Norepinephrine turnover in brown adipose tissue of young rats: effects of rearing temperature.

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)

Increasing age impairs the thyroid hormone response to overfeeding.

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.

Infrared spectroscopic studies of detergent-solubilized uncoupling protein from brown-adipose-tissue mitochondria.

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.

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).

Brown adipose tissue thermogenesis above the lower critical temperature.

Heat-acclimated rats show lighter IBAT deposit with different gross composition and lower GDP-binding than controls at thermoneutrality. A thermal disactivation of the tissue is then inferred. Cafeteria regime increased IBAT mass and GDP-binding when offered to rats at a thermoneutral ambient temperature. These results indicate that BAT thermogenesis at thermoneutrality is not the lowest one of the tissue and that diet-induced thermogenesis can take place even at thermoneutrality.

Maternal brown fat metabolism returns to control level by four weeks postweaning in rats.

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.

Sulfhydryl groups of the uncoupling protein of brown adipose tissue mitochondria. Distinction between sulfhydryl groups of the H+ channel and the nucleotide binding site.

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.

A glycerol-3-phosphate dehydrogenase null mutant in BALB/cHeA mice.

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.

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.

Uncoupling protein and its mRNA in brown adipose tissue of newborn rabbits.

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.

Rare fatty acids in brown fat are substrates for thermogenesis during arousal from hibernation.

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.

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.

Brown adipose tissue in lean and fat selection lines of sheep identified by immunodetection of uncoupling protein in western blots of tissue homogenates.

1. Uncoupling protein (UCP) was purified from perirenal adipose tissue of 2-day-old lambs by a procedure involving Triton solubilization and hydroxyapatite treatment. It has an apparent Mr of 34,000. 2. Rabbit anti-sheep UCP and rabbit anti-rat UCP each cross-reacted with both rat and sheep UCP in Western blots, indicating that the major antigenic determinants of the sheep UCP and rat UCP are similar. 3. In Western blots, the anti-sheep UCP showed tissue specificity by detecting a band corresponding to UCP only in brown adipose tissue, but not in heart or liver homogenates. 4. The Western blotting procedure was used to analyse sheep tissues. UCP was detected in samples of perirenal, omental, back and lymph node fat from 2-day-old lambs, but not in heart, liver, muscle or kidney samples. 5. UCP was not detected in any tissue samples from 34-day-old or 7-month-old lambs. 6. Comparison of the amount of UCP in perirenal fat of 2-day-old lambs from lean, fat and control selection lines, using the Western blotting procedure, showed no apparent difference.

The carnitine-deprived newborn rabbit: a potential model to study carnitine deficiency.

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.

Detection of brown adipose tissue uncoupling protein mRNA in adult patients by a human genomic probe.

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.

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.

Neuropeptides in interscapular and perirenal brown adipose tissue in the rat: a plurality of innervation.

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.

Biochemical characterization of brown adipose tissue beta-adrenergic receptor.

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.