Leptin immunoexpression and innervation in rat interscapular brown adipose tissue of cold-acclimated rats: the effects of L-arginine and L-NAME

The aim of the present study was to explore the effect of nitric oxide on leptin immunoexpression and innervation in interscapular brown adipose tissue (IBAT) of room- and cold- acclimated rats. Animals acclimated both to room-temperature (22 +/- 1 degrees C) and cold (4 +/- 1 degrees C) were treated with L-arginine, a substrate for nitric oxide synthases (NOSs), or N?-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOSs, for 45 days. Leptin expression and localization in brown adipocytes was studied by immunohistochemistry, and innervation stained by the Bodian method. Strong leptin immunopositivity was observed in brown adipocytes cytoplasm of all room-acclimated groups, but nuclear leptin positivity was found only in L-NAME treated rats. In cold-acclimated control and L-NAME treated rats leptin immunopositivity was absent, while L-arginine treatment reversed the cold-induced suppression of leptin expression. Comparing to control, L-arginine, and even more L-NAME, at 22 +/- 1 degrees C induced greater innervation. In conclusion, L-arginine treatment changes leptin expression pattern on cold in rat IBAT.     

Effect of temperature on lipoprotein lipase and lipogenic enzyme activities in brown adipose tissue of hypophysectomized rats

It has been shown that the same modifications on the composition of brown adipose tissue (BAT) which are normally induced following cold stimulation are also observed in hypophysectomized rats acclimated either at 28 degrees C or 15 degrees C. To test the possibility of BAT stimulation in hypophysectomized rats, we have determined some enzymatic activities known to modulate the energy supply to that organ. Seven week old Long-Evans rats were hypophysectomized. Three weeks later, they were exposed to either 28 degrees C or 15 degrees C ambient temperature for five or six weeks. Hypophysectomized rats were compared to age matched or weight matched controls. Total lipoprotein lipase activity (LPL) (triglyceride uptake) was enhanced in BAT of 28 degrees C hypophysectomized rats compared to controls. Cold acclimation led to a large increased activity. Total LPL activity was comparable in BAT of hypophysectomized and control rats. Total malic enzyme and glucose-6-phosphate dehydrogenase activities (in situ lipogenesis) were doubled in BAT of 28 degrees C hypophysectomized compared to controls. A large enhancement was observed in BAT of either 15 degrees C control or 15 degrees C hypophysectomized rats. Among the studied organs (liver, white adipose tissue, heart, BAT) hypophysectomy promotes the three enzyme activities only in BAT. These variations were discussed with relation to the effect of hypophysectomy on brown adipose tissue at 15 degrees C and 28 degrees C.     

Differential response of rat brown and white adipose tissue to environmental or nutritional stress.

1. In vivo fatty acid synthesis by brown adipose tissue was enhanced in rats exposed to cold (5 degrees C) or altitude (4300 m) for 7 days but was unaltered in rats exposed to heat (35 degrees C) for an equivalent period. In vivo fatty acid synthesis by white adipose tissue was depressed by cold exposure while altitude and heat exposure had no effect. 2. In vitro, CO2 production and lipid synthesis were elevated in brown adipose tissue from rats fasted for 4 days. Refeeding (4 days) such rats reversed these effects, leading to depressed values relative to those of control rats. In contrast, these metabolic events in white adipose tissue were decreased by fasting and increased compared to controls during subsequent refeeding.     

Interaction between exercise training and cold acclimation in rats

Five groups of 10 rats were used. Group A included sedentary rats kept at 24 degrees C, group B exercised-trained rats and group C rats exposed at -15 degrees C for 2 h every day and kept at 24 degrees C for the remaining time. These 3 groups were kept on this regimen for 10 weeks. In addition group D was acclimated to cold (2 h.d-1 at -15 degrees C) for 6 weeks and subsequently deacclimated at 24 degrees C for 4 weeks. Group E was also acclimated to cold for 6 weeks and during the deacclimation, at 24 degrees C period which lasted 4 weeks, the animals were exercised 2 h per day. Following the 10 week experimental period all animals were sacrificed and DNA and protein content of the IBAT as well as its total mass were measured. The results show significant increases in the cold adapted group. Exercise training which had no effect on brown adipose tissue IBAT at room temperature, caused an accelerated reduction in weight, DNA and protein content of the BAT in rats previously acclimated to cold. In spite of this, the thermogenic response to noradrenaline was significantly enhanced in the group which exercised during the deacclimation period. It is suggested that tissues other than IBAT may explain this enhanced heat production capacity.     

Effect of unilateral surgical denervation of brown adipose tissue on uncoupling protein mRNA level and cytochrom-c-oxidase activity in the Djungarian hamster

The bilateral lobe of interscapular brown adipose tissue of the Djungarian hamster was unilaterally denervated in order to study the role of the sympathetic innervation for maintenance and cold-induced increase of non-shivering thermogenesis. Denervation decreased the noradrenaline content of brown adipose tissue to less than 9% of the intact contralateral pad. This low noradrenaline level was maintained for 1–14 days after denervation. First, to study the role of the sympathetic innervation of brown adipose tissue in the maintenance of the high thermogenic capacity characteristic of the cold acclimated state, brown adipose tissue was denervated in hamsters either kept at thermoneutrality or acclimated to 5°C ambient temperature for 4 weeks. Cold-acclimated hamsters had elevated levels of uncoupling protein messenger ribonucleic acid (8.1-fold) and cytochrom-c oxidase-activity (3-fold). Denervation of brown adipose tissue decreased uncoupling protein-messenger ribonucleic acid level and cytochrom-c-oxidase-activity as compared to the intact pad in thermoneutral and in cold-acclimated hamsters. However, in cold-acclimated hamsters uncoupling protein-messenger ribonucleic acid level and cytochrom-c-oxidase-activity in denervated brown adipose tissue both were maintained on an elevated 6-fold higher levels as compared to thermoneutral controls. Second, to study the role of the sympathetic innervation of brown adipose tissue in the cold-induced increase in thermogenic capacity, hamsters were denervated prior to cold acclimation and responses were measured after 3 and 14 days of cold exposure. Uncoupling protein-messenger ribonucleic acid level and cytochrom-c-oxidase-activity of intact brown adipose tissue increased after 14 days cold acclimation. Denervation did not completely prevent a cold-induced 1.5-fold increase of cytochrom-c-oxidase-activity and a 3.2-fold increase of the uncoupling protein-messenger ribonucleic acid level in denervated brown adipose tissue after 14 days of cold acclimation. In conclusion, high levels of uncoupling protein-messenger ribonucleic acid and cytochrom-c-oxidase activity in brown adipose tissue of cold-acclimated hamsters can partially be maintained without intact sympathetic innervation, suggesting a considerable contribution of trophic factors not requiring sympathetic innervation for maintenance. The cold-induced increase of cytochrom-c-oxidase activity and expression of uncoupling protein-messenger ribonucleic acid largely depends upon sympathetic innervation of brown adipose tissue.Abbreviations ANOVA analysis of variance - BAT brown adipose tissue - COX cytochrom-c-oxidase - HPLC high performance liquid chromatography - mRNA messenger ribonucleie acid - NA noradrenaline - T _a ambient temperature - UCP uncoupling protein     

Effects of exposure temperature on brown adipose tissue uncoupling protein mRNA levels

The effect of temperature on the amount of uncoupling protein mRNA in rat brown adipose tissue was examined after 1 and 14 days of exposure to cold. The relative amounts after 1 day, compared with rats kept at a thermoneutral temperature of 28 degrees C, were 3.2 at 19 degrees C, 3.3 at 11 degrees C, and 2.1 at 3 degrees C. This suggests that in warm-acclimated rats, a maximal response to a cold stimulus in brown adipose tissue is reached by 19 degrees C. In contrast to these results, the relative amounts of uncoupling protein mRNA after 14 days of cold exposure, compared with rats left at 28 degrees C, were 1.2 at 19 degrees C, 1.9 at 11 degrees C, and 2.1 at 3 degrees C. Since it is known that the amount of uncoupling protein in cold-acclimated rats increases continuously with decrease in temperature, the amount of protein reflects the mRNA levels during later times but not the initial time of exposure to cold.     

Evidence from immunoblotting studies on uncoupling protein that brown adipose tissue is not present in the domestic pig

Adipose tissues and other tissues of the pig have been examined for the presence of the mitochondrial "uncoupling protein," characteristic of brown adipose tissue, in order to assess whether brown fat is present in this species. Mitochondria were prepared from various tissues and the proteins separated on the basis of molecular weight by sodium dodecyl sulphate--polyacrylamide gel electrophoresis. Immunoblotting procedures were then used to probe for uncoupling protein, employing a rabbit anti-(rat uncoupling protein) serum. Pigs were examined at 4 days, 4 weeks, and 8 weeks of age. No evidence for the presence of uncoupling protein was found at any of these ages. The protein was, however, readily detected in brown adipose tissue from rats, mice, golden hamsters, guinea pigs, Richardson's ground squirrel, and lambs. An additional group of pigs was acclimated to the cold (10 degrees C) for a period of 10 days prior to the examination of tissues, but again uncoupling protein was not detected in any tissue. These results indicate that uncoupling protein is either absent from adipose tissues of the pig or is present at such a low concentration that it is unlikely to support thermogenesis. It is concluded that the pig does not contain adipose tissue that is functionally "brown;" adipose tissues in this species appear to be exclusively "white."     

Brown adipose tissue heat production in heat acclimated and perchlorate treated rats

The noradrenaline-induced energy dissipation rate was measured with a direct microcalorimeter in brown adipose tissue taken from rats acclimated to 34 degrees C (HA), perchlorate treated (PC) and heat acclimated-perchlorate treated (HAPC). The response to 10(-7) M NA was reduced by 45%, 47% and 86% in HA, PC and HAPC groups, respectively, as compared to a control group kept at 24 degrees C. In the same groups, the response to 10(-6) M NA was reduced by 34%, 7% and 64%, respectively. The specific activity of the soluble alpha-glycerophosphate dehydrogenase in brown fat from HA rats was reduced by 50%, whereas it was not altered in the PC animals. It is concluded that the sensitivity to noradrenaline of the brown adipose tissue thermogenic mechanisms is decreased in hypothyroidism, and that the acclimation temperature and the thyroid status per se each have a different influence on brown adipose tissue function.     

Glycerokinase activity and lipolysis regulation in brown adipose tissue of cold acclimated rats.

Glycerol release by brown adipocytes from constant cold adapted rats was not stimulated by norepinephrine. On the contrary, the release was stimulated in rats adapted to a nycthemeral fluctuatiing temperature from 5 degrees to 28 degrees C. Glycerokinase activity was greatly increased in brown adipose tissue by cold adptation ; there was no change in the liver. However this increased activity cannot entirely explain the lack of norepinephrine stimulation of glycerol release in the brown adipose tissue of cold adapted rats.     

Hypertension during chronic exposure to cold: comparison between Sprague-Dawley and Long-Evans strains.

It is now well established that chronic exposure of rats to cold (5-6 degrees C) induces an elevation of systolic, diastolic, and mean blood pressures and cardiac hypertrophy within 3 weeks. Since rats of the Long-Evans (LE) strain are known to be resistant to the induction of deoxycorticosterone salt induced hypertension, their cardiovascular responses to chronic exposure to cold were compared with those of rats of the Sprague-Dawley (SD) strain. The results of these studies revealed clear differences between the LE and SD strains of rats. Thus, rats of the SD strain had a significant elevation in their blood pressure; a significantly increased urinary output of norepinephrine and epinephrine; a significantly greater dipsogenic responsiveness to acute administration of angiotensin II, and significant increases in weights of the heart, kidneys, adrenals, and brown adipose tissue compared with their warm-adapted controls. All of these changes are characteristic of rats acclimated to cold. In contrast, rats of the LE strain appear to be less responsive to cold in that blood pressure failed to rise as sharply and to attain as high a level. Furthermore, urinary outputs of norepinephrine and epinephrine were significantly lower in cold-treated rats of the LE strain compared with cold-treated rats of the SD strain, but dipsogenic responsiveness to angiotensin II was unchanged. Although increases in the weight of the previously mentioned organs were also observed in cold-treated rats of the LE strain compared with their warm-adapted controls, weights of the heart and interscapular brown adipose tissue of both groups were significantly less than those of counterparts of the SD strain.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of nitric oxide in remodeling of capillary network in rat interscapular brown adipose tissue after long-term cold acclimation

Cold exposure has been shown to increase blood flow in interscapular brown adipose tissue (IBAT). The aim of the present study was to evaluate the role of the L-arginine-nitric oxide (*NO) pathway on IBAT capillary network remodeling and its possible correlation with superoxide anion radical (O2(*-)). In the rats that received L-arginine (2.25%) or NG-nitro-L-arginine methyl ester (L-NAME, 0.01%) as a drinking liquid and maintained at room (22+/-1 degrees C) or low (4+/-1 degrees C) temperature for 45 days, IBAT capillaries were analyzed by stereology and observed by light and electron microscopy. Additionally, endothelial *NO synthase (eNOS) expression, nitrotyrosine immunoreactivity and both copper zinc superoxide dismutase (CuZnSOD) enzyme activity and immunohistochemical localization were examined. Stereological analyses of IBAT show that the capillary volume density, as well as capillary-to-brown adipocytes ratio, are increased in cold. L-arginine treatment increases, while L-NAME decreases both parameters, compared to respective controls. Those changes were accompanied by capillary dilatation observed by light and electron microscopy. The activity of CuZnSOD is lower in control cold-acclimated rats, as well as in both L-arginine-treated groups, when compared to control animals acclimated to room temperature. L-NAME treatment attenuates the effects both of cold and L-arginine on CuZnSOD and increases immunopositivity for CuZnSOD in room temperature-acclimated rats. Our results show that *NO induces remodeling of the IBAT capillary network by angiogenesis, and presumably that interaction with O2(*-) has a role in that modulation. The increased eNOS expression accompanied by an increased nitrotyrosine immunoreaction observed in both L-arginine-treated groups compared to corresponding controls strengthens this hypothesis.     

Thermogenic capacity of the brown adipose tissue of developing rats; effects of rearing temperature

A chronological study was performed to investigate the postnatal development of the thermogenic capacity of the brown adipose tissue (BAT) comparing rats born and reared at 16 degrees C (cold) or 28 degrees C (control). Mitochondrial mass, cytochrome-c-oxidase activity (index of oxidative capacity) and GDP binding to mitochondria (uncoupling test) were investigated in rats from 1 to 33 days of age. Specific cytochrome-c-oxidase activity was the same in both groups during the first week, then increased in the cold group and decreased in controls; from the 9th day it was always twice as high in the former as in the latter. Specific binding of GDP to mitochondrial proteins remained almost constant in control rats during the first week contrasting with a rapid increase in that for cold rats. Afterwards it decreased in both groups until weaning but remained five times as high in cold rats as in control rats. As growth of BAT is faster and mitochondrial content greater in cold reared rats, the capacity of the tissue for thermogenesis appeared to be greatly temperature dependent soon after birth and during the entire suckling period. However the mechanisms of this stimulation remain to be elucidated.     

Thyroid-stimulating hormone receptor in brown adipose tissue is involved in the regulation of thermogenesis

C.RF- Tshr(hyt/hyt) mice have a mutated thyroid-stimulating hormone receptor (TSHR), and, without thyroid hormone supplementation, these mice develop severe hypothyroidism. When hypothyroid Tshr(hyt/hyt) mice were exposed to cold (4 degrees C), rectal temperature rapidly dropped to 23.9 +/- 0.40 degrees C at 90 min, whereas the wild-type mice temperatures were 37.0 +/- 0.15 degrees C. When we carried out functional rat TSHR gene transfer in the brown adipose tissues by plasmid injection combined with electroporation, there was no effect on the serum levels of thyroxine, although rectal temperature of the mice transfected with pcDNA3.1/Zeo-rat TSHR 90 min after cold exposure remained at 34.6 +/- 0.34 degrees C, which was significantly higher than that of Tshr(hyt/hyt) mice. Transfection of TSHR cDNA increased mRNA and protein levels of uncoupling protein-1 (UCP-1) in brown adipose tissues, and the weight ratio of brown adipose tissue to overall body weight also increased. Exogenous thyroid hormone supplementation to Tshr(hyt/hyt) mice restored rectal temperature 90 min after exposure to cold (36.8 +/- 0.10 degrees C). These results indicate that not only thyroid hormone but also thyroid-stimulating hormone (TSH)/TSHR are involved in the expression mechanism of UCP-1 in mouse brown adipose tissue. TSH stimulates thermogenesis and functions to protect a further decrease in body temperature in the hypothyroid state.     

Effect of food restriction on cold adaptability of rats

To determine the role of the nutritional state in nonshivering thermogenesis during cold adaptation, cold adaptability was compared between cold-adapted (5 degrees C for 4-5 weeks) rats fed ad libitum and cold-adapted rats pair fed with warm controls having the same food intake. Cold-adapted pair-fed rats suffered a significant loss in body weight during cold exposure. However, brown adipose tissue (BAT) in both cold-adapted ad libitum fed and cold-adapted pair-fed rats was enlarged to the same extent as compared with that in control rats. Fat-free dry matter in BAT also increased in cold-adapted ad libitum fed and cold-adapted pair-fed rats to the same extent. Cold tolerance as assessed by the change in the colonic temperature at -5 degrees C was improved relative to control rats and was the same for cold-adapted ad libitum fed and cold-adapted pair-fed rats. Nonshivering thermogenesis as estimated by the noradrenaline-induced increase in oxygen consumption was significantly greater in the cold-exposed rats and there was no significant difference between cold-adapted ad libitum fed and cold-adapted pair-fed rats. These results suggest that an improved cold tolerance by means of nonshivering thermogenesis in brown adipose tissue is closely related to the low temperature itself but not the increased food intake which occurred in the cold.     

Cold-induced alterations of phospholipid fatty acyl composition in brown adipose tissue mitochondria are independent of uncoupling protein-1

The recruitment process induced by acclimation of mammals to cold includes a marked alteration in the acyl composition of the phospholipids of mitochondria from brown adipose tissue: increases in 18:0, 18:2(n-6), and 20:4(n-6) and decreases in 16:0, 16:1, 18:1, and 22:6(n-3). A basic question is whether these alterations are caused by changes in the concentration of uncoupling protein-1 (UCP1) or the thermogenesis it mediates-implying that they are secondary effects-or whether they are an integrated, independent part of the recruitment process. This question was addressed here using wild-type and UCP1-ablated C57BL/6 mice acclimated to 24 degrees C or 4 degrees C. In wild-type mice, the phospholipid fatty acyl composition of mitochondria from brown adipose tissue showed the changes in response to cold that were expected from observations in other species and strains. The changes were specific, as different changes occurred in skeletal muscle mitochondria. In UCP1-ablated mice, cold acclimation induced acyl alterations in brown adipose tissue that were qualitatively identical and quantitatively similar to those in wild-type mice. Therefore, neither the increased content of UCP1 nor mitochondrial uncoupling altered the effect of cold on acyl composition. Cold acclimation in wild-type mice had little effect on phospholipid acyl composition in muscle mitochondria, but cold-acclimation in UCP1-ablated mice caused significant alterations, probably due to sustained shivering. Thus, the alterations in brown adipose tissue phospholipid acyl composition are revealed to be an independent part of the recruitment process, and their functional significance for thermogenesis should be elucidated.     

Mitochondrial respiration in muscle and liver from cold-acclimated hypothyroid rats.

The effects of long-term cold exposure on muscle and liver mitochondrial oxygen consumption in hypothyroid and normal rats were examined. Thyroid ablation was performed after 8-wk acclimation to 4 degrees C. Hypothyroid and normal controls remained in the cold for an additional 8 wk. At the end of 16-wk cold exposure, all hypothyroid rats were alive and normothermic and had normal body weight. At ambient temperature (24 degrees C), thyroid ablation induced a 65% fall in muscle mitochondrial oxygen consumption, which was reversed by thyroxine but not by norepinephrine administration. After cold acclimation was reached, suppression of thyroid function reduced muscle mitochondrial respiration by 30%, but the hypothyroid values remained about threefold higher than those in hypothyroid muscle in the warm. Blockade of beta- and alpha1-adrenergic receptors in both hypothyroid and normal rats produced hypothermia in vivo and a fall in muscle, liver, and brown adipose tissue mitochondria respiration in vitro. In normal rats, cold acclimation enhanced muscle respiration by 35%, in liver 18%, and in brown adipose tissue 450% over values in the warm. The results demonstrate that thyroid hormones, in the presence of norepinephrine, are major determinants of thermogenic activity in muscle and liver of cold-acclimated rats. After thyroid ablation, cold-induced nonshivering thermogenesis replaced 3,5,3'-triiodothyronine-induced thermogenesis, and normal body temperature was maintained.     

3':5'-Cyclic-AMP phosphodiesterase activities in white and brown adipose tissues of cold-acclimated rats.

3':5'-Cyclic-AMP phosphodiesterase (PDE) (EC 3.1.4.17) activity was measured in interscapular brown adipose tissue (BAT) and in white epididymal adipose tissue of rats acclimated to constant or fluctuating cold. Experiments were carried out on isolated adipocytes or tissue homogenates. In brown or white adipose tissue or isolated adipocyte homogenates, two different apparent Km values were found according to the substrate (cAMP) concentration. The low Km was at about 10(-6) M and the high one at about 10(-4) M. The apparent V of the high Km enzyme was about 10-fold higher than the V of the low Km enzyme. Cold acclimation to constant or fluctuating cold did not modify appreciably the Km or V values. For low substrate concentrations (10(-6)-10(-8) M), the specific activity of PDE expressed per milligram of protein was decreased in BAT adipocytes of the two groups of cold-acclimated rats, compared to controls. Inversely, it was increased in total tissue homogenates. These variations were smaller in fluctuating cold than in constant cold-acclimate rats. They could, in part, induce the increases in lipolysis and in blood flow observed in the BAT of cold-acclimated rats.     

Effects of cold acclimation on the activity of lipoprotein lipase in adipose tissues of genetically obese Zucker rats

The activity of lipoprotein lipase (LPL) was studied in interscapilar brown adipose tissue (BAT), epididymal white adipose tissue (WAT) and in the heart of lean and obese adult Zucker rats maintained at 22 degrees C or adapted to cold (10 degrees C). In WAT the specific activity per gram of tissue was lower in obese than in lean rats but the total activity within the tissue was three-fold higher. Cold acclimation did not modify total activity in either lean or obese rats. In BAT, but not in the heart, both specific and total activities were lower in obese than in lean animals. They were enhanced in both tissues following cold acclimation. Six-hour fasting led to a decrease in specific activity in WAT of lean rats but had no effect in obese animals; an increase was observed in BAT and heart of both genotypes. Insulin administration has no effect on activities in WAT in either 22 or 10 degrees C adapted obese rats. Norepinephrine administration stimulates LPL activity in BAT and heart of all groups. It is concluded that the lack of development of obesity previously observed in obese rats following cold acclimation is not due to a decreased capacity of lipid uptake by WAT. It might in part be due to an increased lipid oxidation in BAT.     

Brown fat thermogenesis in cold-acclimated rats is not abolished by the suppression of thyroid function

The effects of long-term cold exposure on brown adipose tissue (BAT) thermogenesis in hypothyroid rats have been examined. Thyroid ablation was performed in normal rats after 2 mo of exposure to 4 degrees C, when BAT hypertrophy and thermogenic activity were maximal. After ablation, hypothyroid and normal controls remained in the cold for 2 additional months. At the end of the 4-mo cold exposure, all untreated hypothyroid rats were alive, had normal body temperature, and had gained an average 12.8% more weight than normal controls. Long-term cold exposure of hypothyroid rats markedly increased BAT weight, mitochondrial proteins, uncoupling protein (UCP)-1, mRNA for UCP-1, and oxygen consumption to levels similar to those seen in cold-exposed normal rats. The results indicate that thyroid hormones are required for increased thermogenic capacity to occur as an adaptation to long-term cold exposure. However, cold adaptation can be maintained in the absence of thyroid hormone.     

Effect of breeding temperature on the growth and composition of brown fat in the newborn rat]

The weight and the lipid, water and norepinephrine contents of the interscapular brown adipose tissue from 1, 3, 7, 11, 14 and 21 day aged rats were measured. The animals were maintained at an ambiant temperature of 16 degrees, 23 degrees or 28 degrees C from birth. It is concluded that nonshivering thermogenesis is not necessary after 3 days of age in animals kept at 28 degrees C and after 11 days of age in the ones kept at 23 degrees C. However that thermogenesis persists for all the suckling period in those kept at 16 degrees C.