activation of an adipogenic program in adult myoblasts with age

Mechanisms of Ageing and Development

123 (2002) 649–661

Activation of an adipogenic program in adult myoblastswith age

Jane M. Taylor-Jones a, Robert E. McGehee b, Thomas A. Rando d,Beata Lecka-Czernik a, David A. Lipschitz a, Charlotte A. Peterson a,c,*

a Department of Geriatrics, Donald W. Reynolds Center on Aging, Uni�ersity of Arkansas for Medical Sciences,629 South Elm Street, Little Rock, AR 72205, USA

b Department of Pediatrics, Uni�ersity of Arkansas for Medical Sciences, 629 South Elm Street, Little Rock, AR 72205, USAc Central Arkansas Veterans Health Care System, Little Rock, AR 72205, USA

d GRECC, VA Palo Alto Health Care System, and Department of Neurology and Neurological Sciences,Stanford Uni�ersity School of Medicine, Stanford, CA 94305, USA

Received 13 August 2001; received in revised form 26 October 2001; accepted 14 November 2001

Abstract

Myoblasts isolated from mouse hindlimb skeletal muscle demonstrated increased adipogenic potential as a functionof age. Whereas myoblasts from 8-month-old adult mice did not significantly accumulate terminal markers ofadipogenesis regardless of culture conditions, myoblasts from 23-month-old mice accumulated fat and expressed genescharacteristic of differentiated adipocytes, such as the fatty acid binding protein aP2. This change in differentiationpotential was associated with a change in the abundance of the mRNA encoding the transcription factor C/EBP�,and in the relative abundance of PPAR�2 to PPAR�1 mRNAs. Furthermore, PPAR� activity appeared to beregulated at the level of phosphorylation, being more highly phosphorylated in myoblasts isolated from youngeranimals. Although adipogenic gene expression in myoblasts from aged animals was activated, presumably in responseto PPAR� and C/EBP�, unexpectedly, myogenic gene expression was not effectively repressed. The Wnt signalingpathway may also alter differentiation potential in muscle with age. Wnt-10b mRNA was more abundantly expressedin muscle tissue and cultured myoblasts from adult compared with aged mice, resulting in stabilization of cytosolic�-catenin, that may potentially contribute to inhibition of adipogenic gene expression in adult myoblasts. The changesreported here, together with those reported in bone marrow stroma with age, suggest that a default program may beactivated in mesenchymal cells with increasing age resulting in a more adipogenic-like phenotype. Whether thischange in differentiation potential contributes to the increased adiposity in muscle with age remains to be determined.Published by Elsevier Science Ireland Ltd.

Keywords: Skeletal muscle; Aging; Myoblasts; Myogenic differentiation; Adipogenic differentiation

www.elsevier.com/locate/mechagedev

* Corresponding author. Tel.: +1-501-526-5826; fax: +1-501-526-5817.E-mail address: [email protected] (C.A. Peterson).

0047-6374/02/$ - see front matter. Published by Elsevier Science Ireland Ltd.

PII: S 0 0 47 -6374 (01 )00411 -0

mailto:[email protected]

J.M. Taylor-Jones et al. / Mechanisms of Ageing and De�elopment 123 (2002) 649–661650

1. Introduction

Skeletal muscle repair and regeneration arelargely mediated by satellite cells, myoblasts thatreside between the sarcolemma and basal laminaof muscle fibers in postnatal muscle and remainthroughout adult life (Snow, 1977). Followingmuscle injury, satellite cells are stimulated to pro-liferate, migrate to the site of injury and fuse withexisting fibers or fuse and differentiate to formnew fibers de novo, thereby regenerating damagedor degenerating fibers (Carlson and Faulkner,1983; Grounds and Yablonka-Reuveni, 1993;Bischoff, 1994). Furthermore, during work-in-duced muscle hypertrophy, mature muscle fibersaccumulate new nuclei as myoblasts divide andsubsequently fuse with adjacent fibers (Darr andSchultz, 1987; Rosenblatt et al., 1994). Althoughsatellite cells retain their myogenic potential eveninto old age (Allen et al., 1980), deficits in satellitecell function with age have been described. My-oblasts cultured from old rats displayed a longerlag period prior to entering the cell cycle thancells from younger rats (Dodson and Allen, 1987;Johnson and Allen, 1993). Moreover, myoblastsshowed a steady decline in replicative capacity invitro as the age of the donor increased (Schultzand Lipton, 1982; Webster and Blau, 1990; Re-nault et al., 2000) that appears to be linked totelomere shortening (Decary et al., 1997). Thus,alterations in myoblast function during aging maycontribute to diminished capacity for regenerationand muscle hypertrophy.

Myoblast differentiation is regulated by themyogenic basic helix-loop-helix (bHLH) tran-scription factor family (MyoD, Myf5, myogenin,and MRF4) (Lassar et al., 1994; Sabourin andRudnicki, 2000). MyoD and Myf5 accumulate inproliferating, undifferentiated myoblasts, whereasmyogenin and MRF4 expression increase duringdifferentiation. MyoD and myogenin are overex-pressed in skeletal muscle from old animals, par-ticularly during regeneration (Marsh et al., 1997;Musaro et al., 1995). Although all four membersare capable of activating muscle-specific gene ex-pression in a variety of cell types, effectively con-verting them into myoblasts, the activity of themyogenic bHLH regulators can be overcome in

myoblasts in vitro so that they can be induced totransdifferentiate into other cells types. Treatmentof C2C12 mouse myoblasts with BMP-2 resultedin accumulation of alkaline phosphatase and cal-cification characteristic of osteoblast differentia-tion (Katagiri et al., 1994). Primary mousemyoblasts engineered to secrete BMP-2 have beenshown to be converted to an osteogenic lineageand contribute to bone formation if transplantedinto SCID mice (Lee et al., 2000). On the otherhand, myoblasts are converted to adipocyte-likecells by treatment with thiazolidinediones, potentactivators of the transcription factor peroxisomeproliferator-activated receptor � (PPAR�)(Grimaldi et al., 1997; Teboul et al., 1995). Al-though PPAR� clearly activates adipocyte-specificgene expression (Tontonoz et al., 1994a; Mandrupand Lane, 1997), it has been hypothesized thatPPAR� normally regulates fatty acid metabolismin skeletal muscle, as well as in adipose tissue(Lapsys et al., 2000). However, overexpression ofPPAR� and C/EBP�, also an activator ofadipocyte-specific genes, stimulates adipogenicdifferentiation in C2C12 myoblasts (Hu et al.,1995). More recently it was suggested that Wnt-10b may be involved in the inhibition of adipo-genic differentiation, as disruption of Wntsignaling caused transdifferentiation of myoblastsinto adipocytes (Ross et al., 2000). In the absenceof Wnt signaling, �-catenin is part of a complexthat targets it for rapid degradation (Kikuchi,2000). Wnt ligands bind to transmembrane recep-tors of the Frizzled family and in response tocanonical Wnt signaling, �-catenin is releasedfrom the complex, stabilized and translocated tothe nucleus where it regulates the transcription ofgenes that promote myogenesis (Cossu andBorello, 1999; Ridgeway et al., 2000; Hoppler etal., 1996) and inhibit adipogenesis (Ross et al.,2000). Thus, mesodermal cell fate is controlled byboth positive and negative regulatory mecha-nisms. As muscle tissue normally containsadipocytes and preadipocytes, the potential con-tribution of myoblast conversion to an adipogeniccell type that would increase fat content in mus-cle, in vivo, has not been described.

An inverse relationship between adipogenic andosteoblastic potential has been documented in the

J.M. Taylor-Jones et al. / Mechanisms of Ageing and De�elopment 123 (2002) 649–661 651

bone marrow (Beresford et al., 1992; Gimble etal., 1996; Kajkenova et al., 1997). In addition, exvivo cultures of bone marrow isolated from thesenescence accelerated mouse-P6 (SAMP6),demonstrated decreased osteoblastogenesis andincreased adipogenesis (Jilka et al., 1996). Theseresults suggest a switch in the differentiation pro-gram of multipotent mesenchymal progenitorsduring aging and provides a potential explanationfor the association of decreased bone formationwith the increased adiposity of the bone marrowseen with advancing age in animals and humans(Moore and Dawson, 1990; Robey and Bianco,1999). Although the increase in inter- and intra-muscular fat that occurs as a function of age(Pahor and Kritchevsky, 1998) likely involveschanges in muscle metabolism, most notably lessefficient fatty acid oxidation, results presentedhere suggest that altered myoblast potential mayalso contribute to increased adipogenesis in mus-cle during aging.

2. Materials and methods

2.1. Isolation and culture of primary myoblasts

Myoblasts were isolated from the tibialis ante-rior of four adult (8 month) and four aged (23month) DBA/2JNIA mice as described (Randoand Blau, 1994). Results shown are from my-oblasts pooled from groups of adult or groups ofaged mice, although comparable results were ob-tained from analysis of individual animals. Prolif-erating myoblasts were plated on collagen-coatedplates (Sigma, St. Louis, MO) and maintained inMyoblast Growth Media (MGM) containingHam’s F-10 media (BioWhittaker, Walkersville,MD) supplemented with 20% fetal bovine sera(FBS) (BioWhittaker), 0.5% pen-strep (Gibco-BRL, Grand Island, NY), and 5 ng/ml bFGF(Promega, Madison, WI). Cells were serially pre-plated to yield pure populations (�98%) of my-oblasts. For differentiation, myoblasts werecultured on E-C-L Attachment Matrix (UpstateBiotechnology, Lake Placid, NY) or Matrigel(Becton Dickinson, Bedford, MA), grown toconfluence in MGM, then switched to Myoblast

Differentiation Media (MDM) consisting ofDMEM media (GiboBRL) with 2% horse sera(Hyclone, Logan, UT) and 0.5% pen-strep. Thistreatment regimen was performed at least threetimes on each isolate. Myogenic differentiationwas assayed by myotube formation and mysoinheavy chain expression after 72 h in MDM (seebelow).

2.2. Acti�ation and assessment of the adipogenicprogram

Proliferating myoblasts were grown to �95%confluence in MGM then switched to AdipocyteInducing Media (AIM) composed of MGM sup-plemented with a cocktail of IBMX (115 mg/mliso-butylmethylxanthine (Sigma); 5×10−4 Mdexamethasone (Sigma); and 25 U/ml insulin(Novolin, Clayton, NC) (McGehee et al., 1993)for 3 days with daily feeding. On day 4, AIM wasreplaced with MGM supplemented with insulinonly. Cultures were harvested 24 h later for RNAisolation. For AIM cultures receiving rosiglita-zone (5–20 �M final concentration, GlaxoWel-come, Durham, NC), treatment began on day 2with daily feeding until harvest. Induction ofadipogenic differentiation was performed at leastthree times and using duplicate plates, adipocytedifferentiation was assessed by Oil Red-O staining(see below).

2.3. Northern analysis of RNA and RT-PCR

Myoblasts isolated from different aged micewere harvested for RNA as described (Dupont-Versteegden et al., 2000). Ten �g of total RNAwas resolved on 1% denaturing agarose gels andribosomal bands visualized with ethidium bro-mide (Gibco/BRL) using the Stratagene (La Jolla,CA) Eagle Eye imaging system for normalizationof RNA loading using Scion Image (NIH) soft-ware. Northern blotting was performed as de-scribed (Dupont-Versteegden et al., 2000) withradioactively labeled [�-dCTP 32P] (New EnglandNuclear, Boston, MA) probes, generated usingthe AMBION Decaprime II kit (Austin, TX).Hybridized filters were exposed to FUJI MedicalX-ray Film RX (Stamford, CT) and signals quan-


titated using Scion Image (NIH) software. cDNAprobes included MyoD, myogenin, and actin(Hughes et al., 1993) and lipoprotein lipase (LPL)(Holm et al., 1988). Blots shown are representa-tive and were repeated at least three times.

Using the Clontech (Palo Alto, CA) AdvantageRT-for-PCR and Advantage cDNA PCR kits,RT-PCR was performed as described in the usermanual for semi-quantitative analysis using gycer-aldehyde-3-phosphate (GAPDH) as the positivecontrol for sample normalization. Reactions werecarried out using an Applied Biosystems (Nor-walk, CT) GeneAMP PCR System 2400. Cyclenumber and annealing temperatures were empiri-cally determined for each set of primers (see be-low). PCR products were resolved on 2% agarosegels by combining GibcoBRL UltraPure agaroseand FMC (Rockland, ME) NuSieve GTG agarose1:1. To insure the amplification reactions werewithin the linear range, PCR products were visu-alized with ethidium bromide on the ChemiIm-ager Imaging System 5500 (Alpha InnotechCorporation, San Leandro, CA) to determine sig-nal saturation. Figures shown are representativeand the experiments was repeated a minimum ofthree times. Primers and PCR conditions were asfollows:

aP2(5�-GGGATTTGGTCACCATCCG, 3�-CC-AGCTTGTCACCATCTCG) GenBank cK02109, 62 °C annealing, 32 cycles, 204 bpproduct.C/EBP�(5�-GCCGCCTTCAACGACGAG, 3�-TGGCCAGGCTGTAGGTGCAT) GenbankcNM–007678, 57 °C annealing, 30 cycles, 449bp product.PPAR�1(5� -TTCTGACAGGACTGTGTGAC-AG, 3�-ATAAGGTGGAGATGCAGGTTC)(Gimble et al., 1996), 55 °C annealing, 27 cy-cles, 354 bp product.PPAR�2(5�-GCTGTTATGGGTGAAACTC-TG, 3�-same as for PPAR�1) (Gimble et al.,1996), 62 °C annealing, 35 cycles, 351 bpproduct.Wnt-10b(5�-CTGCCACTGTCGTTTCCAC-TG, 3�-AGACCCTTTCAACAACTGAACG)(personal communication, J.O. Mason, Univer-sity of Edinburgh, UK), 60 °C annealing, 32cycles, 660 bp product.

GAPDH(5�-ATTGGGAAGCTTGTCATCAA-CG, 3�-CACCCTGTTGCTGTAGCCGT) Gen-bank c32599, 60 °C annealing, 23 cycles, 781bp product.

2.4. Western analysis

Whole cell extracts were prepared as described(McGehee et al., 1993). Fifty �g protein sampleswere resolved on a 12% SDS/PAGE gel and west-ern blotting was performed as described(Kiyokawa et al., 1994). Filters were stained withPonceau S to verify that an equal mass of proteinwas loaded per lane (data not shown). Primaryantibody against PPAR� (Santa Cruz Biotechnol-ogy, Santa Cruz, CA) was diluted 1:167 and thesecondary antibody, an HRP-conjugated goatanti-rabbit (Pierce, Rockford, IL), was diluted1:5000. For �-catenin analysis, whole cell extractswere ultracentrifuged into cytosolic and mem-brane-bound fractions (Young et al., 1998). West-ern blots were performed as described aboveloading 15 �g of protein per sample. Primaryantibody against �-catenin (BD TransductionLaboratories, Lexington, KY) was diluted 1:500.The secondary antibody was the same as de-scribed for PPAR�, and used at the same concen-tration. The ChemiGlow chemiluminescentsubstrate kit (Alpha Innotech Corporation) wasused in conjunction with the ChemiImager Imag-ing System (Alpha Innotech Corporation) to visu-alize the protein bands.

2.5. Immunocytochemistry

For desmin antibody staining (Sigma), cultureswere fixed in neutral buffered formalin for 15 minfollowed by 1 h incubation with primary anti-body, diluted 1:40. The secondary antibody, anAP-conjugated rat anti rabbit IgG (Zymed, SouthSan Francisco, CA), was added at a dilution of1:200. The reaction was visualized using the Vec-tor Alkaline Phosphatase substrate Kit IV(Burlingame, CA).

Myogenic differentiation was assessed by stain-ing for myosin heavy chain using antibodyA4.1025 as described (Sarbassov et al., 1995).Lipid accumulation in myoblast cultures and in


frozen tissue sections was detected by Oil Red-Ostaining following fixation in 3.7% formaldehydeas described (Patel and Lane, 2000). In somecases, counterstaining with 3% methyl green orhematoxylin was performed. Slides were visual-ized using a Nikon E600 microscope with Cool-Snap camera (Tokyo, Japan).

2.6. Statistical analysis

Cultures were treated a minimum of three timeswith each of the differentiation regimens. North-ern, western and PCR analyses were performed atleast three times from each batch of cells. All datawere log2 transformed prior to analysis. ANOVAwas used to determine the significance (P�0.5) ofthe data from all experiments. Fold differencesare indicated in the text and are presented asmeans�S.D in the figure legends.

3. Results

Following isolation from hindlimb skeletalmuscles, primary cultures were maintained in my-oblast growth medium (MGM) to enrich for my-oblasts. The purity of the myoblast cultures wasdetermined immunocytochemically with a mono-clonal antibody recognizing desmin, an intermedi-ate-filament protein expressed specifically inmyoblasts (George-Weinstein et al., 1993). Cul-tures from the adult (8 month, Ad) and aged (23month, Ag) mice were indistinguishable morpho-logically and more than 98% of the cells weredesmin positive in each population (Fig. 1A andB). Myoblasts were induced to undergo myogenicdifferentiation, characterized by fusion into multi-nucleated myotubes, by culture in low serum (my-oblast differentiation medium, MDM). Myoblastsfrom both adult (Fig. 1C) and aged (Fig. 1D)

Fig. 1. Purification and differentiation of mouse myoblasts isolated from 8 month (A and C) and 23 month (B and D) old mice. Bothpopulations of myoblasts cultured in MGM reacted strongly with an antibody recognizing desmin (A and B). Following 3 days inmyoblast differentiation medium (MDM), myoblasts from both adult (C) and aged (D) animals had fused to form myotubes thatexpressed myosin heavy chain.


Fig. 2. Total RNA isolated from myoblasts obtained fromadult (Ad) and aged (Ag) animals prior to differentiation(MGM) and following 3 days in low serum (MDM), wereanalyzed by northern blot with the indicated probes. Myogenicmarkers were expressed at comparable levels in different agedmyoblasts, whereas LPL mRNA was overexpressed an averageof 6.22�1.37-fold in old myoblasts. 18S ribosomal RNA wasused as a control for RNA loading.

the abundance decreased during myogenic differ-entiation in both populations, LPL mRNA con-tinued to be overexpressed by more than four-foldin myoblasts from old animals. This observationprompted examination of additional markers of

animals fused efficiently and expressed high levelsof myosin heavy chain. The extent of myoblastdifferentiation was monitored at the RNA levelby northern analysis (Fig. 2). Although slightvariation in accumulation of muscle-specific geneproducts was observed between myoblast isolates,results of Analysis of Variance (ANOVA) indi-cated that no reproducible differences were appar-ent between myoblasts from adult and agedanimals in MGM or MDM (Fig. 2). MyoDmRNA abundance dropped, whereas myogeninand sarcomeric actin mRNAs increased compara-bly upon exposure to MDM (Fig. 2). Thus, thetwo populations of myoblasts possesed apparentlyequivalent myogenic potential.

One difference observed between myoblastsfrom different aged animals was in LPL mRNAabundance. LPL mRNA was expressed at morethan six-fold higher levels in myoblasts from agedthan adult animals in MGM (Fig. 2). Although

Fig. 3. Myoblasts isolated from 8-month-old mice (A) andfrom 23-month-old mice (B and C) following 24 h in MDMstained with Oil Red-O, counterstained with methyl green. OilRed-O staining, indicated by the arrows, was observed inmononucleated myoblasts (B), and in myoblasts undergoingfusion into myotubes (C) derived from the older animals.Fig. 4. Oil Red-O staining of myoblasts following culture inadipocyte inducing medium (AIM). Fat accumulation wasaugmented in myoblasts isolated from older (B) comparedwith younger (A) mice.


Fig. 5. Total RNA isolated from myoblasts obtained from 8month (Ad) and 23 month (Ag) mice following culture inMGM, AIM, and MDM were analyzed by RT-PCR. Primersets and conditions used to generate the specific productsindicated are described in the Methods. C/EBP� was 6.57�2.41-fold overexpressed in aged myoblasts in MGM. aP2mRNA accumulated to significantly higher levels in agedmyoblasts under all culture conditions (6.54�0.56, MGM;4.96�0.48, AIM; 6.15�2.13, MDM).

scripts encoding both PPAR�1 and PPAR�2 accu-mulated in myoblasts from adult and old mice (Fig.5). PPAR�1 mRNA was detected at lower cyclenumber than PPAR�2 in all cases, demonstratingthat it was more abundantly expressed. However,the relative abundance of PPAR�2 to PPAR�1 wasconsistently higher in myoblasts from older ascompared with younger animals in all cultureconditions (Fig. 5). Similarly, mRNA encoding themarker of terminally differentiated adipocytes,aP2, was expressed at more than six-fold higherlevels in myoblasts from aged animals.

Although the relative abundance of PPAR�1 toPPAR�2 may influence activity, the fact thatPPAR�1 and PPAR�2, together with C/EBP�mRNAs accumulated in myoblasts from youngeranimals in AIM, but aP2 did not, suggest thattranscription factor activity may be controlledpost-transcriptionally in this system. We attemptedto modulate PPAR� activity by treating myoblastswith the specific ligand and activator of PPAR�, thethiadolazindione rosiglitazone. At concentrationsbetween 5 and 20 �M, rosiglitazone did not increaseexpression of adipogenic markers. As shown inFigs. 5 and 6A, aP2 mRNA was highly expressedin myoblasts from old animals cultured in MGMand AIM, and addition of rosiglitazone did notaugment expression. Furthermore, rosiglitazonehad no effect on the expression of myogenic mark-ers (Fig. 6B). Myogenin mRNA was upregulated inAIM compared with MGM, although to a lesserextent than in MDM, in both adult and oldmyoblasts, and rosiglitazone did not dampen thisresponse.

adipogenesis. Oil Red-O staining to monitor accu-mulation of neutral triglycerides revealed that my-oblasts from adult animals demonstratedessentially no staining (Fig. 3A), whereas Oil Red-O positive droplets were abundant in myoblastsfrom aged animals (Fig. 3B). That triglycerideaccumulation was occurring in myoblasts, as op-posed to other contaminating cell types, was evi-denced by the fact that myoblasts undergoingfusion into myotubes were Oil Red-O positive (Fig.3C). To augment fat accumulation, myoblasts werecultured under conditions reported to induceadipogenic differentiation in preadipocyte cell lines(McGehee et al., 1993). Myoblasts from olderanimals responded to culture in the presence ofadipocyte inducing medium (AIM) by increasingtriglyceride accumulation (Fig. 4B) to a muchgreater extent than those from younger animals(Fig. 4A).

To determine if the adipogenic potential ofmyoblasts from old animals is, in fact, increased,we next examined the expression of genes encodingtranscription factors that regulate adipogenesis andterminal differentiation markers of adipocytes (Fig.5). C/EBP� mRNA was expressed at more thansix-fold higher levels in myoblasts from olderanimals compared with those from younger ani-mals in MGM. Expression was comparable inmyoblasts from different aged animals cultured inAIM and comparable but reduced in MDM. Tran-

Fig. 6. Total RNA isolated from myoblasts obtained from 8month (Ad) and 23 month (Ag) mice following culture inMGM, AIM, AIM supplemented with rosiglitazone (ros,5�M), and MDM were analyzed by RT-PCR (A) and north-ern blot (B). Neither the marker of adipocyte differentiation(aP2) nor the myogenic marker (myogenin, MyoG) was af-fected by up to 20 �M rosiglitazone. 18S ribosomal RNA wasused as a control for RNA loading.


Fig. 7. Protein extracts derived from myoblasts purified from 8 month (Ad) and 23 month (Ag) mice following culture in MGM,AIM, and MDM were analyzed by western blot using an antibody recognizing both PPAR�1 and 2. Differences in proteinabundance and phosphorylation state were apparent.

As PPAR� activity has also been shown to bemodulated by phosphorylation (Hu et al., 1996;Han et al., 2000; Lazennec et al., 2000), we nextinvestigated PPAR� by western analysis to detectdifferences in phosphorylation state. Analysis ofmyoblast protein extracts derived from adult ani-mals cultured in AIM and MDM demonstratedthat PPAR�2 was hyperphosphorylated comparedwith those from old mice, suggesting thatPPAR�2 was likely to be inactive in myoblastsfrom younger animals (Fig. 7). However, underthe other culture conditions, PPAR� phosphory-lation could not account for the fact that aP2mRNA was expressed at high levels only in oldmyoblasts (Fig. 5). This may be due to the factthat adipogenic gene expression in myoblasts notonly requires functional PPAR�, but also suffi-cient C/EBP�, which was not readily detectable inadult myoblasts cultured in MGM (Fig. 5).

Additional pathways may regulate adipogenicgene expression in primary myoblasts. Fig. 8Ashows that Wnt-10b mRNA accumulation wasinversely correlated to the expression of aP2mRNA. In myoblasts cultured in AIM, the mostpermissive environment for adipogenic differenti-ation, whereas aP2 mRNA expression was greaterin aged compared with adult myoblasts, Wnt-10bmRNA was 2.5-fold more abundant in adult com-pared with aged myoblasts. The activity of theWnt pathway was affected by altered Wnt-10bgene expression with age as demonstrated by athree-fold higher accumulation of cytosolic �-catenin, a downstream target of the Wnt pathway,in adult myoblasts (Fig. 8B). The Wnt pathwaymay also be involved in regulating adipogenicpotential in muscles in vivo with age, as Wnt-10bmRNA preferentially accumulated in muscle iso-

lated from adult compared with aged animals(Fig. 8C). In the analysis of whole muscle, thevast majority of RNA was contributed by themuscle fibers as opposed to satellite cells, suggest-

Fig. 8. Analysis of the Wnt signaling pathway. (A) Total RNAisolated from myoblasts obtained from 8 month (Ad) and 23month (Ag) mice cultured in AIM were analyzed by RT-PCRusing primers and reaction conditions specific for aP2 andWnt-10b mRNAs. aP2 and Wnt-10b mRNA abundance wasinversely correlated (aP2, 6.89�1.53 overexpressed in agedmyoblasts; Wnt-10b, 2.5�0.05 overexpressed in adult my-oblasts). (B) Protein extracts derived from myoblasts isolatedfrom 8 month (Ad) and 23 month (Ag) mice cultured in AIMwere differentially centrifuged to separate cytosolic and mem-brane fractions, followed by western blot analysis using anantibody recognizing �-catenin. Free �-catenin accumulated to2.95�0.045 higher levels in adult myoblasts. (C) Total RNAisolated from the tibialis anterior muscle of 8 month (Ad) and23 month (Ag) mice were analyzed by RT-PCR using primersand reaction conditions specific for Wnt-10b and gyceralde-hyde-3-phosphate (GAPDH) mRNAs. Differences in Wnt-10bmRNA accumulation were apparent with age.


Fig. 9. Cryostat sections of human vastus lateralis muscle froma 41-year-old (A) and a 78-year-old (B) stained with Oil Red-Oand counterstained with hematoxylin. Oil Red-O positivefibers were abundant in muscle from the older individual.

aging such that genes typical of an adipogenicphenotype are expressed. Results presented utilizedmass cultures of essentially pure and comparablymyogenic cells based on desmin, MyoD and myo-genin expression and fusion potential. Cultureswere not maintained for the time required, or underthe growth conditions to permit, a small subpopu-lation of cells to overgrow the myoblast populationand generate the magnitude of adipogenic responseobserved. Furthermore, the fact that adipogenicmarkers were expressed in myoblasts from agedanimals simultaneously undergoing myogenic fu-sion (Fig. 3), support the idea that a change inmyoblast potential had occurred with age. How-ever, we cannot rule out the possibility that themyoblast populations are heterogeneous and thatonly a subset of myoblasts isolated from agedanimals exhibited altered differentiation potential.For example, the relative contribution of the re-cently described side population of muscle-derivedstem cells (Gussoni et al., 1999; Jackson et al., 1999;Beauchamp et al., 1999; Seale et al., 2000) iscurrently unknown. These cells appear to be veryrare in muscle and, although they have beendemonstrated to be able to differentiate into othercells types dependent on location, they have onlybeen shown to give rise to satellite cells in muscle(Gussoni et al., 1999; Jackson et al., 1999; Lee etal., 2000). Clarification of this point awaits detailedclonal analysis currently underway. In any case, ourdata indicate a stable change in the in vitro differ-entiation potential of myoblasts as a function ofage. This potential is clearly influenced by a varietyof agents including growth factors, substrate, den-sity, and passage number. Thus, cues from theaging muscle environment may determine if thisprogram is activated in vivo.

Adipocyte-specific gene expression is primarilycontrolled by PPAR� and C/EBP� (Lowell, 1999;Loftus and Lane, 1997; Mandrup and Lane, 1997).Two isoforms of PPAR�, derived from differentpromoters and alternative splicing have been iden-tified (Chen et al., 1993; Tontonoz et al., 1994a;Zhu et al., 1995). Previously it has been reportedthat whereas PPAR�1 has a fairly broad tissuedistribution, PPAR�2 is restricted to adipose tissue(Mukherjee et al., 1996; Tontonoz et al., 1994b).Furthermore, a bone marrow cell line that ex-

ing that the well-documented increased accumula-tion of intra- and intermuscular fat observed inhumans with age (reviewed in Pahor andKritchevsky, 1998, and illustrated in Fig. 9) may bedue, at least in part, to a change in gene expression,regulated by crosstalk between multiple signalingpathways.

4. Discussion

Numerous experimental systems have demon-strated the potential of stem cells to give rise to avariety of differentiated cell types. This may beadvantageous in particular cases of injury or dis-ease, but would have unfavorable consequences ifstem cells resident within a tissue differentiateinappropriately. Our data suggest that myoblastpotential may change during the normal course of


pressed PPAR�1 did not express markers of ter-minal adipocyte differentiation until stably trans-fected with a PPAR�2 expression construct(Lecka-Czernik et al., 1999). We show here thatmyoblasts express both PPAR�1 and PPAR�2mRNAs. The abundance of PPAR�2 relative toPPAR�1 mRNA was highest in myoblasts fromaged animals, potentially contributing to in-creased adipogenic potential in those cells. Sur-prisingly, adipogenesis was not augmented byexposure of either adult or aged cells to rosiglita-zone, a synthetic, high affinity ligand forPPAR�s. This is in contrast to reports using amyoblast cell line and myoblasts isolated from1-day-old mice (Grimaldi et al., 1997; Teboul etal., 1995), suggesting that PPAR� activity inadult primary myoblasts may be regulated dis-tinctly. That cellular context influences PPAR�-ligand activity has been demonstrated (Camp etal., 2000), and it was hypothesized that this maybe due to variations in abundance of specificcofactors. It is possible that PPAR�2 in agedmuscle is activated by endogenous ligands andcofactors unique to, or at least overexpressed, inthat environment. In vitro, even in the apparentabsence of exogenous ligand, the combination ofPPAR� and C/EBP� expressed in aged myoblastsappears sufficient to promote adipogenic gene ex-pression.

Although adipogenic gene expression in my-oblasts from aged animals was activated, myo-genic gene expression was not effectivelyrepressed. This was unexpected, as PPAR� andC/EBP� have been shown to inhibit myogenicdifferentiation by downregulating the bHLHfamily of transcription factors that is functionallyseparate from their ability to stimulate adipogen-esis (Hu et al., 1995). Furthermore, in osteoblas-tic progenitors, PPAR�2 downregulates theosteoblast-specific transcription factor Osf2/Cbfa1, thereby inhibiting osteoblastic differentia-tion and promoting adipogenic differentiation(Lecka-Czernik et al., 1999). It is possible thatsignaling pathways in myoblasts inhibit specificfunctions of PPAR� and/or C/EBP�. For exam-ple, mitogen-activated protein kinase (MAPK)activation appears to promote myogenic differen-tiation (Sarbassov and Peterson, 1998), whereas

adipogenesis is inhibited through MAPK-medi-ated phosphorylation of PPAR� (Jaiswal et al.,2000; Hu et al., 1996; Han et al., 2000). ThatPPAR�2 appears hyperphosphorylated in my-oblasts from younger animals supports this idea.However, it was recently reported that phospho-rylation of PPARs by protein kinase A enhancedactivity (Lazennec et al., 2000), suggesting thatthe site of phosphorylation within the proteinmust be considered. The Wnt signaling pathwayhas been reported to repress adipogenesis in my-oblasts through inhibition of the expression ofPPAR� and C/EBP�, likely mediated by Wnt-10b (Ross et al., 2000). We found an inverserelationship between Wnt-10b mRNA, free �-catenin, and adipogenesis, consistent with theidea that the Wnt signaling pathway inhibits ter-minal adipocyte gene expression. However, if thisis the case, Wnt-10b regulates PPAR� and C/EBP� activity posttranslationally in these cells. Itseems equally likely that in aged myoblasts, acti-vation of PPAR� and C/EBP� activity leads todownregulation of Wnt-10b, permitting adipoge-nesis to proceed. Although the mechanism ofaction of the Wnt pathway is unclear, the factthat Wnt-10b mRNA is overexpressed in muscletissue derived from younger compared with olderanimals, suggests that downregulating the path-way with age may provide a permissive environ-ment for adipogenic gene expression indifferentiated muscle fibers, as well as in satellitecells from older animals.

In conclusion, our data suggest that a balanceexists between differentiation programs in my-oblasts, controlled by the relative abundance andactivity of an array of myogenic and adipogenictranscription factors, and that this balance ap-pears shifted with age. Whether this shift con-tributes to the increased adiposity in muscle withage remains to be determined. Given the parallelsbetween changes reported here and those thatoccur in bone marrow stroma with age, it ispossible that over time, mesenchymal stem cellpopulations are gradually replaced with cells withincreased adipogenic potential or that activationof an adipogenic gene program is common toaging mesenchymal cells.


Acknowledgements

We thank Elena Moerman for excellent techni-cal assistance and Dr Edward D. Bearden forstatistical analysis. This work was supported bygrants to C.A. Peterson from the National Insti-tutes on Aging (AG00724 and AG13009), toR.E.M. from the National Cancer Institute(CA78845) and to T.A. Rando from the Depart-ment of Veterans Affairs and the American Fed-eration for Aging Research.

References

Allen, R.E., McAllister, P.K., Masak, K.C., 1980. Myogenicpotential of satellite cells in skeletal muscle of old rats.Mech. Ageing Dev. 13, 105–109.

Beauchamp, J.R., Morgan, J.E., Pagel, C.N., Partridge, T.A.,1999. Dynamics of myoblast transplantation reveal a dis-crete minority of precursors with stem cell-like propertiesas the myogenic source. J. Cell Biol. 144, 1113–1122.

Beresford, J.N., Bennet, J.H., Devlin, C., Leboy, P.S., Owen,M.E., 1992. Evidence for an inverse relationship betweenthe differentiation of adipocytic and osteogenic cells in ratmarrow stromal cell cultures. J. Cell Sci. 102, 341–351.

Bischoff, R., 1994. Myology. In: Engel, A.G., Franzini-Arm-strong, C. (Eds.), The Satellite Cell and Muscle Regenera-tion. McGraw Hill, New York, pp. 97–118.

Camp, H.S., Li, O., Wise, S.C., Hong, Y.H., Frankowski,C.L., Shen, X., Vanbogelen, R., Leff, T., 2000. Differentialactivation of peroxisome proliferator-activated receptor-gamma by troglitazone and rosigliatazone. Diabetes 49,539–547.

Carlson, B.M., Faulkner, J.A., 1983. The regeneration ofskeletal muscle fibers following injury: a review. Med. Sci.Sports Exer. 15, 187–198.

Chen, F., Law, S., O’Malley, B.W., 1993. Identification of twomPPAR related receptors and evidence for the existence offive subfamily members. Biochem. Biophys. Res. Commun.196, 671–677.

Cossu, G., Borello, U., 1999. Wnt signaling and the activationof myogenesis in mammals. EMBO J. 18, 6867–6872.

Darr, K.C., Schultz, E., 1987. Exercise-induced satellite cellactivation in growing and mature skeletal muscle. J. Appl.Physiol. 63, 1816–1821.

Decary, S., Mouly, V., Hamida, C.B., Sautet, A., Barbet, J.P.,Butler-Browne, G.S., 1997. Replicative potential andtelomere length in human skeletal muscle: implications forsatellite cell-mediated gene therapy. Hum. Gene Ther. 8,1429–1438.

Dodson, M.V., Allen, R.E., 1987. Interaction of multiplicationstimulating activity/rat insulin-like growth factor II withskeletal muscle satellite cells during ageing. Mech. AgeingDev. 39, 121–128.

Dupont-Versteegden, E.E., Murphy, R.J.L., Houle, J.D., Gur-ley, C.M., Peterson, C.A., 2000. Mechanisms leading torestoration of muscle size with exercise and transplantationafter spinal cord injury. Am. J. Physiol. 279, C1677–C1684.

George-Weinstein, M., Foster, R.F., Gerhart, J.V., Kaufman,S.J., 1993. In vitro and in vivo expression of alpha 7integrin and desmin define the primary and secondarymyogenic lineages. Dev. Biol. 156, 209–229.

Gimble, J.M., Robinson, C.E., Wu, X., Kelly, K.A., Ro-driguez, B.R., Kliewer, S.A., Lehmann, J.M., Morris,D.C., 1996. Peroxisome proliferator-activated receptor-gamma activation by thiazolidinediones induces adipogen-esis in bone marrow stromal cells. Mol. Pharmacol. 50,1087–1094.

Grimaldi, P.A., Teboul, L., Inadera, D., Gaillard, E., Amri,Z., 1997. Trans-differentiation of myoblasts to adipoblasts:triggering effects of fatty acids and thiazolidinediones.Prostaglandins Leukotrienes Essential Fatty Acids 57, 71–75.

Grounds, M.D., Yablonka-Reuveni, Z., 1993. Molecular andcell biology of skeletal muscle regeneration. In: Partridge,T. (Ed.), Molecular and Cell Biology of Muscular Dystro-phy. Chapman & Hall, London, pp. 210–256.

Gussoni, E., Soneoka, Y., Strickland, C.D., Buzney, E.A.,Khan, M.K., Flint, A.F., Kunkel, L.M., Mulligan, R.C.,1999. Dystrophin expression in the mdx mouse restored bystem cell transplantation. Nature 401, 390–394.

Han, J., Hajjar, D.P., Tauras, J.M., Feng, J., Gotto, A.M.,Nicholson, A.C., 2000. Transforming growth factor-b1(TGF-b1) and TGF-b2 decrease expression of CD36, thetype B scavenger receptor, through mitogen-activatedprotein kinase phosphorylation of peroxisome proliferator-activated receptor-g. J. Biol. Chem. 275, 1241–1246.

Holm, C., Kirchgessner, T.G., Svenson, K.L., Fredikson, G.,Nilsson, S., Miller, C.G., Shively, J.E., Heinzmann, C.,Sparkes, R.S., Mohandas, T., et al., 1988. Hormone-sensi-tive lipase: sequence, expresion, and chromosomal localiza-tion to 19 cent-q13.3. Science 241, 1503–1506.

Hoppler, S., Brown, J.D., Moon, R.T., 1996. Expression of adominant-negative Wnt blocks induction of MyoD inXenopus embryos. Genes Dev. 10, 2805–2817.

Hu, E., Tontonoz, P., Spiegelman, B.M., 1995. Transdifferen-tiation of myoblasts by the adipogenic transcription factorsPPAR gamma and C/EBP alpha. Proc. Natl. Acad. Sci.USA 92, 9856–9860.

Hu, E., Kim, J.B., Sarraf, P., Spiegelman, B.M., 1996. Inhibi-tion of adipogenesis through MAP kinase-mediated phos-phorylation of PPARgamma. Science 274, 2100–2103.

Hughes, S.M., Taylor, J.M., Tapscott, S.J., Gurley, C.M.,Carter, W.J., Peterson, C.A., 1993. Selective accumulationof MyoD and myogenin mRNAs in fast and slow adultskeletal muscle is controlled by innervation and hormones.Development 118, 1137–1147.

Jackson, K.A., Mi, T., Goodell, M.A., 1999. Hematopoieticpotential of stem cells isolated from murine skeletal mus-cle. Proc. Natl. Acad. Sci. USA 96, 14482–14486.


Jaiswal, R.K., Jaiswal, N., Bruder, S., Mbalaviele, G., Mar-shak, D.R., Pittengers, M.F., 2000. Adult human mes-enchymal stem cell differentiation to the osteogenic oradipogenic lineage is regulated by mitogen-activatedprotein kinase. J. Biol. Chem. 275, 9645–9652.

Jilka, R.L., Weinstein, R.S., Takahashi, K., Parfitt, A.M.,Manolagas, S.C., 1996. Linkage of decreased bone masswith impaired osteoblastogenesis in a murine model ofaccelerated senescence. J. Clin. Invest. 97, 1732–1740.

Johnson, S.E., Allen, R.E., 1993. Proliferating cell nuclearantigen (PCNA) is expressed in activated rat skeletal mus-cle satellite cells. J. Cell. Physiol. 154, 39–43.

Kajkenova, O., Lecka-Czernik, B., Gubrij, I., Hauser, S.P.,Takahashi, K., Parfitt, A.M., Jilka, R.L., Manolagas, S.C.,Lipschitz, D.A., 1997. Increased adipogenesis andmyelopoiesis in the bone marrow of SAMP6, a murinemodel of defective osteoblastogenesis and low turnoverosteopenia. J. Bone Min. Res. 12, 1772–1779.

Katagiri, T., Yamaguchi, A., Komaki, M.E.A., 1994. Bonemorphogenetic protein-2 converts the differentiation path-way of C2C12 myoblasts into the osteoblast lineage. J. CellBiol. 127, 1755–1766.

Kikuchi, A., 2000. Regulation of b-catenin signaling in theWnt pathway. Biochem. Biophys. Res. Comm. 268, 243–248.

Kiyokawa, H., Richon, V.M., Rifkind, R.A., Marks, P.A.,1994. Supression of cyclin-dependent kinase 4 during in-duced differentiation of erythroleukemia cells. Mol. CellBiol. 11, 7195–7203.

Lapsys, N.M., Kriketos, A.D., Lim-Fraser, M., Poynten,A.M., Lowy, A., Furler, S.M., Chisholm, D.J., Cooney,G.J., 2000. Expression of genes involved in lipidmetabolism correlate with peroxisome proliferator-acti-vated receptor gamma expression in human skeletal mus-cle. J. Clin. Endocrinol. Metab. 85, 4293–4297.

Lassar, A.B., Skapek, S.X., Novitch, B., 1994. Regulatorymechanisms that coordinate skeletal muscle differentiationand cell cycle withdrawal. Curr. Opin. Cell Biol. 6, 788–794.

Lazennec, G., Canaple, L., Saugy, D., Wahli, W., 2000. Acti-vation of peroxisome proliferator-activated receptors(PPARs) by their ligands and protein kinase A activators.Mol. Endocrinol. 14, 1962–1975.

Lecka-Czernik, B., Gubril, I., Moerman, E.J., Kajkenova, O.,Lipschitz, D.A., Manolagas, S.C., Jilka, R.L., 1999. Inhibi-tion of Osf2/Cbfa1 expression and terminal osteobast dif-ferentiation by PPARgamma2. J. Cell Biochem. 74,357–371.

Lee, J.Y., Qu-Petersen, Z., Cao, B., Kimura, S., Jankowski,R., Cummins, J., Usas, A., Gates, C., Robbins, P., Wernig,A., Huard, J., 2000. Clonal isolation of muscle-derivedcells capable of enhancing muscle regeneration and bonehealing. J. Cell Biol. 150, 1085–1100.

Loftus, T.M., Lane, M.D., 1997. Modulating the transcrip-tional control of adipogenesis. Cur. Opin. Gen. Dev. 7,603–608.

Lowell, B.B., 1999. PPARgamma: an essential regulator ofadipogenesis and modulator of fat cell function. Cell 99,239–242.

Mandrup, S., Lane, M.D., 1997. Regulating adipogenesis. J.Biol. Chem. 272, 5367–5370.

Marsh, D.R., Criswell, D.S., Carson, J.A., Booth, F.W., 1997.Myogenic regulatory factors during regeneration of skele-tal muscle in young, adult and old rats. J. Appl. Physiol.83, 1270–1275.

McGehee, R.E., Ron, D., Brasier, A.R., Habener, J.F., 1993.Differentiation-specific element: a cis-acting developmentalswitch required for the sustained transcriptional expressionof the angiotensinogen gene during hormonal-induced dif-ferentiation of 3T3-L1 fibroblasts to adipocytes. Mol. En-docrinol. 7, 551–560.

Moore, S.G., Dawson, K.L., 1990. Red and yellow marrow inthe femur: age-related changes in appearance at MR imag-ing. Radiology 175, 219–223.

Mukherjee, R., Jow, L., Croston, G.E., Paterniti, J.R., 1996.Identification,characterization,and tissue distribution ofhuman peroxisome proliferator-activated receptor (PPAR)isoforms PPARgamma2 versus PPARgamma1 and activa-tion with retinoid X receptor agonists and antagonists. J.Biol. Chem. 272, 8071–8076.

Musaro, A., Cusella de Angelis, M.G., Germani, A., Cic-carelli, C., Molinaro, M., Zani, B.M., 1995. Enhancedexpression of myogenic regulatory genes in aging skeletalmuscle. Exp. Cell Res. 221, 241–248.

Pahor, M., Kritchevsky, S., 1998. Research hypotheses onmuscle wasting, aging, loss of function and disability. J.Nutr. Health Aging 2, 97–100.

Patel, Y.M., Lane, M.D., 2000. Mitotic clonal expansionduring preadipocyte differentiation: calpain-mediatedturnover of p27. J. Biol. Chem. 275, 17653–17660.

Rando, T.A., Blau, H.M., 1994. Primary mouse myoblastpurification, characterization, and transplantation for cell-mediated gene therapy. J. Cell Biol. 125, 1275–1287.

Renault, V., Piron-Hamelin, G., Forestier, C., DiDonna, S.,Decary, S., Hentati, F., Saillant, G., Butler-Browne, G.S.,Mouly, V., 2000. Skeletal muscle regeneration and themitotic clock. Exp. Gerontol. 35, 711–719.

Ridgeway, A.G., Petropoulos, H., Wilton, S., Skerjanc, I.S.,2000. Wnt signaling regulates the function of MyoD andmyogenin. J. Biol. Chem. 275, 32398–32405.

Robey, P.G., Bianco, P., 1999. Cellular mechanisms of age-re-lated bone loss. In: Rosen, C., Bilezikan, J.P. (Eds.), TheAging Skeleton. Academic Press, San Diego, CA Ref Type:Serial (Book, Monograph).

Rosenblatt, J.D., Yong, D., Parry, D.J., 1994. Satellite cellactivity is required for hypertrophy of overloaded adult ratmuscle. Muscle Nerve 17, 608–613.

Ross, S.E., Hemati, N., Longo, K.A., Bennett, C.N., Lucas,P.C., Erickson, R.L., MacDougald, O.A., 2000. Inhibitionof adipogenisis by Wnt signaling. Science 289, 950–953.

Sabourin, L.A., Rudnicki, M.A., 2000. The molecular regula-tion of myogenesis. Clin. Genet. 57, 16–25.


Sarbassov, D.D., Peterson, C.A., 1998. Insulin receptor sub-strate-1 and phosphatidylinosital 3-kinase regulate extra-cellular signal-regulated kinase-dependent and-independent signaling pathways during myogenic differen-tiation. Mol. Endocrinol. 12, 1870–1878.

Sarbassov, D.D., Stefanova, R., Grigoriev, V.G., Peterson,C.A., 1995. Role of insulin-like growth factors and myo-genin in the altered program of proliferation and differenti-ation in NFB4 mutant muscle cell line. Proc. Natl. Acad.Sci. USA 92, 10874–10878.

Schultz, E., Lipton, B.H., 1982. Skeletal muscle satellite cells:changes in proliferation potential as a function of age.Mech. Ageing Dev. 20, 377–383.

Seale, P., Sabourin, L.A., Girgis-Gabardo, A., Mansouri, A.,Gruss, P., Rudnicki, M.A., 2000. Pax7 is required for thespecification of myogenic satellite cells. Cell 102, 777–786.

Snow, M.H., 1977. The effect of aging on satellite cells isskeletal muscles of mice and rats. Cell Tissue Res. 185,399–408.

Teboul, L., Gaillard, D., Staccini, L., Inadera, H., Ez-Zoubir,A., Grimaldi, P.A., 1995. Thiazolidinediones and fatty

acids convert myogenic cells into adipose-like cells. J. Biol.Chem. 270, 28183–28187.

Tontonoz, P., Hu, E., Graves, R.A., Budavari, A.I., Spiegel-man, B.M., 1994a. mPPARg2: tissue specific regulator ofan adipocyte enhancer. Genes Dev. 8, 1224–1234.

Tontonoz, P., Hu, E., Spiegelman, B.M., 1994b. Stimulationof adipogenesis in fibroblasts by PPARg2, a lipid-activatedtranscription factor. Cell 79, 1147–1156.

Webster, C., Blau, H.M., 1990. Accelerated age-related declinein replicative life-span of Duchenne muscular dystrophymyoblasts: implications for cell and gene therapy. Som.Cell Mol. Gen. 16, 557–565.

Young, C.S., Kitamura, M., Hardy, S., Kitajewksi, J., 1998.Wnt-1 induces growth, cytosolic beta-catenin, and Tcf/Leftranscriptional activation in Rat-1 fibroblasts. Mol. Cell.Biol. 18, 2474–2485.

Zhu, Y., Qi, C., Korenberg, J.R., Chen, X.N., Noya, D., Rao,M.S., Reddy, J.K., 1995. Structural organization of mouseperoxisome proliferator-activated receptor gamma(mP-PAR) gene: alternative promoter use and different splicingyield two mPPARgamma isoforms. Proc. Natl. Acad. Sci.USA 92, 7921–7925.

activation of an adipogenic program in adult myoblasts with age

Documents