Anti-diabetic drugs inhibit obesity-linked phosphorylation of PPARγ by Cdk5

Journal name:
Nature
Volume:
466,
Pages:
451–456
Date published:
DOI:
doi:10.1038/nature09291
Received
Accepted

Abstract

Obesity induced in mice by high-fat feeding activates the protein kinase Cdk5 (cyclin-dependent kinase 5) in adipose tissues. This results in phosphorylation of the nuclear receptor PPARγ (peroxisome proliferator-activated receptor γ), a dominant regulator of adipogenesis and fat cell gene expression, at serine 273. This modification of PPARγ does not alter its adipogenic capacity, but leads to dysregulation of a large number of genes whose expression is altered in obesity, including a reduction in the expression of the insulin-sensitizing adipokine, adiponectin. The phosphorylation of PPARγ by Cdk5 is blocked by anti-diabetic PPARγ ligands, such as rosiglitazone and MRL24. This inhibition works both in vivo and in vitro, and is completely independent of classical receptor transcriptional agonism. Similarly, inhibition of PPARγ phosphorylation in obese patients by rosiglitazone is very tightly associated with the anti-diabetic effects of this drug. All these findings strongly suggest that Cdk5-mediated phosphorylation of PPARγ may be involved in the pathogenesis of insulin-resistance, and present an opportunity for development of an improved generation of anti-diabetic drugs through PPARγ.

At a glance

Figures

  1. Specific fat cell gene dysregulation by the Cdk5-mediated S273 phosphorylation of PPAR[ggr].
    Figure 1: Specific fat cell gene dysregulation by the Cdk5-mediated S273 phosphorylation of PPARγ.

    a, In vitro CDK assays performed using Cdk5/p35 with either WT or S273A mutated PPARγ. IB, immunoblot. Prefix p indicates phosphorylated moiety. b, Phosphorylation of PPARγ in differentiated 3T3-L1 adipocytes stimulated with TNF-α for the indicated times. c, Phosphorylation of PPARγ in cells expressing scrambled (Scr.) or CDK5 shRNA stimulated with indicated cytokines. NT, no treatment. d, Staining of PPARγ-null fibroblasts expressing WT or S273A mutant PPARγ with Oil Red O. Diff., differentiated. e, Gene expression in these cells was analysed by real-time quantitative PCR (qPCR) for expression of various genes (n = 3). f, mRNA expression in transplanted fat pads was analysed by qPCR (n = 5). Error bars, s.e.m.; *P<0.05, **P<0.01.

  2. Cdk5-mediated phosphorylation of PPAR[ggr] is increased in fat tissues of mice fed a high-fat diet.
    Figure 2: Cdk5-mediated phosphorylation of PPARγ is increased in fat tissues of mice fed a high-fat diet.

    a, White adipose tissue (epididymal) from mice on high-fat diet (HFD) for the indicated time was analysed with pSer273 PPARγ and PPARγ antibodies. b, Epididymal (Epi.) or inguinal (Ing.) fat tissue from 13weeks HFD mice was analysed with pSer273 antibody.

  3. Anti-diabetic PPAR[ggr] ligands block CDK5-mediated phosphorylation of PPAR[ggr].
    Figure 3: Anti-diabetic PPARγ ligands block CDK5-mediated phosphorylation of PPARγ.

    a, TNF-α-induced phosphorylation of PPARγ in 3T3-L1 adipocytes expressing either WT or Q286P mutant of PPARγ treated with rosiglitazone and/or GW9662. b, c, In vitro CDK5 assay with either rosiglitazone or MRL24. d, Transcriptional activity of a PPAR-derived reporter gene in response to rosiglitazone or MRL24 (n = 3). e, Microarray analysis of differentiated PPARγ-null fibroblasts expressing WT (NT, rosiglitazone or MRL24 treated) or S273A mutant PPARγ. f, mRNA expression of genes regulated by the phosphorylation of PPARγ in epididymal fat tissue of mice fed with either chow or HFD for 13weeks (n = 5). Error bars, s.e.m.; *P<0.05, **P<0.01, ***P<0.001.

  4. Differential HDX mass spectrometry data for PPAR[ggr]-ligand-binding domain (LBD) with and without rosiglitazone and MRL24.
    Figure 4: Differential HDX mass spectrometry data for PPARγ-ligand-binding domain (LBD) with and without rosiglitazone and MRL24.

    a, Histograms showing the per cent reduction in HDX for helix 3 (H3; IRIFQGCQF), the β-sheet region (ISEGQGFMTRE), helix 12 (H12; QEIYKDLY) and the helix 2–helix 2′ link region containing the site of CDK5 phosphorylation (H2–H2′; KTTDKSPFVIYDM). Values are calculated relative to the measured deuterium (D) value (%) for apo PPARγ-LBD (n = 4; error bars, s.e.m.; **P<0.01, ***P<0.001). b, HDX data for the four peptides of interest are plotted over the structures of PPARγ-LBD bound with rosiglitazone (left, PDB 2PRG) and MRL24 (right, PDB 2Q5P). Percentage reduction in HDX relative to unliganded receptor is coloured according to the key. Red circles, Ser273 residue of PPARγ.

  5. Correlation between the inhibition of phosphorylation and improvement of insulin sensitivity by anti-diabetic PPAR[ggr] ligands.
    Figure 5: Correlation between the inhibition of phosphorylation and improvement of insulin sensitivity by anti-diabetic PPARγ ligands.

    a, Glucose-tolerance tests in 16-week HFD mice treated with vehicle, rosiglitazone or MRL24 (n = 10). b, Phosphorylation of PPARγ in white adipose tissue (WAT). c, The expression of gene sets regulated by PPARγ phosphorylation in WAT (error bars, s.e.m.; *P<0.05, **P<0.01, ***P<0.001). d, Changes of phosphorylation of PPARγ in human fat biopsies from patients that were treated with rosiglitazone for 6 months. ‘Before’, non-treated; ‘after’, 6 months treatment. e, Correlation between the changes of PPARγ phosphorylation normalized to total PPARγ protein and the changes of glucose infusion rate measured by clamp. The data are presented as percentage change after 6 months of rosiglitazone treatment.

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Gene Expression Omnibus

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Author information

  1. These authors contributed equally to this work.

    • Alexander S. Banks,
    • Jennifer L. Estall &
    • Shingo Kajimura

Affiliations

  1. Department of Cancer Biology and Division of Metabolism and Chronic Disease, Dana-Farber Cancer Institute and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Jang Hyun Choi,
    • Alexander S. Banks,
    • Jennifer L. Estall,
    • Shingo Kajimura,
    • Pontus Boström,
    • Dina Laznik,
    • Jorge L. Ruas &
    • Bruce M. Spiegelman
  2. Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, Florida 33458, USA

    • Michael J. Chalmers,
    • Theodore M. Kamenecka &
    • Patrick R. Griffin
  3. Department of Medicine, University of Leipzig, Liebigstr. 20, Leipzig, Germany

    • Matthias Blüher

Contributions

J.H.C. and B.M.S. conceived and designed the experiments. J.H.C., A.S.B., J.L.E., S.K., P.B., D.L., J.L.R., M.J.C., T.M.K, M.B. and P.R.G performed the experiments. All authors analysed the data. J.H.C., A.S.B., J.L.E. and B.M.S. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Correspondence to:

Microarray data have been deposited in Gene Expression Omnibus: GSE22033.

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    This file contains Supplementary Figures 1-16 with legends and Supplementary Tables 1-4.

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