Detailed information on the hydrogen bonding networks formed by fenofibrate, rosiglitazone, and MHY908.

<p>Detailed information on the hydrogen bonding networks formed by fenofibrate, rosiglitazone, and MHY908.</p

MHY908 improved hepatic steatosis in db/db mouse livers.

<p>(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.</p

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

<p>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.</p

Synthesis of MHY908.

<p>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%.</p

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

<p>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.</p

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

<p>(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.</p

Activation of mTOR by MHY1485.

<p>Western blot analysis was performed to detect the change of total protein level and levels of phosphorylated forms of mTOR and 4E-BP1 reflecting the activity of mTOR. Ac2F cells were treated with MHY1485 of different concentrations and rapamycin 5 µM as a positive control for 1 h. Bars represent the phospho-mTOR(Ser2448)/mTOR ratio and the phospho-4E-BP1(Thr37/46)/4E-BP1 ratio normalized with the ratio from untreated samples, respectively. β-Actin blot is shown to verify the same amount of protein loaded. The blots were quantified by densitometry expressed as mean±SD (*p<0.05; n = 3).</p

Increase of the LC3II/LC3I ratio and LC3II-positive vacuoles by MHY1485.

<p>Western blot analysis was performed to detect LC3II and LC3I. Ac2F cells were treated with different concentrations of MHY1485 and rapamycin 5 µM as a positive control for 6 h. Bars represent the LC3II/LC3I ratio calculated by normalizing the LC3II/LC3I ratio from MHY1485-treated or rapamycin-treated samples with the LC3II/LC3I ratios from untreated samples (A). Control cells treated with same volume of vehicle or MHY1485 (2 µM) for 1, 6 or 12 h were collected. Bars represent the LC3II/LC3I ratio calculated by normalizing the LC3II/LC3I ratio from MHY1485-treated samples with LC3II/LC3I ratio from control samples at 1 hour (B). β-Actin blot is shown to verify the same amount of protein loaded. The blots were quantified by densitometry expressed as mean±SD (*p<0.05, **p<0.01, ***p<0.001; n = 3). Live-cell confocal microscopic images of AdGFP-LC3-transfected Ac2F cells treated with 2 µM MHY1485 for 1, 6 or 12 h are shown. The images show the GFP-LC3-positive vacuoles (upper, green), corresponding phase contrast images (middle) and merged images (bottom). Scale bar, 20 µm.</p

Brief scheme of the synthetic process of MHY1485.

<p>Brief scheme of the synthetic process of MHY1485.</p

Failure of the increase of autophagic flux.

<p>Western blot analysis was performed with samples from cells treated in 2 µM MHY1485 for 6 h. The lysosomotropic agents bafilomycin A1 (bafA1, 10 nM) and chloroquine (100 µM) were applied 1 h before the cell harvest to measure the autophagic flux (A, B). Bars represent the LC3II/LC3I ratio normalized with the LC3II/LC3I ratio from untreated samples. β-Actin blot is shown to verify the same amount of protein loaded. The blots were quantified by densitometry expressed as mean±SD (**p<0.01; n = 3). C and D show the immunoblots of p62 and beclin-1 in samples from the cells treated with MHY1485 or the same amount of vehicle for different times. The blots were quantified by densitometry and were normalized with the control sample at 1 h expressed as mean±SD (*p<0.05; n = 3).</p