Identification of a proliferator-activated receptor-γ antagonist for the treatment of type 2 diabetes mellitus

Abstract

In the present study, a novel antagonist of the peroxisome proliferator-activated receptor-γ (PPARγ) was screened and identified, and a cell-based evaluation of the biological activity of this PPARγ antagonist was conducted. The aim of the study was to produce results that may provide a foundation for the development of a novel compound in the treatment of type 2 diabetes mellitus. Since obesity is the main cause of insulin resistance and type 2 diabetes, identifying a new reagent that is able to inhibit adipocyte differentiation and lipid accumulation is a feasible method of developing novel anti-diabetes drugs. The PPARγ antagonist was screened using a mammalian one-hybrid system and transcriptional activation. The effects of the compound on adipocyte differentiation were investigated by staining the preadipocytes with Oil Red O. In addition, the effects of the compound on the expression levels of genes associated with lipid metabolism were detected using quantitative polymerase chain reaction on differentiated mature 3T3-L1 adipocytes. As a PPARγ antagonist, N-((1H-benzo[d]imidazol-2-yl)methyl) aniline (Compound Q) was shown to depress the transcriptional activity and coactivator recruitment of PPARγ, as well as preadipocyte differentiation, in a concentration-dependent manner. The compound was also shown to decrease the expression levels of genes associated with PPARγ-regulated lipid metabolism. In conclusion, the compound screening platform was demonstrated to be valid, and the present study identified a novel PPARγ antagonist that was shown to effectively reduce the rate of adipocyte differentiation and the expression of genes associated with lipid metabolism.

Keywords: lipid metabolism-related genes; peroxisome proliferator-activated receptor-γ antagonist; type 2 diabetes.

Figure 1

(A) Structural formula of Compound Q. Compound Q was shown to depress the activity of Ros-activated (B) PPARγ-LBD-Luc and (C) RXRα:PPARγ-PPRE-Luc in a concentration-dependent manner. Ros, a known PPARγ agonist, is able to activate PPARγ-LBD and increase the transcription of PPRE-Luc. As compared with Ros treatment, the combination of Compund Q and Ros decreased Ros-induced luciferase activity in the mammal one-hybrid and transcriptional activation systems, suggesting that Compund Q is a PPARγ antagonist and can inhibit the transcriptional activity of PPARγ. ^###P<0.001 compared to DMSO group; ^**P<0.01 and ^***P<0.001, respectively, compared to Ros group. Ros, rosiglitazone; DMSO, dimethyl sulfoxide; PPARγ, peroxisome proliferator-activated receptor-γ; Luc, luciferase; PPRE, peroxisome proliferator response element; RXRα, retinoid X receptor-α; CQ, Compound Q.

Figure 2

Compound Q was shown to reduce the rate of adipocyte differentiation in a concentration-dependent manner. (A) DMSO; (B) Ros; (C) Compound Q (1 μM); (D) Compound Q (10 μM); and (E) Compound Q (20 μM). DMSO was used as a negative control, while Ros was used as a positive control, since the drug is known to significantly enhance adipocyte differentiation. Ros, rosiglitazone; DMSO, dimethyl sulfoxide. Magnification, ×200.

Figure 3

Differentiated mature adipocytes were cultured for 24 h in a medium containing Compound Q at various concentrations, a positive control (Ros) or a negative control (DMSO). Compound Q was shown to decrease the expression levels of (A) FAS, (B) C/EBPα, (C) aP2 and (D) HMG-CoA in a concentration-dependent manner, while Ros was found to increase the expression levels. ^###P<0.001, ^*P<0.05, ^**P<0.01 and ^***P<0.001. Ros, rosiglitazone; DMSO, dimethyl sulfoxide; FAS, fatty acid synthase; C/EBPα, CCAAT/enhancer-binding protein-α; aP2, adipocyte fatty acid binding protein 2.