Figure 1.
Effects of 53 novel chemicals on the viability of 3T3-L1 preadipocytes.
3T3-L1 preadipocytes were treated with or without each of the test compounds at a final concentration of 10 µM for 24 h. The cell viability was measured by MTS assay. Data are mean ± SE from three independent experiments with three replicates. *P<0.05 vs. control (no chemical).
Figure 2.
Effects of 53 novel chemicals on intracellular triglyceride (TG) synthesis during 3T3-L1 adipocyte differentiation.
3T3-L1 preadipocytes were induced to differentiate with induction medium containing MDI (M, IBMX (0.5 mM); D, dexamethasone (0.5 µM); I, insulin (5 µg/ml)), insulin (5 µg/ml), and FBS (10%) in the presence or absence of 10 µM of the test compounds for 8 days. On day 8, the cellular TG contents were quantified by AdipoRed assay. Values are mean ± SE of data from three independent experiments with three replicates. *P or #P<0.05 and **P or ##P<0.01 vs. control (no chemical).
Figure 3.
Effect of KMU-3, a novel synthetic derivative of gallic acid, on lipid accumulation during 3T3-L1 adipocyte differentiation.
(A,B) 3T3-L1 preadipocytes were induced to differentiate with induction medium containing MDI, insulin, and FBS in the presence or absence of KMU-3 (A) or gallic acid (GA) (B) at the indicated concentrations for 8 days. On day 8, the cellular lipid contents were assessed by Oil Red O staining. Phase-contrast images of the cells were also taken after the treatment (lower panels in A or B). Each picture in (A) and (B) is a representative of three independent experiments. (C) 3T3-L1 preadipocytes were induced to differentiate with induction medium containing MDI, insulin, and FBS in the presence or absence of KMU-3 at the indicated concentrations for 8 days. On day 8, the cellular TG contents were quantified by AdipoRed assay. Values are mean ± SE of data from three independent experiments with three replicates. *P<0.05 vs. control (no chemical). (D) The chemical structure of KMU-3 and GA.
Figure 4.
Effect of KMU-3 on expressions and/or activities of C/EBP-α, C/EBP-β, PPAR-γ, and STATs during 3T3-L1 adipocyte differentiation.
(A,B,C) 3T3-L1 preadipocytes were induced to differentiate with induction medium containing MDI and insulin in the presence or absence of KMU-3 (10 µM) and harvested at day 2 and 5, respectively. The cellular protein and mRNA at the indicated time point were extracted and analyzed by Western blot (A, C) and RT-PCR (B) analysis, respectively. Each picture in (A), (B), and (C) is a representative of three independent experiments. (D) and (E) The densitometry data of (A) and (B), respectively, that show C/EBP-α, C/EBP-β, and PPAR-γ protein and mRNA levels normalized to actin protein and mRNA levels as percentage of the value at 0 day. (F) The densitometry result of (C) that shows p-STAT-3, p-STAT-1 and p-STAT-5 protein levels normalized to total expression levels of each protein as percentage of the value at 0 day. *p<0.05 compared to the value of day 0. #p<0.05 compared to the value of KMU-3 free control at the indicated days.
Figure 5.
Effect of KMU-3 on expressions and/or activities of STATs, JAKs, Src, SHP-1, and ERK-1/2 in 3T3-L1 preadipocytes incubated with MDI and/or IBMX.
(A) 3T3-L1 preadipocytes were treated with induction medium containing MDI in the presence or absence of KMU-3 (10 µM) and harvested at the time of 45 min, 2, 8, and 48 h, respectively. The cellular protein at the indicated time point was extracted and analyzed by Western blot analysis. (B) 3T3-L1 preadipocytes were treated without (N) or with IBMX (0.5 mM), dexamethasone (Dex, 0.5 µM), and insulin (Ins, 5 µg/ml), respectively, for 2 h. The cellular protein was then extracted and analyzed by Western blot analysis. (C) 3T3-L1 preadipocytes were treated with IBMX (0.5 mM) in the presence or absence of KMU-3 (10 µM) for 2 h. The cellular protein was then extracted and analyzed by Western blot analysis. Each picture in (A), (B), and (C) is a representative of three independent experiments. (D) The densitometry result of (A) that shows p-STAT-1, p-STAT-3, p-STAT-5, p-JAK-1, p-JAK-2, p-Src, SHP-1, p-ERK-1, and p-ERK-2 protein levels normalized to total expression levels of STAT-1, STAT-3, STAT-5, JAK-1, JAK-2, Src, ERK-1 or ERK-2 protein as percentage of the value at the time 0. (E) and (F) The densitometry data of (B) and (C), respectively, that show p-STAT-3 protein levels normalized to total expression levels of STAT-3 protein as percentage of the value at the same time. *p<0.05 compared to the value at the time 0. #p<0.05 compared to the value of KMU-3 free control at the indicated times.
Figure 6.
Effect of KMU-3 or AG490 on lipid accumulation and/or STAT-3 phosphorylation during 3T3-L1 adipocyte differentiation.
(A) 3T3-L1 preadipocytes were induced to differentiate with induction medium containing MDI, insulin, and FBS in the presence or absence of KMU-3 as described in Materials and Methods (a to g) at the indicated time points (2, 5, 8 days). The cellular lipid contents at each time point were assessed by Oil Red O staining. (B,C) 3T3-L1 preadipocytes were induced to differentiate with induction medium containing MDI, insulin, and FBS in the presence or absence of AG490, a JAK-2/STAT-3 inhibitor, at the indicated concentrations for 8 days. On day 8, cellular lipid accumulation and triglyceride (TG) contents were assessed by Oil Red O staining (B) and AdipoRed assay (C), respectively. Phase-contrast images of the cells were also taken after the treatment (lower panel in B). (D) 3T3-L1 preadipocytes were treated without or with MDI in the presence or absence of AG490 at the indicated concentrations for 2 h. The cellular protein was then extracted and analyzed by Western blot analysis. (E) The densitometry result of (D) in which phosphorylation levels of STAT-3 were quantified with total expression levels of STAT-3. *p<0.05 compared to the value obtained at the same time point (2 h) in the absence of any drug.
Figure 7.
Effect of KMU-3 on mRNA expressions of adipcoyte-specific genes and adipokines during 3T3-L1 adipocyte differentiation.
(A) 3T3-L1 preadipocytes were induced to differentiate with induction medium containing MDI, insulin, and FBS in the presence or absence of KMU-3 (10 µM), and harvested at day 2, 5, and 8, respectively. The cellular mRNA at each time point was extracted and analyzed by RT-PCR analysis. Each picture is a representative of three independent experiments. (B) The densitometry of (A) in which mRNA expression levels of FAS, aP2, adiponectin, leptin, RBP-4, and RANTES were quantified with those with action, respectively. *p<0.05 compared to the value obtained at the same time point (day 5) in the absence of KMU-3.