Cross adaption between stress and cold in rats

Three-hour immobilization stress was imposed on male adult rats of Wistar strain by restraining them on a board 6 days a week for 1–8 weeks. The stressed rats showed less body weight gain during the experiment compared to the controls. These stressed animals manifested an improved cold tolerance as shown by no significant fall in colonic temperature in the cold at –5° C for 300 min during the experimental period, while the colonic temperature of the controls fell progressively. Nonshivering thermogenesis as assessed by noradrenaline-induced increase in oxygen consumption was significantly potentiated in the stressed rats. The weight and protein content of the intercapsular brown adipose tissue (BAT) increased and BAT mitochondria were more packed in the stressed rats. Plasma insulin, insulin/glucagon molar ratio and thyroxine levels were lowered in the stressed rats, while the plasma triiodothyronine level remained unchanged. Removal of interscapular BAT led to a loss of improved cold tolerance and a significant reduction of nonshivering thermogenesis in the stressed rats.These results indicate that repetitive stress may induce cross adaptation between stress and cold through an enhanced capacity of nonshivering thermogenesis mediated, at least in part, via stimulation of BAT function.     

Cold-induced changes in glucagon of brown adipose tissue

In order to evaluate the role of glucagon in brown adipose tissue (BAT) function under cold or stress, the changes in immunoreactive glucagon of BAT and plasma as well as uptake and metabolism of radioactive glucagon (125I-G) in this tissue were studied in rats. Glucagon per g fresh tissue was higher in the dorso-cervical BAT than in the interscapular BAT. In warm controls (WC), acute cold exposure (-5 degrees C, 15 min) (CE) or stress (immobilization, 30 min) (AS) elevated glucagon of both sites of BAT as well as plasma. In cold-acclimated animals (CA), the resting levels of BAT glucagon, but not plasma glucagon, were higher than WC. CE caused elevation of plasma glucagon, but not BAT glucagon in CA. AS did not affect glucagon levels in both plasma and BAT in CA. Cold acclimation did not influence 125I-G uptake by BAT, but resulted in a rather lower 125I-G level in plasma and liver. The present results suggest that BAT is a target tissue for glucagon to cause nonshivering thermogenesis in response to cold or stress and that turnover of glucagon is enhanced by cold acclimation.     

Effects of cold and immobilization stress on noradrenaline turnover in brown adipose tissue of rat

Noradrenaline (NA) turnover of the interscapular brown adipose tissue (BAT) was determined in order to evaluate a role of sympathetic NA of this tissue in an enhanced nonshivering thermogenesis which had been previously evidenced in the repetitively stressed rats by immobilization (daily 3-h immobilization for 4 weeks) and the cold-acclimated ones (5 degrees C, 4 weeks). The disappearance rate of NA from the BAT following blockade of NA synthesis with alpha-methyl-p-tyrosine was adopted for estimation of NA turnover of the tissue. Cold acclimation increased both fractional turnover rate (%/h) (k) and turnover rate (ng/(g BAT.h)). Repetitive immobilization stress also elevated turnover rate, but not k. In the warm non-stressed controls acute cold exposure to -5 degrees C and acute immobilization stress elevated the turnover rate. The effect of cold exposure was significantly greater than that of immobilization stress for both indices of NA turnover. In the cold-acclimated rats acute cold exposure increased k as well as turnover rate, but not acute immobilization stress. In the repetitively immobilized rats both acute cold exposure and acute immobilization stress elevated k and turnover rate. These results indicate that immobilization enhances sympathetic activity of thermogenic tissue, BAT. The results also suggest that the extent of sympathetic participation is not necessarily the same between the cold-acclimated and the stressed rats.     

Role of sympathetic nervous system in immobilization- and cold-induced brown adipose tissue thermogenesis in rats

The role of the sympathetic nervous system in 10-min cold (5 degrees C)- or 2-min immobilization-induced thermogenesis in brown adipose tissue (BAT) was studied in warm (25 degrees C)-acclimated rats. Both cold- and immobilization-stresses increased heat production (M), interscapular brown adipose tissue temperature ( Tbat ), and colonic temperature ( Tcol ). Resulting from both stresses, the increase in Tbat was greater than that in Tcol , the differences (delta Tbat ) becoming approximately 0.48 and 0.46 degrees C by the cold exposure and the immobilization, respectively. After sympathectomy, Tbat and delta Tbat did not change on immobilization but increased significantly on the cold exposure. Delta Tbat was 0.31 degrees C in the sympathectomized rats at the end of the cold exposure period. Immobilization-induced BAT thermogenesis may be mainly controlled by the sympathetic nervous system. On the other hand cold-induced BAT thermogenesis seems to be controlled by certain hormonal factors as well as the sympathetic nervous system.     

Repeated immobilization stress increases uncoupling protein 1 expression and activity in Wistar rats

Repeat immobilization-stressed rats are leaner and have improved cold tolerance due to enhancement of brown adipose tissue (BAT) thermogenesis. This process likely involves stress-induced sympathetic nervous system activation and adrenocortical hormone release, which dynamically enhances and suppresses uncoupling protein 1 (UCP1) function, respectively. To investigate whether repeated immobilization influences UCP1 thermogenic properties, we assessed UCP1 mRNA, protein expression, and activity (GDP binding) in BAT from immobilization-naive or repeatedly immobilized rats (3 h daily for 4 weeks) and sham operated or adrenalectomized (ADX) rats. UCP1 properties were assessed before (basal) and after exposure to 3 h of acute immobilization. Basal levels of GDP binding and UCP1 expression was significantly increased (140 and 140%) in the repeated immobilized group. Acute immobilization increased GDP binding in both naive (180%) and repeated immobilized groups (220%) without changing UCP1 expression. In ADX rats, basal GDP binding and UCP1 gene expression significantly increased (140 and 110%), and acute immobilization induced further increase. These data demonstrate that repeated immobilization resulted in enhanced UCP1 function, suggesting that enhanced BAT thermogenesis contributes to lower body weight gain through excess energy loss and an improved ability to maintain body temperature during cold exposure.     

Nitric oxide and thermogenic function of brown adipose tissue in rats

To clarify the effects of cold acclimation and immobilization stress adaptation of rats on nitric oxide (NO) activity in interscapular brown adipose tissue (BAT), we incubated neatly diced (1-mm(3) blocks) BAT in a metabolic chamber for respiration, measured oxygen consumption using a Clark electrode, and estimated NO release in the buffer medium by measuring nitrite plus nitrate (NO(x)) using the Griess method (diazotization reaction). The production of NO(x) in the buffer medium confirmed that BAT releases NO, as there is no other source of NO(x) in the system. The NO activity was observed in the basal condition and increased with noradrenaline stimulation, showing a correlation with oxygen consumption in the warm (25 degrees C)-acclimated control rats. Cold acclimation (5 degrees C, 5 weeks) or immobilization stress adaptation (3 h daily, 25 degrees C, 5 weeks) caused enhanced NO activity in the basal condition in comparison with the control. We suggest that NO is involved in enhancement of the thermogenic functions of BAT in rats.     

Effects of glucagon and noradrenaline on the blood flow through brown adipose tissue in temperature-acclimated rats

After 30 min infusion of glucagon or noradrenaline, blood flow through brown adipose tissue (BAT) from various sites was investigated with the aid of 113Sn-labeled microspheres under hexobarbital anesthesia in cold-acclimated (CA), heat-acclimated (HA), and warm control (WC) rats. Glucagon increased cardiac output in both CA and HA, while noradrenaline increased it in HA but not in CA. Blood flow through BAT as well as the fractional distribution of cardiac output to BAT increased by glucagon dose-dependently and reached a maximum level in a dose of 2 micrograms/min. These glucagon-induced responses were significantly higher in CA and smaller in HA as compared with WC. Noradrenaline in a dose of 2 microgram/min caused larger responses than glucagon in all groups. Glucagon- or noradrenaline-induced blood flow per unit weight of BAT increased or tended to increase by cold acclimation. These results suggest that an in vivo enhanced glucagon-induced thermogenesis in cold-acclimated BAT is partly due to an increased blood flow through this tissue.     

Brown adipose tissue and thermogenesis in hypophysectomized rats in relation to temperature acclimation

The aim of this work was to test the role of pituitary dependent hormones in cold-induced non-shivering thermogenesis. In the 28°C-acclimated rat, hypophysectomy inhibited body growth and led to an atrophy of thyroid and adrenals. In brown adipose tissue (BAT) some alterations were induced which are usually observed after cold acclimation of the animal: increase in relative weight, decreases in the relative amount of lipids, increases in the amounts of protein and DNA and modification of the proportions of several phospholipid fatty acids; moreover, basal lipolysis, in vitro, was enhanced to the same extent as that following cold acclimation of the normal rat. The in vivo stimulation by norepinephrine (NE) of O₂ consumption (test for nonshivering thermogenesis) and of fatty acid release into blood were suppressed.Progressive cold acclimation of the hypophysectomized rats at 15°C led to a hypertrophy of BAT to the same extent as in the sham-operated animals. The in vivo sensitivity to NE was partially restored. The results suggest that hypophysectomy does not suppress the ability to acclimate to moderate cold by means of BAT dependent non-shivering thermogenesis. However, the low ability to produce heat seems to indicate that pituitary or pituitary-dependent hormones are necessary to optimize the cold stimulation of brown fat thermogenesis.     

Effects of chronic administration of noradrenaline and glucagon on in vitro brown adipose tissue thermogenesis.

The in vitro brown adipose tissue (BAT) oxygen consumption stimulated by noradrenaline (NA) or glucagon (G) was significantly lower in chronically NA-treated rats and that of G-treated rats did not differ as compared with that of vehicle-treated control animals. In vitro thermogenic response of BAT in NA-treated rats was consistent with that induced by cold acclimation.     

Metabolic cold acclimation after repetitive intermittent cold exposure in rat

Repetitive intermittent cold exposure (5 degrees C, 6 h/day, 4 weeks) (ICE) resulted in the same cold adaptability as assessed by an enhanced cold tolerance (less drop of colonic temperature at -5 degrees C) and nonshivering thermogenesis (NST) (greater noradrenaline-induced heat production) as that elicited by continuous cold exposure (5 degrees C, 4 weeks) (CA) in rats. Although shorter intermittent (5 degrees C, 2 h/day, 4 weeks) (ICE-2 hr) as well as shorter continuous (5 degrees C, 1 week) (CA-1 wk) cold exposure effected an improved cold adaptability, the magnitude of cold tolerance and NST was smaller as compared with that in CA and ICE. The cold deacclimation process as reflected on the decreased NST did not differ between CA and ICE. Food intake was less in ICE than CA, while increase in body weight during the acclimation period was greater in the former. Increase in adrenal weight was greater in CA than ICE, but plasma corticosterone level did not differ among warm controls (WC), CA, and ICE in resting state (after 18-20 h at warm control temperature of 25 degrees C). Weights of interscapular and dorsocervical brown adipose tissue (BAT) increased to the same degree in CA and ICE. Plasma glucagon level in resting state did not differ among groups, while BAT glucagon levels significantly increased in CA and ICE, but they were higher in dorsocervical site than interscapular site in all acclimated states. Acute cold exposure (-5 degrees C, 15 min) caused increases in plasma corticosterone, glucagon levels, and in BAT glucagon levels in all acclimated groups. The extent of increase was significantly less for plasma glucagon in CA, while plasma corticosterone increased similarly in all groups. These results indicate that repetitive short-term cold exposure could elicit the same cold adaptability as that induced by continuous exposure, but requiring only one-fourth of the time of continuous cold exposure. Moreover, it is suggested that glucagon is involved in both CA and ICE, but the same extent of cold adaptability can be obtained in the less energy-requiring and less stressful state in ICE.     

Lasting consistency of cold adaptability in rats reared in cold for many generations

Wistar rats were successively reared in cold at 5 degrees C from 1969 to 1984. The historical changes observed in these rats were reported. The cold-adapted rats reared in cold for 8 to 11 successive generations (C8-11G) were examined on their cold tolerance and non-shivering thermogenesis. C8-11G rats showed greater nonshivering thermogenesis than that of the warm-adapted control group (W), and rats exposed to cold for periods of 2 to 8 weeks (C). The nonshivering thermogenesis of C8-11G rats was diminished to a similar level to that of W and C rats by administration of a ganglion blocker, of reserpine, or of beta-adrenoceptor blocker. The de-adapted rats reared in warm at 25 degrees C for 3 generations after being reared for many generations in cold (DA-3G) showed much more nonshivering thermogenesis as compared to W rats. Cold tolerance of DA-3G rats was at a level of intermediate between that of W and C rats. Brown adipose tissue (BAT) weight of DA-3G rats was similar to that of C rats, while chemical composition of BAT in DA-3G rats differed from that of C and W rats.     

Brown adipose tissue glyceroneogenesis is activated in rats exposed to cold

We have previously found that glyceroneogenesis is very active in brown adipose tissue (BAT) and increases in fasted, diabetic and high-protein-diet-fed rats, situations of reduced thermogenic activity. To understand better the role of glyceroneogenesis in BAT glycerol-3-phosphate (G3P) generation, we investigated its activity during cold exposure (10 days at 4°C), a condition in which, in contrast to the above situations, BAT thermogenesis is markedly activated. Rates of total (from all sources) BAT fatty acid (FA) synthesis and rates of incorporation of glucose carbon into BAT glyceride-FA and -glycerol in vivo were markedly increased by cold exposure. Cold exposure induced a marked increase in BAT glyceroneogenic activity, evidenced by (1) increased rates of non-glucose carbon incorporation into glyceride-glycerol in vivo and of [1-¹⁴C]-pyruvate incorporation into glyceride-glycerol in vitro, and (2) a threefold increase in phosphoenolpyruvate carboxykinase activity. Most of the glyceride-glycerol synthesized by BAT via glyceroneogenesis or from glucose was used to esterify preformed FA. This use was markedly increased by cold exposure, in parallel with a pronounced activation of BAT lipoprotein lipase activity. In conclusion, during cold exposure BAT glyceroneogenesis is markedly activated, contributing to increase the generation of G3P, which is mostly used to esterify preformed FA.     

Cold-induced and diet-induced thermogenesis in progesterone-treated rats

Both cold-acclimated rats and rats at thermoneutrality received 1.5 mg/day of progesterone over a period of 15 days by means of two subcutaneously implanted Silastic capsules. Progesterone treatment increased total food intake and body mass gain in both groups of treated animals when compared with their controls at the same ambient temperature. However, the interscapular brown adipose tissue (BAT) of the treated rats showed the same thermogenic activity (assessed by GDP-binding), mass and gross composition as that of their respective controls. If it is assumed that enhanced food intake is the physiological drive for diet-induced thermogenesis, it could be concluded that progesterone inhibits diet-induced thermogenesis at thermoneutrality, but has no effect in cold-induced thermogenesis. However, if the physiological drive for diet-induced thermogenesis is not enhanced food intake, but an imbalance in the diet, then given that the same diet was offered to all animals thoroughout the experimental period, it could be that progesterone does not affect BAT, either at thermoneutrality or in the cold.     

The effect of non-esterified long-chain fatty acids on blood flow and thermogenesis in brown adipose tissue in the young dog

In vitro experiments have demonstrated that increasing the molar ratio of extracellular non-esterified fatty acids (NEFA) to albumin stimulates thermogenesis in brown adipocytes. To test these results, in vivo blood flow and local temperature were measured in perirenal brown adipose tissue (BAT) in puppies during thermogenesis induced by increasing the plasma NEFA: albumin ratio by injection of intralipid and heparin. Plasma NEFA concentration increased to 1.5 mmol X 1-I and plasma NEFA:albumin ratio to 4. Concomitantly, the whole body oxygen consumption rose on average about 100% above baseline level. Plasma noradrenaline concentration increased about three-fold and plasma adrenaline concentration about six-fold. The BAT temperature increased by an average of 0.9 degrees C. However, since BAT blood flow was simultaneously reduced by about 50%, it can be calculated that the local heat production was also reduced. Consequently, the increase in whole body oxygen consumption was not due to stimulation of BAT thermogenesis. It is concluded that in vivo assessment of BAT thermogenesis requires concomitant measurements of both local BAT temperature and blood flow.     

Enhanced formation of cyclic AMP after cold acclimation in the rat

Plasma cAMP response to glucagon was enhanced after cold acclimation. Cold-acclimated rats also showed an enhanced production of hepatic cAMP in response to glucagon. In both brown and white fat tissues, cAMP contents increased after cold acclimation. Enhancement of cAMP formation in the liver and fat tissues may partly contribute to an enhanced nonshivering thermogenesis during cold acclimation.     

Competition and cooperation among huddling infant rats

Huddling is expressed by infant rats and continues to be an important behavior throughout adulthood. As a form of behavioral thermoregulation, huddling is thought to play an essential role in compensating for inadequate physiological thermoregulation early in development. Infant rats, however, are capable of heat production shortly after birth using brown adipose tissue (BAT) and exhibit thermogenesis in the huddle, suggesting that huddling does not obviate the need for endothermy during cold exposure. In the present experiment, 4-pup huddles of infant rats (2- or 8-day-olds) were exposed to two subthermoneutral temperatures, and BAT thermogenesis was inhibited in 0, 2, or 4 of the rats in each huddle. Inhibition of BAT thermogenesis compromised the pups' ability to maintain huddle temperature, but surprisingly did not result in enhanced huddling at either age. These results suggest that effective huddling during cold exposure requires the thermal resources provided by endothermy. Furthermore, the heat provided by BAT appears to shape behavioral interactions in the huddle during development.     

Pituitary and autonomic responses to cold exposures in man

This review presents hormonal responses to various cold exposures and their calorigenic effects in man and some animals. Previous studies in rats have shown that cold exposures activate the hypothalamic-pituitary-thyroid axis. Increased thyroid hormone concentrations lead to heat production via general stimulation of metabolism (obligatory thermogenesis) and possibly via activation of thyroid hormone receptors and uncoupling protein 1 (UCP 1) and deiodinase enzyme genes in the brown adipose tissue (BAT). In human subjects long-term cold exposures do not seem to activate the pituitary-thyroid axis, but rather accelerate the elimination of triiodothyronine (T3), leading to low serum concentrations of free T3 hormone. In corollary to this a hypothyreotic condition with increased serum thyroid-stimulating hormone and impaired mood and cognitive performance can be observed after long-term cold exposures such as wintering. During cold exposures the sympathetic nerve system is activated and noradrenaline is released to blood circulation and to BAT, where it leads to production of cAMP, lipolysis and free fatty acids. Free fatty acids open the mitochondrial proton channel protein in BAT. Protons enter the mitochondria and inhibit ATP synthesis (uncoupling). By this way energy is transformed into heat (facultatory or adaptive thermogenesis). In adult human subjects the amount of BAT is small and adaptive thermogenesis (non-shivering thermogenesis) has a smaller role. UCP 1 with other uncoupling proteins may have other functions in the control of body weight, sugar balance and formation of reactive oxygen species.     

Independence of ultrasonic vocalization and thermogenic responses in infant rats

Ultrasonic vocalizations (USV) normally accompany brown adipose tissue (BAT) thermogenesis in infant rats exposed to cold. BAT activation (measured by implanted thermistors) was pharmacologically blocked with hexamethonium (20 mg/kg ip) in 12-13-day-old pups, but they nevertheless showed normal USV responses to cold. Activation of BAT in warm pups by norepinephrine (800 micrograms/kg sc) failed to elicit USV. It is concluded that BAT activation is neither necessary nor sufficient for USV production. To evaluate how tightly the two responses may be coupled centrally, rat pups deprived of nutrients for 24 hr, in which sympathetic activation is known to be inhibited centrally (Bignall, Heggeness, & Palmer, 1975), were studied. These pups vocalized with the same latency in response to cold as normals but failed to show evidence of concurrent BAT activation. It is concluded that USV and BAT thermogenesis are normally elicited together by cold but are not tightly linked physiologically.     

Contributions of baroreceptor reflex to the hypothermic effect of intraventricular angiotensin II in rats

The mechanisms of the hypothermic effect of angiotensin II (AII) injected into the lateral ventricle were investigated in unanesthetized rats at an ambient temperature of 18 degrees C. Mean blood pressure (BP), heart rate (HR), metabolic rate (M), colonic temperature (Tcol), and temperatures of the interscapular brown adipose tissue (TBAT), and the tail skin (Tsk) were continuously monitored. AII at a dose of 5 micrograms produced a sharp and marked elevation in BP accompanied by bradycardia, and a decrease of M and Tco1 in the sinoaortic baroreceptor intact rats. The difference between TBAT and Tcol decreased significantly, which suggests a suppression of nonshivering thermogenesis of the BAT. Tsk was not changed by the AII injection. After sinoaortic denervation, however, the decrease in Tcol and M with AII injection was significantly reduced despite a marked elevation in BP. In addition, intravenous arginine-vasopressin antagonist pretreatment suppressed the elevation in BP and the decrease in HR, Tcol, and M after AII injection. From these results, it is concluded that the hypothermia which occurred after AII injection into the lateral ventricle can be largely attributed to the baroreflexive suppression of M, and to some extent to the direct effect on the thermoregulatory center in rats.     

Adaptive changes in the thermogenesis of rats by cold acclimation and deacclimation

Male Wistar rats, aged 6 weeks, were maintained at 25 degreesC for 9 to 11 weeks (W group), at 10 degreesC for 9 to 11 weeks (C group), and at 25 degreesC for 2 weeks after exposure to 10 degreesC for 9 weeks (D group). Thermogenesis at 10 degreesC was significantly greater than at 25 degreesC. Thermogenesis per body mass in the C group was greater than in the W and D groups. The RQ value at 10 degreesC was greater than at 25 degreesC in the W group, whereas the opposite was observed in the C and D groups. It is suggested that a large part of enhanced thermogenesis, caused by cold acclimation for 9 weeks, is lost because of a decreased secretion of calorigenic hormones, in spite of a slight decrease in BAT mass, during deacclimation for 2 weeks.