Potent anti-diabetic effects of MHY908, a newly synthesized PPAR α/γ dual agonist in db/db mice

Abstract

Peroxisome proliferator-activated receptor (PPAR) α/γ dual agonists have been developed to alleviate metabolic disorders and have the potential to be used as therapeutic agents for the treatment of type 2 diabetes. In this study, we investigated the effects of a newly synthesized PPAR α/γ dual agonist, 2-[4-(5-chlorobenzo [d] thiazol-2-yl) phenoxy]-2-methylpropanoic acid (MHY908) on type 2 diabetes in vitro and in vivo. To obtain initial evidence that MHY908 acts as a PPAR α/γ dual agonist, ChIP and reporter gene assays were conducted in AC2F rat liver cells, and to investigate the anti-diabetic effects and molecular mechanisms, eight-week-old, male db/db mice were allowed to eat ad libitum, placed on calorie restriction, or administered MHY908 (1 mg or 3 mg/kg/day) mixed in food for 4 weeks. Age-matched male db/m lean mice served as non-diabetic controls. It was found that MHY908 enhanced the binding and transcriptional activity of PPAR α and γ in AC2F cells, and it reduced serum glucose, triglyceride, and insulin levels, however increased adiponectin levels without body weight gain. In addition, MHY908 significantly improved hepatic steatosis by enhancing CPT-1 levels. Remarkably, MHY908 reduced endoplasmic reticulum (ER) stress and c-Jun N-terminal kinase (JNK) activation in the livers of db/db mice, and subsequently reduced insulin resistance. The study shows MHY908 has beneficial effects on type 2 diabetes by simultaneously activating PPAR α/γ and improving ER stress, and suggests that MHY908 could have a potent anti-diabetic effect as a PPAR α/γ dual agonist, and potential for the treatment of type 2 diabetes.

Figure 1. Synthesis of MHY908.

Reagents and conditions: (a) Ethyl-bromoisobutyrate, 1N-NaOEt, EtOH, reflux, 14 h, 72%; (b) Na₂S₂O₅, DMF, 80°C, 11 h, 31%; (c) 1N-NaOH, 1,4-dioxane, rt, 17 h, 79%; (d) 1N-NaOH, 1,4-dioxane, rt, 4 h, 99.9%; (e) NaOAc, AcOH, reflux, 1 h, 40%.

Figure 2. MHY908 functioned as a PPAR α/γ dual agonist.

(A) The chemical structure of MHY908. (B) ChIP assay for PPARs binding to PPRE of the PPAR α or γ promoter. The extracted genomic DNA was subjected to immunoprecipitation using an antibody against PPAR α and PPAR γ or IgG as a negative control. Amplification derived from unprecipitated chromatin is shown (input). (C) MHY908 induced the transcriptional activities of PPAR α and γ, respectively. One-factor ANOVA was used to determine the significances of differences: *** p<0.001, ** p<0.01 and * p<0.05 versus the PC DNA. (D) The docking simulation between PPAR α or γ and their activators or MHY908. The Grey zone indicates the active site. Magenta indicates MHY908, and cyan indicates fenofibrate or rosiglitazone (positive controls). The binding energies of compounds for PPAR α were −9.10 kcal/mol (MHY908) and −8.80 kcal/mol (fenofibrate) or and for PPAR γ were −8.88 kcal/mol (MHY908) and −8.03 kcal/mol (rosiglitazone).

Figure 3. Effect of MHY908 on body weight, plasma glucose, TG, and insulin levels in db/db mice.

Mice were treated for 4/kg/day in food. Food intakes and body weights were similar in the Con group and MHY908- treated groups (n = 8/group, eight-weeks old). (A) Changes in body weights. (B) A series of plasma profiles from CR and MHY908 treated db/db mice. One-factor ANOVA was used to determine the significances of differences: *** p<0.001, ** p<0.01 and * p<0.05 versus the Con group. Lean; db/m mice, Con; db/db mice, CR; calorie restriction, 1 mg; MHY 908 1 mg/kg/day, 3 mg; MHY 908 3 mg/kg/day.

Figure 4. MHY908 improved hepatic steatosis in db/db mouse livers.

(A) A histological examination based on hematoxylin-eosin staining showed marked fatty changes in the livers of db/db mice. (Original magnification 100 X) (B) Accumulated lipids in the livers of MHY908-treated db/db mice. One-factor ANOVA was used to determine the significances of differences: *** p<0.001, ** p<0.01 and * p<0.05 versus the Con group. (C) CPT-1 expression was assessed by immunohistochemistry (Original magnification 200 X). Lean; db/m mice, Con; db/db mice, CR; calorie restriction, 1 mg; MHY 908 1 mg/kg/day, 3 mg; MHY 908 3 mg/kg/day.

Figure 5. MHY908 attenuated ER stress induced insulin resistance in the livers of db/db mice.

(A) Results of Western-blot analysis of the livers of db/db mice; the ER stress markers p-IRE, p-PERK, and p-JNK are shown. CR and MHY908 decreased ER stress marker protein levels in db/db livers, and (B) CR and MHY908 improved insulin signaling in db/db livers. Lean; db/m mice, Con; db/db mice, CR; calorie restriction, 1 mg; MHY 908 1 mg/kg/day, 3 mg; MHY 908 3 mg/kg/day.

Figure 6. MHY908 modulated serum leptin and adiponectin levels in db/db mice.

(A) Serum concentrations of leptin (n = 6) (B) Serum concentrations of adiponectin (n = 6) One-factor ANOVA was used to determine the significances of differences: *** p<0.001, ** p<0.01 and * p<0.05 versus the Con group. Lean; db/m mice, Con; db/db mice, CR; calorie restriction, 1 mg; MHY 908 1 mg/kg/day, 3 mg; MHY 908 3 mg/kg/day.

Figure 7. Possible mechanism of the effects of MHY908 on overnutrition-induced insulin resistance.

We suggest that overnutrition induced lipid accumulation and led to insulin resistance by increasing ER stress, and that MHY908 downregulated ER stress and improved insulin signaling in the livers of db/db mice.