An Advanced Glycation End Product (AGE)-Receptor for AGEs(RAGE) Axis Restores Adipogenic Potential of SenescentPreadipocytes through Modulation of p53 Protein Function*

Received for publication, July 10, 2012, and in revised form, November 12, 2012 Published, JBC Papers in Press, November 13, 2012, DOI 10.1074/jbc.M112.399790

Chih-Yu Chen‡, Allison Martorano Abell‡, Yang Soo Moon§, and Kee-Hong Kim‡1

From the ‡Department of Food Science, Purdue University, West Lafayette, Indiana 47906 and the §Department of Animal Scienceand Biotechnology, Gyeongnam National University of Science and Technology, 33 Dongjin-ro, Jinju 660-758, Republic of Korea

Background: The impaired adipogenic potential is a hallmark of preadipocyte senescence.Results: Senescent preadipocytes treated with advanced glycation end products (AGEs) resumed adipogenesis in vitro andex vivo.Conclusion:AGE-induced expression of the receptor for AGEs (RAGE) and AGE-RAGE-dependent inhibition of p53 functionrestore adipogenesis in senescent preadipocytes.Significance: Glycated proteins could participate in aging-impaired adipose development.

The impaired adipogenic potential of senescent preadi-pocytes is a hallmark of adipose aging and aging-related adiposedysfunction. Although advanced glycation end products (AGEs)derived from both foods and endogenous nonenzymatic glyca-tion andAGE-associated signaling pathways are known to play akey role in aging and its related diseases, the role of AGEs inadipose aging remains elusive.We show a novel pro-adipogenicfunction of AGEs in replicative senescent preadipocytes andmouse embryonic fibroblasts, as well as primary preadipocytesisolated from aged mice. Using glycated bovine serum albumin(BSA) as a model protein of AGEs, we found that glycated BSArestores the impaired adipogenic potential of senescent preadi-pocytes in vitro and ex vivo. However, glycated BSA showed noeffect on adipogenesis in nonsenescent preadipocytes. TheAGE-induced receptor for AGE (RAGE) expression is requiredfor the pro-adipogenic function of AGEs in senescent preadi-pocytes. RAGE is required for impairment ofp53 expression andp53 function in regulating p21 expression in senescent preadi-pocytes. We also observed a direct binding between RAGE andp53 in senescent preadipocytes. Taken together, our findingsreveal a novel pro-adipogenic function of theAGE-RAGEaxis inp53-regulated adipogenesis of senescent preadipocytes, provid-ing new insights into aging-dependent adiposity by diet-drivenand/or endogenous glycated proteins.

Preadipocytes from aged adipose tissue display impaired adi-pogenic potential and increased pro-inflammatory cytokinesecretion (1, 2). This is accompanied by cellular senescence ofpreadipocytes as evidenced by increased levels of senescence-

associated�-galactosidase (SA-�-gal)2 activity, reactive oxygenspecies (ROS), and p53, a tumor suppressor protein, expression(3), suggesting a positive correlation between senescence ofpreadipocytes and adipose dysfunction. Analogous to agedpreadipocytes, the preadipocytes isolated from obese animalsand humans appear to exhibit senescence-like phenotypes andimpaired adipogenic potential with elevated sensitivity toinflammatory cytokine-induced macrophage-like phenotypes(3–7). Although understanding the precise role of senescentpreadipocytes in adipose aging still warrants further investiga-tion, facilitated conversion of senescent preadipocytes to adi-pocytes appears to contribute to adipose development duringaging.AGEs are a heterogeneous group of macromolecules that are

formedbynonenzymatic glycation of proteins, lipids, or nucleicacids (7). Although exogenous AGEs aremainly generated dur-ing tobacco smoking and the Maillard reaction (a nonenzy-matic browning reaction between amino acids and reducingsugars mostly in heat-processed foods), endogenous AGEs areformed through the interaction between proteins and glucosederivatives, such as methylglyoxal and glyoxal in circulation.Endogenous AGEs are virtually formed in all tissues, particu-larly during hyperglycemic conditions (8). Excess AGE intakeand chronic accumulation of AGE-related glycated proteins intissues are suggested to further potentiate the aging processresulting in impairedmitochondrial function and decreased lifespan inCaenorhabditis elegans andmice (9–11). Glycated pro-teins are largely known to interact with an AGE-interactingpattern recognition receptor known as RAGE. An AGE-RAGEaxis contributes to aging-associated inflammation, oxidativestress, and the development of some of the chronic diseases

* This work was supported in part by a study abroad scholarship from theTaiwan Government (to C. Y. C.), a start-up fund and a Ralph W. and GraceM. Showalter Trust Award from Purdue University (to K. H. K.), and Next-Generation BioGreen 21 Grant PJ007981 from Technology DevelopmentProgram for Agriculture and Forestry, Korea (to Y. S. M.).

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-BankTM/EBI Data Bank with accession number(s) NM_07425.

1 To whom correspondence may be addressed. Tel.: 765-496-2330; Fax: 765-494-7953; E-mail: keehong@purdue.edu.

2 The abbreviations used are: SA-�-gal, senescence-associated �-galactosid-ase; AGE, advanced glycation end product; C/EBP�/�, CCAAT/enhancer-binding protein�/�; CML, carboxymethyl lysine; FAS, fatty-acid synthase;GA, glyceraldehyde; MEF, mouse embryo fibroblast; MG, methylglyoxal;ORO, Oil Red O; PPAR�, peroxisome proliferator-activated receptor�;RAGE, receptor for AGE; ROS, reactive oxygen species; SV, stromal vascular;DCHF-DA, 2�,7�-dichlorodihydrofluorescein diacetate; HP, high passage;LP, low passage.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 53, pp. 44498 –44507, December 28, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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such as atherosclerosis (12), diabetes complications, andAlzheimer disease (13). In addition, AGEs in high fat diets areshown to potentiate high fat diet-induced metabolic andinflammatory disorders such as obesity, insulin resistance, andcardiac disease (14, 15). Conversely, reduction ofAGE intake (9,10), inhibition of AGE formation (15, 16), or alleviation ofRAGE expression and function (17–20) was suggested to be aneffective approach to attenuate AGE-RAGE axis-associatedmetabolic and inflammatory disorders. However, the preciserole of the AGE-RAGE axis in adipose tissue development dur-ing aging is unknown.Here, we found that exogenousAGEswere able to restore the

adipogenic potential of replicative senescent preadipocytes,whereas AGEs showed no effect on adipogenesis of young prea-dipocytes. This is through AGE-induced RAGE expression andits interaction with p53, thereby abrogating senescence-associ-ated p53 function in preadipocytes. These results reveal a novelpro-adipogenic function of AGEs in adipose aging in which theAGE-RAGE axis modulates the function of senescence-in-duced p53, resulting in restoring the impaired adipogenicpotential of aged adipose tissue.

EXPERIMENTAL PROCEDURES

Materials and Reagents—Bovine serum albumin (BSA) aswell as methylglyoxal (MG) (21) were purchased fromMP Bio-medicals, and glyceraldehyde (GA) was from Sigma. Insulin,dexamethasone, 3-isobutyl-1-methylxanthine, propidiumiodide, and RNase A were purchased from Sigma. Fetal bovineserum (FBS) was purchased from PAA (Dartmouth, MA). Dul-becco’s modified Eagle’s medium (DMEM), penicillin/strepto-mycin, and sodium pyruvate were from VWR (Radnor, PA).TRIzol� reagent and SuperScriptII were purchased from Invit-rogen. A protein assay kit was obtained from Bio-Rad.AGE Preparation—Twenty mg/ml of BSA dissolved in 0.2%

(w/v) Na3N containing phosphate-buffered saline (PBS) wasincubatedwith 100mMMGor 100mMGAat 37 °C for 6 days togenerate MG-BSA or GA-BSA, respectively. The glycated BSA(i.e. MG-GSA and GA-BSA) was then filtered through a filterwith a pore size of 0.22 �m followed by dialysis at 4 °C againstwater to remove any free form ofMG or GA. The glycated BSAwas then aliquoted and stored at �20 °C.Preadipocyte Culture and Differentiation—3T3-L1 preadi-

pocytes were purchased from American Type Culture Collec-tion (ATCC, Manassas, VA). Cells were cultured and differen-tiated as described previously (22). Replicative senescence of3T3-L1 preadipocytes was introduced by performing a serialpassaging of 70–80% confluent cells. To determine the effect ofBSA or glycated BSA on adipogenesis of 3T3-L1 cells, BSA,MG-, or GA-BSA was either added during the course of adipo-genesis or at day�2 to day 0 prior to the initiation of adipogen-esis. In a RAGE neutralization study, 10�g/ml anti-RAGE anti-body (Santa Cruz Biotechnology Inc., Santa Cruz, CA) wasadded to the differentiating 3T3-L1 cells fromday 0 to day 2. Toexamine the effect of inhibition of p53 function on adipogenesisof senescent 3T3-L1 preadipocytes, cells were pretreated withthe indicated concentrations of pifithrin-� (Calbiochem) andBSA or glycated BSA at day �3 to day �2 and day �2 to day 0,respectively, prior to the initiation of adipogenesis. The lipids

accumulated in differentiated preadipocytes at day 8 werestained with Oil Red O (ORO) as described previously (22, 23).Mouse Embryo Fibroblasts (MEFs), Primary Stromal Vascu-

lar (SV) Cell Culture, and Animal Study—MEFs purchasedfrom ATCC were cultured in 10% FBS/DMEM with 1% peni-cillin/streptomycin. Passage numbers of 1–2 and 7–10 wereconsidered as LP and HP, respectively. The cells were differen-tiated to adipocytes as described previously (24). Inguinal andepididymal adipose tissues were dissected from 6-week-old and1-year-old chow diet-fed male C57BL/6Nmice for the study inFig. 1H and 14-week-old chow diet-fed or high fat diet (60%calories from fat)-fed mice for the studies in Fig. 1I. Twelve-week-old and 1-year-old chow diet-fed male C57BL/6N micewere used for the studies in Fig. 3,C andD. All procedures wereapproved by Purdue University Animal Care and Use Commit-tee. Primary adipose tissues isolated from these mice weredigestedwith collagenase to isolate the SV fraction as describedpreviously (25). The SV fraction containing primary preadi-pocytes was cultured in 10% FBS/DMEM, and subjected to adi-pogenesis in 10% FBS/DMEMcontaining 1�g/ml insulin, 5�M

dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 2.5�g/ml rosiglitazone for 3 days, followed by incubation in 10%FBS/DMEM supplemented with 1 �g/ml insulin for 5 days.SA-�-gal Staining—3T3-L1 cells fixed with 3.7% formalde-

hyde overnight at 4 °C were incubated with freshly preparedstaining buffer (pH6.0, 150mMNaCl, 2mMMgCl2, 40mMcitricacid, 5 mM sodium phosphate, 5 mM K3Fe(CN)6, and 5 mM

K4Fe(18)6) containing 1mg/ml 5-bromo-4-chloro-indolyl-�-D-galactopyranoside (X-gal) (IBI Scientific, New Haven, CT) for18h at 37 °C.At the endof the incubation, cellswere rinsedwithPBS and were photographed under bright field (26). SA-�-gal-positive cells were quantified by Quantity One 4.5 software.Measurement of Reactive Oxygen Species—Levels of intracel-

lular ROS in LP and HP preadipocytes were measured by load-ing the cells with 40 �M 2�,7�-dichlorodihydrofluoresceindiacetate (DCHF-DA) (Cayman Chemical) for 30 min at 37 °C.Intracellular DCHF-DA oxidized to fluorescent 2�,7�-dichloro-dihydrofluorescein was monitored by the fluorescence micro-scope (Leica Microsystems). For quantification of the intracel-lular 2�,7�-dichlorodihydrofluorescein, cells grown in a 96-wellplate incubated with DCHF-DA for 30 min were subjected to afluorescence microplate reader (SpectraMax Gemini EM,Molecular Devices) at an excitation wavelength of 485 nm andemission of 535 nm.Immunoblotting—3T3-L1 cells were subjected to immuno-

blot analysis as described previously (22). Primary antibodiesused in the study include phospho-Akt (Ser(P)-473) and totalAkt (Epitomics, Burlingame, CA), carboxymethyl lysine (CML)(R&D system, Minneapolis, MN), p21 (Genscript, ScotchPlains, NJ), phospho-p53 (Ser-15) (Cell Signaling, Beverly,MA), RAGE, p53, �-actin, and �-tubulin (Santa Cruz Biotech-nology), and secondary HRP-conjugated mouse and rabbitantibodies (The Jackson Laboratory, Bar Harbor, ME). Proteinbands were detected with Pierce ECL Plus Western blottingreagents by autoradiography. Film was scanned and processedusing the ImageJ software, version 1.45S, from National Insti-tutes of Health, for quantification of protein band intensity bynormalization to the band intensity of �-actin or �-tubulin.

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Immunofluorescence—3T3-L1 preadipocytes were seeded oncoverslips overnight and then treated with or without glycatedBSA for 24 h. The cells were fixed with 3.7% formaldehyde for30 min and permeabilized in 0.2% Triton-contained PBS for 20min. One percent BSA-contained PBST solution (0.1% Tween20 in PBS) was used for blocking, and the cells were incubatedwith RAGE antibody (1:50). Primary antibody binding wasdetected using a fluorescein-conjugated secondary antibody(Santa Cruz Biotechnology). 4�-6-Diamidino-2-phenylindole(DAPI) (Calbiochem) was used for nuclear staining. RAGE andDAPI signals from the cells were visualized by LSM 710 confo-cal microscopy (Carl Zeiss, Inc.).Immunoprecipitation—To examine the interaction between

RAGE and p53, 3T3-L1 cells treated with BSA or glycated BSAwere lysed with radioimmunoprecipitation assay buffer. RAGEand p53 in cell lysates were incubated with 2 �g of anti-RAGEand anti-p53 antibody, respectively, or control IgG at 4 °C for1 h, followed by immunoprecipitation with protein A/G PLUS-agarose beads (Santa Cruz Biotechnology) for 30 min at 4 °C.The beads were washed with cold PBS four times and boiledwith the sample buffer (60 mM Tris, pH 6.8, 10% glycerol, 2%SDS, 0.1% bromphenol blue, and 0.1 M dithiothreitol) for SDS-PAGE and immunoblot analysis with the indicated antibodies.Chromatin Immunoprecipitation (ChIP)—ChIP was per-

formed as described previously (27) with some modifications.3T3-L1 cells were cross-linked with formaldehyde at 1.5% inPBS for 15min and neutralizedwith 125mMglycine. The nucleiextracted from these cells were subjected to sonication to pro-duce the chromatin fragments from 200 to 1,000 bp 5� with20-s bursts using the sonifierW-150 type (G. Heinemann, Ger-many). For chromatin preparation, the isolated nuclei wereprecleared with 20 �l of protein A/G PLUS-agarose beads for30min at 4 °C. 2% of the chromatin solutionwas used as “input”in Fig. 5, and the remainder was incubated with p53 antibody (2�g) or a control IgG overnight at 4 °C. The immune complexeswere then recovered by incubating with 40 �l of protein A/GPLUS-agarose beads for 2 h at 4 °C. The complexes were thensubjected to 1% SDS, 100mMNaHCO3, and 200mMNaCl over-night at 67 °C to reverse cross-linking. DNA was then purifiedusing a PCR purification kit (Qiagen Inc., Chatsworth, CA) andwas subjected to reverse transcription (RT)-PCR analysis toamplify mouse p21 promoter sequences (�2927 to �2595)using a primer set as follows: forward, 5�-CGGAGACCAGCAGCAAAATCG-3�, and reverse, 5�-TGACACATACACACCCC AGG CAC-3� (28).PCR Analyses—Total mRNAs from cells or tissues were

extracted with TRIzol� (Invitrogen). The cDNA was synthe-sized by SuperScriptII system. A real time PCR was performedusing a SYBR premixed Taq reactionmixture in a thermocycler(Applied Biosystems, Foster City, CA). Values were normalizedby �-actin expression. Data were analyzed using the ��CTmethod. RT-PCRwas performed usingMJMini Personal Ther-mal Cycler (Bio-Rad). The sequences of primers used in realtime PCRwere as follows: PPAR� (forward, 5�-CCCAATGGTTGC TGA TTA CAA AT-3�, and reverse, 5�-CTA CTT TGATCG CAC TTT GGT ATT CT-3�); C/EBP� (forward, 5�-GGTTTA GGG ATG TTT GGG TTT TT-3�, and reverse, 5�-AAGCCC ACT TCA TTT CAT TGG T-3�); C/EBP� (forward,

5�-AGCGGCTGCAGAAGAAGGT-3�, and reverse, 5�-GGCAGC TGC TTG AAC AAG TTC-3�); FAS (forward, 5�-GCCACCCACCGTCAGAAG-3�, and reverse, 5�-TGTCACATCAGC CAC TTG AGT GT-3�); adiponectin (forward, 5�-GATGCA GGT CTT CTTG GTC CTA A-3�, and reverse, 5�-GGCCCT TCA GCT CCT GTC A-3�); IL-6 (forward, 5�-TCG GAGGCTTAATTACACATGTTC-3�, and reverse, 5�-TGC CATTGC ACA ACT CTT TTC T-3�); RAGE (forward, 5�-AACACAGCCCCCATCCAA-3�, and reverse, 5�-GCTCAACCAACA GCT GAA TGC-3�); p53 (forward, 5�-GCT GCC CCCACC ATG AG-3�, and reverse, 5�-AAT TTC CTT CCA CCCGGA TAA-3�); p21 (forward, 5�-AAT TGG AGT CAG GCGCAG AT-3�, and reverse, 5�-TCC TGT TTA CCC CAG CTCTGA A-3�); �-actin (forward, 5�-AGA TGA CCC AGA TCATGTTTGAGA-3�, and reverse, 5�-CACAGCCTGGATGGCTAC GT-3�). The sequences of primers used in RT-PCR wereas follows: PPAR� (forward, 5�-CCA CCA ACT TCG GAATCAGCT-3�, and reverse, 5�-CTA CTT TGA TCGCAC TTTGGT ATT CT-3�); FAS (forward, 5�-ACC ACT GCA TTGACGGCCGG-3�, and reverse, 5�-GGG TCAGGCGGGAGACCG AT-3�); adiponectin (forward, 5�-GGC CGT TCT CTTCAC CTA CG-3�, and reverse, 5�-AGA TGG AGG AGC ACAGAG CC-3�); RAGE (forward, 5�-CAG GGT CAC AGA AACCGG-3�, and reverse, 5�-ATT CAG CTC TGC ACG TTCCT-3�); �-actin (forward, 5�-TGA CGG GGT CAC CCA CACTGTGCCCATCTA-3�, and reverse, 5�-CTAGAAGCATTTGCG GTG GAC GAT GGA GGG-3�).Lentiviral Transduction—A lentivirus-based shRNA transfer

plasmid (pLKO.1) was a gift from Dr. Xiaoqi Liu (Purdue Uni-versity). All the procedures were described previously (29). Thetargeting sequence of mouse RAGE (GenBankTM accessionnumberNM_07425) is GGGAAGGAGGTCAAGTCCAAC.To generate lentivirus, 293T cells were co-incubated with 4 �gof pLKO.1-RAGE, 4 �g of pHR�-CMV-R8.20vpr, and 2 �g ofpHR�-CMV-VSV-G using Lipofectamine 2000 reagents (Invit-rogen). 24 h post-transfection, viruses were then harvestedevery 12 h until 72 h. The harvested media containing virusparticles were filtered though a 0.45-�m pore size filter andcentrifuged at 170,000 rpm for 90 min. 3T3-L1 cells wereinfected with the viral pellets in the presence of 10 �g/ml Poly-brene. After 24 h of viral transduction, cells were selected with1 mg/ml puromycin for at least 3 days.Statistical Analysis—All data are presented as means � S.E.

Statistical analysis was performed using SAS 9.0 software. One-way analysis of variance was used to determine significance oftreatment effect and interactions. Significant differencesbetween group means were assessed by Dunnett’s multiplecomparison. p � 0.05 was considered significant.

RESULTS

RAGE Expression Positively Correlates with ReplicativeSenescence-impaired Adipogenesis in Vitro, Adipose TissueAging, and Obesity—Preadipocytes in adipose tissue from agedanimals and humans have been shown to have impaired adipo-genic potency (1, 30). Because continuous cell division is knownto promote replicative senescence of cells in vitro (31–33), wefirst examined the effect of replicative senescence on the adipo-genic ability of 3T3-L1 murine preadipocytes and their cellular

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senescence bymeasuring the levels of SA-�-gal activity, mRNAlevels of p53 and IL-6, and ROS production. Preadipocytes atpassage number 15 (high passage (HP)) showed impaired adi-pogenesis as judged by ORO staining (Fig. 1A) and quantitativeanalysis of adipocyte marker gene expressions such as adi-ponectin and PPAR� (Fig. 1B) when compared with those fromadipocytes at passage number 5 (low passage (LP)). Theimpaired adipogenesis ofHP preadipocytes was correlatedwith

delayed cell cycle progress during the mitotic clonal expansionof adipogenesis (data not shown).Moreover, these HP cells dis-played a significant increase in SA-�-gal activity (Fig. 1C) andmRNA levels of p53 and IL-6 (Fig. 1D) compared with thosefrom LP preadipocytes. Replicative senescence of 3T3-L1 prea-dipocytes is associatedwith enhancedROSproduction (Fig. 1E)and impaired insulin-induced Akt phosphorylation (Fig. 1F).These results collectively suggested that a serial passage in3T3-L1 preadipocytes yields senescent characteristics withimpaired adipogenic potential in vitro. The RAGE gene hasbeen reported to be expressed in various tissues such as mousebone marrow in an aging-dependent manner (34). In addition,aging is reported to be positively associated with accumulationof AGEs. AGEs have the intrinsic fluorescence (i.e. pentosidine)and are chemically characterized by modification of lysine res-idues primarily to CML (35, 36). To further examine whetherthe AGE-RAGE axis is associated with adipose aging, we nextmeasured the levels of intracellular AGEs, as judged by detect-ing the presence of CML-modified proteins and/or RAGE insenescent adipocytes and adipose tissue isolated from aged orobese mice. Differentiated HP senescent preadipocytes exhib-ited a higher level of RAGE than that in LP adipocytes withreduced levels of adiponectin and PPAR� compared with thosein LP adipocytes (Fig. 1G). Similarly, elevated levels of theCML-modified proteins RAGE and p53 were also observed inthe inguinal fat pads isolated from 1-year-old mice comparedwith those in 6-week-old chow-fed mice (Fig. 1H). Moreover,high fat diet-induced obese mice displayed elevated levels ofCML-modified proteins, RAGE and aP2, an adipocyte markerprotein, in epididymal fat pads compared with those in leanmice (Fig. 1I), as well as RAGE mRNA (Fig. 1J). Overall, ourresults indicate that a serial passage in 3T3-L1 preadipocytes isassociated with development of senescent characteristics. Fur-thermore, adipose aging and obesity appear to be correlatedwith an elevated level of AGE-RAGE interaction.Glycated BSA Restores Impaired Adipogenic Potential of

Senescent Preadipocytes—Toexamine the effect ofAGEs on theadipogenic potential of senescent preadipocytes, we firstexposed senescent preadipocytes to glycated BSA during adi-pogenesis as depicted in Fig. 2A. Glycated BSA generated byincubation of BSAwith glucose derivatives such asMG andGAis fluorescent, a characteristic of AGEs (37), and this has beenused as in vitro model of AGEs in many studies (38, 39). BothBSA and glycated BSA, such as MG-BSA and GA-BSA at theconcentration of 300 �g/ml, showed no additional effect onadipogenesis of LP preadipocytes as judged by both ORO (Fig.2A,middle panel) and quantification ofOROstained lipids (Fig.2B). Consistent with Fig. 1A, senescent HP preadipocytes failedto differentiate when the cells were treated with BSA during 8days of adipogenesis (Fig. 2A). Interestingly, treatments ofsenescent HP preadipocytes with 300 �g/ml MG-BSA or GA-BSA resulted in �2.5- and 3.5-fold induction of intracellularlipid droplet accumulation, respectively, compared with BSA-treated control cells (Fig. 2, A and B). In accordance withincreased lipid accumulation, senescent HP preadipocytes dif-ferentiated in the presence of MG-BSA or GA-BSA showeddramatically increased levels of adipocytemarker genes such asPPAR�, C/EBP�, adiponectin, FAS, and leptin (Fig. 2C) as well

FIGURE 1. Replicative senescence-impaired adipogenic potential of3T3-L1 preadipocytes and a positive correlation of AGE-RAGE axis withadipose tissue aging and adiposity. A, ORO staining of differentiated3T3-L1 preadipocytes (Pre) with different passage numbers (e.g. p.5, p.10, andp.15) for 8 days. B, gene expression analysis of adiponectin and PPAR� in dif-ferentiated preadipocytes with passage number of �5 (LP) and �15 (HP) byreal time PCR. C, SA-�-galactosidase activity assay in LP and HP 3T3-L1 prea-dipocytes as shown in the overall view (top panel) and microscopic image(middle panel, �100 magnification) and quantification of SA-�-gal-positivecells (bottom panel). D, real time PCR analysis of mRNA levels of p53 and IL-6 inLP and HP preadipocytes. E, levels of ROS in LP and HP preadipocytes weredetected by DCHF-DA assay. The fluorescent signals in these cells wereviewed by fluorescence microscopy (top panel, �100 magnification) andquantified by a fluorescence microplate reader (bottom panel). F, LP and HP3T3-L1 preadipocytes were treated with or without 167 nM insulin for 30 or 60min. The levels of phosphorylated Akt (p-Akt(Ser473)), total Akt, and �-actin inthese cells were analyzed by immunoblot assay. G, RT-PCR analysis of RAGE,adiponectin, and PPAR� in preadipocytes (D0) and differentiated adipocytes(D9) of LP and HP 3T3-L1 cells. H, immunoblot analysis of inguinal fat padisolated from 6-week-old and 1-year-old chow diet-fed male mice (left panel),and quantification of intensity of the major CML-modified protein (�55 kDa)and RAGE and p53 bands (right panel). I, immunoblot analysis of epididymalfat pads isolated from 14-week-old chow diet-fed lean and high fat diet-fedobese male mice (left panel) and quantification of intensity of the major CMLmodified protein (�55 kDa) and RAGE and aP2 bands (right panel). J, real timePCR analysis of RAGE in epididymal fat pads isolated from lean and obesemice. Bars represent mean � S.E. (n � 3–5). *, p � 0.05; **, p � 0.01; ***, p �0.001. All experiments were repeated at least 2–3 times.

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as RAGE mRNA (Fig. 2D). However, glycated BSA showed noeffect on C/EBP� in differentiated HP preadipocytes (Fig. 2C).We next examined whether pre-exposure of senescent HPpreadipocytes to glycated BSA is necessary for restoring theimpaired adipogenic potential of senescent preadipocytes. Asdepicted in Fig. 3A, a 48-h pretreatment (i.e. day�2 to day 0) ofHP preadipocytes with MG-BSA or GA-BSA resulted in anincrease of lipid droplet accumulation after 8 days of differen-tiation. We further confirmed the pro-adipogenic function ofglycated BSA in replicative senescentMEF cells and cells in theSV fraction of adipose tissues isolated from mice of differentages, such as 12 weeks old and 1 year old. Although LP MEFcells (i.e. passage 1–2) were normally differentiated with accu-mulated lipid droplets regardless of the treatments, HP MEFcells (i.e. passage 7–10) showed impaired adipogenesis asjudged by ORO staining, and this impaired adipogenic abilitywas restored when theHPMEF cells were pretreated withMG-BSAorGA-BSA for 48 h prior to induction of adipogenesis (Fig.3B). Furthermore, 1-year-old primary inguinal SV cells pre-treated withMG-BSA or GA-BSA for 48 h resulted in accumu-lation of lipid droplets after 8 days of adipogenesis, whereasglycated BSA showed no effect on adipogenesis in primaryinguinal SV cells isolated from 12-week-old mice as judged byORO staining (Fig. 3C) and gene expression analysis adipocytemarkers such as PPAR�, adiponectin, and FAS (Fig. 3D). Col-lectively, these results supported the hypothesis that AGEs

restore the adipogenic potential of aged preadipocytes both invitro and ex vivo.RAGE Is Required for the Pro-adipogenic Effect of Glycated

BSA in Senescent Preadipocytes—Our findings of the glycatedBSA-restored adipogenic potential of senescent preadipocyteswith an elevated level ofRAGE led us to hypothesize that RAGEmediates AGE-induced adipogenesis in senescent preadi-pocytes. To test this, we first confirmed the effect of glycatedBSA on the protein levels of RAGE in HP preadipocytes. Wefound that senescent HP preadipocytes treated with MG-BSAor GA-BSA for 48 h displayed an �2- or 3-fold increase inRAGE level, respectively (Fig. 4A, left panel). Immunofluores-cence of glycated BSA-treated HP preadipocytes further con-firmed induction of RAGE by glycated BSA detected predomi-nantly in the cytoplasm and nucleus (Fig. 4A, right panel). Wenext investigated whether the increase in RAGE by glycatedBSA is required for AGE-induced adipogenesis in HP preadi-pocytes. To ascertain this, we utilized neutralizing antibodyagainst RAGE.We found that glycated BSA-induced adipogen-esis of senescent HP preadipocytes was blunted by neutralizingthe antibody against RAGE (Fig. 4B). Moreover, a lentiviralknockdown of RAGEmRNA in senescent HP preadipocytes asconfirmed by PCR (Fig. 4C, left panel) and immunoblot (Fig.4C, right panel) analyses abrogated glycated BSA-induced adi-pogenesis in senescent HP 3T3-L1 cells (Fig. 4D) with reducedlevels of PPAR� and FAS after 8 days of differentiation (Fig. 4E).These findings suggested that RAGE is required for AGE-in-duced restoration of the adipogenic potential of senescentpreadipocytes.AGE-activated RAGE Binds to p53 and Inhibits p53 Expres-

sion in Senescent Preadipocytes—Given that a tumor suppres-sor p53 has been shown to modulate adipose tissue functionand senescence (40), we attempted to examine whether p53modulates AGE-RAGE-induced adipogenesis in senescentpreadipocytes. First, we examined the effect of glycated BSA onthe protein levels of p53 and p21, a downstream target of p53, inHP preadipocytes. As expected, senescent HP preadipocytestreated with glycated BSA showed an increase in RAGE (Fig.5A). This was accompanied by reduced levels of p53 and phos-phorylated p53 (Fig. 5A), suggesting a suppressive role of gly-cated BSA in p53 expression in senescent preadipocytes. Wenext investigated whether RAGE expression is required for gly-cated BSA-suppressed protein levels of p53 and p21 in senes-cent preadipocytes. As shown in Fig. 5A, a lentiviral knockdownof RAGE in senescent preadipocytes reversed glycated BSA-suppressed total and phosphorylated forms of p53 and p21.Consistent with this, glycated BSA also suppressed mRNA lev-els of p53 and p21 in control senescent preadipocytes, whereasthis was reversed in MG- or GA-BSA-treated RAGE knock-down senescent preadipocytes (Fig. 5B). Apparently, glycatedBSA-suppressed p21 expression was through altered DNAbinding ability of p53 to the promoter region of p21 in MG- orGA-BSA-treated control senescent preadipocytes as judged byChIP assay (Fig. 5C). However, RAGE knockdown resulted inincreased DNA binding ability of p53 to the p21 promoter inboth BSA- and glycated BSA-treated senescent preadipocytes(Fig. 5D). Because our finding in Fig. 4A demonstrated the pres-ence of RAGE in the cytosolic space of senescent preadipocytes,

FIGURE 2. Glycated BSA promotes adipogenesis of senescent preadi-pocytes. A, schematic of BSA or glycated BSA (MG- and GA-BSA, 300 �g/ml)treatment of LP and HP 3T3-L1 preadipocytes during an 8-day of adipogene-sis (top panel). Representative ORO-stained images of these cells (bottompanel). B, quantification of ORO-stained 3T3-L1 cells in A. Real time PCR anal-ysis (C) and RT-PCR analysis (D) of genes in HP preadipocytes differentiated inthe presence or absence of 300 �g/ml BSA or glycated BSA. Bars representmean � S.E. (n � 3). *, p � 0.05; **, p � 0.01; ***, p � 0.001. All experimentswere repeated at least 2–3 times.

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we further sought the direct binding between RAGE and p53,and the effect of glycated BSA on the RAGE-p53 binding. Ourimmunoprecipitation assay revealed a physical interactionbetween RAGE and p53 in senescent preadipocytes, and thiswas not affected by the treatment with glycated BSA (Fig. 5D).Consistent with our finding of AGE-impaired p53 function insenescent preadipocytes, differentiating senescent preadi-pocytes pretreated with different concentrations of pifithrin-�,a p53 inhibitor, for 24 h followed by a 48-h treatment withglycated BSAprior to the initiation of adipogenesis resulted in adose-dependent increase in adipogenesis as judged by OROstaining (Fig. 5E). Overall, our results indicate that AGE-RAGE-induced adipogenesis in senescent preadipocytes isthrough AGE-dependent impairment of p53 expression andp53 function in senescent preadipocytes. Interestingly, we alsodemonstrated a direct interaction between RAGE and p53 insenescent preadipocytes, but its role in adipogenesis and thefunction of senescent preadipocytes still remain elusive.

DISCUSSION

Senescent preadipocytes are known to exhibit reduced repli-cative and adipogenic capacity, increased pro-inflammatoryresponses, and susceptibility to lipotoxicity during aging (3).Muchof our current understanding of the biological function ofAGEs has been largely limited to their actions in diabetic andcardiovascular complications during aging through AGE-RAGE-induced oxidative stress and inflammation (41, 42). TheAGE-RAGE pathway has been shown to be associated withaging of humans and animals (14, 43), as well as the cellulardifferentiation programof various cell types (44, 45).Moreover,RAGE has shown to participate in high fat diet-induced

complications such as cardiac inflammation (15), athero-sclerosis (46), renal implication (17) with potential adiposity.However, the role of the AGE-RAGE axis in adipose agingremains unknown.Here, we report a novel function of AGEs in restoring

impaired adipogenic potential of senescent preadipocytesthrough RAGE-dependent modulation of p53 function viaRAGE-dependent suppression of p53 and possibly by a physicalinteraction between RAGE and p53 as depicted in Fig. 6. Ourfindings thus infer that senescent preadipocytes are likely toresume adipogenesis when these cells are exposed to diet-driven exogenous and endogenously generated glycated pro-teins through altered p53 expression and function, therebyimpacting adipose development during aging.A number of intriguing questions have been brought forth by

our findings. First, mechanisms by which RAGE, a membrane-bound protein, is found in the cytosol and interacts with p53 insenescent preadipocytes should be determined. Althoughmostof the predicted and experimentally tested proteolytic cleavagesites in RAGE are found in the extracellular domain to generatesoluble forms of RAGE, it has been reported to also be detectedin the intracellular space in various cell types such as musclesatellite cells, astrocytes, and amyloid-�-stimulated murinecortical neuron (47–49). Increasing evidence points to the roleof proteolytic cleavage in regulating the function of RAGE iso-forms in both the extracellular and intracellular space (50).Interestingly, aging-dependent truncation of RAGE and itsdetection in the cytosol of humanmuscle satellite cells has beenreported (47). Moreover, generation of the truncated form ofRAGE in aged human muscle satellite cells appears to be

FIGURE 3. Pretreatment of glycated BSA leads to an induction of adipogenesis in senescent HP 3T3-L1 preadipocytes, MEF, and inguinal SV cells. A,schematic of glycated BSA (MG- and GA-BSA, 300 �g/ml) pretreatment of HP 3T3-L1 preadipocytes prior to an 8-day adipogenesis (top panel). RepresentativeORO-stained image (middle panel) and quantification of ORO-stained HP adipocytes pretreated with BSA or glycated BSA (bottom panel) are shown. Repre-sentative microscopic image of ORO-stained LP (passage number 2) and HP (passage number 7) MEF cells (B) and primary SV cells isolated from inguinal fatpads of 12-week-old and 1-year-old chow diet-fed male mice (C) were pretreated with BSA or glycated BSA prior to induction of adipogenesis. Cells were thenstained with ORO and subjected to microscopic imaging (�100 magnification). D, RT-PCR analysis of differentiated inguinal SV cells in C. Bars represent mean �S.E. (n � 3). **, p � 0.01; ***, p � 0.001.

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dependent on proteosomal activity. Thus, it would be plausibleto assume that AGE-induced binding between RAGE and p53in senescent preadipocytes is likely through AGE-dependentproteolytic cleavage of RAGE by not-yet-knownmechanism(s).More studies are necessary to test this hypothesis.Second, although the understanding of the mechanism

underlying AGE-RAGE axis-regulated p53 expression and p53function in senescent preadipocytes remains elusive, accumu-lated evidence suggests an important role for RAGE and p53 incell proliferation and senescence. An inhibitory function ofRAGE in p53 expression and p53 function has been reported intumor and osteoblastic cells (51, 52). In linewith this, RAGEhasbeen recently proposed to regulate both autophagy and apopto-sis (53–55). Additionally, RAGE knockdown has been shown tolower degradation and ubiquitination of p53, thereby promot-ing its nuclear translocation in murine osteoblastic cells (52).

On the one hand, p53 has been known to inhibit adipogenesisand to be involved in maintaining the proper function of theadipose tissue (56) and 3T3-L1 cells (57), possibly through sup-pression of the key adipogenic transcriptional factor such asPPAR� (58). On the other hand, activated p53 is known to beinvolved in the development of early aging in adipose tissuewith reduced adipose tissue deposition and adipose inflamma-tion in animals (23, 60, 61). p53 appears to modulate adiposeaging because inhibition of p53 function in adipose tissue hasbeen reported to suppress senescence-like phenotypes withimproved insulin sensitivity (61). Collectively, these studiesindicate that future study should be directed to understandingthe role of RAGE in p53-dependent cross-talk betweenautophagy and apoptosis in adipose aging. It should also benoted that RAGE knockdown in senescent preadipocytesresulted in a slight increase in p53 protein (Fig. 5A) but adecrease in p53mRNA (Fig. 5B) in the absence of glycated BSAtreatment. Although more studies are needed to address thisdiscrepancy, this result suggests a possibility of the potentialrole of RAGE in regulating p53 protein stability.Nevertheless, our findings define a novel pro-adipogenic

function of AGEs in senescent preadipocytes. Although ourstudy is largely based on in vitro and ex vivo studies, we wouldpredict that a chronic dietary intake of AGEs would positivelycontribute to adipose development during aging. Serum AGElevels in humans appear to be influenced by diet-driven AGEintake. For example, administration of single AGE-rich dietresulted in an increase in serum AGE levels from �10 to 80units/ml in normal subjects and to �120 units/ml in diabeticsubjects after several h of ingestion, where 1 unit is defined to beequivalent to �10 �g of CML-BSA (62). Accordingly, serumAGEs levels found in this studywould be in the range of 0.8–1.2mg/ml glycated BSA. In addition, Sandu et al. (14) reported thatnormalmice fed a high fat diet containing 995.4 units/mgAGEsfor 6 months resulted in elevation of AGE levels in the serumand adipose tissue at 124 � 7 units/ml and 400 units/mg (i.e.�1.24 mg of CML-BSA/ml and �4 mg of CML-BSA/mg),respectively. Supporting this notion, a recent study demon-strated thatmice fed a standard chow diet supplementedwith 1mg of MG-BSA/g of diet for 18 months showed elevated AGElevels in the serum as well as in adipose tissue to 0.43 mg ofCML-BSA/ml and 190 nmol of MG/g of tissue, respectively(63). Collectively, these studies suggest that the AGE concen-tration we used in our study is likely to be achievable in the seraof humans and animals after a chronic oral administration of anAGE-rich diet.Recent studies showed that chronic AGE-rich standard diet

feeding studies in animals showed an increase in body weightwith no report on the change in adipose mass with elevatedinsulin resistance (14, 64, 65). Conversely, a long term of feed-ing study of mice with AGE-restricted standard diet resulted inreduced bodyweight gain with enhanced insulin sensitivity andlife span at the end of the study (66). Moreover, Monden et al.(59) andCai et al. (63) recently demonstrated a causative role ofdietary AGEs and RAGE expression in adiposity, and its relatedinsulin resistance and cellular anti-oxidant system. Cai et al.(63) further showed that oral administration of an AGE-richdiet promotes insulin resistance in mice by severe deficiency of

FIGURE 4. RAGE is required for AGE-induced adipogenesis in senescentpreadipocytes. A, immunoblot analysis (top left panel) and quantification(bottom left panel) of RAGE in LP and HP 3T3-L1 preadipocytes exposed to BSA(300 �g/ml) or MG-BSA or GA-BSA (300 �g/ml) for 48 h. Immunofluorescentimages of RAGE (green) and DAPI-stained nucleus (blue) of HP 3T3-L1 preadi-pocytes treated with or without 300 �g/ml BSA, MG-BSA, or GA-BSA for 48 h(right panel). Scale bars, 20 �m. B, ORO staining (left panel) and quantitativeORO staining analyses (right panel) of HP 3T3-L1 preadipocytes differentiatedfor 8 days after pretreatment with 300 �g/ml BSA or glycated BSA from day�2 to day 0 in the presence or absence of 10 �g/ml anti-RAGE antibody. C, HP3T3-L1 preadipocytes infected with control lentivirus (sh-Control) or lentiviruscontaining RAGE-specific shRNA (sh-RAGE) for 24 h were subjected to RT-PCRanalysis of RAGE mRNA (left panel). Immunoblot analysis of RAGE in lentivirus-infected HP 3T3-L1 preadipocytes treated with 300 �g/ml BSA, MG-BSA, orGA-BSA for 48 h (right panel). Lentivirus-infected HP preadipocytes were pre-treated with 300 �g/ml BSA, MG-BSA, or GA-BSA for 48 h followed by an 8-dayadipogenesis. These cells were subjected to ORO staining analysis (D) and realtime PCR analysis of RAGE, PPAR�, and FAS (E). Pre, preadipocytes; Adipo, adi-pocytes; N.S., not significant. Bars represent mean � S.E. (n � 3). *, p � 0.05; **,p � 0.01; ***, p � 0.001. #, p � 0.05 versus BSA. All experiments were repeatedat least 2–3 times.

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an anti-AGE advanced glycation receptor1 (AGER1) and SIRT1in peripheral tissues with impaired glucose uptake, alteredinsulin signaling pathway, and inflammatory activation in adi-

pose tissue (63). Although these elegant studies clearly indi-cated a novel pathogenic role of AGEs and an altered RAGElevel in obesity and type 2 diabetes, the direct role of the AGE-RAGE axis in aging-dependent impairment of adipogenicpotential in aged preadipocytes and its contribution to aging-related metabolic and inflammatory dysfunction are unknown.To our knowledge, our study is the first report that AGEs areable to restore the senescence-impaired adipogenic potential ofaged preadipocytes. These findings implicate that AGE-in-duced adipogenesis in senescent preadipocytes is likely to con-tribute to exacerbating aging-related adiposity.In conclusion, our finding of the pro-adipogenic function of

AGE-RAGE axis in senescent preadipocytes through a mecha-nism by which RAGE suppresses p53 function and expressionmay provide new insights into the aging-dependent adiposityby glycated proteins derived from the diet and/or chronicallydysregulated metabolic conditions such as hyperglycemia.

Acknowledgment—We thank members in the Kim laboratory forcomments.

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FIGURE 5. Binding between AGE-activated RAGE and p53 inhibits p53 expression in senescent preadipocytes. LP 3T3-L1 preadipocytes, control (sh-Control), and RAGE-knockdown (sh-RAGE) HP preadipocytes treated with or without 300 �g/ml BSA or MG-BSA/GA-BSA for 48 h were subjected to immunoblotanalysis of RAGE, phosphorylated p53 (p-p53 (Ser15)) and p21 (A), and gene expression analysis of p53 and p21 (B). ChIP assay (C) and immunoprecipitation (D)of the HP preadipocytes used in A. IP, immunoprecipitation. IB, immunoblot. E, confluent HP preadipocytes were treated with pifithrin-� (PFT-�) at 0, 10, and50 �M for 24 h followed by an exposure to 300 �g/ml BSA or MG-BSA/GA-BSA for additional 48 h in the absence of pifithrin-�. These cells were subjected toadipogenesis for 8 days. Lipid accumulation in differentiated HP preadipocytes was detected by ORO staining (left panel) and a quantitative ORO staininganalysis (right panel). Bars represent mean � S.E. (n � 3). *, p � 0.05; **, p � 0.01; ***, p � 0.001. N.S., not significant. ##, p � 0.01 and ###, p � 0.001 versus BSA.All experiments were repeated at least 2–3 times.

FIGURE 6. Proposed function of AGE-RAGE axis in adipogenesis of senes-cent preadipocytes. Activated RAGE by binding with proteins glycated withglucose derivatives in senescent preadipocytes triggers inhibition of p53function (e.g. transcriptional activation of p21) through a direct binding tocytosolic p53. However, the mechanism by which RAGE is released frommembrane to cytosol upon interaction with glycated proteins remains elu-sive. In addition, the AGE-RAGE axis results in suppression of the p53 mRNAlevel in senescent preadipocytes by an unknown mechanism. The AGE-RAGEaxis-regulated p53 function and expression, in turn, allow senescent preadi-pocytes to restore the impaired adipogenic potential.

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Pro-adipogenic Role of AGE-RAGE in Senescent Preadipocytes

DECEMBER 28, 2012 • VOLUME 287 • NUMBER 53 JOURNAL OF BIOLOGICAL CHEMISTRY 44507

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Chih-Yu Chen, Allison Martorano Abell, Yang Soo Moon and Kee-Hong Kimp53 Protein Function

Restores Adipogenic Potential of Senescent Preadipocytes through Modulation of An Advanced Glycation End Product (AGE)-Receptor for AGEs (RAGE) Axis

doi: 10.1074/jbc.M112.399790 originally published online November 13, 20122012, 287:44498-44507.J. Biol. Chem. 

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VOLUME 287 (2012) PAGES 44498 – 44507DOI 10.1074/jbc.A112.399790

An advanced glycation end product (AGE)-receptor for AGEs (RAGE) axis restores adipogenic potential of senescentpreadipocytes through modulation of p53 protein function.Chih-Yu Chen, Allison Martorano Abell, Yang Soo Moon, and Kee-Hong Kim

PAGE 44501:

Panels H, I, and J in Fig. 1 were omitted inadvertently. The correct figure is shown below.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 15, p. 10949, April 12, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

APRIL 12, 2013 • VOLUME 288 • NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 10949

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Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice ofthese corrections as prominently as they carried the original abstracts.

VOLUME 287 (2012) PAGES 44498 – 44507DOI 10.1074/jbc.A114.399790

An advanced glycation end product (AGE)-receptor for AGEs (RAGE) axis restores adipogenic potential of senescentpreadipocytes through modulation of p53 protein function.Chih-Yu Chen, Allison Martorano Abell, Yang Soo Moon, and Kee-Hong Kim

PAGES 44504 AND 44505:

Panels D and E in Fig. 4 and panel E in Fig. 5 were inadvertently omitted from the final version of the article. The missing panels have now beenprovided. This correction does not change the interpretation of the results or the conclusions.

FIGURE 4

FIGURE 5

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 16, p. 11570, April 18, 2014© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

11570 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 16 • APRIL 18, 2014

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