As alluded to above, adipose tissue is heterogeneous, containing numerous cell types. White adipocytes are unilocular, containing one large lipid droplet and few mitochondria, whereas brown adipocytes are multilocular and have numerous mitochondria Cousin et al.

More recently, a third adipocyte, the beige cell has been identified Himms-Hagen et al. Beige adipocytes exhibit an intermediary phenotype and are referred to as paucilocular, as these cells contain more than one lipid droplet and multiple mitochondria Himms-Hagen et al.

Brown and beige adipocytes display distinct genetic fingerprints and importantly unlike brown adipocytes that show high basal expression of thermogenic genes such as UCP1 , beige adipocytes only exhibit these genes in response to activating stimuli including cold and β-adrenoceptor agonists Walden et al.

In addition to UCP1-dependent thermogenesis, beige adipocytes produce heat through futile creatine Fig. In mice, proteomic analyses revealed a beige adipocyte-specific arginine-creatine metabolic pathway Kazak et al.

Futile creatine cycling is important in beige adipocyte thermogenesis in ADP-depleted states, wherein this pathway drives the hydrolysis of ATP and thus increases oxygen consumption Kazak et al.

Cold exposure for 1 week increased the expression of both creatine kinase U-type, mitochondrial Ckmt1 and creatine kinase S-type, mitochondrial Ckmt2 in inguinal beige adipocytes, indicative of an upregulation in creatine cycling.

Furthermore, treatment with the β3-adrenoceptor agonist, CL , induced Ucp1 -expressing beige adipocytes in the inguinal fat depot, as well as Ucp1 -negative, Ckmt2 -positive beige adipocytes in epididymal fat Bertholet et al.

Murine beige adipocytes also produce heat via futile calcium cycling and the activation of SERCA2b Ikeda et al. Inhibition or downregulation of SERCA2b in inguinal adipose tissue attenuates the noradrenaline-induced increase in oxygen consumption Ikeda et al.

To date, the vast majority of studies have utilised UCP1 protein or mRNA expression as a marker for beige adipocytes, and thus the abundance and importance of these cells have likely been underestimated.

It is now apparent that multiple pathways contribute to thermogenesis in these unique beige cells. The role of beige adipocytes in determining thermogenesis and energy expenditure in larger mammals including sheep and pigs, however, requires further interrogation. Schematic diagram depicting the futile creatine cycle.

Within mitochondria, thermogenesis can occur in the ADP-depleted state via creatine cycling. Creatine is phosphorylated by creatine kinase CK and then dephosphorylated. The responsible phosphatase is currently unidentified.

The process of creatine dephosphorylation is thermogenic via the hydrolysis of ATP. Control of adaptive thermogenesis is mediated by the hypothalamus, and while only explained briefly here, has been well detailed by Morrison Morrison In rodents, when exposed to cold, thermosensory inputs act via the median preoptic area to stimulate the dorsomedial hypothalamus DMH to increase sympathetic nerve activity to BAT through the rostral raphe pallidus rRPa Hermann et al.

Development of Designer Receptor Exclusivity Activated by Designer Drugs DREADD technology has allowed for further characterisation of these temperature-sensitive neurons. Use of DREADDS suggests that activation of GABA neurons in the preoptic area has little effect on body temperature or energy expenditure Yu et al.

Indeed, these studies show that leptin-receptor-expressing neurons in the preoptic area are integral to ambient temperature-induced metabolic food intake and energy expenditure adaptations Yu et al.

Within the preoptic area, there is clearly topographical neuronal organisation as cold exposure increases c-Fos levels in GABA neurons within the ventral part of the lateral preoptic area Zhao et al. Optogenetic inhibition of this subset of GABA neurons causes hyperthermia, whereas activation of the same reduces body temperature Zhao et al.

Thus, within the preoptic area, there is an integrated network of neurons, including both GABA-ergic and leptin responsive cells, capable of sensing changes in skin temperature and modifying thermogenic output.

In addition to the aforementioned temperature-sensitive pathway, metabolic factors such as leptin, insulin and ghrelin modulate thermogenic activity via hypothalamic appetite-regulating peptides.

Blood-borne factors can diffuse across the blood brain barrier via fenestrated capillaries and act directly on neurons in the arcuate nucleus Banks Importantly, two distinct sets of neurons are found in the arcuate nucleus, being either orexigenic or those that elicit satiety.

The POMC neurons are activated by leptin Elias et al. A second population of neurons contain neuropeptide Y NPY and agouti-related protein AgRP , which stimulate food intake in response to direct stimulation by ghrelin Kamegai et al.

NPY exerts an immediate effect to stimulate food intake, primarily via action at Y1 receptors in the PVN Kask et al. On the other hand, AgRP acts as an inverse agonist at the MC4R to stimulate food intake Nijenhuis et al.

Hypothalamic appetite-regulating peptides exert reciprocal control to modulate food intake and energy expenditure, in particular BAT and muscle thermogenesis Verty et al.

Indeed, pseudorabies-tracing studies show that appetite-regulating neurons of the hypothalamus ultimately project to neural networks controlling sympathetic outflow to peripheral tissues including BAT Bamshad et al.

As mentioned earlier, activation of the SNS and the release of catecholamines, in particular, noradrenaline are fundamental to BAT thermogenesis. Indeed, genetic deletion of all three β-adrenergic receptors βAR in brown adipocytes of mice causes profound obesity by negating thermogenesis Bachman et al.

Interestingly, in humans, isoprenaline a non-specific βAR treatment increases energy expenditure without an associated activation of BAT Vosselman et al. Similarly, blockade of the βAR with propranolol had no effect on cold-induced BAT thermogenesis in humans Wijers et al.

This lack of effect, however, is likely due to receptor specificity as both isoprenaline and propranolol show preferential agonistic and antagonistic affinity to the β1AR and β2AR, respectively. Indeed, in healthy lean men, administration of the β3AR-specific agonist, mirabregon, activates BAT and causes a concurrent increase in resting metabolic rate Cypess et al.

Together, these studies highlight that in humans, the β3AR is essential to catecholamine-mediated BAT thermogenesis. In addition to catecholamines, thyroid hormones TH are notable endocrine regulators of BAT activity.

Brown adipocytes contain the deiodinase type 2 DIO2 enzyme, allowing for local conversion of thyroxine T4 to triiodothyronine T3 Carvalho et al. In rodents, TH act directly at nuclear thyroid hormone receptors located in brown adipocytes to transcriptionally upregulate UCP1 expression Weiner et al.

Furthermore, clinical data demonstrate that BAT activity is higher in the subclinical hyperthyroid state than in the hypothyroid state Broeders et al.

Although the classical action of T3 is thought to be peripherally mediated, more recent studies have shown that TH can also act centrally within the hypothalamus to regulate BAT thermogenesis.

Tanycytes in the mediobasal hypothalamus express DIO2 and thus convert T4 to T3 Coppola et al. Furthermore, in mice, intracerebroventricular administration of T3 increases BAT thermogenesis via reduced hypothalamic levels of AMP kinase AMPK and subsequent activation of the SNS Lopez et al.

Indeed, sub-chronic 6 days central administration of T3 leads to browning of WAT in mice Alvarez-Crespo et al. To date, much of the work defining the regulation of thermogenesis and its contribution to energy balance has been in rodents.

This has provided invaluable information and understanding of the neuroendocrine mechanisms that control thermogenesis. More recently, a number of large animal models have been employed including pigs and sheep, which provide further insight into the role of thermogenesis in long-term regulation of body weight in mammalian species.

It is well recognised that pigs lack a functional UCP1 protein Hou et al. Pigs, specifically those belonging to the Suidae species, do not have exons 3—5 of the UCP1 gene, rendering animals prone to hypothermia-induced death as neonates Berg et al.

With only exons 1 and 2, UCP1 can still be transcribed; however, protein translation does not occur Hou et al. Hence, previous histological studies failed to detect UCP1 protein immunoreactivity at baseline Rowlatt et al. It has since been proposed that pigs do indeed possess functional BAT; however, adaptive thermogenesis occurs via UCP1-independent mechanisms Ikeda et al.

Indeed, recent work comparing cold-tolerant Tibetan pigs to cold-sensitive Bama pigs has provided direct evidence of adaptive thermogenesis in subcutaneous sWAT and perirenal WAT Lin et al.

Furthermore, morphological studies show that in response to cold exposure, subcutaneous adipose tissue displays evidence for beige cell recruitment with an increase in multilocular adipocytes, increased mitochondrial DNA copy number and increased expression of PGC1a and the beige cell marker CD Wu et al.

Furthermore, in Tibetan pigs cold tolerant , cold exposure increased the expression of the UCP3 gene and protein in isolated subcutaneous adipocytes and this is associated with increased uncoupled respiration, providing evidence to suggest an increase in UCP3-driven thermogenesis Lin et al.

Role of thermogenesis in determining cold tolerance in pigs. Tibetan pigs are cold tolerant and this coincides with the recruitment of beige adipocytes in subcutaneous WAT in response to cold exposure.

Although pigs do not that express functional uncoupling protein UCP 1, adipocytes exhibit UCP3 and this mediates mitochondrial uncoupling and adipose tissue thermogenesis. The contribution of UCP3 to brown fat thermogenesis has been contentious and appears to be dependent on the species studied.

In mice, earlier work suggested that BAT thermogenesis was dependent on UCP1 Matthias et al. Despite this, hamsters that lack functional UCP3 specifically in brown adipocytes have increased propensity to weight gain, which is indicative of a reduction in energy expenditure Fromme et al.

Although innate differences in UCP3 expression in adipose tissue of pigs have been linked to cold tolerance, to date, there are no data on BAT-specific UCP3 function and the control of body weight in this species.

In addition to UCP3-associated uncoupling and thermogenesis, recent data suggest that SERCA-driven beige cell thermogenesis also occurs in pigs. Indeed, the work by Ikeda et al. Ikeda et al. Retroviral expression of PRDM in subcutaneous porcine adipocytes increases the expression of beige-cell-specific markers including CIDEA and TMEM26 Ikeda et al.

Furthermore, decreased SERCA2b expression reduced basal and noradrenaline-induced oxygen consumption and extracellular acidification rates in isolated pig adipocytes Ikeda et al.

Thus, it is now clear that adipose tissue thermogenesis and the associated energy expenditure are not solely mediated via UCP1 and mitochondrial uncoupling, but in fact, a number of cellular pathways, across both adipose tissue and skeletal muscle, act in concert to determine total thermogenic potential.

In lambs, the expression of UCP1 is maximal in perirenal adipose tissue on the first postnatal day, rapidly declining with the expansion of WAT Symonds , Pope et al.

Mapping of UCP1 mRNA in lambs shows abundant expression in sternal and retroperitoneal adipose depots compared to omental fat, which is a predominantly WAT depot Symonds et al.

Indeed, adult sheep retain UCP1 expression in both sternal and retroperitoneal fat and this coincides with post-prandial heat production, albeit this response is greater in the sternal fat depot Henry et al.

This coincides with the expression of UCP1 protein, where UCP1-positive brown-like adipocytes were only detectable in sternal adipose tissue of adult ewes Henry et al. Data logger temperature probes have been employed to measure longitudinal heat production in multiple tissues to index thermogenic output in sheep.

Sheep are a grazing species and therefore do not display typical meal-associated excursions such as changes in ghrelin secretion. Despite this, temporal food restriction in sheep entrains a pre-prandial rise in ghrelin Sugino et al. Furthermore, post-prandial thermogenesis in both skeletal muscle and retroperitoneal adipose depots is markedly enhanced by intracerebroventricular infusion of leptin Henry et al.

Thus, in spite of relatively low levels of UCP1 in adult sheep, skeletal muscle and specific adipose depots retain thermogenic capacity. Over recent years, we have utilised the sheep to dissect the differential roles of adipose tissue and skeletal muscle thermogenesis in the long-term control of body weight, which is discussed in detail in the following section.

Similar to other species, ovine body weight can be readily manipulated through dietary management Henry et al. Sheep are ruminants and thus body weight is increased through feeding a high-energy diet enriched in lupin grain and oats.

Diet-induced obesity, however, is not associated with any change in heat production in adipose tissues or skeletal muscle of sheep Henry et al. On the other hand, long-term food restriction and low body weight are associated with a homeostatic decrease in thermogenesis in sternal and retroperitoneal adipose tissue and skeletal muscle Henry et al.

Importantly, similar to humans, the reduction in thermogenesis caused by food restriction and low body weight is still evident at one year post-weight loss, which suggests that homeostatic changes in thermogenesis contribute to impaired weight loss and increased long-term weight regain Henry et al.

Effect of chronic food restriction and weight loss on adaptive thermogenesis in ewes. Tissue temperature recordings show that caloric restriction and low body weight cause a homeostatic decrease in night time thermogenesis in ovariectomised ewes. This metabolic adaptation occurs in both sternal adipose tissue adipose tissue enriched in uncoupling protein 1 and skeletal muscle and to a lesser extent in retroperitoneal adipose tissue.

The reduction in thermogenesis is associated with increased expression of neuropeptide Y NPY in the arcuate nucleus and melanin-concentrating hormone MCH in the lateral hypothalamus.

The homeostatic reduction in thermogenesis is coordinated by the hypothalamus. Long-term weight loss in ovariectomised ewes increases the expression of the orexigenic neuropeptides NPY in the arcuate nucleus and melanin-concentrating hormone MCH in the lateral hypothalamus LH to increase hunger and reduce energy expenditure Henry et al.

Regarding the anorexigenic melanocortin pathway, the effect of low body weight on the expression of POMC is controversial with data showing a decrease Backholer et al. This is not surprising since POMC is the precursor to multiple neuropeptides, only one of which includes aMSH and the ultimate end product is dependent on post-translational processing Mountjoy On the other hand, increased Agrp and Npy expression and reduced Pomc mRNA have been observed in rodents Bi et al.

Thus, weight-loss-induced changes in hypothalamic gene expression are likely to reduce thermogenesis, whilst causing a concurrent increase in hunger drive. This represents a homeostatic mechanism to protect against weight loss and promote weight regain in calorie-restricted individuals.

Animals were originally selected for innate differences in adiposity by measuring back fat thickness and two lines were created via selective breeding strategies. A key feature of the genetically lean and obese sheep is an inherent difference in the growth hormone GH axis, where lean animals have increased mean GH concentration in plasma and an associated increase in pituitary gland weight Francis et al.

The increase in pituitary gland weight is primarily due to a greater number of cells in the lean animals Francis et al.

Furthermore, expression of GH and the GH secretagogue receptor GHSR is greater in genetically lean sheep, indicating differential responses to ghrelin, an agonist of the GHSR French et al.

This suggests that innate differences in the set-point of the GH axis may underpin differences in adiposity in the genetically lean and obese sheep; however, this is only one aspect that could contribute to this phenotype.

Interestingly, food intake is similar in genetically lean and obese sheep as is the expression of POMC, Leptin Receptor and NPY in the arcuate nucleus. On the other hand, lean animals have elevated post-prandial thermogenesis in retroperitoneal adipose tissue and this coincides with increased expression of UCP1 in this tissue Henry et al.

The divergence in thermogenesis is specific to adipose tissue since post-prandial thermogenesis is similar in genetically lean and obese animals Henry et al. Despite similar expression of appetite-regulating peptides in the arcuate nucleus of the hypothalamus, genetically lean sheep have increased expression of MCH and pre-pro-orexin ORX in the LH compared to obese animals Anukulkitch et al.

While both neuropeptides are considered orexigenic Shimada et al. Deletion of MCH in mice results in hypophagia and a lean phenotype Shimada et al. Orexin is critical in the embryonic development of BAT in mice Sellayah et al.

Thus, increased expression of ORX in the LH of lean sheep may be an important physiological determinant of increased thermogenesis in retroperitoneal fat and the associated changes in adiposity.

It is widely recognised that there is marked variation in the glucocorticoid response to stress or activation of the hypothalamo-pituitary adrenal HPA axis Cockrem , Walker et al.

The activity of the HPA axis in response to stress is impacted on by age Sapolsky et al. Nonetheless, in any given population individuals can be characterised as either high HR or low LR glucocorticoid responders Epel et al. It is important to note that female LR and HR sheep have similar basal plasma cortisol concentration and divergence in glucocorticoid secretion only occurs in response to ACTH or stress Lee et al.

Previous studies have suggested that obesity itself causes perturbation of the HPA axis with impaired glucocorticoid-negative feedback Jessop et al. Furthermore, cortisol directly impacts on metabolic function; however, this will not be addressed in the current review.

Initial studies in rams show that high cortisol response to adrenocorticotropin ACTH is associated with lower feed-conversion efficiency Knott et al.

Furthermore, in rams, adiposity is correlated to cortisol responses to ACTH Knott et al. More recent work shows that identification of high HR and low LR cortisol responders in female sheep can predict altered propensity to gain weight when exposed to a high-energy diet, where HR gain more adipose tissue than LR Lee et al.

Thus, at least in female sheep, data suggest that cortisol responses can be used as a physiological marker that predicts propensity to become obese. Previous studies in women suggest that HR eat more after a stressful episode than LR Epel et al.

Furthermore, HR individuals display preference for foods of high fat and sugar in response to psychological stress Tomiyama et al. Similarly, in ewes, baseline food intake is similar in LR and HR, but HR eat more following either psychosocial barking dog or immune lipopolysaccharide exposure stressors Lee et al.

In addition to altered food intake, HR ewes have reduced thermogenesis in skeletal muscle only; in response to meal feeding, post-prandial thermogenesis in skeletal muscle is greater in LR than in HR Lee et al. This again exemplifies divergence in the control of adipose tissue and skeletal muscle thermogenesis Fig.

Schematic depiction of the altered metabolic phenotype in animals selected for either high or low cortisol responsiveness. Sheep are characterised as either high HR or low LR cortisol responders when given a standardised dose of adrenocorticotropic hormone.

Animals characterized as HR have increased propensity to become obese, which is associated with perturbed control of food intake and reduced energy expenditure. Post-prandial thermogenesis in skeletal muscle is decreased in HR compared to LR ewes.

Furthermore, food intake in response to stress is greater in HR than in LR and the former are resistant to the satiety effect of alpha-melanocyte stimulating hormone aMSH.

High-cortisol-responding animals have reduced expression of the melanocortin 4 receptor MC4R in the paraventricular nucleus of the hypothalamus PVN. We propose that the decreased levels of MC4R underpin the altered metabolic phenotype and increased propensity to become obese when compared to LR.

For example, at baseline in the non-stressed resting state, HR individuals show an overall upregulation of the HPA axis, with increased expression of CRF and arginine vasopressin, but reduced expression of oxytocin in the PVN Hewagalamulage et al.

In addition to altered expression of genes within the HPA axis, a key neuroendocrine feature of the LR and HR animals is altered expression of the MC3R and MC4R in the PVN Fig. Reduced MC4R expression coincides with the development of melanocortin resistance.

Central infusion of leptin reduces food intake in both LR and HR animals, but intracerebroventricular infusion of aMSH reduces food intake in LR only. Thus, reduced MC4R expression appears to be central to the metabolic phenotype of HR that confers increased propensity to become obese in HR individuals Fig.

Interestingly, gene expression of NPY , AgRP and POMC in the arcuate nucleus is equivalent in LR and HR Hewagalamulage et al. Hence, differences in the control of food intake and thermogenesis are most likely manifest at the level of the melanocortin receptor. Indeed, previous work in sheep has shown the MC4R to be central in mediating the reduction in food intake caused by immune challenge Sartin et al.

Furthermore, in rodents, direct injection of the melanocortin agonist melanotan II into the ventromedial nucleus of the hypothalamus increases skeletal muscle thermogenesis Gavini et al.

We propose that reduced expression of the MC4R in HR animals underpins the metabolic phenotype wherein food intake is relatively increased in response to stress and reduced post-prandial thermogenesis in skeletal muscle is associated with propensity to become obese.

Historically, thermogenesis was considered to primarily occur in brown adipocytes and was solely driven by UCP1. It is now recognised that beige adipocytes and skeletal muscle also contribute to total thermogenic capacity and that thermogenesis is differentially regulated in these tissues.

Indeed, in beige adipocytes, thermogenesis occurs via three distinct mechanisms, with these being UCP1-driven mitochondrial uncoupling, futile creatine cycling and futile calcium cycling. On the other hand, in skeletal muscle, thermogenesis is associated with UCP3 and futile calcium cycling.

Unlike rodents, large mammals including sheep and pigs do not contain a defined or circumscribed brown fat depot but have dispersed brown adipocytes within traditionally white fat depots. Large animals have provided invaluable insight into alternative mechanisms of thermogenesis.

The sheep has been particularly useful in delineating the differential role of adipose tissue and skeletal muscle in the control of body weight. Furthermore, sheep models have allowed characterisation of the neuroendocrine pathways that may contribute to altered thermogenesis.

We have shown that in sheep, both skeletal muscle and BAT differentially contribute to thermogenesis and therefore total energy expenditure. Changes in thermogenesis, however, do not exclusively associate with altered gene expression at the level of the arcuate nucleus.

Indeed, decreased MC4R expression in HR animals and reduced orexin expression in the genetically obese animals coincide with altered thermogenic output. This review highlights the importance of the use of large animal models to ascertain the contribution and control of thermogenesis in multiple tissues and the relative role in the regulation of body weight.

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review. This work was supported by Australian Research Council grant number DP and National Health and Medical Research Council grant number APP Animal Science 63 — Journal of Pathology and Bacteriology 91 — Obesity Reviews 19 — Molecular Metabolism 5 — Neuroendocrinology 91 — Biochimica et Biophysica Acta — Astrup A Thermogenesis in human brown adipose tissue and skeletal muscle induced by sympathomimetic stimulation.

Acta Endocrinologica Supplement S9 — S American Journal of Physiology E — E Science — Neuroendocrinology 91 27 — Journal of Biological Chemistry — Bal NC , Maurya SK , Sopariwala DH , Sahoo SK , Gupta SC , Shaikh SA , Pant M , Rowland LA , Bombardier E , Goonasekera SA , et al.

Nature Medicine 18 — Journal of Neuroendocrinology 29 e Balthasar N , Dalgaard LT , Lee CE , Yu J , Funahashi H , Williams T , Ferreira M , Tang V , McGovern RA , Kenny CD , et al. Cell — American Journal of Physiology R — R Banks WA Characteristics of compounds that cross the blood-brain barrier.

BMC Neurology 9 S3 — S3. American Journal of Physiology: Endocrinology and Metabolism E — E Disease Models and Mechanisms 9 — Cellular and Molecular Life Sciences 73 — PLoS Genetics 2 e Cell Metabolism 25 e — Lancet — Circulation Research — American Journal of Physiology: Regulatory, Integrative and Comparative Physiology R — R International Journal of Obesity and Related Metabolic Disorders 17 Supplement 3 S78 — S Blondin DP , Daoud A , Taylor T , Tingelstad HC , Bezaire V , Richard D , Carpentier AC , Taylor AW , Harper ME , Aguer C , et al.

Journal of Physiology — International Journal of Obesity 37 — PLoS ONE 11 e Progress in Neuro-Psychopharmacology and Biological Psychiatry 35 — Metabolism 64 — Physiology Reviews 84 — Carey AL , Pajtak R , Formosa MF , Van Every B , Bertovic DA , Anderson MJ , Eikelis N , Lambert GW , Kalff V , Duffy SJ , et al.

Diabetologia 58 — Endocrinology — Cinti S The adipose organ: morphological perspectives of adipose tissues.

Proceedings of the Nutrition Society 60 — Claret M , Smith MA , Batterham RL , Selman C , Choudhury AI , Fryer LG , Clements M , Al-Qassab H , Heffron H , Xu AW , et al.

Journal of Clinical Investigation — Clement K , Vaisse C , Lahlou N , Cabrol S , Pelloux V , Cassuto D , Gourmelen M , Dina C , Chambaz J , Lacorte JM , et al. Nature — Cockrem JF Individual variation in glucocorticoid stress responses in animals.

General and Comparative Endocrinology 45 — Diabetes 64 — Coppola A , Liu Z , Andrews Z , Paradis E , Roy M-C , Friedman JM , Ricquier D , Richard D , Horvath TL , Gao X-B , et al. Cell Metabolism 5 21 — Journal of Cell Science — Neuron 24 — Clinical Science 69 — Cypess AM , Lehman S , Williams G , Tal I , Rodman D , Goldfine AB , Kuo FC , Palmer EL , Tseng Y-H , Doria A , et al.

New England Journal of Medicine — Cypess AM , Weiner LS , Roberts-Toler C , Elia EF , Kessler SH , Kahn PA , English J , Chatman K , Trauger SA , Doria A , et al.

Cell Metabolism 21 33 — Nature Medicine 19 — Dalgaard K , Landgraf K , Heyne S , Lempradl A , Longinotto J , Gossens K , Ruf M , Orthofer M , Strogantsev R , Selvaraj M , et al. Bioscience Reports 25 — Neuron 23 — Psychoneuroendocrinology 26 37 — Farooqi IS Monogenic human obesity.

Frontiers of Hormone Research 36 1 — New Zealand Journal of Agricultural Research 41 — However, despite their large fraction of fast-twitch fibers, even small rodents make use of muscle NST. As demonstrated by Jensen et al. Further, Pant et al. Thus, the fiber type composition of skeletal muscles in small mammals is flexible and can be adjusted to thermoregulatory requirements.

Currently, there are, however, insufficient studies on a large enough range of species of different size to obtain a clear picture of the impact of body mass on muscle NST capacity. The term endothermy is often taken to imply a pattern of homeothermic endothermy, i.

However, many endothermic mammals and birds are actually abandoning homeothermy during challenging periods and undergo heterothermic phases during which they reduce T b and metabolic rate in a state of torpor Ruf and Geiser, Up to date at least species of heterothermic mammals and birds have been identified Ruf and Geiser, and it is now widely accepted that heterothermy is a plesiomorphic, ancient trait from which homeothermy has evolved Grigg et al.

The mammalian ancestor was likely a small, nocturnal insectivorous animal that regularly used torpor Luo et al. In placental heterotherms UCP1 plays an important role during rewarming from torpor Nedergaard and Cannon, A study on UCP1-ablated mice has shown that although the lack of UCP1-mediated NST does not impair the expression of a full torpor bout i.

However, the mice were kept at warm conditions prior to the experiment, which prevents the cold-induced increase of muscle NST reported in later studies Bal et al. Therefore, animals likely had to primarily rely on shivering thermogenesis and were not able to use muscle NST for rewarming.

It would be interesting to see if cold acclimatization of animals prior to the experiment would lead to a differing result. Nevertheless, these data suggest that uncoupling of the proton gradient in BAT might have evolved to allow for a more rapid arousal and reduced energetic costs for rewarming, because slow rewarming increases the time spent at high metabolic rates Oelkrug et al.

If UCP1-ablated mice have lower rewarming rates this should also be the case for monotremes and marsupials. Unfortunately, there is no comprehensive comparison between rewarming rates of marsupiala and placentalia that also take into account differences in T a and T b.

Among known heterothermic mammals, i. These subzero T b s are known from at least eight species Ruf and Geiser, , e. Low tissue temperatures pose a problem because of Arrhenius effects, i. These Arrhenius effects may hamper, or at least significantly slow down, rewarming to euthermia.

Theoretically, this problem could be overcome by local heating of a small thermogenic tissue, i. This is one of the properties of BAT and therefore BAT is likely not only increasing the speed of arousals from torpor, but was also important for rewarming from torpor at low T b s.

Even if the temperatures at earth were warmer at the time of BAT evolution, animals will still have experienced daily and yearly fluctuations, similar to daily fluctuations in tropical habitats seen today.

Not surprisingly then, the local heating of BAT can be even detected by thermal imaging of skin e. Arguably then, the evolution of BAT was especially beneficial for heterothermic placental mammals, as it allowed them to tolerate lower levels of T b as a result of the enormous reduction of metabolic rate during hibernation and torpor Ruf and Geiser, This likely enhanced adaptive radiation of placental mammals and their ability to overwinter in the north-temperate and arctic zones.

These are climates that are significantly colder than those inhabited by marsupials and monotremes, among which a preference for warm habitats ranging from rainforests to deserts is an ancestral trait Mitchell et al.

If the evolution of BAT indeed facilitated the use of hibernation, the lack of BAT in birds may help to explain why there is only a single bird species known to truly hibernate Jaeger, ; Woods and Brigham, , although a number of birds show shallow daily torpor Ruf and Geiser, This suggests that the bird thermoregulatory phenotype, compared with the typical small mammal, is characterized by a high degree of homeothermy, little heterothermy, and high exercise performance.

Given their lack of BAT, it seems surprising then that muscle NST, or a combination of muscle NST and shivering, is sufficient to allow many birds to maintain very high 42°C T b even during cold exposure.

In cold-acclimated ducklings skeletal-muscles were identified as the major site of NST Duchamp and Barre, The fact that muscle NST seems more efficient in birds might be partially related to the better insulation of feathers in comparison with mammalian hair Aschoff, While birds can decrease their conductance enormously during cold exposure due the air trapped in the rigid feather structure, mammalian hair is softer and less suitable to trap air as an insulation barrier McNab, However, we suggest that there is another main reason for a lack of selective advantages of a BAT-like tissue in birds, which—to our knowledge—has never been considered before: skeletal muscles in birds already reach metabolic rates that are at least twice as high as in exercising small mammals, and can be as high as 8—18 times BMR Butler et al.

The rate-limiting step causing this difference between mammals and birds is the much greater capacity of avian skeletal muscles to take up circulating fatty acids reviewed in Jenni-Eiermann, It seems logical then that placental mammals, possessing BAT, would much benefit from enhanced fatty acid import, compared with skeletal muscle cells.

This is indeed ensured by the function of lipoprotein lipase, which, along with other enzymes, allows the massive import of fatty acids into BAT during thermogenesis at significantly higher rates than into skeletal muscle cells of placental mammals Heldmaier et al.

Interestingly, when the capacity of fatty acid import into muscle cells was increased by overexpression of lipoprotein lipase in transgenic mice, this improved cold resistance—independent from BAT thermogenesis—and elevated muscular fatty acid oxidation Jensen et al.

As noted by Jensen et al. Arguably, these differences in fuel import capacity into thermogenic tissues may well-explain the absence of BAT in birds, as well as the lower thermogenic capacity of marsupials and monotremes.

In summary, it seems that muscle NST may have been an important step in the evolution of endothermy. Endothermy is clearly facilitated by increasing mitochondrial membrane surface Else and Hulbert, and activity of the sodium-potassium pump, which is the greatest contributor to BMR Clarke et al.

However, apart from shivering, muscle NST via SERCA ATP hydrolysis was probably the first metabolic pathway in mammals solely used for thermogenesis. Interestingly, after mixed support for this model from smaller studies over the last decades, strong evidence for the aerobic capacity model comes from a recent comprehensive phylogenetically informed study ranging from fish and amphibians to birds and mammals, which shows that there is in fact a positive correlation between maximum and resting metabolic rates in mammals, and that this pattern is a result of natural selection Nespolo et al.

These findings again point to an important role of enhanced muscle function and metabolism for the emergence of endothermy. One of the reasons why the importance of muscle NST may have been underestimated in the past but see, e. In contrast, activation of UCP1-mediated NST occurs prior to the onset of shivering Böckler and Heldmaier, which makes it easier to identify as a separate mechanism.

However, even NST in BAT can also occur simultaneously with shivering thermogenesis Böckler and Heldmaier, Arguably then, the evolution of endothermy was not characterized by switches from one to another, possibly improved, metabolic pathway.

Instead it seems that increasing levels of endothermy were achieved by recruiting additional mechanisms of thermogenesis to muscular work during locomotion, including specialized shivering thermogenesis, increases in mitochondrial density and membrane leakage, increases in sodium-potassium pump activity, shifts in SERCA activity toward NST.

Highly endothermic mammals living in cold environments apparently can use all of these mechanisms simultaneously. There are several possible selective advantages to this last evolutionary step, the additional recruitment of UCP1-mediated NST.

As already pointed out previously Rowland et al. Further, we suggest that the evolution of BAT in addition to muscle NST was related to heterothermy being predominant among early endothermic mammals.

This is because, in comparison with large muscles, a small dedicated thermogenic tissue such as BAT is much more suited to rapidly warm up and escape limiting Arrhenius effects of low tissue temperature during hibernation and torpor in harsh habitats.

Finally, we argue that additional mechanisms for NST are not required by animals that have enhanced capacities to fuel muscle NST by high rates of fatty acid import. Such a group of endotherms are birds, which probably evolved this superior fuel transport capacity as an adaptation to flight.

This would explain why birds have high endothermic capacities, despite the absence of BAT. JN wrote the first draft of the manuscript.

All authors added text, discussed, and edited the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Anderson, D. A micropeptide encoded by a putative long non-coding RNA regulates muscle performance. Cell , — doi: CrossRef Full Text Google Scholar. Anderson, K. Multi-Omic Analysis of Hibernator Skeletal Muscle and Regulation of Calcium Handling.

Master of Science MSc thesis, University of Minnesota. Proteogenomic analysis of a hibernating mammal indicates contribution of skeletal muscle physiology to the hibernation phenotype. Proteome Res. PubMed Abstract CrossRef Full Text Google Scholar.

Andrews, M. Advances in molecular biology of hibernation in mammals. Bioessays 29, — Arnold, W. Ecophysiology of omega fatty acids: a lid for every jar. Physiology 30, — Asahi, M.

Aschoff, J. Thermal conductance in mammals and birds: its dependence on body size and crcadian phase. Part A Physiol. Babu, G. Differential expression of sarcolipin protein during muscle development and cardiac pathophysiology. Bal, N. Increased reliance on muscle-based thermogenesis upon acute minimization of brown adipose tissue function.

Sarcolipin is a newly identified regulator of muscle-based thermogenesis in mammals. Barbot, T. Chakraborti and N.

Dhalla Cham: Springer , — Barnes, B. Freeze avoidance in a mammal: body temperatures below 0°C in an arctic hibernator. Science , — Beard, L. Reproduction in the short-beaked echidna, Tachyglossus aculeatus : field observations at an elevated site in South-east Queensland.

New South Wale , 89— Google Scholar. Bennett, A. Endothermy and activity in vertebrates. Berg, F. The uncoupling protein 1 gene UCP1 is disrupted in the pig lineage: a genetic explanation for poor thermoregulation in piglets.

PLoS Genet. Berthon, D. Metabolic changes associated with sustained hr shivering thermogenesis in the newborn pig. Part B Biochem.

Shivering thermogenesis in the neonatal pig. Thermal Biol. Bicudo, J. Thermogenesis in birds. Block, B. Thermogenesis in muscle. Characterization of the sarcoplasmic reticulum proteins in the thermogenic muscles of fish.

Cell Biol. Böckler, H. Interaction of shivering and non-shivering thermogenesis during cold exposure in seasonally-acclimatized Djungarian hamsters Phodopus sungorus. Brauch, K. Digital transcriptome analysis indicates adaptive mechanisms in the heart of a hibernating mammal.

Genomics 23, — Buser, K. Effect of cold environment on skeletal muscle mitochondria in growing rats. Cell Tissue Res.

Butler, J. The effects of sarcolipin over-expression in mouse skeletal muscle on metabolic activity. Butler, P. Behaviour and physiology of Svalbard barnacle geese Branta leucopsis during their autumn migration. Avian Biol. Cannon, B. Brown adipose tissue: function and physiological significance.

Carey, F. A brain heater in the swordfish. Chaffee, R. Studies on thermogenesis in brown adipose tissue in temperature-acclimated Macaca mulatta. Clarke, R. Acta , — Clausen, T. Significance of cation transport in control of energy metabolism and thermogenesis. PubMed Abstract Google Scholar.

Cornelius, F. Modulation of Na,K-ATPase and Na-ATPase activity by phospholipids and cholesterol. steady-state kinetics. Biochemistry 40, — da Costa, D. PubMed Abstract CrossRef Full Text. Dawson, W. Seasonal acclimatization to temperature in cardueline finches.

A , — de Meis, L. Regulation by ADP. IUBMB Life 57, — Duchamp, C. Skeletal muscle as the major site of nonshivering thermogenesis in cold-acclimated ducklings. Dumonteil, E. Cell Physiol. Else, P. Emre, Y. Avian UCP: the killjoy in the evolution of the mitochondrial uncoupling proteins. Foster, D.

Nonshivering thermogenesis in the rat. Measurements of blood flow with microspheres point to brown adipose tissue as the dominant site of the calorigenesis induced by noradrenaline. Canadian J. Fritsches, K. Warm eyes provide superior vision in swordfishes.

Gaudry, M. Inactivation of thermogenic UCP1 as a historical contingency in multiple placental mammal clades. Geiser, F. Daily torpor and thermoregulation in antechinus Marsupialia : influence of body mass, season, development, reproduction, and sex.

Oecologia 77, — Metabolic rate and body temperature reduction during hibernation and daily torpor. Seasonality of torpor and thermoregulation in three dasyurid marsupials. B , — The relationship between body mass and rate of rewarming from hibernation and daily torpor in mammals.

Seasonal changes in the critical arousal temperature of the marsupial Sminthopsis crassicaudata correlate with the thermal transition in mitochondrial respiration. Life Sci. Hibernation and daily torpor in Australian mammals.

Giroud, S. Membrane phospholipid fatty acid composition regulates cardiac SERCA activity in a hibernator, the Syrian hamster Mesocricetus auratus.

PLOS ONE 8:e Graier, W. Cell Calcium 44, 36— Graves, H. Behavior and ecology of wild and feral swine Sus scrofa. Grigg, G. The evolution of endothermy and its diversity in mammals and birds. Barnes and H. Carey Fairbanks, AK: Institute of Arctic Biology; University of Alaska , — Augee Chipping Norton, NSW: Roy.

Hasselbach, W. Die Calciumpumpe der Erschlaffungsgrana des Muskels und ihre Abhängigkeit von der ATPSpaltung. Über den Mechanismus des Calciumtransports durch die Membranen des sarcoplasmatischen Reticulums.

Hayward, J. Evolution of brown fat: its absence in marsupials and monotremes. Heaton, G. Brown-adipose-tissue mitochondria: photoaffinity labelling of the regulatory site of energy dissipation.

Heldmaier, G. Metabolic adjustments during daily torpor in the Djungarian hamster. Herpin, P. Development of thermoregulation and neonatal survival in pigs. Livestock Product. Effect of cold exposure on energy metabolism in the young pig.

Hoppeler, H. Normal mammalian skeletal muscle and its phenotypic plasticity. Hughes, D. Molecular evolution of UCP1 and the evolutionary history of mammalian non-shivering thermogenesis.

BMC Evol. Hulbert, A. Life, death and membrane bilayers. On the importance of fatty acid composition of membranes for aging. Membranes as possible pacemakers of metabolism. Mechanisms underlying the cost of living in animals. Inesi, G. Cell Commun. Jaeger, E. Further observations on the hibernation of the poor-will.

Condor 51, — Jastroch, M. Marsupial uncoupling protein 1 sheds light on the evolution of mammalian nonshivering thermogenesis. Genomics 32, — Uncoupling protein 1 in fish uncovers an ancient evolutionary history of mammalian nonshivering thermogenesis Physiol. Genomics 22, — Jenni-Eiermann, S.

Energy metabolism during endurance flight and the post-flight recovery phase. Jensen, D. Increased thermoregulation in cold-exposed transgenic mice overexpressing lipoprotein lipase in skeletal muscle: an avian phenotype?

Lipid Res. Kabat, A. Non-shivering thermogenesis in a carnivorous marsupial, Sarcophilus harrisii , the the absence of UCP1. Kammersgaard, T. Hypothermia in neonatal piglets: interactions and causes of individual differences. Ketzer, L. Heart Circul. Koteja, P.

Energy assimilation, parental care and the evolution of endothermy. Ser B Biol. Lin, J. Cold adaptation in pigs depends on UCP3 in beige adipocytes. Lovegrove, B. The evolution of endothermy in Cenozoic mammals: a plesiomorphic-apomorphic continuum. Luo, Z.

A Jurassic eutherian mammal and divergence of marsupials and placentals. Nature , — MacLennan, D. Malignant hyperthermia. Mall, S. Maurya, S. Sarcolipin is a key determinant of the basal metabolic rate, and its overexpression enhances energy expenditure and resistance against diet-induced obesity.

McNab, B. An analysis of the body temperatures of birds. Condor 68, 47— Temperature regulation and oxygen consumption in the Philippine tarsier Tarsius syrichta. Mitchell, K. Molecular phylogeny, biogeography, and habitat preference evolution of marsupials.

Mitidieri, F. Montigny, C. S-Palmitoylation and S-Oleoylation of rabbit and pig sarcolipin. Morrissette, J. CrossRef Full Text. Nedergaard, J. Preferential utilization of brown adipose tissue lipids during arousal from hibernation in hamsters.

Nespolo, R.

Furthermore, in mice, intracerebroventricular administration of T3 increases BAT thermogenesis via reduced hypothalamic levels of AMP kinase AMPK and subsequent activation of the SNS Lopez et al. Indeed, sub-chronic 6 days central administration of T3 leads to browning of WAT in mice Alvarez-Crespo et al.

To date, much of the work defining the regulation of thermogenesis and its contribution to energy balance has been in rodents.

This has provided invaluable information and understanding of the neuroendocrine mechanisms that control thermogenesis.

More recently, a number of large animal models have been employed including pigs and sheep, which provide further insight into the role of thermogenesis in long-term regulation of body weight in mammalian species.

It is well recognised that pigs lack a functional UCP1 protein Hou et al. Pigs, specifically those belonging to the Suidae species, do not have exons 3—5 of the UCP1 gene, rendering animals prone to hypothermia-induced death as neonates Berg et al.

With only exons 1 and 2, UCP1 can still be transcribed; however, protein translation does not occur Hou et al. Hence, previous histological studies failed to detect UCP1 protein immunoreactivity at baseline Rowlatt et al. It has since been proposed that pigs do indeed possess functional BAT; however, adaptive thermogenesis occurs via UCP1-independent mechanisms Ikeda et al.

Indeed, recent work comparing cold-tolerant Tibetan pigs to cold-sensitive Bama pigs has provided direct evidence of adaptive thermogenesis in subcutaneous sWAT and perirenal WAT Lin et al.

Furthermore, morphological studies show that in response to cold exposure, subcutaneous adipose tissue displays evidence for beige cell recruitment with an increase in multilocular adipocytes, increased mitochondrial DNA copy number and increased expression of PGC1a and the beige cell marker CD Wu et al.

Furthermore, in Tibetan pigs cold tolerant , cold exposure increased the expression of the UCP3 gene and protein in isolated subcutaneous adipocytes and this is associated with increased uncoupled respiration, providing evidence to suggest an increase in UCP3-driven thermogenesis Lin et al.

Role of thermogenesis in determining cold tolerance in pigs. Tibetan pigs are cold tolerant and this coincides with the recruitment of beige adipocytes in subcutaneous WAT in response to cold exposure.

Although pigs do not that express functional uncoupling protein UCP 1, adipocytes exhibit UCP3 and this mediates mitochondrial uncoupling and adipose tissue thermogenesis. The contribution of UCP3 to brown fat thermogenesis has been contentious and appears to be dependent on the species studied.

In mice, earlier work suggested that BAT thermogenesis was dependent on UCP1 Matthias et al. Despite this, hamsters that lack functional UCP3 specifically in brown adipocytes have increased propensity to weight gain, which is indicative of a reduction in energy expenditure Fromme et al.

Although innate differences in UCP3 expression in adipose tissue of pigs have been linked to cold tolerance, to date, there are no data on BAT-specific UCP3 function and the control of body weight in this species.

In addition to UCP3-associated uncoupling and thermogenesis, recent data suggest that SERCA-driven beige cell thermogenesis also occurs in pigs.

Indeed, the work by Ikeda et al. Ikeda et al. Retroviral expression of PRDM in subcutaneous porcine adipocytes increases the expression of beige-cell-specific markers including CIDEA and TMEM26 Ikeda et al. Furthermore, decreased SERCA2b expression reduced basal and noradrenaline-induced oxygen consumption and extracellular acidification rates in isolated pig adipocytes Ikeda et al.

Thus, it is now clear that adipose tissue thermogenesis and the associated energy expenditure are not solely mediated via UCP1 and mitochondrial uncoupling, but in fact, a number of cellular pathways, across both adipose tissue and skeletal muscle, act in concert to determine total thermogenic potential.

In lambs, the expression of UCP1 is maximal in perirenal adipose tissue on the first postnatal day, rapidly declining with the expansion of WAT Symonds , Pope et al. Mapping of UCP1 mRNA in lambs shows abundant expression in sternal and retroperitoneal adipose depots compared to omental fat, which is a predominantly WAT depot Symonds et al.

Indeed, adult sheep retain UCP1 expression in both sternal and retroperitoneal fat and this coincides with post-prandial heat production, albeit this response is greater in the sternal fat depot Henry et al.

This coincides with the expression of UCP1 protein, where UCP1-positive brown-like adipocytes were only detectable in sternal adipose tissue of adult ewes Henry et al. Data logger temperature probes have been employed to measure longitudinal heat production in multiple tissues to index thermogenic output in sheep.

Sheep are a grazing species and therefore do not display typical meal-associated excursions such as changes in ghrelin secretion.

Despite this, temporal food restriction in sheep entrains a pre-prandial rise in ghrelin Sugino et al. Furthermore, post-prandial thermogenesis in both skeletal muscle and retroperitoneal adipose depots is markedly enhanced by intracerebroventricular infusion of leptin Henry et al.

Thus, in spite of relatively low levels of UCP1 in adult sheep, skeletal muscle and specific adipose depots retain thermogenic capacity.

Over recent years, we have utilised the sheep to dissect the differential roles of adipose tissue and skeletal muscle thermogenesis in the long-term control of body weight, which is discussed in detail in the following section.

Similar to other species, ovine body weight can be readily manipulated through dietary management Henry et al. Sheep are ruminants and thus body weight is increased through feeding a high-energy diet enriched in lupin grain and oats. Diet-induced obesity, however, is not associated with any change in heat production in adipose tissues or skeletal muscle of sheep Henry et al.

On the other hand, long-term food restriction and low body weight are associated with a homeostatic decrease in thermogenesis in sternal and retroperitoneal adipose tissue and skeletal muscle Henry et al. Importantly, similar to humans, the reduction in thermogenesis caused by food restriction and low body weight is still evident at one year post-weight loss, which suggests that homeostatic changes in thermogenesis contribute to impaired weight loss and increased long-term weight regain Henry et al.

Effect of chronic food restriction and weight loss on adaptive thermogenesis in ewes. Tissue temperature recordings show that caloric restriction and low body weight cause a homeostatic decrease in night time thermogenesis in ovariectomised ewes.

This metabolic adaptation occurs in both sternal adipose tissue adipose tissue enriched in uncoupling protein 1 and skeletal muscle and to a lesser extent in retroperitoneal adipose tissue.

The reduction in thermogenesis is associated with increased expression of neuropeptide Y NPY in the arcuate nucleus and melanin-concentrating hormone MCH in the lateral hypothalamus.

The homeostatic reduction in thermogenesis is coordinated by the hypothalamus. Long-term weight loss in ovariectomised ewes increases the expression of the orexigenic neuropeptides NPY in the arcuate nucleus and melanin-concentrating hormone MCH in the lateral hypothalamus LH to increase hunger and reduce energy expenditure Henry et al.

Regarding the anorexigenic melanocortin pathway, the effect of low body weight on the expression of POMC is controversial with data showing a decrease Backholer et al. This is not surprising since POMC is the precursor to multiple neuropeptides, only one of which includes aMSH and the ultimate end product is dependent on post-translational processing Mountjoy On the other hand, increased Agrp and Npy expression and reduced Pomc mRNA have been observed in rodents Bi et al.

Thus, weight-loss-induced changes in hypothalamic gene expression are likely to reduce thermogenesis, whilst causing a concurrent increase in hunger drive. This represents a homeostatic mechanism to protect against weight loss and promote weight regain in calorie-restricted individuals.

Animals were originally selected for innate differences in adiposity by measuring back fat thickness and two lines were created via selective breeding strategies. A key feature of the genetically lean and obese sheep is an inherent difference in the growth hormone GH axis, where lean animals have increased mean GH concentration in plasma and an associated increase in pituitary gland weight Francis et al.

The increase in pituitary gland weight is primarily due to a greater number of cells in the lean animals Francis et al. Furthermore, expression of GH and the GH secretagogue receptor GHSR is greater in genetically lean sheep, indicating differential responses to ghrelin, an agonist of the GHSR French et al.

This suggests that innate differences in the set-point of the GH axis may underpin differences in adiposity in the genetically lean and obese sheep; however, this is only one aspect that could contribute to this phenotype.

Interestingly, food intake is similar in genetically lean and obese sheep as is the expression of POMC, Leptin Receptor and NPY in the arcuate nucleus. On the other hand, lean animals have elevated post-prandial thermogenesis in retroperitoneal adipose tissue and this coincides with increased expression of UCP1 in this tissue Henry et al.

The divergence in thermogenesis is specific to adipose tissue since post-prandial thermogenesis is similar in genetically lean and obese animals Henry et al. Despite similar expression of appetite-regulating peptides in the arcuate nucleus of the hypothalamus, genetically lean sheep have increased expression of MCH and pre-pro-orexin ORX in the LH compared to obese animals Anukulkitch et al.

While both neuropeptides are considered orexigenic Shimada et al. Deletion of MCH in mice results in hypophagia and a lean phenotype Shimada et al. Orexin is critical in the embryonic development of BAT in mice Sellayah et al.

Thus, increased expression of ORX in the LH of lean sheep may be an important physiological determinant of increased thermogenesis in retroperitoneal fat and the associated changes in adiposity.

It is widely recognised that there is marked variation in the glucocorticoid response to stress or activation of the hypothalamo-pituitary adrenal HPA axis Cockrem , Walker et al. The activity of the HPA axis in response to stress is impacted on by age Sapolsky et al.

Nonetheless, in any given population individuals can be characterised as either high HR or low LR glucocorticoid responders Epel et al. It is important to note that female LR and HR sheep have similar basal plasma cortisol concentration and divergence in glucocorticoid secretion only occurs in response to ACTH or stress Lee et al.

Previous studies have suggested that obesity itself causes perturbation of the HPA axis with impaired glucocorticoid-negative feedback Jessop et al. Furthermore, cortisol directly impacts on metabolic function; however, this will not be addressed in the current review.

Initial studies in rams show that high cortisol response to adrenocorticotropin ACTH is associated with lower feed-conversion efficiency Knott et al. Furthermore, in rams, adiposity is correlated to cortisol responses to ACTH Knott et al. More recent work shows that identification of high HR and low LR cortisol responders in female sheep can predict altered propensity to gain weight when exposed to a high-energy diet, where HR gain more adipose tissue than LR Lee et al.

Thus, at least in female sheep, data suggest that cortisol responses can be used as a physiological marker that predicts propensity to become obese. Previous studies in women suggest that HR eat more after a stressful episode than LR Epel et al.

Furthermore, HR individuals display preference for foods of high fat and sugar in response to psychological stress Tomiyama et al. Similarly, in ewes, baseline food intake is similar in LR and HR, but HR eat more following either psychosocial barking dog or immune lipopolysaccharide exposure stressors Lee et al.

In addition to altered food intake, HR ewes have reduced thermogenesis in skeletal muscle only; in response to meal feeding, post-prandial thermogenesis in skeletal muscle is greater in LR than in HR Lee et al.

This again exemplifies divergence in the control of adipose tissue and skeletal muscle thermogenesis Fig. Schematic depiction of the altered metabolic phenotype in animals selected for either high or low cortisol responsiveness.

Sheep are characterised as either high HR or low LR cortisol responders when given a standardised dose of adrenocorticotropic hormone. Animals characterized as HR have increased propensity to become obese, which is associated with perturbed control of food intake and reduced energy expenditure.

Post-prandial thermogenesis in skeletal muscle is decreased in HR compared to LR ewes. Furthermore, food intake in response to stress is greater in HR than in LR and the former are resistant to the satiety effect of alpha-melanocyte stimulating hormone aMSH. High-cortisol-responding animals have reduced expression of the melanocortin 4 receptor MC4R in the paraventricular nucleus of the hypothalamus PVN.

We propose that the decreased levels of MC4R underpin the altered metabolic phenotype and increased propensity to become obese when compared to LR. For example, at baseline in the non-stressed resting state, HR individuals show an overall upregulation of the HPA axis, with increased expression of CRF and arginine vasopressin, but reduced expression of oxytocin in the PVN Hewagalamulage et al.

In addition to altered expression of genes within the HPA axis, a key neuroendocrine feature of the LR and HR animals is altered expression of the MC3R and MC4R in the PVN Fig.

Reduced MC4R expression coincides with the development of melanocortin resistance. Central infusion of leptin reduces food intake in both LR and HR animals, but intracerebroventricular infusion of aMSH reduces food intake in LR only.

Thus, reduced MC4R expression appears to be central to the metabolic phenotype of HR that confers increased propensity to become obese in HR individuals Fig. Interestingly, gene expression of NPY , AgRP and POMC in the arcuate nucleus is equivalent in LR and HR Hewagalamulage et al.

Hence, differences in the control of food intake and thermogenesis are most likely manifest at the level of the melanocortin receptor. Indeed, previous work in sheep has shown the MC4R to be central in mediating the reduction in food intake caused by immune challenge Sartin et al.

Furthermore, in rodents, direct injection of the melanocortin agonist melanotan II into the ventromedial nucleus of the hypothalamus increases skeletal muscle thermogenesis Gavini et al. We propose that reduced expression of the MC4R in HR animals underpins the metabolic phenotype wherein food intake is relatively increased in response to stress and reduced post-prandial thermogenesis in skeletal muscle is associated with propensity to become obese.

Historically, thermogenesis was considered to primarily occur in brown adipocytes and was solely driven by UCP1. It is now recognised that beige adipocytes and skeletal muscle also contribute to total thermogenic capacity and that thermogenesis is differentially regulated in these tissues.

Indeed, in beige adipocytes, thermogenesis occurs via three distinct mechanisms, with these being UCP1-driven mitochondrial uncoupling, futile creatine cycling and futile calcium cycling. On the other hand, in skeletal muscle, thermogenesis is associated with UCP3 and futile calcium cycling.

Unlike rodents, large mammals including sheep and pigs do not contain a defined or circumscribed brown fat depot but have dispersed brown adipocytes within traditionally white fat depots. Large animals have provided invaluable insight into alternative mechanisms of thermogenesis. The sheep has been particularly useful in delineating the differential role of adipose tissue and skeletal muscle in the control of body weight.

Furthermore, sheep models have allowed characterisation of the neuroendocrine pathways that may contribute to altered thermogenesis. We have shown that in sheep, both skeletal muscle and BAT differentially contribute to thermogenesis and therefore total energy expenditure.

Changes in thermogenesis, however, do not exclusively associate with altered gene expression at the level of the arcuate nucleus. Indeed, decreased MC4R expression in HR animals and reduced orexin expression in the genetically obese animals coincide with altered thermogenic output.

This review highlights the importance of the use of large animal models to ascertain the contribution and control of thermogenesis in multiple tissues and the relative role in the regulation of body weight. The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.

This work was supported by Australian Research Council grant number DP and National Health and Medical Research Council grant number APP Animal Science 63 — Journal of Pathology and Bacteriology 91 — Obesity Reviews 19 — Molecular Metabolism 5 — Neuroendocrinology 91 — Biochimica et Biophysica Acta — Astrup A Thermogenesis in human brown adipose tissue and skeletal muscle induced by sympathomimetic stimulation.

Acta Endocrinologica Supplement S9 — S American Journal of Physiology E — E Science — Neuroendocrinology 91 27 — Journal of Biological Chemistry — Bal NC , Maurya SK , Sopariwala DH , Sahoo SK , Gupta SC , Shaikh SA , Pant M , Rowland LA , Bombardier E , Goonasekera SA , et al.

Nature Medicine 18 — Journal of Neuroendocrinology 29 e Balthasar N , Dalgaard LT , Lee CE , Yu J , Funahashi H , Williams T , Ferreira M , Tang V , McGovern RA , Kenny CD , et al. Cell — American Journal of Physiology R — R Banks WA Characteristics of compounds that cross the blood-brain barrier.

BMC Neurology 9 S3 — S3. American Journal of Physiology: Endocrinology and Metabolism E — E Disease Models and Mechanisms 9 — Cellular and Molecular Life Sciences 73 — PLoS Genetics 2 e Cell Metabolism 25 e — Lancet — Circulation Research — American Journal of Physiology: Regulatory, Integrative and Comparative Physiology R — R International Journal of Obesity and Related Metabolic Disorders 17 Supplement 3 S78 — S Blondin DP , Daoud A , Taylor T , Tingelstad HC , Bezaire V , Richard D , Carpentier AC , Taylor AW , Harper ME , Aguer C , et al.

Journal of Physiology — International Journal of Obesity 37 — PLoS ONE 11 e Progress in Neuro-Psychopharmacology and Biological Psychiatry 35 — Metabolism 64 — Physiology Reviews 84 — Carey AL , Pajtak R , Formosa MF , Van Every B , Bertovic DA , Anderson MJ , Eikelis N , Lambert GW , Kalff V , Duffy SJ , et al.

Diabetologia 58 — Endocrinology — Cinti S The adipose organ: morphological perspectives of adipose tissues. Proceedings of the Nutrition Society 60 — Claret M , Smith MA , Batterham RL , Selman C , Choudhury AI , Fryer LG , Clements M , Al-Qassab H , Heffron H , Xu AW , et al.

Journal of Clinical Investigation — Clement K , Vaisse C , Lahlou N , Cabrol S , Pelloux V , Cassuto D , Gourmelen M , Dina C , Chambaz J , Lacorte JM , et al. Nature — Cockrem JF Individual variation in glucocorticoid stress responses in animals.

General and Comparative Endocrinology 45 — Diabetes 64 — Coppola A , Liu Z , Andrews Z , Paradis E , Roy M-C , Friedman JM , Ricquier D , Richard D , Horvath TL , Gao X-B , et al. Cell Metabolism 5 21 — Journal of Cell Science — Neuron 24 — Clinical Science 69 — Cypess AM , Lehman S , Williams G , Tal I , Rodman D , Goldfine AB , Kuo FC , Palmer EL , Tseng Y-H , Doria A , et al.

New England Journal of Medicine — Cypess AM , Weiner LS , Roberts-Toler C , Elia EF , Kessler SH , Kahn PA , English J , Chatman K , Trauger SA , Doria A , et al.

Cell Metabolism 21 33 — Nature Medicine 19 — Dalgaard K , Landgraf K , Heyne S , Lempradl A , Longinotto J , Gossens K , Ruf M , Orthofer M , Strogantsev R , Selvaraj M , et al. Bioscience Reports 25 — Neuron 23 — Psychoneuroendocrinology 26 37 — Farooqi IS Monogenic human obesity.

Frontiers of Hormone Research 36 1 — New Zealand Journal of Agricultural Research 41 — Domestic Animal Endocrinology 18 — Journal of Animal Science 84 — American Journal of Physiology: Physiological Genomics 38 54 — Gaborit B , Venteclef N , Ancel P , Pelloux V , Gariboldi V , Leprince P , Amour J , Hatem SN , Jouve E , Dutour A , et al.

Cardiovascular Research 62 — Diabetes, Obesity and Metabolism 16 97 — Hara J , Beuckmann CT , Nambu T , Willie JT , Chemelli RM , Sinton CM , Sugiyama F , Yagami K , Goto K , Yanagisawa M , et al. Neuron 30 — Heaton JM The distribution of brown adipose tissue in the human. Journal of Anatomy 35 — Journal of Animal Science 93 — Journal of Chemical Neuroanatomy 13 1 — Neuroendocrinology 27 44 — Clinical Pediatrics 49 — American Journal of Physiology: Cell Physiology C Biochemical and Biophysical Research Communications — Ikeda K , Kang Q , Yoneshiro T , Camporez JP , Maki H , Homma M , Shinoda K , Chen Y , Lu X , Maretich P , et al.

Neuroscience — Ito M , Gomori A , Ishihara A , Oda Z , Mashiko S , Matsushita H , Yumoto M , Ito M , Sano H , Tokita S , et al. Nature Genetics 16 Jespersen NZ , Larsen TJ , Peijs L , Daugaard S , Homoe P , Loft A , de Jong J , Mathur N , Cannon B , Nedergaard J , et al.

Cell Metabolism 17 — Journal of Clinical Endocrinology and Metabolism 86 — Journal of the American College of Nutrition 21 55 — Diabetes 50 — British Journal of Pharmacology — Kazak L , Chouchani Edward T , Jedrychowski Mark P , Erickson Brian K , Shinoda K , Cohen P , Vetrivelan R , Lu Gina Z , Laznik-Bogoslavski D , Hasenfuss Sebastian C , et al.

Handbook of Experimental Pharmacology — Domestic Animal Endocrinology 34 — Nature Genetics 19 — International Journal of Obesity 38 — FASEB Journal 28 35 — Psychoneuroendocrinology 47 — Experimental and Clinical Endocrinology and Diabetes Supplement 1 S4 — S6.

International Journal of Obesity 35 Journal of Molecular Cell Biology 9 — Locke AE , Kahali B , Berndt SI , Justice AE , Pers TH , Day FR , Powell C , Vedantam S , Buchkovich ML , Yang J , et al.

Lopez M , Varela L , Vazquez MJ , Rodriguez-Cuenca S , Gonzalez CR , Velagapudi VR , Morgan DA , Schoenmakers E , Agassandian K , Lage R , et al. Nature Medicine 16 — In Current Protocols in Pharmacology , chapter 5, unit 5.

Hoboken, NJ, USA : Wiley. Metabolism 41 — UCP2 or UCP3 do not substitute for UCP1 in adrenergically or fatty acid-induced thermogenesis. Biology of Reproduction 49 — Temperature 3 — Montague CT , Farooqi IS , Whitehead JP , Soos MA , Rau H , Wareham NJ , Sewter CP , Digby JE , Mohammed SN , Hurst JA , et al.

Animal Science 65 93 — Morrison SF Central neural control of thermoregulation and brown adipose tissue. Autonomic Neuroscience: Basic and Clinical 14 — International Journal of Obesity and Related Metabolic Disorders 19 — All mouse strains used in this study were born at the expected Mendelian ratios with normal fertility.

Musclin neutralizing Ab preparation was performed by ABclonal Technology Co. Through epitope prediction, the peptide sequence C-HSKKRFGIP-Nle-DRIGRNR corresponding to aa of Musclin was synthesized and used for immunizing rabbits to generate antibodies.

Polyclonal antibodies against Musclin were obtained from inoculated rabbits. Antibodies were purified using affinity chromatography on columns containing the corresponding peptides.

Female mice were only used for the cold exposure study shown in Fig. The mouse age and sex information for each experiment has also been included in the corresponding figure legend. ExpiF cell line was obtained from Thermo Fisher.

AAV cell line was purchased from Agilent. HEKT, ExpiF, and AAV cell lines were authenticated and routinely used in our previous studies 2 , 59 , 60 , HUVEC cell line was kindly provided by Dr. Nan Xu Henan University , and has been authenticated and successfully used in their previous study AAV production and purification were performed by ChuangRui Bio Lianyungang, China.

Culture media were replaced with DMEM plus 0. Cell lysates were gently transferred onto the top layer, and the remaining volume of the ultracentrifuge tube was filled with cell lysis buffer.

The viral titer was determined by qPCR assay with a standard curve generated by serial dilutions of the AAV shuttle vector. Mice were granted free access to pre-chilled food and water during the whole assay.

Core body temperature was monitored at indicated time points using a portable intelligent digital thermometer TH During this period, metabolic parameters including mouse body temperature, body weight, blood glucose, as well as plasma TG and NEFA levels were monitored.

Body surface temperature was measured using a thermal imaging camera FLIR Systems, Tsc InfraRed Camera. Mice were anesthetized with isoflurane and quietly laid on a whiteboard with their back up, followed by image capturing with the camera anchored at the same height for all mice in the same batch.

FLIR Tools 5. Food and water were freely accessible to mice. O 2 consumption, CO 2 production, energy expenditure, total locomotor activity, and food intake were monitored under both normal conditions and under adrenergic stimulation.

The metabolic parameters for each mouse were measured two days before CL injection and were continued to be monitored for one more day after CL injection. Mouse running performance was assessed using the treadmill running system from Columbus Instruments.

The inclination angle was level. Total running distance and running time were recorded at the point when the mice reached exhaustion. After full differentiation, myotubes were subject to RNA isolation and gene expression analysis.

ExpiF cells were transfected with pcDNA3. The XF96 microplate was then loaded into a Seahorse XFe96 analyzer for equilibration and determination of the basal respiration rate. OCRs were recorded and the ATP production, maximal respiration, and the spare respiratory capacity-dependent OCRs were calculated.

Cell viability was also quantified using the CCK8 kit Beyotime for normalization. The XF Cell Mito Stress test kit Agilent was used in this assay. Seahorse Wave Desktop and Controller 2.

An NMR analyzer NIUMAG, QMNH was applied to measure the body fat and lean mass. An enzyme-linked immunosorbent assay ELISA kit from Crystal Chem was used to measure plasma insulin concentration. For the detection of Musclin levels in human plasma, a Human Musclin ELISA kit CUSABIO, CSB-Eh was applied.

For the detection of ANP levels in mouse plasma, a mouse ANP ELISA kit Elabscience, E-EL-Mc was used. For the detection of CNP levels in mouse plasma, a Mouse CNP ELISA kit Beijing Sino-UK Institute of Biological Technology, HY-NE was applied.

Mouse tissues including liver, skeletal muscle, BAT, eWAT, and iWAT were dissected, fixed in formalin, followed by embedding in paraffin and cutting for tissue sections at μm for liver, skeletal muscle, and BAT, or μm for eWAT and iWAT.

Tissue sections were then stained with hematoxylin and eosin, and subjected to image acquisition using NIS Elements F 4. The cell size of iWAT was quantified using Image J 1. The SEAP-binding assay was performed as described Firstly, the vectors expressing SEAP or SEAP-Musclin fusion protein were transiently transfected into HEKT cells.

The sections were washed with PBS containing 0. CellSens Standard Olympus was used for image acquisition and data collection. Total RNA from WAT was isolated using a commercially available kit TIANGEN Biotech, DP Total RNA from other tissues including skeletal muscle, heart, brain, small intestine, liver, BAT, bone, and cultured cells was isolated following the standard method using TRIzol.

RNA was then reverse transcribed using HiScript II Q RT SuperMix Vazyme, R , followed by qPCR analysis using SYBR Green Roche. The qPCR primers used are listed in Supplementary Data. Sequencing libraries were constructed from total RNA using SMART-RNAseq Library Prep Kit Hangzhou KaiTai, AT In brief, mRNA was isolated from total RNA with Sera-Mag Magnetic Olido dT particles, and then chemically fragmented.

And the cDNA libraries were subsequently amplified using the KAPA high-fidelity DNA polymer. Quality of the libraries was validated by the Bioanalyzer Agilent Technologies. Subsequently, high-throughput sequencing was performed using a NovaSeq Illumina. Raw reads were filtered with fastp V0.

Filtered data were then aligned with HISAT2 V2. p13 for human data. The FPKM fragments per kilobase of exon per million fragments mapped was calculated using StringTie V2.

For iWAT from cold-acclimated and room temperature-housed control WT mice, RNA sequencing was performed in Majorbio Bio-pharm Biotechnology Co. Shanghai, China. The library was prepared using TruSeqTM RNA Sample Prep Kit Illumina. Shortly, mRNA was isolated by oligo dT beads and then fragmented.

After quantified by TBS, paired-end RNA sequencing was performed using the NovaSeq Illumina. The raw reads were processed similarly and the TPM transcripts per million reads values were used to determine gene expression levels. For human scWAT, RNA sequencing was performed in BGI Shenzhen, China.

Similarly, mRNA was purified using oligo dT -attached magnetic beads and was then fragmented. cDNA was generated by random hexamer-primed reverse transcription, and end-repaired through incubation with a-Tailing Mix and RNA Index Adapters. The obtained cDNA fragments were amplified using PCR followed by purification with Ampure XP Beads, which was validated on the Bioanalyzer Agilent Technologies.

Distinctively, further heat-denaturation and circularization by the splint oligo sequence of the PCR products from previous step were needed to get the final library. Pair end base reads were generated for analysis. The sequencing data was filtered with SOAPnuke V1. p12 with HISAT2 V2.

Expression level of gene FPKM value was calculated by RSEM V1. For all these sequencing data, differential expression analysis was performed using the Deseq2 v. Gene Ontology GO and pathway grouping and enrichment studies were performed by clusterProfiler V3.

Html Results were visualized by ggplot2 V3. html 70 and pheatmap V1. Protein lysates from skeletal muscles and adipose tissues, or whole-cell lysates from cultured cells were quantified using BCA protein assay Beyotime.

The final results were visualized with chemiluminescence ECL western blotting substrates. A proximity-dependent biotin identification BioID assay was used to identify the receptors for Musclin on the plasma membrane of adipocytes.

The agarose beads were pelleted by centrifugation and then subjected to five times washing with washing buffer, followed by protein denaturation, SDS-PAGE gel electrophoresis, and Coomassie blue staining or silver staining Pierce Silver Stain Kit , Thermo Fisher.

The gel fragments in pulldown samples containing differential protein bands as compared to control were collected for electrospray ionization tandem M.

analysis on a Thermo Finnigan LTQ Orbitrap Instrument Proteome Discoverer version 1. The mass spectrometry proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE 74 partner repository with the dataset identifier PXD HEKT cells were transiently transfected with plasmids expressing control or Musclin.

Finally, cells and culture media were harvested separately. Cells were subjected to total protein lysate preparation.

The obtained protein pellet was washed in cold acetone, air-dried, and finally resuspended in SDS-containing lysis buffer.

The input and IP samples were subjected to immunoblotting using antibodies against Musclin Abcam , Flag Sigma, A, M2 , or HSL Cell signaling, s. Cell proteins were quantified using a BCA protein assay kit Beyotime for normalization.

Tissue weight was used for normalization. A standard chromogen method was used to measure the tissue non-heme iron described as previously described All statistical analyses were performed using GraphPad Prism 9 software.

n values represent biological replicates for cell experiments, or mouse number for in vivo animal studies, or human subject number unless otherwise indicated. Specific details for the n value are noted in each figure legend.

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

RNA-Seq data of iWAT from cold-acclimated mice and their controls are available in SRA database under accession code: PRJNA The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 74 partner repository with the dataset identifier PXD , which can be freely accessed.

The images representing human participants, mouse models, skeletal muscle, myotube, adipose tissue, RNA-Sequencing, qPCR analysis, culture dish, and adipocyte shown in Figs. Source data are provided with this paper. Meng, Z. et al. Glucose sensing by skeletal myocytes couples nutrient signaling to systemic homeostasis.

Cell 66 , — e Article CAS PubMed PubMed Central Google Scholar. Baf60c drives glycolytic metabolism in the muscle and improves systemic glucose homeostasis through Deptor-mediated Akt activation.

Diabetes 63 , — Uncoupling exercise bioenergetics from systemic metabolic homeostasis by conditional inactivation of Baf60 in skeletal muscle. Diabetes 67 , 85—97 Article CAS PubMed Google Scholar.

Pedersen, B. Muscles, exercise and obesity: skeletal muscle as a secretory organ. Febbraio, M. Who would have thought—myokines two decades on.

Article PubMed Google Scholar. Whitham, M. Extracellular vesicles provide a means for tissue crosstalk during exercise. Cell Metab.

Giudice, J. Muscle as a paracrine and endocrine organ. Article CAS Google Scholar. Priest, C. Inter-organ cross-talk in metabolic syndrome. Ishibashi, J. Beige can be slimming. Science , — Petrovic, N.

Chronic peroxisome proliferator-activated receptor gamma PPARgamma activation of epididymally derived white adipocyte cultures reveals a population of thermogenically competent, UCP1-containing adipocytes molecularly distinct from classic brown adipocytes. Rosen, E.

What we talk about when we talk about fat. Cell , 20—44 Ikeda, K. The common and distinct features of brown and beige adipocytes. Trends Endocrinol. Wu, J. Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human. Cell , — Matthew, H.

Brown and beige fat: development, function and therapeutic potential. Article Google Scholar. Townsend, K. Brown fat fuel utilization and thermogenesis. UCP1-independent signaling involving SERCA2b-mediated calcium cycling regulates beige fat thermogenesis and systemic glucose homeostasis.

Med 23 , — Kaisanlahti, A. Browning of white fat: agents and implications for beige adipose tissue to type 2 diabetes.

Biochem 75 , 1—10 Carobbio, S. Brown and beige fat: From molecules to physiology and pathophysiology. Acta Mol. Cell Biol. lipids , 37—50 Blüher, M. Obesity: global epidemiology and pathogenesis. Wang, Q. The hepatokine Tsukushi gates energy expenditure via brown fat sympathetic innervation.

Article PubMed PubMed Central Google Scholar. Xiong, X. Landscape of intercellular crosstalk in healthy and NASH liver revealed by single-cell secretome gene analysis. Cell 75 , — Thomas, G. Osteocrin, a novel bone-specific secreted protein that modulates the osteoblast phenotype.

Nishizawa, H. Musclin, a novel skeletal muscle-derived secretory factor. Kazak, L. A creatine-driven substrate cycle enhances energy expenditure and thermogenesis in beige fat. Genetic depletion of adipocyte creatine metabolism inhibits diet-induced thermogenesis and drives obesity.

Ablation of adipocyte creatine transport impairs thermogenesis and causes diet-induced obesity. Roux, K. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.

Li, J. CAS PubMed Google Scholar. Tsai, T. Sim, J. P2X1 and P2X4 receptor currents in mouse macrophages. Shen, W. Interaction of rat hormone-sensitive lipase with adipocytelipid-binding protein. Natl Acad. USA 96 , — Article ADS CAS PubMed PubMed Central Google Scholar.

Bostrom, P. A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.

Nature , — Article ADS PubMed PubMed Central Google Scholar. Rao, R. Meteorin-like is a hormone that regulates immune-adipose interactions to increase beige fat thermogenesis. Roberts, L. β-Aminoisobutyric acid induces browning of white fat and hepatic β -oxidation and is inversely correlated with cardiometabolic risk factors.

Moffatt, P. Osteocrin is a specific ligand of the natriuretic peptide clearance receptor that modulates bone growth. Watanabe-Takano, H. Mechanical load regulates bone growth via periosteal Osteocrin.

Cell Rep. Zhou, R. Endocrine role of bone in the regulation of energy metabolism. Bone Res. Ataman, B. Evolution of Osteocrin as an activity-regulated factor in the primate brain. Subbotina, E. Musclin is an activity-stimulated myokine that enhances physical endurance.

USA , — Szaroszyk, M. Skeletal muscle derived Musclin protects the heart during pathological overload. Li, Y. Role of musclin in the pathogenesis of hypertension in rat. PLoS One 8 , e Miyazaki, T. A new secretory peptide of natriuretic peptide family, osteocrin, suppresses the progression of congestive heart failure after myocardial infarction.

Hu, C. Osteocrin attenuates inflammation, oxidative stress, apoptosis, and cardiac dysfunction in doxorubicin-induced cardiotoxicity. Kattih, B. Low circulating musclin is associated with adverse prognosis in patients undergoing transcatheter aortic valve implantation at low-intermediate risk.

Heart Assoc. Zhong, Y. Decreased plasma musclin levels are associated with potential atrial fibrillation in non-diabetic patients. Sternberg, E. Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene.

CAS PubMed PubMed Central Google Scholar. Bothe, G. Selective expression of Cre recombinase in skeletal muscle fibers. Genesis 26 , — Kita, S. Competitive binding of musclin to natriuretic peptide receptor 3 with atrial natriuretic peptide. Zois, N. Natriuretic peptides in cardiometabolic regulation and disease.

Bae, C. Adipocyte-specific expression of C-type natriuretic peptide suppresses lipid metabolism and adipocyte hypertrophy in adipose tissues in mice fed high-fat diet. Katafuchi, T. Peptides 31 , — Levy, J. Transferrin receptor is necessary for development of erythrocytes and the nervous system.

Hentze, W. Balancing acts: molecular control of review mammalian iron metabolism. Zhang, Z. Adipocyte iron levels impinge on a fat-gut crosstalk to regulate intestinal lipid absorption and mediate protection from obesity.

Senyilmaz, D. Regulation of mitochondrial morphology and function by stearoylation of TFR1. Qiu, J. Transferrin receptor functionally marks thermogenic adipocytes.

Cell Dev. Eguchi, J. Transcriptional control of adipose lipid handling by IRF4. Kong, Q. BAF60a deficiency in macrophage promotes diet-induced obesity and metabolic inflammation.

Diabetes 71 , — Wang, R. Dietary intervention preserves beta cell function in mice through CTCF-mediated transcriptional reprogramming. Sun, Y. Mitochondrial TNAP controls thermogenesis by hydrolysis of phosphocreatine.

Zhang, P. Neuregulin 4 suppresses NASH-HCC development by restraining tumor-prone liver microenvironment. Hu, W. High phosphate impairs arterial endothelial function through AMPK-related pathways in mouse resistance arteries. Acta Physiol. Müller, H. Placental lactogen-I PL-I target tissues identified with an alkaline phosphatase—PL-I fusion protein.

Kim, D. HISAT: a fast spliced aligner with low memory requirements. Methods 12 , — Li, R. SOAP: short oligonucleotide alignment program. Bioinformatics 24 , — Li, B. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome.

Love, M. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. Yu, G. clusterProfiler: an R package for comparing biological themes among gene clusters.

OMICS: A J. Wickham, H. ggplot2: Elegant Graphics for Data Analysis Springer International Publishing, Chen, T. The genome sequence archive family: toward explosive data growth and diverse data types.

CNCB-NGDC Members and Partners. Database resources of the national genomics data center, China National Center for Bioinformation in Nucleic Acids Res. Han, J. The CREB coactivator CRTC2 controls hepatic lipid metabolism by regulating SREBP1. Article ADS CAS PubMed Google Scholar. Perez-Riverol, Y.

The PRIDE database resources in a hub for mass spectrometry-based proteomics evidences. Rebouche, C. Microanalysis of non-heme iron in animal tissues. Biochem Biophys. Methods 58 , — Download references.

The authors thank Drs. Dante Neculai Zhejiang University , Christopher R Wood Zhejiang University , and Zhe Yu Zhang Zhejiang University for critical proofreading and editing of the manuscript.

We thank the Meng lab members for the helpful discussion and technical support for this study. We also thank Drs. Peng Wu and Wei Chen Zhejiang University and Dr. Cheng Ma the Core Facilities of Zhejiang University School of Medicine for technical support.

We also appreciate Dr. Nan Xu Henan University for providing the HUVECs purchased from ATCC. This work was supported by grants from the National Natural Science Fund for Excellent Young Scholars of China , the National Key Research and Development Programme of China YFA, YFC , the Training Program of the Major Research Plan of the National Natural Science Foundation of China , the National Natural Science Foundation of China , , the Zhejiang Provincial Natural Science Foundation of China LZ21H, LHDMD22H , the Construction Fund of Medical Key Disciplines of Hangzhou OO , the Innovative Institute of Basic Medical Sciences of Zhejiang University, and the Fundamental Research Funds for the Central Universities to Z.

This study was also supported by grants from the National Natural Science Foundation of China , the Zhejiang Provincial Natural Science Foundation of China LQ21C , and the China Postdoctoral Science Foundation M to S. This work was also supported by grants from the National Key Research and Development Programme of China YFC, YFC , the National Natural Science Foundation of China , , the Key Disciplines of Medicine Innovation discipline, CX24 and the Fundamental Research Funds for the Central Universities XZZX to J.

The authors gratefully acknowledge the support from K. Wong Education Foundation. Department of Pathology and Pathophysiology and Department of Cardiology of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

Key Laboratory of Disease Proteomics of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, China. Chronic Disease Research Institute, Zhejiang University School of Public Health, Hangzhou, China.

Life Sciences Institute and Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, USA. Department of Biology and Chemistry, College of Science, National University of Defense Technology, Changsha, China. Department of Endocrinology and Metabolism, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

Department of Endocrinology, The Second Affiliated Hospital of Soochow University, Suzhou, China. The Second Affiliated Hospital, School of Public Health, Zhejiang University School of Medicine, Hangzhou, China.

Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, China. Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

You can also search for this author in PubMed Google Scholar. conceived and designed the research. Lv, X. Li, H. performed the experiments. conducted the bioinformatics analysis.

provided critical reagents for this study. contributed to the discussion and data interpretation. contributed to the discussion. wrote the manuscript with help from the other authors. Correspondence to Jun-Fen Fu or Zhuo-Xian Meng.

Nature Communications thanks the anonymous reviewer s for their contribution to the peer review of this work. Peer reviewer reports are available. Open Access This article is licensed under a Creative Commons Attribution 4. Reprints and permissions.

Jin, L. Nat Commun 14 , Download citation. Received : 02 March Accepted : 27 June Published : 19 July Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative. By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily. Skip to main content Thank you for visiting nature. nature nature communications articles article.

Download PDF. Subjects Homeostasis Metabolic syndrome Obesity. Abstract Skeletal muscle and thermogenic adipose tissue are both critical for the maintenance of body temperature in mammals.

Results Identification of Musclin as a risk factor for human obesity We firstly developed a transcriptomic screening strategy to uncover any novel muscle-enriched secreted factors that may be important in the pathogenesis of obesity and its associated metabolic disorders Fig. Full size image.

Discussion Skeletal muscle, the central organ for postprandial glucose disposal, is critical for controlling the glucose metabolism and energy homeostasis of the whole-body 1 , 2 , 3 , 4.

Methods Ethics statement All animal studies were performed in compliance with the Guide for the Use and Care of Laboratory Animals by the Medical Experimental Animal Care Committee of Zhejiang University.

Human study Human biological samples including skeletal muscle from a total of 54 donors; 41 male and 13 female; data shown in Fig. Adeno-associated virus production and transduction AAV production and purification were performed by ChuangRui Bio Lianyungang, China.

Infrared imaging Body surface temperature was measured using a thermal imaging camera FLIR Systems, Tsc InfraRed Camera. Treadmill running assay Mouse running performance was assessed using the treadmill running system from Columbus Instruments.

Preparation of Fc-tagged Musclin protein from ExpiF cells ExpiF cells were transfected with pcDNA3. Metabolic measurements An NMR analyzer NIUMAG, QMNH was applied to measure the body fat and lean mass.

Histological analysis Mouse tissues including liver, skeletal muscle, BAT, eWAT, and iWAT were dissected, fixed in formalin, followed by embedding in paraffin and cutting for tissue sections at μm for liver, skeletal muscle, and BAT, or μm for eWAT and iWAT.

SEAP-Musclin binding assay The SEAP-binding assay was performed as described RNA isolation and RT-qPCR Total RNA from WAT was isolated using a commercially available kit TIANGEN Biotech, DP Immunoblotting analysis Protein lysates from skeletal muscles and adipose tissues, or whole-cell lysates from cultured cells were quantified using BCA protein assay Beyotime.

BioID and mass spectrometry A proximity-dependent biotin identification BioID assay was used to identify the receptors for Musclin on the plasma membrane of adipocytes. Preparation of protein fractions from cell culture media HEKT cells were transiently transfected with plasmids expressing control or Musclin.

Tissue iron assay A standard chromogen method was used to measure the tissue non-heme iron described as previously described Statistical analysis All statistical analyses were performed using GraphPad Prism 9 software. Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Code availability All software and codes used in this study are open-source and publicly available. References Meng, Z. Article CAS PubMed PubMed Central Google Scholar Meng, Z.

Article CAS PubMed Google Scholar Pedersen, B. Article CAS PubMed Google Scholar Febbraio, M. Article PubMed Google Scholar Whitham, M. Article CAS PubMed Google Scholar Giudice, J. Article CAS Google Scholar Priest, C.

Article PubMed Google Scholar Ishibashi, J. Article CAS PubMed PubMed Central Google Scholar Petrovic, N. Article CAS PubMed Google Scholar Rosen, E. Article CAS PubMed PubMed Central Google Scholar Ikeda, K. Article CAS PubMed PubMed Central Google Scholar Wu, J. Article CAS PubMed PubMed Central Google Scholar Matthew, H.

Article Google Scholar Townsend, K. Article CAS PubMed Google Scholar Ikeda, K. Article CAS PubMed PubMed Central Google Scholar Kaisanlahti, A. Article CAS PubMed Google Scholar Carobbio, S. Article CAS PubMed Google Scholar Blüher, M. Article PubMed Google Scholar Wang, Q. Article PubMed PubMed Central Google Scholar Xiong, X.

Article CAS PubMed PubMed Central Google Scholar Thomas, G. Article CAS PubMed Google Scholar Nishizawa, H. Article CAS PubMed Google Scholar Kazak, L.