Disparate effects of fenfluramine on thermogenesis in brown adipose tissue in the rat.

It has been suggested that fenfluramine, a clinically used appetite suppressant, can also promote weight loss by augmenting energy expenditure, as indicated by increased whole-body O2 consumption (VO2) and mitochondrial GDP binding in brown adipose tissue (BAT) of fenfluramine-treated rats. To further investigate a possible involvement of BAT in the drug's metabolic effects, 113Sn-labelled microspheres were injected into the left cardiac ventricle of conscious rats 70-80 min after intraperitoneal delivery of 20 mg/kg fenfluramine (DL-mixture) or saline vehicle. At 28 degrees C ambient temperature, fenfluramine augmented resting whole-body VO2 and increased the microsphere entrapment in BAT, indicating enhanced blood flow and metabolism. At 20 degrees C ambient temperature, the expected increase in BAT blood flow associated with nonshivering thermogenesis was observed in control rats, but in fenfluramine-treated rats the increase in BAT blood flow was severely attenuated, and VO2 and body temperature were reduced. The stimulatory effect of fenfluramine on BAT metabolism was not prevented by urethane anesthesia but did not occur if the tissue was denervated. These blood flow measurements corroborate previous reports, based on GDP-binding assays, that fenfluramine treatment can augment thermogenesis in BAT by effects mediated through the innervation of the tissue. However, the data also indicate that this calorigenic effect is dependent on ambient temperature being near thermoneutrality and that in a cool environment the drug inhibits BAT thermogenesis.     

High-energy diets produce different effects on fatty acid synthesis in brown adipose tissue, white adipose tissue and liver in the rat

The influence of feeding rats a high-energy diet for 7 days on fatty acid synthesis in brown adipose tissue, white adipose tissue and liver of the rat was investigated. The incorporation of 3H2O and [U-14C]glucose into fatty acid was measured in vivo. The rats fed the high-energy diets had higher rates of fatty acid synthesis in white adipose tissue than the controls fed on chow, while fatty acid synthesis in brown adipose tissue and liver was either decreased or unchanged relative to that of controls fed on chow. After an oral load of [U-14C]glucose the incorporation of radioactivity into tissue fatty acid was several-fold higher in brown adipose tissue than in white adipose tissue in rats fed on chow. In rats fed the high-energy diets, incorporation of radioactivity into fatty acid in brown adipose tissue was decreased while that into white adipose tissue was either increased (Wistar rats) or unchanged (Lister rats).     

Studies on plasma free-fatty-acid metabolism and triglyceride synthesis of brown adipose tissue in vivo during cold-induced thermogenesis of the newborn rabbit.

Parameters of plasma free fatty acid metabolism (pool size, half time, disappearance rate, turnover time and absolute turnover rate), the influx of plasma free fatty acids into the glycerides of brown adipose tissue and the pathway of triglyceride synthesis in brown adipose tissue (glycerol-1-phosphate versus monoglyceride pathway) were examined after intravenous injection of [1-14C]palmitate in newborn rabbits. In the thermoneutral environment of 35 degrees C the turnover rate of plasma free fatty acids was 10.20 mumol/min per 100 g body weight and its flux into the glycerides of brown adipose tissue 0.367 mumol/min per 100 g body weight. Cold exposure at an ambient temperature of 20 degrees C caused a decrease to 5.84 mumol/min and 0.207 mumol/min per 100 g body weight, respectively. Both under basal conditions at an ambient temperature of 35 degrees C and under cold-induced thermogenesis at an ambient temperature of 20 degrees C triglyceride synthesis in brown adipose tissue ran through the glycerol 1-phosphate pathway.     

长爪沙鼠褐色脂肪组织和肝脏产热特征的季节性变化

长爪沙鼠（Meriones unguiculatus）是一种栖息于典型草原和荒漠草原非冬眠群居性的小型哺乳动物。为研究其产热功能的季节变化,我们分别在2003年秋季（9月下旬）、冬季（11月下旬）、2004年春季（3月底-4月下旬）和夏季（7月下旬）,分别测定了其体重、褐色脂肪组织和肝脏的重量、线粒体总蛋白含量和细胞色素c氧化酶活力,以及褐色脂肪组织中解偶联蛋白1（Uncoupling protein1,UCPl）的含量等。结果显示：除雄鼠的体重显著高于雌鼠外,其它各项指标均无性别差异。体重和褐色脂肪组织的重量都在冬季较高,显著高于夏季,而肝脏的重量在夏季显著高于其它季节。褐色脂肪组织和肝脏的线粒体蛋白含量和细胞色素c氧化酶活力以及UCP1含量,都在冬季较高,夏季较低。这些结果表明：在野外条件下,褐色脂肪组织和肝脏在细胞水平上产热能力的提高和UCP1含量的增加,是长爪沙鼠抵御寒冷的重要方式。     

Post-natal development of brown adipose tissue in the rat bred at 23 degrees C or 28 degrees C.

In view to study the effects of thermal environment on the development and the thermogenic activity of interscapular brown adipose tissue (BAT), young rats born at 23 degrees C or 28 degrees C were sacrificed at 1, 3, 7, 11, 14 or 21 days after birth. The rate of increase in animal weight was quite the same at both temperatures up to the 14th day. The development of BAT and its contents in lipids, in water and in noradrenaline indicate that the energetic activity of the tissue is greatly stimulated in rats kept at 23 degrees C up to the 11th day. It is concluded that in rats bred in the habitual thermal conditions (23 degrees C), the occurrence of non shivering thermogenesis (NST) is important during the period of ten days after birth; in the following period NST could be progressively replaced by other thermoregulatory processes.     

Lipogenesis in genetically diabetic (db/db) mice: developmental changes in brown adipose tissue, white adipose tissue and the liver

Developmental changes in lipogenesis have been examined in interscapular brown adipose tissue (BAT), epididymal white adipose tissue and the liver of genetically diabetic (db/db) mice and their normal siblings. Lipogenesis was measured in vivo with 3H2O, from weaning (21 days of age) until 20 weeks of age. Hyperinsulinaemia was evident in db/db mice at all ages. Low rates of lipogenesis were observed at weaning in tissues of both groups of mice, but the rate rose rapidly in the first few days post-weaning. In normal mice, peak lipogenesis was obtained in each tissue at 4-5 weeks of age, and there were no major changes (on a whole-tissue basis) thereafter. A different developmental pattern was apparent in db/db mice. The rate of lipogenesis in BAT rose sharply after weaning, reaching a peak at 26 days of age (several times higher than normal mice), and then falling rapidly such that by 45 days of age it was lower than in normal mice; at age 20 weeks lipogenesis in BAT of the diabetic animals was negligible. In white adipose tissue of the db/db mutants lipogenesis (per tissue) reached a maximum at 5 weeks of age, and fell substantially between 10 and 20 weeks of age. Hepatic lipogenesis in the db/db mice rose progressively from weaning until 8 weeks of age, and then decreased. Except at weaning, hepatic lipogenesis (per tissue) was much greater in db/db mice than in normal mice, and the liver was a more important site of lipogenesis in diabetic mice than in normals, accounting for up to 60% of the whole-body total. In contrast, BAT accounted for a considerably smaller proportion of whole-body lipogenesis in db/db mice than in normal mice. It is concluded that there are major developmental differences in lipogenesis between tissues of db/db mice, and between diabetic and normal animals. The data suggest that there is an early and preferential development of insulin resistance in BAT of the db/db mutant.     

Fatty acid synthesis in liver and adipose tissue of normal and genetically obese (ob/ob) mice during the 24-hour cycle.

1. The synthesis of long-chain fatty acids de novo was measured in the liver and in regions of adipose tissue in intact normal and genetically obses mice throughout the daily 24h cycle. 2. The total rate of synthesis, as measured by the rate of incorporation of 3H from 3H2O into fatty acid, was highest during the dark period, in liver and adipose tissue of lean or obese mice. 3. The rate of incorporation of 14C from [U-14C]glucose into fatty acid was also followed (in the same mice). The 14C/3H ratios were higher by a factor of 5-20 in parametrial and scapular fat than that in liver. This difference was less marked during the dark period (of maximum fatty acid synthesis). 4. In normal mice, the total rate of fatty acid synthesis in the liver was about twofold greater than that in all adipose tissue regions combined. 5. In obese mice, the rate of fatty acid synthesis was more rapid than in lean mice, in both liver and adipose tissue. Most of the extra lipogenesis occurred in adipose tissue. The extra hepatic fatty acids synthesized in obese mice were located in triglyceride rather than phospholipid. 6. In adipose tissue of normal mice, the rate of fatty acid synthesis was most rapid in the intra-abdominal areas and in brown fat. In obese mice, all regions exhibited rapid rates of fatty acid synthesis. 7. These results shed light on the relative significance of liver and adipose tissue (i.e. the adipose 'organ') in fatty acid synthesis in mice, on the mino importance of glucose in hepatic lipogenesis, and on the alterations in the rate of fatty acid synthesis in genetically obese mice.     

DNA synthesis in mouse brown adipose tissue is under beta-adrenergic control

The rate of DNA synthesis in mouse brown adipose tissue was followed with injections of [3H]thymidine. Cold exposure led to a large increase in the rate of [3H]thymidine incorporation, reaching a maximum after 8 days, whereafter the activity abruptly ceased. A series of norepinephrine injections was in itself able to increase [3H]thymidine incorporation. When norepinephrine was injected in combination with the alpha-adrenergic antagonist phentolamine or with the beta-adrenergic antagonist propranolol, the stimulation was fully blocked by propranolol. It is suggested that stimulation of DNA synthesis in brown adipose tissue is a beta-adrenergically mediated process and that the tissue is an interesting model for studies of physiological control of DNA synthesis.     

Changes in the lipogenic response to feeding of liver, white adipose tissue and brown adipose tissue during the development of obesity in the gold-thioglucose-injected mouse.

Lipogenic response to feeding was measured in vivo in liver, epididymal white adipose tissue (WAT) and interscapular brown adipose tissue (BAT), during the development of obesity in gold-thioglucose (GTG)-injected mice. The fatty acid synthesis after a meal was higher in all tissues of GTG-treated mice on a total-tissue basis, but the magnitude of this increase varied, depending on the tissue and the time after the initiation of obesity. Lipogenesis in BAT from GTG mice was double that of control mice for the first 2 weeks, but subsequently decreased to near control values. In WAT, lipogenesis after feeding was highest 2-4 weeks after GTG injection, and in liver, lipid synthesis in fed obese mice was greatest at 7-12 weeks after the induction of obesity. The post-prandial insulin concentration was increased after 2 weeks of obesity, and serum glucose concentration was higher in fed obese mice after 4 weeks. These results indicate that increased lipogenesis in GTG-injected mice may be due to an increase in insulin concentration after feeding and that insulin resistance (assessed by lipogenic response to insulin release) is apparent in BAT before WAT and liver.     

Developmental changes in fatty acid synthesis in interscapular brown adipose tissue of lean and genetically obese (ob/ob) mice.

Fatty acid synthesis was measured in vivo with 3H2O in interscapular brown adipose tissue of lean and genetically obese (ob/ob) mice. At 26 days of age, before the development of hyperphagia, synthesis in brown adipose tissue was higher in the obese than in the lean mice; synthesis was also elevated in the liver, white adipose tissue and carcass of the obese mice. At 8 weeks of age, when hyperphagia was well established, synthesis remained elevated in all tissues of the obese mice, with the exception of brown adipose tissue. Elevated synthesis rates were not apparent in brown adipose tissue of the obese mice at 14 days of age, nor at 35 days of age. These results demonstrate that brown adipose tissue in ob/ob mice has a transitory hyperlipogenesis at, and just after, weaning on to a low-fat/high-carbohydrate diet. Once hyperphagia has developed, by week 5 of life, brown adipose tissue is the only major lipogenic tissue in the obese mice not to exhibit elevated rates of fatty acid synthesis; this suggests that insulin resistance develops much more rapidly in brown adipose tissue than in other lipogenic tissues of the ob/ob mouse.     

Great adaptability of brown adipose tissue mitochondria to extreme ambient temperatures in control and cold-acclimated gerbils as compared with mice

1. The gerbil (Gerbillus campestris) is a desert rodent able to tolerate high (38 degrees C) and low (-20 degrees C) ambient temperatures, probably due to both its low resting metabolic rate in hot environment and its high peak metabolic rate in cold. 2. Measurement of mitochondrial state IV respiration and cytochrome-oxidase activity (COX) were made in interscapular brown adipose tissue (IBAT), liver and hind limb muscles of gerbils and mice of nearly equal body mass, acclimated for 4 weeks at cold ambient temperature (CA) or reared at thermoneutrality (TN). 3. The most striking difference between these two animal species appears to be in IBAT mitochondria: in TN animals, the level of state IV respiration and COX activity was lower in gerbils than in mice, but the cold acclimation-induced increase in these parameters was greater in gerbils than in mice. 4. Alternatively, in gerbils as in mice, cold acclimation induced a reduction in muscle mitochondrial COX activity. No important change due to cold acclimation was observed in liver mitochondria, either in gerbils or in mice. 5. As compared with mice, the lower state IV respiration in IBAT mitochondria from TN gerbils may explain their low RMR, whereas the higher COX activity of IBAT mitochondria from CA gerbils may explain their higher PMR. 6. As a result of this great adaptability of BAT mitochondria, the gerbil seemed to be able to live in a wide range of ambient temperatures in its natural habitat.     

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 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.     

A dramatic accumulation of glycogen in the brown adipose tissue of rats following recovery from cold exposure

In a morphological study of brown adipose tissue (BAT) of rats returned after exposure to cold (+5 degrees C) to neutral temperature (+25 degrees C), striking periodic acid Schiff staining was observed, indicating substantial glycogen accumulation. Enzymatic analysis revealed that the glycogen content increased from the 4.05 +/- 0.51 (micromol glucose unit per gram of tissue, mean +/- SE) control value to 57.3 +/- 9.66 when the animals were returned to neutral temperature for 24 h after a 1-week cold period. Glycogen repletion was also observed in liver and skeletal muscle; however, the glycogen levels in these tissues never exceeded the control values. The accumulation of glycogen in the BAT started by the 3rd hour of replacement and peaked by the 24th hour. This glycogen was readily utilized during the next short cold exposure of the animals. The plasma leptin concentration was reduced at the cold temperature. The hexokinase II activity in the BAT increased to 29.3 +/- 1.46 vs the 11.8 +/- 1.06 control (mU/mg protein +/- SE) after a 1-week cold exposure and this level was maintained during the return to neutral temperature. The total glycogen synthetase (GStot) and the glycogen synthetase a activity also increased after a 1-week cold exposure and increased further during the replacement. The level of GStot reached 26.9 +/- 1.39 vs 9.54 +/- 1.43 control by the 24th hour of replacement. At the same time, the glycogen phosphorylase a activity declined during the replacement. The concentration of glucose 6-phosphate (an activator of GS) decreased in the cold but returned to normal during the replacement. These changes in the BAT are in favor of glycogen synthesis.     

The central efferent mechanism of brown adipose tissue thermogenesis induced by preoptic cooling

This study was performed to investigate central efferent mechanisms for brown adipose tissue thermogenesis. In unanesthetized rats, the effects of local anesthesia of the ventromedial hypothalamus, anterior hypothalamus, and lateral hypothalamus were observed on the brown adipose tissue thermogenesis induced by preoptic cooling. Rats had a thermode, thermocouple, and bilateral injection cannulae chronically implanted in the hypothalamus and a thermocouple beneath the interscapular brown adipose tissue. The experiments were done at an ambient temperature of 24-25 degrees C. Preoptic cooling increased brown adipose tissue and colonic temperatures without shivering. Injecting lidocaine bilaterally into the ventromedial hypothalamus during preoptic cooling reduced brown adipose tissue temperature (Tbat). The mean maximum decrease of Tbat was 0.51 +/- 0.26 degrees C and occurred 5-8 min after lidocaine injection. When lidocaine was injected into the anterior hypothalamus, Tbat increased. The mean maximum increase of Tbat was 0.85 +/- 0.29 degrees C and occurred 4-9 min after lidocaine injection. In the lateral hypothalamus, lidocaine had no effect on Tbat. Tbat was not influenced by injection of saline into the ventromedial, anterior, or lateral hypothalamus. The efferent pathway from preoptic to brown adipose tissue may thus traverse the medial part of hypothalamus. The ventromedial hypothalamus facilitates and anterior hypothalamus inhibits brown adipose tissue thermogenesis induced by preoptic cooling.     

Regulation of carnitine palmitoyltransferase activity in the liver and brown adipose tissue in the newborn rat: effect of starvation and hypothermia

The overt activity of hepatic carnitine palmitoyltransferase (CPT1) increased during the last day of gestation in the foetus and after prolonged starvation in the newborn kept at 37 degrees C. Its sensitivity to inhibition by malonyl-CoA decreased during the perinatal period studied. Brown fat CPT1 increased under the same experimental conditions. However, its sensitivity to malonyl-CoA remains unchanged. Hypothermia at 24 degrees C decreased in the liver and increased in brown adipose tissue CPT1 activity in response to fasting. Glucose injection at birth decreased CPT1 activity in the liver but did not have any effect in the presence of mannoheptulose. This effect of glucose was non-significant in brown adipose tissue.     

Utilization of methylmalonate for the synthesis of branched-chain fatty acids by preparations of chicken liver and sheep adipose tissue.

1. The utilization of methyl[2-14C]malonyl-CoA for fatty acid synthesis was investigated using synthetase preparations from chicken liver and sheep adipose tissue. 2. The rate of fatty acid synthesis from acetyl-CoA and malonyl-CoA was greatly diminished in the presence of methylmalonyl-CoA. 3. In the absence of malonyl-CoA, methylmalonyl-CoA was utilized for fatty acid synthesis only very slowly by the synthetase from sheep adipose tissue and not at all by that from chicken liver. 4. Despite the inhibitory effect of methylmalonyl-CoA on fatty acid synthesis from malonyl-CoA, it was utilized by the synthetase preparations from both species to produce a complex mixture of methyl-branched fatty acids.     

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.     

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.