian species using an ELISA-technique. It was found that the antibody preparations readily cross-reacted with different species, but that the affinity of the antibody preparations was greater towards the homologous species than the other species. The reactivity of an antibody preparation towards mitochondria from different tissues from the homologous species was also tested, and the exclusive occurrence of thermogenin in BAT could be confirmed.
Acclimatization↗
The molecular basis for adrenergic desensitization in hamster brown adipose tissue: uncoupling of adenylate cyclase activation.
A crude membrane fraction was prepared from hamster brown adipose tissue. Extensive washing of the crude membranes was crucial for the appearance of specific beta-adrenergic receptor binding as assessed by (-)-[3H]dihydroalprenolol. Adrenergic agents competed for the specific binding sites with beta 1-specificity. Binding characteristics were very similar to those earlier found in intact cells, supporting our previous finding that a single (non-tumour) mammalian cell may contain as many as 60,000 beta-adrenergic receptors. Desensitization in situ (i.e. chronic norepinephrine stimulation due to cold acclimation) only marginally affected the number of beta 1-receptors and their affinity (Ki) for norepinephrine. Total (fluoride-stimulated) adenylate cyclase increased somewhat, but the Kact for norepinephrine slightly decreased. Thus the ratio Ki/Kact was rather unaffected by cold acclimation. However, the fraction of adenylate cyclase which could be stimulated by norepinephrine decreased drastically. GTP introduced a low-affinity form (for agonist) of the receptor. The form observed in isolated cells must primarily be the high-affinity form. The basis for desensitization must reside in a diminished ability to transfer the signal from the receptor to the cyclase. This change may be molecularly located in the N-protein or in its interaction with the receptor.
Acclimatization↗
Beta-adrenergic stimulation of lipoprotein lipase in rat brown adipose tissue during acclimation to cold.
Lipoprotein lipase activity in adult rats was investigated in animals subjected to cold and to different hormonal treatments. In contrast to changes in tissue wet weight and total protein content, which showed a lag time of about 1 day, lipoprotein lipase activity was markedly (fourfold) increased after only 4 h in the cold. Total lipoprotein lipase activity reached a plateau already after 1-3 days, whereas wet weight and protein content did not plateau until 3 wk. Neither insulin nor glucose injections could mimic the cold-induced increase in lipoprotein lipase activity seen after 4 h. However, the effect of norepinephrine injections was identical to the effect of cold. The beta-agonist isoprenaline was as effective as norepinephrine, whereas the alpha-agonist phenylephrine had no effect. The beta-antagonist propranolol inhibited the cold-induced increase in lipoprotein lipase activity. It is concluded that, in contrast to white adipose tissue, brown adipose tissue lipoprotein lipase is stimulated in vivo by a beta-adrenergic mechanism and that it is this beta-adrenergic mechanism that is responsible for the rapid recruitment of lipoprotein lipase during cold exposure.
Adipose Tissue, Brown↗
Preferential utilization of brown adipose tissue lipids during arousal from hibernation in hamsters.
The participation of brown adipose tissue in the arousal process of golden hamsters was studied. The utilization of lipids in different depots of brown adipose tissue was followed gravimetrically. From both the interscapular and the cervical brown adipose tissue depots, 28 mg of lipid were lost during arousal; there was no measurable loss of lipid from the white adipose tissue depots. The total weight of eight identified depots of brown adipose tissue in nonhibernating, cold-acclimated hamsters was estimated to be 1,700 mg, of which 475 g were lipid. It is calculated that a total of 255 mg lipid disappeared from brown adipose tissue during arousal; this lipid is theoretically capable of giving rise to 2.4 kcal (9.9 kJ) of heat. It is concluded that the heat produced by the combustion of the lipid that disappeared from the brown adipose tissue during the arousal process could be the major source of the heat needed to rewarm the hamster from hibernating to euthermic body temperatures.
Adipose Tissue↗
Quantitative differentiation of alpha- and beta-adrenergic respiratory responses in isolated hamster brown fat cells: evidence for the presence of an alpha 1-adrenergic component.
The respiratory (thermogenic response of brown fat cells has been investigated for differentiation between alpha- and beta-adrenergic components. The relative sensitivity of the cells generally followed the pattern of the EC50 for isoprenaline less than norepinephrine = epinephrine much less than phenylephrine and the response to all these agonists was much more sensitive to propranolol than to phentolamine. Based on these criteria the response was primarily beta 1. However, the biphasic nature of the dose-response curves and the antagonist inhibition curves indicated additionally the presence of an alpha-component. Inhibition studies demonstrated the IC50 series: prazosin less than phentolamine less than yohimbine, indicating that the alpha-component is of the alpha 1-subtype. The effects of selective alpha- and beta-stimulation were additive. The maximal oxygen consumption of isolated hamster brown fat cells was composed of an 80% beta 1 adrenergic component and a 20% alpha 1 adrenergic component. Different mechanisms (beta 1 through cyclic AMP and alpha 1 possibly through Ca2+) and perhaps different purposes (e.g. short-term and long-term regulation, respectively) may explain the coexistence of two stimulatory adrenergic responses in one cell type.
Adipose Tissue, Brown↗
Identification of [3H]prazosin binding sites in crude membranes and isolated cells of brown adipose tissue as alpha 1-adrenergic receptors.
The alpha 1-receptor selective adrenergic antagonist [3H]prazosin was used to study adrenergic binding sites in crude membranes and isolated cells from hamster brown adipose tissue. The antagonist labelled a site which fulfilled the criteria for being the alpha 1-receptor which participates in mediation of a part of the norepinephrine-induced respiration (thermogenesis) in intact cells. The similarity between the characteristics of the binding site in crude membrane fractions and in isolated brown fat cells suggested that the site is of postsynaptic origin. In equilibrium binding studies [3H]prazosin bound with very high affinity (Kd = 0.4 nM), and the maximal binding capacity was 72 fmol/mg protein and 207 fmol/10(6) cells, equal to 120 000 receptors per cell. The kinetically calculated Kd had a value of 0.17 nM, in good agreement with that determined in the equilibrium binding experiments. The relative potencies of adrenergic agents to displace [3H]prazosin revealed a typical alpha 1-specificity: WB-4101 = prazosin greater than phentolamine greater than dihydroergocryptine much greater than yohimbine greater than propranolol for antagonists, and L-phenylephrine = L-norepinephrine = L-epinephrine greater than L-isoprenaline greater than D-norepinephrine for agonists. Thus stereoselectivity was also shown. The actual Ki values for antagonists were closely similar in crude membranes and isolated cells whereas the alpha 1-receptor showed a 10-20 times higher affinity for agonists in the cellular preparation than in the crude membranes. The Ki values for the different antagonists and agonists derived from binding studies in isolated cells were compared with the IC50 and EC50 values for these agents obtained from studies on alpha 1-mediated cellular effects. It is suggested that tight coupling exists between alpha 1-receptor occupancy and alpha 1-mediated effects.
Adipose Tissue, Brown↗
Development of brown fat cells in monolayer culture. I. Morphological and biochemical distinction from white fat cells in culture.
The ability of cells from the stromal-vascular fraction of rat brown adipose tissue to develop into adipocytes in primary cell cultures was investigated. Comparison was made with precursor cells isolated by the same procedure from the white adipose tissue of the same animals and cultured in parallel under identical conditions. The culture procedure used allowed the cells isolated from both tissues to rapidly proliferate and differentiate. During the first week in culture the brown fat cells grew to confluence and accumulated fat in a multilocular way. During the second week, further fat was accumulated, but the cells remained multilocular. Analysis of the parallel white fat cell cultures revealed clear differences between the two adipocyte types, although the rates of cell growth were identical. Measurement of the size of the cellular lipid inclusions as a function of the time in culture indicated a much higher number of fat droplets larger than 30 micron in the white adipocytes. Moreover, after isolation of pelleting fractions of both cultured cell types, comparative functional analysis of their mitochondria by oxygen consumption measurement, as well as direct cytochrome-c-oxidase determinations, showed a significantly higher amount of mitochondria in the brown fat cell fractions than in the white fat cell fractions. It was concluded that mature brown fat contains precursor cells which can proliferate and develop into adipocytes in monolayer cell culture and which have inherent characteristics distinct from those of white fat precursor cells.
Adipose Tissue↗
Effects of dietary essential fatty acids on active thermogenin content in rat brown adipose tissue.
Rats were fed diets providing either 0.3% (low EFA), 3% (control) or 10% (high EFA) of the energy as essential fatty acids. (All diets provided 20 energy percent of fat.) Both the experimental groups had a lower body weight than the controls. Small differences were found between the groups in many traditional parameters of brown adipose tissue activity (wet weight, protein content, mitochondrial content, cytochrome c oxidase activity). A specific increase in the mitochondrial concentration of the 32,000-dalton GDP-binding protein thermogenin was observed in the high EFA group (from 0.30 to 0.45 nmol/mg). When the thermogenin content of the animal was expressed per gram body weight, the content was more than doubled (from 22 to 47 pmol/g body weight) in the high EFA group, but was unaltered in the low EFA group. It is concluded that the effect of low EFA is nonspecific and due to a general decrease in health status. The effects of high EFA are, however, specific and resemble the changes observed in animals exhibiting diet-induced thermogenesis. It is suggested that the animals fed high amounts of essential fatty acids are in a state of decreased metabolic efficiency and this may be at least in part an explanation for their low body weight.
Adipose Tissue, Brown↗
External esophageal construction remembered.
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Adult↗
A statewide evaluation of long term care.
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Humans↗
Exclusive occurrence of thermogenin antigen in brown adipose tissue.
Thermogenin is the purine-nucleotide binding polypeptide in brown adipose tissue mitochondria (Mr 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 less than 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.
Adipose Tissue↗
Desensitisation of beta-adrenergic responsiveness in vivo. Decreased coupling between receptors and adenylate cyclase in isolated brown-fat cells.
Chronic catecholamine stimulation in vivo of brown adipose tissue during acclimation of hamsters to cold does not result in any alteration of beta-adrenergic receptor number or affinity when determined in isolated adipocytes by (-)-[3H]dihydroalprenolol binding. Norepinephrine displacement of (-)-[3H]dihydroalprenolol showed the same Ki for both groups. However, the slope of the displacement curve was shallower for cells from cold-acclimated animals than for controls. Cyclic AMP accumulation was stimulated by norepinephrine in cells from both groups of animals, although the dose-response curve for cells from cold-acclimated animals was shifted to the right and the maximum value obtained was less than half that found in cells from control animals. The slope of the curve was again lower. Other catecholamines stimulated cAMP accumulation with an order of potency in agreement with a response mediated through beta 1-adrenergic receptors. The dose-response curve for norepinephrine-stimulated oxygen consumption was also shifted to the right for cells from cold-acclimated animals, although the maximal respiration was only slightly reduced. The slope factor was again decreased. The results are interpreted in terms of a reduced coupling between the beta-receptor and the metabolic response in isolated brown adipocytes from cold-acclimated animals as a result of chronic catecholamine stimulation in vivo.
Acclimatization↗