The therapeutic potential of inhibiting PPARγ phosphorylation to treat type 2 diabetes

A promising approach for treating type 2 diabetes mellitus (T2DM) is to target the Peroxisome Proliferator-Activated Receptor γ (PPARγ) transcription factor, which regulates the expression of proteins critical for T2DM. Mechanisms involved in PPARγ signaling are poorly understood, yet globally increasing T2DM prevalence demands improvements in drug design. Synthetic, nonactivating PPARγ ligands can abolish the phosphorylation of PPARγ at Ser273, a posttranslational modification correlated with obesity and insulin resistance. It is not understood how these ligands prevent phosphorylation, and the lack of experimental mechanistic information can be attributed to previous ambiguity in the field as well as to limitations in experimental approaches; in silico modeling currently provides the only insight into how ligands block Ser273 phosphorylation. The future availability of experimental evidence is critical for clarifying the mechanism by which ligands prevent phosphorylation and should be the priority of future T2DM-focused research. Following this, the properties of ligands that enable them to block phosphorylation can be improved upon to generate ligands tailored for blocking phosphorylation and therefore restoring insulin sensitivity. This would represent a significant step forward for treating T2DM. This review summarizes current knowledge of the roles of PPARγ in T2DM as well as the effects of synthetic ligands on the modulation of these roles. We hypothesize potential factors that contribute to the reduction in recent developments and summarize what has currently been done to shed light on this critical field of research. A promising approach for treating type 2 diabetes mellitus (T2DM) is to target the Peroxisome Proliferator-Activated Receptor γ (PPARγ) transcription factor, which regulates the expression of proteins critical for T2DM. Mechanisms involved in PPARγ signaling are poorly understood, yet globally increasing T2DM prevalence demands improvements in drug design. Synthetic, nonactivating PPARγ ligands can abolish the phosphorylation of PPARγ at Ser273, a posttranslational modification correlated with obesity and insulin resistance. It is not understood how these ligands prevent phosphorylation, and the lack of experimental mechanistic information can be attributed to previous ambiguity in the field as well as to limitations in experimental approaches; in silico modeling currently provides the only insight into how ligands block Ser273 phosphorylation. The future availability of experimental evidence is critical for clarifying the mechanism by which ligands prevent phosphorylation and should be the priority of future T2DM-focused research. Following this, the properties of ligands that enable them to block phosphorylation can be improved upon to generate ligands tailored for blocking phosphorylation and therefore restoring insulin sensitivity. This would represent a significant step forward for treating T2DM. This review summarizes current knowledge of the roles of PPARγ in T2DM as well as the effects of synthetic ligands on the modulation of these roles. We hypothesize potential factors that contribute to the reduction in recent developments and summarize what has currently been done to shed light on this critical field of research. The Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-modulated transcription factors belonging to the nuclear receptor superfamily of proteins and have critical roles in controlling many processes in the human body. The PPARs are sorted into three subtypes: PPARα, PPARβ/δ, and PPARγ. They differ in their ligand specificity, transcriptional activity, and tissue expression profiles. Of the three subtypes, PPARγ has attracted the greatest research efforts for its roles in a number of diseases (1Semple R.K. Chatterjee V.K. O'Rahilly S. PPAR gamma and human metabolic disease.J. Clin. Invest. 2006; 116: 581-589Crossref PubMed Scopus (662) Google Scholar, 2Chandra M. Miriyala S. Panchatcharam M. PPARγ and its role in cardiovascular diseases.PPAR Res. 2017; 2017: 6404638Crossref PubMed Scopus (53) Google Scholar), and exists in two isoforms; PPARγ1 and PPARγ2, which differ by an additional 30 amino acids at the N-terminus of PPARγ2 (3Braissant O. Foufelle F. Scotto C. Dauça M. 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Importance of the 5'-flanking region.J. Biol. Chem. 1997; 272: 25252-25259Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Coregulatory proteins are recruited to the PPARγ/RXRα complex to either transcribe or repress the gene downstream of the PPRE. These large coregulatory proteins recruit chromatin remodelers and are classed as either coactivators, e.g., Steroid Receptor Coactivator (SRC) or Nuclear Receptor Coactivating Proteins (NCoA), or corepressors, such as Nuclear Receptor Corepressor (NCoR) or Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor (SMRT) (16Jepsen K. Rosenfeld M.G. Biological roles and mechanistic actions of co-repressor complexes.J. Cell Sci. 2002; 115: 689-698Crossref PubMed Google Scholar). Coactivator recruitment unwinds chromatin to expose the target gene, providing access for the transcriptional machinery to promote transcription. 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Because of these critical roles played by PPARγ, a large effort has been invested in developing synthetic ligands specific to PPARγ, which can reverse various disease states, including type 2 diabetes, inflammation (18Elzahhar P.A. Alaaeddine R. Ibrahim T.M. Nassra R. Ismail A. Chua B.S.K. Frkic R.L. Bruning J.B. Wallner N. Knape T. von Knethen A. Labib H. El-Yazbi A.F. Belal A.S.F. Shooting three inflammatory targets with a single bullet: Novel multi-targeting anti-inflammatory glitazones.Eur. J. Med. Chem. 2019; 167: 562-582Crossref PubMed Scopus (17) Google Scholar, 19Chang M.R. Ciesla A. Strutzenberg T.S. Novick S.J. He Y. Garcia-Ordonez R.D. Frkic R.L. Bruning J.B. Kamenecka T.M. Griffin P.R. Unique polypharmacology nuclear receptor modulator blocks inflammatory signaling pathways.ACS Chem. Biol. 2019; 14: 1051-1062Crossref PubMed Scopus (6) Google Scholar, 20Capra V. Bäck M. Barbieri S.S. Camera M. Tremoli E. Rovati G.E. 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Spiegelman B.M. mPPAR gamma 2: Tissue-specific regulator of an adipocyte enhancer.Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (1977) Google Scholar). Adipose tissues secrete various cytokines and adipokines, such as adiponectin, leptin, TNF-α, IL-6, and resistin. These proinflammatory molecules have significant effects on insulin resistance and the development of obesity (26Fantuzzi G. Adipose tissue, adipokines, and inflammation.J. Allergy Clin. Immunol. 2005; 115 (quiz 920): 911-919Abstract Full Text Full Text PDF PubMed Scopus (1877) Google Scholar). The roles of PPARγ are most prominent in white adipose tissue where the receptor is most heavily expressed, but it is also expressed in the liver and muscle, where it has a direct impact on insulin sensitivity. These critical roles of PPARγ in the development of T2DM make PPARγ an attractive drug target for the treatment and management of T2DM. In the absence of a ligand, PPARγ-controlled genes have a basal level of expression and this level can be altered by synthetic ligands binding to PPARγ (Fig. 1). Full agonist ligands systematically turn on genes under the transcriptional control of PPARγ by causing subtle structural changes in the protein to promote coactivator binding. Partial agonists exhibit a similar effect but to a lesser degree, which somewhat promotes coactivator binding to turn on some genes above basal levels. Nonactivating ligands, such as antagonists or inverse agonists, modulate PPARγ by binding with high affinity but without increasing gene expression levels. In the case of inverse agonists, they modulate PPARγ by reducing basal gene expression through the recruitment of corepressors. A well-studied full agonist of PPARγ is Rosiglitazone (Avandia), an effective insulin sensitizer that was generously prescribed to treat T2DM; yearly sales of Avandia reached US $3.3 billion in 2006 (27Nissen S.E. 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Antagonists and inverse agonists of PPARγ have shown the most promise for treating T2DM for their capacity to normalize insulin sensitivity but without the side effects seen for full or partial agonists. They have been extensively characterized in structure and biochemical properties (37Frkic R.L. Marshall A.C. Blayo A.L. Pukala T.L. Kamenecka T.M. Griffin P.R. Bruning J.B. PPARγ in complex with an antagonist and inverse agonist: A tumble and trap mechanism of the activation helix.iScience. 2018; 5: 69-79Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 38Marciano D.P. Kuruvilla D.S. Boregowda S.V. Asteian A. Hughes T.S. Garcia-Ordonez R. Corzo C.A. Khan T.M. Novick S.J. Park H. Kojetin D.J. Phinney D.G. Bruning J.B. Kamenecka T.M. Griffin P.R. Pharmacological repression of PPARγ promotes osteogenesis.Nat. Commun. 2015; 6: 7443Crossref PubMed Scopus (76) Google Scholar, 39Goldstein J.T. Berger A.C. Shih J. Duke F.F. Furst L. Kwiatkowski D.J. Cherniack A.D. 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In order for this to occur, it is imperative that a better understanding of PPARγ is developed. Posttranslational modifications (PTMs) of PPARγ, such as phosphorylation, deacetylation, ubiquitination, and SUMOylation, are adding to the complexity of its modulation (47Choi S.S. Park J. Choi J.H. Revisiting PPARγ as a target for the treatment of metabolic disorders.BMB Rep. 2014; 47: 599-608Crossref PubMed Scopus (60) Google Scholar). Of significant clinical relevance is the phosphorylation of the receptor at specific sites to effect modulation of various processes. A recently identified PTM of PPARγ is phosphorylation at Tyr78, which regulates the expression of cytokines and chemokines and has roles in adipocyte inflammation (48Choi S. Jung J.E. Yang Y.R. Kim E.S. Jang H.J. Kim E.K. Kim I.S. Lee J.Y. Kim J.K. Seo J.K. Kim J.M. Park J. Suh P.G. Choi J.H. 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The PPARγ phosphorylation site that is of greatest clinical relevance is at Ser273 (Ser245 in PPARγ isoform 1 nomenclature), located within the LBD of PPARγ (Fig. 2). Critically, high levels of phosphorylated Ser273 (pSer273) are observed in obesity and insulin resistance (43Choi J.H. Banks A.S. Kamenecka T.M. Busby S.A. Chalmers M.J. Kumar N. Kuruvilla D.S. Shin Y. He Y. Bruning J.B. Marciano D.P. Cameron M.D. Laznik D. Jurczak M.J. Schürer S.C. et al.Antidiabetic actions of a non-agonist PPARγ ligand blocking Cdk5-mediated phosphorylation.Nature. 2011; 477: 477-481Crossref PubMed Scopus (396) Google Scholar, 50Banks A.S. McAllister F.E. Camporez J.P. Zushin P.J. Jurczak M.J. Laznik-Bogoslavski D. Shulman G.I. Gygi S.P. Spiegelman B.M. An ERK/Cdk5 axis controls the diabetogenic actions of PPARγ.Nature. 2015; 517: 391-395Crossref PubMed Scopus (198) Google Scholar). This PTM is key for treating T2DM, as the phosphorylation status of Ser273 appears be a key contributor to the onset of T2DM. There is a well-studied correlation between high levels of PPARγ phosphorylated at Ser273 with increased obesity and insulin resistance. In order to investigate the mechanism behind this, a fibroblast cell line harboring PPARγ ΔS273A was established as a useful tool for understanding the effects of abolishing Ser273 phosphorylation (51Choi J.H. Banks A.S. Estall J.L. Kajimura S. Boström P. Laznik D. Ruas J.L. Chalmers M.J. Kamenecka T.M. Blüher M. Griffin P.R. Spiegelman B.M. Anti-diabetic drugs inhibit obesity-linked phosphorylation of PPARgamma by Cdk5.Nature. 2010; 466: 451-456Crossref PubMed Scopus (669) Google Scholar). The authors utilized this mutant to investigate the downstream transcription of a set of genes hypothesized to have roles in the onset of T2DM. Some noteworthy genes whose transcription was increased for the ΔS273A mutant protein include: i) CD36, a membrane protein involved in fatty acid uptake into muscle and adipose tissue (52Pepino M.Y. Kuda O. Samovski D. Abumrad N.A. Structure-function of CD36 and importance of fatty acid signal transduction in fat metabolism.Annu. Rev. Nutr. 2014; 34: 281-303Crossref PubMed Scopus (275) Google Scholar); ii) adiponectin, a hormone that plays crucial roles in preventing insulin resistance and T2DM (53Achari A.E. Jain S.K. Adiponectin, a therapeutic target for obesity, diabetes, and endothelial dysfunction.Int. J. Mol. Sci. 2017; 18: 1321Crossref PubMed Scopus (499) Google Scholar); iii) adipsin, an adipokine that improves the function of insulin-producing β-cells (54Lo J.C. Ljubicic S. Leibiger B. Kern M. Leibiger I.B. Moede T. Kelly M.E. Chatterjee Bhowmick D. Murano I. Cohen P. Banks A.S. Khandekar M.J. Dietrich A. Flier J.S. 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Thrap3 docks on phosphoserine 273 of PPARγ and controls diabetic gene programming.Genes Dev. 2014; 28: 2361-2369Crossref PubMed Scopus (42) Google Scholar) which identified a key mechanism connecting the two events. It was discovered that thyroid hormone receptor-associated protein 3 (Thrap3) recognizes and selectively binds to PPARγ when it is phosphorylated at Ser273. Upon binding to PPARγ, this coregulatory protein causes differential expression of a set of genes that promotes insulin resistance, which can le