Cold acclimation in food-restricted rats

Food intake, body weight and brown adipose tissue (BAT) mass and composition of rats exposed at 6 degrees C either with food ad libitum or food-restricted were compared with those of rats in the thermoneutral zone, with food ad libitum. Cold acclimation with food ad libitum increases food intake and prevents body weight gains. IBAT (interscapular BAT) increases its mass and changes its composition after 3 weeks of cold exposure. Cold acclimation with food restriction produces a progressive decrease in body weight. IBAT mass increases after 3 weeks but changes in composition occur sooner. It is concluded that the overfeeding that accompanies cold acclimation is not necessary for non-shivering thermogenesis in BAT.     

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.     

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.     

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.

Restricted food intake limits brown adipose tissue hypertrophy in cold exposure

Two factors that may determine brown adipose tissue (BAT) hypertrophy during conditions of increased metabolic heat production are increased food intake and increased sympathetic nervous system (SNS) activity. Since these two proceed pari passu during cold exposure, their independent contributions to BAT hypertrophy are unknown. To examine the role of each, we limited the food intake of a group of cold exposed rats by pair feeding them to warm exposed control rats and then compared the pair fed rats to ad lib fed cold exposed animals. Restricted food intake limited absolute BAT hypertrophy (0.226 +/- 0.01 g. vs 0.488 +/- 0.02 g, pair fed vs ad lib, P less than 0.01), BAT as per cent body weight (0.189 +/- 0.12 vs 0.252 +/- 0.012, P less than 0.01) and BAT protein content (34.4 +/- 3.8 vs 48.9 +/- 2.6 mg, P less than 0.01) despite evidence of quantitatively similar activation of the SNS in BAT in both groups. We conclude that increased food intake contributes to BAT hypertrophy in cold exposure independent of sympathetic activity.     

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.     

寒冷诱导产热时大鼠棕色脂肪能量代谢相关蛋白谱的变化

棕色脂肪组织(BAT)的生理作用与白色脂肪显著不同,它以产热的形式释放能量而不是将能量以ATP的形式储存．线粒体是在能量代谢和维持细胞稳态中具有重要功能的细胞器．为了更好地了解棕色脂肪中的能量代谢过程,运用双向电泳及质谱相结合的技术,分离了大鼠白色和棕色脂肪线粒体,对其差异蛋白质谱进行了系统分析和鉴定．参与脂肪和氨基酸代谢、三羧酸循环及线粒体呼吸链的蛋白质在棕色脂肪线粒体中的表达明显高于白色脂肪线粒体,在寒冷诱导下这些蛋白质的表达进一步上调．此外,参与辅酶Q合成的一系列COQ 基因在棕色脂肪中经寒冷适应后表达明显上调．该研究表明,辅 酶Q合成的增高在非颤栗性产热中具有重要作用,为进一步了解棕色脂肪特异性的能量代谢提供了新的思路．     

Comparative studies on lipogenic enzyme activities in brown adipose tissue and liver of the rat during starvation-refeeding transition and cold exposure

1. The effect of starvation-refeeding transition and cold exposure on the activity of lipogenic enzymes in brown adipose tissue (BAT) and liver from rats was compared. 2. Starvation caused a decrease of lipogenic enzyme activities in BAT and liver. 3. Refeeding of the animals with a high carbohydrate diet caused an increase of lipogenic enzymes in these tissues. 4. Cold exposure (4 degrees C for 30 days) led to the increase of BAT enzyme activities to the values observed in rats fed a high carbohydrate diet. 5. Under the same conditions the activity of hepatic lipogenic enzymes also increased but never reached the values observed in the liver of rats fed with a high carbohydrate diet. 6. Therefore BAT and liver lipogenic enzymes showed, in general, a similar pattern of variation under identical nutritional conditions, but substantial differences between these two organs occurred as far as the response to cold exposure was concerned. 7. The experiments also revealed that in the control animals BAT displayed a higher lipogenic potential than the liver.     

Cold exposure increases glucose utilization and glucose transporter expression in brown adipose tissue.

When rats were exposed to a cold environment (4 degrees C) for 10 days, tissue glucose utilization was increased in brown adipose tissue (BAT), a tissue specified for non-shivering thermogenesis, but not in skeletal muscle. Cold exposure also caused an increase in the amount of GLUT4, an isoform of glucose transporters expressed in insulin-sensitive tissues, in parallel with an increased cellular level of GLUT4 mRNA. In contrast to BAT, no significant effect of cold exposure was found in skeletal muscle. The results suggest the cold-induced increase in glucose utilization by BAT is attributable, at least in part, to the increased expression of GLUT4.     

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.     

Unmasking of GDP binding sites on hamster brown adipose tissue mitochondria and uncoupling protein.

1. A rapid unmasking of GDP binding sites on brown adipose tissue (BAT) mitochondria was observed when hamsters acclimatized to 28 degrees C were exposed to a temperature of 4 degrees C for 2 hr. 2. No rapid unmasking of GDP binding sites was observed when hamsters housed at 22 degrees C were briefly exposed to 4 degrees C. 3. The amount of GDP bound to BAT mitochondria from hamsters increased during 2 weeks of exposure to 4 degrees C, but did not change between 2 weeks and 30 days of cold exposure. 4. Incubation of mitochondria with 10 mM Mg2+ prior to the GDP binding assay increased the subsequent GDP binding to BAT mitochondria from hamsters housed at 28, 22 or 4 degrees C, albeit to different degrees. 5. The amount of GDP bound to uncoupling proteins isolated from untreated and Mg(2+)-treated mitochondria of hamsters and rats was measured. Scatchard analyses of the binding of GDP to purified uncoupling protein indicate that increases in the number of binding sites due to Mg2+ treatment of mitochondria do not change the affinity of the protein for GDP.     

Effects of running training on in vitro brown adipose tissue thermogenesis in rats

Brown adipose tissue (BAT) is a major site of nonshivering thermogenesis (NST) during cold acclimation for most mammals. Repetitive nonthermal stress such as immobilization has been shown to enhance the capacity of NST as cold acclimation. In the present study, the effects of running training, another type of nonthermal stress, were investigated on in vitro thermogenesis and the cellularity of interscapular BAT in rats. The rats were subjected to treadmill running for 30 min daily at 30 m/min under 8° inclination for 4–5 weeks. In vitro thermogenesis was then measured in minced tissue blocks incubated in a Krebs-Ringer phosphate buffer containing glucose and albumin at 37° C, using a Clark type oxygen electrode. The trained rats showed less body weight gain during the experiment. The weights of BAT and epididymal white adipose tissue were smaller in the trained rats. Noradrenaline- and glucagon-stimulated oxygen consumption were also significantly smaller in the trained rats. The tissue DNA level was greater in the trained rats, but the DNA content per tissue pad did not significantly differ. The results indicate that running training reduces BAT thermogenesis, possibly as an adaptation to conserve energy substrates for physical work.     

Alterations of glucose-dependent insulinotropic polypeptide (GIP) during cold acclimation

Cold acclimation is initially associated with shivering thermogenesis in skeletal muscle followed by adaptive non-shivering thermogenesis, particularly in brown adipose tissue (BAT). In response, hyperphagia occurs to meet increased metabolic demand and thermoregulation. The present study investigates the effects of cold (4 ± 1 °C) acclimation and hyperphagia on circulating and intestinal levels of gastric inhibitory polypeptide (GIP) in rats. Pair fed animals were used as additional controls in some experiments. Cold acclimation for 42 days significantly (p<0.01) increased daily food intake. There was no corresponding change in body weight. However, body weights of pair fed cold exposed rats were significantly (p<0.01) reduced compared to controls and ad libitum fed cold exposed rats. By day 42, non-fasting plasma glucose was increased (p<0.05) by chronic cold exposure regardless of food intake. Corresponding plasma insulin concentrations were significantly (p<0.01) lower in pair fed cold exposed rats. Circulating GIP levels were elevated (p<0.05) in ad libitum fed cold acclimated rats on days 18 and 24, but returned to normal levels by the end of the study. The glycaemic response to oral glucose was improved (p<0.01) in all cold exposed rats, with significantly (p<0.05) elevated GIP responses in ad libitum fed rats and significantly (p<0.05) reduced insulin responses in pair fed rats. In keeping with this, insulin sensitivity was enhanced (p<0.05) in cold exposed rats compared to controls. By the end of the study, cold acclimated rats had significantly (p<0.01) increased BAT mass and intestinal concentrations of GIP and GLP-1 compared to controls, independent of food intake. These data indicate that changes in the secretion and actions of GIP may be involved in the metabolic adaptations to cold acclimation in rats.     

Cold adaptation and thyroid hormone metabolism.

Resting oxygen consumption and energy expenditure is sensitive to slight alterations in thyroid function. This means that timing and magnitude of cold adaptation would to some extent depend on thyroid function. Local thyroid hormone metabolism is important for energy expenditure and dissipation of heat in special tissues. Recruitment of brown adipocytes and upregulation of uncoupling protein 1 in mitochondria depends on high tissue T3 concentrations. Most of this T3 is derived from local 5' deiodination of T4. Brown fat is vital for cold exposed mice and rats, and may be important for temperature adaptation in human neonates. The role of thyroid hormone metabolism in adult human cold adaptation has not been finally clarified. Hypothetically, cold exposure may enhance T3 production by deiodination of T4 in skeletal muscle, which may enhance heat production in muscle via a change in muscle fiber type. Another hypothetical possibility is recruitment of brown adipocytes embedded in white adipose tissue in human adults. Understanding cold adaptation in human adults may lead to development of new drugs against obesity.     

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)     

Comparative subcellular fractionation of control and cold-adapted rat brown and white adipose tissue with special reference to peroxisomal and mitochondrial distributions

A new technique for single-step subcellular fractionation of adipose tissue homogenates by analytical sucrose density gradient centrifugation in a vertical pocket reorientating rotor is described. The density gradient distributions of mitochondrial and peroxisomal marker enzymes in brown and white adipose tissue of control and cold exposed rats are compared. The equilibrium density of brown fat mitochondria was found to be significantly increased compared with white fat mitochondria. GDP binding activity was localized solely to the mitochondria in both control and cold-adapted brown adipose tissue. Brown and white fat mitochondria fractions were isolated by differential centrifugation and the specific activities of various enzymes in the homogenate and mitochondrial preparations determined. The specific activity of creatine kinase in brown adipose tissue was found to be ten-fold higher than in white fat and subcellular fractionation studies showed the activity to have an exclusively cytosolic distribution in both tissues. GDP binding activity and some of the mitochondrial enzymes showed, in brown adipose, a striking increase in total activity in cold adapted rats compared to control animals. For some enzyme activities there was a small increase when expressed per mg tissue or per mg mitochondrial protein. When expressed per mg DNA i.e. per cell, there was a reduced specific activity of the mitochondrial and peroxisomal enzymes in both brown and white adipose tissue on cold adaptation.     

Antioxidant properties of UCP1 are evolutionarily conserved in mammals and buffer mitochondrial reactive oxygen species

Mitochondrial uncoupling reduces reactive oxygen species (ROS) production and appears to be important for cellular signaling/protection, making it a focus for the treatment of metabolic and age-related diseases. Whereas the physiological role of uncoupling protein 1 (UCP1) of brown adipose tissue is established for thermogenesis, the function of UCP1 in the reduction of ROS in cold-exposed animals is currently under debate. Here, we investigated the role of UCP1 in mitochondrial ROS handling in the Lesser hedgehog tenrec (Echinops telfairi), a unique protoendothermic Malagasy mammal with recently identified brown adipose tissue (BAT). We show that the reduction of ROS by UCP1 activity also occurs in BAT mitochondria of the tenrec, suggesting that the antioxidative role of UCP1 is an ancient mammalian trait. Our analysis shows that the quantity of UCP1 displays strong control over mitochondrial hydrogen peroxide release, whereas other factors, such as mild cold, nonshivering thermogenesis, oxidative capacity, and mitochondrial respiration, do not correlate. Furthermore, hydrogen peroxide release from recoupled BAT mitochondria was positively associated with mitochondrial membrane potential. These findings led to a model of UCP1 controlling mitochondrial ROS release and, presumably, being controlled by high membrane potential, as proposed in the canonical model of “mild uncoupling”. Our study further promotes a conserved role for UCP1 in the prevention of oxidative stress, which was presumably established during evolution before UCP1 was physiologically integrated into nonshivering thermogenesis.