Non-shivering thermogenesis and brown adipose tissue activity in essential fatty acid deficient rats.

The effects of essential fatty acid (EFA) deficiency on energetic metabolism and interscapular brown adipose tissue (BAT) activity were examined in the cold acclimated rat. Weanling male Long-Evans rats were fed on a low fat semipurified diet (control diet, 2% sunflower oil; EFA deficient diet, 2% hydrogenated coconut oil) for 9 weeks. They were exposed at 5 degrees C for the last 5 weeks. In EFA deficient rats, compared to controls, growth retardation reached 22% at sacrifice. Caloric intake being the same in the two groups, it follows that food efficiency was decreased by 40%. Resting metabolism in relation to body surface area was 25% increased. Calorigenic effect of norepinephrine (NE) in vivo (test of non-shivering thermogenesis) underwent a marked decrease of 34%. BAT weight was 21% decreased but total and mitochondrial protein content showed no variation. A 26% increase in purine nucleotide binding per BAT (taken as an index of thermogenic activity) was observed, suggesting that the enhancement in resting metabolism observed was mainly due to increased BAT thermogenesis. However, BAT mitochondria respiratory studies which are more direct functional tests showed a marked impairment of maximal O2 consumption of about 30% with palmitoyl-carnitine or acetyl-carnitine (both in presence of malate) or with alpha-glycerophosphate as substrate. It is likely that this impaired maximal BAT oxidative capacity may explain the impaired NE calorigenic effect in vivo. A possible increase in mitochondrial basal permeability is also discussed.     

Full expression of uncoupling protein gene requires the concurrence of norepinephrine and triiodothyronine

We have examined the uncoupling (UCP) protein gene expression in euthyroid and hypothyroid rats. UCP mRNA levels were estimated by northern blot analysis of total RNA from brown adipose tissue (BAT). Stimuli were endogenous (cold) and exogenous norepinephrine (NE), isoproterenol, T3, and T4. While the euthyroid rats UCP mRNA levels increase 2- to 3-fold by 2 h after NE or overnight cold exposure, these stimuli and isoproterenol are ineffective in hypothyroid rats. One single dose of T4, equal to the daily production rate, brings about a normal response in hypothyroid rats exposed to cold overnight. Hypothyroid rats recover their responsiveness to NE approximately 4 h after a receptor saturating dose of T3. On the other hand, such a dose of T3 induces a 3- to 4-fold increase in UCP mRNA levels in hypothyroid rats without the need of exogenous NE, and this response is not reduced by raising ambient temperature to thermoneutrality (28 C). However, the following evidence indicates that T3 requires adrenergic input to stimulate the accumulation of UCP mRNA: first, euthyroid animals maintained at 28 C do not respond to such a treatment. Second, when T3 was injected to hypothyroid rats with unilaterally denervated BAT, only the intact side responded to T3 with an elevation of the UCP mRNA levels, but both sides remained responsive to T3 + NE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of glycerokinase in brown adipose tissue is stimulated by the sympathetic nervous system

The effect of cold exposure (4 degrees C) or prolonged norepinephrine infusion on the activity and mRNA levels of glycerokinase (GyK) was investigated in rat interscapular brown adipose tissue (BAT). Cold exposure for 12 and 24 h induced increases of 30% and 100%, respectively, in the activity of BAT GyK, which was paralleled by twofold and fourfold increase in enzyme mRNA levels. BAT hemidenervation resulted in reductions of 50% and 30% in GyK activity and in mRNA levels, respectively, in denervated pads from rats kept at 25 degrees C, and suppressed in these pads the cold-induced increases in both GyK activity and mRNA levels. The increase in GyK activity induced by cold exposure was not affected by phenoxybenzamine, but was markedly inhibited by previous administration of propranolol or actinomycin D. BAT GyK activity did not change significantly after 6 h of continuous subcutaneous infusion of norepinephrine (20 microg/h), but increased twofold and fourfold after 12 and 24 h, with no further increase after 72 h of infusion. Norepinephrine infusion also activated mRNA production, but the effect was comparatively smaller than that on enzyme activity. beta-Adrenergic agonists also stimulated GyK activity with the following relative magnitude of response: CL316243 (beta(3)) > isoproterenol (non-selective) > dobutamine (beta(1)). In vitro rates of incorporation of glycerol into glyceride-glycerol were increased in BAT from rats exposed to cold. The data suggest that in conditions of a sustained increase in BAT sympathetic flow there is a stimulation of GyK gene expression at the pretranslational level, with increased enzyme activity, mediated by beta-adrenoreceptors, mainly beta(3).     

Novel form of lipolysis induced by leptin.

Hyperleptinemia causes disappearance of body fat without a rise in free fatty acids (FFA) or ketones, suggesting that leptin can deplete adipocytes of fat without releasing FFA. To test this, we measured FFA and glycerol released from adipocytes obtained from normal lean Zucker diabetic fatty rats (+/+) and incubated for 0, 3, 6, or 24 h in either 20 ng/ml recombinant leptin or 100 nM norepinephrine (NE). Whereas NE increased both FFA and glycerol release from adipocytes of +/+ rats, leptin increased glycerol release in +/+ adipocytes without a parallel increase in FFA release. In adipocytes of obese Zucker diabetic fatty rats (fa/fa) with defective leptin receptors, NE increased both FFA and glycerol release, but leptin had no effect on either. Leptin significantly lowered the mRNA of leptin and fatty acid synthase of adipocytes (FAS) (p < 0.05), and up-regulated the mRNA of peroxisome proliferator-activated receptor (PPAR)-alpha, carnitine palmitoyl transferase-1, (CPT-1), and acyl CoA oxidase (ACO) (p < 0.05). NE (100 nM) also lowered leptin mRNA (p < 0.05) but did not affect FAS, PPARalpha, ACO, or CPT-1 expression. We conclude that in normal adipocytes leptin directly decreases FAS expression, increases PPARalpha and the enzymes of FFA oxidation, and stimulates a novel form of lipolysis in which glycerol is released without a proportional release of FFA.     

Uptake of serum free fatty acids by lipids of the brown adipose tissue]

The flow rate of serum free fatty acids (FFA) into the lipids of brown adipose tissue (BAT) of newborn rabbits was determined by intravenous injection of [14C]-1-palmitate. For a normal 7 day old animal during acute cold exposure the flow rate is (1 hour in 20 degrees C ambient temperature) 0.209 mumol/minute, that is 3.6% of the serum FFA turnover. Prolonged cold exposure only induced an increase in FFA influx if the lipid depot had been depleted (48 hours starvation in 20 degrees C). Consequently, the BAT takes up serum FFA for heat production only after mobilisation of its lipid stores. It is supposed that the mechanism of the uptake of serum FFA by the BAT is connected with their esterification to triglycerides. The phospholipids of BAT which are not only membrane bound lipids are characterized by a high metabolism.     

Cooperation between BAT and WAT of rats in thermogenesis in response to cold, and the mechanism of glycogen accumulation in BAT during reacclimation

Rats were exposed to cold and then reacclimated at neutral temperature. Changes related to fatty acid and glucose metabolism in brown and white adipose tissues (BAT and WAT) and in muscle were then examined. Of the many proteins involved in the metabolic response, two lipogenic enzymes, acetyl-coenzyme A carboxylase (ACC) and ATP-citrate lyase, were found to play a pervasive role and studied in detail. Expression of the total and phosphorylated forms of both lipogenic enzymes in response to cold increased in BAT but decreased in WAT. Importantly, in BAT, only the phosphorylation of the ACC1 isoenzyme was enhanced, whereas that of ACC2 remained unchanged. The activities of these enzymes and the in vivo rate of FFA synthesis together suggested that WAT supplies BAT with FFA and glucose by decreasing its own synthetic activity. Furthermore, cold increased the glucose uptake of BAT by stimulating the expression of components of the insulin signaling cascade, as observed by the enhanced expression and phosphorylation of Akt and GSK-3. In muscle, these changes were observed only during reacclimation, when serum insulin also increased. Such changes may be responsible for the extreme glycogen accumulation in the BAT of rats reacclimated from cold.     

Sympathetic activation of glucose utilization in brown adipose tissue in rats.

The effects of norepinephrine (NE) infusion and surgical denervation or electrical stimulation of the sympathetic nerves on 2-deoxyglucose (2-DG) uptake in interscapular brown adipose tissue (BAT) were investigated in vivo in rats to obtain direct evidence for sympathetic control of glucose utilization in this tissue. 2-DG uptake was rather low in fasted rats, but after refeeding it increased in the BAT as well as the heart, skeletal muscle, and white adipose tissue, in parallel with an increase in plasma insulin level. Cold exposure also enhanced 2-DG uptake in the BAT without the increase in plasma insulin level, while it had no appreciable effect on 2-DG uptake in other tissues. Sympathetic denervation greatly attenuated the stimulatory effect of cold exposure on 2-DG uptake in BAT, but it did not affect the increased 2-DG uptake after refeeding. Electrical stimulation of the sympathetic nerves entering BAT or NE infusion produced a marked increase in 2-DG uptake in BAT without noticeable effects in other tissues. beta-Adrenergic blockade, but not alpha-blockade, abolished the increased 2-DG uptake in BAT. It was concluded that glucose utilization in BAT is activated directly, independently of the action of insulin, by sympathetic nerves via the beta-adrenergic pathway.     

Effect of cold acclimation on norepinephrine stimulated oxygen consumption in muscle

Summary The purpose of this study was to determine the effect of norepinephrine (NE) on oxygen consumption ( ) of perfused (constant flow) muscle in cold acclimated (CA) and control rats. Infusion of NE for a five minute period caused an increase in of similar magnitude in both groups. Infusion of NE for 30 min resulted in an elevated steady state in the cold acclimated group, while the control group showed only an initial increase in followed by a continual decline during the remainder of the 30 min infusion period. These results suggest that when rats are challenged by cold exposure, the magnitude of the initial muscle response to NE by control and acclimated rats is the same, but a useful sustained higher muscle oxygen consumption is found only in the cold acclimated animals.Abbreviations CA cold acclimated - NE norepinephrine     

Activity of antioxidant enzymes in rat skeletal muscle and brown fat: effect of cold and propranolol

1. Effects of acute (6 h) and chronic (21 day) cold (6°C) exposure, as well as propranolol (15 mg/kg) on the activities of CuZnSOD, MnSOD and catalase in the rat skeletal muscle (SM) and brown adipose tissue (BAT), which are important sites of cold-induced thermogenesis, were investigated.

2. The changes in the activity of antioxidant enzymes were tissue specific and dependent on the duration of cold exposure. Thus, in the SM of acutely cold exposed rats, the activity of all antioxidant enzymes studied was elevated, whereas in the BAT the activity of both SOD_s decreased and that of catalase remained unchanged. In cold acclimated rats, the activity of all the three enzymes was increased in the BAT whereas in the SM, CuZnSOD activity was enhanced, MnSOD activity decreased and catalase activity returned to the control level.

3. Propranolol also differently altered the antioxidant enzyme activity in SM and BAT, alterations being dependent on the acclimation temperature. Thus, in room acclimated rats propranolol decreased the activity of all antioxidant enzymes in SM but did not affect those in BAT. However, in the SM propranolol prevented the elevation of MnSOD and catalase activities, induced by acute cold. In cold acclimated rats propranolol inhibited CuZnSOD activity in both SM and BAT but increased that of MnSOD.

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.     

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     

冷驯化条件下中缅树鼩脂肪组织代谢产物的变化

为探究冷驯化条件下中缅树鼩(Tupaia belangeri)白色脂肪组织(WAT)和褐色脂肪组织(BAT)的差异代谢物变化,本研究采集对照组和冷驯化28天组中缅树鼩的WAT和BAT,采用非靶向代谢组液相色谱—质谱联用检测技术分析其差异代谢物含量变化.结果 表明,冷驯化组较对照组WAT中有7种差异代谢物显著上调;BAT...     

Organ blood flow and brown adipose tissue oxygen consumption during noradrenaline-induced nonshivering thermogenesis in the Djungarian hamster

During NA-induced NST blood flow through BAT increased from 0.18 ml min-1 to 3.21 ml min-1 in 23 degrees C acclimated (equals thermoneutrality) and from 0.61 ml min-1 to 9.67 ml min-1 in outdoors (-2 to 12 degrees C Ta) acclimated Djungarian hamsters. In 23 degrees C acclimated hamsters this increase was accomplished by a diversion of blood flow from visceral organs without a change in cardiac output (19.7 versus 20.5 ml min-1 before and after NA). In outdoors acclimated hamsters we also observed a redistribution of blood flow from the viscera to BAT. In addition, cardiac output increased from 24.3 to 38.8 ml min-1. Metabolic rate of BAT in situ was determined from organ blood flow and the (A-V)O2 of blood across the interscapular BAT. BAT of outdoor acclimated hamsters showed a significantly higher metabolism in comparison to 23 degrees C acclimated hamsters (81.1 versus 30.4 mlO2h-1). Furthermore, this calculation revealed that 28% (23 degrees C acclimated hamsters) and 61% (outdoors acclimated hamsters) of total NST were located in BAT of Phodopus sungorus.     

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.     

Effects of rearing temperature on sympathoadrenal activity in young adult rats

Animals reared at 18 degrees C exhibit enhanced innervation of brown adipose tissue (BAT) and greater cold tolerance as adults, yet gain more weight when fed an enriched diet compared with rats reared at 30 degrees C. To explore this paradox, sympathoadrenal activity was examined using techniques of [(3)H]norepinephrine ([(3)H]NE) turnover and urinary catecholamine excretion in male and female rats reared until 2 mo of age at 18 or 30 degrees C. Gene expression in BAT was also analyzed for several sympathetically related proteins. Although [(3)H]NE turnover in heart did not differ between groups, [(3)H]NE turnover in BAT was consistently elevated in the 18 degrees C-reared animals, even 2 mo after removal from the cool environment. Gene expression for uncoupling proteins 1 and 3, GLUT-4, leptin, and the alpha(1A)-adrenergic receptor was more abundant in BAT and the increase in epinephrine excretion with fasting suppressed in 18 degrees C-reared animals. These studies demonstrate that obesity consequent to exposure to 18 degrees C in early life occurs despite tonic elevation of sympathetic input to BAT. Diminished adrenal epinephrine responsiveness to fasting may play a contributory role.     

Inducible beige adipocytes improve impaired glucose metabolism in interscapular BAT-removal mice

Inducible beige adipocytes are emerging as an interesting issue in obesity and metabolism research. There is a neglected possibility that brown adipocytes are equally activated when external stimuli induce the formation of beige adipocytes. Thus, the question is whether beige adipocytes have the same functions as brown adipocytes when brown adipose tissue (BAT) is lacking. This question has not been well studied. Therefore we determine the beneficial effects of beige adipocytes upon cold challenge or CL316243 treatments in animal models of interscapular BAT (iBAT) ablation by surgical denervation. We found that denervated iBAT were activated by cold exposure and CL316243 treatments. The data show that beige adipocytes partly contribute to the improvement of impaired glucose metabolism resulting from denervated iBAT. Thus, we further used iBAT-removal animal models to abolish iBAT functions completely. We found that beige adipocytes upon cold exposure or CL316243 treatments improved impaired glucose metabolism and enhanced glucose uptake in iBAT-removal mice. The insulin signaling was activated in iBAT-removal mice upon cold exposure. Both the activation of insulin signaling and up-regulation of glucose transporter expression were observed in iBAT-removal mice with CL316243 treatments. The data show that inducible beige adipocytes may have different mechanisms to improve impaired glucose metabolism. Inducible beige adipocytes can also enhance energy expenditure and lipolytic activity of white adipose tissues when iBAT is lacking. We provide direct evidences for the beneficial effect of inducible beige adipocytes in glucose metabolism and energy expenditure in the absence of iBAT in vivo.     

The role of insulin in norepinephrine turnover and thermogenesis in brown adipose tissue after acute cold-exposure

The role of insulin in norepinephrine turnover (NE) and thermogenesis in brown adipose tissue (BAT) after acute cold-exposure was studied using streptozocin (STZ)-induced diabetic rats. NE turnover was estimated by the NE synthesis inhibition technique with alpha-methyl-p-tyrosine. BAT thermogenesis was estimated by measuring mitochondrial guanosine-5'-diphosphate (GDP), cytochrome oxidase activity and mitochondrial oxygen consumption in BAT at an ambient temperature of 22 degrees C and during a six-hour cold-exposure at 4 degrees C. In insulin-deficient diabetic rats, the NE turnover, mitochondrial GDP binding, cytochrome oxidase activity and mitochondrial oxygen consumption in BAT at 22 degrees C were significantly reduced, compared with those of control rats. Treatment of STZ-induced diabetic rats with insulin prevented a decrease in NE turnover and BAT thermogenesis. Acute cold-exposure increased the NE turnover of BAT in insulin-deficient diabetic rats. The BAT thermogenic response to acute cold-exposure, however, did not occur in insulin-deficient diabetic rats. These results suggest that insulin is not essential in potentiating NE turnover in BAT after acute cold-exposure, but is required for cold-induced thermogenesis.     

Lipolysis in brown adipose tissue of cold- and heat-acclimated hamsters

Rates of release of free fatty acids (FFA) and glycerol to the incubation medium by brown adipose tissue (BAT) slices isolated from heat-acclimated (H), cold-acclimated (C), and control (N) hamsters in the absence or presence of epinephrine (E) were studied. Rates of FFA and glycerol release by tissue slices isolated from H and N animals were similar. In tissue slices isolated from C animals rate of release of FFA and glycerol was three times as high. Addition of E to the incubation medium (200 microgram/ml) had no effect on the rate of FFA and glycerol release of slices from C animals, but tripled the rates of slices from N, resulting in similar values for the two groups. In slices from H animals the rate of release was lower than in the other two groups, increasing only 1.5-fold. Pretreatment of N animals with triiodothyronine (T3; 0.8 microgram/100 g daily for 7 days) doubled the rates of FFA and glycerol release. Addition of E to the medium affected both pretreated and nontreated slices similarly. Two possible mechanisms by which temperature acclimation controls the lipolytic rate of BAT are suggested by 1) the concentration of specific enzymes and 2) cellular metabolites and hormones which activate existing systems. It seems that both operate in temperature-acclimated hamsters.