Fig 1.
Effects of LFE or FSB on rosiglitazone- and pioglitazone-induced adipocyte differentiation.
3T3-L1 cells were treated with rosiglitazone (50 μM) or pioglitazone (10 μM) in the presence or absence of LFE (10 μg/ml, A-C) or FSB (1 μg/ml, D-F). Six days after the induction of adipocyte differentiation, cells were stained with Oil Red O solution and visualized under an optical microscope (A and D). Absorbance at 490 nm of solutions eluted after Oil Red O staining was used to quantify the extent of adipocyte differentiation (B and E). The mRNA expressions of PPARγ target genes were determined by qPCR (C and F). Experiments were repeated three times in triplicate, and results are presented as means ± SDs. #P<0.05 vs. control; *P<0.05, **P<0.01, ***P<0.001 vs. rosiglitazone or pioglitazone alone.
Fig 2.
Effects of LFE or FSB on PPARγ transactivation activity and cofactor recruitment.
HEK293T cells were transfected with pFA-Gal4-PPARγ-LBD and pFR-Luc reporter vector, and incubated with rosiglitazone (1 μM) with or without LFE or FSB at different concentrations for 18 h. Transactivation activities were assessed by measuring luminescence (A). The effects of LFE or FSB on PPARα and PPARδ were examined after cotransfecting HEK293T cells with pFA-Gal4-PPARα-LBD or pFA-Gal4-PPARδ-LBD. Transactivation was stimulated with the selective PPARα agonist GW7647 or the selective PPARδ agonist GW0742; GW6741 (a selective antagonist of PPARα) and GSK0660 (a selective antagonist of PPARδ) were used as positive controls (B, C). To examine the profiles of cofactor recruitments, HEK293T cells were transiently cotransfected with either pM-SRC-1 (D) or pBind-NCoR-1 (E) together with pVP-PPARγ and pFR-Luc using X-tremeGENE9 DNA transfection reagent. Cells were grown for 24 h in the presence or absence of rosiglitazone with LFE or FSB. The recruitments of the cofactor and corepressor are expressed as fold induction relative to luciferase activity. Experiments were repeated three times in triplicates, and results are presented as means ± SDs. #P<0.05 vs. control; *P<0.05, **P<0.01, ***P<0.001 vs. rosiglitazone alone. The binding mode of FSB on PPARγ was presented (F). FSB was represented gray elementary color. The red line is the hydrogen binding between the side chain of R357 and FSB. The protein molecule is shown as ribbon cartoon except the interacting residues with FSB.
Fig 3.
In vivo effects of LFE on ob/ob mice.
LFE (100 or 300 mg/kg; n = 10 per group) was orally administered once daily for 8 weeks to 6-week-old ob/ob mice. Body weights (A left), food intakes (A right), and plasma glucose levels (B) were measured weekly. After 8 weeks of treatment, OGTT (C) and ITT (D) were carried out. Fasting plasma glucose levels after 8 weeks treatment were measured (E). Experiments were repeated twice, and results are presented as means ± SDs. *P<0.05, **P<0.01 vs. vehicle (0.05% CMC).
Fig 4.
Effects of LFE on fat tissues in ob/ob mice.
After 8 weeks of LFE administration, total fat weights were measured (A). The mRNA expressions of PPARγ target genes were determined by qPCR in subcutaneous and visceral fats (B). The protein levels of PPARγ and C/EBPα were detected by western blotting of subcutaneous and visceral fats (C). The mRNA expressions of UCP-1 in subcutaneous and visceral fats were determined by qPCR (D). The mRNA levels of proinflammatory markers, adiponectin and resistin were determined by qPCR (E and F). *P<0.05, **P<0.01, ***P<0.001 vs. vehicle (0.05% CMC).
Fig 5.
Effects of LFE on hepatic steatosis in ob/ob mice.
After 8 weeks of LFE administration, plasma levels of ALT and AST, and TG were determined using commercial kits (A). Liver tissues were frozen and tissue sections were stained with either H&E or Oil Red O, and visualized under an optical microscope (B). Hepatic TG contents were measured using commercial kit (C). Plasma levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) were measured using ELISA kits (D). Hepatic mRNA levels of IL-1β and IL-6 were measured (E). *P<0.05, **P<0.01, ***P<0.001 vs. vehicle (0.05% CMC).
Fig 6.
In vivo effects of LFE on KKAy mice.
LFE (100 mg/kg, 300 mg/kg; n = 10 per group) was administered once daily for 8 weeks to 6-week-old KKAy mice. Body weights (A left), food intakes (A right), and plasma glucose levels (B) were measured weekly. After 8 weeks of treatment, OGTT (C) and ITT (D) were carried out. Experiments were repeated twice, and results are presented as means ± SDs. *P<0.05, **P<0.01 vs. vehicle (0.05% CMC).
Fig 7.
Effects of LFE on fat tissues in KKAy mice.
After 8 weeks of LFE administration, total fat weights were measured (A). The mRNA expressions of PPARγ target genes were determined by qPCR in subcutaneous and visceral fats (B). The protein levels of PPARγ and C/EBPα were detected by western blotting of subcutaneous and visceral fats (C). The mRNA levels of proinflammatory markers were determined by qPCR in subcutaneous and visceral fats (D). *P<0.05, **P<0.01 vs. vehicle (0.05% CMC).
Fig 8.
Effects of LFE on hepatic steatosis in KKAy mice.
After 8 weeks of LFE administration, plasma levels of ALT and AST, and TG were determined using commercial kits (A). Liver tissues were frozen and tissue sections were stained with H&E or Oil Red O, and examined under an optical microscope (B). Hepatic TG contents were measured using commercial kit (C). Plasma levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) were measured using ELISA kits (D). Hepatic mRNA levels of IL-1β and IL-6 were measured (E). *P<0.05, **P<0.01, ***P<0.001 vs. vehicle (0.05% CMC).