cells

Review The Remaining Mysteries about Brown Adipose Tissues

Miwako Nishio 1 and Kumiko Saeki 1,2,*

1 Department of Laboratory Molecular Genetics of Hematology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo 113-8510, Japan; [email protected] 2 Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo 162-8655, Japan * Correspondence: [email protected]; Tel.: +81-3-3202-7181

 Received: 30 September 2020; Accepted: 4 November 2020; Published: 10 November 2020 

Abstract: Brown (BAT), which is a thermogenic fat tissue originally discovered in small hibernating mammals, is believed to exert anti- effects in humans. Although evidence has been accumulating to show the importance of BAT in metabolism regulation, there are a number of unanswered questions. In this review, we show the remaining mysteries about BATs. The distribution of BAT can be visualized by nuclear medicine examinations; however, the precise localization of human BAT is not yet completely understood. For example, studies of 18F-fluorodeoxyglucose PET/CT scans have shown that interscapular BAT (iBAT), the largest BAT in mice, exists only in the neonatal period or in early infancy in humans. However, an old anatomical study illustrated the presence of iBAT in adult humans, suggesting that there is a discrepancy between anatomical findings and imaging data. It is also known that BAT secretes various metabolism-improving factors, which are collectively called as BATokines. With small exceptions, however, their main producers are not BAT per se, raising the possibility that there are still more BATokines to be discovered. Although BAT is conceived as a favorable tissue from the standpoint of obesity prevention, it is also involved in the development of unhealthy conditions such as cancer cachexia. In addition, a correlation between browning of mammary and progression of breast cancers was shown in a xenotransplantation model. Therefore, the optimal condition should be carefully determined when BAT is considered as a measure the prevention of obesity and improvement of metabolism. Solving BAT mysteries will open a new door for health promotion via advanced understanding of metabolism regulation system.

Keywords: brown adipose tissue; BATokine; interscapular BAT; trapezius muscle; cachexia; extracellular vesicles

1. Introduction Brown adipose tissues (BATs), which contribute to non-shivering , has been attracting attention as a target for therapeutic development against obesity and metabolic disorders. BATs are required for the maintenance of body temperature in a cold environment in small mammals, which suffer from large heat loss due to high surface/volume ratios. BATs also contribute to rapid thermogenesis after hibernation. In mice, the presence of BATs has been anatomically determined in specific sites of the body including in interscapular, cervical, axillary, perirenal, and mediastinal regions [1,2] as well as in mammary gland [3]. Humans also have functional BATs [4–7], whose distribution has been visualized by 18F-fluorodeoxyglucose (18F-FDG)-PET/CT examinations as fat depots with high glucose uptake capacities. In adult humans, BATs are detected in deep neck regions, supraclavicular regions, axillary regions, paravertebral regions, periaortic regions, and suprarenal regions (Figure1). The localization of BATs can be summarized as “alongside of large arteries” and “in

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Cells 2020regions, 9, 2449 (Figure 1). The localization of BATs can be summarized as “alongside of large arteries” and2 of 15 “in peri- spaces”, both of which receive abundant adrenergic signals. Since adrenergic stimuli are required for the maintenance of BATs, this distribution pattern of BATs seems highly peri-adrenal gland spaces”, both of which receive abundant adrenergic signals. Since adrenergic stimuli reasonable. are required for the maintenance of BATs, this distribution pattern of BATs seems highly reasonable. The amounts of BAT, which tend to reduce with age [8], are inversely correlated with obesity The amounts of BAT, which tend to reduce with age [8], are inversely correlated with obesity [9–11] [9–11] and glucose intolerance [12]. In humans, interscapular BAT (iBAT) is detectable in newborns andand glucose young intolerance suckling infants [12]. Inby humans,18F-FDG-PET/CT interscapular examinations. BAT (iBAT) There is, is detectablehowever, a indifference newborns in the and 18 youngdepth suckling of iBAT infants between by miceF-FDG-PET and humans./CT examinations. In mice, iBAT Therelocates is, in however, the subcutaneous a difference space in thebeneath depth of iBATthe layer between of white mice adipose and humans. tissue (WAT), In mice, whereas iBAT iBAT locates locates in the beneath subcutaneous the trapezius space muscle beneath in the layerhumans. of white Although adipose the tissue reason (WAT), for the whereas disappearance iBAT locates of iBAT beneath in humans the trapezius during muscle early infancy in humans. is Althoughunknown, the the reason presence for the of iBAT disappearance in inter-muscular of iBAT spaces in humans (i.e., between during the early trapezius infancy muscle is unknown, and the the presencerhomboid of muscles) iBAT in may inter-muscular be disadvantageous spaces (i.e., for betweenlong-term the maintenance trapezius muscleof iBAT and because the rhomboid skeletal muscles)muscles may produce be disadvantageous substantial heat. for long-term maintenance of iBAT because skeletal muscles produce substantialIt is heat. known that transplantation of murine BAT [13] or human pluripotent stem -derived brownIt is known that (hBA) transplantation [14] improves of murine and BAT glucose [13] ormetabolism human pluripotent [13,14] and stemprevents cell-derived obesity brown[13]. adipocytes Therefore, (hBA) approaches [14] improves to augmenting lipid and BAT glucose or preventing metabolism its age-dependent [13,14] and prevents reduction obesity may be [13 ]. Therefore,an effectual approaches strategy to for augmenting the control BAT of obesity. or preventing It is also its believed age-dependent that metabolism-improving reduction may be an eeffectsffectual strategyof BAT for is the executed control via of secreted obesity. factors, It is alsowhich believed are collectively that metabolism-improving termed BATokines. Up etoff ectsnow, of several BAT is factors have been reported to serve as BATokines including growth factor 21 (Fgf21) [15,16], executed via secreted factors, which are collectively termed BATokines. Up to now, several factors have Interleukin 6 (Il6) [13], Growth differentiation factor (Gdf15) [17], morphogenetic protein 8b been reported to serve as BATokines including Fibroblast growth factor 21 (Fgf21) [15,16], Interleukin 6 (Bmp8b [18], Angiopoietin-like 8 (Angptl8) [19], Neuregulin 4 (Nrg4) [20,21], Slit guidance ligand 2 (Il6) [13], Growth differentiation factor (Gdf15) [17], Bone morphogenetic protein 8b (Bmp8b [18], (Slit2) [22], Ependymin related 1 (Epdr1) [23], and Phospholipid transfer protein (Pltp) [24]. Except Angiopoietin-likefor Angptl8 and8 Nrg4 (Angptl8), however, [19 ],the Neuregulin expression levels 4 (Nrg4) of those [20,21 factors], Slit are guidance low in BATs, ligand implying 2 (Slit2) that [22 ], Ependyminthey serve related as autocrine/paracrine 1 (Epdr1) [23], and factors Phospholipid involved in transfer the regulation protein (Pltp)of BAT [ 24functions]. Except rather for Angptl8 than and hormonesNrg4, however, that mediate the expression inter-organ levels communications. of those factors Although are low biological in BATs, significance implying that of enhanced they serve as autocrineAngptl8 /releaseparacrine by activated factors involved BAT remains in the unclear regulation (reviewed of BAT in functions[25]), Nrg4-Bmp8b rather than axis is suggested that mediateto be inter-organ involved in communications.promoting sympathetic Although innervation biological of BAT significance [26]. On ofthe enhanced other hand, Angptl8 hBA secretes release a by activatedlow molecular BAT remains weight unclear factor (≈800 (reviewed Da) that in enhances [25]), Nrg4-Bmp8b insulin secretion axis isby suggested pancreatic tobeta be cells involved [27]. It in promotingis also shown sympathetic that hBA innervation secretes a ofvariety BAT [of26 extracellular]. On the other vesicles hand, (EVs) hBA [17], secretes which a lowmay molecularserve as weightBATokines factor ( as800 in the Da) case that of enhances WAT [28]. insulin secretion by pancreatic beta cells [27]. It is also shown ≈ that hBAAlthough secretes BAT a variety is generally of extracellular accepted as vesicles a favorable (EVs) fat [tissue,17], which hyperactivation may serve of as BAT BATokines may induce as in the caseunfavorable of WAT outcomes. [28]. In Apoe-deficient mice, hyperactivation of BAT by acute cold exposure promotes atheroscleroticAlthough BAT plaque is generally growth accepted and instability as a favorable [29]. Involvement fat tissue, ofhyperactivation BAT in the development of BAT may of cancer induce unfavorablecachexia outcomes.is suggested In (reviewedApoe-deficient in [30–33]). mice, It hyperactivation is even suggested of that BAT browning by acute (i.e., cold acquisition exposure promotesof BAT- like phenotypes) of breast cancer cells and neighboring cells promotes the progression of breast cancers atherosclerotic plaque growth and instability [29]. Involvement of BAT in the development of cancer [34], warning that activation of BAT should be kept within an appropriate level when BAT-focused cachexia is suggested (reviewed in [30–33]). It is even suggested that browning (i.e., acquisition of therapeutic development against obesity is considered. BAT-like phenotypes) of breast cancer cells and neighboring cells promotes the progression of breast In the following sections, the above-mentioned points are explained in detail for in-depth cancersdiscussion [34], warningabout remaining that activation mysteries of of BATBATs. should be kept within an appropriate level when BAT-focused therapeutic development against obesity is considered.

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FigureFigure 1. 1. BATs are are located located alongside alongside of large of arteries large arteriesand in peri-adrenal and in peri-adrenal gland spaces. gland(a) A 18FDG- spaces. 18 (a)APET/CTFDG-PET examination/CT examination result of a young result adult of a with young acute adult cold withexposure acute at 19 cold °C exposurefor 2 h. Adopted at 19 ◦fromC for 2 h. Adopted from Figure 7B in Reference [14].; (b) Schematic presentation of body BAT localization in humans. BATs are located in deep neck regions, supraclavicular regions, axillary regions, paravertebral regions, and periaortic regions, which can be summarized as alongside of large arteries, as reported in Cells 2020, 9, 2449 3 of 15

the case of deep neck BAT [35], and in suprarenal regions, which have been determined as peri-adrenal gland spaces [36]. These regions receive abundant adrenergic signals, which contribute to the maintenance of BAT. In the following sections, the above-mentioned points are explained in detail for in-depth discussion about remaining mysteries of BATs.

2. The Remaining Mysteries about BAT

2.1. Localization of BAT Historically, the presence of murine BATs was anatomically determined while the existence of human BATs was recognized by 18F-FDG-PET/CT examinations. Therefore, there was once a controversy as to whether the body distribution of BATs was equivalent between small and large mammals. The findings obtained by nuclear medicine examinations in mice has resolved the dispute. Single photon emission computed tomography (SPECT)/CT scans using a lipid probe, (123/125I)-b-methyl-p-iodophenyl-pentadecanoic acid, along with 18F-FDG-PET/CT scans revealed the existence of additional murine BATs that had previously been recognized as unique to humans. Therefore, it is currently accepted that mice and humans share a high degree of topological similarity of BATs [37]. The remaining question is regarding iBAT, which is the largest BAT. While iBAT continues to exist throughout life in mice, it is detectable only in the neonatal period and early infancy in humans by 18F-FDG-PET/CT scans. Because distinct results were obtained from SPECT/CT-based imaging and 18F-FDG-PET/CT-based imaging of BATs in mice [37], it cannot be easily concluded that adult humans lack iBAT only by the findings of 18F-FDG-PET/CT scans. More than 100 years ago, an anatomical study illustrated the presence of BAT-like tissue in interscapular regions in both newborns and adults in humans [38]. At that time, the presence of BAT in humans was not known and the author called the organ as “interscapular gland”, which show the identical characteristics to iBAT. The author described that it: (1) has a dark brown color; (2) is a paired organ; (3) is lobulated; and (4) is well supplied with blood vessels, all of which correspond to the characteristic of iBAT. The author wrote that “Were it not for the colour and definite outline of the gland, it might be easily mistaken for fat”, suggesting that this organ is not a gland but “a brown colored fat”. Sketches of the of this organ further indicated its resemblance with that of BAT. The author also described that “This gland in man is the homologue of the so-called hibernating gland in the rodents”, strongly suggesting that it is iBAT. Interestingly, it was described that this organ showed projections to neck and clavicular regions, indicating that iBAT makes a complex with deep Cells 2020, 9, x FOR PEER REVIEW 4 of 15 neck BAT and supraclavicular BAT (Figure2).

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FigureFigure 2. The2. The existence existence of of iBATiBAT inin adultadult humans. humans. (a) ( aAn) An anatomical anatomical study study of humans of humans reported reported the the presencepresence of “a brown colored fat depot” beneath beneath the the trapezius trapezius muscle muscle (Tr) (Tr) [38]. [38 ].It Itwas was described described that that thisthis fat “extends fat “extends horizontally horizontally toward toward the centre the centre of the interscapularof the interscapular region, region, where it where forms it a somewhat forms a T-shapedsomewhat mass T-shaped over and mass between over and the between rhomboidii the rhomboidii muscles” and muscles” “is entirely and “is covered entirely bycovered the trapezius by the muscle”trapezius [38]. muscle” This brown [38]. fat This depot brown reportedly fat depot possesses reportedly morphological possesses morphological and histological and characteristics histological characteristics of BATs [38]. Figure was created by referring to Figure 2 in Reference [38]. (b) Schematic presentation of iBAT in adult humans drawn according the descriptions in Reference [38].

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Figure 3. Development of BAT during embryogenesis. (a) Schematic presentation of the development of BAT. Brown progenitors are differentiated from Myf5-positive myotomes, which are dorsal portions of dermatomyotomes. Myf5-positive cells also consist of back muscle progenitors. Dermatomyotomes are differentiated from somites, which are derived from paraxial . Figure was created by referring to Figure 2 in Reference [40]. (b) Schematic presentation of the localization and migration of the trapezius muscle progenitors and those of brow adipocytes. Figure was created by referring to Figure 1 in Reference [39]. (c) Schematic presentation of relative positional relationship between the trapezius muscle (Tz) and iBAT in mouse (left) and human (right). Figure was created by referring to Figure 1 in Reference [39].

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of BATs [38]. Figure was created by referring to Figure2 in Reference [ 38]. (b) Schematic presentation of iBAT in adult humans drawn according the descriptions in Reference [38]. Intriguingly, human iBAT is located beneath the trapezius muscle, whereas murine iBAT is located in the subcutaneous space beneath the WAT layer. The difference in the depth of iBAT between mice and humans may come from the difference in the time of migration of the progenitor of the trapezius muscle and that of brown adipocytes(a during) embryogenesis. It is known that(b) the trapezius muscle progenitors migrateFigure from the2. The third existence and fourth of iBAT pharyngeal in adult humans. arches ( toa) upperAn anatomical back regions study of [39 humans], while reported brown the adipocyte progenitorspresence arise of “a from brown a posteriorcolored fat partdepot” of beneath somite the and trapezius migrate muscle to dorsal(Tr) [38]. spaces It was described (reviewed that in [40]). If the migrationthis fat “extends of brown horizontally adipocyte toward progenitors the centre precedes of the interscapular that of the region, trapezius where muscle, it forms iBAT a will reach subcutaneoussomewhat T-shaped spaces mass and over be and located between beneath the rhomboidii WAT as muscles” in the and case “is of entirely mice. covered On the by other the hand, if the migrationtrapezius of muscle” the trapezius [38]. This muscle brown progenitorsfat depot reportedly precedes possesses that of brownmorphological adipocyte and progenitors,histological iBAT characteristics of BATs [38]. Figure was created by referring to Figure 2 in Reference [38]. (b) will be located beneath the trapezius muscle as in the case of humans (Figure3). Currently, information Schematic presentation of iBAT in adult humans drawn according the descriptions in Reference [38]. about iBAT in adult humans is limited. For detailed assessment of human iBAT, nuclear medicine examinations using a new probe are required.

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FigureFigure 3. Development3. Development of of BAT BAT during during embryogenesis. embryogenesis. ( aa)) Schematic Schematic presentation presentation of of the the development development of BAT.of BAT. Brown Brown adipocyte adipocyte progenitors progenitors are are di differentiatedfferentiated from Myf5-positive Myf5-positive myotomes, myotomes, which which are are dorsaldorsal portions portions of of dermatomyotomes. dermatomyotomes. Myf5-positiveMyf5-positive cells cells also also consist consist of of back back muscle muscle progenitors. progenitors. DermatomyotomesDermatomyotomes are are di differentiatedfferentiated from from somites, somites, which are are derived derived from from paraxial paraxial mesoderm. mesoderm. FigureFigure was was created created by referring by referring to Figure to Figure2 in Reference 2 in Reference [ 40]. (b ) [40]. Schematic (b) Schematic presentation presentation of the localization of the andlocalization migration and of the migration trapezius of musclethe trapezius progenitors muscle andprogenitors those of and brow those adipocytes. of brow adipocytes. Figure was Figure created was created by referring to Figure 1 in Reference [39]. (c) Schematic presentation of relative positional by referring to Figure1 in Reference [ 39]. (c) Schematic presentation of relative positional relationship relationship between the trapezius muscle (Tz) and iBAT in mouse (left) and human (right). Figure between the trapezius muscle (Tz) and iBAT in mouse (left) and human (right). Figure was created by was created by referring to Figure 1 in Reference [39]. referring to Figure1 in Reference [39].

2.2. BATokines

Since BAT-depleted mice developed morbid obesity [41] and glucose and lipid metabolic disorders [42], BAT plays indispensable roles in obesity prevention and metabolism improvement. Nevertheless, ucp1-deficient mice, which lack BAT-dependent thermogenesis activities, did not undergo obesity under ambient temperature [43]. Therefore, functions other than thermogenesis per se including Cells 2020, 9, x FOR PEER REVIEW 5 of 15

2.2. BATokines Since BAT-depleted mice developed morbid obesity [41] and glucose and lipid metabolic disorders [42], BAT plays indispensable roles in obesity prevention and metabolism improvement. Nevertheless,Cells 2020, 9, 2449ucp1-deficient mice, which lack BAT-dependent thermogenesis activities,5 of 15did not undergo obesity under ambient temperature [43]. Therefore, functions other than thermogenesis per se includingsecreted factor-mediated secreted factor-mediated effects contribute effects to obesity contribute prevention to obesity (reviewed prevention in [8]). Several (reviewed factors in [8]). Severalhave factors been reportedhave been to servereported as BATokines to serve as including BATokines Fgf21 including [15,16], Il6 Fgf21 [13], Gdf15 [15,16], [17 Il6], Bmp8b [13], Gdf15 [18], [17], Bmp8bAngptl8 [18], [Angptl819], Nrg4 [19], [20,21 Nrg4], Slit2 [20,21], [22], Epdr1 Slit2 [23 [22],], and Epdr1 Pltp [ 24[23],]. Regarding and Pltp Fgf21, [24]. Il6,Regarding Bmp8b, Slit2, Fgf21, Il6, Bmp8b,Epdr1, Slit2, Pltp, Epdr1, and Gdf15A,Pltp, and BAT Gdf15A, is not theBAT major is not producer the major as aproducer gene expression as a gene database expression indicates database indicates(Figure (Figure4a–d,g–i). 4a–d,g–i). It was reported It was reported that activated thatBAT activated expressed BAT Fgf21 expressed at a level Fgf21 comparable at a level with comparable that with ofthat the of liver the [liver15]. However, [15]. However, the gene the expression gene expression database database shows that shows the main that source the main of Fgf21 source is not of Fgf21 the liver but the (Figure4a), which is compatible to the finding of Kuroda et al. [ 44]. It was is not the liver but the pancreas (Figure 4a), which is compatible to the finding of Kuroda et al. [44]. even shown that cold exposure or β3-adrenergic stimulation caused a significant induction of Fgf21 It was even shown that cold exposure or β3-adrenergic stimulation caused a significant induction of mRNA levels in BAT without a concomitant increase in FGF21 plasma level [16]. Therefore, Fgf21 may Fgf21serve mRNA as an levels autocrine in BAT/paracrine without BATokine. a concomitant Regarding Il6increase (Figure 4inb) FGF21 and Gdf15 plasma (Figure level4c), BAT [16]. is Therefore, not Fgf21the may main serve producer, as an either. autocrine/paracrine Nevertheless, it was BATokine. shown that Regarding intraperitoneally Il6 (Figure transplanted 4b) and iBAT-derived Gdf15 (Figure 4c), BATIl6 induced is notIl6 theexpression main in producer, the host BATs either. [13], suggesting Nevertheless, that iBAT-derived it was shown Il6 serves that as intraperitoneally a mediator transplantedof inter-BAT iBAT-derived functional association Il6 induced in the Il6 body. expression Since Gdf15 in the and host Il6 create BATs a mutually[13], suggesting gene-inducing that iBAT- derivedcycle Il6 [17 serves], it seems as a that mediator Gdf15 is of also inter-BAT involved in functional this association. association Regarding in Angptl8the body. and Since Nrg4T, Gdf15 BAT is and Il6 createthe a mainmutually producer gene-inducing (Figure4e,f). However,cycle [17], biological it seems significance that Gdf15 of enhancedis also involved expression in Angptl8this association. in BAT remains elusive [25]. Moreover, Angptl3/8-deficient mice show upregulated thermogenesis [45], Regarding Angptl8 and Nrg4T, BAT is the main producer (Figure 4e,f). However, biological suggesting that Angptl8 may serve as a negative feedback regulator involved in modulating rather significance of enhanced expression Angptl8 in BAT remains elusive [25]. Moreover, Angptl3/8- than upregulating metabolism. On the other hand, NRG4 is suggested to contribute to the promotion deficientof sympathetic mice show innervation upregulated together thermogenesis with Bmp8 [[45],26]. suggesting that Angptl8 may serve as a negative feedback Lowregulator molecular involved weight in modulating substances, such rather as than triiodothyronine, upregulating retinaldehyde, metabolism. retinoicOn the other acids, hand, NRG4free is fattysuggested acids, and to contribute lactate, also serveto the as promotion BATokines toof contribute sympathetic to the innervation improvement together of metabolism with Bmp8 [26]. (reviewed in [46]). Although the molecular structure is not yet determined, there is a low molecular Lowweight molecular molecule weight that enhances substances, basal such insulin as secretiontriiodothyronine, in the culture retinaldehyde, supernatant retinoic of hBA acids, [27]. free fatty Furtheracids, and investigations lactate, also are required serve as for BATokines the discovery to of contribute additional BATokines.to the improvement of metabolism (reviewed2.3. Adverse in [46]). Effects Although of BAT the molecular structure is not yet determined, there is a low molecular weight molecule that enhances basal insulin secretion in the culture supernatant of hBA [27]. Further Since the rediscovery of human BATs in 2009 [4–7], they have been expected as an excellent investigations are required for the discovery of additional BATokines. therapeutic target for the treatment of obesity. However, BATs may also exert adverse effects under inappropriately activated conditions.

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0 Iris BAT MEF WAT Lens Liver Lung Bone Heart Ovary Testis Retina Uterus Spleen Cornea Eyecup Bladder NK cells NK Prostate Pituitary Placenta Stomach Microglia Pancreas Mast cells Mast Amygdala Epidermis Spinal Spinal cord Cerebellum Osteoclasts T cells T CD4+ cells T CD8+ lymph nodes lymph Bone marrowBone Ciliary bodies Ciliary Mast cells Mast IgE Hippocampus Adrenal gland Adrenal T cells foxP3+ T Intestine large Salivarygland Olfactorybulb Intestine small Lacrimalgland Umbillical Umbillical cord Hypothlalamus Sleletal muscle Sleletal Dorsal striatum Dorsal Follicular B Follicular cells Osteoblast day 5 day Osteoblast B cells GL7- KLH GL7- Bcells Cereberal cortex Cereberal B cells GL7+ KLH GL7+ B cells B cells GL7- Alum GL7- B cells Osteoblast 14 day Osteoblast 21 day B cells GL7+ Alum GL7+ B cells Nucleus Accubens Nucleus Dorsal rootDorsal ganglia Thymotyte SP CD4+ Thymotyte SP CD8+ Thymotyte SP Mammary gland Mammarylact BM0hr B cells marginal zonemarginal B cells Granulocyte mac1+gr1+ Granulocyte Macrophage BM 2hr LPS BM Macrophage2hr LPS BM Macrophage6hr Hematopoietic stemcells Hematopoietic Granulo progenitor mono Thymotyte DP CD4+CD8+Thymotyte DP Macrophage BM 24hr BMLPS 24hr Macrophage Mast cellsMast IgE+antigen 1hr cellsMast IgE+antigen 6hr Retinal pigment Retinal epithelium Cereberal cortex Cereberal prefrontal Mega erythrocyte progenitor Mega Common myeloidprogenitor Common Dendritic cellsDendritic CD8a- myeloid Mammary glandnon-lactating Mammary Macrophage peri LPS thioperi0hrs MacrophageLPS thioperi1hrs MacrophageLPS thioperi7hrs MacrophageLPS Dendritic cells CD8a+ Dendritic lymphoid Dendritic cells plasmacytoid B220+cells Dendritic plasmacytoid (a)

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0 Iris BAT MEF WAT Lens Liver Lung Bone Heart Ovary Testis Retina Uterus Spleen Kidney Cornea Eyecup Bladder NK cellsNK Pituitary Prostate Placenta Stomach Microglia Pancreas Mast cellsMast Amygdala Epidermis Spinal cord Spinal Cerebellum Osteoclasts T cells CD4+ T cells CD8+ T lymph nodeslymph Bone marrowBone Ciliary bodies Ciliary Mast cellsMast IgE Hippocampus Adrenal gland Adrenal cells foxP3+ T Intestine large Salivarygland Olfactory bulb Intestine small Lacrimal gland Umbillical cord Umbillical Hypothlalamus Sleletal muscle Sleletal Dorsal striatum Dorsal Follicular BFollicular cells Osteoblast day 5 Osteoblast day B cells GL7- KLH BGL7- cells Cereberal cortex Cereberal B cells GL7+ KLHGL7+ B cells B cells GL7- Alum GL7- B cells 14 Osteoblast day 21 Osteoblast day B cells GL7+ Alum BGL7+ cells Nucleus Accubens Nucleus Dorsal root Dorsal ganglia Thymotyte CD4+ SP Thymotyte CD8+ SP Macrophage BM 0hr Macrophage Mammary glandMammary lact B cells marginal zone marginal B cells Granulocyte Granulocyte mac1+gr1+ Macrophage BM 2hr BMLPS 2hr Macrophage BMLPS 6hr Macrophage Hematopoietic stem Hematopoietic cells Granulo mono progenitor Thymotyte DP CD4+CD8+ DP Thymotyte Macrophage BMLPS Macrophage 24hr Mast cellsMast IgE+antigen 1hr cellsMast IgE+antigen 6hr Retinal pigmentRetinal epithelium Cereberal cortexCereberal prefrontal Mega erythrocyteprogenitor Mega Common myeloid progenitor Common myeloid Dendritic Dendritic cells myeloid CD8a- Mammary glandMammary non-lactating Macrophage peri LPS thioperi 0hrs MacrophageLPS thioperi 1hrs MacrophageLPS thioperi 7hrs MacrophageLPS DendriticcellsCD8a+ lymphoid Dendritic cells plasmacytoid B220+cells Dendritic plasmacytoid (b)

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0 Iris BAT MEF WAT Lens Liver Lung Bone Heart Ovary Testis Retina Uterus Spleen Kidney Cornea Eyecup Bladder NK cells NK Prostate Pituitary Placenta Stomach Microglia Pancreas Mast cells Mast Amygdala Epidermis Spinal cord Cerebellum Osteoclasts T cells T CD4+ cells T CD8+ lymph nodes lymph Bone marrowBone Mast Mast cells IgE bodiesCiliary Hippocampus Adrenal gland Adrenal T cellsfoxP3+ T Intestine large Salivary gland Olfactorybulb Intestinesmall Lacrimalgland Umbillical Umbillical cord Hypothlalamus Sleletal muscleSleletal Dorsal striatum Dorsal Follicular BFollicular cells Osteoblast day 5 Osteoblastday B cells GL7- KLH BGL7- cells Cereberal Cereberal cortex B cells GL7+ GL7+ KLH B cells B cells GL7- Alum GL7- B cells 14Osteoblastday 21Osteoblastday B cells GL7+ Alum GL7+ B cells Nucleus Accubens Nucleus Dorsal rootDorsal ganglia Thymotyte CD4+ SP Thymotyte CD8+ SP Macrophage BM Macrophage0hr amr glandlact Mammary B cells marginal zone Bmarginal cells Granulocyte mac1+gr1+ Macrophage BM 2hr BMLPS Macrophage2hr BMLPS Macrophage6hr Hematopoietic Hematopoietic stemcells Granulo mono progenitor Thymotyte DP CD4+CD8+Thymotyte DP Macrophage BMLPS Macrophage24hr Mast cells Mast IgE+antigen 1hr cells Mast IgE+antigen 6hr Retinal pigmentRetinal epithelium Cereberal Cereberal cortex prefrontal Mega erythrocyte Mega progenitor Common myeloid myeloid Commonprogenitor Dendritic cellsDendritic CD8a- myeloid Mammary glandMammary non-lactating arpae perithio LPS Macrophage0hrs perithio LPS Macrophage1hrs perithio LPS Macrophage7hrs Dendritic cellsDendritic CD8a+ lymphoid Dendritic cells plasmacytoid B220+ cellsDendritic plasmacytoid (c)

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0 Iris BAT MEF WAT Lens Liver Lung Heart Bone Ovary Testis Retina Uterus Spleen Kidney Cornea Eyecup Bladder NK cells NK Prostate Pituitary Placenta Stomach Microglia Pancreas Epidermis cells Mast Amygdala Spinal Spinal cord Cerebellum Osteoclasts T cells T CD4+ cells T CD8+ lymph nodeslymph Bone marrowBone Ciliary bodiesCiliary Mast cells Mast IgE Hippocampus Adrenal gland Adrenal T cells T foxP3+ Intestine large Salivary gland Olfactorybulb Intestine small Lacrimal gland Umbillical cordUmbillical Hypothlalamus Sleletal muscle Sleletal Dorsal striatum Dorsal Follicular BFollicular cells Osteoblast day 5 day Osteoblast B cells GL7- KLH GL7- B cells Cereberal cortex Cereberal B cells GL7+ KLH GL7+ B cells B cells GL7- Alum GL7- Bcells 14 Osteoblast day 21 Osteoblast day B cells GL7+ Alum GL7+ B cells Nucleus Accubens Nucleus Dorsal Dorsal rootganglia ThymotyteCD4+ SP ThymotyteCD8+ SP Mammary glandMammarylact BM0hr Macrophage B cells marginal zone marginal B cells Granulocyte mac1+gr1+ Granulocyte Macrophage BM LPS Macrophage2hr BM LPS Macrophage6hr Hematopoietic stem Hematopoietic cells Granulo progenitor mono Thymotyte DP CD4+CD8+ThymotyteDP Macrophage BM 24hr LPSBM Macrophage24hr Mast cells Mast 1hr IgE+antigen cells Mast 6hr IgE+antigen Retinal pigment Retinal epithelium Cereberal cortex Cereberal prefrontal Mega erythrocyteprogenitor Mega Common myeloid progenitor myeloid Common Dendritic cellsDendritic myeloid CD8a- Mammary gland Mammarynon-lactating Macrophage perithio0hrs LPSMacrophage perithio1hrs LPSMacrophage perithio7hrs LPSMacrophage Dendritic cells CD8a+ Dendritic lymphoid Dendritic cells plasmacytoid B220+ cellsDendritic plasmacytoid (d)

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0 Iris BAT MEF WAT Lens Lung Liver Bone Heart Ovary Testis Retina Uterus Spleen Kidney Cornea Eyecup Bladder NK cellsNK Prostate Pituitary Placenta Stomach Microglia Pancreas Mast cellsMast Amygdala Epidermis Spinal Spinal cord Cerebellum Osteoclasts T cells T CD4+ cells T CD8+ lymph lymph nodes Bone marrowBone Ciliary bodiesCiliary Mast cellsMast IgE Hippocampus Adrenal gland Adrenal T cells T foxP3+ Intestine large Salivarygland Olfactory bulb Intestine small Lacrimal gland Umbillical cordUmbillical Hypothlalamus Sleletal muscle Sleletal Dorsal Dorsal striatum Follicular B Follicular cells Osteoblast day 5 Osteoblast day B cells GL7- KLH GL7- B cells Cereberal Cereberal cortex B cells GL7+ KLHGL7+ B cells B cells GL7- Alum GL7- B cells 14 Osteoblast day 21 Osteoblast day B cells GL7+ Alum GL7+ B cells Nucleus Accubens Nucleus Dorsal Dorsal root ganglia Thymotyte SP CD4+ ThymotyteSP CD8+ ThymotyteSP BMMacrophage 0hr Mammary glandMammary lact B cells marginal zonemarginal Bcells Granulocyte mac1+gr1+ Macrophage BM 2hr LPSBM Macrophage 2hr LPSBM Macrophage 6hr Hematopoietic Hematopoietic stem cells Granulo progenitor mono Thymotyte DP CD4+CD8+Thymotyte DP Macrophage BMLPS 24hr Macrophage Mast cellsMast IgE+antigen 1hr cellsMast IgE+antigen 6hr Retinal pigmentRetinal epithelium Cereberalcortex prefrontal Mega erythrocyte progenitorMega Common myeloid Common progenitor myeloid Dendritic Dendritic cells CD8a- myeloid Mammary glandMammary non-lactating Macrophage peri LPS thioperi Macrophage0hrs LPS thioperi Macrophage1hrs LPS thioperi Macrophage7hrs LPS Dendritic cells CD8a+ lymphoid Dendritic cells plasmacytoid B220+Dendritic cells plasmacytoid (h)

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0 Iris BAT MEF WAT Lens Liver Lung Bone Heart Ovary Testis Retina Uterus Spleen Kidney Cornea Eyecup Bladder NK cells NK Prostate Pituitary Placenta Stomach Microglia Pancreas Mast cells Mast Amygdala Epidermis Spinal Spinal cord Cerebellum Osteoclasts T cellsCD4+ T cellsCD8+ T lymph nodes lymph Bone marrowBone Ciliary bodies Ciliary Mast cells Mast IgE Hippocampus Adrenal gland Adrenal cellsfoxP3+T Olfactorybulb Intestine large Salivary gland Intestinesmall Lacrimal gland Umbillical cordUmbillical Hypothlalamus Sleletal muscle Sleletal Dorsal striatum Dorsal Follicular Follicular Bcells Osteoblast day 5 Osteoblastday B cells GL7- GL7- KLH B cells Cereberal cortex Cereberal B cells GL7+ KLH GL7+ B cells B cells GL7- Alum GL7- B cells Osteoblastday 14 Osteoblastday 21 B cells GL7+ Alum GL7+ B cells Nucleus Accubens Nucleus Dorsal rootDorsal ganglia Thymotyte SP ThymotyteCD4+ SP ThymotyteCD8+ SP Macrophage BM0hr Macrophage Mammary glandMammary lact B cells marginal zonemarginal B cells Granulocyte mac1+gr1+ Granulocyte Macrophage BM 2hr BMLPS2hr Macrophage BMLPS6hr Macrophage Hematopoietic stemHematopoietic cells Granuloprogenitor mono Thymotyte DP CD4+CD8+Thymotyte DP Macrophage BM 24hr BMLPS24hr Macrophage Mast cells Mast 1hr IgE+antigen cells Mast 6hr IgE+antigen Retinal pigment Retinal epithelium Cereberalcortex prefrontal Mega erythrocyte Mega progenitor Common myeloid Common progenitor myeloid Dendritic cellsDendritic myeloid CD8a- Mammary glandMammarynon-lactating Macrophage peri LPS thioperi0hrs LPSMacrophage thioperi1hrs LPSMacrophage thioperi7hrs LPSMacrophage Dendritic cells Dendritic CD8a+ lymphoid Dendritic cells plasmacytoid B220+ Dendritic cells plasmacytoid (i)

FigureFigure 4. Gene 4. Geneexpression expression profiles profiles of BATokine of BATokine genes genes in in murine murine tissues tissues for Fgf21Fgf21( a(a),),Il6 Il6(b (),b),Gdf15 Gdf15 (c), Bmp8b(c ), (dBmp8b), Angptl8(d), Angptl8 (e), Ngr4(e), Ngr4 (f), Slit2(f), Slit2 (g),( gEpdr1), Epdr1 (h(h) ) and and Pltp( i ().i). The The information information regarding regarding gene gene expressionsexpressions in each in each BATokine BATokine gene gene was was retrieved retrieved by searchingsearching the the data data in BioGPSin BioGPS database database [47] using[47] using a dataset of GeneAtlas MOE430, gcrma [48]. Although this dataset contains not only the gene expression a dataset of GeneAtlas MOE430, gcrma [48]. Although this dataset contains not only the gene data of murine tissues but also those of cell lines, only the former were used in creating graphs. expression data of murine tissues but also those of cell lines, only the former were used in creating graphs.There are several reports that suggest possible adverse effects of BATs. Under arteriosclerosis-prone conditions with lipid metabolism disorders, hyperactivation of BATs may cause disease exacerbation 2.3. Adverseas reported Effects in ofApoe BAT-deficient mice, which suffer from atherosclerotic plaque growth and instability when BATs are activated by acute cold exposure [29]. Involvement of BAT in the development of Sincecancer the cachexia rediscovery was also of reported human (reviewed BATs in in2009 [30– 33[4–7],]). It they is known have that been IL6 expected (reviewed as in [an49– excellent51]) therapeuticand GDF15 target (reviewed for the treatment in [52–54]) of serve obesity. as key However, mediators BATs of cancer may cachexia. also exert Interestingly, adverse effects IL6 and under inappropriatelyGDF15 are theactivated two “mutually conditions. inducing BATokines” that work in an autocrine/paracrine manner [17]. ThereIt seems are possible several that reports cancer-derived that suggest circulating possible IL6 adverse or GDF15 effects may triggerof BATs. the Under activation arteriosclerosis- of BATs. proneIf conditions the concentrations with lipid of plasma metabolism IL6 and GDF15 disorders, exceed hyperactivation the critical value, positive of BATs feedback may cause cycles ofdisease exacerbationinter-BAT as mutual reported activations in Apoe-deficient would be created mice, which to induce suffer cancer from cachexia atherosclerotic as a result ofplaque uncontrolled growth and catabolism. BATs may even be involved in the progression of cancer per se. In a xenotransplantation instability when BATs are activated by acute cold exposure [29]. Involvement of BAT in the model, where human breast cancer cells were transplanted to mice, BA-selective gene inductions development of cancer cachexia was also reported (reviewed in [30–33]). It is known that IL6 (reviewed in [49–51]) and GDF15 (reviewed in [52–54]) serve as key mediators of cancer cachexia. Interestingly, IL6 and GDF15 are the two “mutually inducing BATokines” that work in an autocrine/paracrine manner [17]. It seems possible that cancer-derived circulating IL6 or GDF15 may trigger the activation of BATs. If the concentrations of plasma IL6 and GDF15 exceed the critical value, positive feedback cycles of inter-BAT mutual activations would be created to induce cancer cachexia as a result of uncontrolled catabolism. BATs may even be involved in the progression of cancer per se. In a xenotransplantation model, where human breast cancer cells were transplanted to mice, BA-

Cells 2020, 9, 2449 9 of 15 were observed in both transplanted grafts and host microenvironmental cells [34]. It was shown that BA-like phenotype was acquired in early stages of xenografts [34]. Since depletion of BA-tilted cells, which were positive for UCP1 or MYF5, significantly reduced tumor development [34], the acquisition of BA phenotypes is not a result but a cause of cancer progression. Whether this phenomenon is specific to breast cancers or shared among various cancers is not known. There are some affinities between mammary and BATs. First, BATs are known to emerge in mammary glands during postnatal development [3]. Secondly, there is a resemblance in the gene expression profile between BATs and mammary glands as they are categorized into the same group in BioGPS [47]. Thirdly, both BATs and mammary fat pads, but not other WATs, are independent of cebpa gene in their development. Cebpa-deficient mice, which die immediately after birth, are rescued by cebpa gene expression in the liver with the albumin promoter. These mice show intact BATs and mammary fat pads, while they lack subcutaneous, epidydimal and perirenal WATs [55]. Although an involvement of BATs in the progression of other cancers remains elusive, risk assessment should be carefully performed when BAT-based anti-obesity therapeutic development is considered.

2.4. BAT as a Producer of Extracellular Vesicle (EVs) Not only soluble factors but also EVs serve as mediators of inter-organ communications. The concept of EVs covers a wide variety of microparticles including secretory autophagosomes [56] as well as exosomes, apoptotic bodies, and oncosomes. It is widely accepted that BAT-derived exosomes contribute to metabolism regulation (reviewed in [57,58]). They can even ameliorate metabolic syndromes in mice with high-fat diet [59]. Fat tissues, especially BATs, are recognized as important sources of circulating miRNAs, which are involved in regulating gene expressions of other tissues including the liver [60]. It was also reported that brown adipocyte-derived exosomal miR-132-3p suppressed hepatic Srebf1 expression to attenuate lipogenic gene expression [61]. Since WAT-derived EVs are known to contribute to lipase-independent lipid release [28], BAT-derived EVs may also serve as lipid releasers. Although current information about BAT-derived large-sized EVs is limited, hBA is known to secret various kinds of EVs including exosomes and mitochondrial marker-positive large sized EVs [17]. It was recently reported that cell-free functional mitochondria are detected in circulating blood [62]. Since brown adipocytes contain abundant mitochondria, BAT may possibly contribute to systematic mitochondrial supply by secreting functional mitochondria into circulation.

3. Discussions and Future Perspective Ever since the rediscovery of human BATs, very little light has been shed on adult iBAT. Although 18F-FDG-PET/CT examinations are incapable of visualizing iBAT in adult humans, its existence was shown by anatomical studies. Interestingly, digital infrared thermal imaging (DITI) of healthy individuals taken from the back side often illustrates that the region covering the trapezium muscle including its tendinous parts is one of the spaces with the hottest dermal temperatures [63]. Because tendinous parts do not produce so much heat compared to muscular parts, the trapezius muscle per se may not be the heat source, but rather, certain tissues existing over or beneath the trapezius muscle would be responsible. Since there are no specific tissues over the trapezius muscle, tissues existing just beneath the trapezius muscle might be the heat producer. It was reported that iBAT of adult humans “extends horizontally from the scapular spine to the center of the interscapular region to form a T-shaped mass over and between the rhomboidii muscles and is entirely covered by the trapezius muscle” [38]. Therefore, iBAT may be responsible, at least in part, for the high thermogenesis in the above-mentioned region. Future studies are required for detailed characterization of iBAT in adult humans. To obtain insight into the precise distribution of BATs in human body, new approaches are required in addition to the conventional imaging technique such as 18F-FDG-PET. We previously reported that intravenously administrated carbon nanotubes coated with poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB-CNTs) provided high-quality images of BATs in Cells 2020, 9, 2449 10 of 15

miceCells [ 642020]., 9 We, x FOR found PEER REVIEW that PMB-CNTs adhered specifically to the endothelial cells 10 of in 15 fat tissues. Therefore, they provide clear images of BATs through near infrared photographing. We also reportedphotographing. that CNTs We coated also reported with phospholipid that CNTs coated polyethylene with phospholipid glycol (PLPEG-CNTs) polyethylene glycol provide (PLPEG- images ofCNTs) BATs byprovide a diff imageserent mechanism. of BATs by a different We showed mechanism. that PLPEG-CNTs We showed that specifically PLPEG-CNTs extravasated specifically from BATextravasated from and theBAT extent capillaries of extravasation and the extent was of augmented extravasation by fastingwas augmented [65], suggesting by fasting that [65], CNTs willsuggesting provide an that excellent CNTs will tool provide for imaging an excellent BATs in tool accordance for imaging with BATs their activities.in accordance Regarding with their CNTs, however,activities. there Regarding are concerns CNTs, in however, terms of theresafety are for concerns clinical usage. in terms Development of safety for of clinical safe probes usage. that Development of safe probes that extravasate from BAT capillaries in proportion to its activity may extravasate from BAT capillaries in proportion to its activity may provide a feasible technique to image provide a feasible technique to image the distribution of human BATs. the distribution of human BATs. Although mice and humans share a high degree of topological similarity of BATs [37], there may Although mice and humans share a high degree of topological similarity of BATs [37], there may be be some differences in the mode of the contributions to metabolism regulation. It is known that, in some differences in the mode of the contributions to metabolism regulation. It is known that, in contrast contrast to murine BATs, rat BATs show enhanced lipid accumulations despite upregulated lipid to murineoxidization BATs, and rat thermogenesis BATs show enhanced under cold lipid environments accumulations [66]. despite This is upregulateddue to amplified lipid uptake oxidization of free and thermogenesisfatty acid (FFA), under which cold was environments produced by [WAT66]. This as a result is due of to enhanced amplified lipolysis uptake [66]. of free In addition, fatty acid cold (FFA), whichacclimation was produced did not by enhance WAT as a fat result oxidation of enhanced in rat lipolysis WATs [despite66]. In addition, upregulated cold lipolysis acclimation and did nottriacylglycerol enhance fat oxidation resynthesis in rat[66], WATs which despite indicates upregulated that beige lipolysis adipose andtissues triacylglycerol may not play resynthesis significant [ 66], whichroles indicates in heat production that beige or adipose energy expenditure tissues may under not play cold significant environments. roles Although in heat production it remains elusive or energy expenditurewhether human under cold BATs environments. are analogous Although to rat BATs it remains or murine elusive BATs, whether the human system BATs for metabolism are analogous to ratregulation BATs or generally murine BATs,depends the on system the body for metabolismsize of the species regulation reflecting generally surface/volume depends on ratios. the body Since size of thebody species wights reflecting of rats (~800 surface g) are/volume larger ratios. than those Since of body mice wights (~40 g), of rat rats BATs (~800 would g) are provide larger thanuseful those of miceinformation (~40 g), when rat BATs considering would providedetailed usefulcharacteristics information of human when BATs. considering In this regard, detailed we characteristics observed ofan human interesting BATs. phenomenon In this regard, that we b-adrenergic observed anstimuli interesting enhanced phenomenon lipid accumulations that b-adrenergic (Figure 5a) stimuli in enhancedhuman embryonic lipid accumulations -derived (Figure brown5a) in adipocytes human embryonic (hES-BA) stem despite cell-derived augmented brown mitochondrial adipocytes (hES-BA)respiration despite and thermogenesis augmented mitochondrial [14]. Since the respirationculture medium and thermogenesisof hES-BA contained [14]. abundant Since the FFA culture [14], FFA uptake by hES-BA was satisfactorily upregulated by b-adrenergic stimuli as in the case of medium of hES-BA contained abundant FFA [14], FFA uptake by hES-BA was satisfactorily upregulated rat BATs under cold environments (Figure 5b). by b-adrenergic stimuli as in the case of rat BATs under cold environments (Figure5b).

(a) (b)

FigureFigure 5. 5.BAT BAT activation activation enhances enhances thethe formation of of lipid lipid droplets droplets in in rats rats and and humans. humans. (a) (hES-BAa) hES-BA [14] [14] waswas treated treated with with isoproterenol isoproterenol (100(100 nM)nM) for 4 h and phase phase contrast contrast micrographs micrographs were were taken taken before before andand after after the the treatment. treatment. (b (b) )Schematic Schematic presentation presentation the effects the eff ofects cold of acclimation cold acclimation of rats [64] of ratsand those [64] and thoseof b-adrenergic of b-adrenergic stimuli stimuli of hES-BA. of hES-BA. In both In both cases, cases, lipid lipid accumulation accumulation is augmented is augmented as a asresult a result of of upregulatedupregulated FFA FFA uptake uptake by by activated activated brownbrown adipocytes.

ManyMany of theof the approaches approaches to identify to identify BATokines BATokines have have been beentaken taken on the on assumption the assumption that BATokine that secretionsBATokine are secretions upregulated are upregulated under conditions under conditions where BAT where activities BAT activities are enhanced. are enhanced. However, However, this may this may not always be the case. We observed that the titer of insulin secretion-stimulating molecule not always be the case. We observed that the titer of insulin secretion-stimulating molecule that exists that exists in the supernatant of hES-BA [27] was not influenced by b-adrenergic stimuli, suggesting in the supernatant of hES-BA [27] was not influenced by b-adrenergic stimuli, suggesting that this that this molecule is continuously secreted to regulate basal metabolic rate. Interestingly, this molecule is continuously secreted to regulate basal metabolic rate. Interestingly, this molecule augments molecule augments basal insulin secretion without being affected by glucose concentrations in basal insulin secretion without being affected by glucose concentrations in culture medium [27]. culture medium [27]. BATokines may contribute to metabolism regulation in various modes of actions, some of which are dependent on BAT activity and others are not.

Cells 2020, 9, 2449 11 of 15

BATokines may contribute to metabolism regulation in various modes of actions, some of which are dependent on BAT activity and others are not. BATs may be a double-edged sword, exerting favorable effects under obesity-related conditions but providing unbeneficial outcome under cancer-related conditions. To understand the optimal condition of BATs toward health promotion, tools that precisely reflect the activity and the volume of BATs in human body is required. For example, identification of the specific serum markers that indicate the total BAT volumes and active BATs volumes may be useful. To advance our understanding of BATs and apply the correct information of human BATs to clinical purposes, we have to solve at least the following three questions: (1) What animals (e.g., mice and rats) are the best model to understand the involvement of human BATs in metabolism regulation? (2) What are the genuine BATokines that are professionally involved in inter-tissue communications for metabolism regulation? (3) What molecules serves as serum markers of human BATs that provide the quantitative value for the total BAT volume and that for active BAT volume? There remain a number of mysteries about human BATs, which should be explored toward advanced understanding of BATs to make the best use of them for the improvement of human health.

4. Conclusions Human BAT is basically equivalent to that of mice in terms of body distribution and functions. To obtain more detailed and precise information regarding the distribution of BAT in humans, other imaging techniques than 18FDG-PET/CT are required. Although BAT is believed to exert anti-obesity effects via secreted factors, genuine BATokines that are specifically produced by BAT to regulate the functions of distant organs have not yet been identified. Since there is a possibility that BAT promotes cancer progression, safety tests should be carefully performed when BAT is considered as a therapeutic target against obesity.

Author Contributions: Conceptualization, M.N. and K.S.; graph creation using BioGPS data, K.S.; Writing—original draft preparation, K.S.; and Writing—review and editing, M.N. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: Authors have no conflict of interest to be declared.

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