REVIEW ARTICLE

Brown adipose tissue and novel therapeuticapproaches to treat metabolic disorders

SABINIANO ROMAN, AHMAD AGIL, MACARENA PERAN, EDUARDO ALVARO-GALUE,FRANCISCO J. RUIZ-OJEDA, GUMERSINDO FERN�ANDEZ-V�AZQUEZ, and JUAN A. MARCHAL

GRANADA AND JA�EN, SPAIN; SHEFFIELD, UK; AND WINSTON SALEM, NC

From the Biopathology and R

(IBIMER), Centre for Biomedical

Granada, Spain; Tissue Engine

Institute, University of Sheffield

Pharmacology and Neurosciences

Biosanitary Institute of Granad

Unversity/University of Granada,

Health Sciences, University of J

Institute for Regenerative Med

Medicine, Winston Salem, NC;

Technology, Centre for Biome

Granada, Granada, Spain; Hospi

Spain; Department of Human

In humans, 2 functionally different types of adipose tissue coexist: white adiposetissue (WAT) and brown adipose tissue (BAT). WAT is involved in energy storage,whereas BAT is involved in energy expenditure. Increased amounts of WAT maycontribute to the development of metabolic disorders, such as obesity-associatedtype 2 diabetes mellitus and cardiovascular diseases. In contrast, the thermogenicfunction of BAT allows high consumption of fatty acids because of the activity ofuncoupling protein 1 in the internal mitochondrial membrane. Interestingly, obesityreduction and insulin sensitization have been achieved by BAT activation-regeneration in animal models. This review describes the origin, function, and differ-entiation mechanisms of BAT to identify new therapeutic strategies for the treatmentof metabolic disorders related to obesity. On the basis of the animal studies, novelapproaches for BAT regeneration combining stem cells from the adipose tissuewith active components, such as melatonin, may have potential for the treatmentof metabolic disorders in humans. (Translational Research 2014;-:1–16)

Abbreviations: 18F-FDG PET ¼ 18F-fluorodeoxyglucose positron-emission tomography; ADSCs ¼adipose-derived stem cells; aP2 ¼ adipocyte fatty-acid-binding protein; AR ¼ adrenergicreceptor; ASCs ¼ adult stem cells; BAT ¼ brown adipose tissue; BMI ¼ body mass index; BMP¼ bonemorphogenic protein; C/EBP¼ cytosine-enhancer-binding protein; cAMP¼adenosine30, 50-cyclicmonophosphate;CNS¼central nervous system;CT¼computed tomography; ESCs¼ embryonic stem cells; FAs¼ fatty acids; FFAs ¼ free FAs; FNDC5 ¼ fibronectin type III domain-containing protein 5; HP¼ hypothalamus; MEL¼melatonin; MSC¼mesenchymal stem cell; MT¼ melatonin receptor; NE ¼ norepinephrine; Necdin ¼ postmitotic neuron-specific growthsuppressor; p107 ¼ retinoblastoma-like protein 1; PGC-1a ¼ coactivator 1a of PPAR; PKA ¼cAMP-dependent protein kinase A; PKA-CREB ¼ PKA-cAMP response element-binding;PPARg ¼ peroxisome proliferator-activated receptor g; PRDM16 ¼ positive regulatorydomain 16; Rb ¼ retinoblastoma protein; Runx2 ¼ osteogeneic key transcriptional factor; RXR¼ retinoid X receptor; SCs¼ stem cells; SNS¼ sympathetic nervous system; SRC¼ steroid recep-

egenerative Medicine Institute

Research, University of Granada,

ering Group, Kroto Research

, Sheffield, UK; Department of

Institute, Faculty of Medicine,

a (ibs.GRANADA), Hospitals

Granada, Spain; Department of

a�en, Ja�en, Spain; Wake Forest

icine Wake Forest School of

Institute of Nutrition and Food

dical Research, University of

tal University La Paz, Madrid,

Anatomy and Embryology,

Biosanitary Institute of Granada (ibs.GRANADA), Hospitals

Unversity/University of Granada, Granada, Spain.

Submitted for publication June 11, 2014; revision submitted October

16, 2014; accepted for publication November 4, 2014.

Reprint requests: Juan A. Marchal, Department of Human Anatomy

and Embryology, Faculty of Medicine, University of Granada,

Avenida de Madrid 11, E-18012 Granada, Spain and Ahmad Agil,

Department of Pharmacology, Faculty of Medicine, University of

Granada, Spain; e-mail: jmarchal@ugr.es.

1931-5244/$ - see front matter

� 2014 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/j.trsl.2014.11.002

1

Translational Research2 Roman et al - 2014

tor coactivators 1, 2 and 3; T2DM¼ type 2 diabetes mellitus; TGs¼ triglycerides; UCP1¼ uncou-pling protein 1; WAT ¼ white adipose tissue; Wnt ¼ embryogenic signaling pathway

INTRODUCTION

Adipose tissue is one of the largest organs in the bodyand plays an important role in central energy balanceand lipid homeostasis.1 Two types of adipose tissueare found in mammals, white adipose tissue (WAT)and brown adipose tissue (BAT).WAT functions to storeenergy, whereas BAT specializes in energy expendi-ture.2

InWAT cells, energy is stored via synthesis of triglyc-erides (TGs) that accumulate in lipid vesicles. WAT iscomposed of visceral and subcutaneous fat and repre-sents 10% of healthy body weight.3,4 Excess WATis related to several metabolic disorders. Althoughvisceral fat is less sensitive to insulin than subcuta-neous fat, both fat tissues play a role in the deve-lopment of type 2 diabetes mellitus (T2DM) andcardiovascular complications.5-8 In addition, an incre-ase in fatty acids (FAs), derived from excessive WATenergy storage, leads to an increased liver glucoseoutput and consequent production of atherogenic lipidssuch as very low-density lipoproteins.9

In contrast, BAT plays an important thermogenicfunction in neonatal mammals, rodents, and hiberna-tors, helping to counteract the cold stress of birth.2,3

In adult mammals, BAT has the capacity to modulateenergy balance by metabolizing FAs and dissipatingthe energy produced as heat.10 The ability of BAT toburn fat could be used as a novel therapeutic strategyto combat obesity and metabolic diseases.Therefore, the goal of this review is to identify thera-

peutic approaches with the potential to regenerate BATin humans to treat metabolic disorders.After a brief overview of BAT in terms of its origin, its

physiological properties, and the key molecular signalsfor BAT differentiation, this review summarizes thepathways currently used in the research communityfor BAT regeneration in animals and new routes thatneed to be investigated.

ORIGIN AND ROLE OF BAT

It was believed that white and brown adipocytes arisefrom the same precursor cells.11 However, DNA micro-array studies revealed that brown adipocytes do notshare a progenitor with white adipose cells but ratherhave the same origin as skeletal muscle cells.12,13

Lineage-tracing experiments suggested a model inwhich tripotent cells in the central dermomyotomegive rise to dermis, epaxial muscle, and brown fat.14

BAT precursor cells express myogenic factor 5, suggest-

ing their close localization to skeletal muscle cells dur-ing fetal development.15

Brown fat depots were also found in the WAT of theSiberian dwarf hamster, in which 10% of adipocytes ex-press the specific BAT marker uncoupling protein 1(UCP1).16 Other authors reported a 17% increase inbrown fat within WAT after administering adrenergicagonists. They observed a rise in the number of brownadipocytes but not in preadipocytes, suggesting thatbrown adipocytes can differentiate from mature whiteadipocytes.The fact that brown fat depots can be found in a pool

of white adipocyte precursors16 and that this populationcan be increased by administration of adrenergic ago-nists led to the hypothesis that 2 distinct brown adipo-cyte lineages exist.17 One shares precursors withskeletal muscle cells and localizes to interscapular areasand skeletal muscle, and the other derives from whiteadipocytes and localizes in the WAT itself.1

BAT is responsible for nonshivering thermogenesis tomaintain body temperature in cold environments18 andcan be found in rodents and newborn humans, mainlyin interscapular, paraspinal, and supraclavicular sites(Fig 1). BAT is highly vascularized and innervated incomparison with WAT1 and is composed of brown adi-pocytes, which contain multilocular lipid droplets andlarge numbers of mitochondria (Fig 1). In a cold atmo-sphere, the hypothalamus (HP) drives the release ofnorepinephrine (NE) in BAT via sympathetic nervoussystem (SNS) activation19 (Fig 1). The high innervationof BAT allows rapid stimulation of the adipocytemembrane. The adrenergic receptor (AR) is a7-transmembrane G protein–coupled receptor. On acti-vation, lipolysis is stimulated via the adenosine 30,50-cy-clic monophosphate (cAMP)-dependent protein kinaseA (PKA) signaling pathway.20

Free FAs (FFAs) derived from TG lipolysis via cyto-chrome c oxidase activation (Fig 2) stimulate mitochon-drial biogenesis in brown adipocyte nuclei.1 These FFAsundergo b-oxidation, and respiratory chain proteinsfrom the mitochondrial internal membrane generate aproton electrochemical gradient between the mitochon-drial matrix and the intermembrane space.21 The pres-ence of UCP1, which belongs to a superfamily ofanion carrier proteins, in the internal membrane of themitochondria mediates the re-entry of protons into themitochondria and dissipates energy as heat instead ofproducing adenosine triphosphate22 (Fig 2). The highnumber of mitochondria in brown adipocytes allows

Fig 1. Localization, function, and morphologic characteristics of

BAT. HP, hypothalamus; SNS, sympathetic nervous system; BAT,

brown adipose tissue; NE, norepinephrine; BAP, brown adipose pro-

genitor; BA Cell, brown adipose cell.

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the expenditure of large amounts of FFAs. In addition,the high vascularization of this tissue enhances thetransfer of the thermogenic effect throughout thebody. Finally, the thermogenic property of BAT mayresult in metabolization of enough TGs to have an effecton body weight,23 reducing obesity, and the associatedrisk of developing diabetes.24,25

UCP1 has only been found in BATand is therefore anideal marker for this tissue. It represents 10% of BATmitochondrial membrane proteins, whereas UCP2 andUCP3, which belong to the same family of transmem-brane proteins, represent only 0.01%–0.1%.1

In rodents, increased expression of UCP1 induced bypharmacologic agents or genetic manipulations hasbeen shown to reduce obesity and improve insulin sensi-tivity.1 In contrast, the absence of UCP1 leads to micewith cold intolerance and easy weight gain.26 However,other studies have reported UCP1-independent thermo-genesis in UCP1-deficient mice that is sufficient tomaintain body temperature and body weight duringcold exposure after an adaptation period.1

The following 2 main functions of BAT have beenproposed27:

(1) Prenatal BAT from progenitors common to skel-etal muscle maintains body temperature.

(2) Postnatal BAT from WAT progenitors maintainsbody weight.

MOLECULAR SIGNATURE OF BROWN ADIPOCYTES

Peroxisome proliferator–activated receptor g andcytosine–enhancer-binding protein. The 2 main tran-scriptional factors involved in the adipogenesis of brownand white adipocytes are peroxisome proliferator–activated receptor g (PPARg) and cytosine–enhancer-binding protein (C/EBP).28-31 Impairment of PPARg

or C/EBP in mice reduces BAT recruitment.32 Thisfact suggests that both factors are necessary foradipogenic differentiation.32 However, specifically forBAT differentiation, other factors may be neededbecause PPARg and C/EBPa have been used to inducemesenchymal stem cell (MSC) differentiation toward awhite adipose lineage.33-35

PPARg activation induces the expression of a largenumber of genes involved in lipid and glucose meta-bolism. It can be activated by antidiabetic drugs suchas thiazolidinediones and dietary or intracellular FAsand metabolites (prostaglandins).36,37 Active PPARgdimerizes with retinoid X receptor (RXR) and bindsspecific DNA sequences to promote the transcriptionof several genes, such as C/EBPa, which binds to andis a coactivator of PPARg (Fig 3). This heterodimerbinds protein-containing positive regulatory domain16 (PRDM16) to stimulate the differentiation of brownfat cells.4 In addition, PPARg induces its coactivator 1a(PPARg coactivator 1a [PGC-1a]), which exerts posi-tive feedback on PPARg and promotes UCP1 expres-sion38 (Fig 3).PPARg has 3 main functions as follows: (1) adipocyte

differentiation, (2) increase lipid storage, and (3) antidi-abetic effects. The third function results from the tran-scription of the FA-binding protein (aP2), which is aregulator of adipokines (leptin and adiponectin) andother proteins (lipoprotein lipase and adipose differenti-ation related protein) that can modulate lipid meta-bolism and insulin response.39 In addition, PPARg canbind nuclear factor kB instead of RXR, inhibiting thetranscription of the inflammatory cytokine tumor necro-sis factor a and interleukin 140 (Fig 3).

PPARg coactivator 1a. PGC-1a was the first proteinfound to interact with PPARg to mediate brown fatcell differentiation.41 PGC-1a is a coactivator ofPPARg in brown adipocytes and a regulator of energybalance38,41 (Fig 3). Its expression is highly inducedby noradrenaline and PPARg and RXR agonists. Aftercold induction and subsequent AR stimulation, PGC-1a expression is induced by PKA-cAMP responseelement binding (PKA-CREB) and is responsible fornonshivering thermogenesis.42,43 In addition, othercAMP-dependent proteins, such as aP2 and activatingtranscription factor 4, have been proven to beimportant regulators of PGC-1a and, therefore, oflipid metabolism and thermogenesis.44,45

PGC-1a induces mitochondrial biogenesis byenhancing the expression of UCP1, respiratory chainproteins, Krebs cycle proteins, and FA oxidative en-zymes.46-48 Interestingly, loss of both PGC-1a andPGC-1b from brown preadipocytes in culture inhibitsbrown fat characteristics, for example, mitochon-

Fig 2. Molecular machinery of brown adipocytes. Activation of intercellular protein cascade driving nuclear tran-

scription and generation of proton electrochemical gradient from respiratory chain proteins in the mitochondria.

BA Cell, brown adipocyte cell; NE, norepinephrine; AC, adenylyl cyclase; ATP, adenosine triphosphate; cAMP,

adenosine 30,50-cyclic monophosphate; PKA, cAMP-dependent protein kinase A; FA, fatty acids; PPAR gamma,

peroxisome proliferator–activated receptor g; RXR, retinoid X receptor; RNA Pol II, ribonucleic acid polymerase

II; COX, cytochrome C oxidase; Cyt C, cytochrome C; UCP1, uncoupling protein 1; ATP synthase, adenosine

triphosphate synthase.

Translational Research4 Roman et al - 2014

dria biogenesis and their function, without affectingadipogenesis.49

There are 3 types of steroid receptor coactivators(SRCs) in the nuclear membrane that can modulatePGC-1a activity as follows (Fig 3): (1) SRC-1 reinforcesPGC-1a coactivation of PPARg; (2) SRC-2 inhibits PGC-1a coactivation of PPARg;50 and (3) SRC-3 inhibitsPGC-1a acetylation by GCN5 (histone acetyl trans-ferase),51 leading to mitochondrial biogenesis.52,53

There are also embryogenic factors such as nuclearreceptor–binding protein 140, which inhibits PGC-1a–mediated mitochondrial biogenesis and UCP1expression54 (Fig 3). When receptor-binding protein140 is genetically removed, BAT depots appear inWAT.55-57 Tumor-suppressor proteins, such as retino-blastoma protein (Rb) and retinoblastoma-like protein1 (p107), also inhibit PGC-1a54 (Fig 3). Rb activatesp107, which in turn has a direct inhibitory effect on

PGC-1a.58 Nevertheless, deficient pRb MSCs can bedifferentiated in adipocytes with the brown fat pheno-type (high mitochondrial content and expression ofPGC-1a and UCP1).59

PR domain containingprotein 16. PRDM16 was identi-fied as 1 of the 3 most expressed genes in brown adipo-cytes.38 PRDM16 is a zinc-finger transcription factor. Inaddition to having 2 points of the zinc-finger DNA-binding domain, it has a PR domain with intrinsichistone methyltransferase activity.60-62

Various studies in mice myocytes and white preadipo-cytes reported that this protein is the switch for intercon-verting lineage to brown adipocytes.4,63 Nevertheless,when the DNA-binding domain is mutated, differentia-tion into brown adipocytes still occurs. It is believed thatPRDM16 acts as a mediator of PGC-1a and PPARg.4

Therefore, in contrast to PPARg, PRDM16 is sufficientbut not necessary for the differentiation of brown

Fig 3. Nuclear machinery of brown adipocytes. External or intracel-

lular activation of nuclear receptors and consequent transcriptional

modifications required to synthesize specific proteins for BAT differ-

entiation and parallel mechanisms for decreasing inflammation. FA,

fatty acids; TZD, thiazolidinedione; PPARg, peroxisome prolifera-

tor–activated receptor g; RXR, retinoid X receptor; PRDM16, PR

domain containing protein 16; PGC-1a, coactivator 1a of PPAR; C/

EBPa, CCAAT–enhancer-binding protein a; FoxC2, forkhead box

C2; cAMP, adenosine 30,50-cyclic monophosphate; RIP140, nuclear

receptor–binding protein; Rb, retinoblastoma protein; p107,

retinoblastoma-like protein 1; CtBP, C-terminal–binding proteins;

RNA Pol II, ribonucleic acid polymerase II; WAT, white adipose tis-

sue; UCP1, uncoupling protein 1; BAT, brown adipose tissue; aP2,

adipocyte fatty acid–binding protein; LPL, lipoprotein lipase;

ADRP, adipose differentiation related protein; Adipo, adipokines;

NF-kB, nuclear factor kB; TNF-a, tumor necrosis factor a; IL-1, inter-

leukin 1; SRC-1, -2, and -3, steroid receptor coactivators; GCN5, his-

tone acetyltransferase.

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adipocytes.3 When PRDM16 was removed after em-bryologic development, BAT differentiation decreased,suggesting that other PRDMs may stimulate the sameprocess.63

Exogenous PRDM16 in white preadipocytes led to anincrease in PPARg, PGC-1a, and UCP1 expres-sions.38,64 PRDM16 binds and stimulates PPARg andPPARa when these are bound to RXR (Fig 3). Further-more, PRDM16 directly interacts with PGC-1a, stimu-lating the interaction between PGC-1a and PPARg(Fig 3) and forming a PRDM16-PPARg-PGC-1a com-plex.15 In addition, it was observed that PRDM16 canbind forkhead box C2 to potentiate the cAMP-dependent thermogenic program (Fig 3).65 This pro-motes BAT differentiation in WAT by increasing theexpression of UCP1 and other mitochondrial genes.66

Brown preadipocytes with suppressed PRDM16exhibit a skeletal muscle cell phenotype.64 In contrast,

ectopic PRDM16 in myocytes causes brown fat celldifferentiation.3 PRDM16 prevents skeletal muscledifferentiation by inhibition of protein 1 of myogenicdifferentiation, myogenin, and myosin heavy chainexpression.64 Finally, PRDM16 also suppresses WATdifferentiation by binding the C-terminal–binding pro-teins 1 and 2 corepressors, which inhibit resistin andangiotensinogen gene expressions64 (Fig 3).

Early transcription factors. Bone morphogenetic pro-teins (BMPs) have a key role in the embryogenic devel-opment of BAT in rodents.67 BMP regulation hasnot been demonstrated in vivo, but treatment withBMP2, BMP4, BMP6, and BMP7 resulted in lipidaccumulation in preadipocytes in vitro (Fig 4). BMPhas an effect at all BAT differentiation levels as seenin MSCs derived from adipose tissue. BMP7 inducesthe expression of all early regulators of BAT such asPRDM16, PGC-1a, UCP1, PPARg, and C/EBP. Inaddition, BMP7 was shown to be specific to BAT.BMP7 knockout mice embryos are UCP1-deficient butshow WAT differentiation. Conversely, injection ofadenovirus BMP7 in these mice produced BATbut not WAT development, with a correspondingincrease in energy consumption and lack of weightgain. BMP4 inhibits UCP1 expression in brownpreadipocytes and, similar to BMP2, promotes WATdifferentiation (Fig 4).BMPs stimulate the key transcriptional factor associ-

ated with osteoblast differentiation (Runx2) (Fig 4).However, Runx2 is inhibited when BMP2, BMP6, andBMP7 are expressed in brown preadipocytes.68 BMP7also inhibits factors suppressing prenatal adipogenicdifferentiation in BAT-derived MSCs and in brown pre-adipocytes, such as postmitotic neuron-specific growthsuppressor (necdin), adipogenic inhibitor preadipo-cyte factor 1, and the embryogenic signaling pathway(Wnt).67,69

In vivo, after subcutaneous injection of BMP7-treatedMSCs, an increased number of brown adipocytes weredetected based on PPARg, C/EBP, PRDM16, andUCP1 expressions. In addition, it was found that theresulting increase in energy consumption and body tem-perature was not related to exercise or food intake.67

BAT in humans. Until recently, it was believed that hu-man BAT was only present in newborns and subse-quently undergoes hyperplasia and hypertrophy so thatit is absent in adults. Specifically, BAT activity wasconsidered to decline after the age of 2 years, when itsmetabolic activity reaches maximum values, furtherdecreasing in adolescents and being nonfunctional inadults. Nevertheless, significant amounts of BAT havebeen observed at the base of the neck in human adults,and it can be activated by exposure to cold. In fact, thepresence of UCP1 has been observed in BAT from

Fig 4. Adipogenic, myogenic, dermogenic, and osteogenic differentiation from commonMSCs. Specific positive

or negativeMSCmarkers are in black and between parentheses. Differentiation factors are shown in green, and the

name of each cell type is in blue. (*): The differentiating factors for brown preadipocytes and brown adipocytes are

the same as those for visceral white adipocyte differentiation. (?): Unknown.MSC, mesenchymal stem cell; Myf5,

myogenic factor 5; BMP, bone morphogenic protein; CD34, cell-surface antigen and stem cell marker with adhe-

sion function; CD44, cell-surface antigen and stem cell marker with adhesion andmigration functions; Pref-1, pre-

adipocyte factor 1; PPARg, peroxisome proliferator–activated receptor g; aP2, adipocyte fatty acid–binding

protein; Prdm16, PR domain containing protein 16; necdin, postmitotic neuron-specific growth suppressor;

FoxC2, forkhead box C2; PGC-1a, coactivator 1a of PPAR; UCP1, uncoupling protein 1; C/EBP, CCAAT–

enhancer-binding proteins; Runx2, key transcriptional factor associated with osteoblast differentiation; RXR,

retinoid X receptor; TZD, thiazolidinedione; FA, fatty acids. For interpretation of the references to color in this

figure legend, the reader is referred to the web version of this article.

Translational Research6 Roman et al - 2014

patients with pheochromocytoma and hibernoma54,70

using 18F-fluorodeoxyglucose (18F-FDG) positron-emission tomography fused to computed tomography(CT).3 This methodology has been widely used todetect metastases by capturing 18F-FDG in cells withactive accumulated (not metabolized) glucose thatwere subsequently localized by CT. Patients withpheochromocytomas showed higher levels of active BATin several locations of the body that disappeared aftertumor resection.71 The authors of another recent study ofpatients with pheochromocytomas investigated whetherwhite-to-brown transdifferentiation can occur in thisunique model of adrenergic hyperstimulated human fat.The results indicated that in adult humans, pure whiteomental fat subjected to adrenergic stimulation showsdistinct WAT-BAT plasticity. Under these conditions,white adipocytes seem to be able to undergo a processof direct transformation into brown adipocytes, and thewhole tissue undergoes a general rearrangementtoward BAT.72

In addition, BAT has also been detected in one-thirdof patients with thyroid diseases.73 In another study,18F-FDG positron-emission tomography fused to CTmethods were used to analyze the presence of BATin 10 lean (body mass index; BMI , 25) and 14 obese(BMI . 25) subjects under neutral (22�C) and cool(16�C) temperature conditions. BAT activity was de-tected in 23 (96%) of the subjects at 16�C but notat 22�C and was significantly higher in the lean group,indicating that BMI is inversely proportional to theamount of BAT.74 In a comparative study, BAT wasfound in 76 of 1013 women (7.5%) vs 30 of 959men (3.1%), indicating that BAT activation was2-fold higher in females than in males.75 Moreover,UCP1 gene polymorphisms have been associatedwith obesity and diabetes, highlighting the rele-vance of UCP1 in maintaining an appropriate energybalance.In conclusion, although BAT has lost its function for

evolutionary reasons, it seems to remain present in

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humans with higher activity in younger and thinner in-dividuals.76 Recent studies using tomography and CThave demonstrated the presence of active BAT depotsin healthy adult humans that respond to cold inductionand whose presence was inversely correlated to BMIand age.77 However, it has not yet been establishedwhether adult humans possess ‘‘classical’’ interscapularBAT analogous to that found in mice and infants.78

Recent studies in adult humans have identified a transi-tional form of BAT that exhibits intermediate morpho-logic and functional characteristics between BAT andWAT.79,80

Current therapeutic approaches. A mouse model hasshown that genetic suppression of BAT increasesobesity and predisposition to the development of meta-bolic diseases.26 Therefore, genetic interventions orpharmacologic therapies have emerged with the aimof increasing BAT depots. These studies have shownpositive effects in rodent models with metabolicdisorders, such as prevention of weight gain, reductionof FFAs in plasma, and increased insulin sensitivity.Nevertheless, genetic interventions are currently notwell controlled, and their adverse effects are not wellunderstood.In animal models, b3-ARs increase energy expendi-

ture after being activated by NE.81,82 These receptorsare widely abundant in rodents, and b3-adrenergicagonists have been shown to reduce fat depots,improve insulin resistance, and cause lipid catabolismin skeletal muscle fibers.83,84 b3-adrenergic agonistsstimulate TG hydrolysis in brown adipocytes found inrodent WAT by increasing UCP1 expression.15 There-fore, BAT differentiation has been found in WAT depotsafter b3-adrenergic stimulation. Furthermore, Grujicet al.85 demonstrated that a significant thermogeniceffect requires b3-AR activation in white adipocytes,leading to an increase in oxygen consumption and insu-lin secretion and a reduction in food intake. Similar toNE, b3-adrenergic agonists cause very rapid activationof b3-AR, observed within only 10 minutes.85 This im-plies that de novo protein synthesis is not required, andthermogenesis must be activated by FFA derived fromTG hydrolysis.Alternatively, pharmacologic therapies can have

adverse effects. Thyroid receptor activation has a ther-mogenic effect similar to b3-AR with the complicationof inducing hyperthyroidism.86,87

In animals, BMP7 has a role in regulating MSC dif-ferentiation into the adipogenic linage. Because BMPshave been implicated in metabolic diseases, it ispostulated that manipulation of these pathways mighthave therapeutic applications for obesity and T2DMin humans. However, combining these factors withhigh-fat diet animal models has shown an improve-

ment in adipogenesis but no effect on blood glucoseor insulin resistance.88 The mechanism of activationof BAT differentiation by BMP7 is not well under-stood. In addition, its role in bone formation needsto be further investigated before clinical use. Drugsthat increase PRDM16 in myoblasts or white preadi-pocytes may be a more promising approach.63 Never-theless, similar to BMP7, there is a need for a greaterunderstanding of the role of PRDM16, given that itmay affect differentiation of other tissues such asWAT and skeletal muscle. Another promising thera-peutic strategy may be the stimulation of cAMP inwhite adipocytes, which has been shown to augmentPGC-1a levels.89 Alternatively, the antidiabeticthiazolidinediones have the greatest effect on BATdifferentiation among pharmacologic agents. Theyare used as insulin sensitizers and are specific PPARgagonists.3

Studies with animal models have demonstrated a‘‘browning process’’ consisting of the occurrence ofbrown-like adipocytes, called ‘‘brite’’ (brown-in-white)or ‘‘beige’’ adipocytes in WAT depots, in response tospecific stimuli such as chronic cold exposure, b-adren-ergic stimulation, and other pharmacologic and nutri-tional agents.80,90 Nevertheless, in lean animals, BATrecruitment or activation by cold or pharmacologicagents does not affect body weight, and the increasein their metabolic rate is compensated by an increasein food intake. Only in obese animals does BATrecruitment directly induce weight loss and increaseUCP1 expression in BAT,82 mechanisms which needto be better understood. Furthermore, few studies havedemonstrated impaired BAT activation after cold expo-sure91,92 or sympathomimetics93 and insulin adminis-tration.92

Finally, in humans, it has been reported that b3-adren-ergic agonists have no significant effect.94 However,genetic obesity in humans is associated with modifiedb3-ARs. Obese Pima Indians and Japanese individualshave a variant of b3-AR; in addition, in obese Westernpatients, the same deficiency in these receptors predis-pose patients to the development of T2DM.95 Toimprove drug therapies regulating lipid metabolism inobesity, better knowledge of cellular mechanismsinvolved in b3-AR activation in human brown adipo-cytes is necessary. Likewise, PPARg does not have thesame effects in humans and rodents. PPARa is the keyfactor in central human adipogenic differentiation andis activated by fibrates, which are administered topatients with hypertriglyceridemia. New PPARa ago-nists combined with lipolytic agents may be a good op-tion to reduce WAT.94

Cold temperature seems to be the most effective wayto activate BAT in humans. In addition, a recent study

Translational Research8 Roman et al - 2014

has established that �250 g of fully functional BAT inhumans contributes 20% of whole body basal energyconsumption.74

NOVEL THERAPEUTIC APPROACHES

Catecholamines. Treatment of genetically and diet-induced obese rats with b3-adrenergic agonistsameliorates their pathologic condition. Moreover, UCP1-positive brown adipocytes are observed in areas of WATbecause of the direct transdifferentiation of differentiatedunilocular adipocytes. Catecholamines can also stimulateBAT differentiation and UCP1 expression in rats.96

In vitro studies have shown that catecholamines,epinephrine and NE play an important role in thestimulation of brown preadipocyte proliferation anddifferentiation to mature brown adipocytes.97

In human adults, catecholamines mediate effects onenergy balance by improving the efficiency of carbohy-drate metabolism, increasing lipolysis, and reducingadipogenesis. However, this effect is poorly observedin the subcutaneous adipose tissue of obese subjects.Recently, activation of and increases in the amounts ofBAT because of long-lived catecholamine metaboliteshave been shown when comparing healthy subjectsand patients with pheochromocytoma, a neuroendocrinetumor that intermittently secretes excessive amounts ofcatecholamines. This, together with the inverse relation-ship between BAT activity and WAT amount, suggeststhe important role of ARs in the upregulation of BATand its effect on human obesity.97

Other tissues may also be involved in the thermogeniceffect.98 Systemic infusion of catecholamines (isopren-aline) for nonselective b-adrenergic stimulation at highconcentrations did not activate BAT in humans. Energyexpenditure was increased to the same extent as coldexposure; however, this therapeutic approach may nothave an effect on BAT differentiation and lipolysisand therefore on obesity and metabolic syndrome.

Irisin hormone. In humans, PGC-a controls mitochon-drial biogenesis and oxidative metabolism in manydifferent cell types. Inmuscle tissue, PGC-a is stimulatedby exercise, activating mitochondrial biogenesis, angio-genesis, and type IIa and type I fibers, which are moreoxidative and high-endurance fibers.99 Furthermore, abeneficial effect has been shown when PGC-a isoverexpressed in muscle, including resistance to age-related obesity and diabetes in transgenic mice.100

Bostr€om et al101 have shown that PGC-a stimulatesthe expression of several muscle genes, including fibro-nectin type III domain-containing protein 5 (FNDC5),which encodes a type I membrane protein that is cleavedto yield a protein of 112 amino acids that is secreted intothe blood. This end product is called irisin hormone, and

it has been related to a significant increase in total bodyenergy expenditure and resistance to obesity-linkedinsulin resistance in mice. FNDC5 is particularly abun-dant in heart muscle. In addition, heart-derived natri-uretic peptides activate the white adipose thermogenicprogram.102

Exercise has the capacity to improve metabolic statusin patients with obesity and T2DM. Irisin may be asignal from organs involved in high-energy expendingactivity, such as skeletal muscle and heart, to use energystored in adipose tissue after exercise. Basal plasmalevels of irisin increased after 10 weeks of regular exer-cise in humans. In addition, irisin administered viaadenoviral vectors also greatly improves glucose ho-meostasis in mice fed a high-fat diet and activates thethermogenic program through increased energy expen-diture and development of brown fat-like cells in WAT,the latter determined by increased UCP1 expression.101

Other researchers also proved the capacity of irisin toinduce ‘‘browning’’ and to increase energy expenditurein humans, so this hormone has the potential to reduceobesity.103 The authors of a recent study examined therelationship among FNDC5 gene expression and circu-lating irisin levels with obesity, insulin sensitivity, andT2DM using 243 adipose tissue and 34 muscle tissue bi-opsies from a group of Caucasians with differentdegrees of obesity. The results showed that circulatingirisin could induce browning of human adipose tissue,leading to improved function and capacity of BAT andenhancing FNDC5 gene expression in adipose tissue.Moreover, the authors demonstrated reduced circulatingirisin concentration and FNDC5 gene expression in ad-ipose tissue and muscle from obese and T2DM subjects,suggesting a loss of brown fat-like characteristics. Thus,they speculate that irisin is produced in a positive feed-back loop by adipose tissue itself.104

The possibility of using this hormone as a therapeuticagent for obesity and T2DM is quite feasible. Irisin ishighly conserved in all mammalian species, with100% of homology between mouse and humans, sothis hormone could potentially be produced via recom-binant DNA technology.

Photoperiods and melatonin. NE and epinephrine arethe main neurotransmitters secreted after cold stimula-tion in the HP and lead to increased heart rate, releaseof glucose from energy stores, and increase in bloodpressure. This is a mechanism to enhance metabolic ac-tivity and maintain the body temperature. It operates viathe SNS by activating ARs.105

In the HP, proopiomelanocortin is believed to play animportant role in linking adipose functions to obesityand metabolic syndrome.105 This polypeptide precursoracts as a multifunctional prohormone in the HP, pitui-tary gland, and melanocytes. Melanocortin hormones,

Fig 5. Effects of MEL on energy balance favoring and inhibiting BAT

formation. MEL, melatonin; BAT, brown adipose tissue; UCP1, un-

coupling protein 1.

Translational ResearchVolume -, Number - Roman et al 9

which include adrenocorticotropin and melanocyte-stimulating hormone, are the main end product of proo-piomelanocortin. These hormones directly induceinsulin resistance, promote a proinflammatory adipo-kine profile, and stimulate UCP1 expression inadipocytes.105 Inhibition of melanocortin receptorspromoted lipid uptake, TG synthesis, and lipidaccumulation in WAT. In addition, although the activa-tion of central nervous system (CNS) melanocortin-stimulated lipid mobilization, its inhibition led toinsulin and glucose uptake in WAT but decreased useof glucose in skeletal muscle and BAT.106

Photoperiods also affect the level of the HP and areknown to exert some effect on adipose tissue.107 Ani-mals with higher amounts of BAT, such as rodents andhibernators, show seasonal changes in adipose tissuethat may be regulated by photoperiods. In Siberiandwarf hamsters, short day induction was associatedwith a BMI decrease and BAT activation throughincrease in NE levels.108 In the natural habitat, thechanging photoperiod predicts changes in ambient tem-perature and food availability, which are important reg-ulatory factors for nonshivering thermogenesis. Energypreservation mechanisms and metabolic changes to pre-pare for a harsh environment offer evident survival ben-efits. The regulatory effects of photoperiod are strongerthan those of reduced ambient temperature in somecases. Similar to cold stimulation, short photoperiodexposure led to BAT development in Djungarian ham-sters and white rats. Moreover, the effect on weightloss associated with short photoperiod exposure disap-peared when the pineal gland was surgically removedfrom Djungarian hamsters. Interestingly, body weightloss in Djungarian hamsters has been induced by admin-istration of exogenous melatonin (MEL) in longphotoperiod-exposed animals.107

MEL is a phylogenetically ancient molecule109

with many important functions as an autocoid andparacoid tissue factor, hormone, and sexual-selectionsignal.110,111 MEL is mostly secreted by the pinealgland (Fig 5), reaching the highest physiological levelsat night, and its secretion is substantially greater inwinter than in summer. MEL biosynthesis in the pinealgland declines with age,112 and this decline has beencorrelatedwith increased visceral adipose tissue in smallmammals.113 Pinealectomized Syrian hamsters showeda deficit in BAT formation that may be because of thedisappearance of the MEL rhythm, the signal that iden-tifies seasonal photoperiodic changes.112 Likewise,MEL production increases in response to cold exposurein hypertensive and Wistar-Kyoto rats.114 An elevatedand prolonged MEL signal during short photoperiodsis consistently related to the upregulation of BAT in hi-bernators. MEL administered in anterior HP nuclei and

the CNS produced a 59% increase in the interscapularBAT mass, whereas subcutaneous injection of the sameamount of MEL failed to induce a BAT increase of thatmagnitude.115 In addition, short day-induced Siberianhamsters reduced their body fat through SNS activity af-ter an increase in MEL107 (Fig 5). MEL receptors (MT),especially MT1, were found in the CNS,116 leading tothe hypothesis that MEL activates the SNS, whichthen induces the expression of BAT-specific markers.MEL may have a direct effect on BAT given that MTs

are present on the membranes of brown adipocytes inSyrian hamsters. Similar to b-ARs, MT1/MT2 are Gprotein–coupled receptors. However, in contrast to theeffects of NE, MTs reduce intracellular cAMP levels,PKA activity, and CREB phosphorylation. These recep-tors can also be coupled to Gq proteins, leading toactivation of phospholipase C, which increases diacyl-glycerol in brown adipocytes. Diacylglycerol activatesprotein kinase C and triggers the upregulation ofCREB. Activation of MTs in BAT also enhances thestimulatory effects of NE.117

Adipocyte differentiation is inhibited by MEL,reducing TG accumulation118,119 (Fig 5) and inhibitingthe expression of several adipocyte differentiationmarkers, such as PPARg, aP2, C/EBPa, C/EBPb, peril-ipin, and adiponectin.119 In contrast, MEL enhances os-teogenesis through Runx2 expression.120 Furthermore,in another study MEL increased leptin secretion, WATformation, and food intake121 (Fig 5). Additionally,it has been demonstrated that MEL inhibits nuclear fac-tor kB, leading to protection against oxidative stress122

(Fig 5), which may have a positive effect on the mito-chondria of BAT cells. This could be a direct effect

Translational Research10 Roman et al - 2014

because MEL was oxidized by mitochondrial cyto-chrome C in cell culture and in vivo.123

A study in Siberian hamsters demonstrated that MELdirectly affected peripheral tissue by inhibiting mito-chondrial genome expression in brown adipocytes(Fig 5). MEL may inhibit mitochondrial transcriptionfactor 1, causing the downregulation of mitochondrialgenome expression and nuclear respiratory factors 1and 2, which are in turn the regulators of mitochondrialtranscription factor 1. However, the same study foundno changes in UCP1 gene expression124 (Fig 5). Despitethese controversial results, the effects of MEL onobesity and metabolic disorders are encouraging giventhat it has been demonstrated that MEL restrains bodyweight gain without changing food intake.113,124

Additionally, our recent findings indicate that MELalso has great potential to prevent diabetes byreducing body weight and chronic inflammation125-127

and inducing browning of subcutaneous WAT inZ€ucker diabetic fatty rats.128 Finally, mounting evi-dence, obtained mainly in rodents, suggests that MELmight increase energy expenditure by activating non-shivering thermogenesis in BAT.110

Stem cells and tissue engineering. Stem cells (SCs)have long-term replicative and self-renewal potential,maintaining undifferentiated properties, and multili-neage differentiation ability.129

Adult SCs (ASCs) or tissue-derived SCs maintain thesteady-state functioning of a cell, that is, homeostasis,and, with limitations, replace cells that die because ofinjury or disease. ASCs are present in most tissuesand usually divide to generate progenitor or precursorcells, which then differentiate into ‘‘mature’’ cell typesthat have characteristic shapes and specialized func-tions.130 Their multilineage differentiation capacityhas now been extensively characterized.131-133 Theyare able to proliferate and differentiate into tissues ofmesenchymal, endodermal, and ectodermal origin,leading to speculation about their pluripotency.134,135

Adipose tissue is the main source of multipotent SCs.Adipose-derived stem cells (ADSCs) can be easily andsafely isolated from patients, that is, via lipoaspirate,131

and can be differentiated in vitro into tissues from the 3embryonic layers. Recent studies described ADSCs aspericytes that are attached to the endothelial cells ofblood vessels.136 In adipose tissue, these cells havebeen described as preadipocytes.137 These perivascularcells are found in different tissues (eg, skeletal muscle,BAT, WAT, tissue, pancreas, and placenta) and havethe potential to differentiate along mesenchymallineages.4,12,63,138,139

Cell-therapy strategies to improve metabolic disor-ders are based on the implantation of brown adipocytecells in the interscapular areas to regenerate BAT in hu-

mans. Adult progenitor cells can be induced to differen-tiate into BATand are easily expanded in the laboratoryfor transplantation. In fact, ASCs have been success-fully used for breast surgery reconstruction140,141 andmyocardial regeneration after infarction.142

Progenitors of WAT and skeletal muscle as well asmature brown adipocytes have been transplanted in ro-dents to regenerate BAT. Although transplantation ofbone marrow–derived SCs or skin fibroblasts did notform adipose tissue pads,143 injection of ADSCs144

and differentiated adipocytes145 formed BAT pads.C3H10T1/2 multipotent progenitor cells (embryonicfibroblasts) that differentiated into brown adipocytesexpressing UCP1 after BMP7 treatment have alsobeen successfully used to form both multilocularbrown and unilocular white adipocytes when implantedinto UCP1-null mice.67 Other authors have inducedin vitro brown adipocyte differentiation of primarymyoblasts and skin fibroblasts using PRDM16.38,63 Inhumans, ASCs isolated from skeletal muscle have alsobeen differentiated into brown adipocytes expressingUCP1.146 A recent study showed that local administra-tion of BMP2 leads to the expansion, migration, and dif-ferentiation of progenitor cells from the peripheralnerve perineurium to brown adipose-like cells.147

Furthermore, as already described in this review,MEL, a physiological molecule, can stimulate ASC dif-ferentiation into BAT precursor cells, and it may alsohave the potential to recruit BATand activate nonshiver-ing thermogenesis.Nevertheless, studies on the efficacy of transplanting

preadipocytes and mature brown adipocytes have notoffered conclusive results. Other studies based on acell-therapy approach subcutaneously implanted plurip-otent SCs in mice. Cells were differentiated beforebeing implanted by gene transfection using adeno-virus148 or by culturing the cells with transcriptionfactors.149 Although embryonic stem cells gave rise toBAT pads with mature functional properties,148 inducedpluripotent SCs had a beneficial effect on lipid meta-bolism.149

BAT has been successfully transplanted in rodents us-ing small clusters. However, large grafts of adipose tis-sue (WAT and BAT) have shown to undergo necrosispost-transplantation.150,151 The use of a syntheticmatrix as a scaffold greatly improves the viability ofbrown adipose cells after transplantation.152-155 Incomparison with injected cells, ASCs and preadi-pocytes previously seeded on a matrix of polyglycolicacid and-or collagen scaffolds showed decreasednecrosis and improved vascularization.156,157

Other factors are also important for the survival anddifferentiation of transplanted grafts. Grafts of BATtransplanted from obese mice to thin mice showed

Fig 6. Therapeutic potential for ASC transplantation and beneficial effects in the regeneration of BAT. BAT,

brown adipose tissue; WAT, white adipose tissue; ASCs, adult stem cells; miRNA, microRNA; BMP7, bone

morphogenetic protein 7; PPARg, peroxisome proliferator–activated receptor g; PGC-1a, coactivator 1a of

PPAR; PRDM16, PR domain containing protein 16; RXR, retinoid X receptor.

Translational ResearchVolume -, Number - Roman et al 11

changes in cell morphology from large adipocytes withunilocular lipid droplets and sparse mitochondria tosmall adipocytes with multilocular lipid droplets anddense mitochondria. This study indicated the influenceof the host environment on this conversion, whichmay be stimulated by variations in FA composition.158

More recently, subcutaneous transplants of BAT instreptozotocin-induced diabetic mice restored euglyce-mia, reduced tissue inflammation, and reversed clinicaldiabetes markers. The authors proposed that the com-bined action of several adipokines re-established

chronic glycemic homeostasis in the absence of insu-lin.158

Ultimately, the long-term effect of increasing BAT isexpected to improve energy expenditure, leading toweight loss and increased insulin sensitivity.

CONCLUSIONS

Several studies have investigated the clinical rele-vance of BAT as a therapeutic target for patients atrisk of developing metabolic diseases. However,

Translational Research12 Roman et al - 2014

upregulation via transcription factors and the role of thedifferent molecular factors in regenerating and-orstimulating BAT need to be better understood to avoidundesirable effects in humans. Alternatively, novel ther-apeutic approaches for BAT regeneration have beeninvestigated recently in animal models, with intenseresearch efforts on the following topics:

(1) Direct autologous ASCs transplantation into BATdepots (Fig 6).

(2) Transplantation of brown adipocytes differenti-ated from preadipocytes after being treated withspecific factors, such as PPARg, PRDM16,BMP7, or retinoic acid159 (Fig 6).

(3) Transplantation of ASCs genetically manipulatedwith specific BAT transcription factors. This hasbeen proposed to promote adipogenesis by inhib-iting differentiation into unwanted cell line-ages160 (Fig 6). The use of adenoviral vectorshas been tested for this purpose in animals,although liposomes would be more suitable inhumans because they may trigger a lower inflam-matory response when releasing the RNA intothe cell161 (Fig 6).

(4) Use of hydrogel, polymer (lactic-co-glycolic acid)and-or collagen scaffolds111,162,163 previouslyseeded and cultured with brown adipocytes. Inaddition, these materials may be previously coatedwith angiogenic, antifibrotic, antiapoptotic, andanti-inflammatory factors to improve the survivalof the engineered adipose tissuegrafts164-166 (Fig 6).

(5) Manipulation of photoperiods that could be usedto induce BAT formation. MEL is naturally pro-duced in the body in response to the perceptionof light and may play a valuable therapeutic rolein the differentiation of ASCs into brown adipo-cytes.

ACKNOWLEDGMENTS

Conflicts of Interest: The authors report no conflictsof interest. All authors have read the journal’s policyon conflicts of interest. All authors have read the jour-nal’s authorship agreement.This work was supported in part by grants from

the Consejer�ıa de Econom�ıa, Innovaci�on y Ciencia(Junta de Andaluc�ıa, the Ministerio de Econom�ıa yCompetitividad (grant number SAF 2013-45752-R)excellence project, grant number CTS-6568) and the In-stituto de Salud Carlos III (Fondo de Investigaci�on Sani-taria, European fund for regional development(FEDER) funds, grant number PI10/02295).The authors gratefully acknowledge Emma Guti�errez

Gonz�alez for the design of the illustrations.

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