Unusually high mitochondrial alpha glycerophosphate dehydrogenase activity in rat brown adipose tissue.

Brown adipose tissue of the rat has been found to have an unusually high activity of mitohondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) when assayed both by a histochemical staining procedure and by a quantitative biochemical method with isolated mitochondria. In contrast to succinic, glutamic, and beta-hydroxybutyrate dehydrogenases, all mitochondrial enzymes, the activity of alpha-GPD in brown fat was 10 times that in liver, more than 20 times that in white adipose tissue, and 9 times that in kidney. The soluble NAD-linked alpha-GPD was also higher in brown adipose tissue than in white adipose tissue, liver, or kidney, but the differences were much less marked. The possible importance of the high activity of mitochondrial alpha-GPD in the regulation of synthesis of esterified lipid and in thermogenesis in brown fat is discussed.

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 (Houstek, J., et al. (1988) Biochim. Biophys. Acta 935, 19-25).

Hybridization in situ studies of angiotensinogen gene expression in rat adrenal and lung.

Both rat adrenal and lung contain low levels of angiotensinogen mRNA, as shown by Northern blot and nuclease S1 analyses of RNA extracted from these tissues. We sought to identify the cellular localization of angiotensinogen mRNA in these two tissues using hybridization in situ of tissues obtained from both control rats and rats administered a combination of dexamethasone, ethynylestradiol, and T3. For the adrenal of hormone-treated rats, angiotensinogen mRNA was identified in periadrenal fibroblast-like cells and brown adipose tissue. For control rats, positive hybridization was obtained for fibroblast-like cells immediately adjacent to the adrenal capsule, but not for periadrenal brown adipose tissue. No hybridization was obtained for cells of the adrenal cortex, medulla, capsule or vessels. For the lung of hormone treated, but not control rats, angiotensinogen mRNA was identified in perivascular and peribronchial fibroblast-like cells and brown adipose tissue in the lung hilum. No hybridization was obtained for pulmonary parenchyma, bronchi, or vessels. These results confirm the widespread tissue distribution of angiotensinogen mRNA, and provide further evidence for the formation of angiotensin within tissues by mechanisms independent of the circulating renin-angiotensin system.

Location and regulation of rat angiotensinogen messenger RNA.

The presence of angiotensinogen messenger RNA (mRNA) was detected in rat vascular and adipose tissue. Angiotensinogen mRNA in rat aorta was localized in the adventitia and surrounding adipose tissue, and not in the vascular smooth muscle. Freshly dispersed and cultured endothelial and aortic smooth muscle cells did not contain detectable amounts of angiotensinogen mRNA. In addition to periaortic adipose tissue, angiotensinogen mRNA was present in other fat depots of both brown and white types. To examine regulation of angiotensinogen gene expression, Sprague-Dawley rats were treated with angiotensin converting enzyme inhibitor or underwent bilateral nephrectomy. Relative levels of angiotensinogen mRNA in brown adipose tissues increased dramatically by 48 hours after bilateral nephrectomy. However, only one source of brown adipose tissue showed increased angiotensinogen mRNA levels after animals were treated for 5 days with converting enzyme inhibitor. In addition, angiotensinogen was released into the medium from incubated adipose tissues with levels increasing over a 2-hour period. These results demonstrate that angiotensinogen is synthesized by adipose tissue in the rat and may play a role in the function of this tissue.

Prostaglandins E2 and F alpha levels in white and brown adipose tissues of cold acclimated rats as measured by a new micromethod.

A new micro-method was used to determine the effects of cold acclimation of rats on the levels of prostaglandin E2 and F alpha in both white and brown adipose tissues. Whereas PGF alpha levels were significantly higher than PGE2 levels in white fat, no difference between the amounts of the two prostaglandins was observed in brown fat. In both tissues, cold acclimation did not induce any change in prostaglandin levels.

Occurrence of an active regulatory mechanism of protein synthesis during starvation and refeeding in Bombyx mori fat body.

The origin of the amino acids which participate in protein synthesis at the recovery from starvation have been determined in the fat body from Bombyx mori larvae. Endogeneous amino acids have been labelled with [3H] leucine and ingested ones with [14C] leucine, allowing their discrimination in the organism. 22 minutes after refeeding, proteosynthetic activity of the fat body, estimated by the polysome level, is increased 2.5 fold. Endogeneous leucine represents more than 90 p. cent of the leucine present in nascent polypeptides. Free leucine pools of the fat body and of hemolymph increase, mainly through the release of endogeneous leucine. It is therefore concluded that refeeding with amino acids induces the production of a signal or critical factor, responsible for the increase in proteosynthetic activity in the fat body.

Increased alpha 1-adrenergic receptor density in brown adipose tissue of cold-acclimated rats and hamsters.

Binding sites for [3H]prazosin were characterized in crude membrane fractions from rat brown adipose tissue. Based on agonist (norepinephrine approximately equal to phenylephrine much greater than isoprenaline) and antagonist (prazosin much greater than yohimbine greater than propranolol) potencies to compete with [3H]prazosin, the binding sites were identified as alpha 1-receptors, not previously described in rat brown adipose tissue. As the [3H]prazosin binding sites could be observed in isolated brown fat cell preparations, they were probably postsynaptic. The effect of cold acclimation was studied in crude membrane fractions from control and cold-acclimated (4 degrees C) rats and hamsters. Cold acclimation did not change the affinity of the receptor for agonists and antagonists, but there was a significant increase in the number of alpha 1-receptors (per mg protein), both in rat (100% increase) and hamster (40% increase) brown fat. Based on these results and on earlier results on beta-receptors from this and other laboratories, it is suggested that activation of brown adipose tissue is associated with an increase in the relative density of alpha 1-receptors (i.e. in the alpha 1/beta ratio) and an increased significance of alpha 1-adrenergic pathways for the function of the tissue.

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.

Long-term effects of lateral hypothalamic lesions on brown adipose tissue in rats.

A classic feature of animals with lateral hypothalamic (LH) lesions is their regulation of body weight at sub-normal levels. The present studies were done to determine whether this is associated with enhanced thermogenic activity of their brown adipose tissue (BAT). Three groups of young chow-fed male Holtzman rats were formed: (1) animals receiving bilateral radiofrequency heat lesions of the dorsal LH and then permitted free access to chow (LH rats); (2) non-lesioned animals that were pair-fed (PF) to the lesioned rats during a 2 week post-operative recovery period (Phase 1); (3) non-lesioned, ad lib fed (NORM) controls. After Phase 1, each group was divided and permitted free access to chow alone or an additional selection of palatable, novel food items (a "cafeteria" diet) for 2-3 weeks (Phase 2) to stimulate diet-induced thermogenesis in BAT. Finally, half of each sub-group was exposed to 4 degrees C for 15 hr to stimulate nonshivering thermogenesis in BAT. During Phase 1 LHs and PFs ate 50% less than NORMs. This resulted in a weight deficit of 16% for LHs and 12% for PFs. After the additional period of feeding palatable foods (Phase 2) LHs collectively weighed 14% less than NORMs whereas previously PFs had a weight deficit of only 4%. They gained less weight than NORMs or PFs despite a similar energy intake. LHs had small deposits of gonadal white adipose tissue [both total amount and expressed per metabolic body mass (kg 0.75)]. The weight of interscapular BAT was less in the LHs but its concentration of protein (mg/g) was higher.(ABSTRACT TRUNCATED AT 250 WORDS)

Meal associated changes in brown fat thermogenesis and glycogen.

Data indicate a close association between a decrease in feeding-induced brown adipose tissue (BAT) thermogenesis and an increase in food consumption. The present study examines the hypothesis that feeding-induced BAT thermogenesis, or feeding-induced changes in BAT glycogen, a mobile form of energy store and a correlate of BAT thermogenesis, may modulate feeding behavior. We report that propranolol, which completely abolished meal-induced BAT thermogenesis, did not evoke intake of a larger meal. Though BAT glycogen concentration is a sensitive measure of the state of feeding, on a meal to meal basis it does not correlate with hunger and satiety. Hence the hypothesis is not supported by the current data. We also report that meal-induced BAT hypertrophy and glycogen deposition can be dissociated from meal-induced BAT thermogenesis.

Immunological detection of cDNA clones encoding the uncoupling protein of brown adipose tissue: evidence for an antigenic determinant within the C-terminal eleven amino acids.

Poly(A)+RNA was isolated from brown adipose tissue of cold acclimated rats and a fraction enriched for uncoupling protein mRNA was used to generate a cDNA library in pBR 322. Immunological screening of 1,500 colonies with an affinity-purified antiserum against the uncoupling protein yielded five positive clones, pUCPrat1-5. Clone pUCPrat2 encoded the C-terminal 54 amino acids of rat uncoupling protein and exhibited 90% amino acid homology with the hamster protein. Clones pUCPrat3-5 encoded only the C-terminal 11 amino acids suggesting that an antigenic determinant lies within this sequence.

Adaptive changes in the concentration of the mitochondrial 'uncoupling' protein in brown adipose tissue of hamsters acclimated at different temperatures.

The effect of acclimation at different temperatures on the activity of interscapular brown adipose tissue has been investigated in the hamster, a hibernator. Between 31 degrees and 4 degrees C the cytochrome oxidase activity of the tissue increased 4- to 5-fold, mitochondrial GDP binding per mg of mitochondrial protein doubled, and the amount of uncoupling protein rose from 1.7% to 5.4% of total mitochondrial protein. It is concluded that there are clear adaptive changes induced by temperature in brown adipose tissue of the hamster, but the changes are limited in comparison with those in the mouse.

The effect of temperature acclimation on the spin-lattice relaxation time of brown adipose tissue.

Cold acclimation induced an increase of spin-lattice relaxation time (T1) of proton nuclear magnetic resonance in rat brown adipose tissue with concomitant increase of water content, while heat acclimation did not effect T1 of this tissue, although its water content was significantly decreased. Temperature acclimation did not affect either T1 or water content of neck muscle.

Effects of exercise and intermittent cold exposure on shivering and nonshivering thermogenesis in rats.

The effects of both exercise training and intermittent cold acclimation on heat production (shivering and nonshivering thermogenesis (NST)) in rats were studied. Warm-acclimated rats (housed at 24 degrees C, WA) and intermittently cold-acclimated rats (exposed daily to -5 degrees C for 2 h, CA) were forced to run (25 m.min-1 for 1 h) every day (WA-T and CA-T). WA and CA left sedentary (WA-S and CA-S) served as controls. Norepinephrine (NE)-induced thermogenic capacity assessed from the increment of oxygen consumption (VO2) and colonic temperature (Tc) were measured 4 weeks after commencing acclimation and exercise training. The thermogenic capacity was greater in CA than in WA. However, in WA, WA-T responded to NE less than WA-S, whereas the response of CA-T and CA-S did not differ. Wet weight of interscapular brown adipose tissue (IBAT) and its protein (and dry matter--regarded to be highly representative of protein) content were larger in CA than in WA. Respective sedentary and exercised groups of rats had similar IBAT protein (and dry matter) content although tissue weight was lighter in WA-T than in WA-S. Lipid content of IBAT was also larger in CA than in WA. IBAT of WA-T had less lipid compared to that of WA-S while no difference was seen between CA-S and CA-T. Shivering activity during acute cold (4 degrees C) exposure was less in CA compared to WA and there was no difference between respective groups of exercised and sedentary rats. Propranolol, a blocker of NE-dependent NST, eliminated the difference in shivering among these four groups. When exposed to severe cold (-10 or -20 degrees C), the fall in Tc of rats fasted for 18 h was greater in WA than in CA. CA-T showed a greater decrease in Tc than CA-S during -20 degrees C exposure while it did not differ during -10 degrees C exposure. On the other hand, Tc of WA-T and WA-S did not differ significantly during either cold exposure period. These results suggest that exercise training in rats housed at 24 degrees C suppresses NE-dependent NST whereas another nonshivering thermogenic mechanism (NE-independent) may compensate this suppression. However, NE-dependent NST of WA-S and WA-T did not parallel protein (dry matter) content of IBAT; no difference existed in IBAT protein between these two groups.(ABSTRACT TRUNCATED AT 400 WORDS)

Possible role of polyamines in the function of brown adipose tissue.

In order to ascertain possible involvement of polyamines in the physiological regulation of brown fat function, effect of temperature acclimation on the polyamine contents of this tissue and effects of polyamines on the noradrenaline-induced thermogenesis is isolated brown adipocytes were investigated in rats. Daily urinary excretion of polyamines measured collectively for spermidine and spermidine per body weight was decreased in heat acclimation and increased in cold acclimation. Polyamine concentrations per fresh weight of brown fat showed extremely low values compared with those of other tissues previously reported. Putrescine and spermidine contents per fat-free dry matter of brown fat were decreased in cold acclimation, but were not affected in heat acclimation. Spermidine and spermine inhibited the noradrenaline-induced thermogenesis of brown adipocytes dose-dependently. These results suggest that polyamines regulate the heat production of brown adipose tissue in an inhibitory way and cold acclimation potentiates heat production of this tissue by reducing polyamine levels.

Distribution and metabolism of 2,4,5,2', 5'-pentachlorobiphenyl.

Single doses of 2,4,5,2',5-pentachlorobiphenyl uniformly labeled with 14-C have been administered intravenously and orally to mice. Whole-body autoradiograms and scintillation counting of tissue samples have shown that most radioactivity leaves the circulation for the tissues within one hour. Peak concentrations varied, being highest in brown fat, which after 24 hours comprised the major reservoir of the unchanged compound in the body. Radioactivity disappeared rather rapidly drom most other tissues, although the longest retention occurred in bronchial epithelium and some parts of the renal tubules. The excretion of radioactivity was mainly through the bile, into feces, with a half-time of six days. There was little unchanged compound in the feces, the major metabolite was a hydroxylated derivative, both free and conjugated.

[Variability in several ecologic-physiologic traits of different populations of the Asiatic Arctic suslik Citellus parryi]. / Izmenchivost' nekotorykh ékologo-fiziologicheskikh priznakov raznykh populiatsii aziatskogo arkticheskogo suslika Citellus parryi

In several geographic populations of the Arctic ground squirrel C. parryi, studies have been made on changes in oxygen consumption during decrease of the ambient temperature from 25 to 3-4 degrees, thermal preference, hemoglobin content of the blood and composition of the adipose tissue (both brown and white, subcutaneous and visceral). Significant shifts of these indices were found. In animals from various parts of the species area, different sensitivity to cooling was found, as indicated by determinations of oxygen consumption at different temperatures and the prefered temperature: different hemoglobin content of the blood was also found together with differences in the level of two unsaturated fatty acids - the oleic and linoleic ones. Comparison of these data with similar results obtained on various populations of the Siberian ground squirrel C. undulatus revealed obvious differences between these close species with respect to the indices studied.