N-Acetylfarnesylcysteine Is a Novel Class of Peroxisome Proliferator-activated Receptor γ Ligand with Partial and Full Agonist Activity in Vitro and in Vivo

Kavita Bhalla,Jeffry Stock,William Twaddel,Geoffrey D Girnun,Edwin Pozharski,Jang Hyun Choi,Bor Jang Hwang,Ruby Dewi,Lihui Ou,John Mclenithan

doi:10.1074/jbc.m111.257915

Abstract

The thiazolidedione (TZD) class of drugs is clinically approved for the treatment of type 2 diabetes. The therapeutic actions of TZDs are mediated via activation of peroxisome proliferator-activated receptor γ (PPARγ). Despite their widespread use, concern exists regarding the safety of currently used TZDs. This has prompted the development of selective PPARγ modulators (SPPARMs), compounds that promote glucose homeostasis but with reduced side effects due to partial PPARγ agonism. However, this also results in partial agonism with respect to PPARγ target genes promoting glucose homeostasis. Using a gene expression-based screening approach we identified N-acetylfarnesylcysteine (AFC) as both a full and partial agonist depending on the PPARγ target gene (differential SPPARM). AFC activated PPARγ as effectively as rosiglitazone with regard to Adrp, Angptl4, and AdipoQ, but was a partial agonist of aP2, a PPARγ target gene associated with increased adiposity. Induction of adipogenesis by AFC was also attenuated compared with rosiglitazone. Reporter, ligand binding assays, and dynamic modeling demonstrate that AFC binds and activates PPARγ in a unique manner compared with other PPARγ ligands. Importantly, treatment of mice with AFC improved glucose tolerance similar to rosiglitazone, but AFC did not promote weight gain to the same extent. Finally, AFC had effects on adipose tissue remodeling similar to those of rosiglitazone and had enhanced antiinflammatory effects. In conclusion, we describe a new approach for the identification of differential SPPARMs and have identified AFC as a novel class of PPARγ ligand with both full and partial agonist activity in vitro and in vivo.

Highlights

More than 20 million people in the United States have type 2 diabetes mellitus (T2DM).2 T2DM is a chronic metabolic dis
PPAR␥ agonists are associated with numerous side effects, which have prompted the development of selective PPAR␥ modulators (SPPARMs)
To identify differential SPPARMs, we developed a gene expression-based approach based on a method described previously [60] This approach enables the identification of both full and partial agonist effects on PPAR␥ depending on the genes of interest

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Chemicals—3T3-L1 preadipocytes were obtained from ATCC. PPAR␥ knock-out mouse embryonic fibroblasts (MEFPPAR␥KO) were obtained from Dr Evan Rosen (Beth Israel Deaconess Medical Center, Boston MA) [11]. Adipocyte Differentiation—3T3-L1 cells were subjected to a differentiation protocol as described previously in the absence or presence of AFC or rosiglitazone [20]. Western Blot Analysis—Western blotting for phosphorylated and nonphosphorylated PPAR␥ was performed as described previously by Hauser et al [21]. Phosphorylation of the CDK5 site on PPAR␥ at serine 273 was performed as previously described [22]. 3T3-L1 fibroblasts expressing PPAR␥ were pretreated with rosiglitazone, MRL24, or AFC with the indicated doses and incubated with TNF-␣. Western blotting for serine 112 (or 82 of PPAR␥1)phosphorylated and nonphosphorylated PPAR␥ was performed as described previously by Hauser et al [21]. Ligand Binding Assay—Histidine-tagged PPAR␥-LBD fusion construct (a gift from Dr John Schwabe, Medical Research Council, UK) was expressed in E. coli as described previously [24]. Counting of adipocytes was performed blinded by a pathologist (Dr Twaddel)

RESULTS

Control Rosiglitazone AFC

DISCUSSION