The role of BAT in cardiometabolic disorders and aging

Best Practice & Research Clinical Endocrinology & Metabolism xxx (2016) 1e17

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The role of BAT in cardiometabolic disorders and aging Denis P. Blondin, PhD, Postdoctoral Fellow,  C. Carpentier, MD, Director of Diabetes, Obesity and Dr Andre Cardiovascular Complications Axis of the Centre de recherche du Centre hospitalier universitaire de Sherbrooke, CIHR-GSK Chair on Diabetes * Department of Medicine, Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke, Universit e de Sherbrooke, Sherbrooke, Canada

a r t i c l e i n f o Article history: Available online xxx Keywords: energy metabolism brown adipose tissue aging cardiometabolic disorders obesity diabetes

The demonstration of the presence of metabolically active brown adipose tissue (BAT) in adult humans using positron emission tomography (PET) over the past decade has lead to the rapid development of our knowledge regarding the role of BAT in energy metabolism in animal models and in humans. Although animal models continue to provide highly valuable information regarding the mechanisms regulating BAT development, mass and metabolic functions, these studies led to many assumptions that have been at best only partially verified in humans so far. Combined to some limitations of the current investigation approaches used in humans, this has lead to speculation on the potential role of BAT dysfunction in the development of cardiometabolic disorders and on the potential of BAT metabolic activation to treat these conditions. Here we propose a critical review of the evidence for the implication of BAT in cardiometabolic health. © 2016 Elsevier Ltd. All rights reserved.

bec, J1H 5N4, * Corresponding author. Centre de recherche du centre hospitalier universitaire de Sherbrooke, Sherbrooke, Que Canada. Fax: þ1 819 564 5292. E-mail address: [email protected] (A.C. Carpentier). http://dx.doi.org/10.1016/j.beem.2016.09.002 1521-690X/© 2016 Elsevier Ltd. All rights reserved.

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Introduction The recent rediscovery of brown adipose tissue (BAT) in adult humans has rejuvenated the interest for its possible physiological and pathophysiological role. Retrospective positron emission tomography (PET) imaging studies using the glucose analogue 18F-fluorodeoxyglucose (18FDG) rapidly provided evidence for lower prevalence rate and smaller volume of spontaneously active BAT in ageing, obesity and diabetes [1e4]. Combined with the unambiguous demonstration of the astounding thermogenic potential of BAT in rodents to combat cold [5e8] and even prevent or reverse diet-induced obesity [9], diabetes [10] and other cardiometabolic complications [11], this led most to view BAT as an ideal target to shift the chronic caloric balance to the negative side to prevent or treat obesity, and/or to reduce plasma glucose and lipid levels [11e14]. As always with exciting discoveries, there have been unverified assumptions taken for granted that led to perhaps too premature hope that BAT activation is an important key for the treatment of obesity and its cardiometabolic complications. This review aims to provide a critical appraisal of the state of knowledge about BAT physiological and pathophysiological roles in humans. BAT energy metabolism and the effect of ageing and cardiometabolic health BAT is a specialized tissue found abundantly in interscapular, subscapular, axillary, paravertebral, mediastinal, perirenal, and periaortic regions of many placental mammals, whose function is to produce heat [15]. BAT may contribute more than 50% of non-shivering thermogenesis in cold-adapted small mammals [16,17], allowing these animals to live in below thermoneutral environments without relying on the shivering process to produce heat [18,19]. Morphologically, brown adipocytes are characterized by their rich innervation and vascularization as well as the presence of numerous large spherical mitochondria surrounded by several small lipid droplets [20]. Its defining characteristic, however, and the source of its outstanding thermogenic function is conferred by UCP1 [21], a mitochondrial protein uniquely expressed in this tissue which uncouples mitochondrial respiration from ATP synthesis thus releasing the potential energy from substrate oxidation as heat [22]. BAT activation and recruitment is largely under the control of the sympathetic nervous system [23e25]. The release of norepinephrine by the sympathetic nerve terminals that innervate BAT, acts on the b-adrenergic receptors (b-AR) present on the cell surface of brown adipocytes, activating the adenylyl cyclase/cAMP/protein kinase A (PKA) signaling cascade required for the hydrolysis of intracellular TG. The mobilized long chain fatty acids released through this hydrolysis then activate UCP1 thereby initiating the UCP1-mediated uncoupling process [26]. There are several lines of evidence from rodent models that suggest that hydrolysis of intracellular TG is essential for BAT thermogenesis. For example, mice lacking adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL), the TG lipases that sequentially catalyse the first two steps in TG hydrolysis, suggest that UCP1 activation is largely dependent upon ATGL. In contrast to HSL-deficient mice that exhibit an accumulation of TG and diglycerides but normal cold-induced thermogenesis [27], ATGL-deficient mice show severe thermoregulatory defects [28]. Similarly, mice with an adiposespecific loss of carnitine palmitoyltransferase 2 (CPT2) [29], necessary for the initiation of b-oxidation of long-chain fatty acids, or ablation of all PKA phosphorylation sites on perilipin 1 which prevents the action of lipases [30], show BAT thermogenic dysfunction. Rats acutely and chronically exposed to the cold (10  C) given nicotinic acid (NiAc), an inhibitor of intracellular TG lipolysis, show a suppression in interscapular BAT oxidative metabolism and uptake of both circulating glucose and fatty acids [31]. This demonstrates that, in rodents, fatty acids derived from intracellular TG are essential for BAT thermogenesis, despite its tremendous ability to take up circulating glucose, NEFA and dietary fatty acids [11e14,31]. Further, recent evidence in rats suggests that increases in cold-induced uptake of circulating glucose and NEFA are accompanied by an upregulation in the expression of genes involved in glucose and fatty acid transport as well as glycogenesis, de novo fatty acid synthesis and fatty acid esterification [31]. That inhibition of BAT intracellular lipolysis resulted in a significant decrease in glucose and NEFA uptake suggests that TG hydrolysis is tightly coupled to anabolism [32e34]. In rodents, fatty acids derived from circulating triglyceride-rich lipoproteins (TRL), such as chylomicrons and VLDL, are the main source of fatty acids stored as TG in BAT [35]. BAT of mice exposed to the cold or treated with a b3-AR agonist can very efficiently take up injected TRL-mimicking particles Please cite this article in press as: Blondin DPThe role of BAT in cardiometabolic disorders and aging, Best Practice & Research Clinical Endocrinology & Metabolism (2016), http://dx.doi.org/10.1016/ j.beem.2016.09.002

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[11,13,14], consistent with the known induction of lipoprotein lipase (LPL) expression during cold and high fat feeding-induced BAT activation [36e38]. TRL are largely taken up by BAT as fatty acids following LPL-mediated intravascular lipolysis, while core remnants or whole particles are also taken up but primarily with larger chylomicron-sized particles (>150 nm) and to a progressively greater extent at lower environmental temperatures (7  C > 21  C > 28  C). The local lipolysis of TG-rich lipoproteins by tissues expressing LpL (e.g. heart, muscle, BAT and WAT), leads to the resulting TRL remnants becoming enriched with circulating apoliproteins, including apolipoprotein E (apoE). These apoE-enriched TRL remnants are carried to the liver where they are taken up by hepatocytes through the interaction between apoE and the LDL-receptor (LDLR), LDLR-related protein-1 and proteoglycans [39]. A study performed in a hyperlipidemic mouse model clearly illustrates the necessary interaction between the liver and BAT in order for the latter to confer its anti-atherogenic potential, such that the clearance of TRL by stimulated BAT must be matched by the ability of the liver to clear apoE-enriched lipoprotein remnants via this apoE-LDLR interaction [11]. The absence of the latter, such as what is found in Apoe / and Ldlr / mice, results in atherosclerotic plaque growth [11,40]. To date, human studies have shown that circulating glucose and fatty acids are taken up by stimulated BAT, representing ~1% and ~0.25% of plasma glucose and NEFA turnover respectively [41e45]. The rapid increase in BAT radio-density during acute cold exposure suggests that intracellular TG are the primary fuel to sustain BAT thermogenesis. In contrast to rodent studies, the ability of BAT to take up dietary fatty acids has never been examined in humans. It is possible that the capacity of BAT to clear circulating substrates is greatest after a cold stimulus, when the replenishment of intracellular TG likely occurs, but this also requires confirmation. What is less clear is the effect of obesity and diabetes on BAT intracellular TG utilization. It is possibly impaired given the impairment in catecholamine-induced NEFA mobilisation and insulin resistance in white adipose tissues (WAT) observed in these conditions [46]. Effect of catecholamines/sympathomimetics Blunted lipolytic action of catecholamines in WAT is an early event in obesity in humans [47] and in BAT of obese Zucker rats [48] and high-fat, high-sucrose fed mice [49], due to a reduction in b-AR density. Epidemiological studies in humans demonstrate an inverse relationship between BAT prevalence and body mass index or percent adiposity [1,2,50,51] which may reflect a resistance to catecholamines. We recently showed that sympathetically-induced WAT lipolysis and total BAT oxidative metabolism are closely associated in humans [43]. However, BAT lipolysis and oxidative metabolism did not appear impaired in overweight individuals exposed to mild cold [42]. Others have shown that BAT glucose uptake is significantly reduced in obese [52e55] and morbidly obese [56,57] compared to lean individuals exposed to mild cold or ingesting a sympathomimetic (ephedrine [55]) but increases following a 10 d cold acclimation [53,54] or bariatric surgery [57]. However, these improvements in BAT glucose uptake are likely a reflection of decrease in TG content following the intervention rather than an improvement in lipolytic action of catecholamines (e.g. [53]). Both ATGL and HSL protein expression in WAT are decreased in obese, insulin-resistant state, which is associated with the degree of insulin-resistance and hyperinsulinemia in obesity [58]. It is unclear whether this decreased expression in ATGL and HSL extends to human BAT as well. The use of b-blockers has been successful in limiting BAT activation [59,60], whereas the systemic infusion of a non-selective b-AR agonist [61] or administration of sympathomimetic agents such as ephedrine [55,62] have proven to be ineffective at activating BAT. To date, cold exposure has been by far a more effective strategy to stimulate BAT and comes without the cardiovascular risk associated with the use of high doses of non-selective b-agonists or sympathomimetic agents. The explanations for the lack of effectiveness of b-AR agonists and sympathomimetic agents in activating BAT include the relatively low doses used and the limited bioavailability of the agents at the brown adipocytes. More recently, ingestion of a b3-AR agonist was used as a more BAT-specific activating agent unlikely to evoke undesirable cardiovascular effects [63]. Ingesting 200 mg of mirabegron (b3-AR agonist) increased 18 FDG in BAT compared to ingesting a placebo, but the uptake was not nearly as significant as cold exposure and at the dose ingested still evoked increased cardiovascular responses expected of a b-AR agonist. Further, at the dose given, the resulting uptake of 18FDG uptake may simply be the result of Please cite this article in press as: Blondin DPThe role of BAT in cardiometabolic disorders and aging, Best Practice & Research Clinical Endocrinology & Metabolism (2016), http://dx.doi.org/10.1016/ j.beem.2016.09.002

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activation of b2-AR in the BAT vasculature, consequently increasing BAT blood flow and glucose uptake, without increasing thermogenesis (see section BAT thermogenesis). Effect of insulin In addition to catecholamine signaling, activation of insulin and insulin-like growth factor-1 (IGF-1) pathways are critical for complete differentiation of brown adipocytes [64]. Epidemiological studies have shown that the prevalence of BAT is significantly lower in individuals with than those without T2D [1]. Prospective cold exposure studies have similarly shown that the net BAT glucose uptake is significantly lower in individuals with T2D [42], which using the current definition criteria (see Section Measuring BAT presence and metabolism) suggests there may be lower 18FDG-positive BAT volume in these individuals. The lower net glucose uptake and 18FDG-positive BAT volume in T2D certainly is compatible with the demonstrated insulin stimulation of BAT glucose uptake [52,65]. Insulin-stimulated glucose uptake in BAT has been well characterized by the insulin/phosphoinositide 3-kinase/phosphoinositidedependent kinase-1/Akt (PI3K-PDK1-Akt) signaling pathway stimulating the rapid translocation of glucose transporter 4 (GLUT4) to the cell membrane [66,67]. This insulin-stimulated glucose uptake in BAT appears to be a result of increased glucose extraction by the tissue, as BAT blood flow does not increase under insulin-stimulation [65]. Glucose is also taken up by BAT through insulin-independent mechanisms. NE-induced b3-AR stimulation increases glucose uptake in BAT of mice [68] and in cultured brown adipocytes in the absence of insulin [69e71] through GLUT1 rather than GLUT4 [72]. b3AR-stimulated glucose uptake in BAT occurs through two cAMP-mediated signaling pathways: 1) an increase in transcription and de novo synthesis of GLUT1; and 2) mTOR complex 2 (mTORC2)-stimulated translocation of the newly synthesized GLUT1 to the cell membrane [73]. Given that BAT glucose uptake is lower in obese individuals under both cold- and insulin-stimulated states, it is possible that both insulin-dependent and b3-AR-stimulated glucose uptake in BAT are impaired. Further work is required to determine the factors leading to lower BAT glucose uptake in obesity and T2D. Age Age is one of the most significant determinants of BAT activity in all mammals, from rodents [74] to primates [75] and humans [76], independent of adiposity. BAT mass and activity is highest in infant humans and progressively decrease with age [76]. One possible explanation for this age-associated decrease in BAT activity or presence may be related to catecholamine-resistance and thus decrease in b-AR-mediated lipolysis with increasing age, an effect documented in WAT of humans [77,78] and BAT in rats [79]. This age-related lipolytic resistance to catecholamines in WAT may be due to reduced expression of b2-AR in adipocytes [80], similar to what is found in white adipocytes isolated from obese individuals [81]. Alternatively, age-related decrease in BAT activity may also occur as a result of decrease in critical proteins involved in BAT lipolysis, such as ATGL or HSL. The latter has been shown to occur in WAT of obese, insulin resistant individuals [58]. Clearly, further work is needed to determine whether BAT thermogenesis is impaired with age or whether there is simply a reduced uptake of glucose and ‘whitening’ phenotype such as what has been observed in overweight men [42]. Acclimation/acclimatization status Humans chronically or intermittently exposed to a cold stress exhibit modified physiological responses to a cold stimulus. These responses are a result of cold acclimatization or cold acclimation. The former is defined by the IUPS Thermal Commission as physiological changes naturally occurring as a result of the climate in which the organism is exposed to (e.g. seasonal, geographical, working conditions) [82]. The latter refers to physiological changes induced experimentally in a controlled laboratory/ clinical setting [82]. Female Korean breath-hold divers (Ama), performing daily dives in water at ~10  C during winter months, given a continuous infusion of NE (0.15 mg/kg2/min) showed a ~8% increase in oxygen consumption compared to pre-injected values [83]. Similarly, four men of European descent infused with NE at various rates before and after a 29 week residence in Antarctica in late winter [84] demonstrated a ~10% increase in oxygen consumption at the highest NE infusion rates (0.15 and Please cite this article in press as: Blondin DPThe role of BAT in cardiometabolic disorders and aging, Best Practice & Research Clinical Endocrinology & Metabolism (2016), http://dx.doi.org/10.1016/ j.beem.2016.09.002

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0.30 mg/kg/min). These NE-induced increases in oxygen consumption are assumed to reflect BAT thermogenesis, but no direct measure of BAT oxidative metabolism was made to confirm this assumption. Other seasonal acclimatization studies provided indirect evidence that perhaps BAT thermogenesis is recruited. In one instance, the cold response of 5e6 young men were examined monthly for one year and showed a significant reduction in shivering during the winter months [85], which by definition represents an increase in NST (ie. potentially BAT thermogenesis). Necropsies performed on outdoor workers have suggested that repeated cold exposures can result in greater BAT mass, compared to indoor workers of the same age [86], but it is impossible to assess the thermogenic role of this BAT mass in the absence of measures of BAT metabolic activity. Some of the earliest cold-acclimation studies (see [87,88] for review) support the hypothesis of increased BAT thermogenesis upon repeated cold exposure. This was best demonstrated by Davis et al. [89] who showed that exposing six unacclimated men to 12e14  C air, 8 h per day for 31 consecutive days suppressed mean shivering activity by 80%, implying an increase in NST. It is only recently that direct evidence has begun to emerge regarding changes in BAT volume and activity on whole-body metabolism in a cold-acclimated state. Using 18FDG PET, some have shown that daily cold exposure for as little as 10 days to as long as one month can increase the volume of BAT taking up glucose (assumed to represent BAT mass) by 40e45% [44,90,91]. Only one study to date has directly measured changes in BAT oxidative capacity and thus the true thermogenic change of the tissue following cold-acclimation [44]. In that study we showed that six unacclimated men after 2 h of daily cold exposure (10  C water in LCS) for 4-weeks increased total BAT oxidative metabolism 2.2-fold. Despite similar whole-body thermogenic rates in the unacclimated and acclimated states, shivering intensity however did not significantly change as a result of cold acclimation. Combined, the cold acclimatization and acclimation data suggest that BAT mass and activity can change significantly as a result of chronic or intermittent cold exposure in humans. What is less clear is the metabolic impact of these changes. Two recent studies have shown improvements in fasting glycemia [54] and whole-body insulin sensitivity [53] in obese and diabetic individuals, respectively, following a 10-day cold acclimation protocol. However, the data clearly shows that these changes are largely a result of improvements in skeletal muscle glucose clearance rather than BAT, as evidenced by enhanced skeletal muscle GLUT4 translocation thus increasing skeletal muscle glucose uptake compared to only marginal contribution of BAT to glucose uptake. With skeletal muscle accounting for 50% of the glucose clearance compared to ~1% by BAT during mild cold exposure [43], it is clear that cold-induced improvement in glucose homeostasis is largely mediated by skeletal muscle, not by BAT metabolic activation. Measuring BAT presence and metabolism Despite indications for the presence of BAT in histopathological examinations performed on coldexposed outdoor workers [86], the presence of functional BAT in adult humans was only recently widely acknowledged with the development and wide use of 18FDG PET/CT [92e95]. 18FDG is transported in a cell through the same transporters as glucose and phosphorylated by hexokinase to form 18 FDG-6-phosphate. 18FDG-6-phosphate does not participate any further in glycolysis and is subsequently trapped within the cell and accumulates in proportion to the glycolytic rate in tissues that do not display significant glucose-6-phosphate dephosphorylation activity [96]. It was demonstrated that the supraclavicular and paravertebral adipose tissue depots that become 18FDG-positive upon cold exposure are BAT [97,98] as these 18FDG-positive fat depots displayed biomolecular characteristics of classical BAT described in animal models [2,24,97,98], including the expression of proteins such as UCP1, deiodinase iodothyronine type II (DIO2), PPAR-g coactivator 1a (PGC1a), PR domain-containing 16 (PRDM16) and b3-AR that drive BAT thermogenesis [98]. Because obtaining biopsies to confirm the presence, characterization and ex vivo function of BAT is not feasible under non-surgical conditions in humans, 18FDG uptake in a fat depot upon cold exposure has been considered the ‘gold standard’ in identifying BAT and in many cases accepted for characterizing in vivo BAT function, despite its acknowledged limitations [99]. Despite the ubiquitous use of 18FDG in identifying BAT, there are no universally accepted definition criteria using 18FDG PET/CT. Since the recent emergence of prospective BAT studies in humans, significant advances have been made in the methods used to detect the mass and metabolism of BAT beyond 18FDG PET. These methods along with 18FDG PET will be discussed in Please cite this article in press as: Blondin DPThe role of BAT in cardiometabolic disorders and aging, Best Practice & Research Clinical Endocrinology & Metabolism (2016), http://dx.doi.org/10.1016/ j.beem.2016.09.002

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the following section, describing in particular some of the current limitations in the methods and our interpretation of the related outcomes. BAT mass Identifying the true presence of BAT in humans has proven to be quite difficult. Although, 18FDG uptake in a fat depot upon cold exposure is still considered the ‘gold standard’ in identifying BAT, the defining thresholds used in terms of glucose uptake and adipose tissue identification from CT radiodensity vary tremendously (for review see [100,101]). Further, there are studies which have shown 18 FDG PET to be unreliable to measure BAT thermogenesis [42,65]. For example, we previously showed that 18FDG uptake in BAT is significantly impaired in older, overweight men with and without controlled type 2 diabetes (T2D), despite normal cold-induced BAT oxidative metabolism and NEFA uptake [42]. Using the commonly applied 18FDG PET/CT thresholds to identify BAT of a tissue radiodensity between 30 and 150 Hounsfield units (HU) and 18FDG uptake during cold exposure  1.5 SUV units, one would conclude that BAT volume is lower in the latter two groups compared to lean healthy men. However, if either BAT oxidative metabolism or NEFA uptake were used instead, our conclusion might be different. BAT glucose uptake and thus BAT volume were associated with a lower BAT blood flow and radio-density, indicative of greater TG content. Therefore, 18FDG uptake may be more indicative of lower BAT lipid content and higher vascularisation than oxidative metabolism per se. There are a number of other biomolecular features of brown adipocytes that may be better indicators to define the presence and/or amount of BAT. As indicated above, both BAT oxidative metabolism (measured using [15O]-labelled oxygen or [11C]-acetate) or NEFA uptake (using [18F]-FTHA) may be more reliable tracers to measure BAT volume in different metabolic contexts. However, each have inherent limitations that prevent their use for this application. Both [15O]-labelled oxygen and [11C]containing tracers have short radioactive half-lives (~2 and ~20 min, respectively) and are rapidly metabolized in tissues (i.e. rapid disappearance of the signal in tissues). This rapid metabolism can only be captured using real-time (i.e. dynamic scanning) PET acquisition that currently can only be applied to a limited anatomical location. These tracers thus cannot currently be used to examine whole-body BAT volume. The uptake of [18F]-FTHA is low in BAT relative to other tissues and therefore more difficult to discriminate from WAT [42]. Other SPECT, MRI or MRS methodologies may have future potential in identifying the presence of BAT not only in an active state but also under unstimulated conditions. However, to date, such modalities are either impossible to use in humans, have not been assessed in humans, provide low resolution and specificity (e.g. SPECT) or are susceptible to movement artefacts and partial volume effects (e.g. MRI and MRS) (see Table 1). Measurement of BAT metabolic activity not relying on glucose metabolism As described above, 18FDG reflects BAT glycolytic rate. BAT, when stimulated, is highly lipolytic and requires the long chain fatty acids released upon catecholamine-mediated lipolysis of intracellular TG to both activate UCP1 and serve as a substrate. Inhibiting intracellular lipolysis through various means suppresses BAT thermogenesis [28e31] and is not rescued by the uptake of circulating glucose [31]. Further, in hyperinsulinemic conditions, BAT can take up glucose without producing heat [65]. As discussed above, BAT 18FDG and BAT oxidative metabolism are dissociated in older, overweight individuals without and with T2D [43]. In fact, BAT glucose uptake is a more reliable indicator of BAT blood flow [42,52,65] and may even influence tissue perfusion [102]. If not 18FDG, what other methodologies may be applied to study BAT activity and metabolism? Both [15O]-labelled oxygen and [11C]-acetate PET have been used to quantify BAT oxidative metabolism in humans (see Table 1) [41e45,52,65,103,104]. Both require on-site cyclotrons and dynamic acquisition and modeling. [15O]-labelled oxygen with PET remains the ‘gold standard’ to measure tissue oxygen consumption, but can be quite cumbersome to produce and use. [11C]-acetate provides a direct measure of Krebs cycle kinetics (i.e. tissue VCO2). This method is based on the following assumptions [105]: 1) acetate enters the Krebs cycle freely after rapid conversion into acetyl-CoA; 2) other acetate metabolic pathways (e.g. de novo lipogenesis) are relatively slow compared to the Krebs cycle carbon fluxes; 3) carbon fluxes into the Krebs cycle through acetyl-CoA is directly coupled to the production of Please cite this article in press as: Blondin DPThe role of BAT in cardiometabolic disorders and aging, Best Practice & Research Clinical Endocrinology & Metabolism (2016), http://dx.doi.org/10.1016/ j.beem.2016.09.002

Modality PET/SPECT [18F]-FDG

[18F]-FTHA

BAT outcomes

Advantages

Limitations

Effect of ageing

Effect of obesity

Effect of diabetes

References

Volume of activity Glucose uptake Relative glucose uptake (SUV) Net glucose uptake (dynamic scanning)

 Broadly available in PET centers  Current ‘gold-standard’ for the identification and quantification of BAT metabolic activity  Quantification of relative (static scanning with SUV) or absolute (dynamic scanning) BAT glucose uptake  Quantification of nonesterified fatty acid (i.v. administration) or dietary fatty acid (oral administration) uptake in BAT  Better correlation with BAT oxidative metabolism with aging without or with type 2 diabetes than glucose uptake  Potential to assess unstimulated BAT volume

 Uptake increased by insulin administration and reduced by age and obesity, independent of BAT oxidative metabolism  Cannot discriminate between oxidative and non-oxidative BAT glucose metabolism  Exposure to radiation  Cannot discriminate between oxidative and non-oxidative BAT fatty acid metabolism  Exposure to radiation

YY

YY

YYY

[41e44,52 e54,56,57,100]

YY

YY

YYY

YY

YY

YYY

↔

↔

↔

?

?

?

?

?

?

[134]

↔

↔

↔

[41e44]

↔

↔

↔

?

?

?

[135]

?

?

?

[45,103]

?

?

?

[45,52,65,103]

Y

?

?

[136e138]

?

?

?

[139]

Nonesterified fatty acid uptake Dietary fatty acid uptake

[18F]-FBnTP

Mitochondrial content

[11C]-acetate

Blood flow

Oxidative metabolism

[11C]-MRB

 Easy to use in conjunction with measurement of oxidative metabolism  Direct measure of Krebs cycle kinetics (tissue VCO2)

[ O]-O2

Norepinephrine receptor ligand Oxidative metabolism

 Potential to assess unstimulated BAT volume  Gold-standard measure of tissue VO2

[15O]-H2O

Blood flow

[99mTc]-MIBI

Mitochondrial content Tissue perfusion Tissue perfusion

 Direct measure of perfusion  Easy and inexpensive to produce  Widely available

15

99m

[

Tc]tetrofosmin

 Widely available

 Not assessed in humans  Rapid washout upon cold or b3 adrenergic stimulation  Surrogate measure of blood flow  Requires dynamic acquisition and modeling  Surrogate measure of tissue VO2  Requires dynamic acquisition and modeling  Not assessed in humans  Cumbersome to produce and use  Requires dynamic acquisition and modeling  Requires dynamic acquisition and modeling  Lower resolution and lower specificity than PET  Lower resolution and lower specificity than PET  Limited experience in human BAT investigation

[41,42,45]

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(continued on next page)

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Table 1 Methodologies to determine BAT presence and metabolism.

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Modality

BAT outcomes

Advantages

Limitations

Effect of ageing

Effect of obesity

Effect of diabetes

References

[123I]-MIBG

Catecholaminemetabolizing tissues imaging

 Widely available

 Lower resolution and lower specificity than PET  Iodine administration to prevent thyroid damage

?

?

?

[138,140]

 Absence of ionizing radiation  Excellent soft-tissue contrast  Better spatial and temporal resolution than PET  Direct measure of BAT fat fraction relative to water content  Absence of ionizing radiation  Excellent soft-tissue contrast  Better spatial and temporal resolution than PET

 Movement artefacts  No quantification of energy substrate metabolic rate  Quantification of fat is not absolute

?

?

?

[113]

 No better than [18F]FDG PET to determine BAT activation  Limited experience

?

?

?

[113]

 Effect of blood flow on water spectra (blood flow s thermogenesis)  Limited to selected voxels  Highly susceptible to partial volume effects  Reflects tissue perfusion  Don't know for sure that it is dissolving in lipids, rather than water intracellular water compartment  Not assessed in humans  Limited to selected voxels  Partial volume effects  Not assessed in humans

?

?

?

[114,115]

?

?

?

[141]

?

?

?

[142]

MRI/MRS Water-Fat MRI (T1-weighted imaging)

T2*-weighted imaging

1

H-MRS

129

Xe-MRS

[1-13C]pyruvateMRS

Tissue difference in T2 relaxation time e shorter T2 time reflecting higher iron/ mitochondrial content Quantification of TG content

 Precise determination of triglyceride content relative to water

Tissue perfusion Fat-water fraction MR thermography

 Xe lipophilic, therefore dissolve in BAT lipids.

Based on the detection of hyperpolarized [1-13C]-pyruvate into downstream metabolites upon its oxidation

 Direct determination of pyruvate metabolism in tissues

may

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Table 1 (continued )

‘BAT temperature’

Thermistors

‘BAT temperature’

MR-based

‘BAT temperature’

 Non-invasive  Absence of ionizing radiation  Can be low cost (for low resolution and sensitivity)  Can perform static or dynamic acquisition  Non-invasive  Absence of ionizing radiation  Low cost  Temporally sensitive  3D spatial resolution

 Measures skin temperature  Not useful for assessment of deep tissues  Costly for high-resolution and sensitivity camera  2D, not 3D resolution  Measures skin temperature  Not useful for assessment of deep tissues  Poor temperature accuracy (þ/ 6  C)  Low temporal and spatial resolution

↔

YY

?

[116 e118,143,144]

?

?

?

[101,119]

?

?

?

PET: Positron emission tomograph; SPECT: Single-photon emission computed tomography; [18F]FDG: [18F]-fluorodeoxyglucose; [18F]FTHA: 14(R,S)-[18F]-fluoro-6-thia-heptadecanoic acid; [18F]FBnTP: [18F]-flurobenzyl triphenyl phosphonium; [11C]MRB: (S,S) e [11C]O-methylreboxetine; [99mTc]MIBI: [99mTc]-methoxyisobutylisonitrile; [123I]MIBG: [123I]metaiodobenzylguanidine; MRI: Magnetic resonance imaging; MRS: Magnetic resonance spectroscopy.

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Thermometry Infrared

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reducing equivalents; 4) the Krebs cycle contribution to the production of reducing equivalents is stable and accounts for approximately two thirds of total production; and 5) the production of reducing equivalents is tightly coupled to oxygen consumption. [11C]-acetate may also be used as a surrogate of tissue blood flow either through modelling [105e108] or using peak 11C radioactivity as an index of tissue perfusion [109]. A number of other PET, SPECT and MRI methodologies are available to measure BAT blood flow (see Table 1), the most common, easily producible and inexpensive of which is [15O]labelled water. However, as is described in section 4.3.2, careful interpretation is necessary when measuring BAT blood flow as recent investigations demonstrate that BAT blood flow can be regulated independently of BAT thermogenesis [102,110]. In rodents, circulating fatty acids, particularly those released through intravascular lipolysis of circulating chylomicron and very-low-density lipoprotein (VLDL) by the endothelium-bound enzyme lipoprotein lipase (LPL), are a major source of fatty acids incorporated in BAT intracellular TG [11,13,14,111]. Far less is known, however, regarding the role of BAT in clearing circulating fatty acids in humans. The administration of the long-chain fatty acid tracer [18F]-FTHA may be used to examine NEFA uptake (i.v. administration) or dietary fatty acid uptake (oral administration) in various tissues in humans [112]. To date, two groups have given [18F]-FTHA intravenously during cold exposure and have shown that NEFA uptake by BAT increases by 63% during cold exposure, compared to room temperature [45], but only accounts for 0.25% of plasma NEFA turnover [41]. Interestingly, NEFA uptake by BAT is not impaired in older, overweight individuals with or without well-controlled T2D [42]. The role of BAT in clearing dietary fatty acids in humans remains unknown. One limitation in the use of [18F]-FTHA is that it reflects fatty acid uptake, but it is not possible to determine once taken up in the tissue whether it is directed toward oxidation or non-oxidative disposal. Given the radioactivity exposure when using PET and SPECT, there have been significant efforts made to examine nonionizing methods to study BAT activity and metabolism. Using T1-and T2*weighted MRI, BAT fat fraction and mitochondrial content have been estimated, respectively [113]. Although this approach provides excellent soft-tissue contrast and better spatial and temporal resolution than PET, it is also susceptible to movement artefacts and quantification of BAT fat and mitochondrial content is not absolute. Others have used [1H]-MRS to quantify BAT TG content [114,115]. However, an increase in blood flow as is observed during cold-induced BAT stimulation affects the water peak spectra that serves as the internal comparative control, thus influencing the precise quantification of TG content. Finally, since the primary product of BAT stimulation is heat, there has been a trend towards the use of thermometry via static or dynamic infrared thermography, the use of thermistors or MR-based thermography to measure supraclavicular skin temperature as an index of BAT thermogenesis [101,116e119]. Although infrared thermography is certainly more cost effective, non-invasive and nonionizing, it is merely a measure of local skin temperature and certainly not an indicator of BAT thermogenesis. It is not useful for the measurement of deep tissues, appears influenced by BMI and subcutaneous adipose tissue thickness [116] and at best is a reflection of BAT blood flow, not thermogenesis. All of the studies using thermometry to date have compared supraclavicular skin temperature to BAT 18FDG uptake as a reference or as evidence of its effectiveness. This has consistently generated significant associations between these measures. However, the usefulness of these thermographic measurements remains to be determined in individuals suffering from cardiometabolic diseases. With BAT thermogenesis being fueled predominantly by intracellular TG, the application of 1H-MRS in this type of research will prove to be valuable given that it has remained the ‘gold standard’ in the quantification of TG content in other tissues such as skeletal muscle, heart and liver. However, much work remains to be done to apply the approaches previously used to study TG metabolism in other tissues for the purposes of examining BAT metabolism. This is particularly important if examining BAT in older individuals or overweight/obese individuals with or without T2D. A recent study performed in diet-induced obese mice showed an obesity-induced capillary rarefaction in BAT, leading to the development of a WAT-like phenotype with BAT shifting functions towards lipid storage rather than thermogenesis [49]. A similar BAT ‘whitening’ as a result of capillary rarefaction was proposed to explain the greater TG content and lower BAT blood flow seen in older, overweight men with and without T2D [42]. If repeated cold exposure is to be used as a therapeutic strategy to counteract the effects of obesity and diabetes, examining BAT TG metabolism will be critical. Please cite this article in press as: Blondin DPThe role of BAT in cardiometabolic disorders and aging, Best Practice & Research Clinical Endocrinology & Metabolism (2016), http://dx.doi.org/10.1016/ j.beem.2016.09.002

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Thermogenic responses to cold The physiological trigger for BAT thermogenesis is cold exposure. Therefore, cold exposure has been by far the most frequently employed strategy to activate BAT in humans as it comes without the cardiovascular risk associated with the use of high doses of b-agonists or sympathomimetic agents. As a consequence almost all that we know about metabolic activation of BAT in humans rest on coldmediated activation. It is thus important to examine the mechanisms of cold-stimulated thermogenic responses and their potential implication in cardiometabolic diseases. Shivering thermogenesis A common approach used to measure the thermogenic contribution of BAT is to cool participants until the presence of shivering is visually observed, then increasing the water temperature by 1e2  C, thereby resulting in what some consider maximal non-shivering thermogenesis and consequently maximal BAT activity [120]. The assumption then is that the cold-induced thermogenesis produced at this temperature is entirely attributable to BAT. However, even muscle activity carefully quantified by surface EMG cannot detect deep muscle shivering and, therefore, underestimates total cold-activated muscle metabolic activity and shivering thermogenesis [41,43]. There is currently no evidence to support the assumption that BAT can be activated without triggering any shivering response anywhere in the body. Obviously, the shivering response participates to the cold-induced thermogenic response and confounds the interpretation of the role of BAT in this process, especially given the large bulk of skeletal muscles compared to the small volume of BAT. Data from a recent study using [15O]O2 PET imaging [45] suggests that, assuming a mean tissue-specific oxygen consumption of skeletal muscle during a mild cold exposure (1.2  resting metabolic rate) of 0.34 mL/100 g/min, a 72 kg man with a muscle mass estimated as 42% of body mass (30 kg, see [121]), shivering thermogenesis would represent ~40% of whole-body energy expenditure (or 821 kcal/day), a doubling of the resting muscle contribution seen at room temperature. Interestingly, although cold acclimation results in a near abolishment of shivering thermogenesis in favour of BAT thermogenesis in rodents, such a phenomenon has not been shown in humans using EMG [44]. The predominant metabolic role of skeletal muscles over BAT during cold exposure has been well documented for systemic glucose metabolism [43,53]. For example, despite careful attempts to minimize [43] or even avoid [53] shivering, two independent groups demonstrated that cold-induced stimulation of glucose disappearance from circulation is due to skeletal muscles, with a very minor role played by BAT. In addition to demonstrating that at least acute metabolic activation of BAT does not provide great hope to serve as a treatment of hyperglycemia, these results clearly show the tight relationship between BAT metabolic activation and shivering upon cold exposure. BAT thermogenesis Increase in BAT sympathetic nervous system (SNS) drive leads to the release of norepinephrine (NE) from sympathetic postganglionic nerve terminals that act on b3-adrenergic receptors (AR) found at the cell surface of brown adipocytes [122]. The b3-adrenergic stimulation activates the adenylyl cyclase/cAMP/protein kinase A (PKA) signaling cascade required for the hydrolysis of intracellular TG, thereby mobilizing fatty acids that serve as both activators and metabolic substrates fueling BAT thermogenesis [15,123]. The long-chain fatty acids released following lipolysis activate UCP1 [26] which uncouples oxidative phosphorylation thereby dissipating the mitochondrial proton gradient and releasing the potential energy from substrate oxidation as heat. Deficiencies in critical steps involved in either intracellular TG lipolysis or b-oxidation of the subsequently released long-chain fatty acids result in severe thermoregulatory defects in rodents (e.g. [28,29]), which demonstrates that b-AR stimulation of intracellular lipolysis is essential for BAT thermogenesis. Although estimating maximal BAT thermogenesis in rodents is routinely performed simply by intraperitoneal injection of norepinephrine and quantifying the rise in oxygen consumption, maximal adrenergic stimulation is not possible in humans given the associated cardiovascular risk. Consequently, estimating the contribution of BAT thermogenesis to whole-body thermogenesis in humans has been Please cite this article in press as: Blondin DPThe role of BAT in cardiometabolic disorders and aging, Best Practice & Research Clinical Endocrinology & Metabolism (2016), http://dx.doi.org/10.1016/ j.beem.2016.09.002

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problematic. Whole-body energy expenditure has been shown to increase marginally (~10%) in response to norepinephrine infusion [83,84,124e129] but may increase by as much as 24% [130]. However, the organs contributing to this increase may include the heart and the liver in addition to BAT. Various attempts have been made to calculate the thermogenic contribution of BAT indirectly based on the assumption that BAT glucose uptake is a reliable indicator of thermogenesis and that a cold-induced rise in energy expenditure above basal metabolic rate is entirely attributed to BATderived non-shivering thermogenesis during mild cold exposure [53,54,56,90]. As described in Section Measuring BAT presence and metabolism, such assumptions are now known to be incorrect as glucose uptake has been shown unequivocally to be an invalid marker of BAT thermogenesis and coldinduced increases in energy expenditure during even mild cold exposures reflect a combination of BAT thermogenesis as well as skeletal muscle shivering and non-shivering thermogenesis [43,131]. More recently, a study using [15O]O2 PET imaging [45] measured BAT oxidative metabolism directly. With mean BAT oxidative metabolism measured as 1.2 mL/100 g/min, and a mean BAT mass of 133 g, BAT thermogenesis could account for only 0.5% of whole-body energy expenditure (103 kcal/day) during mild cold exposure (1.2  resting metabolic rate), substantially lower than the 20% of total energy expenditure commonly cited [132]. BAT thermogenesis is accompanied by an increase in BAT blood flow in order to carry in metabolic substrates and oxygen and carry out the heat and metabolic by-products. Recent investigations, however, suggest that BAT blood flow may be regulated independently of BAT thermogenesis. For example, in mice lacking UCP1 and thus unable to produce heat via BAT thermogenesis [7], NE injection results in a similar rise in BAT blood flow as their wild type counterparts who can produce heat [102]. To investigate this dissociation between BAT perfusion and thermogenesis, other investigators injected salbutamol in mice to stimulate b2-AR which are detectable in BAT depots, but absent in isolated brown adipocytes [110]. They found that b2-AR stimulation increased BAT blood flow but did not increase in vitro oxygen consumption of isolated brown adipocytes. These results suggest that the release of NE from sympathetic postganglionic nerve terminals in BAT may act not only on b3-AR to stimulate BAT lipolysis and subsequently thermogenesis, but also on b2-AR to increase tissue blood flow in parallel. Thus increases in BAT blood flow and BAT thermogenesis may both occur independently as a result of sympathetic stimulation. There are contexts in which tissue blood flow and glucose uptake may increase in the absence of BAT thermogenesis, as in hyperinsulinemia [65] and/or during hyperglycemia [102]. With b2-AR being the most abundant b-AR in human BAT (63% of total bAR mRNA [133]), it is possible that b2-AR predominates over a-AR in BAT vasculature and thus under NE stimulation induce a vasodilatory response, as others have hypothesized [110]. More research is required to determine the mechanisms that increase BAT blood flow under thermal and non-thermal BAT stimulations. However, what is clear is that BAT blood flow may increase due to the release of vasoactive molecules of various origins including postganglionic sympathetic nerve terminals, vascular smooth muscles, vascular endothelium, or brown adipocytes and therefore is not a reliable indicator of BAT thermogenesis. Conclusion Most of what we know about the potential role of BAT in cardiometabolic diseases in humans is based on BAT glucose metabolism. Emerging evidence suggest an ageing, obesity and diabetesassociated reduction in BAT glucose uptake without clear defects in BAT NEFA or oxidative metabolism. BAT glucose metabolism is likely more related to intracellular BAT TG content and blood flow than to BAT oxidative metabolism per se. Data from animal models and humans suggest that intracellular TG is the main oxidative fuel for BAT. Resistance to insulin and catecholamine actions are likely explanations for ageing and/or obesity-related reduction in BAT glucose metabolism, but whether these mechanisms also affect BAT thermogenic rate is unknown. Muscle shivering cannot be experimentally dissociated from BAT metabolic activation using current cold exposure protocols in humans. Muscle metabolic activation assumes a dominant role over BAT in cold-induced stimulation of glucose disappearance and, likely, in cold-induced thermogenesis in humans. The role of BAT in humans to metabolize circulating TG and dietary fat, or its contribution to diet-induced thermogenesis awaits the final results of ongoing studies. Thus, it is still too early to determine whether BAT metabolic activation Please cite this article in press as: Blondin DPThe role of BAT in cardiometabolic disorders and aging, Best Practice & Research Clinical Endocrinology & Metabolism (2016), http://dx.doi.org/10.1016/ j.beem.2016.09.002

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may improve postprandial dyslipidemia or significantly contribute to the treatment of obesity, insulin resistance and diabetes. Chronic activation of BAT thermogenesis for the treatment of obesity and cardiometabolic disorders remains a potential option as an adjunct therapy to lifestyle and other known effective interventions. However, there are still outstanding issues about BAT physiological and pathophysiological roles in humans that need to be resolved before we can accurately delineate the true therapeutic potential of this approach. Duality of interests/disclosures A.C.C. has had research contracts with the following companies: Amsterdam Molecular Therapeutics (now UniQure), Innodia, Sanofi-Aventis, GlaxoSmithKline, Bristol-Myers Squibb, Pfizer, AstraZeneca, Aventis, Novartis, Neurocrine, Merck-Frosst, Schering, Roche. He is also a research consultant for UniQure. Conflict of interest The authors have declared that no conflict of interest exists related to the content of this manuscript. Acknowledgements A.C.C is the recipient of the Canadian Institutes of Health Research-GlaxoSmithKline (CIHR-GSK) Chair in Diabetes. DPB is a recipient of a CIHR Postdoctoral fellowship. References [1] Ouellet V, Routhier-Labadie A, Bellemare W, et al. Outdoor temperature, age, sex, body mass index, and diabetic status determine the prevalence, mass, and glucose-uptake activity of 18F-FDG-Detected BAT in humans. J Clin Endocrinol Metab 2011;96:192e9. [2] Cypess AM, Lehman S, Williams G, et al. Identification and importance of brown adipose tissue in adult humans. N Engl J Med 2009;360:1509e17. [3] Lee P, Greenfield JR, Ho KK, et al. A critical appraisal of the prevalence and metabolic significance of brown adipose tissue in adult humans. Am J Physiol Endocrinol Metab 2010;299:E601e6. [4] Au-Yong IT, Thorn N, Ganatra R, et al. Brown adipose tissue and seasonal variation in humans. Diabetes 2009;58: 2583e7. [5] Enerback S, Jacobsson A, Simpson EM, et al. Mice lacking mitochondrial uncoupling protein are cold-sensitive but not obese. Nature 1997;387:90e4. [6] Golozoubova V, Hohtola E, Matthias A, et al. Only UCP1 can mediate adaptive nonshivering thermogenesis in the cold. Faseb J 2001;15:2048e50. [7] Golozoubova V, Cannon B, Nedergaard J. UCP1 is essential for adaptive adrenergic nonshivering thermogenesis. Am J Physiol Endocrinol Metab 2006;291:E350e7. [8] Oufara S, Barre H, Rouanet JL, et al. Adaptation to extreme ambient temperatures in cold-acclimated gerbils and mice. Am J Physiol 1987;253:R39e45. [9] Feldmann HM, Golozoubova V, Cannon B, et al. UCP1 ablation induces obesity and abolishes diet-induced thermogenesis in mice exempt from thermal stress by living at thermoneutrality. Cell Metab 2009;9:203e9. [10] Guerra C, Navarro P, Valverde AM, et al. Brown adipose tissue-specific insulin receptor knockout shows diabetic phenotype without insulin resistance. J Clin Invest 2001;108:1205e13. *[11] Berbee JF, Boon MR, Khedoe PP, et al. Brown fat activation reduces hypercholesterolaemia and protects from atherosclerosis development. Nat Commun 2015;6:6356. [12] Stanford KI, Middelbeek RJ, Townsend KL, et al. Brown adipose tissue regulates glucose homeostasis and insulin sensitivity. J Clin Invest 2013;123:215e23. *[13] Khedoe PP, Hoeke G, Kooijman S, et al. Brown adipose tissue takes up plasma triglycerides mostly after lipolysis. J Lipid Res 2015;56:51e9. *[14] Bartelt A, Bruns OT, Reimer R, et al. Brown adipose tissue activity controls triglyceride clearance. Nat Med 2011;17:200e5. [15] Cannon B, Nedergaard J. Brown adipose tissue: function and physiological significance. Physiol Rev 2004;84:277e359. [16] Foster DO, Frydman ML. Tissue distribution of cold-induced thermogenesis in conscious warm- or cold-acclimated rats reevaluated from changes in tissue blood flow: the dominant role of brown adipose tissue in the replacement of shivering by nonshivering thermogenesis. Can J Physiol Pharmacol 1979;57:257e70. [17] Heldmaier G, Buchberger A. Sources of heat during nonshivering thermogenesis in Djungarian hamsters: a dominant role of brown adipose tissue during cold adaptation. J Comp Physiol B 1985;156:237e45. [18] Himms-Hagen J. Brown adipose tissue thermogenesis: interdisciplinary studies. FASEB J 1990;4:2890e8. [19] Klingenspor M. Cold-induced recruitment of brown adipose tissue thermogenesis. Exp Physiol 2003;88:141e8.

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