β3-Adrenergic regulation of leptin, food intake, and adiposity is impaired with age

With age, there are increases in adiposity, leptin mRNA in white adipose tissue (WAT), and serum leptin levels in rats. Beta3-adrenergic agonists are anti-obesity agents in rodents, and activation of beta3-adrenergic receptors (beta3AR) mediates an acute decrease in food consumption, increased thermogenesis in brown adipose tissue (BAT), increased lipolysis in WAT, and suppression of leptin gene expression and serum leptin levels. Because beta3AR signal transduction is impaired with age in BAT and WAT, beta3AR-mediated regulation of leptin, feeding behavior, and adiposity may also be impaired with age. To test this hypothesis, we infused young (6 month) and old (24 month) F344 x BN rats with the beta3AR agonist CL316,243 (1 mg kg(-1) day(-1)) using osmotic minipumps for 7 days. Food intake, body mass, adiposity, and serum leptin, as well as leptin, uncoupling protein 1 (UCP1), and lipoprotein lipase (LPL) mRNA in BAT or WAT were determined. In young rats, CL316,243 infusion reduced body mass and adiposity, suppressed serum leptin and leptin mRNA levels in WAT, induced UCP1 in WAT, and increased UCP1 and LPL mRNA levels in BAT. Moreover, treatment with CL316,243 was associated with a marked but transient suppression of food intake. Most of the responses elicited in the old rats with CL316,243 treatment were qualitatively similar to those in the young rats, but blunted in magnitude. Beta3AR activation of adenylyl cyclase was also reduced in the aged rats. These data suggest that, although CL316,243 is an effective agent in aged rats, the maximum responses are reduced, suggesting that the impaired beta3AR signal transduction with age is a major determining factor in the reduced effectiveness of CL316,243 in older rats.     

Leanness of Lou/C rats does not require higher thermogenic capacity of brown adipose tissue

Lou/C rats, an inbred strain of Wistar origin, remain lean throughout life and therefore represent a remarkable model of obesity resistance. To date, the exact mechanisms responsible for the leanness of Lou/C rats remain unknown. The aim of the present study was to investigate whether the leanness of Lou/C rats relies on increased thermogenic capacities in brown adipose tissue (BAT).Results showed that although daily energy expenditure was higher in Lou/C than in Wistar rats, BAT thermogenic capacity was not enhanced in Lou/C rats kept at thermoneutrality as demonstrated by reduced thermogenic response to norepinephrine in vivo, similar oxidative activity of BAT isolated mitochondria in vitro, similar levels of UCP1 mRNA and lower abundance of UCP1 protein in interscapular BAT depots. Relative abundance of β₃-adrenergic receptor mRNA was lower in Lou/C BAT while that of GLUT4, FABP or CPT1 was not altered. Activity-related energy expenditure was however considerably increased at thermoneutrality as Lou/C rats demonstrated an impressively high spontaneous running activity in voluntary running wheels. Prolonged cold-exposure (4 °C) depressed the spontaneous running activity of Lou/C rats while BAT thermogenic capacity was increased as reflected by rises in BAT mass, oxidative activity and UCP1 expression.It is concluded that the leanness of Lou/C rats cannot be ascribed to higher thermogenic capacity of brown fat but rather to, at least in part, increased locomotor activity. BAT is not deficient in this rat strain as it can be stimulated by cold exposure when locomotor activity is reduced suggesting some substitution between these thermogenic processes.     

Thermogenesis in brown adipose tissue with age: Post-receptor activation by forskolin

₃-Adrenergic-stimulated thermogenesis in brown adipose tissue (BAT) is diminished with age. ₃-Adrenergic receptors are positively coupled to adenylyl cyclase in BAT. To determine whether thermogenesis, in response to direct activation of adenylyl cyclase, is also impaired with age, we examined whole body oxygen consumption, mitochondrial guanosine diphosphate (GDP) binding and BAT mitochondrial uncoupling protein (UPC) mRNA levels in 4- and 24-month-old F-344 rats following forskolin administration. We also examined the forskolin-induced change in body temperature in 4-month-old rats. In some instances, the results were compared with administration of the specific ₃-adrenergic agonist, CGP-12177. Forskolin (3.5 mg/kg) increased oxygen consumption but decreased body temperature. In subsequent experiments the BAT was unilaterally denervated. In these rats, the forskolin-(1.8 mg/kg) stimulated increase in oxygen consumption was similar in young and old rats. Forskolin increased GDP binding and UCP mRNA levels in both the denervated and innervated BAT pads. The increases were equal or greater in the BAT from senescent rats. These findings, coupled with our previous report of an impaired CGP-12177-stimulated increase in GDP binding in senescent rats, suggests ₃-adrenergic-stimulated, but not post-receptor-stimulated, thermogenesis is diminished with age.     

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.     

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.     

Influence of Thyrotrophin-Releasing Hormone on Thermoregulatory Adaptation after Birth in Near-Term Lambs Delivered by Caesarean Section

We investigated the hypothesis that exogenous stimulation with thyrotrophin-releasing hormone (TRH) immediately prior to umbilical cord clamping can improve thermoregulatory adaptation after birth in near-term lambs delivered by Caesarean section. Lambs received an umbilical vein injection of saline +/- TRH (8 microg) prior to cord clamping. The rate of change in colonic temperature and oxygen consumption after birth were not influenced by TRH, but TRH-treated lambs exhibited a greater incidence of shivering compared with controls over the first hour of neonatal life. Two and a half hours after birth, TRH-treated lambs possessed brown adipose tissue (BAT) with a higher thermogenic activity (i.e. GDP binding to mitochondrial protein), but their BAT had a reduced DNA content and they had less hepatic glycogen than control lambs. TRH administration had no effect on iodothyronine 5' deiodinase activity in BAT and liver, or on plasma concentrations of total triiodothyronine, thyroxine, cortisol or free fatty acids. Three TRH-treated but no control lambs, failed to establish continuous breathing, so tissues from these treated lambs together with time-matched controls were sampled 25 min after birth. These 'non-surviving' TRH-treated lambs had very high plasma catecholamine concentrations, but their lung weights were similar to controls. 'Surviving' TRH-treated lambs possessed lungs with less DNA than non-surviving TRH-treated lambs. It is concluded that umbilical vein injection of TRH prior to umbilical cord clamping increases the recruitment of both shivering and non-shivering thermogenesis after birth.     

Effects of the beta-adrenergic agonist, ritodrine, and insulin on plasma potassium concentrations in fetal lambs.

Prolonged infusion of the beta 2-adrenergic agonist, ritodrine, into sheep during late pregnancy decreased maternal plasma K+ from 3.6 to 2.5 mmol l-1 during the first 6-8 h of infusion, as it does during tocolysis in women. This decrease was not accompanied by significant change in fetal plasma K+ concentration. Ritodrine infusion (1-3.5 micrograms kg-1 min-1) directly into the fetus also did not decrease fetal plasma K+ significantly. In contrast, insulin (2.5 mU kg-1 min-1), infused together with glucose (3.6 mg kg-1 min-1) directly into the fetus decreased fetal plasma K+ concentration by 0.8 mmol l-1 within 1 h. The results suggest immaturity in beta 2-adrenergic receptor regulation of electrogenic K+ uptake by muscle in fetal lambs.     

Yohimbine reduces neuropathology induced by ketamine/xylazine anesthesia.

Ketamine, an NMDA receptor antagonist, is commonly used in combination with xylazine, an alpha 2-adrenergic receptor agonist, to induce surgical anesthesia in birds and other vertebrates. Problems associated with this anesthetic combination include impaired thermoregulation, compounded by the inability to feed while anesthesia lasts (approximately 6 h after a single dose), and the ketamine-induced vacuolization of large cortical neurons. In the zebra finch, yohimbine (an alpha 2-adrenergic receptor agonist) counters the effects of ketamine/xylazine anesthesia, speeding recovery after the surgical procedure has been completed. In addition, administration of yohimbine reduces the formation of vacuoles in large cortical neurons and in neuropil. Yohimbine administration should be considered following all procedures involving ketamine/xylazine anesthesia.     

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.     

Comparison of DNA synthesis in white and brown adipose tissue in rats with ventromedial hypothalamic lesions

Ventromedial hypothalamic (VMH) lesions cause excessive fat accumulation in white adipose tissue (WAT), and brown adipose tissue (BAT); however, little information is available on whether or not cell proliferation occurs in WAT and BAT after VMH lesioning. In this study, we determined the DNA content and thymidine incorporation in unilateral parametrial WAT and interscapular BAT 0, 1, 3, and 7 days after VMH lesioning, and examined the mechanism of increased DNA content in WAT. In rats with VMH lesions, the weight of WAT and BAT had increased significantly at 7 days, and the DNA content and thymidine incorporation of WAT had increased significantly at 3 days and continued to increase for up to 7 days, while those of BAT did not increase for as long as 7 days after VMH lesioning. Restricted food intake according to the pair-feeding method partially inhibited the increased DNA content in WAT. The increased DNA content in WAT was mostly restored but not completely by the administration of anti-insulin antibody, and by administration of propranolol, a -adrenergic blocker. The results demonstrated that VMH lesions induced DNA synthesis in WAT early after VMH lesioning, but did not induce DNA synthesis in BAT, and suggested that either hyperinsulinemia or a -adrenergic receptor mechanism or both may be responsible for the increased DNA content in WAT.     

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.     

Effects of UCP3 genotype, temperature and muscle type on energy turnover of resting mouse skeletal muscle

Uncoupling protein 3 (UCP3) is a mitochondrial transporter protein which, when over-expressed in mice, is associated with increased metabolic rate, increased feeding and low body weight. This phenotype probably reflects the increased levels of UCP3 partially uncoupling mitochondrial respiration from cellular ATP demands. Consistent with that, mitochondria isolated from muscles of mice that over-express UCP3 are less tightly coupled than those from wild-type mice but the degree of uncoupling is not modulated by likely physiological regulatory factors. To determine whether this also applies to intact muscle fibres, we tested the hypothesis that UCP3 constitutively (i.e. in an unregulated fashion) uncouples mitochondria in muscles from mice that over-expressed human UCP3 (OE mice). The rate of heat production of resting muscles was measured in vitro using bundles of fibres from soleus and extensor digitorum longus muscles of OE, wild-type (WT) and UCP3 knock-out mice. At 20 degrees C, the only significant effect of genotype was that the rate of heat production of OE soleus (3.04+/-0.16 mW g(-1)) was greater than for WT soleus (2.31+/-0.05 mW g(-1)). At physiological temperature (35 degrees C), the rate of heat production was independent of genotype and equal to the expected in vivo rate for skeletal muscles of WT mice. We conclude that at 35 degrees C, the transgenic UCP3 was not constitutively active, but at 20 degrees C in slow-twitch muscle, it was partially activated by unknown factors. The physiological factor(s) that activate mitochondrial uncoupling by UCP3 in vivo was either not present or inactive in resting isolated muscles.     

Orexin-a regulates body temperature in coordination with control of arousal state

Orexin-a regulates body temperature in coordination with control of arousal state     

Uncoupling protein 3 expression and intramyocellular lipid accumulation by NMR following local burn trauma

Burn trauma is a clinical condition accompanied by muscle wasting that severely impedes rehabilitation in burn survivors. Mitochondrial uncoupling protein 3 (UCP3) is uniformly expressed in myoskeletal mitochondria and its expression has been found to increase in other clinical syndromes that, like burn trauma, are associated with muscle wasting (e.g., starvation, fasting, cancer, sepsis). The aim of this study was to explore the effects of burn trauma on UCP3 expression, intramyocellular lipids, and plasma-free fatty acids. Mice were studied at 6 h, 1 d and 3 d after nonlethal hindlimb burn trauma. Intramyocellular lipids in hindlimb skeletal muscle samples collected from burned and normal mice were measured using 1H NMR spectroscopy on a Bruker 14.1 Tesla spectrometer at 4 degrees C. UCP3 mRNA and protein levels were also measured in these samples. Plasma-free fatty acids were measured in burned and normal mice. Local burn trauma was found to result in: 1) upregulation of UCP3 mRNA and protein expression in hindlimb myoskeletal mitochondria by 6 h postburn; 2) increased intramyocellular lipids; and 3) increased plasma-free fatty acids. Our findings show that the increase in UCP3 after burn trauma may be linked to burn-induced alterations in lipid metabolism. Such a link could reveal novel insights into how processes related to energy metabolism are controlled in burn and suggest that induction of UCP3 by burn in skeletal muscle is protective by either activating cellular redox signaling and/or mitochondrial uncoupling.     

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.     

Differential peripheral expansion and in vivo antigen reactivity of alpha/beta and gamma/delta T cells emigrating from the early fetal lamb thymus

The concentrations of different lymphocyte subsets in the blood of lambs which had been thymectomized (Tx) in utero between days 67-75 of fetal gestation were measured at birth and at various intervals during the first year of life. Compared to thymus-intact (Ti) controls, Tx lambs were severely depleted of both alpha/beta and gamma/delta T cells at birth (less than 10% of control levels). The majority of the residual alpha/beta T cells present in Tx lambs at birth were CD4+CD8-. As the Tx lambs aged, the concentration of alpha/beta T cells in blood increased steadily to reach levels around 50% of control values. In contrast, the circulating gamma/delta T cells did not expand in Tx animals and remained barely detectable throughout the observation period, although these cells accounted for 30%-60% of the T cells in the blood of Ti lambs. The expansion of alpha/beta but not gamma/delta T cells was also reflected in changes in the cellular composition of solid lymphoid organs in Tx lambs. B cell numbers were similar in both groups at birth but Tx lambs were persistently B lymphopenic from 3 weeks of age onwards. The alpha/beta T cells that had expanded in Tx lambs responded to stimulation with bacterial antigens in a way that was qualitatively similar to the response in Ti lambs. By contrast, the few gamma/delta T cells in Tx lambs responded abnormally. Our results show that although sheep alpha/beta and gamma/delta T cells are equally thymus dependent during ontogeny, the early fetal thymic emigrants which establish the two T cell lineages in the periphery have strikingly different antigen reactivities and capacities for self-renewal and expansion.     

Functional and anatomical characterization of brown adipose tissue in heart failure with blood oxygen level dependent magnetic resonance

Recent studies have suggested that brown adipose tissue (BAT) plays an important role in obesity, insulin resistance and heart failure. The characterization of BAT in vivo, however, has been challenging. No technique to comprehensively image BAT anatomy and function has been described. Moreover, the impact on BAT of the neuroendocrine activation seen in heart failure has only recently begun to be evaluated in vivo. The aim of this study was to use MRI to characterize the impact of heart failure on the morphology and function of BAT. Mice subjected to permanent ligation of the left coronary artery were imaged with MRI 6 weeks later. T₂ weighted MRI of BAT volume and blood oxygen level dependent MRI of BAT function were performed. T₂* maps of BAT were obtained at multiple time points before and after administration of the β₃ adrenergic agonist CL 316 243 (CL). Blood flow to BAT was studied after CL injection using the flow alternating inversion recovery (FAIR) approach. Excised BAT tissue was analyzed for lipid droplet content and for uncoupling protein 1 (UCP1) mRNA expression. BAT volume was significantly lower in heart failure (51 ± 1 mm³ versus 65 ± 3 mm³; p < 0.05), and characterized by a reduction in lipid globules and a fourfold increase in UCP1 mRNA (p < 0.05). CL injection increased BAT T₂* in healthy animals but not in mice with heart failure (24 ± 4% versus 6 ± 2%; p < 0.01), consistent with an increase in flow in control BAT. This was confirmed by a significant difference in the FAIR response in BAT in control and heart failure mice. Heart failure results in the chronic activation of BAT, decreased BAT lipid stores and decreased BAT volume, and it is associated with a marked decrease in ability to respond to acute physiological stimuli. This may have important implications for substrate utilization and overall metabolic homeostasis in heart failure. Copyright © 2016 John Wiley & Sons, Ltd.