Thermogenic Brown Fat in Humans: Implications in Energy Homeostasis, Obesity and Metabolic Disorders

Abstract

In mammals including humans, there are two types of adipose tissue, white and brown adipose tissues (BATs). White adipose tissue is the primary site of energy storage, while BAT is a specialized tissue for non-shivering thermogenesis to dissipate energy as heat. Although BAT research has long been limited mostly in small rodents, the rediscovery of metabolically active BAT in adult humans has dramatically promoted the translational studies on BAT in health and diseases. It is now established that BAT, through its thermogenic and energy dissipating activities, plays a role in the regulation of body temperature, whole-body energy expenditure, and body fatness. Moreover, increasing evidence has demonstrated that BAT secretes various paracrine and endocrine factors, which influence other peripheral tissues and control systemic metabolic homeostasis, suggesting BAT as a metabolic regulator, other than for thermogenesis. In fact, clinical studies have revealed an association of BAT not only with metabolic disorders such as insulin resistance, diabetes, dyslipidemia, and fatty liver, but also with cardiovascular diseases including hypertension and atherosclerosis. Thus, BAT is an intriguing tissue combating obesity and related metabolic diseases. In this review, we summarize current knowledge on human BAT, focusing its patho-physiological roles in energy homeostasis, obesity and related metabolic disorders. The effects of aging and sex on BAT are also discussed.

INTRODUCTION

We have been facing a worldwide pandemic of obesity, which is closely associated with not only musculoskeletal disorders but also common diseases such as diabetes mellitus, dyslipidemias, fatty liver, hypertension, and arteriosclerosis. Moreover, obesity predisposes to adverse outcomes in some diseases, as in the example of the increased mortality in patients with COVID-19. Obesity is the state of excessive accumulation of triglyceride (TG) in adipose tissues because of a prolonged positive energy balance. In mammals including humans, there are two types of adipose tissue, white and brown adipose tissues (BATs). The two tissues are similar in their major population of adipocyte having intracellular lipid droplets, but are quite different in the physiological functions. White adipose tissue (WAT) is the primary site of energy storage, while BAT is a specialized tissue for non-shivering thermogenesis (NST) to dissipate energy as heat.

Although BAT research has long been limited mostly in small rodents, the rediscovery of metabolically active BAT using fluorodeoxyglucose (FDG)-positron emission tomography (PET) and computed tomography (CT) in adult humans [1,2,3,4] has dramatically accelerated the translational studies on BAT in health and diseases. It is now established that BAT, through its thermogenic and energy dissipating activities, plays a role in the regulation of body temperature, energy expenditure (EE), and body fatness [5,6]. Moreover, over a past decade, increasing evidence has demonstrated that BAT cross talks with some peripheral tissues and controls their functions, systemic homeostasis of energy and metabolic substrates, suggesting BAT as a metabolic regulator beyond thermogenesis [7,8,9]. In this review, we summarize current knowledge on human BAT, with reference to its patho-physiological roles in energy homeostasis, obesity and metabolic diseases.

BROWN FAT AS A SITE OF ENERGY EXPENDITURE BY NON-SHIVERING THERMOGENESIS

In general, daily energy consumption is roughly divided into three components: basal/resting EE, exercise/physical activity-associated EE, and NST. A representative example of NST is cold-induced thermogenesis (CIT), an increased EE seen after cold exposure. It is now well-established that BAT is a site of CIT, and BAT-dependent CIT, together with skeletal muscle shivering, plays a role in the maintenance of body temperature under cold conditions. In fact, BAT thermogenesis is activated by acute cold exposure, and mice deficient of uncoupling protein 1 (UCP1), a key mitochondrial molecule for BAT thermogenesis, are cold-intolerant and cannot maintain their body temperature after acute cold exposure [10,11]. Similarly, in humans, cold exposure activates BAT as reflected as increased uptake of FDG and fatty acid derivatives on PET imaging (Fig. 1A) [1,2,4,12,13,14,15,16]. Moreover, the cold-induced metabolic activation of BAT correlates positively with CIT (Fig. 1B, 1C) [12,17,18], confirming a significant role of BAT for CIT in humans. Now, cold-induced FDG uptake is used as a surrogate of activity and amount of BAT in humans.

Fig. 1. Brown adipose tissue (BAT), energy expenditure (EE), and non-shivering thermogenesis. (A) BAT detected by ¹⁸F-FDG-PET/CT after acute cold exposure. (B) Basal EE under a warm condition. (C) Cold-induced thermogenesis (CIT) after acute cold exposure. (D) Diet-induced thermogenesis (DIT) after meal intake. ^*p<0.05, ^**p<0.01. Adapted from Yoneshiro et al (Obeisty [Silver Spring] 2011;19:13-6) [12] and constructed from Hibi et al (Int J Obes [Lond] 2016;40:1655-61) [29].

EE above the basal metabolic rate in response to meal intake is referred to as the “specific dynamic action of food” or “diet-induced thermogenesis (DIT)”, which is another component of NST [19,20]. As mentioned above, cold exposure is the most potent and physiological stimulus to activate BAT, but BAT-dependent CIT would be very low in our daily life under well-controlled conditions with the presence of clothing and heating systems. In contrast, DIT is observed after every meal intake independently of environmental temperature, and estimated as about 10% of ingested energy of food. DIT is usually divided into two components: obligatory and facultative thermogenesis. Obligatory thermogenesis refers to the obligatory response including digestion, absorption, and storage of ingested nutrients, whereas facultative thermogenesis refers to the additional responses to obligatory thermogenesis and may be closely related to BAT activation. Activation of BAT after food intake has been proved in small rodents. For example, a single meal ingestion produced a rapid increase in tissue respiration [21], glucose utilization and fatty acid synthesis in intact BAT of rats, but to a much lower extent in surgically denervated BAT [22]. Moreover, EE after food intake is lower in UCP1-deficient mice than in wild-type mice [23]

In humans, the possible contribution of BAT thermogenesis to DIT was suggested by Nagai et al [24], who showed that single nucleotide polymorphism (SNP) in the UCP1 gene is associated with reduced DIT. Rediscovery of BAT in adult humans has prompted further studies to test whether BAT thermogenesis is activated after single meals. Some studies using FDG-PET/CT revealed a reduced or unexpectedly low FDG uptake into BAT after meal intake [25,26]. Although these results seem conflicting with the idea of postprandial activation of BAT thermogenesis, they can be explained by increased insulin-stimulated FDG uptake into skeletal muscle, which reduces FDG bioavailability for BAT, which in turn leads to underestimation of BAT activity [26].

This limitation of FDG-PET/CT is overcome by measuring oxygen uptake using ¹⁵O[O₂]-PET and blood flow using ¹⁵O[H₂O]-PET, which are direct indicators of thermogenesis and mitochondrial substrate oxidation [27]. Din et al [28] demonstrated that oxygen consumption and blood flow in BAT rose immediately after meal intake to an extent comparable to those observed after cold exposure. To confirm the role of BAT in DIT, we measured whole-body EE continuously for 24 hours in healthy humans using a human calorimeter [29,30]. When the participants were divided into BAT-positive and -negative groups according to the result of FDG-PET/CT examination, there was no significant difference in body composition and basal EE between the two groups. However, EE after meals was significantly higher in the BAT-positive group (9.7% of the total energy intake) than in the BAT-negative group (6.5%) (Fig. 1D), suggesting that about 3% is BAT-dependent. All these results indicate that BAT contributes to DIT, at least in part, in humans.

MECHANISMS OF BROWN-FAT ASSOCIATED NON-SHIVERING THERMOGENESIS

The mechanism of cold-induced activation of BAT has extensively been investigated in vivo and in vitro, and is schematically depicted in Fig. 2. When animals are exposed to cold temperatures, cold is perceived by temperature sensors, transient receptor potential (TRP) channels, which are membrane proteins that transmit information about changes in the environment such as temperature, touch, pain, osmolarity, and naturally occurring substances. Cold-activated TRP on sensory neurons on the body surface transmit information to the brain and increase the activity of sympathetic nerves (SNs). Noradrenaline (NA) released from SN endings stimulates brown adipocytes via the β-adrenergic receptor (βAR) and triggers intracellular events including hydrolysis of TG, oxidation of resulting fatty acids, and activates UCP1.

Fig. 2. Neuro-endocrine mechanisms of cold- and diet-induced brown fat thermogenesis.βAR: β-adrenergic receptor, CCK: cholecystokinin, SCTR: secretin receptor, N: nerve, NA: noradrenaline, TGR5: G-protein-coupled bile acid-activated receptor, TRP: transient receptor potential channel, UCP1: uncoupling protein 1.

When animals are exposed to cold temperatures for a long time, they adapt to their surroundings by increasing the number of brown adipocytes and the amount of UCP1 through the proliferation of interstitial preadipocytes and matured adipocytes [31,32]. In addition to BAT hyperplasia, prolonged cold exposure gives rise to an apparent induction of UCP1-positive adipocytes in WAT. This type of adipocytes, termed “beige” or “brite” cells, is developmentally distinct from “classical” brown adipocytes [33,34], and has the thermogenic potential in response to βAR stimulation [35,36]. Thus, chronic cold exposure results in increased EE through the persistent activation and recruitment of classical brown adipocytes and beige cells, and the consequent “browning” of WAT and body fat reduction. As the UCP1-positive human adipose depot consists of a mixture of brown and beige adipocytes [37,38,39], here we refer to it collectively as BAT and thermogenic adipocytes.

Several mechanisms/factors have been proposed to participate in postprandial BAT activation. Based on the principal role of the SN-βAR axis for CIT, it is conceivable that this axis is also a key mechanism in BAT-associated DIT (Fig. 2). In fact, in both experimental animals and humans, the plasma levels of NA and tissue NA turnover are low during fasting but increases immediately after food intake [40,41,42,43]. We [22] found in rats that postprandial metabolic activation of BAT was diminished either after surgical severing of SNs entering BAT or by giving a meal through a gastric tube. LeBlanc et al [40] and Diamond et al [44] showed in humans and dogs that responses in oxygen consumption, and plasma levels of NA and insulin shortly after food intake were substantially reduced when food was administered through a stomach tube. These results collectively suggest a significant role of sympathetic activation triggered by oropharyngeal taste sensation in BAT-associated DIT.

Food intake evokes a rapid release of various gastrointestinal hormones, some of which are likely factors participating in DIT. Li et al [45] found abundant expression of the secretin receptor in murine brown adipocytes, and demonstrated that secretin activates UCP1- and secretin receptor-dependent thermogenesis in vitro and in vivo. They also confirmed in humans that the increment of plasma secretin levels induced by a single meal positively correlated with BAT activity, and that secretin infusion increased FDG uptake in BAT. In addition to secretin, other gut hormones such as cholecystokinin and glucagon-like peptide 1 were suggested to triggers BAT thermogenesis in small rodents through the vagal afferent and sympathetic nervous system [46,47]. A stimulatory role of the vagal afferent in BAT thermogenesis was also reported in humans [48]. Another humoral factor may be bile acids, which are secreted into the intestinal lumen in response to meal intake and modified by gut flora. Bile acids are now recognized as a metabolic regulator, affecting multiple functions, in addition to lipid-digestive functions, to regulate energy metabolism, as well as glucose and lipid metabolism [49]. In fact, bile acids were reported to activate BAT thermogenesis in mice and humans [50,51,52]. Thus, it is conceivable that these multiple gut derived factors, together with the SN system, participate in BAT-associated DIT, but further studies are needed to uncover the detailed neuroendocrine mechanisms of DIT in humans.

BROWN FAT AS A REGULATOR OF ENERGY BALANCE AND BODY FAT

Consistent with the significant role of BAT in NST and short-term regulation of EE, there is substantial evidence for BAT as a long-term regulator of EE and body fatness: almost all obese model animals express lower levels of UCP1 in BAT, while mice over-expressing UCP1 are leaner [53,54]. Mice lacking UCP1 get obese when they are kept at thermoneutral temperatures [55] or on a high-fat diet [56].

In humans, studies on SNP in some BAT-related genes have suggested a significant contribution of BAT thermogenesis to regulation of energy balance and body fatness. For example, Trp64Arg mutation in the β3AR gene and A3826G mutation in the UCP1 gene are associated with higher body fat content, lower metabolic rate, and smaller weight loss via treatment with low-calorie diets [57,58,59,60]. Consistently, retrospective readings of FDG-PET/CT in thousands of patients have revealed that BAT prevalence is lower in patients with higher BMI [3,61,62,63,64]. Prospective studies in healthy participants also demonstrated that BAT-negative subjects are higher in their adiposity-related parameters than BAT-positive subjects (Fig. 3A) [1,2,65,66,67]. Thus, the inverse relationship between BAT and body fatness is well confirmed (Fig. 3B). The apparent association between BAT prevalence and adiposity, however, is to be carefully evaluated, because these are considerably influenced by age. In fact, the mean age is lower in the BAT-positive participant group than the BAT-negative group (Fig. 3A). Detailed analysis revealed that the prevalence of cold-activated BAT is more than 50% in young subjects of the twenties, decreased with age, and in less than 10% of the fifties and sixties (Fig. 3C) [67]. A strong impact of age on BAT prevalence has also been reported in various clinical studies [3,61,62]. The aging process produces increased fat mass and decreased lean mass, therefore, it is possible that age-related accumulation of body fat is associated with decreased BAT activity. This is supported by the findings that the adiposity-related parameters increased with age in the BAT-negative group, while they remained unchanged from the twenties to forties in the BAT-positive group (Fig. 3D) [67]. BAT-associated NST is rather small (<100 kcal/day) in our daily life (Fig. 1C, 1D), but it is maximally 36,500 kcal/year, equivalent to 4 kg fat, which may be enough to explain the age-related accumulation of body fat. Such age-related changes in BAT will be discussed again in a latter section.

Fig. 3. Age-related changes in brown fat and body fatness. The prevalence and activity of brown adipose tissue (BAT) was assessed by FDG-PET/CT combined with acute cold exposure. (A) Mean age and obesity-related parameters in subjects with detectable BAT (BAT+) and those without it (BAT-). (B) Inverse correlation between BAT activity and visceral fat area. (C) Effects of age on BAT prevalence and visceral fat area. (D) Age-related accumulation of visceral fat in subjects with detectable BAT (BAT+) and those without it (BAT-). (E) The thermogenic activity of BAT is high during neonatal periods, but decreases with age in some individuals who get obese. In contrast, other individuals who keep BAT during their adulthood do not get obese, suggesting that BAT is protective against age-related accumulation of body fat, and that its reactivation/recruitment is effective for combating obesity. ^*p<0.05, ^**p<0.01. Constructed from Saito et al (Diabetes 2009;58:1526-31) [1] and Yoneshiro et al (Obesity [Silver Spring] 2011;19:1755-60) [67].

ACTIVATION AND RECRUITMENT OF BROWN FAT AS AN ANTI-OBESITY REGIMEN

The finding that BAT is protective against body fat accumulation has encouraged the search how to activate or recruit BAT (Fig. 3E). This is particularly intriguing because people with lower or undetectable BAT activities are more obese and to be treated. As noted in previous sections, cold is the most physiological and powerful stimulus for activation and recruitment of BAT. Cold exposure elicits increased proliferation and differentiation of classical brown adipocyte but also a remarkable induction of beige adipocytes in WAT. Some genes expressed selectively in mouse beige cells are also highly expressed in human supraclavicular fat deposits identified as BAT by FDG-PET/CT [37,68,69]. Lee et al [70] reported that preadipocytes isolated from human supraclavicular fat were capable of differentiating into UCP1-positive adipocytes in vitro. Moreover, we found that BAT activity in humans is remarkably increased during winter in individuals who showed undetectable activities in summer [1]. All these facts suggest that human BAT is largely composed of beige cells and inducible in response to appropriate stimulation. In fact, when men with undetectable or low BAT activity were kept in a cold environment for 2–6 hours every day for several weeks, their BAT activity was significantly increased while body fat was decreased [71,72,73] (Fig. 4A, 4B). More importantly, the change in BAT activity was negatively correlated with those in body fat content [71] (Fig. 4C). These results indicate that human BAT can be induced and/or recruited, and is involved in reducing body fat.

Fig. 4. Recruitment of brown fat and reduction of body fat by repeated cold exposure. Participants were exposed to either a cold (Cold) or warm (Control) condition for 2 hours every day for 6 weeks. (A) Brown adipose tissue (BAT) activity assessed by FDG-PET/CT. (B) Changes in body fat. (C) Correlation between the changes in body fat and BAT activity. Adapted from Yoneshiro et al (J Clin Invest 2013;123:3404-8) [71].

Although daily cold exposure thus can recruit human BAT, it would seem difficult to increase exposure to cold in daily life. As shown in Fig. 2, cold stimulus is received by TRP. Among the members of the TRP family, TRPM8 and TRPA1 are the most likely receptor candidates sensitive to low temperatures [74]. The mean activation temperatures of TRPA1 and TRPM8 are around 20℃, being comparable with those applied in human studies to activate BAT. Accordingly, chemical activation of these receptors would mimic the effects of cold exposure. Actually, there are various ingredients in food acting as agonists for these TRPs [74], a representative of which is menthol, a cooling and flavor compound in mint, acting on TRPM8. TRPA1 is activated by allyl- and benzyl-isothiocyanates, pungent elements in mustard and Wasabi (Japanese horseradish). Among the TRP agonists, the most extensively studied is capsaicin, a pungent principle of chili pepper, which is a potent agonist for a nociceptive receptor TRPV1. Both animal and human studies have demonstrated that capsaicin and its nonpungent analogue (capsinoids) increase BAT thermogenesis through the activation of TRPV1 and the SN system, and decrease body fat [71,75,76,77,78]. Thus, these food ingredients activating TRPs may be promising as an anti-obesity regimen easily applicable in daily life [79,80].

Pharmacological activation of BAT thermogenesis targeting β3AR expressed abundantly in adipocytes has long been expected as an anti-obesity regimen, while the β3AR agonists so fat developed showed, more or less, undesirable cardiovascular side effects in humans [81,82]. Among them, mirabegron may be promising, because it induces WAT browning with minimal side effects. Clinically, mirabegron is widely used for the treatment of overactive bladder, because it relaxes the smooth muscle of the bladder where β3AR is expressed [83]. Cypess et al [84] reported acute mirabegron treatment increases FDG uptake into BAT, in parallel with an increase in EE, heart rate, and blood pressure, at a high dose (200 mg/kg), but only slightly at clinically approved doses (50 mg/kg). Following studies, however, demonstrated that chronic treatment with 50 mg/kg mirabegron induces UCP1 in WAT and improved glucose homeostasis [85,86,87]. It is to be noted in these studies that chronic mirabegron treatment resulted in undetectable change in body fatness, but improved glucose tolerance and insulin sensitivity, suggesting beneficial effects of beige adipocytes on systemic metabolism as discussed in the next section.

BROWN FAT REGULATES SYSTEMIC METABOLISM BEYOND THERMOGENESIS

Increasing evidence has suggest that BAT has an impact on systemic metabolism, independent of that on the regulation of EE and body fatness. For instance, mouse studies have shown that transplantation of BAT or brown adipocytes results in improved glucose tolerance and increased insulin sensitivity [88,89]. In humans, we [66] found in healthy adults that blood glucose and HbA1c levels are lower in individuals with higher BAT activities, BAT being an independent determinant of these parameters (Fig. 5A). These results seem compatible with retrospective analyses of patient data showing blood glucose as a determinant of BAT prevalence [3,62,63,64]. Chondronikola et al [90] reported that mild cold exposure increased whole-body glucose disposal, plasma glucose oxidation, and insulin sensitivity in men with significant amounts of BAT, but not in those without detectable BAT. Improved insulin sensitivity in parallel with BAT recruitment after cold acclimation was also shown in healthy men [91,92] and in patients with type 2 diabetes mellitus [93]. In connection with the beneficial effects of BAT on insulin sensitivity, interesting is that BAT actively metabolizes branched chain amino acids (BCAA) to lower their plasma levels in humans [94]. Since the accumulation of intracellular BCAA is known to inhibit insulin signaling through mTOR activation, impaired insulin sensitivity often seen in obesity and aging may be attributable to increased circulating BCAA resulting from reduced BAT activity [95].

Fig. 5. Impact of brown fat on blood parameter and cardiometabolic diseases. (A) Blood parameters in healthy subjects. HDL-Cho: high-density lipoprotein cholesterol, LDL-Cho: low-density lipoprotein cholesterol, TG: triglyceride. Constructed from Matsushita et al (Int J Obes [Lond] 2014;38:812-7) [66]. (B) Cardiometabolic diseases. AF: atrial fibrillation/atrial flutter, CAD: coronary artery disease, CHF: congestive heart failure, CVD: cerebrovascular disease, T2DM: type 2 diabetes. Closed circles show a significant association with BAT independent of body adiposity, age, and sex. Adapted from Becher et al (Nat Med 2021;27:58-65) [64].

Very recently, Seki et al [96] reported that the BAT-associated changes in systemic glucose metabolism result in a marked suppression of tumor. Exposure of tumor-bearing mice to cold conditions inhibits the growth of various types of solid tumor. The cold-induced tumor suppression is ablated by surgical removal of BAT and feeding on a high-glucose diet, and in UCP1-deficient mice. Mechanistically, cold-induced BAT activation results in increased systemic glucose metabolism and decreased blood glucose, which impedes the glycolysis-based metabolism in cancer cells. A pilot study in a patient with Hodgkin’s lymphoma showed a markedly reduced FDG uptake into the tumor tissue after mild cold exposure, but more human studies are needed to strengthen its clinical relevance.

BAT has also significant effects on systemic lipid metabolism. The main energy source of BAT thermogenesis is fatty acids derived from intracellular TG and incorporated from circulation. BAT activation by cold exposure markedly accelerates clearance of plasma TG-rich lipoproteins due to increased uptake into BAT, and corrects hyperlipidemia [97]. Similar effects were also found by pharmacologic activation of BAT with aß3AR agonist, which improves dyslipidemia, and more importantly, protects hyperlipidemic model mice from atherosclerosis [98]. Activation of BAT thermogenesis increases high-density lipoprotein (HDL) levels, promoting HDL cholesterol turnover [86,99]. All these facts suggest a direct contribution of BAT to the regulation of blood lipoprotein metabolism.

The effects of BAT on systemic metabolism would be closely related to the etiology of various metabolic and cardiovascular diseases [100]. Becher et al [64] categorized 53,475 patients by presence or absence of BAT, and found improved profiles of blood glucose, TG, and HDL-cholesterol in individuals with BAT, as already reported in healthy humans. Notably, BAT independently correlated with lower rates of type 2 diabetes, dyslipidemia, hypertension, coronary artery disease, and congestive heart failure (Fig. 5B). These results are well consistent with previously reported association between BAT and diabetes [62] and also with a 5-year follow-up study [101] showing that BAT activity correlated with lower carotid intima-media thickness and higher carotid elasticity. An association of BAT with hepatic steatosis was also reported in humans [102,103,104].

BROWN FAT-DERIVED ENDOCRINE FACTORS AFFECTING SYSTEMIC METABOLISM

As mentioned above, even small amounts of BAT influence the broad range of systemic metabolisms. Mechanistically, some of the effects may be attributed to the relatively high metabolic activity of BAT itself, as in the case of the lowering effect on TG-rich lipoprotein and BCAA; however, other effects seem difficult to be explained solely by its own activity. In this connection, it is interesting that BAT secretes various molecules into extracellular fluid, collectively called as batokine (BATkine), which may mediate the effects of BAT on other tissues [8,105] (Fig. 6).

Fig. 6. Endocrine actions of brown fat-derived factors, BATkines: 12,13-diHOME,12,13-dihydroxyoctadecaenoic acid, FA: fatty acid, FGF21: fibroblast growth factor 21, IL-6: interleukin-6, miR: microRNA, NGF: nerve growth factor, NRG: neuregulin 4, VEGF: vascular endothelial growth factor.

Activated BAT secretes some growth factors, such as vascular endothelial growth factor and nerve growth factors, which promote angiogenesis and SN growth associated with tissue hyperplasia [106,107,108]. In addition to these paracrine factors, BAT secretes a wide variety of polypeptides into circulating blood. Among them, interleukin-6 (IL-6) may be a likely BATkine to regulate systemic glucose metabolism. In the study of BAT transplantation, Stanford et al [109] found that the improved metabolic profile was lost when the BAT used for transplantation was obtained from IL-6-deficient mice. Qing et al [110] reported that acute stress induces IL-6 secretion from BAT, which is the required instructive signal for mediating hyperglycemia through hepatic gluconeogenesis.

Another candidate of BATkines is fibroblast growth factor 21 (FGF21). FGF21 is released abundantly from the liver, but it is also released from activated BAT and contributes to increased circulating levels [111,112,113]. FGF21 is known as an important regulator of systemic metabolism and whole-body energy balance; it increases energy expenditure by inducing BAT thermogenesis and WAT browning, while it suppresses sugar intake by actin on the ventromedial hypothalamus. FGF21 stimulates glucose utilization in WAT, hepatic fatty acid oxidation, and peripheral lipoprotein catabolism [114,115]. Ruan et al also [116] reported that brown adipocyte-specific FGF21 knockout impaired the effects of adenosine A2A receptor agonism in attenuating hypertensive cardiac remodeling, suggesting an endocrine role of BAT in controlling hypertensive cardiac remodeling though the release of FGF21. Despite many studies in mice, however, it remains to be investigated whether FGF21 secreted from BAT, compared with that from the liver, plays key physiological roles in humans.

In addition, many polypeptides such as neureglin4 (NRG4) and myostatin were identified as BATkines [117,118], but a comprehensive overview is beyond the scope of this article and found in other literatures [8,105].

MicroRNAs (miRNAs) may serve as signals between BAT and other tissues. miRNAs produced in adipocytes have a role in the differentiation and function of adipocyte itself and are also secreted through exosomes and taken-up into other cells [119]. Thomou et al [120] reported that mice with adipose-tissue-specific deletion of the miRNA-processing enzyme exhibit a substantial decrease in the levels of circulating exosomal miRNAs, including miRNA-99b, and that the transplantation of BAT restores the circulating miRNA level, improves glucose tolerance, and suppresses hepatic FGF21 expression. These results suggest that exosomal miRNA-99b is secreted from BAT into blood circulation, acts on the liver, and suppresses hepatic FGF21 expression. It was also reported that circulating levels of miR-92a-3p and miRNA-122 are inversely correlated with BAT activity in humans [121,122].

Some lipid molecules are also suggested as active BATkines. Oxylipins, molecules derived from the oxidation of polyunsaturated fatty acids, are attracted interest because of their action as intercellular signaling molecules and involvement in the regulation of many cell and tissue responses. Particularly interesting is a linoleic acid derivative 12,13-dihydroxyoctadecaenoic acid (12,13-diHOME) secreted from BAT upon cold exposure. 12,13-diHOME activates BAT in autocrine manner to enhance thermogenesis, resulting in decreased levels of serum TGs [123]. Moreover, 12,13-diHOME secretion from BAT is enhanced by exercise and increases fatty acid uptake in muscle [124]. Very recently, Sugimoto et al [125] reported that cold- and β3AR agonist-activated BAT produces maresin 2, a member of the anti-inflammatory pro-resolving mediators synthesized from docosahexaenoic acid, and reduces inflammation in obesity in part by targeting macrophages in the liver. A role of BAT metabolism of succinate, an intermediate of the tricarboxylic cycle, has also been suggested in liver inflammation. Cold exposure produces substantial and selective accumulation of succinate in BAT, which is sufficient to elevate UCP1-dependent thermogenesis and sequester elevated circulating succinate [126,127]. In contrast, without UCP1, BAT exhibits a diminished capacity to clear succinate from the circulation, and elevated extracellular succinate in liver tissue that drives inflammation through the action on liver resident stellate cells and macrophages [128]. As such, BAT may regulate liver immune cell infiltration and pathology through succinate metabolism.

In addition, several small molecules were identified as BATkine candidates: for example, 3-methyl-2-oxovaleric acid, 5-oxoproline and β-hydroxyisobutyric acid synthesized in and released from brown adipocytes induce thermogenic gene expression in white adipocytes and energy metabolism in skeletal myocytes [129]. Thus, BATkines are likely to mediate the beneficial effects of activated BAT on other tissues/organs, but their pathophysiological roles in humans are largely elusive to date.

AGE-RELATED CHANGES IN BROWN FAT FUNCTIONS

As mentioned above, BAT is a promising tissue combating obesity and cardiometabolic diseases. It is also true, however, that the amount and activity of human BAT decline remarkably with age, while the prevalence of these diseases increases. Accordingly, it is important to understand the mechanisms/factors of age-related decline of BAT and how to re-activate and recruit BAT in elderly [130].

Ageing is associated with a decline of both classical and beige adipocytes, reflected by increased intracellular lipid accumulation, reduced mitochondrial and UCP1 content, and reduced ability of beige adipocyte induction [131,132]. There is a heterogenous population of beige adipocyte progenitor cell, which is maintained during postnatal life by its ability to proliferate and differentiate in response to beiging signals [133]. Aging is associated with not only an impaired ability of beige adipocyte progenitor cell to proliferate, which leads to a marked decrease in the progenitor cell number, but also a decreased differentiating ability of the progenitor cell [134,135,136].

The age-related change of BAT is regulated by multiple intrinsic factors, including a longevity gene Sirtuin 1 [137], CD81 [138], and the FSTL1 gene encoding the follistatin-like one glycoprotein [139]. Mitochondrial dysfunctions are also likely to link intrinsically to age-related decline of BAT thermogenic function [140]. Tajima et al [141] reported that mitochondrial lipoylation is reduced in aged BAT, but its enhancement by alipoic acid supplementation restores BAT function, thereby preventing age-associated obesity and glucose intolerance.

Among various neuro-endocrine mechanisms/factors, the SN-bAR system may be critical. In aged animals, sympathetic signaling to BAT is greater both at thermoneutrality and during cold exposure [142,143], whereas BAT cell proliferation induced by cold exposure is substantially attenuated [144]. These facts imply that ageing is associated with the decreased sensitivity of BAT to sympathetic stimulation. Consistent with this idea, there is a report demonstrating decreased β3AR mRNA expression and impaired cellular responses to adrenergic stimulation in BAT of aged mice [145]. This seems well compatible with the observations in humans that SN activity increases with age, while thermogenic response to cold decreases [146]. We demonstrated a significant impact of SNP in the β3AR gene on the age-related decline in human BAT [60]. Bahler et al [147] reported, however, using imaging technics with a ¹²³I-labelled NA analogue and ¹⁸F-FDG, that both sympathetic drive and BAT activity are lower in older men than in younger men. Thus, further studies are needed to draw out the precise mechanism underlying the age-related decline in BAT in terms of SN activity.

Another likely mechanism is related to the production of pro-inflammatory mediators by macrophages infiltrating in adipose tissue [148]. It is known that the number of M1, but not M2 macrophage, in adipose tissue increases in obesity. While anti-inflammatory resident M2 macrophages are stimulatory to beige adipocyte induction [149], pro-inflammatory M1 macrophages impair brown adipocyte activity and beige adipogenesis [150,151,152]. Pro-inflammatory cytokines such as tumor necrosis factor a and interleukin-1β induce a decreased viability of brown adipocytes accompanied by a massive reactive oxygen species production and downregulation of thermogenic gene expression [153], and induce apoptosis of brown adipocytes [154]. It is thus likely that increased infiltration of M1 macrophage in WAT facilitates the attenuated browning of WAT, suggesting the suppression of M1 macrophage activity in WAT would be effective to induce beige adipocytes, thereby preventing against obesity and related metabolic disorders.

SEX DIFFERENCES IN BROWN FAT

Ageing is associated with several endocrine changes, including diminished gonadal function. Animal studies have demonstrated sex differences in BAT: for example, compared to BAT of male rats, that of females shows higher mitochondrial density and cristae height, higher UCP1 levels, and higher sensitivity to adrenergic stimulation [155]. Higher ability of WAT browning was also reported in female mice [156]. A comprehensive gene expression analysis revealed higher UCP1 expression in WAT of female mice [157]. Consistently, human studies have demonstrated sex difference in CIT and BAT throughout the life span [158,159]. In children, the activity and mass of BAT change slightly with age along with sexual maturation, being higher in prepubertal girls than in boys [160]. In adults, although the prevalence of BAT detected by FDG-PET scans markedly declines with age, its sex difference persists, being higher in women than in men [3,61,62,63,161,162]. Moreover, higher UCP1 expression and browning potential of WAT in women were reported [163,164]. It is to be careful, however, that the sex difference in BAT detected by FDG-PET may not always reflect the difference in the amount and/or intrinsic activity of BAT. In almost all clinical studies in patients so far reported, FDG-PET was carried out at room temperatures without maximizing BAT activity by cold exposure. Considering sexual dimorphism of thermic, metabolic and cardiovascular responses to cold exposure [165], as well as cold sensation [166], it is possible that the above-mentioned sex difference in human BAT may be due to the difference in the sensitivity to cold stimulus. In fact, several prospective studies in healthy participants revealed no significant sex difference in the prevalence and activity of BAT estimated under cold conditions [15,167,168], despite of higher CIT in women.

The sex dimorphism suggests the direct and indirect effects of gonadal hormones on BAT [169]. Estrogens are the most likely hormone to enhance the activity and differentiation of BAT [170]. The stimulatory effects of estrogens are mediated both directly through the action on nuclear receptors (ER) expressed in brown adipocytes [171] and indirectly through the brain-SN system [172]. In contrast to estrogens, androgens were reported to inhibit differentiation and UCP1 expression in brown adipocytes in vitro [173,174]. In vivo studies reported that surgical removal of testis results in increased UCP1 expression in BAT and WAT [175,176]. These results seem consistent with those in human studies that hyperandrogenism in women as in polycystic ovary syndrome (PCOS) is associated with lower BAT activity and obesity [177,178]. Reduced UCP1 expression was also found in androgen-induced PCOS model animals [179,180]. However, in vivo effects of androgens so far reported are controversial. For example, several studies have shown no effects of dihydroxytestosterone treatment on UCP1 expression in BAT of orchiectomized mice or female mice [181,182,183]. Moreover, mice deficient of the nuclear androgen receptor were shown to display a reduced expression of thermogenic genes in BAT and get obese [184]. Thus, the in vivo effects of androgens appear rather complicated, but a part of them may be explained by locally increased estrogen level due to intratissue aromatizaition of androgens in WAT and the brain [185]. In fact, the testosterone-induced reduction of WAT mass in hypogonadal male mice requires ER in the brain [186]. Further studies are needed to uncover the mechanisms and factors responsible to the sex differences in BAT.

CONCLUSION AND FUTURE PERSPECTIVES

As discussed throughout the previous sections, BAT participates in the regulation of whole-body EE, systemic metabolism, and cardiovascular functions in humans. Accordingly, this specific tissue is now recognized as an intriguing therapeutic target of obesity and metabolic disorders such as diabetes mellitus, dyslipidemia, and related cardiovascular diseases. In fact, several drugs and food ingredients targeting BAT have been tested for pharmacological and nutritional therapy of obesity and metabolic syndrome [79,81,187]. Although not discussed in this article, possible contribution of BAT to some beneficial effects of exercise is also suggested, particularly in context with the interaction between skeletal muscle and beige adipocytes [188,189].

Despite these advances in BAT researches, there are some unsolved but critical problems in humans, including the genetic and environmental factors responsible for a remarkable individual difference in the amount and activity of BAT, and the patho-physiological relevance of UCP1-independent thermogenic mechanisms of beige adipocytes [190]. Particularly important is the method used in assessing human BAT. To date, FDG-PET is a standard tool [191]; however, this method has serious limitations, including the enormous cost of devices, radiation exposure, and acute cold exposure, which make repeated measurements difficult and an impediment in basic and clinical studies. Moreover, the uptake of FDG into BAT is not always associated with its thermogenic activity. There is therefore an urgent need to establish less invasive and simpler methods for quantitative assessment of human BAT [192,193,194,195]. This would promote prospective human studies, including longitudinal observations and the development of practical, easy, and effective regimens that can activate and recruit BAT.