In both rodents and humans, BAT is activated by sympathetic nervous system (SNS) stimulation [13] through the release of norepinephrine and its non-selective agonism on β1, 2, and 3 adrenergic receptors (ADRs) on various BAT tissues [14]. Downstream activation of the βADRs increases the release of stored nutrients such as free fatty acids that upregulate Ucp1 in BAT and can also induce browning in WAT. Moreover, recent research indicates that Ucp1 is dispensable to increasing EE due to browning through the Ucp1 independent futile creatine cycling [15]. While stimulation by β3 agonists activates BAT in rodents [16, 17], there is conflicting evidence regarding which βADR subtype is the primary activator of human BAT [18–21]. In both rodents [22] and humans [23], BAT activation through cold exposure increases fatty acid uptake through the activation of βADRs. Obese and T2D individuals, who could benefit from increased EE through BAT activation, generally lack the presence of metabolically active BAT. Nevertheless, both rodent and human studies have investigated several pharmacological agents that activate BAT, through various mechanisms, including direct and indirect βADRs. However, these studies have shown differing levels of success. In the next section we will review BAT activators and discuss the relevance of the pathways involved.

Various pharmacological activators have been studied in rodents to upregulate BAT activity for weight loss purpose. The activation of BAT results in the maintenance of thermal homeostasis and disposal of excess metabolites [1]. 2-4-dinitrophenol (DNP), a component of explosives, wood preservation solutions, and herbicides, was commercially used as a weight loss medication for 5 years in the 1930s [24]. While not a direct activator of BAT, DNP induces extreme increases in metabolic rate and thus weight loss through widespread mitochondrial uncoupling [24]. Interestingly, these mechanisms appear to be independent to Ucp1 pathways. DNP supplemented drinking water increased metabolic rate and induced fat and total body mass loss in diet-induced obese (DIO) female C57BL/6J mice held at thermoneutrality (30°C) after 9 weeks [25]. Surprisingly, DNP treatment decreased mRNA and protein levels of Ucp1, which could be reflective of reduced BAT function even in the face of increased EE.

Berberine (BBR), a plant derived anti-obesity medication, increases adipose triglyceride lipase (ATGL) expression and basal rates of TG lipolysis in adipose tissue [26]. Daily intraperitoneal (i.p.) BBR administration of 1.5 mg/kg for 6 weeks to obese C57BL/6 male mice increased BAT thermogenesis and EE [27]. Interestingly, this BBR treatment also increased BAT volume, glucose uptake, and Ucp1 and Prdm16 expression levels in these obese male mice [27]. Similarly, 4 weeks of BBR i.p. injections at a higher dose of 5 mg/kg/day in obese male C57BL/6J mice increased EE, lipid oxidation, BAT ¹⁸F-FDG uptake, BAT mitochondrial content, UCP1 protein expression, and BAT transcription factor genes such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1-α) that regulate energy metabolism [28]. This indicates that BBR can induce BAT activation through mechanisms other than direct βADR agonism. However, the direct pathways through which BBR increases BAT remains unclear. Other plant derived agents include Withania somnifera extract (WSE), and withaferin A (WFA) a major constituent of WSE. Dietary supplementation of 0.5% WSE to a HFD for 10 weeks increased mRNA expression of genes involved in thermogenesis and mitochondrial biogenesis, and reduced lipid droplet size in BAT of DIO male C57BL/6J mice [29, 30]. Oral supplementation (1.5 mg/kg) of WFA for 7 days in DIO male C57BL/6J mice increased EE, UCP1 protein and mRNA levels in BAT, and phosphorylation of p38 and ERK_1/2 in WAT indicative of browning. Interestingly, WFA treated mice closely resembled the phenotypic results observed in the metabolically healthy low-fat diet-fed group [30]. While not providing direct evidence of BAT activation, these studies suggest that WSE and WSA may influence mitochondrial biogenesis and Ucp1 expression in both BAT and WAT.

Resveratrol, a polyphenol produced in the skin of fruits such as grapes and berries, has been investigated for its protective effects against weight gain [31, 37] and obesity related diseases. It has been extensively reviewed [32–35]. However, in brief, the changes in adipose tissue are suggested to act through WAT browning by promoting AMPK phosphorylation, which increases PGC1α and sirtuin 1 (SIRT1) production. This induction leads to mitochondrial biogenesis and browning, rather than direct BAT activation [36, 37]. This is supported by morphological changes in WAT to BAT-like lipid droplets, and increased BAT mass [38]. Alternately, resveratrol may adjust gut microbiota composition that improves intestinal barrier dysfunction and reduces inflammation [39]. While resveratrol supplementation offers benefits in improving metabolic conditions, there is little evidence supporting direct BAT activation. The interaction between resveratrol and adipose tissue appears to occur through WAT browning rather than BAT activation.

Several studies have evaluated βADR agonists to increase BAT activity in rodents. BAT in rodents expresses functional β3 and β1 ADR, with β3ADR now being well-established to directly upregulate BAT processes [40]. In obese female C57BL/6 (ob/ob) mice, oral administration of β3ADR agonists BRL 26830A, BRL 33725A, and BRL 35135A for 4 weeks showed a decrease in weight gain with no change in food intake, and BAT UCP1 protein expression [41].Wilson S et al reported that lean male Sprague-Dawley rats given single i.p injections of 5 mg/kg of BRL 26830A increased EE while simultaneously increasing lipid oxidation in BAT, thus resulting in a substantial decrease in BAT lipid droplet size [42]. In sedentary obese male Zucker rats, gastric cannula administration of BRL 35135 increased mitochondrial guanosine diphosphate (GDP) binding, an indicator of increased Ucp1 uncoupling, mitochondrial protein content, and reduced WAT mass compared to those that received either vehicle or an ⍺₂ ADR agonist [43]. Both acute and chronic treatment with the selective β3ADR agonist ICI D7114 also suggest BAT activation in rats. In male Wistar rats, ICI D7114 increased EE and mitochondrial GDP binding in BAT suggesting BAT activation, however this was associated with an increase in heart rate [44]. As chronic tachycardia increases risk of cardiovascular events, a human safety trial is warranted [45]. To note, ICI D7114 had no effect on weight loss or changes in fat mass in male Sprague-Dawley rats [46]. Due to technological limitations in these studies, it is difficult to attribute metabolic changes to BAT activation alone.

Recently, there has been a shift towards investigating non-sympathomimetic methods for BAT activation, as sympathomimetics could inadvertently stimulate the central nervous system and non-target organs. Peroxisome proliferator-activated receptor γ (PPARγ) is a transcriptional regulator necessary for the maturation of brown adipocytes [47]. It promotes BAT adipocyte differentiation and lipid storage [48]. PPARγ agonists have been used in T2D treatment to improve insulin sensitization but can result in weight gain. Interestingly, PPARγ mediated increases in oxygen consumption and fatty acid oxidation have been repeatedly shown in vitro [49, 50]; however, these effects are not observed in vivo. In fact, opposite effects are seen in both humans [51] and rodents [52], where PPARγ activation increases lipid storage. While PPARγ agonism is an exciting concept for BAT activation in vitro, the inability to replicate increases in EE and lipid oxidation in vivo makes its use in treating metabolic complications beyond insulin sensitization unlikely.

Signalling from the angiotensin II (AngII) has regulatory effects on energy homeostasis and can act through PPARγ activation to promote adipocyte differentiation and insulin sensitivity. However the role of AngII signalling specifically in BAT is less clear [53]. Recently, the use of angiotensin type 2 receptor (AT2R) agonism has been explored as an alternate method of BAT activation [54]. C21, an AT2R agonist, was not protective against weight gain from HFD feeding in male C57BL/6J mice. However, C21-treated mice showed increased BAT mass and brown adipocyte differentiation despite being fed a HFD compared to mice fed a regular chow and treated with C21. While direct measures of BAT activity were not taken, protein levels of UCP1 and electron transport chain complexes I, II, III, and IV were upregulated with C21 treatment in HFD fed mice independent to changes in ATP synthase. This may suggest increased thermogenic capacity in an obese state, however further investigation is required to determine if AT2R agonism can improve metabolic outcomes such as glucose or lipid levels and if there is indeed BAT activation.

Metabokines produced from branch chain amino acid catabolism including 3-methyl-2-oxovaleric acid (MOVA), 5-oxyproline (5OP), and β-hydroxyisobutyric acid (BHIBA) significantly increase the expression of genes associated with BAT thermogenesis and lipid metabolism [55]. DIO mice treated with either MOVA, 5OP, or BHIBA via drinking water for 17 weeks showed increased EE with no changes in overall activity or food intake. Additionally, these metabokines showed higher levels of mitochondrial biogenesis markers and ¹⁸F-FDG uptake in BAT. MOVA and 5OP given together reduce fat mass by nearly 25% and improve glucose tolerance. The TCA cycle intermediate succinate is another potential metabolic signal of BAT activation [56]. Obese mice provided with succinate supplemented drinking water for 4 weeks had markedly decreased body weight, and improved glucose tolerance independent of changes in food intake. This response appears to be driven through Ucp1 mediated thermogenesis as Ucp1 KO mice abolishes this positive effect. These results highlight the potential of non-sympathomimetic BAT activation.

Sympathomimetics are known to increase EE, however it is unclear if this is mediated by BAT activation in humans. Ephedrine, approved for treating hypotension, exerts its effects by inhibiting neuronal reuptake of norepinephrine allowing more time for it to act on postsynaptic βADRs [57]. Acute oral ingestion of 1 mg/kg body weight ephedrine in fasted healthy men increased perirenal BAT blood flow after 30 and 60 min in some participants [58]. This corresponded with an increase in both perirenal BAT and overall body temperature, circulating glucose and oxygen consumption by 19% after 60 min, and reduced RER after 150 min with no change in circulating non-esterified fatty acids (NEFAs) or glycerol levels. Both systolic blood pressure and heart rate increased over the duration of the experiment. A higher dose of 2.5 mg/kg body weight of ephedrine increased ¹⁸F-FDG uptake into supraclavicular BAT in lean fasted men, but not in obese or placebo groups [19]. This also corresponded with increased BAT activation in lean but not obese participants and resulted in no change in core body temperature or plasma NEFA levels. Ephedrine also increased EE and circulating glucose, but also increased systolic blood pressure. In contrast to acute treatment, chronic treatment with 1.5 mg/kg ephedrine orally for 28 days in fasted metabolically healthy men results in no change in resting EE, respiratory exchange ratio (RER), and a decrease in systolic blood pressure and BAT activity at the end of treatment [59]. Cypess et al reported that lean male and females that received an intramuscular injection of 1 mg/kg ephedrine and subjected to cold exposure (14°C) maintained their temperature without shivering [60]. Both cold and ephedrine increased metabolic rate by 79 and 136 kcal/day, respectively, and decreased RER indicating more use of fatty acids as fuels, but no changes were observed between interventions. Systolic and diastolic blood pressure increased with cold and ephedrine treatment but only ephedrine increased heart rate [60]. Ephedrine elevated plasma glucose, NEFA, lactate, and insulin levels and no change in ¹⁸F-FDG uptake compared to cold and placebo groups. Remarkably, this study included female participants and no sex-based differences were reported in response to ephedrine administration.

While the reasoning for using ephedrine as a BAT activator is logical, these results are conflicting and may suggest shifting focus to other approaches. With increases in EE and BAT activity, heart rate and blood pressure also increase with ephedrine. This makes the individual contribution to BAT difficult to discern as a systemic increase in blood flow and nonspecific βADR activation could account for much of this increase [18]. Additionally, the inability for ephedrine to stimulate BAT activity in obese individuals and its reduction in potency of BAT stimulation with chronic treatment make its use in treating obesity unlikely. It is also important to consider the extent of EE afforded by treatments that would lead to meaningful weight loss in obese individuals. Although ephedrine increased EE, this minimal increase may not be clinically relevant. Moreover, individuals with obesity did not respond to ephedrine for reasons unknown thereby rendering ephedrine an unlikely candidate to expend excess stored calories in humans.

Other approaches include direct βADR agonists that mimic SNS stimulation. Mirabegron, a β3ADR agonist used to treat overactive bladder [61], has been explored as a sympathomimetic BAT activator. Higher than the approved dose for hyperactive bladder, which is 50 mg, mirabegron shows promising effects to upregulate BAT activity. Healthy men selected with cold induced detectable BAT given an acute dose of 200 mg mirabegron showed an increase in BAT activity measured by ¹⁸F-FDG uptake in all 12 participants [62]. Compared to placebo, there was higher resting metabolic rate, plasma glucose and NEFA, heart rate, and systolic blood pressure. However, these cardio stimulatory effects were lower compared to ephedrine [19, 60]. Interestingly, there were no changes in ¹⁸F-FDG uptake in subcutaneous WAT, liver, or skeletal muscle, except for BAT that showed detectable changes in ¹⁸F-FDG uptake with mirabegron treatment. A dose dependent effect on BAT activation was found when comparing between 50 and 200 mg mirabegron in healthy young men with cold activated detectable BAT [20]. Of note, only the 200 mg dose increased EE in these men with no changes in plasma glucose or insulin and increases in NEFA levels were dose dependent possibly through β3ADR mediated lipolysis. Heart rate and systolic blood pressure were higher with the 200 mg dose. 150 mg/day mirabegron treatment for 10 weeks increased UCP1 and CIDEA protein expression in subcutaneous WAT to a greater extent than cold treatment, however there was no change in PCG1α, a known indicator of mitochondrial biogenesis or mitochondrial DNA content, which is activated by βADR in rodents [63]. No change in heart rate or blood pressure were seen indicating that cardiovascular effects may only be an acute side effect. Chronic treatment with 100 mg mirabegron for 4 weeks increases BAT activity and volume in healthy women, particularly in those with initially lower BAT amount [64]. While there were acute changes with EE and RER, after 4 weeks there were no differences from baseline. At this lower dose, heart rate and systolic blood pressure on day 1 were higher to a greater degree than in subjects from previous studies [20, 62, 63]. The increase in NEFA levels after chronic mirabegron treatment was decreased compared to day 1 and increased fasting plasma high density lipoprotein content after 4 weeks suggesting alterations to whole body lipid metabolism. In lower doses, mirabegron did not elicit any effects on BAT activation, however the higher 100–200 mg dose cause an increase in BAT activity. Similar results were shown with acute 50 and 200 mg mirabegron increased EE, however, only 200 mg increased BAT blood flow, oxidative metabolism, and ¹⁸F-FDG uptake [18]. While β3ADR signalling is assumed to activate BAT, 50 mg dose showed no change in BAT activity. Similar to previous observations, higher doses of mirabegron increased heart rate and systolic blood pressure, which indicate nonselective βADR agonism by mirabegron and alternative βADR subtype activation of BAT [18]. Contrasting results have emerged regarding the abundance of βADR subtype in human BAT. β3ADR is reported as being the most abundant followed by with β2ADRs in vivo [65, 66]. Conversely, others report almost non-existent β3ADR mRNA expression in vitro in human BAT [67]. Differentiated human BAT adipocytes in vitro treated with formoterol, a highly selective β2ADR agonist, showed a similar increase in oxygen consumption rate (OCR) as norepinephrine, and higher than mirabegron [18]. Additionally, this effect was inhibited when formoterol was used in conjunction with a β2ADR antagonist. In vivo, formoterol has been shown to increase resting EE, and decrease RER with no change in heart rate or blood pressure in lean men, however no measures of BAT activity were performed [68]. β2ADRs have been shown to increase EE and rates of lipolysis in non-human models [69], however, if human BAT is indeed activated by β2ADR agonism, these alterations of EE and RER could potentially be mediated by BAT. However, these studies have yet to be explored.

Direct sympathomimetics effectively increase BAT EE but show limited efficacy in treating obesity by upregulating BAT thermogenesis. Mirabegron increases EE and BAT activity, but its cardio stimulatory effects could potentially be dangerous for certain populations. Notably, females and other target demographic of overweight and obese individuals who are already at a higher risk of hypertension and other cardiovascular complications.

Effects of aging on BAT is largely attributed to changes in immune and senescent cell activity as well as mitochondrial dysfunction. With aging, the immune system becomes more unregulated, and there is an increase in pro-inflammatory M1 macrophage activation within adipose tissue [192]. Additionally, there is an increased presence of senescent immune cells in BAT which damage adipose tissue by secreting inflammatory cytokines. Senescent immune cells reduce expression of thermogenic markers such as Ucp1 and Ppargc1a, and also downregulate gene expression of adipose RNA binding motif 3 (RBM3) which prevents sympathetic innervation of the BAT [193]. Senescent T cells also promote BAT whitening in aging mice by secreting IFN- γ, which inhibits brown adipocyte differentiation [194]. Mitochondrial dysfunction increases with age and leads to a greater oxidative stress. Cui et al found that 20-month-old mice have less expression of BAT thermogenic genes including Ucp1 and Ppargc1a in contrast to 6-week-old mice [195]. These changes in BAT were accompanied by a decrease in antioxidants, indicating higher age-related oxidative stress. Inducing oxidative stress using hydrogen peroxide in brown adipocytes also reduced the quantity and morphology of mitochondria; however, these effects were rescued by antioxidant treatments [195]. In humans, similar declines in BAT mass can be observed due to age, with some studies observing that the decline is more prevalent in males than females [9, 196]. Intriguingly, a puberty-related rise in BAT mass has been described in children over the age of 10, suggesting that BAT development peaks with sexual maturity and musculoskeletal development, coinciding with an increase in growth and sex-related hormones [197–199]. However, despite this rise, a decline in BAT activity persists into adulthood, likely attributed to greater body fat accumulation. The mechanisms underlying the regulation at the level of brown adipocytes remain unclear [200].

Sex based differences in BAT morphology and activity deserve attention. Although the majority of studies have included males, there are notable differences in BAT function between males and females [201]. Female rats have been shown to have greater BAT mass, mitochondrial content, and Ucp1 expression compared to male rats [202, 203]. PET/CT scanning has shown that females may have lower BAT volume but similar activity compared to male [204]. Conversely, other studies have shown that females have greater detectable BAT than males [205, 206]. Sex based differences in BAT presence are age-related, with higher BAT activity in females at younger ages. However, this difference becomes obsolete in post-menopausal women, indicating that changes in sex hormone levels may contribute to BAT function [201]. Recently, Blondin et al, [181] has demonstrated that BAT oxidative metabolism and glucose uptake is greater in premenopausal women in comparison to postmenopausal women but the change in BAT tissue radiodensity, which is an indirect lipid content in BAT was not altered between groups of women. Transcription of the batokine BMP8B, which aids in modulating BAT for thermogenesis, is promoted in female mice with estradiol 2 (E2, the main circulating form of estrogen) treatment, whereas ovariectomy drastically decreases BMP8B expression in female mice [207], and decreases thermogenic activity and Ucp1 expression [208]. The mechanisms of estrogen induced increases in thermogenic activity may be through the activation of the estrogen receptor α leading to subsequent norepinephrine stimulated lipolysis. In contrast, testosterone levels lowers the rate of lipolysis [209]. Estrogen can also influence BAT activity via sympathetic nervous system stimulation to increase UCP1 protein expression and thermogenic activity [210]. The in vivo effects of androgens on BAT activity are less clear. Removal of testes, the primary location of testosterone production, increases UCP1 protein expression in BAT implying a blunting effect of testosterone on BAT thermogenic ability [211]. However, it is difficult to determine if changes in BAT function are directly mediated by testosterone signalling or by the conversion of testosterone to E2 in the tissue that may be modulating BAT activity [201]. It is important to consider the complexities and importance of sex-based genetic and hormonal influences on BAT function when investigating potential BAT activators to combat obesity, to meet the needs of all individuals who could benefit from these therapies. A comprehensive discussion of the effects of biological sex on BAT function, morphology, and growth has been discussed elsewhere [201, 212, 213].

Evidence that exercise influences BAT activity is more prevalent in rodents than in humans. In obese male mice, 4 weeks of daily aerobic exercise increased BAT mass while also upregulating genes involved in glucose and lipid metabolism, however only serum glucose levels were reduced following training, whereas serum lipid and cholesterol were unchanged [214].

Exercise can also stimulate browning of WAT. Male Swiss rats that underwent 8 weeks of aerobic or resistance training [215], had significantly higher Ucp1 protein, Ppargc1a, and Cidea gene expression in both inguinal and retroperitoneal WAT [216]. Moreover, swim training in Sprague-Dawley rats fed a HFD demonstrate reductions in weight but does not alter the thermogenic profile in BAT. Instead, it promotes myogenic protein markers, suggesting that BAT may adopt a muscle-like oxidative function during exercise rather than a thermogenic function [216]. Conflicting evidence on the influence of exercise on BAT has emerged, as studies in humans fail to replicate findings seen in rodents. Recent research in sedentary humans demonstrates that exercise has no impact on BAT activation [217]. In the ACTIBATE trial, no changes in BAT volume or F-FDG uptake were identified across control, moderate-exercise, and vigorous-exercise groups following 24 weeks of combined endurance and resistance exercises [217]. Moreover, various studies in both male and female endurance athletes report lower BAT activity and volume compared to non-athletes counterparts, challenging the notion that BAT and exercise are complementary [218, 219].

Exercise also stimulates the release of hormones or activating factors, referred to as exerkines, that can influence BAT activity. Of note is AMPK, a key regulator of skeletal muscle metabolism. In relation to BAT, AMPK activation increases Ucp1 expression in differentiated brown adipocytes derived from mice undergoing 4 weeks of voluntary aerobic training [220]. Exercise also improves efficiency of exerkines such as FGF-21 in obese mice by increasing the expression of KLB receptors in BAT [221]. However, not all exerkines affect BAT as seen with irisin. After acute swimming interventions, male mice show increased FDNC5 but no changes in Ppargc1a, Ucp1 gene expression, or irisin protein levels in BAT [222]. In humans, the effects of exerkines are more varied. A recent study assessing 16 exerkines, including FGF-21, lactate, and irisin found that endurance activities increased FGF-21, reduced lactate, but failed to detect irisin or measure any changes [223]. This study, which had limitations due to its small sample size and inclusion of only female participants, concluded that exercise altered the circulation of exerkines other than irisin [223]. However, only changes in lactate levels were associated with changes in BAT volume [223]. Overall, exercise and BAT activity shows that this relationship is less reproducible in humans than in rodents. Although exercise and BAT thermogenesis have positive effects on energy expenditure and metabolism that result in weight loss, they work independently rather than in tandem. The impact of exercise likely outweighs any influence of BAT thermogenesis, as exercise demands a greater utilization of energy substrates and restricts blood flow to adipose tissue overall, whereas it stimulates or maintains muscle mass that makes up the majority of body weight as least in healthy humans.

Ucp1 is a key protein involved in facilitating thermogenesis in BAT. However, evidence suggests that Ucp1 is only functional when it is activated. This is evident when mice with 50 times higher amount of Ucp1 are placed on a high fat/sucrose diet. Despite higher UCP1, animals experience weight gain at a similar rate to controls [224]. Only upon injection of β3ADR, mice with higher UCP1 display greater EE, indicating how activation of Ucp1 is key to producing any thermogenic effects. Similarly, Ucp1 ^−/− and WT mice exhibit similar levels of EE and susceptibility to DIO at thermoneutrality. However, with noradrenaline treatment Ucp1 was activated in WT mice leading to greater EE [225]. This highlights that thermogenic activity in BAT does not occur ubiquitously and requires on-going activation of Ucp1. Therapeutic interventions must achieve sustained activation of Ucp1 for pharmacological efficacy. Current options capable of Ucp1 activation, such as βADR agonists, have inherent limitations as they increase heart rate and blood pressure [20].

On the other hand, Ucp1 is dispensable for thermogenesis evident from recent, studies that have identified non-shivering thermogenic processes that act independently of Ucp1 [10, 226], one of these being calcium cycling present in beige adipose tissue. Calcium (Ca²⁺) is released and sequestrated by the sarco-endoplasmic reticulum (SERCA) through SERCA2b in beige adipocytes [10, 227]. This cycling process releases heat as a by-product and utilizes ATP to fuel SERCA2b uptake of Ca²⁺ into SERCA [10]. Ca²⁺ cycling is activated either by β-ADR stimulation [10] or cold-exposure [10, 228], and this process seems to be protective against DIO at thermoneutrality in both WT and Ucp1 ^−/− mice [229].

Another UCP1-independent ATP-dependent mechanism of thermogenesis is creatine substrate futile cycling where phosphocreatine (PCr) is shuttled from the mitochondria towards the endoplasmic reticulum (ER) to be converted to ATP and creatine (Cr) by creatine kinase (CK) [11]. This cycle of ATP production through the PCr/CK cycle, which primarily takes place in beige adipocytes, provides fuel for calcium cycling, as SERCA2b present in the endoplasmic reticulum of cells can utilize ATP to take up Ca²⁺ by the support of cytosolic CK, leading to the dissipation of heat [11]. Kazek et al demonstrated that reductions in creatine induced by β-guanidinopropionic acid were linked to reductions in oxidative metabolism in both iWAT and BAT following β-3ADR agonist treatments [15]. Additionally, they found that among Ucp1 ^−/− mice, those treated with β-GPA had reduced ability to maintain body temperature after cold exposure (4°C) compared to vehicle controls. The absence of creatine kinase B (Ckb), a key regulator of creatine cycling, in mice leads to decreases EE and increased risk of obesity [230]. This shows that in the absence of Ucp1, Cr cycling serves as a compensatory measure to maintain thermogenesis. This is corroborated by adipocyte-selective inactivation of Ckb that diminishes thermogenesis and predisposes to obesity [15]. Recently, demonstrated in mice with adipocyte-selective deletion of either Ucp1 or Ckb are euthermic which worsens cold intolerance making mice hypothermic. This suggests that thermogenic adipocytes use redundant, non-paralogous proteins to maintain body temperature [231]. Although further studies are required for comprehensive understanding, this suggests that while Ucp1 plays a significant role in thermogenesis, it is not indispensable.

It is important to consider the availability or “recruitability” of BAT and the amount it contributes to whole body EE when assessing its potential as a therapeutic strategy to manage obesity. We know that BAT is relatively less abundant in human adults, in both lean and obese populations compared to rodents [9]. Early studies in human BAT indicated that CIT (16°C) was significantly reduced in individuals that were obese compared to lean individuals [232]. More recently, when quantifying changes following CIT (16°C), lean males still seem to achieve greater BAT activity with a 17% increase in basal metabolic rate, whereas obese males only experienced an increase by 6% [144]. Among healthy males and females, following mild CIT (15.5°C), BAT only attributed to a modest 15–25 kcal/day increase in EE [233] and another study assessing EE following cold stimulus at 6°C found that BAT only contributed to a 10 ± 5 kcal/day increase in EE [234]. Additionally, the exact contribution of BAT activity to EE is questionable where cervio-thoracic muscles, as observed in PET-CT scans, have indicated greater impacts to cold-induced EE than BAT [234]. Interestingly, no linear relationship between BAT and whole-body EE has been indicated, and BAT contributions only represented 1% of the total whole-body changes in EE after CIT [234]. Although BAT can contribute towards whole body EE, which is especially apparent in rodents, the evidence thus far is insufficient to contribute weight reduction in obese human populations. It is also important to explore the benefits of BAT activation beyond solely increasing EE to aid in weight loss. BAT activation may provide protection against metabolic dysregulation such as hyperglycemia and hyperlipidemia associated with obesity. BAT activation using CL, 316243 (β3ADR agonist) over 10 weeks in E3L and CETP mice that closely resemble human lipoprotein metabolism robustly lowered plasma TG and total cholesterol levels while maintaining identical food intake [235]. These mice also showed significant reduction in atherosclerotic lesion size after 10 weeks of treatment further demonstrating the protective effects of highly functional BAT. Cold exposure increases glucose uptake in BAT [236, 237] even in fasted rats with low insulin levels [238] indicating sympathetic induced glucose uptake. While glucose is not thought to constitute much of the thermogenic substrate, it may be used in the intracellular synthesis of triglycerides for thermogenesis [3]. In metabolically healthy men with functional BAT, cold exposure significantly increased glucose uptake into BAT, resting energy expenditure, and glucose and lipid oxidation [239]. However cold exposure has little effect in healthy individuals without detectable BAT even during cold exposure. Given that the prevalence of BAT is low, maximizing tissue activity is challenging and may not have an overwhelming effect on energy expenditure to offset obesity, and might not be responsive across all individuals and patient populations. Thus, we conclude that it is not yet adequately reported in humans that BAT is an effective therapeutic target to induce enough weight loss to protect against the adverse effects of obesity but may be a tissue that can detect and influence energy metabolism.