HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 18/12/1477 04-1944fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MATSUSUE, K. Articles by GONZALEZ, F. J. Search for Related Content PubMed PubMed Citation Articles by MATSUSUE, K. Articles by GONZALEZ, F. J.

PPARß/ potentiates PPAR-stimulated adipocyte differentiation

KIMIHIKO MATSUSUE^*,1, JEFFREY M. PETERS^,1 and FRANK J. GONZALEZ^*,2

^* Laboratory of Metabolism, National Cancer Institute, Bethesda, Maryland, USA; and
Department of Veterinary Science and The Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, Pennsylvania, USA

²Correspondence: Laboratory of Metabolism Building 37, Room 3106 National Cancer Institute Bethesda, MD 20892, USA. E-mail: fjgonz{at}helix.nih.gov

SPECIFIC AIMS

Based on evidence from genetic and pharmacological studies there is strong evidence that PPAR has a central role in mediating adipocyte differentiation, yet some reports suggest that PPARß may be involved in this pathway. To specifically determine the role of PPARß in adipocyte differentiation, preadipocytes derived from wild-type and PPARß null mice were examined.

PRINCIPAL FINDINGS

1. Adipocytes from wild-type mice exhibited morphology consistent with adipocyte terminal differentiation whereas adipocytes from PPARß null mice failed to differentiate after 7 days of culture in standard differentiation medium
Morphological appearance of adipocytes cultured in standard differentiation medium with a PPAR ligand showed that troglitazone significantly enhanced lipid accumulation in wild-type cells compared with cells cultured in the absence of troglitazone, and this effect was marginally reduced in PPARß null adipocytes. The morphological appearance of adipocytes cultured in standard differentiation medium with PPARß ligand L165041 revealed minimal differences in overt lipid accumulation in wild-type cells compared with cells cultured in the absence of L165041, while no differences between PPARß null adipocytes were observed in the presence or absence of L165041. Morphological changes observed in cultured adipocytes were also confirmed by Oil Red O staining and quantification of intracellular triglycerides.

2. To determine whether the expression of mRNAs encoding proteins that regulate terminal differentiation of adipocytes contribute to the observed differences in adipocyte morphology, lipid accumulation and apparent differentiation between wild-type and PPARß null adipocytes, Northern blot analysis was performed using RNA from preadipocytes and adipocytes cultured in the absence or presence of the standard differentiation medium, with and without a PPARß or PPAR ligand
PPARß null adipocytes exhibited changes in mRNAs encoding markers of adipocyte differentiation in response to standard differentiation medium that were consistent with the morphological differences (Oil Red O staining and triglyceride accumulation). Treatment of wild-type adipocytes with the standard differentiation medium and the PPARß ligand L165041, resulted in enhanced expression of mRNAs known to be involved in adipocyte differentiation and this effect was significantly diminished in PPARß null cells (Fig. 1 ). Treatment of wild-type adipocytes with standard differentiation medium and PPAR ligand troglitazone, resulted in accelerated and enhanced expression of mRNAs known to be involved in adipocyte differentiation and this effect was only partially reduced in the PPARß null cells.

View larger version (112K): [in this window] [in a new window]   Figure 1. Expression of adipocyte markers is impaired in PPARß null adipocytes in response to differentiation signals. Wild-type (+/+) and PPARß null (–/–) adipocytes were cultured in the absence (A) or presence (B) of standard differentiation medium including IDX, with and without 0.5 µM L165041 or 10 µM troglitazone (TGZ). RNA was isolated before (as preadipocytes of low confluence) and 0, 1, 2, 3, 5, and 7 days after adding inducers. Quantification of hybridization signals was performed using a PhosphorImager and are expressed as the fold change (after normalization with 18 s levels) relative to band in wild-type adipocytes (underlined). The exposure time is 72 h (A) or 36 h (B).

CONCLUSIONS AND SIGNIFICANCE

PPAR and PPARß have both been implicated in molecular signaling that mediates adipocyte differentiation. Whereas the role of PPAR is well established in this process, the specific role of PPARß is less certain. The current study reveals that in presence of standard differentiation medium, PPARß is required for maximal adipocyte differentiation as PPARß null adipocytes exhibit significantly impaired lipid accumulation and expression of adipose differentiation marker mRNAs. This suggests that in the absence of PPARß expression, PPAR and other factors required for adipogenesis (e.g., C/EBPs, etc.) have impaired signaling for maximal terminal differentiation. Co-treatment of adipocytes with standard differentiation medium and PPARß ligand L165041 increased terminal differentiation in wild-type adipocytes. This is consistent with previous reports demonstrating a similar phenotype in response to PPARß activation by fatty acids and L165041. Since these effects were absent in PPARß null adipocytes, this shows that PPARß is required to mediate, in part, changes in gene expression associated with adipocyte differentiation and lipid accumulation. Treatment of wild-type adipocytes with the PPARß ligand L165041 did not enhance adipocyte differentiation in the absence of standard differentiation medium, and terminal differentiation of embryonic fibroblasts was not induced by L165041. These results are consistent with the lack of induction of aP2 in differentiating 3T3-L1 cells in response to L165041 at concentrations below 30 µM that specifically activate PPARß. This suggests that other factors in the culture medium are required to facilitate adipocyte differentiation, and are modulated by PPARß signaling. It is possible that increased activity of cAMP-dependent pathways contribute to this effect, as suggested by others. These results show that PPARß is required for maximal differentiation of adipocytes through mechanisms that might include interactions with PPAR or C/EBPs. The significantly impaired adipocyte differentiation observed in PPARß null cells in the present study suggests that reduced adiposity found in PPARß null mice may be due in part to reduced differentiation of adipocytes during early development.

Adipocyte differentiation induced by activation of PPAR is markedly influenced by PPARß. Since expression of PPAR is significantly lower in PPARß null adipocytes, these results support the idea that direct or indirect regulation of PPAR by PPARß may in part explain the observed differences in phenotype and suggest that PPARß is required for maximal induction of differentiation induced by activation of PPAR. Others have suggested that PPARß can function to repress PPAR- or PPAR-mediated alterations in target gene expression. Results from the present studies are inconsistent with this idea, as ligand activation of PPAR in the absence of PPARß expression did not lead to enhanced expression of any of the PPAR target genes. Ligand activation of PPAR in PPARß null adipocytes, typically led to lower or similar expression, of PPAR target genes (aP2, perilipin, ADRP and CD36). While increased expression of PPARß in cell lines can lead to inhibition of PPAR target genes in some cases, deletion of PPARß is not necessarily associated with enhanced expression of PPAR target genes. The reason for this disparity is uncertain but could be due to differences in cell lines vs. primary cells used for the present analysis.

Others have shown increased expression of aP2, CD36/FAT and ADRP mRNA in response to either PPAR or PPARß activation. The present studies are consistent with these results, as expression of aP2, CD36/FAT, ADRP, and perilipin mRNA were all increased in differentiating adipocytes in response to ligand activation of both PPARß and PPAR in the presence of differentiation-conditioned culture medium. Expression of adipocyte marker mRNAs was markedly accelerated, and enhanced, in response to ligand activation of PPAR compared with cells treated with L165041. Increased expression of aP2, CD36/FAT, perilipin, and ADRP were significantly reduced in PPARß null adipocytes as a result of treatment with either L165041 or troglitazone. These results show that while PPARß participates in mediating changes in target genes associated with adipocyte differentiation, increased expression still occurs in the absence of PPARß; albeit at relatively lower levels. As troglitazone was more effective at inducing differentiation in wild-type cells than PPARß null cells, and ligand activation of PPARß was less effective at inducing differentiation, this suggests that PPAR has a more predominant role in mediating these alterations in gene expression. Activation of PPARß in adipocytes in the absence of standard differentiation medium did not increase mRNAs encoding proteins required for terminal differentiation in either genotype. The influence of PPARß is indicated by the finding that ligand activation of PPAR in the absence of standard differentiation medium markedly increased expression of PPAR, ADRP, and aP2 wild-type adipocytes, which was significantly lower in PPARß null adipocytes. Terminal differentiation of embryonic fibroblasts was not induced in either genotype by L165041 but was induced in wild-type mice by troglitazone, an effect that was significantly reduced in the absence of PPARß expression. These results demonstrate that PPARß is required for maximal adipocyte differentiation, PPAR has a more prominent role in this process, and PPARß is required for optimal expression of PPAR in adipocytes.

To account for one possible role of PPARß in adipocyte differentiation, it was suggested that PPARß could indirectly regulate PPAR expression, which in turn modulates changes in gene expression required for differentiation. Results from the current studies support this idea as expression of PPAR is significantly lower in PPARß null cells and induction of adipogenic marker mRNAs, known to be regulated at least in part by PPAR, is significantly lower in PPARß null adipocytes. While regulation of PPAR expression is not well characterized, it is known that C/EBP binding sites are located in the promoter of PPAR2 and expression of PPAR can be increased by co-transfection of either C/EBP or C/EBP. C/EBP mRNA levels are also significantly lower in PPARß null adipocytes after treatment with standard differentiation medium, which suggests that regulation of C/EBP may be indirectly influenced by PPARß. Further work is necessary to determine how PPARß modulates expression of these two transcription factors with critical roles in adipocyte differentiation, but evidence from these studies support the idea that PPARß influences expression of both PPAR and C/EBP.

It has been suggested that PPARß may regulate adipogenesis by modulating clonal expansion prior to terminal adipocyte differentiation as ligand activation of PPARß causes increased cell proliferation of preadipocytes. PPAR-retinoblastoma (Rb) -histone deacetylase 3 (HDAC3) complex was shown to modulate PPAR activity and adipocyte differentiation, as inhibition of HDAC3 activity stimulates this PPAR-dependent event. Although not examined in this work, it is possible that PPARß interacts in this regulation by unknown mechanisms, possibly through Rb or HDAC3-dependent pathways that, in turn, modulate PPAR transcriptional activity required for adipocyte differentiation.

Results from these studies demonstrate a requirement for PPARß in adipocyte differentiation and show that ligand activation of PPARß in the presence of standard differentiation medium causes differentiation mediated in part by PPARß. As the level of differentiation induced by activation of PPAR was substantially reduced in the absence of PPARß expression, the results support the idea that PPARß is required for maximal stimulation of adipogenesis. It is clear that PPARß and PPAR interact in mediating adipocyte differentiation through molecular pathways that converge with the regulation of similar target genes, and likely through other unidentified independent pathways (Fig. 2 ).

View larger version (23K): [in this window] [in a new window]   Figure 2. Evidence is provided showing that PPAP-mediated adipocyte differentiation is potentiated by PPARß, suggesting that PPARß could modulate PPAR expression and/or PPAR target genes required for adipocyte differentiation.

FOOTNOTES

¹ These authors contributed equally to the work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1944fje; doi: 10.1096/fj.04-1944fje

This article has been cited by other articles:

				Y. H. Yu, S. C. Wu, W. T. K. Cheng, H. J. Mersmann, and S. T. Ding Ectopic expression of porcine peroxisome proliferator-activated receptor {delta} regulates adipogenesis in mouse myoblasts J Anim Sci, January 1, 2008; 86(1): 64 - 72. [Abstract] [Full Text] [PDF]

				J. A. Jorgensen, D. Zadravec, and A. Jacobsson Norepinephrine and rosiglitazone synergistically induce Elovl3 expression in brown adipocytes Am J Physiol Endocrinol Metab, November 1, 2007; 293(5): E1159 - E1168. [Abstract] [Full Text] [PDF]

				L. Andrulionyte, P. Peltola, J.-L. Chiasson, M. Laakso, and for the STOP-NIDDM Study Group Single Nucleotide Polymorphisms of PPARD in Combination With the Gly482Ser Substitution of PGC-1A and the Pro12Ala Substitution of PPARG2 Predict the Conversion From Impaired Glucose Tolerance to Type 2 Diabetes: The STOP-NIDDM Trial. Diabetes, July 1, 2006; 55(7): 2148 - 2152. [Abstract] [Full Text] [PDF]

				H. E. Marin, M. A. Peraza, A. N. Billin, T. M. Willson, J. M. Ward, M. J. Kennett, F. J. Gonzalez, and J. M. Peters Ligand Activation of Peroxisome Proliferator-Activated Receptor {beta} Inhibits Colon Carcinogenesis. Cancer Res., April 15, 2006; 66(8): 4394 - 4401. [Abstract] [Full Text] [PDF]

				O. S. Gardner, B. J. Dewar, and L. M. Graves Activation of Mitogen-Activated Protein Kinases by Peroxisome Proliferator-Activated Receptor Ligands: An Example of Nongenomic Signaling Mol. Pharmacol., October 1, 2005; 68(4): 933 - 941. [Abstract] [Full Text] [PDF]

				D. J. Kim, I. A. Murray, A. M. Burns, F. J. Gonzalez, G. H. Perdew, and J. M. Peters Peroxisome Proliferator-activated Receptor-{beta}/{delta} Inhibits Epidermal Cell Proliferation by Down-regulation of Kinase Activity J. Biol. Chem., March 11, 2005; 280(10): 9519 - 9527. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 18/12/1477 04-1944fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MATSUSUE, K. Articles by GONZALEZ, F. J. Search for Related Content PubMed PubMed Citation Articles by MATSUSUE, K. Articles by GONZALEZ, F. J.