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Caffeine inhibits adipogenic

Caffeine inhibits adipogenic differentiation of primary adipose-derived stem cells and bone marrow stromal

cells

Shu-Hui Su a ,Huey-Wen Shyu b ,Yao-Tsung Yeh b ,Kuan-Ming Chen c ,Hua Yeh b ,Shu-Jem Su b ,?

Institute of Medical Sciences,College of Medicine,Tzu Chi University,Hualien,Taiwan

Department of Medical Laboratory Science and Biotechnology,School of Medicine and Health Sciences,Fooyin University,No.151,Chinhsueh Rd.,Ta-liao,Kaohsiung 83101,Taiwan c

Department of Biochemistry,College of Medicine,Kaohsiung Medical University,Kaohsiung 80708,Taiwan

a r t i c l e i n f o Article history:

Received 14January 2013Accepted 16May 2013

Available online 30May 2013Keywords:Caffeine

Adipose-derived stem cell Bone marrow stromal cell Adipogenesis

a b s t r a c t

Caffeine consumption has been related to loss of body weight and modulates lipid metabolism.However,impacts of caffeine on adipogenic differentiation have not been well determined yet.The present study evaluated the effects of caffeine on adipogenesis using primary rat adipose-derived stem cells (ADSCs)and a mouse bone marrow stromal cell line (M2-10B4)in vitro .ADSCs and M2-10B4were continuously exposed to caffeine (0.1–1mM)during adipogenic differentiation for 7and 12days,respectively.Oil red O and Nile red staining showed that caffeine reduced lipid droplet and adipocyte levels in both cell types.In addition,Nile red staining and FACScan ?ow cytometry showed that caffeine dose-dependently decreased adipocyte differentiation from 20%to 50%of the control ADSCs and M2-10B4cells.Caffeine decreased the expression of adipogenesis-related genes including peroxisome proliferator-activated receptor-c ,CCAAT/enhancer-binding protein-a ,adipocyte lipid binding protein,lipoprotein lipase,leptin,and TNF a in a dose-dependent manner.Rather,low concentration of caffeine (0.1mM)signi?cantly increased IL-6expression,but unexpectedly inhibited that at a concentration more than 0.3mM.Taken together,caffeine was able to effectively inhibit adipogenic differentiation of ADSCs and M2-10B4cells partly through its inhibition of adipogenesis-related factors.

1.Introduction

Caffeine (1,3,7-trimethylxanthine)is a plant alkaloid found in coffee,tea,chocolate,cola,and soft drinks.It is commonly con-sumed around the world,especially in Europe and North America,as a part of the normal diet (Putnam et al.,2007).Interestingly,caf-feine has been proposed for weight loss and maintenance for a long time,and is a non-selective antagonist for adenosine receptors (Jacobson,2009).Several studies have shown that caffeine reduces body weight and adipose tissue weight in animal models (Hursel and Westerterp-Plantenga,2010;Yun et al.,2008).Caffeine increases energy expenditure by inhibiting phosphodiesterase-induced degradation of intracellular cyclic adenosine monophos-phate (Dulloo,1993)and decreases energy intake by reducing food intake (Racotta et al.,1994).On the other hand,caffeine is also the most popularly consumed drug,which was concocted to amphet-amine-type stimulants (ATSs)tablets such as Ya–Ba to increase their weight and volume and to enhance the stimulating effect of methamphetamine (Puthaviriyakorn et al.,2002).Additional caf-feine consumed in the form of caffeinated beverages is usually ta-

ken with ATS tablets to reduce fatigue and drowsiness.Nevertheless,acute caffeine intake may also increase heart rate and alter blood pressure (Karatzis et al.,2005).It is importantly noted that effects of caffeine depend on dose;at low doses,caffeine shows antioxidant effects (Lee,2000),while at high doses,it may increase neurotoxicity in striatum via the release of dopamine from striatal nerve endings,leading to the subsequent generation of reactive oxygen species (Golembiowska et al.,2009).Accord-ingly,caffeine has been reported to have genotoxic and mutagenic potentials (Fernandez et al.,2003;Grice,1987;Nehlig and Debry,1994;Ritter et al.,2005).

Prevalence of obesity and associated metabolic disorders has been major public health concerns.Obesity results from excessive energy intake and low energy expenditure,and is a risk factor for lifestyle-related diseases such as hypertension,atherosclerosis,diabetes,osteoarthritis,and certain cancers (Poulos et al.,2010).Development of adipose tissue involves formation of new adipo-cytes from undifferentiated preadipocytes,resulting in an increase of adipocyte size.Because bone marrow adipocytes inhibit osteo-blast proliferation (Jilka,2002)and disrupt normal blood supply to the bone tissue (Laroche,2002),treatments that inhibit marrow adipogenesis and decrease bone marrow adipocyte populations will have positive effects on bone health and prevention of adiposity.

Corresponding author.Tel.:+88677811151x5412.

E-mail address:sc096@https://www.wendangku.net/doc/9b7585428.html,.tw (S.-J.Su).

Bone marrow mesenchymal stem cells(BMSCs)or bone marrow mesenchymal stromal cells can differentiate into multiple cell lin-eages,including osteoblasts,adipocytes,chondrocytes,and muscle cells,under speci?c conditions(Baksh et al.,2004).Adipose-derived stem cells(ADSCs),which can also differentiate into adipo-cytes,chondrocytes,and osteoblasts,contribute to adipose tissue-derived cytokine secretion.The phenotypic and functional characteristics of ADSCs are very similar to those of BMSCs(Kilroy et al.,2007).Both ADSCs and BMSCs can differentiate into multiple mesenchymal lineages,thus providing a unique model for better understanding of the early differentiation events.

Adipogenesis is a complex process,involving the proliferation of precursor cells,their commitment to adipogenic lineage,and ter-minal differentiation.Adipogenic differentiation requires activa-tion by several transcription factors,including CCAAT/enhancer-binding protein-a(C/EBP-a),peroxisome proliferator-activated receptor gamma(PPAR c),and PPAR c target genes such as adipo-cyte lipid binding protein(aP2)and lipoprotein lipase(LPL)(Jan-derova et al.,2003).PPAR c is a key transcription factor that is involved in lipid metabolism and adipocyte differentiation;it acts synergistically with C/EBP-a to coordinate the adipocyte differen-tiation cascade(Rosen et al.,2009).Notably,leptin mRNA is in-creased during differentiation,with the highest levels observed at the end of adipogenic differentiation(Janderova et al.,2003). Cytokines released from preadipocytes can function as the initia-tors of adipogenesis or osteogenesis.Previous studies have shown that IL-6and TNF a are expressed and upregulated in adipose tis-sues from obese subjects(Hotamisligil et al.,1993;Kern et al., 2001).These cytokines may augment adipocyte differentiation (Kim et al.,2011).In addition,differentiated cells show key fea-tures of adipocytes such as expression of speci?c molecular mark-ers and accumulation of lipid droplets in the cytoplasm(Xiong et al.,2005).

Previous studies have shown that caffeine suppresses intracel-lular lipid accumulation in3T3-L1mature adipocytes(Nakabay-ashi et al.,2008)and inhibits insulin-stimulated glucose uptake but increases lipolytic effects in adipose cells(Akiba et al.,2004; Nakabayashi et al.,2008).Li et al.(2010)further reported that caf-feine signi?cantly stimulates lipolysis in adipocytes.However,the effects of caffeine on adipogenesis and the process of preadipocyte differentiation into adipocytes have not been determined yet. Regarding with caffeine commonly consumed in diet and drug can modulate body weight and lipid metabolism,and can be de-tected in various biological tissues such as the liver,brain,adipose tissue,and bone marrow(Cartiser et al.,2011;Serra et al.,2011), we thus speculated that caffeine might exert its effects on ADSCs and bone marrow stromal cells to modulate adipogenic differenti-ation.The present study investigated the effects and partial under-lying mechanisms of caffeine on adipogenic differentiation using primary rat ADSCs and bone marrow stromal cells(M2-10B4)as model.The results of our study provide direct evidence that caf-feine exerts antiadipogenic effects by downregulating adipogene-sis-related factors.

2.Materials and methods

2.1.Materials

Caffeine,b-glycerophosphate(b GP),ascorbic acid,3-isobutyl-1-methylxanthine(IBMX),dexamethasone,insulin,Oil Red O,Nile red and troglitazone(Tro)were obtained from Sigma(MO,USA). Dulbecco’s modi?ed Eagle’s medium(DMEM),DMEM-F12,fetal calf serum(FCS),penicillin,and streptomycin were obtained from Gibco(NY,USA).Collagenase type II was obtained from Worthing-ton(NJ,USA).2.2.Preparation and culture of primary rat adipose-derived stem cells (ADSCs)

Primary rat ADSCs from rat adipose tissue were isolated and cultured as previously described(Gimble and Guilak,2003),with minor modi?cations.The fatty tissue around the kidneys and testi-cles of4-weeks-old Sprague-Dawley rats was separated.After re-moval of visible blood vessels,the tissue was?nely minced with scissors and digested with collagenase type II(0.15%w/v)in Krebs-Ringer-bicarbonate HEPES buffer(0.11g/L CaCl2,0.25g/L MgSO4,0.84g/L NaHCO3,7g/L NaCl,0.55g/L KH2PO4,7.15g/L HEPES and10g/L BSA,1ml of200l M adenosine;pH7.4)for 60min at37°C with shaking.The?oating adipocytes were sepa-rated by centrifugation at1200rpm for5min.ADSCs(1?106-cells)were plated in tissue culture?asks in DMEM supplemented with10%FBS,100U/mL penicillin,and0.1mg/mL streptomycin, at37°C in a humidi?ed atmosphere containing5%CO2.The culture medium was changed every3days and the primary cells were pas-saged twice before being used for differentiation assays.The cell surface CD29,CD44and CD90protein markers of ADSCs were pos-itively expressed and analyzed by?ow cytometry at P3and the fol-lowing criteria were used to characterize the ADSCs.The P3cells passaged at a density of4?104/cm2were used for the experiments.

2.3.Culture of M2-10B4mouse bone marrow stromal cell line

The M2-10B4mouse bone marrow stromal cell line,obtained from American Type Culture Collection(Rockville,MD,USA),was derived from the bone marrow stromal cells of a(C57BL/ 6J?C3H/HeJ)F1mouse.These cells were cultured in DMEM med-ium containing10%heat-inactivated FBS supplemented with1mM sodium pyruvate,100U/mL penicillin,and100U/mL streptomycin at37°C in a humidi?ed atmosphere containing5%CO2.Upon reaching80–90%con?uence,the cells were detached using tryp-sin/EDTA,counted,and subcultured at a density of4?105cells/ 100mm dish.

2.4.Cell viability assay

Cells(2?104cells)were seeded in each well of a24-well cul-ture plate(Corning,New York,USA)and grown at37°C in a5% CO2incubator.After overnight incubation,the cells were treated with caffeine(0.1–1mM)in adipogenic medium for24h,and then cell number was counted with crystal violet elution assay for via-bility,and expressed as a percentage of the corresponding control group.

2.5.Induction of adipogenic differentiation

For adipocyte differentiation,ADSCs were cultured in adipo-genic differentiation medium containing DMEM supplemented with0.25mM3-isobutyl-1-methylxanthine,1mM dexametha-sone,and1l mol/L insulin(MDI medium)in the presence or ab-sence of caffeine(0.1–1mM)for12days.M2-10B4cells were cultured in adipogenic differentiation medium containing DMEM supplemented with2l M troglitazone(Tro medium)in the pres-ence or absence of caffeine(0.1–1mM)for7days.Media and treat-ments were changed every3days.

2.6.Lipid droplet staining with Oil red O

Cell monolayers were rinsed twice with PBS and?xed with10% formaldehyde for30min at room temperature.After washing with distilled water two times,cells were stained with?ltered3mg/mL Oil red O dissolved in60%isopropanol for10min at room

S.-H.Su et al./Toxicology in Vitro27(2013)1830–18371831

temperature.The plates were placed on an orbital shaker for 1h and then rinsed with 70%ethanol to remove the background Oil red O stain (Ramirez-Zacarias et al.,1992;Sanchez-Hidalgo et al.,2007).

2.7.Quanti?cation of adipocyte number by ?ow cytometry

Adipocytes were quanti?ed after adipogenic differentiation using lipophilic Nile red ?ow cytometry analysis (Backesjo et al.,2009;Gimble et al.,1995;Peng et al.,2011;Smyth and Wharton,1992).In brief,cells were carefully trypsinized,centrifuged for 10min at 200g at 4°C,washed twice with PBS and ?xed with 10%formaldehyde at 4°C.For ?ow cytometry,the cells were stained with 1l g/mL Nile red for 30min at 37°C.The samples were then analyzed with a FACScan ?ow cytometer (Beckman Coulter),and Nile red ?uorescence was measured on the FL2emis-sion channel using a 575-nm band-pass ?lter.Data analysis was performed using the WinMDI 2.9software.

2.8.Total RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA was extracted from differentiated cells resuspended in REzolTM reagent (PROtech Technologies,Taiwan)according to the manufacturer’s protocol.For RT-PCR,approximately 2l g of to-tal RNA was converted to single-stranded DNA using a commercial cDNA synthesis kit (Promega Corporation).Aliquots of the cDNA were ampli?ed with Ampli-Taq DNA polymerase (Yeastern Bio-tech)and primers speci?c to PPAR-c ,C/EBP-a ,Leptin,LPL,IL-6and TNF a .The ampli?ed PCR products were analyzed by electro-phoresis through a 2%agarose gel,which was photographed.The housekeeping gene GAPDH was used as the internal control.The intensity of each band was calculated by densitometry analysis,and the results expressed as a percentage of the density of the cor-responding GAPDH gene band.Primers used were as follows:PPAR c (forward:TCA GGT TTG GGC GAA TGC and reverse:GCT GGT CGA TAT CAC TGG AGA TC),C/EBP a (forward:CGC CTT CAA CGA CGA GTT CC and reverse:TAG TCA AAG TCA CCG CCG

CCA),

adipogenic differentiation of ADSCs and M2-10B4cells.ADSCs (A)and M2-10B4cells (B)were cultured in a basic and Tro)in the presence or absence of caffeine (0.1–1mM).After 12and 7days of incubation (for ADSCs and differentiation was determined by Oil red O staining of lipid droplets.Morphological changes in cells were Lipid droplets were quanti?ed using isopropanol dissolved Oil red O and by determining absorbance at 490medium in the presence of caffeine (0.1–1mM)for 24h and cell viability was further assessed by crystal violet.experiments.?p <0.05compared with the control.

aP2(forward:GCG TGG AAT TCG ATG AAA TCA and reverse:CCC GCC ATC TAG GGT TAT GA),Leptin(forward:GGA TGA CAC CAA AAC CCT CAT C and reverse:CCG ACT GCG TGT GTG AAA TG), LPL(forward:ATG GAG AGC AAA GCC CTG CTC and reverse:GTT AGG TCC AGC TGG ATC GAG),IL-6(forward:CTG CCT TCC CTA CTT CAC AA and reverse:TGG AAG CAT CCA TCA TTT CT),TNF a (forward:GGA GAA CAG CAA CTC CAG AA and reverse:CAG GAA TGA GAA GAG GCT GA)and GAPDH(forward:CGA TCC CGC TAA CAT CAA AT and reverse:GGA TGC AGG GAT GAT GTT CT).

2.9.Statistical analysis

All quantitative data were expressed as mean±SD.Statistical analysis was performed by SigmaPlot7.0using a paired t-test.Re-sults with p<0.05were considered statistically signi?cant.

3.Results

3.1.Concentration-response effects of caffeine on adipogenic differentiation of ADSCs and M2-10B4cells

Adipocyte differentiation involves dramatic changes in cell morphology and gene expression(Tontonoz et al.,1995).Morpho-logical observations include presence of lipid droplets in adipocyte cytoplasm,which are stained positively with Oil red O.Previous studies have used Nile red?ow cytometry for assessing adipocyte cell numbers(Gimble et al.,1995;Smyth and Wharton,1992).To determine the effects of caffeine on adipogenic differentiation of ADSCs and M2-10B4cells,both the cell types were treated with different concentrations of caffeine(0.1,0.3,and1mM)for12 and7days,respectively.Adipocyte formation was observed using Oil red O staining(Fig.1).Minimal lipid accumulation was ob-served in cells cultured in the basic medium(negative control; Fig.1).ADSCs cultured in adipogenic medium(MDI medium)for 12days showed signi?cantly increased lipid accumulation up to 60%compared with negative controls.Similarly,M2-10B4cells cul-tured in adipogenic medium(Tro medium)for7days showed sig-ni?cantly increased lipid accumulation up to65%compared with negative controls.Caffeine also decreased lipid droplet accumula-tion in a concentration-dependent manner(Fig.1A and B).Nile red staining and?ow cytometry were used to calculate the per-centage of cells that positively stained as adipocytes(Fig2).Fol-lowing ADSCs cultured in the MDI medium for12days,31.8%of the cells were found to be adipocytes;treatment with0.1,0.3,or 1mM caffeine reduced the number of adipocytes to26.7%,23.2%, and17.6%,respectively.Additionally,M2-10B4cells cultured in the Tro medium for7days,34.8%of the cells were found to be adi-pocytes;treatment with0.1,0.3,or1mM caffeine reduced the number of adipocytes to27.8%,24.6%,and19.8%,respectively (Fig.2).This result is consistent with those of adipogenesis studies in3T3-L1preadipocyte cell line(data not shown).Caffeine concen-trations of0.1–0.3mM did not affect ADSC or M2-10B4cell viabil-ity(p>0.05);but29.3%of ADSC and26.5%of M2-10B4cells were reduced in the presence of1mM of caffeine(p<0.05),as deter-mined using crystal violet staining(Fig.1C).Therefore,0.1–0.3mM caffeine reduced the number of ADSCs and M2-10B4cells differentiating into adipocytes(Figs.1and2).At high concentra-tions(1mM),caffeine suppressed cell differentiation partly through inducing cell toxicity.

reduced Nile-red-stained positive cells formation in ADSCs and M2-10B4cells.ADSCs and M2-10B4cells were cultured in a basic medium medium(MDI and Tro)in the presence or absence of caffeine(0.1–1mM).After12and7days of incubation(for ADSCs and M2-10B4,

?xed,and stained with Nile red solution.Percentage of Nile-red-stained cells in the total population of each sample was quanti?ed

results are expressed as the mean±SD of three independent experiments.?p<0.05compared with control.

S.-H.Su et al./Toxicology in Vitro27(2013)1830–18371833

inhibited the expression of adipogenesis-related factors in ADSCs and M2-10B4cells.ADSCs(A)and M2-10B4cells(B)were cultured in an adipogenic medium(MDI and Tro)in the presence or absence of caffeine(0.1–1mM).After12and7days of incubation(for expression of PPAR c,C/EBP-a,LPL,aP2and leptin mRNA was measured by RT-PCR.Expression of PPAR c,C/EBP-a,LPL,aP2and leptin genes All results are expressed as the mean±SD of three independent experiments.?p<0.05compared with control.

target genes which were directly related to lipogenic pathways,including LPL and aP2were reduced.Adipokine leptin mRNA expression was decreased as well.An adipogenic transcription fac-tor C/EBP a expression was suppressed only when treated with 0.3mM caffeine (p <0.05).Similarly,caffeine also signi?cantly and dose-dependently inhibited PPAR c ,aP2,LPL,and leptin mRNA expression in M2-10B4cells as shown in Fig.3B.However,C/EBP a expression was slightly reduced only when treated with 0.3mM caffeine.These ?ndings collectively suggest that caffeine could effectively inhibit adipogenesis-related signaling in both ADSCs and M2-10B4cells (Fig.3).It is noted that leptin transcript is in-creased during differentiation,with the highest levels observed at the end of adipogenic differentiation (Janderova et al.,2003).Re-cent insights into the metabolic and immunological functions of preadipocytes showed that these cells are potent producers of pro-in?ammatory cytokines such as IL-6and TNF a (Tsatsanis et al.,2006).In the present study,ADSCs continued to express IL-6and TNF a in the basic medium (negative control group)(Fig.4).How-ever,ADSCs cultured in adipogenic medium showed reduced IL-6levels than those in the control group (Fig.4).Caffeine resulted in a minor but signi?cant increase in IL-6expression at a concen-tration of 0.1mM in treated cells compared with that in control cells;however,this effect was inhibited at concentrations of 0.3–1mM.Furthermore,caffeine inhibited TNF a expression in a con-centration-dependent manner (Fig.4).4.Discussion

An important ?nding of the present study was that caffeine had suppressive impacts on the adipogenic differentiation of ADSCs and M2-10B4cells.Caffeine reduced the number of lipid droplets,expression of adipogenic genes,and adipocyte formation in a con-centration-dependent manner in both ADSCs and M2-10B4cells.A possible mechanism by which low caffeine concentration (0.1mM)inhibits adipogenic differentiation involves the activation of AMP-activated protein kinase (AMPK)and extracellular signal-regulated kinase (ERK)pathways,which further downregulate adi-pogenic transcriptional pathways.A previous study has shown that caffeine (0.1mM)increases ERK activation (Belibi et al.,2002),whereas activated ERK may inhibit adipogenic differentiation through PPAR c suppression (Jung et al.,2011).Additionally,caf-feine also activates AMPK (Egawa et al.,2011),and AMPK-activat-ing molecules exert antiadipogenic effects by downregulating adipogenic signaling (Ahn et al.,2008;Zhou et al.,2009).Notably,trypan blue dye exclusion and crystal violet staining showed that caffeine concentration of 0.1–0.3mM had no cytotoxic effect on both the cell types,indicating that inhibition of adipogenic differ-entiation and adipocyte formation was not caused by the suppres-sion of cell viability (data not shown).However,1mM caffeine signi?cantly suppressed adipocyte differentiation of both cell types.One explanation for this may be that caffeine induces cell death.The present study provides evidence that caffeine remark-ably inhibited the differentiation of ADSCs and bone marrow stro-mal cells into adipocytes.Caffeine inhibited lipid accumulation and adipocyte formation in a concentration-dependent manner (Figs.1and 2).In addition,caffeine also signi?cantly inhibited adipogene-sis in a concentration-dependent manner,and this inhibition might be resulted from the downregulation of adipogenesis-related genes PPAR c ,aP2,leptin,and LPL (Fig.3).An earlier study had shown that caffeine resulted in a remarkable inhibition of LPL mRNA and enzy-matic activity in 3T3-F442A preadipocytes (Couturier et al.,1998).PPAR c is a ligand-activated transcription factor that plays a cen-tral role in adipocyte gene expression and differentiation (Tonto-noz et al.,1995).PPAR c targets is critical for adipogenesis,and PPAR c target genes are also directly implicated in lipogenic path-ways,including LPL (Mead et al.,2002)and aP2(Stremmel et al.,1985).By contrast,LPL was related to lipolysis,such as triglycer-ide-rich lipoproteins and plasma triglyceride clearance (Davies et al.,2012).

In this study,also it is noted that the caffeine has different reg-ulation and control effects on PPAR c and LPL in two cell types.Although ADSC and M2-10B4have similar characteristics,their sources are different.ADSC is derived from adipose tissue;basi-cally,the gene expressions related to lipogenesis may be higher than bone marrow stromal cell M2-10B4.On the other hand,there are many downstream genes regulated and controlled by PPAR c .LPL is one of them,and LPL not only participates in adipogenesis,but also participates in lipogenesis and lipolysis.In the ADSC,caf-feine signi?cantly inhibited the expression of PPAR c ,but its inhib-itory effect on LPL gene is not apparent.This result shows that the low concentration caffeine in?uences ADSC cells probably by inhibiting adipogenesis to reduce the formation of adipocytes.

expression of adipogenesis-related cytokines in ADSCs.ADSCs were cultured in a basic medium (negative days.After 12days of incubation,total RNA was isolated and analyzed for IL-6and TNF a expression by RT-PCR.GAPDH.All results are expressed as the mean ±SD of three independent experiments.?p <0.05compared

the M2-10B4,it is apparent that the caffeine dose-dependent inhibits PPAR c and LPL genes signi?cantly,suggesting that the caf-feine can inhibit adipogenesis and lipogenesis simultaneously.The expression of aP2gene has similar phenomenon.

A recent report suggested that expression of in?ammatory cyto-kines(TNF a and IL-6)may augment adipocyte differentiation(Kim et al.,2011).In the present study,0.1mM caffeine decreased TNF a expression and slightly increased IL-6expression(Fig.4).Consis-tently,another recent study showed that caffeine downregulated TNF a gene expression in human adipose tissue,thus improving adipose tissue in?ammation during obesity;however,IL-6expres-sion was not changed in this study(Dray et al.,2007).It is noted that IL-6can also stimulate osteoblast differentiation(Taguchi et al.,1998);it will be rationally that increased IL-6results in in-creased osteoblast formation but decreased adipocyte formation. Eventually,caffeine concentrations of0.3–1mM can signi?cantly decrease the expression of cytokines,TNF a and IL-6(Fig.4).De-creased IL-6and TNF a,adipokines secreted by mature adipocyte, may partly re?ect decreased adipocyte differentiation.

An interesting?nding of our previous studies and this study was that a low caffeine concentration(0.1mM)increased osteogenesis (Su et al.,2013)but inhibited adipogenesis(Figs.1–4)in both the cell types.We found that0.1mM caffeine signi?cantly enhanced the expression of Runt-related transcription factor2(Runx2)and Sirtuin1(Sirt1)(Su et al.,2013).Indeed,expression of Runx2and Sirt1in mesenchymal cells inhibited their differentiation into adi-pocytes by blocking the PPAR c2activity(Backesjo et al.,2009; Kobayashi et al.,2000).Furthermore,caffeine concentrations higher than0.3mM have antiadipogenic and antiosteogenic properties, indicating that caffeine may induce stem cell death.

Caffeine in low concentration(0.1–0.3mM=19.4–58.2l g/ml) can effectively inhibit adipogenesis in the dose dependent manner. High concentrations(P1mM)of caffeine have been commonly used in previous in vitro studies(Hashimoto et al.,2004;Okano et al.,2008;Sinchai et al.,2011);however,treatment with high concentrations may not be physiologically applicable for human body.It has been reported that caffeine causes adverse effects at excessively high blood concentrations(>80l g/ml)in humans (Nomura et al.,2005).

The results of the present study show that caffeine has antiadip-ogenic properties.Caffeine suppress the differentiation of ADSCs and BMSCs into adipocytes,which mediated by its inhibition of adipogenesis-related factors.The caffeine concentration(0.1mM) used in general cell cultures is approximately equivalent to that in a cup of coffee containing150mg caffeine(Starbucks latte). Therefore,consuming1cup of coffee containing150mg of caffeine per day is appropriate and may decrease adipocyte differentiation and body weight.However,daily consumption of more than 300mg of caffeine(which may exceed the0.3mM caffeine concen-tration used in cell cultures)through coffee,especially for long periods,may induce stem cell cytotoxicity.

Con?ict of interest

None declared.

Acknowledgement

This study was supported by the National Science Council,Exec-utive Yuan,Taiwan(NSC97-2320-B-242-004).

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