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Aging in both man and rodent is associated with increases in body weight and body fat. In young adult rats, mixed calorie overfeeding increases triiodothyronine production and decreases weight gain efficiency compared with chow diets. This study was undertaken to determine whether 4- and 14-month-old adult rats have similar responses in thyroxine (T4) and triiodothyronine (T3) metabolism during mixed calorie overfeeding. Four- and 14-month-old male Sprague-Dawley rats were each separated into two groups (n = 14): control (CHOW) versus chow + mixed calorie (CAFE) overfeeding for 28 days. The 4-month-old CAFE-fed rats ingested 662 +/- 54 extra kcal over 28 days and gained 147 +/- 7 vs 113 +/- 5 g (P less than 0.01) for their age-matched CHOW group. The 14-month-old CAFE group ingested 309 +/- 45 extra kcal and gained 58 +/- 6 g weight vs -12 +/- 5 g for their age-matched CHOW group (P less than 0.01). Serum T4 concentrations were unchanged during overfeeding or aging. The serum T3 concentration was increased 24% in the 4-month-old CAFE group compared with the age-matched CHOW group (P less than 0.05), but there was no difference in the serum T3 concentration between the 14-month-old CAFE and CHOW groups. The metabolic clearance and production rates of T3 and T4 were decreased in the 14-month-old vs 4-month-old groups (P less than 0.01). The T3 metabolic clearance rate was increased in the CAFE versus CHOW group in the 4-month-old groups (137.3 +/- 11.2 vs 103.0 +/- 10.1 ml/kg/hr, P less than 0.01) but unchanged in the 14-month-old CAFE and CHOW groups (47.6 +/- 6.6 vs 53.4 +/- 5.3 ml/kg/hr, not significant). Liver type I iodothyronine 5'-deiodinase activity increased during overfeeding in the young (63.6%, P less than 0.02) but not in the older rat group. T4 and T3 production rates were decreased in the older rats and did not increase during overfeeding as observed in the young adult rat.

Brown adipose tissue (BAT) has an important role in the thermogenesis of newborn mammals and cold-acclimated rodents. Heat production within the tissue is regulated by the sympathetic nervous system (SNS) via norepinephrine (NE) release. As only little is known of the progressive role of SNS during the developmental period, NE content and turnover were determined in BAT of infant rats 1-33 days of age. The dams and the pups were kept at either 28 or 16 degrees C. It was observed that the NE level in the tissue and the mediator turnover had an almost parallel evolution during the experimental period. In the first week of life, level and turnover of NE were higher in the 28 degrees C group than in the 16 degrees C group. During the second week, the level remained constant in rats kept at 28 degrees C, but the NE turnover slightly decreased. In contrast, a large increase of both parameters was observed in rats kept at 16 degrees C. Then a decrease occurred in both groups until weaning. Subsequently the values remained almost constant. It must be noted that, from the end of the first week, NE content and turnover were always significantly higher in the 16 degrees C group than in the 28 degrees C group. At the end of the first month, the level was 50% higher in cold-acclimated rats and turnover was four times as high as in controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The uncoupling protein of brown-adipose-tissue mitochondria has been purified in the form of mixed micelles with lipid and reduced Triton X-100. This surfactant has the advantage over conventional Triton X-100, that it does not interfere with amide bands in infrared spectra. The structure of the uncoupling protein in micellar form has been examined by Fourier-transform infrared spectroscopy (FTIR). In order to decompose the amide I contour into its components, band-narrowing (Fourier derivation and deconvolution) and band-decomposition techniques have been used. Combining data from spectra taken in H2O and 2H2O media, the following percentage distribution of secondary structure patterns has been obtained: 50% alpha-helix, 28-30% beta-structure; 13-15% beta-turns and 7% unordered. Thermal denaturation of the uncoupling protein has also been monitored by FTIR. In accordance with previous observations of different proteins, thermal denaturation is marked by a shift in the amide I maximum and the appearance of two new peaks in 2H2O, at around 1620 cm-1 and 1685 cm-1. Denaturation occurs in the 40-50 degrees C temperature range, in agreement with studies of GDP-binding capacity. Cooling down the thermally denatured protein produces a new change in its secondary structure; however, the original conformation is not restored. The uncoupling protein possesses a nucleotide-binding site. On addition of GDP, small changes in protein conformation occur, attributable to changes in tertiary structure. However, no detectable effects are seen in the presence or absence of the other physiological regulators, the free fatty acids. The uncoupling protein shares important similarities in its primary structure with other anion carriers of the mitochondrial membrane; one of these, the adenine-nucleotide translocator, has been used in a comparative study, applying the same FTIR techniques described above for the uncoupling protein. Both proteins have a similar proportion of alpha-helix, probably corresponding to the segments spanning the membrane, but the conformation of the polar domains appears to differ.

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

The beta-adrenergic receptor of rodent brown fat plays a key role in the control of energy dissipation by this tissue. The aim of the present study was to further characterize the biochemical properties of this receptor. The beta-receptor of rat interscapular brown adipose tissue plasma membranes was found to bind the beta-adrenergic antagonist [125I]cyanopindolol with a high affinity (KD 67 pM). The [125I]cyanopindolol receptor complex could be solubilized by digitonin and the isoelectric point of the solubilized receptor was found to be 5.8. Brown adipose tissue plasma membranes were labeled with the photoaffinity ligand [125I] cyanopindolol diazirine and labeled membrane proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis and analyzed by autoradiography. Autoradiograms revealed a peptide of 62 kDa whose labeling was stereoselectively displaced by alprenolol and isoproterenol. The beta 1-selective antagonist betaxolol was about 100 times more potent in displacing the labeling of this 62 kDa peptide than the beta 2-selective antagonist ICI 118,551. Based upon these data, it appears that the beta-receptor in brown adipose tissue is a beta 1 subtype with molecular weight of 62 kDa.

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.

After 15 wk on a moderately high-calorie high-fat (CM) diet, 43% of 40 3-mo-old male Sprague-Dawley rats developed diet-induced obesity (DIO) (29% more weight gain), whereas 57% of diet-resistant (DR) rats gained no more weight than 20 chow-fed controls. When switched to chow for another 7 wk, DR rats ate 13% less, gained 55% less weight, and had 49% lower food efficiency, whereas DIO rats ate 4% less but had comparable weight gain and efficiency to controls. DIO rats had 29% more carcass lipid (percent of carcass weight). DIO rat retroperitoneal white adipose pads had 65% more cells that were the same size as those in chow-fed pads; DR rat cells were similar to controls. Both DR and DIO rats increased norepinephrine turnover in their interscapular brown adipose pads by greater than 90%. DIO rats also had 40% lower pancreatic turnover; their plasma insulin levels were 327% of controls after 15 wk on the CM diet and 188% after 7 wk on chow. DR levels were the same as controls at both times. Therefore, regulation of caloric intake, pancreatic sympathetic tone, and plasma insulin levels were three important differences between rats that resisted and those that developed DIO on high-energy diets.

Mersalyl, 5,5'-dithio-bis(2-nitrobenzoate) (Nbs2) and fluorescent Thiolyte DB react with SH groups in the H+ channel (SHc) of the uncoupling protein of brown adipose tissue mitochondria, as inferred from their inhibition of H+ transport. Cl- transport by the uncoupling protein was unaffected. Using these modifiers and N-ethylmaleimide (MalNEt), distinct SH groups (SHB) in the purine nucleotide binding site were identified. Nbs2 reacts more readily with the SHB than with the SHc groups, but mersalyl and Thiolyte DB are more reactive with the SHc groups. MalNEt reacts exclusively with the SHB. GDP inhibition is fully prevented after sufficient modification of the SHB. Pretreatment with p-diazobenzenesulfonate (N2PhSO2) suppresses only 20-25% of fluorescence of Thiolyte-DB-labeled uncoupling protein on SDS/PAGE gels, while MalNEt suppresses 66% and Nbs2 80-90%. Since N2PhSO2 also affects the GDP binding site, these results demonstrate that the N2PhSO2-reactive residue is not identical with the SHB.

The occurrence of neuropeptides in rat brown adipose tissue has been investigated. Immunohistochemical studies on interscapular and perirenal brown fat have demonstrated unequivocally the presence of substance-P (SP)-like, neuropeptide-Y (NPY)-like and calcitonin gene related-peptide (CGRP)-like immunoreactive elements (putative nerves) in adventitial distribution on inter- and intralobular supply arteries and in accompanying nerve bundles. At a more peripheral level, some NPY-like immunoreactive elements and a greater number of CGRP-like immunoreactive elements were observed in the parenchymal field. Somatostatin, bombesin, neurotensin, enkephalin, and vasoactive-intestinal-polypeptide immunoreactivities were not detected. No differences in neuropeptide distribution were noted between interscapular and perirenal brown fat. There is a degree of coincident distribution of SP, NPY and CGRP with that of noradrenergic nervous elements as visualized by condensation histochemistry. Since after 6-hydroxydopamine treatment not all the nerve terminals in rat brown adipose tissue are stigmatized (earlier report), the present results have been discussed in the light of a possible pluralism in innervation of brown adipose tissue.

A solid-phase radioimmunoassay is described for the estimation of the uncoupling protein content of human brown adipose tissue mitochondria, as an index of thermogenic capacity. The concentration of inner mitochondrial membrane uncoupling protein was measured in brown adipose tissue samples from 48 individuals who died suddenly. The uncoupling protein content of axillary adipose tissue was greater than that of perirenal adipose tissue. Variations in brown adipose tissue uncoupling protein content, which would be consistent with changing thermogenic requirements and capacity, were observed in different groups of subjects. Significantly lower concentrations were found in adults and in pre-term and stillborn infants than in older infants and children.

The interscapular brown adipose tissue (IBAT) of cold-acclimated white rats was partially denervated by cutting in most cases four of the five intercostal nerves going to one of its two bilaterally located, lobular pads. After 24 h the functional ability and the degree of residual sympathetic innervation of the pads and of their lobes were assessed (in separate groups of rats): the former by measuring tissue blood flow as an index of sympathetically mediated calorigenesis during exposure of the animals to -6 degrees C, the latter by measuring the residual noradrenaline (NA) content of the tissue. Only the four anterior intercostal nerves contributed significantly to the sympathetic innervation of IBAT pads. On average their contributions were unequal, but there was considerable variation between rats in the relative contribution of each nerve. The measurements on lobes of the pads indicated that the sympathetic fibers supplied by a given intercostal nerve were not distributed uniformly throughout the pad. For example, measurements of NA indicated that on average the fibers of the fourth nerve were four times as abundant in the lateral lobe as in the posterior lobe. But at this level of analysis also there was wide variation between rats. The variation in the participation of each intercostal nerve in the sympathetic innervation of rat IBAT and in the distribution of each nerve's fibers to different parts of the IBAT pad could be of genetic origin or it could reflect a considerable degree of flexibility during development of the innervation of IBAT. Because of the observation variation, it is suggested that if functional responses of IBAT evoked by electrical stimulation of intercostal nerves are to be recorded from a highly localized area of tissue, stimulation of all the nerves probably would be required to obtain responses that are consistent between animals.

The aim of this work was to test the role of pituitary dependent hormones in cold-induced non-shivering thermogenesis. In the 28 degrees C-acclimated rat, hypophysectomy inhibited body growth and led to an atrophy of thyroid and adrenals. In brown adipose tissue (BAT) some alterations were induced which are usually observed after cold acclimation of the animal: increase in relative weight, decreases in the relative amount of lipids, increases in the amounts of protein and DNA and modification of the proportions of several phospholipid fatty acids; moreover, basal lipolysis, in vitro, was enhanced to the same extent as that following cold acclimation of the normal rat. The in vivo stimulation by norepinephrine (NE) of O2 consumption (test for nonshivering thermogenesis) and of fatty acid release into blood were suppressed. Progressive cold acclimation of the hypophysectomized rats at 15 degrees C led to a hypertrophy of BAT to the same extent as in the sham-operated animals. The in vivo sensitivity to NE was partially restored. The results suggest that hypophysectomy does not suppress the ability to acclimate to moderate cold by means of BAT dependent non-shivering thermogenesis. However, the low ability to produce heat seems to indicate that pituitary or pituitary-dependent hormones are necessary to optimize the cold stimulation of brown fat thermogenesis.

Ingested metabolizable energy was partitioned into 3 components--storage, cost of storage, and maintenance--to study energy utilization during 20 days of refeeding following a 3-day fast in adult male Wistar rats. During the refeeding period, one experimental group was refed ad libitum, whereas two others were restricted to approximately 75 and 50%, respectively, of prefast food intake. Energy utilization in the experimental groups was compared with that in nonfasted controls. Energy conservation occurred in all experimental groups during refeeding, and the primary form of that conservation was a decrease in the energy required for maintenance. The decreased requirement for maintenance allowed a greater proportion of ingested energy to be used for restoration of carcass energy. The degree of energy conservation was, in general, proportional to the degree of food restriction during refeeding.

Rats previously undernourished (PU) during the first 3 postnatal weeks gained less weight than controls during the subsequent rehabilitation period, in spite of similar levels of food intake per body weight 0.75. Resting oxygen consumption (ml/min/kg0.75) was significantly elevated (15-22%) in the PU group after 36 days of age but this difference was almost completely abolished by beta-adrenergic blockade with propranolol. PU rats also showed a greater thermogenic activity after noradrenaline (25 micrograms/100 g b.wt.) than controls. The mass of brown adipose tissue (BAT, mg/100 g b.wt.) was reduced by undernutrition but during rehabilitation BAT increased in PU rats to a value greater than that of controls. These results indicate that the low weight gain of PU rats may result partly from a high metabolic rate due to sympathetic activation of BAT.

Sympathoadrenal activity was assessed in adult rats with obesity-producing hypothalamic knife cuts prior to and after the onset of gross obesity by measuring urinary excretion of norepinephrine and epinephrine and by determining rates of norepinephrine turnover in selected organs. Urinary excretion of norepinephrine, as an index of overall sympathetic nervous system activity, was approximately doubled throughout the 4-week study in knife-cut rats, as was intake of the high-fat diet. Three days after knife-cut surgery (before the onset of gross obesity) rates of norepinephrine turnover (ng X organ-1 X hr-1) were 23-33% lower in three of the four organs examined than in the corresponding organs of control rats; rates of norepinephrine turnover were depressed in pancreas, interscapular brown adipose tissue, and abdominal white adipose tissue and unchanged in hearts. Four weeks after surgery when gross obesity was evident, rates of norepinephrine turnover were accelerated in heart (+82%) and pancreas (+63%), but remained low in interscapular brown adipose tissue (-27%) and abdominal white adipose tissue (-28%). Adrenal medullary activity, assessed by urinary excretion of epinephrine, was suppressed within the 1st day after knife-cut surgery and remained suppressed for several weeks. Brown adipose tissue and white adipose tissue appear to be selectively excluded from the generalized activation of the sympathetic nervous system in adult hyperphagic rats with obesity-producing hypothalamic knife cuts. Activation of the sympathetic nervous system was associated with reciprocal suppression of adrenal medullary responses in knife-cut rats.

The usefulness of imidazole-buffered osmium tetroxide as a stain for lipids in transmission electron microscopy has been investigated. Rat liver and other tissues were fixed by perfusion with glutaraldehyde and post-fixed with osmium-imidazole and the appearance of lipid droplets was compared with that after post-fixation in unbuffered aqueous osmium tetroxide or an osmium solution buffered otherwise. Prominent electron-opaque staining of lipid droplets and of lipoprotein particles was noted after post-fixation with 2% osmium-imidazole, pH 7.5, for 30 min. The lipid droplets appeared well circumscribed with no evidence of diffusion. In contrast, the intensity of staining was much less and there was some diffusion around lipid droplets in material post-fixed in aqueous or cacodylate-buffered osmium tetroxide. Spot tests on filter paper revealed that unsaturated fatty acids, especially linolenic and linoleic acids reacted more intensely with osmium-imidazole than with aqueous osmium tetroxide. These findings demonstrate that osmium-imidazole provides an excellent stain for lipids in transmission electron microscopy and that most probably it stains lipids with unsaturated fatty acids.

Weanling male Sprague-Dawley rats received bilateral electrolytic lesions in the ventromedial hypothalamic nuclei (VMNL rats); sham-operated rats served as controls. For 28 post-operative d all animals were fed three equicaloric [16.9 kJ/g (4.03 kcal)] diets with different amounts of macronutrients in each (HCD = high-carbohydrate diet, HFD = high-fat diet and HPD = high-protein diet). VMNL rats selected more than controls from HCD (P less than 0.05) and HFD (P less than 0.01) but similar amounts from the HPD. The total intake from all three diets was greater (P less than 0.001) than that of the controls, but in percent of total diet intakes, VMNL selected proportions comparable to the controls. In terms of macronutient intake, VMNL rats ingested more than controls from each (carbohydrate, P less than 0.001; fat, P less than 0.05; protein, P less than 0.01). Again, in percent of total macronutrient intake, they ingested proportions comparable to the controls. Lee Index (P less than 0.001) was greater and body weight gain/kJ (kcal) smaller (P less than 0.001) in VMNL rats than in controls. However, body weight gains were normal. For the following 14 d, one group of VMNL rats and one control group continued to self-select from the three diets while another VMNL and control group received lab chow [14.2 kJ/g (3.39 kcal)]. Analysis of variance showed a lesion effect for the Lee Index (P less than 0.001) and Lee Index gain /kJ(kcal) (P less than 0.01) but body weight gains and caloric intake were normal among the groups, ie the VMNL rats switched to chow decreased their caloric intake to control levels. On sacrifice, white and brown fat percent protein (P less than 0.001 for both), carcass lipid (P less than 0.001) and protein (P less than 0.01) and plasma insulin (P less than 0.001) showed lesion effects, but there were no differences among the groups in plasma glucose, glycerol, total protein and free-fatty acids. Availability of palatable diets immediately following VMN lesion placement in weanling rats will result in hyperphagia that after one month recedes to normophagia, whether the rats are fed palatable or less palatable diets. Availability of a less palatable diet (chow) following presentation of palatable diets will not result in diminished caloric intake, body weight, obesity and hyperinsulinemia.

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.

Significance of the intercapsular brown adipose tissue (IBAT) in the thermal response to chlorpromazine (CPZ) during acute and chronic cold exposure was studied in rats. Adult male Wistar-Imamichi rats were exposed to 4 degrees C for 1 (cold-exposed) or 30 days (cold-acclimated). Intraperitoneal injection of 10 mg/kg CPZ caused a more marked and lasted hypothermia in acute cold-exposed rats as compared with that seen in control or in cold-acclimatized rats. Hypothermia induced by CPZ was unaffected by removal of IBAT either in control or acute cold-exposed rats. But in cold-acclimated rats, the removal of IBAT potentiated the hypothermia by CPZ. The relative weight of IBAT in cold-acclimated rats was about 3 times heavier than that in control and acute cold-exposed rats. CPZ had no effect on the relative weight of IBAT in all groups examined. Total lipids in IBAT showed no significant changes following CPZ administration in rats of all groups. In acute cold-exposed rats, serum FFA level progressively decreased after CPZ injection. The noradrenaline concentration in IBAT increased after chronic cold exposure and CPZ suppressed this elevation. The results suggest that the thermogenesis related sympathetic activity in the IBAT plays a mediatory role in thermal response to CPZ during cold acclimation.

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.

We studied body composition, white and brown adipose tissue cellularity, lipoprotein lipase activity and metabolic enzyme activity in three groups of rats: nonpregnant controls, lactating dams and nonlactating dams (i.e., dams not permitted to suckle their young). Nine to 11 rats in each group were killed on d 12 postpartum (study d 34) and on d 40 postpartum (study d 62). During lactation, brown fat citrate synthase, beta-hydroxyacylCoA dehydrogenase (HOAD) and lipoprotein lipase activities were significantly lower in the lactating than in the nonlactating dams or virgin controls. Although the nonlactating dams had their pups removed within 24 h after delivery, by d 12 postpartum citrate synthase and HOAD activities were significantly lower than those of nonpregnant controls. By the end of the study there were no differences among the three groups except in the case of HOAD. HOAD activity in the lactation group was significantly lower than in the nonlactation group. White fat cell number in the parametrial depot was significantly increased in the nonlactation groups by d 12 postpartum. In the lactation group, increased white fat cell number in this depot was detectable at d 40 postpartum, when carcass fat stores and fat cells had been repleted. These results demonstrate that both lactating and nonlactating dams undergo cellular hyperplasia, at least in the parametrial depot. This may predispose them to obesity. Also, decreased brown fat metabolism in the nonlactating dams may be contributing to their significantly greater carcass fat content at d 12 postpartum.

Guanosine diphosphate binding to the uncoupling protein of isolated mitochondria of brown adipose tissue in newborn rabbits was measured as an index of thermogenic activity. The binding was 0.281 +/- 0.022 nmol GDP/mg mitochondrial protein at 1 day of age, 0.214 +/- 0.017 at 3 days, 0.428 +/- 0.038 at 5 days, and 0.208 +/- 0.016 at 7 days. The increase in binding between 3 and 7 days of age suggests that the brown fat has an increased thermogenic capacity at that age. In addition, the potential for synthesis of the uncoupling protein was investigated in 1- to 5-day-old newborn rabbits by probing the total cellular ribonucleic acid for the messenger that codes for uncoupling protein. The amount of uncoupling protein messenger was highest at 1 day of age and declined at least until 5 days of age. Because the amount of uncoupling protein messenger decreased as the GDP binding increased, the results suggest that either the initially translated uncoupling protein was unmasked at about 5 days of age or there was a delay in the incorporation of uncoupling protein into the mitochondrial inner membrane, or both.

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.

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.

Binding of (-)-[3H]dihydroalprenolol [( 3H]DHA) to isolated brown adipose tissue (BAT) microsomal membranes was used to estimate beta-adrenoreceptor density, and this was found to be saturable, reversible, and stereospecific. Scatchard analysis indicated a single class of binding sites of equilibrium dissociation constant 2.2 nM, with a maximum number of binding sites of 160-170 fmol/mg protein. These values were unaffected by the age or sex of the animals, and similar Kd values were obtained for binding to heart and lung membranes. The kinetically derived Kd from forward and reverse reactions was 1.4 nM for BAT. Hofstee analysis of displacement of [3H]DHA produced linear plots for propranolol but curvilinear plots for atenolol and ICI 118,551. Atenolol showed 50-fold selectivity for beta 1-receptors and ICI 118,551 50-fold selectivity for beta 2-receptors in heart, lung, and BAT membranes. These plots indicated beta 1:beta 2-receptor ratios of 59:41 for BAT and heart and 25:75 for lung. It is possible that both beta-adrenoreceptor subtypes in BAT may be involved in the activation of thermogenesis in the tissue.

The modifications in weight and composition (lipids, proteins, water) of rat interscapular brown adipose tissue (BAT) were studied along the six first weeks of cold exposure and acclimatization. The variations of noreponephrine content was also investigated. During the first day of cold exposure, the major part of tissue lipids was released. During the following two days there was a fall in lipid and norepinehprine contents and uptake of water. Then, until the end of the first week a rapid repletion occurred. At that moment the relative pass of the tissue and the amounts of its principal components reached values which are not changed during the following weeks. We can conclude that the adaptative changes in the levels of BAT essential components are carried out at the end of the first week of cold exposure, long time before the non shivering thermogenesis is entirely effective.

The apparent size of the monomeric form of uncoupling protein from brown adipose tissue of several mammalian species was compared by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. Including earlier results the apparent molecular mass of the protein from rat was about 32 000 daltons and varied about 1500-2000 daltons from the different sources with the size increasing in the order human less than rat less than or equal to mouse less than hamster less than or equal to rabbit. The size of newly synthesized uncoupling protein was also found to vary among species. However in the two cases examined, rat and rabbit, the precursor and its respective mature monomeric protein had the same apparent size as shown by coelectrophoresis.

Dark-gold sections of osmium tetroxide-fixed, Epon-embedded brown adipose tissue before and after low-temperature oxygen plasma microincineration were examined using a high-resolution scanning transmission electron microscope and an energy-dispersive X-ray spectrometer. Microincineration produced ash patterns which were free of organic matrix, chlorine (from the Epon) and probably osmium (from the fixative). X-ray sensitivity was improved by a factor of 2-4 owing to decreased background, and sulphur, calcium and probably phosphorus were detected in the ash. Fidelity of the ash patterns permitted microanalytical spatial resolution of 0.1 micrometer or better. Oxygen plasma microincineration is thus shown to offer advantages for high resolution X-ray microanalysis of conventionally sectioned biological material. Its future application to shock-frozen, frozen-dried, unstained sections is indicated.

This report describes the novel development of an animal model for neonatal carnitine deficiency using the artificially fed newborn rabbit. Each litter was separated from the mother following the first colostrum feeding and divided into 2 groups, one of which was fed a purified rabbit formula that was essentially free of carnitine; the other received the same formula supplemented with L-carnitine (100 mg/l). At 9-13 d of age, rabbit pups receiving the carnitine-free formula had lower concentrations of total, free and acylcarnitine in plasma and urine, as well as lower total acid soluble carnitine concentrations in liver, muscle, heart and brown adipose tissue than those receiving the same formula supplemented with L-carnitine. Their plasma and tissue levels were also lower, but their urinary carnitine concentrations were higher than those in naturally-raised pups. The findings suggest that the described animal model may prove to be a useful tool for the investigation of certain aspects of neonatal carnitine deficiency.

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.

We investigated whether intermittent cold acclimation and low intensity exercise training, two interventions known to improve thermoregulatory function in young rodents, enhanced cold tolerance in aged C57BL/6J mice. Mice, aged 18 to 20 months, were randomly assigned to four treatment conditions: (a) intermittent cold acclimation (CA) (50 min per day, 5 times per week for 3 weeks at 5 degrees C), (b) submaximal treadmill exercise (EX) (15 m min-1 for 30 min, 5 times per week for 8 weeks, (c) sequential treatment of CA + EX, and (d) control group. Mice were exposed to a 3-hr cold stress test (15 degrees C) prior to and following treatment. CA aged mice maintained normothermia and demonstrated significant increases in oxygen consumption and brown adipose tissue protein concentration compared with controls; enhanced cold tolerance was probably due to increased utilization of nonshivering thermogenesis. EX aged mice had elevated O2 consumption and increased skeletal muscle enzyme activity compared with controls; however, cold tolerance was not enhanced compared with CA mice. The data suggest that intermittent CA effectively reduces hypothermia during cold challenge in aged mice.

Clusters of dense particles were observed irregularly in mitochondrial profiles of shock-frozen brown adipose tissue and liver when cryosectioned at a chamber temperature of -80 degrees C and not if sectioning was performed at -110 degrees C. The particles are rich in Ca and P (Ca/P =0.5); they are therefore not any form of tricalcium orthophosphate (e.g. hydroxyapatite). They apparently form by electrolyte diffusion in the section and therefore cannot be considered as representing the in vivo pool of mitochondrial calcium.

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)

Because brown adipose tissue lipids are the preferred substrate for thermogenesis during arousal from hibernation, the fatty acid composition of brown fat lipids was followed during cold acclimation and during a hibernation bout. In control golden hamsters (living at 22 degrees C), the fatty acid composition of the white adipose tissue closely resembled that of the food, but brown adipose tissue contained more animal-derived fatty acids. As an effect of acclimation to cold, the fatty acid composition of brown adipose tissue changed to resemble that of the food, and no marked differences between white and brown adipose tissue were then evident. During a hibernation bout, a major part of the fatty acids accumulated in brown fat during entry into hibernation consisted of "rare" acids, such as homo-gamma-linoleic acid. Homo-gamma-linoleic, together with eicosadienoic acid and lignoceric acid, was preferentially utilized during the early phase of arousal. During this phase, "bulk" fatty acids, such as linoleic acid, were spared, whereas in late arousal, linoleic acid was the preferred substrate. It was concluded that rare fatty acids are of quantitative significance in brown adipose tissue during hibernation and arousal.

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.

Hypocaloric diet (control diet diluted 1:1 with cellulose) feeding during pregnancy caused a reduction in the body weight of rat pups at birth whereas the main parameters of brown adipose tissue composition and thermogenic activity were unaffected. When prenatally underfed rat pups were nursed by untreated dams eating control diet during lactation, early (day 4 of life) and late (day 13 of life) neonatal body weight was rehabilitated and brown adipose tissue remained essentially unchanged. When prenatally underfed rat pups were nursed by dams that were fed with the hypocaloric diet during lactation, neonatal body weight continued lower at the two mentioned days of life and the overall thermogenic capacity of brown fat (GDP binding/g body weight) was substantially depressed. The reduction of the brown fat thermogenic capacity in these pups is mainly due to a hypotrophy of the mitochondrial component of the tissue as indicated by the lowered cytochrome oxidase activity. Results indicate that mild maternal underfeeding during lactation may depress brown adipose tissue thermogenic function in neonates whereas similar hypocaloric intakes during pregnancy did not alter brown fat thermogenic capacity.

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.

A photodetection device permitting rapid and direct photographic recording of light emission from luminol-based immunodot and Western blotting assays is described in detail. This device permits contact exposure on Polaroid film of luminescent samples on nitrocellulose strips, and thus obviates the need for darkroom and film processing facilities. Immunodetection of mouse alpha-fetoprotein in brown fat homogenate and immunoglobulin E in nonimmune human sera employing luminol-based Western blotting and immunodot assays were used to demonstrate the versatility and operational simplicity of the detection device. The use of the detection device in combination with luminol-based immunoassays resulted in greater signal intensities and increased sensitivity over that attainable with the commonly used chromogenic substrate, 4-chloro-1-naphthol.

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.

A mutation in the BALB/cHeA subline of mice has resulted in loss of the glycerol-3-phosphate dehydrogenase (glycerol-P dehydrogenase) activity in adult tissues. Analysis of F2 offspring segregating for enzyme activity levels and a PvuII restriction fragment length polymorphism in the Gdc-1 gene on Chromosome 15 indicates that the mutation has occurred in or around the structural gene. Southern blot analysis failed to detect any major change due to chromosomal insertion, deletion, or rearrangement of the mutant gene. In vitro measurements of transcription in isolated nuclei failed to detect any difference in transcription between the BALB/cHeA and BALB/cBy strains of mice. However, Northern blot analysis with poly(A+) RNA isolated from brown fat shows that the mutant has about a 50-fold reduction in hybridizable transcripts of altered size. The data suggest that the mutation affected the processing of nuclear transcripts of glycerol-P dehydrogenase. The morphological, physiological, and reproductive characteristics of the mutant mice appear normal. This suggests that alternate pathways of energy metabolism and lipid synthesis exist which obviate the function of glycerol-P dehydrogenase.

A glyoxylic acid method using cryostat sections to demonstrate catecholaminergic fibres of the central nervous system was modified to show the extent of the adrenergic innervation in rat brown adipose tissue. It revealed prominent interlacing fluorescent parenchymal fibres surrounding individual adipocytes. The density of this network of fine fibres was not evident using earlier techniques. The new method also confirmed the dense networks of adrenergic fibres associated with arterial vessels. Its specificity was verified by simultaneously performing radioenzymatic determinations of tissue catecholamine levels and histochemical studies of brown adipose tissue from normal and sympathectomized rats. Chemical sympathectomy with 6-hydroxydopamine resulted in a pronounced decrease in brown adipose tissue and heart catecholamine (noradrenalin and dopamine) levels. Significantly, in brown adipose tissue of sympathectomized animals no fluorescence could be detected in terminal nerves of either the parenchyma or those of vascular smooth muscles. Nevertheless, some intense fluorescence was seen in axon bundles. The findings suggest that catecholamines of the parenchymal innervation form a larger proportion of the total catecholamine content of brown adipose tissue than was previously believed, provide stronger support for direct control of the function of multilocular adipocytes, and also confirm unpublished data reporting considerable dopamine content in brown adipose tissue.

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.

1. Studies on human brown adipose tissue require specific molecular probes. A human genomic library has been screened with a complementary DNA corresponding to the uncoupling protein (UCP) of rat brown adipose tissue mitochondria. 2. Two recombinant phages were isolated, carrying genomic sequences of human UCP. From them we have subcloned a 0.5 kilobase fragment. This fragment, H-Ucp-0.5, contained two intronic regions and two exonic regions. Exonic regions encoded a sequence of 84 amino acids which exhibited a strong homology with central domain at rat UCP. The organization of H-Ucp-0.5 was confirmed by SI mapping analysis. 3. A Southern analysis suggested that the gene is single type in the human, as it is in rodents. 4. In Northern analysis experiments, H-Ucp-0.5 detected a specific 1.8 kb mRNA in human brown adipose tissue obtained from six patients with phaeochromocytoma and from one patient with a hibernoma. This molecular probe is a new, sensitive and reliable tool with which to study human brown adipocytes.

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)