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Biochimica et Biophysica Acta


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Review

White and brown adipose stem cells: From signaling to clinical implications☆
Carolyn Algire 1, Dasa Medrikova 1, Stephan Herzig ⁎
Joint Research Division Molecular Metabolic Control, German Cancer Research Center (DKFZ), Center for Molecular Biology (ZMBH) University of Heidelberg, Network Aging Research,
University Hospital Heidelberg, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Epidemiological studies estimate that by the year 2030, 2.16 billion people worldwide will be overweight
Received 25 July 2012 and 1.12 billion will be obese [1]. Besides its now established function as an endocrine organ, adipose tissue
Received in revised form 28 September 2012 plays a fundamental role as an energy storage compartment. As such, adipose tissue is capable of extensive
Accepted 2 October 2012
expansion or retraction depending on the energy balance or disease state of the host, a plasticity that is
Available online xxxx
unparalleled in other organs and – under conditions of excessive energy intake – significantly contributes
Keywords:
to the afore mentioned obesity pandemic. Expansion of adipose tissue is driven by both hypertrophy and
Adipose tissue hyperplasia of adipocytes, which can renew frequently to compensate for cell death. This underlines the
Progenitor cell importance of adipocyte progenitor cells within the distinct adipose tissue depots to control both energy
Obesity storage and endocrine functions of adipose tissue. Here we summarize recent findings on the identity and
Signaling plasticity of adipose stem cells, the involved signaling cascades, and potential clinical implications of these
cells for the treatment of metabolic dysfunction in obesity. This article is part of a Special Issue entitled
Brown and White Fat: From Signaling to Disease.
© 2012 Published by Elsevier B.V.

1. Introduction advent of the obesity pandemic in “westernized” countries, much at-


tention has been paid to understanding the signals involved in the
Once considered an inert mass of stored energy, the past two de- differentiation of such “energy burning” brown adipocytes. Adipose
cades have seen a surge in interest in the complexity of adipose tissue tissue is capable of extensive expansion or retraction depending on
and its role in disease. In addition to serving as a site for energy stor- the energy balance or disease state of the host, a plasticity that is un-
age, adipocytes secrete proteins involved in inflammation, appetite paralleled in other organs. Expansion of adipose tissue is driven by
regulation, blood pressure control and energy balance [2–5]. Adipo- both hypertrophy and hyperplasia of adipocytes, which can renew
cytes are unique in their ability to store large quantities of lipids frequently to compensate for cell death, suggesting the necessity of
that can be rapidly released and used for energy by other organs, adipocyte progenitor cells within the adipose tissue depot, that are
when necessary; however, excessive adipose tissue, particularly in capable of differentiating into mature and functional adipocytes.
the visceral adipose tissue depot, is associated with increased risk of
insulin resistance, cardiovascular disease and cancer [6–9]. There ex- 2. Developmental origin of adipose tissue
ists two distinct kinds of adipose tissue: white adipose tissue (WAT),
specifically designed for energy storage, and brown adipose tissue Formation of adipose tissue commences shortly after birth in most
(BAT), which derives from a muscle-like precursor and, when activat- rodents and during mid-gestation in higher mammals, including
ed, burns energy under conditions of non-shivering thermogenesis. humans with both types of adipose (white adipose tissue and brown
Through the expression of uncoupling protein 1 (UCP1) BAT uncou- adipose tissue) originating from the mesoderm. While the formation
ples oxidative phosphorylation from the synthesis of ATP, resulting of white adipose tissue commences shortly after birth, brown adipose
in the generation of heat and serves to protect an organism from hy- tissue develops before birth, as its function is to protect a newborn
pothermia. The role of BAT in human physiology has been overlooked against cold after transition from intrauterine environment [11].
in the past; however, since the discovery of metabolically active BAT The generation of mature adipocytes is comprised of two phases:
in adult humans [10], it resumed a place in the spotlight. Given the determination and differentiation. In this process, multipotent stem
cells become adipoblasts that can further differentiate into pre-
adipocytes, cells already committed to become fat cells. Under appro-
☆ This article is part of a Special Issue entitled Brown and White Fat: From Signaling
priate stimulation, in the ultimate phase of differentiation, pre-
to Disease.
⁎ Corresponding author. Tel.: +49 6221 423594; fax: +49 6221 423595.
adipocytes convert to mature, lipid-laden adipocytes.
E-mail address: s.herzig@dkfz.de (S. Herzig). The first phase, determination, involves the commitment of
1
Equal contribution. multipotent stem cells to the adipocyte lineage, at which point the

1388-1981/$ – see front matter © 2012 Published by Elsevier B.V.


http://dx.doi.org/10.1016/j.bbalip.2012.10.001

Please cite this article as: C. Algire, et al., White and brown adipose stem cells: From signaling to clinical implications, Biochim. Biophys. Acta
(2012), http://dx.doi.org/10.1016/j.bbalip.2012.10.001
2 C. Algire et al. / Biochimica et Biophysica Acta xxx (2012) xxx–xxx

cell is referred to as a pre-adipocyte. During the second phase, termi- PPARγ expressing cells were localized to the adipose vasculature and
nal differentiation, the pre-adipocyte undergoes multiple rounds of express the above mentioned Sca-1 and CD34, in addition to known
mitosis before exiting the cell cycle and differentiating into a mature mural cell markers (smooth-muscle actin (SMA), platelet-derived
adipocyte. In the progression from pre-adipocyte to adipocyte, the growth factor receptor beta (PDGFRβ) and chondroitin sulfate proteo-
cell takes on the characteristics and metabolic capabilities of a mature glycan 4 known as NG2), indicating that adipocyte progenitors reside
adipocyte while under the control of a series of tightly regulated tran- in the mural cell compartment of adipose depot. A close relationship
scriptional and morphological changes. WAT is readily available for between newly arising adipocytes and vasculature was also shown
study from human patient samples and experimental animal models; [23] using reporter genes expressed under the VE-cadherin promoter.
however, it is difficult to maintain in vitro and cannot be expanded. VE-cadherin is indispensable for vasculature formation; however, it
Terminal differentiation is more extensively characterized in immor- was also found to be expressed in pre-adipocytes that are character-
talized cell lines, such as the mouse line 3T3-L1 [12,13], which can un- ized by the pre-adipocyte determination marker Zfp423, a multi zinc
dergo one or two rounds of cell division prior to differentiation and finger transcriptional regulator [24]. Together with morphological,
pre-adipocyte cell lines that differentiate without post-confluence immunohistochemical and gene expression analysis these results sup-
mitosis (e.g. C3H10T1/2). Pre-adipocytes maintain the ability to di- port the notion that pericytes (endothelial cells that surround capil-
vide and have been reported to have a turnover rate of up to 4.5% laries) could serve as a source of adipocyte progenitors.
and 5% per day for humans and mice, respectively. Mature adipocytes Despite the widely accepted model that WAT arises exclusively
are widely believed to have lost the ability to divide following the from Myf5 (myogenic regulatory factor) negative progenitors, it has
completion of terminal differentiation, and adipocyte turnover has recently been shown that a deletion of PTEN (phosphatase and tensin
been reported to be up to 10% per year [14]. homolog) in Myf5 positive cells unexpectedly affected adipogenesis of
WAT. Lineage tracing experiments in mice expressing yellow fluores-
2.1. White adipogenesis cent protein in Myf5 positive cells revealed interscapular and retro-
peritoneal WAT derived from Myf5 positive progenitors [25].
2.1.1. Adipose tissue resident progenitors These white depots did not display the BAT gene expression signature,
Adipose tissue is characterized by striking plasticity, namely which indicates that Myf5 positive progenitors give rise to a subset of
the capability of expansion and retraction, depending on changes in white adipocytes. Studies of progenitor cells in humans are primarily
energy supply and demand, respectively. In adulthood, two means based on FACS analysis and immunohistochemistry. Similar to mice,
of adipose tissue expansion exist, hypertrophy and hyperplasia, human adipose tissue-derived stem cells were shown to express
which are complementary to each other. Hypertrophy is character- stem cell markers Sca-1 and CD34 and were localized in close proxim-
ized by the enlargement of adipocytes, while hyperplasia leads to an ity to the vascular network [26]. Furthermore, pericyte marker CD146
increased number of adipocytes. As mature adipocytes have lost the has been detected on adipose-derived progenitors and the adipogenic
ability to divide, hyperplasia of adipose tissue must be achieved by potential of those cells was confirmed after cell sorting [27]. Under an-
differentiation of precursor cells. Adipocyte progenitors derive from giogenic conditions, human adipose tissue explants have angiogenic
pluripotent mesenchymal stem cells (MSC) (Fig. 1), which are capa- capabilities and can form new blood vessels, ex vivo, lined with cells
ble of differentiating into cells of both mesodermal (myocytes, adipo- containing lipid droplets [23]. Overall, recent reports strongly support
cytes, chrondrocytes and osteoblasts) [15,16] and non-mesodermal the endothelial origin of human adipocytes, and propose a close rela-
[17–19] origin. tionship between adipogenic and angiogenic processes.
Adipocyte progenitors arise from the mesoderm and are localized to
areas along blood capillaries [11], indicating that adipose tissue de- 2.1.2. Adipose tissue non-resident progenitors
velops in coordination with vasculature. MSC can be isolated from the Previously, the proposed source of progenitors for formation of
stromal vascular fraction (SVF) of adipose tissue; however, the prolifer- new adipocytes has been restricted to adipose tissue resident cells;
ative and adipogenic capacity of these cells is significantly affected by however, a reason for re-evaluation of this notion has emerged. It
the location of the depot from which they were isolated and diverse was shown that progenitors from other tissues could also contribute
conditions such as aging and obesity [20]. to the generation of new adipocytes [20,28,29]. Bone marrow
In contrast to hematopoietic stem cells, a clear definition of cells containing a marked number of hematopoietic and mesenchymal pro-
considered to be adipose tissue stem cells is lacking; however, genitors appears to be a promising candidate (Fig. 1). After transplan-
Rodeheffer et al. [21] largely contributed to the characterization of adi- tation of a green fluorescent protein (GFP)-labeled bone marrow-
pocyte precursors by applying fluorescence-activated cell sorting derived hematopoietic cell subpopulation into irradiated wild-type
(FACS) on freshly isolated SVF to separate distinct cell subpopulations mice, GFP-expressing adipocytes appeared in the adipose tissue of re-
based on well-defined stem cell markers. Thus, hematopoietic and en- cipient animals [28]. Selection of cells positive for a murine myeloid
dothelial cells were sorted out from the SVF, leading to the creation of marker, CD11b, confirmed the myeloid origin of GFP-labeled adipo-
a lineage-negative (Lin−) population. From the Lin− population, cells cytes. Furthermore, adipocytes originating from the myeloid lineage
positive for antigens CD34 and CD29 were selected as they demonstrat- were characterized by increased expression of inflammatory and che-
ed high adipogenic potential. Finally, based on staining for stem cell an- motactic genes. These cells accumulated preferentially in visceral adi-
tigen 1 (Sca-1) and CD24, two surface markers known to be expressed pose tissue, which is consistent with the increased inflammatory state
on stem cells in different tissues, the progenitor population was defined of this depot, compared to subcutaneous adipose tissue [20].
as: Lin−/CD29+/CD34+/Sca-1+/CD24+, and its adipogenic potential
was confirmed in vivo by injecting Lin−/CD29+/CD34+/Sca-1+/ 2.2. Brown adipogenesis
CD24+ cells into lipodystrophic or high fat diet fed mice, where this
cell population was capable of forming functional adipose tissue. Although conventional brown and white adipocytes differ consid-
In order to identify adipose tissue progenitors and their localiza- erably in function and morphology, they have long been considered to
tion within fat tissue, Tang et al. [22] used lineage tracing based on share a common progenitor. Both adipose tissues originate from MSC;
the labeling of cells that express peroxisome proliferator-activated re- however, brown adipocytes, unlike white adipocytes, are derived
ceptor gamma (PPARγ), the master regulator of adipogenesis. Labeled from the myogenic lineage, characterized by Myf5 expression
cells were found in the SVF and exhibited considerable proliferative (Fig. 1). After deletion of PR domain containing 16 (PRDM16), the
capacity and adipogenic potential, both in culture and after transplan- transcriptional regulator of brown adipocyte determination, in
tation to nude mice. Immunohistochemical examination revealed that brown adipocyte precursors, the resulting cells exhibited a skeletal

Please cite this article as: C. Algire, et al., White and brown adipose stem cells: From signaling to clinical implications, Biochim. Biophys. Acta
(2012), http://dx.doi.org/10.1016/j.bbalip.2012.10.001
C. Algire et al. / Biochimica et Biophysica Acta xxx (2012) xxx–xxx 3

Mesenchymal
Stem Cell

Progenitor Progenitor
Cell Cell

Inducible White Brown Myocyte


Brown Adipocyte Adipocyte
Adipocyte

Hematopoetic
Cell

Fig. 1. Origin of adipocytes. Adipocytes (white, inducible brown and brown) are mainly derived from mesenchymal stem cells of mesodermal origin. White adipocytes are derived
from progenitors of adipogenic lineage or hematopoietic progenitors. Inducible brown adipocytes differentiate directly from adipocyte progenitors or may be produced through
transdifferentiation from white adipocytes. Whether or not these cells can be derived from hematopoietic progenitors is not known. Finally, brown adipocytes originate from pro-
genitors of myogenic lineage.

muscle phenotype, as opposed to the expected white adipocyte brown adipocytes can be recruited in WAT. These de novo recruited
phenotype [30]. In addition, lineage tracing experiments in mice ex- brown adipocytes (“brown-in-white” BRITE or “Beige” cells) are charac-
pressing fluorescently tagged Myf5 revealed that brown adipocyte terized by high mitochondrial content, brownish color and are consis-
progenitors originate from the myogenic lineage, and thus are more tent with conventional brown adipocytes in that they express UCP1
closely related to skeletal muscle than to WAT [30]. miRNA cluster and contribute to energy dissipation. Although morphologically similar,
MiR193b-365 also plays a critical regulatory role in the process of brown adipocytes and inducible brown adipocytes do not share a com-
brown fat/muscle lineage determination as it blocks myogenesis and mon precursor cell [30].
promotes expression of adipogenic genes via interaction with Two possibilities for how inducible brown adipocytes could be
PRDM16 [31]. Given that BAT is highly vascularized, it is not surprising recruited in WAT have been proposed (Fig. 1). The first is referred to
that similar to white adipocytes, brown adipocytes might also be de- as transdifferentiation of already mature white adipocytes into brown
rived from endothelial cells [23]; however, in addition to expressing adipocytes [35]. Recently it has been demonstrated, that the cellular
VE-cadherin, brown pre-adipocytes must be Myf5-positive. composition of white adipose tissue depots is altered following cold
Based on cellular morphology, human brown adipocyte precursor stimulation. The increase in brown adipocytes was accompanied by a
cells were identified to be in close association with vasculature [32], proportional decrease in white adipocytes, without changes in total
which further supports the data obtained from animal studies. Never- adipocyte number. Furthermore, no signs of apoptosis, mitosis or degra-
theless, the characterization and localization of brown adipocyte pro- dation of adipocytes were present [36]. In another report, after
genitors in the human body still need to be elucidated. To date, co-infusion of mice with bromodeoxyuridine (BrDU) and β-adrenergic
human adipose tissue-derived stem cells were shown to differentiate agonist CL316.243, the proportion of BrDU labeled UCP1 positive cells,
to brown adipocytes only after stimulation of PPARγ with its potent indicating recruitment of BRITE cells from progenitors, was significantly
activator rosiglitazone [33] or after lentiviral transduction of mesenchy- higher in epididymal WAT than in inguinal, while UCP1 induction was
mal progenitor cells with transcription factors essential for brown fat higher in inguinal WAT [37]. This observation further supports the
development (PPARγ2, Cebpb, Prdm16) [34]; however, there is little in- occurrence of conversion of mature adipocytes to the BRITE cells. The
formation about the clinical relevance of these observations. second possibility involves differentiation from existing progenitor
cells. Recently it was reported that progenitors of inducible BAT express
2.2.1. Inducible brown fat specific markers, namely CD137 and transmembrane protein 26
Following stimulation of β3-adrenergic receptors, via cold exposure (TMEM26), and displayed increased UCP1 expression compared to
or pharmacological treatment, fat cells morphologically resembling cells negative for those markers [38]. In addition, it was reported that

Please cite this article as: C. Algire, et al., White and brown adipose stem cells: From signaling to clinical implications, Biochim. Biophys. Acta
(2012), http://dx.doi.org/10.1016/j.bbalip.2012.10.001
4 C. Algire et al. / Biochimica et Biophysica Acta xxx (2012) xxx–xxx

the gene expression pattern of human BAT closely resembles inducible BMPs signal through two cell surface receptors, BMPr1 and BMPr2,
BAT, more than conventional BAT, and these findings were consistent in both with serine/threonine kinase activities. Activation of these re-
both mature adipocytes and progenitors [38]. Progenitors of inducible ceptors leads to phosphorylation of BMPr1 kinase which then
BAT were indentified after BrDU was injected into mice treated with activates Smad-1, -5, and -8, leading to the formation of a complex
β3-adrenergic receptor agonist and newly differentiated UCP1 positive with Smad4 that translocates to the nucleus where it regulates
cells were detected in white fat. These results were confirmed by line- gene expression. Expression of a constitutively active BMP receptor
age tracing studies using constitutive and inducible reporter systems, (CA-BMPr1A and CA-BMPr1B) induced commitment to the pre-
and inducible brown adipocyte progenitors were further defined as adipocyte lineage in the absence of BMP-2 and BMP-4 [43]. BMP sig-
Sca-1, CD34 and PDGFRα positive [37], in contrast to white progenitors, naling alters the expression of three cytoskeleton proteins, Lox, Tpt1
that express PDGFRβ and endothelial markers [22]. Interestingly, and αB-crystallin that play important roles in adipocyte lineage
PDGFRα-positive progenitors developed into brown adipocytes under commitment [46]. Commitment to the adipocyte lineage can be erad-
β3-adrenergic stimulation, whereas, in vivo, under conditions of icated by the knockdown of Lox1; however, it is only partially lost fol-
high-fat diet feeding, the cells differentiated into white adipocytes, lowing the loss of Tpt1 and αB-crystallin. Given that pre-adipocytes
which demonstrated the bidirectional potential of differentiation differ dramatically in their morphology from MSC, BMPs may pro-
depending on the external stimuli [37]. mote commitment to pre-adipocytes through the regulation of the
cytoskeleton and consequent effects on cellular morphology.
2.3. Microenvironment

Adipogenic potential of progenitor cells does not depend merely 3.1.2. Wnt signaling
on surface markers expressed but also on the microenvironment in The Wnt family of signaling proteins exerts its effects through the
which they reside. Many studies have demonstrated that adipose frizzled receptor and the low-density lipoprotein-related protein 5/6
tissue-derived stem cells are capable of differentiation into various co-receptor, and can signal through both a “canonical” [47] and a
tissues depending on the differentiation signals provided [39,40]. “non-canonical” pathway [48]. Wnts play a role in both the lineage
For example, selected progenitors were able to reconstitute adipose commitment and differentiation phase of adipogenesis, although
tissue only in lipodystrophic and high-fat diet fed mice, but not in with opposing effects [49,50]. The canonical signaling pathway func-
wild-type chow-fed mice [21] and the fate of bipotential adipocyte tions early during lineage commitment and involves the binding of
progenitors was highly dependent on external nutritional or Wnt to its receptors which promotes the dissociation of the “destruc-
β3-adrenergic stimuli [37] which further supports the importance of tion complex”, comprised of adenomatous polyposis coli, axin and
the microenvironment. It is also probable that not only one subpopu- glycogen synthase kinase (GSK)-3β, thereby permitting the accumu-
lation of adipose progenitors exists and contributes to the renewal lation of β-catenin, that was previously embedded in the complex
and hyperplastic growth of adipose tissue. More likely, adipose pro- [47]. In the nucleus, β-catenin targets transcription factors that are
genitors from different sources could contribute to a pool of stem differentially expressed in MSC (C3H10T1/2) vs. pre-adipocytes
cells in a depot specific manner [20]. Indeed, whereas most inducible (A33, derived from C3H10T1/2), such as R-spondins-2 and -3 [44],
brown adipocytes in abdominal WAT seem to arise from the induc- which are dramatically upregulated in proliferating A33 cells and
tion of adipogenesis from Sca-1, CD34 and PDGFRα positive progeni- are known to activate canonical Wnt signaling, thus implicating
tors, enhanced UCP1 expression in inguinal WAT may involve Wnt signaling in the commitment phase of MSC to the adipocyte lin-
transdifferentiation of existing white adipocytes into a brown adipo- eage. Later in the adipogenic program, Wnt inhibits adipocyte differ-
cyte phenotype [37]. entiation by inhibiting the expression of important adipogenic
transcription factors, PPARγ and CCAAT/enhancer binding protein
3. Signaling pathways in adipogenesis (C/EBP)α, and by doing so, maintains a balance among the myogenic,
osteoblastogenic and adipogenic cell fates [49,51] (Fig. 2).
The complete list of signals involved in the commitment phase of
adipogenesis remains to be described; however, several factors have
been identified that can either promote or inhibit the commitment 3.2. Differentiation of adipocytes from pre-adipocytes
of multipotent MSC to the pre-adipocyte lineage, most notably bone
morphogenic protein (BMP-2 and BMP-4) and Wnt signaling pro- Cells isolated from the SVF of adipose tissue depots can be differen-
teins. Accumulated evidence suggests that external growth factors tiated in culture when stimulated by specified contents of a “differenti-
and signaling molecules converge on the promoters of lineage specific ation cocktail”. In culture, pre-adipocytes will undergo multiple rounds
transcription factors. These external signals tightly regulate the of mitosis until they reach growth arrest, the G1 phase of the cell cycle.
transcriptional program to promote one cell type, while inhibiting an- At this point, differentiation can be induced. Induction initiates a series
other. For example, PPARγ, stimulates adipogenesis while inhibiting of events that requires pre-adipocytes to re-enter the cell cycle, undergo
chondrogenesis and the formation of cartilage, whereas inhibition mitotic clonal expansion until they eventually exit the cell cycle, change
of PPARγ can promote osteogenesis and inhibition adipogenesis their morphology, accumulate cytoplasmic triglycerides and acquire the
[41,42] (Fig. 2). metabolic features and capabilities of mature adipocytes. The differenti-
ation induction cocktail is composed of dexamethasone (a glucocorti-
3.1. Classical pro-adipogenic signals coid), insulin, 3-isobutyl-1-methylxanthine (IBMX) and fetal bovine
serum (FBS). The order in which the cells are exposed to these stimuli
3.1.1. Bone morphogenic protein is crucial for the differentiation process as it was previously shown
Both BMP-2 and BMP-4 have been shown to play a role in the com- that dexamethasone can be added to the differentiation cocktail
mitment of multipotent MSC to the adipocyte lineage. Exposure of the prior to IBMX, but the reverse will impede the differentiation of
dividing MSC cell line C3H10T1/2 to either BMP-4 or BMP-2 will result pre-adipocytes to mature adipocytes [52]. Dexamethasone treatment
in pre-adipocyte-like cells that can be differentiated into adipocytes is important in the initial phases of the induction of differentiation as
when treated with differentiation inducers following growth arrest it activates the transcription factor C/EBPβ. IBMX is a phosphodiesterase
[43,44]. In addition to BMP-2 and BMP-4, BMP-7 has been shown to inhibitor that raises cyclic AMP (cAMP) levels in the cell, leading to the
play a pivotal role in the differentiation of brown adipocytes even in activation of the related transcription factor C/EBPδ. C/EBPβ and δ in
the absence of the required hormonal cocktail [45]. turn induce transcription of C/EBPα and PPARγ [5,53].

Please cite this article as: C. Algire, et al., White and brown adipose stem cells: From signaling to clinical implications, Biochim. Biophys. Acta
(2012), http://dx.doi.org/10.1016/j.bbalip.2012.10.001
C. Algire et al. / Biochimica et Biophysica Acta xxx (2012) xxx–xxx 5

Adipocyte

Preadipocyte

Mesenchymal
Stem Cell

Fig. 2. Signaling pathways in adipogenesis. The proposed pathway from mesenchymal stem cell to mature adipocyte. Following signals from the Wnt and BMP family of proteins,
the MSCs enter the pre-adipocyte lineage (Commitment). Later, differentiation signals promote the transition from pre-adipocyte to mature adipocyte (Differentiation).

3.3. Transcriptional mediators of adipogenesis response element-binding protein-1c (SREBP-1c) and C/EBPβ can in-
crease PPARγ ligand production although these findings did not lead
3.3.1. C/EBPs to the identification of PPARγ agonists [60,61]. Once expressed PPARγ
C/EBP family members are vital for the differentiation of adipo- and C/EBPα positively feedback on each other through their respective
cytes whereby early induction of C/EBPβ and C/EBPδ leads to the C/EBP regulatory elements, this action is presumed to perpetuate the
induction of C/EBPα and PPARγ. Almost immediately after the induc- adipocyte phenotype in mature adipocytes. In addition to its role in
tion of differentiation, cAMP response element binding protein differentiation, PPARγ is crucial for the maintenance of mature adipo-
(CREB) becomes phosphorylated and activates the expression of cytes. For example, expression of dominant negative PPARγ in differen-
C/EBPβ [54]. Approximately 14–16 h after induction, C/EBPβ acquires tiated 3T3-L1 adipocytes leads to de-differentiation and loss of lipid
DNA binding capabilities as the pre-adipocytes re-enter the cell cycle. accumulation [62].
C/EBPβ likely plays a role in mitotic clonal expansion, which is
required for differentiation. The function of C/EBPβ and C/EBPδ 3.4. Differentiation of progenitor cells into brown adipocytes
may be redundant as knockout of C/EBPβ in mice had little effect on
adipose tissue accumulation, whereas the double C/EBPβ−/− C/ Inducible brown adipocytes can arise from multipotent progeni-
EBPδ −/− knockout showed markedly reduced adipose tissue mass, tors, or perhaps even from transdifferentiation of existing white adi-
mostly due to decreased cell number [55]. Early in the differentiation pocytes, in WAT depots, when exposed to certain stimuli [30,63,64].
process, C/EBPβ is phosphorylated twice, by mitogen-activated pro- Brown pre-adipocyte recruitment is controlled by the TGF-β family
tein kinase (MAPK) and GSK3β [56], which induces a conformational of secreted proteins, such as BMP-7 [45], while Wnt signaling re-
change resulting in the dimerization of two monomers of C/EBPβ and presses the differentiation of brown pre-adipocytes into brown adi-
creates a DNA-binding pocket. Once C/EBPβ is capable of binding pocytes. The presence of inducible brown adipocytes can lead to
DNA, 14–16 h post differentiation induction, it leads to the weight loss, improved insulin sensitivity and resistance to high fat
upregulation of C/EBPα and PPARγ which act together as pleiotropic feeding in animal models [65].
transcriptional activators of the large group of genes that produce While the transcription factors encoded by C/EBP and PRDM16
the adipocyte phenotype [5,51]. have been shown to function as key regulators of BAT differentiation,
defining the signaling pathways involved in the de novo recruitment
3.3.2. PPARγ of BAT remains an active area of research.
PPARγ is the master regulator of adipogenesis, as it is both sufficient
and necessary for adipogenesis [5,53]. It exists as three isoforms 3.4.1. Beta adrenergic signaling
(PPARγ1, PPARγ2 and PPARγ3) that are differentially spliced from the The sympathetic nervous system is the major regulator of BAT
same transcribed gene [57,58]. In order to exert effects on peroxisome activity through β-adrenergic signaling [66]. In existing BAT depots,
proliferator response elements, PPARs must form heterodimers with β1-adrenergic signaling promotes proliferation of BAT pre-adipocytes
the retinoid acid receptor (RXR). In primary adipocytes, PPARγ2 is the whereas in animal models, β3-adrenergic receptor activation can pro-
predominant isoform and is more efficient at promoting adipogenesis mote the recruitment of inducible brown adipocytes in WAT depots
than the other isoforms [5]. Endogenous PPARγ ligands include unsatu- and is required for activation of the thermogenic program [67,68]. In
rated and oxidized fatty acids, eicosanoids and prostaglandins. These humans, β3-receptor agonists have not been successful in reducing ad-
agonist ligands promote transcription through the recruitment of iposity, which could result from fewer β3-receptors or lack of inducible
co-activating proteins which then recruit the transcription apparatus brown fat [68]. β3-receptor agonists lead to an increase in intracellular
that induce an open and accessible DNA [59]. Efforts to describe addi- cAMP which activates the lipolytic pathway whereby triglycerides are
tional endogenous PPARγ ligands have been largely unsuccessful; hydrolyzed to free fatty acids (FA) and glycerol. The activation of this
however, studies have found that the transcription factors sterol pathway is critical for BAT activity as it was recently shown that loss

Please cite this article as: C. Algire, et al., White and brown adipose stem cells: From signaling to clinical implications, Biochim. Biophys. Acta
(2012), http://dx.doi.org/10.1016/j.bbalip.2012.10.001
6 C. Algire et al. / Biochimica et Biophysica Acta xxx (2012) xxx–xxx

of desnutrin/ATGL, the first hydrolase in the triglyceride hydrolysis insulin resistance, hypertension, dyslipidemia, and hyperglycemia
pathway, results in the conversion of BAT to a WAT-like tissue [69]. as hallmarks of the so-called metabolic syndrome, eventually culmi-
nating into end-stage diseases such as type 2 diabetes, atherosclero-
3.4.2. COX-2 sis, cardio-vascular failure, and even cancer [78].
Cyclooxigenase-2 (COX-2) is a rate limiting enzyme in the synthesis In life-style intervention programs, there is often an impressive
of prostaglandins, and is downstream of the β-adrenergic signaling short-term success in weight reduction, but this success is often
pathway. COX-2 overexpression and the synthesis of prostaglandins blunted by weight regain after the program's end, which is in the
can shift the differentiation of adipocyte progenitors, isolated from the most cases due to an insufficient patient compliance [79,80]. In par-
epididymal depot, towards a brown adipocyte phenotype in vitro. ticular, existing pharmacotherapy for the management of obesity,
In vivo, COX-2 overexpressing mice and mice injected with a which is primarily aimed at weight loss, weight loss maintenance
β3-adrenergic receptor agonist displayed de novo recruitment of and risk reduction, has only a modest efficacy reflected by a weight
brown adipocytes in the epididymal WAT. The appearance of inducible loss of 5 to 10% of the initial body weight, followed by a weight loss
BAT was associated with increased energy expenditure and resistance plateau under further therapy [81].
to high fat diet induced weight gain [65]. In turn, inhibition of COX-2 Overall, pharmacological strategies have proven to be remarkably
activity impaired cold-induced expression of UCP1 in WAT [65,70]. ineffective against the increasing problem of obesity. Besides the need
In addition to the signals described above, recent advances in the for weight reduction, in recent years there is an increasing awareness
field of brown adipocyte biology have suggested that other factors for obesity related diseases like dyslipidemia, high triglyceride–low
can recruit inducible brown adipocytes. Fibroblast growth factor 21 HDL-syndromes, non-alcoholic fatty liver disease and/or insulin resis-
(FGF-21) is primarily secreted by the liver where it affects glucose ho- tance. However, similar to the therapeutic alternatives for weight re-
meostasis; however, it was shown by Hondares et al. [71] that brown duction, treatments for those conditions are also underdeveloped and
fat can become a source of FGF-21 following activation of the thermo- need urgent improvement [82,83].
genic program. FGF-21 has proven vital for thermogenesis as
FGF-21−/− mice display an inability to adapt to cold. Interestingly, 4.2. Adipose stem cells in anti-obesity therapies
FGF-21 was shown to enhance PGC-1 protein levels, independent of
changes in mRNA, and positively affects the recruitment of adipocytes The described studies and acquired knowledge on adipocyte pro-
with thermogenic capabilities within WAT depots [72]. Böstrom et al. genitors, signaling pathways affecting their cellular and functional
[73] recently reported that mice overexpressing PPARγ co-activator fate, and distinct properties of white vs. brown adipocytes open the
1α (PGC-1α) in skeletal muscle have an increased expression of fi- intriguing possibility that even the regeneration and activation of
bronectin type III domain containing 5 (FNDC5), a membrane protein small amounts of BAT in adult humans/patients would have a signif-
that is cleaved intracellularly and secreted as the peptide hormone icant impact on systemic energy expenditure, thereby representing
irisin. This study reported that irisin acted on mature white adipo- an unprecedented approach to the regulation of body weight and as-
cytes to increase expression of UCP1 and a broad program of brown sociated disorders, while simultaneously counteracting WAT tissue
adipocyte gene expression patterns. In vitro, it has been reported injury as imposed by energy overload and tissue-specific inflamma-
that treatment of murine white pre-adipocytes, isolated from the ep- tion of WAT stores in the obese state.
ididymal depot, with high concentrations of the PPARγ activator Indeed, in humans, it is estimated that even as little as 50 g of
rosiglitazone promoted mitochondriogenesis, expression PGC-1α stimulated and active BAT could account for a 20% daily increase in
and UCP1, an effect that could be augmented still following treatment energy expenditure [84], and that up to 24% of the increase in metab-
with norepinephrine [64]. These cells maintained genuine thermo- olism in lean men produced by ephedrine can be attributed to BAT
genic capacity; however, they did not express the transcription fac- activation [85]. Concomitant to the increase in energy expenditure,
tors known to be associated with classic brown adipocytes, such regeneration and/or activation of BAT can be envisaged to also bene-
PRDM16, LIM homeobox protein 8 (Lhx8) and mesenchyme homeo- ficially influence other clinical parameters associated with obesity,
box 2 (Meox2) [74]. Therefore, these cells may represent an addition- type 2 diabetes and the metabolic syndrome.
al subset of adipocytes that maintain the transcriptional signature of While brown/BRITE adipocytes do not seem to display increased cel-
white adipocytes while possessing the potential to express UCP1 lular insulin sensitivity per se (K. Mössenböck and S. Herzig,
and the brown adipocyte phenotype. unpublished observations), both basal and insulin-stimulated glucose
uptake are substantially elevated as compared with white adipocytes.
4. Therapeutic implications Thus any WAT to BAT transformation or BAT recruitment will increase
the systemic glucose clearance and potentially also improve glucose
4.1. The obesity pandemic tolerance and insulin sensitivity. Indeed, a number of clinical studies
have validated substantial cold-induced glucose uptake in human
Globally, the increase in the prevalence of overweight/obese people subjects as measured by PET studies using 18-fluordeoxyglucose.
has reached a qualified epidemic stage with currently more than one Depending on the experimental setting, cold-stimulated glucose clear-
billion overweight and at least 400 million clinically obese. Type 2 dia- ance through BAT was enhanced between 4 and 15-fold, negatively
betes mellitus and the associated beta-cell failure is up to 90% attribut- correlating with BMI [86,87].
able to weight gain and has increased in parallel with the pandemic of Furthermore, insulin resistance is correlated to WAT inflammation,
obesity. Approximately 197 million people worldwide have impaired and studies on the differential metabolic impact of distinct WAT de-
glucose tolerance, most commonly because of obesity and the associat- pots and their capacities to recruit BAT suggest that WAT to BAT trans-
ed metabolic syndrome, and this number is expected to increase to formation could reduce inflammation and improve insulin resistance
420 million by 2025 [75,76]. [88]. In addition, prolonged treatment of a patient with levothyroxine
Basic risk factors for obesity comprise excessive caloric intake, a (suppressive treatment for thyroid cancer) resulted in dramatic im-
sedentary life-style, as well as genetic predisposition, all of which provement in insulin sensitivity, paralleled by enhanced BAT volume
precipitate in aberrant systemic energy/fat storage in the form of and activity—in spite of extreme insulin resistance due to a mutation
WAT [77]. In this setting, normal WAT function is severely damaged in the insulin receptor gene. Consequently, BAT activation could
as it is compromised by tissue inflammation and aberrant metabolic potentially mediate non-insulin-dependent glucose disposal [89].
properties. Excessive WAT depots can subsequently be made directly Indeed, the thyroid hormone receptor (TR) beta was found to be
responsible for the manifestation of systemic and tissue-specific required for UCP1-dependent adaptive thermogenesis in mice [90],

Please cite this article as: C. Algire, et al., White and brown adipose stem cells: From signaling to clinical implications, Biochim. Biophys. Acta
(2012), http://dx.doi.org/10.1016/j.bbalip.2012.10.001
C. Algire et al. / Biochimica et Biophysica Acta xxx (2012) xxx–xxx 7

“Metabolocentric” Stem Cell Therapy

BAT
ACTIVATION/
RECRUITMENT
=> TZDs
=> β adrenergic
stimulation * BODY WEIGHT
REGULATION
ADIPOCYTE
* GLUCOSE
STEM CELLS TOLERANCE

TISSUE
GENERATION
=> Transplantation
ex-vivo cell ENERGY
cultures HOMEOSTASIS
(CEBP/PRMD16)

Fig. 3. Metabolocentric therapy. Adipocyte stem cells offer the future possibility to control energy homeostasis through either the pharmacological activation/recruitment of brown
adipose tissue (BAT) in vivo or through the ex vivo generation of BAT via gene transfer of critical transcriptional regulators into progenitor cells. TZDs, thiazolidinediones.

potentially mediated largely via central rather than peripheral TR BRITE cells more than murine BAT [38], thereby adding an additional
effects [91]. In addition, fibroblast growth factor (FGF) 21 is produced layer of complexity to translational research in this direction.
by BAT and has been shown to promote beneficial effects on glucose Despite these current obstacles, the description of adipocyte white/
metabolism [92]. Finally, BAT activity controls triglyceride and/or BRITE/brown progenitor isolation protocols for defined cell populations
FA clearance, thereby counteracting hypertriglyceridemia. In patho- in mice provides a promising starting point for clinical-therapeutic re-
physiological settings, (mice fed a high fat diet), cold exposure search in humans in this direction, which may lead to the identification
corrected hyperlipidemia and improved deleterious effects of insulin of cellular pools that could generate/maintain proliferatively competent
resistance, which could be attributed to enhanced BAT activation brown/BRITE adipocytes in humans during the aging process, thereby
[93]. In addition, tracer studies in humans also underlined the poten- maintaining “young” levels of energy expenditure and obesity resis-
tial of BAT to efficiently take up FA and use these substrates in oxida- tance throughout the life-time of an individual. Further clinical research
tive metabolism [94,95]. in this direction is clearly validated in the future (Fig. 3).

4.3. Outlook and challenges Acknowledgements

While the described studies have on the one side demonstrated We apologize to our colleagues whose contributions could not be
the existence and feasibility of human adipocyte progenitors and on cited due to space limitations. We thank members of our lab for crit-
the other side provided clinical evidence for the functionality and ically reading the manuscript and discussions. In particular, we thank
metabolo-regulatory impact of human BAT, actual therapeutic use of Tatiana Golea for administrative and graphical support. Our work is
this resource is currently still in its infancy. Indeed, clinical studies supported by grants from the Deutsche Forschungsgemeinschaft
using beta adrenergic stimuli to activate BAT thermogenesis and and the EU FP7 project DIABAT (HEALTH-F2-2011-278373).
counteract obesity showed limited efficacy and had to be abandoned
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Please cite this article as: C. Algire, et al., White and brown adipose stem cells: From signaling to clinical implications, Biochim. Biophys. Acta
(2012), http://dx.doi.org/10.1016/j.bbalip.2012.10.001