Steroid profiles of brown adipose tissue

In brown adipose tissue of alp-marmot (Marmota marmota), badger (Meles meles) and Wistar rats steroids of C21- and C19-type are identified and quantified. The detection of 3 alpha-hydroxy-5 alpha-pregnan-20-one, 3 alpha-hydroxy-5 beta-pregnan-20-one, 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one, 3 alpha,21-dihydroxy-5 beta-pregnan-20-one and 3 beta,21-dihydroxy-5 alpha-pregnan-20-one is of special interest since sleep-inducing properties have been described with these steroids.     

Subcellular fractionation of brown adipose tissue

The present study proposes a technique, using Metrizamide, which permits the preparation of brown adipose tissue plasma membranes from the crude mitochondria as well as from the crude microsome fraction. These plasma membranes have high relative specific activities of their marker enzyme, 5′-nucleotidase (15 ± 3 and 14 ± 2 respectively) and, particularly those originating in the crude microsomes, are relatively free of mitochondria contamination. This study also shows the influence of the mode of cell disruption on microsome integrity. When cell disruption was achieved by grinding in liquid nitrogen the purified microsome NADPH cytochrome c reductase specific activity was found to be 3.5 times greater than that of microsomes obtained after homogenization of the tissue.     

Glycerokinase activity in human brown adipose tissue

Brown adipose tissue (BAT) is known to be responsible for heat production in newborn and adult hibernating mammals. In rats and mice, BAT has been demonstrated to possess a much higher glycerokinase activity than white adipose tissue (WAT). It has been speculated that this high activity may cause the futile cycle of triglyceride breakdown and resynthesis to be activated, thus contributing to heat production. However, at present very little information is available regarding the location, function, and quantitative importance of BAT in adult human subjects. Our objective in this study was to locate BAT in human subjects and to characterize it biochemically, especially with respect to the enzyme glycerokinase. We have looked for histologically identifiable BAT in 32 human subjects and found it in 12 subjects. Most of the BAT samples were obtained from perirenal adipose depots in children undergoing surgery. Some of the samples were almost totally comprised of BAT cells, whereas others were a mixture of BAT cells and WAT cells. The glycerokinase activity per gram of tissue was higher in BAT than in WAT in all the subjects where the above comparison was made. The activity per mg protein or per microgram DNA was higher in most BAT samples. In one pure BAT specimen, the basal lipolytic rate and the lipoprotein lipase activity were measured and they were both higher in BAT than in the WAT obtained from the same patient. These results show that human brown adipose tissue possesses an enzymatic profile very similar to that of rodent brown adipose tissue.     

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.     

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.     

Cold-induced activation of brown adipose tissue and adipose angiogenesis in mice

Exposure of humans and rodents to cold activates thermogenic activity in brown adipose tissue (BAT). This protocol describes a mouse model to study the activation of BAT and angiogenesis in adipose tissues by cold acclimation. After a 1-week exposure to 4 °C, adult C57BL/6 mice show an obvious transition from subcutaneous white adipose tissue (WAT) into brown-like adipose tissue (BRITE). The BRITE phenotype persists after continuous cold exposure, and by the end of week 5 BRITE contains a high number of uncoupling protein-1-positive mitochondria, a characteristic feature of BAT. During the transition from WAT into BRITE, the vascular density is markedly increased owing to the activation of angiogenesis. In BAT, cold exposure stimulates thermogenesis by increasing the mitochondrial content and metabolic rate. BAT and the increased metabolic rate result in a lean phenotype. This protocol provides an outstanding opportunity to study the molecular mechanisms that control adipose mass.     

Beta-oxidation in peroxisomes of brown adipose tissue.

Peroxisomes and mitochondria from brown adipose tissue of the rat were separated by differential pelleting and isopycnic gradient centrifugation. Both fractions oxidized palmitoyl-CoA with comparable specific activities. Unlike the mitochondrial beta-oxidation the peroxisomal activity was not influenced by carbon monoxide. Peroxisomal beta-oxidation together with carnitine acetyl-transferase, which is also located in peroxisomes, might be involved in chemical thermogenesis by delivering acetyl groups to the mitochondria.     

Morphochemical study of brown adipose tissue during adrenal gland regeneration in the white rat

The interscapular brown adipose tissue in male, white Wistar rats in the 1,3,6,15,30, and 60th day of regeneration of adrenals after their enucleation weas investigated. The tissue slides were stained with H and E, with Masson's triple stain and with Sudan B. The activity of the following dehydrogenses was evaluated: Succinic dehydrogenase, delta 5,3 beta-hydroxysteroid dehydrogenase and the activity of the following enzymes; monoaminooxidase (MAO), Alkaline phosphatase (AlPh) and acid phosphatase (AcPh) was studied. Between the first ad 30th day of regeneration of adrenals morphochemical changes were observed; a great number of cells with eosinophylic cytoplasm appeared and with minute vacuols in their cytoplasm, the blood vessels became more numerous and enlarged and the activity of the enzymes tested was enhanced, instead sudanophilia was weaken and the reaction of corticosteriods significantly intensified. The changes observed may indicate on an enhanced hormonal activity. The brown adipose tissue may function as an endocrine gland which supports the regenerating adrenals of the white rat, enables survival and preservation of homeostasis during the insufficiency of the adrenal cortex after adrenal enucleation. The initiation of the endocrine activity of the brown adipose tissue in the course of regeneration of the adrenals proceeds probably without the direct participation of catecholamines originating from the adrenals.