Sex-associated differences in cold-induced UCP1 synthesis in rodent brown adipose tissue

 The effects of acute and chronic acclimation to cold on uncoupling protein 1 (UCP1) levels, as well as on GDP-binding to mitochondria, cytochrome c oxidase activity and mitochondrial protein concentration in brown adipose tissue (BAT) of intact male and female rats have been analyzed. Results reveal that females rats are more sensitive to cold because their threshold temperature for the thermogenic response is set at a higher value (around 22°C) than that of males (around 18°C), hence leading to differences in BAT UCP1 levels between the sexes at different environmental temperatures. In vitro experiments showed that steroid hormones, β-estradiol, estrone and progesterone, can reduce norepinephrine-induced UCP1 synthesis in brown adipocytes differentiated in primary culture. Thus the different sex-associated response of cold-induced thermogenesis in rats does not appear to be explained by a direct action of sex steroids upon the adipocyte, implying that other factors in the thermogenic regulatory system must be involved. Received: 23 March 1998 / Received after revision: 20 May 1998 / Accepted: 21 May 1998     

Brown adipose tissue response to cafeteria diet-feeding involves induction of the UCP2 gene and is impaired in female rats as compared to males

Noradrenaline-dependent brown adipose tissue (BAT) thermogenesis is activated by the cold and excess energy intake, largely depends on the activity of the uncoupling protein 1 (UCP1), and is mediated mainly through the beta3-adrenoceptor (beta3-AR). We investigated the expression of ucp2, a gene that encodes a putative UCP1-like uncoupling protein, along with that of ucp1 and beta3-ar, in the interscapular BAT (IBAT) of male and female rats chronically fed a cafeteria diet. After 3 months on this diet, male rats attained a 34% excess body mass and showed IBAT hypertrophy and increased IBAT thermogenic potential, in terms of both UCP1 and UCP2 mRNA expression (both by 1.6-fold), UCP1 protein expression (by 1.75-fold) and GDP binding to IBAT mitochondria (by 2.2-fold); female rats attained a larger excess body weight (50%) and their IBAT, although hypertrophied, showed no signs of increased thermogenic potential per gram of tissue. Interestingly, the IBAT of female rats was already activated compared to males. Treatment of mouse brown adipocytes in primary culture with noradrenaline also triggered a dose-dependent increase of the levels of UCP1 mRNA and UCP2 mRNA. Retroregulatory down-regulation of the beta3-AR mRNA levels was found in the two models used. The results support a physiological role for UCP2, along with UCP1, in rodent BAT thermogenesis.     

Synergic effect of overweight and cold on uncoupling proteins expression,a role of alpha(2)/beta(3) adrenergic receptor balance?

The effect of cold exposure, being overweight and their interaction was investigated on the response of uncoupling proteins UCP1, UCP2 and UCP3 and the alpha(2)/beta(3) adrenergic receptor (AR) balance in brown adipose tissue (BAT), as well as the involvement of leptin gene expression in white adipose tissues, in control and overweight male rats of the dietary obesity model known as the post-cafeteria model. UCP1, UCP2 and UCP3 mRNAs were up-regulated by cold, with a synergic effect of cold exposure and being overweight on UCP1 mRNA levels (with the related UCP1 protein response), and with UCP2 mRNA showing a parallel response. Furthermore, the BAT alpha(2)/beta(3) AR ratio was diminished in overweight rats. The results suggest that the UCP1-dependent thermogenic capacity in BAT of post-cafeteria overweight rats has a more sensitive response to cold exposure and that UCP2 and UCP3 could be somehow involved in the thermogenic response but differentially regulated. Moreover, the diminished alpha(2)/beta(3) AR ratio in BAT could be one of the factors involved in the more sensitive response of overweight rats to cold in terms of BAT thermogenesis-related parameters.     

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.     

Unloading does not increase brown-adipose-tissue activity in rat pups

Our previous study has shown that chronic hindlimb suspension leads to an increase in both the thermogenic capacity and the activity in brown adipose tissue (BAT) of adult rats (Yamashita, H., Ohira, Y., Wakatsuki, T., Yamamoto, M., Kizaki, T., Oh-ishi, S., Sato, Y. and Ohno, H. (1995). J. Appl. Physiol. 78: 384-387). In order to examine if unloading also increases the BAT activity in rat pups, the hindlimbs in the suspended pups were unloaded by tail suspension beginning on postnatal day 4 and suspended until day 21. The thermogenic activity (which was assessed by guanosine 5'-diphosphate binding to BAT mitochondria) was markedly lower in 21-day-old suspended pups than in 21-day-old control pups, although there was no difference in uncoupling protein 1 (UCP1) content or UCP1 mRNA expression in the BAT mitochondrial fraction between both pups. Likewise, there was no disparity in either adrenal or thymus mass between the control and suspended pups throughout the experiment. These results suggest that, in contrast to adult rats, chronic hindlimb suspension leads to a decrease in the thermogenic activity in BAT of rat pups possibly for reason that pups are less susceptible to the stress of unloading.     

Sexual dimorphism in the adrenergic control of rat brown adipose tissue response to overfeeding

Gender-related differences in the brown adipose tissue (BAT) response to overfeeding rats on a cafeteria diet were studied by assessing the balance between the expression of beta-adrenoceptors (beta1-, beta2-, beta3-AR) and alpha2A-AR and their relation to the expression of uncoupling proteins (UCP1, UCP2, UCP3). Cafeteria diet feeding for 15 days, which involved a similar degree of hyperphagia in both sexes, led to a greater body weight excess in females than in males and a lower activation of thermogenesis. Gender-related differences were found for different adrenoceptor expression and protein levels, which might explain, in part, sex differences in the thermogenic parameters. The lower expression of alpha2A-AR in females than in males could be responsible for the higher expression of UCP1 and thermogenic capacity under non-hyperphagic conditions. However, in a situation of high adrenergic stimulation--as occurs with overfeeding--as there is a preferential recruitment of the beta3-AR by noradrenaline compared with other adrenergic receptors, the higher levels of beta3-AR in males rats than in females could be responsible for the greater thermogenic capacity and the lesser weight gain in males. Thus, the alpha2/beta3 balance in BAT could be a key in the thermogenic control.     

Effect of Postnatal Age and a β3-Adrenergic Agonist (Zeneca D7114) Administration on Uncoupling Protein-1 Abundance in the Lamb

We examined the effect of time after birth and beta(3)-adrenergic agonist (Zeneca D7114) administration on uncoupling protein-1 (UCP1) abundance and thermoregulation in the lamb. Forty twin lambs, all born normally at term, were maintained at a cold ambient temperature of between 3 and 8 degrees C. At 0.5, 1.75, 5.25, 11.25 and 23.25 h after birth eight sets of twins were fed 20 ml of formula milk +/- 10 mg kg(-1) of beta(3)-adrenergic agonist, and 45 min after feeding brown adipose tissue (BAT) was sampled. Colonic temperature was measured and BAT analysed for UCP1 abundance, GDP-binding to mitochondrial protein (i.e. thermogenic activity) and catecholamine content. Colonic temperature declined between 1.25 and 6 h from 40.2 degrees C to 39.2 degrees C and then increased to 39.8 degrees C at 12 h, but increased after feeding at all ages. UCP1 abundance increased from 1.25 h after birth, to peak at 2 h after birth in controls, compared with 6 h after birth in beta(3)-adrenergic agonist-treated lambs. The level of GDP-binding to mitochondrial protein did not change significantly with age but was increased by beta(3)-adrenergic agonist treatment. The noradrenaline (norepinephrine) content of BAT increased between 1.25 and 12 h after birth, irrespective of beta(3)-adrenergic agonist administration. The total weight of perirenal BAT plus its lipid, protein and mitochondrial protein content declined over the first 6 h of life. UCP1 development continues over the first 24 h of neonatal life, and can be manipulated by beta(3)-adrenergic agonist administration. This may represent one method of improving thermoregulation in newborn lambs. Experimental Physiology (2001) 86.1, 65-70.     

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.     

Hypothalamic neuronal histamine regulates sympathetic nerve activity and expression of uncoupling protein 1 mRNA in brown adipose tissue in rats

To clarify how hypothalamic neuronal histamine regulates peripheral energy expenditure, we investigated the effect of infusion of histamine into the third cerebral ventricle or discrete hypothalamic regions on sympathetic nerve activity and expression of uncoupling protein 1 (UCP1) mRNA in brown adipose tissue (BAT). Infusion of histamine (200 nmol) into the third cerebral ventricle of anesthetized rats significantly increased the electrophysiological activity of sympathetic nerves (P<0.01) and UCP1 mRNA expression in the BAT (P<0.05). Microinjection of histamine (10 nmol) into the paraventricular nucleus (PVN) and preoptic area (POA) produced similar significant increases in BAT sympathetic nerve activity (P<0.01 for each). By contrast, injection of histamine into the ventromedial hypothalamic nucleus or lateral hypothalamic area had no effect. We conclude that hypothalamic neuronal histamine may regulate energy expenditure in BAT through the activation of sympathetic nerves. The PVN and/or POA appear to be the principal hypothalamic sites that mediate the stimulatory effect of histamine on this efferent pathway.     

Chronic l-menthol-induced browning of white adipose tissue hypothesis: A putative therapeutic regime for combating obesity and improving metabolic health

IntroductionObesity constitutes a serious global health concern reaching pandemic prevalence rates. The existence of functional brown adipose tissue (BAT) in adult humans has provoked intense research interest in the role of this metabolically active tissue in whole-body energy balance and body weight regulation. A number of environmental, physiological, pathological, and pharmacological stimuli have been proposed to induce BAT-mediated thermogenesis and functional thermogenic BAT-like activity in white adipose tissue (WAT), opening new avenues for therapeutic strategies based on enhancing the number of beige adipocytes in WAT.HypothesisRecent evidence support a role of l-menthol cooling, mediated by TRPM8 receptor, on UCP1-dependent thermogenesis and BAT-like activity in classical WAT depots along with the recruitment of BAT at specific anatomical sites. l-Menthol-induced BAT thermogenesis has been suggested to occur by a β-adrenergic-independent mechanism, avoiding potential side-effects due to extensive β-adrenergic stimulation mediated by available beta receptor agonists. l-Menthol has been also linked to the activation of the cold-gated ion channel TRPA1. However, its role in l-menthol-induced UCP1-dependent thermogenic activity in BAT and WAT remains undetermined. White adipose tissue plasticity has important clinical implications for obesity prevention and/or treatment because higher levels of UCP1-dependent thermogenesis can lead to enhanced energy expenditure at a considerable extent. We hypothesize that chronic dietary l-menthol treatment could induce TRPM8- and TRPA1-dependent WAT adaptations, resembling BAT-like activity, and overall improve whole-body metabolic health in obese and overweight individuals.ConclusionsThe putative impact of chronic l-menthol dietary treatment on the stimulation of BAT-like activity in classical WAT depots in humans remains unknown. A detailed experimental design has been proposed to investigate the hypothesized l-menthol-induced browning of WAT. If our hypothesis was to be confirmed, TRPM8/TRPA1-induced metabolic adaptations of WAT to BAT-like activity could provide a promising novel therapeutic approach for increasing energy expenditure, regulating body weight, and preventing obesity and its related co-morbidities in humans.     

Dissociation between brown adipose tissue thermogenesis and sympathetic activity in rats with high plasma levels of oestradiol

It has been shown previously that high plasma levels of oestradiol inhibit brown adipose tissue thermogenesis. Since rats and mice show a close association between thermogenic activity in and sympathetic discharge to brown fat, we measured the noradrenaline turnover in rats with high plasma levels of oestradiol to establish whether the observed inhibition of thermogenic activity is brought about by a reduction in the sympathetic drive to brown adipocytes. Oestradiol-filled Silastic capsules were implanted subcutaneously in female rats previously acclimated either to thermoneutrality or to cold. Control rats received empty implants. After 15 days treatment, noradrenaline turnover was measured by blocking its synthesis with -methyl-p-tyrosine. As expected, noradrenaline turnover was higher in cold-acclimated rats than in rats kept at thermoneutrality. The presence of high plasma oestradiol levels did not alter sympathetic activity in any of the treated groups despite reducing thermogenic activity. This result reveals that oestradiol dissociates the thermogenic activity of brown adipose tissue from its sympathetic activation. Such dissociation has never been previously reported in rats, although it seems to be common in Syrian hamsters. However the causative factor in this species is unknown.A preliminary report of this work was presented to the XXXIIth International Congress of Physiological Sciences, Glasgow, August 1993 and appears as abstract No. 287.6/P.     

Cold-induced thermogenesis in hypothyroid rats

Hypothyroidism was induced in adult rats by oral administration of methimazole. Euthyroid and hypothyroid rats were maintained at 23 °C or exposed at 6 °C for 3 weeks. Both euthyroid and hypothyroid rats maintained at 23 °C had similar interscapular brown adipose tissue (BAT) composition and thermogenic activity. Cold-exposed hypothyroid rats showed the same interscapular BAT mass and gross tissue composition as cold-exposed euthyroid animals, but the interscapular BAT of cold-exposed hypothyroid rats did not show the characteristic increase in GDP binding, and the increase in mitochondrial mass was lower than in euthyroid rats. From these results we conclude that thyroid hormones do not influence BAT significantly when thermogenic requirements are moderate, but they participate in the trophic response of the tissue when thermogenic requirements are intense. This thyroid hormone participation in the BAT trophic response occurs at the mitochondrial level, both in quantitative (mitochondrial mass) and qualitative (GDP-binding) aspects.     

Inactivation of brown adipose tissue thermogenesis by oestradiol treatment in cold-acclimated rats

Both cold-acclimated female rats and rats at thermoneutrality received 0.15–0.20 mg daily of 17-oestradiol over 15 days via a Silastic capsule implanted subcutaneously. Controls received empty implants. Comparison between the oestradiol-treated animals and the untreated controls revealed that at thermoneutrality, oestradiol treatment decreased food intage and body weight gain, but did not affect brown adipose tissue (BAT) thermogenesis and composition. By contrast, in cold-acclimated rats, oestradiol treatment did not modify food intake or body weight gain, but it decreased BAT thermogenesis. It is concluded that the effects of oestradiol treatment on BAT depend on the activity of the tissue, i.e. it has no effect on BAT when the tissue is thermogenically inactive, but it decreases cold-induced BAT thermogenesis. It is suggested that oestradiol could be the hormonal factor responsible for the previously observed inactivation of BAT thermogenesis during pregnancy in cold-acclimated rats.     

Brown adipose tissue of rats with obesity-inducing ventromedial hypothalamic lesions

Male and female Holtzman rats were made hyperphagic and obese with bilateral radiofrequency heat lesions of the ventromedial hypothalamic (VMH) area. When VMH rats were maintained at 28 degrees C, their brown adipose tissue (BAT) DNA, protein, and cytochrome oxidase contents were normal although more stored lipid was present, as judged from a threefold increase in wet weight. Thermogenic activity of BAT mitochondria was normal in male VMH rats, as judged from the unchanged level of guanosine diphosphate (GDP) binding (known to be a sensitive index of the functional activity of the thermogenic proton conductance pathway), and reduced in female VMH rats. When rats with VMH lesions were exposed to cold (4 degrees C for 24 h), the visible hyperemia of their BAT and normal large increase in mitochondrial GDP binding indicated normal thermogenic responsiveness. We conclude that the medial nuclei of the hypothalamus and associated afferent or efferent nerve tracts do not represent an essential central nervous system link for cold-induced, sympathetic-mediated activation of BAT thermogenesis. It is possible, however, that diet-induced, sympathetic-mediated activation of BAT function and growth might require an intact VMH region because no enhancement of BAT mitochondrial function normally associated with hyperphagia was detected in these hyperphagic VMH-lesioned animals.     

Oxygen consumption of oestradiol-treated rats

Rectal temperature and oxygen consumption (üüO₂) were monitored in female rats acclimated either to cold or to thermoneutrality and with and without chronic administration of oestradiol. The hormone is known to inactivate brown adipose tissue (BAT) and to reduce its response to noradrenaline (NA). The role of sympathetic control was studied by administering NA or the adrenergic blocker propranolol. Oestradiol treatment did not affect rectal temperature in the states of acclimation to thermoneutrality and to cold, nor did it change the hypothermic response of cold-exposed rats to temporary food deprivation. In the cold-acclimated rats, both controls and oestradiol-treated animals exhibited similar degrees of metabolic reduction after propranolol administration in the cold and similar degrees of metabolic activation by NA at thermoneutrality. Rats acclimated to thermoneutrality showed a larger metabolic response to NA when treated with oestradiol. The results suggest that oestradiol, while inactivating the BAT response to NA, activates the NA responsiveness of other metabolically active tissues in cold-induced thermogenesis. The observation of a greater oxidative capacity in the kidney and the rectus abdominis muscle of oestradiol-treated, cold-acclimated rats would be in line with this proposal.     

Reduced norepinephrine turnover in brown adipose tissue of ob/ob mice

Obese (ob/ob) mice have a lower thermogenic capacity than lean mice. The possible role of brown adipose tissue (BAT) in this defect was investigated. Lean and obese mice were exposed to 33 (thermoneutral), 25, or 14 degrees C for up to 3 wk. BAT cytochrome oxidase activity and numbers of Na+-K+-ATPase enzyme units, enzymes involved in thermogenesis, were similar at 33 or 25 degrees C. Chronic exposure to 14 degrees C increased these enzymes 34 and 62%, respectively, in lean mice and nearly 150% in obese mice. Sympathetic nervous system activity, which stimulates thermogenesis in BAT, was evaluated by measuring norepinephrine (NE) turnover. At 25 degrees C, NE turnover rate in BAT of obese mice was only 40% as rapid as in BAT of lean mice. Chronic exposure to 33 degrees C depressed NE turnover in BAT of lean mice, but not in obese mice, whereas exposure to 14 degrees C accelerated NE turnover in both lean and obese mice. Lower sympathetic nervous system activity in BAT of obese mice at 25 degrees C is likely a major factor in their reduced nonshivering thermogenesis and resultant high efficiency of energy storage.     

Effect of immobilization stress on in vitro and in vivo thermogenesis of brown adipose tissue.

Repetitive intermittent stress such as immobilization has been shown to induce an improved cold tolerance through an enhanced capacity of nonshivering thermogenesis (NST), causing positive cross adaptation between nonthermal stress and cold. In the present study, effect of 3-h-daily immobilization stress for 4-5 weeks was investigated on in vitro and in vivo thermogenesis of interscapular brown adipose tissue (BAT). In vitro thermogenesis was measured in the minced tissue blocks incubated in Krebs-Ringer phosphate buffer with glucose and albumin at 37 degrees C, using a Clark-type oxygen electrode. The stressed rats showed less body weight gain during the experiment. The BAT weight, its protein and DNA contents were significantly greater in the stressed rats. Basal, noradrenaline- and glucagon-stimulated oxygen consumptions were significantly greater in the stressed rats. In vivo thermogenesis was assessed by the changes of temperatures in colon (Tcol), BAT (TBAT), and tail skin (Tsk) induced by noradrenaline or glucagon infusion in the anesthetized rats. Noradrenaline and glucagon increased the TBAT and the extent of increase was greater in the stressed rats. These results indicate that cross adaptation between nonthermal stress and cold may be mediated through an enhanced thermogenic activity of BAT.     

Short photoperiod enhances thermogenic capacity in Brandt's voles

Environmental cues play important roles in the regulation of an animal's physiology and behavior. In the present study, we examined the effects of short photoperiod (SD) on body weight as well as on several physiological, hormonal, and biochemical measures indicative of thermogenic capacity to test our hypothesis that short photoperiod stimulates increases in thermogenesis without cold stress in Brandt's voles. SD voles showed increases in basal metabolic rate (BMR) and nonshivering thermogenesis (NST) during the 4-week photoperiod acclimation. At the end, these voles (SD) had lower body weights, higher levels of cytochrome C oxidase (COX) activity and mitochondrial uncoupling protein-1 (UCP1) contents in brown adipose tissues (BAT), and higher concentrations of serum tri-iodothyronine (T3) and thyroxine (T4) compared to LD voles. No differences were found between male and female voles in any of the above-mentioned measurements. Together, these data indicate that SD experience enhances thermogenic capacity similarly in males and females of Brandt's voles.     

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.     

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.