MATERIALS AND METHODS
MaterialsAntibodies specific for phospho-PPARγ (pPPARγ) Ser273 were purchased from Bioss (Beijing, China). Antibodies against aP2, PPARγ, Akt, pAkt (Ser473) and glucose transporter type 4 (GLUT4) were from Cell Signaling (Shanghai, China). Rosiglitazone, luteolin, GW9662, insulin, wortmanin and tumor necrosis factor (TNF)-α were purchased from Sigma (Shanghai, China).
Preparation of 4-(4′-Methylbenzylidene)-2-hydroxy-3-pinanone1.68 g (10.0 mmol) 2-hydroxy-3-pinanone, 1.44 g (12.0 mmol) p-methyl benzaldehyde, 10 mL tert-butanol, and 0.31 g potassium tert-butoxide were successively added into 50 mL three-neck flask and heated at 70°C for 3h under protection of N2. The reaction was monitored with GC until the conversion ratio of 2-hydroxy-2-pinanone reached 99.3%. Reaction was extracted with 75 mL × 3 ethyl acetate. Saturated brine was used to wash the combined organic layer until neutrality. The residue was dried with Na2SO4 and recrystallized in acetyl acetate–petroleum ether (1 : 2 (v/v)) to get 2.12 g of intermediate 4-(4′-methylbenzylidene)-2-hydroxy-3-pinanone, white crystal, yield 78.4%, purity 99.0%, mp 125.8–126.4°C; IR (KBr, ν cm−1): 3474, 2975, 2920, 2869, 1697, 1611, 1509, 1469, 1052, 1011, 841, 811; 1H-NMR (CDCl3, 500 MHz) δ: 0.84 (s, 3H), 1.38 (s, 3H), 1.39 (s, 3H), 1.83 (d, J = 10.45 Hz, 1H), 2.10 (t, J = 6.1 Hz, 1H), 2.32 (s, 3H), 2.51–2.55 (m, 1H), 3.29 (t, J = 6.1 Hz, 1H), 5.35 (s, 1H), 7.20–7.25 (m, 4H), 7.47 (s, 1H); 13C-NMR (CDCl3, 125 MHz) δ: 20.79, 23.71, 25.10, 26.83, 28.29, 39.50, 42.73, 50.53, 74.68, 129.06, 129.25, 131.82, 132.08, 137.92, 140.16, 201.20; MS (70 eV) m/z (%):270 (11), 252 (6), 237 (4), 227 (29), 212 (9), 201 (21), 199 (6), 185 (5), 171 (38), 165 (8), 157 (14), 143 (31), 141 (20), 129 (25), 115 (23), 105 (32), 99 (100), 91 (12), 79 (12), 65 (6).
Synthesis of WSF-70.68 g (2.5 mmol) of 4-(4′- methylbenzylidene)-2-hydroxy-3-pinanone, 0.21 g (3.0 mmol) of hydroxylamine hydrochloride, 5 mL 20% aqueous NaOH, and 10 mL anhydrous ethanol were added into 50 mL three-neck-flask and heated at 60°C for 1–2 h under N2 protection. Ethyl acetate was used for extraction. The organic layer was dried with Na2SO4 after being washed with saturated brine until neutrality. The residue was recrystallized in cyclohexane-ethyl acetate to get white needle crystal WSF-7, yield 69.8%, purity 99.3%, mp 130.8–131.4°C; IR (KBr, ν cm−1): 3439, 2973, 2922, 2872, 1706, 1517, 1472, 1092, 1043, 839, 756, 733; 1H-NMR (CDCl3, 500 MHz) δ: 0.69 (s, 3H), 0.99 (s, 3H), 1.34 (s, 3H), 1.53 (t, J = 6.05 Hz, 1H), 1.88 (d, J = 10.7 Hz, 1H), 1.98 (t, J = 6.05 Hz, 1H), 2.25 (s, 3H), 2.34–2.38 (m, 2H), 3.57 (d, J = 9.7 Hz, 1H), 5.40 (s, 1H), 7.07 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 7.95 Hz, 2H); 13C-NMR (CDCl3, 125 MHz) δ: 20.64, 23.10, 24.45, 26.59, 26.88, 38.91, 39.66, 46.16, 50.85, 52.71, 75.05, 127.46, 128.22, 133.42, 135.95, 213.02; MS (70 eV) m/z (%): 267 (48), 266 (22), 252 (58), 238 (8), 224 (79), 210 (5), 199 (9), 184 (44), 171 (11), 170 (57), 158 (13), 157 (18), 143 (60), 134 (22), 129 (13), 128 (23), 118 (20), 109 (25), 105 (51), 103 (100), 91 (50), 78 (99), 77 (87), 67 (35), 65 (31), 54 (25), 53 (34), 51 (24).
Cell Differentiation and Treatment3T3-L1 preadipocytes (ATC C) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone) supplemented with 10% fetal bovine serum (FBS) (GIBCO) at 37°C in 5% CO2 and induced to differentiate in DMEM containing 0.5 mM IBMX, 1 mM DEX, 5 mg/mL insulin and 10% FBS. To examine the effect on differentiation, 3T3-L1 preadipocytes were induced to differentiate in the presence of WSF-7 or 0.5 µM rosiglitazone.10) At Day 8 post-differentiation, the cells were stained with Oil Red O.11,12) To examine the effect on adiponectin oligomerization, mature 3T3-L1 adipocytes were incubated in a serum-free medium containing 0.05% bovine serum albumin (BSA) for 16h followed by treatment with WSF-7 alone or together with 25 µM GW9662 for 48 h. To examine the effect on PPARγ phosphorylation at Ser-273, 3T3-L1 adipocytes were pretreated with 50 ng/mL TNF-α for 1 h, followed by treatment with WSF-7 or 0.5 µM rosiglitazone for another hour. 3T3-L1 adipocytes were treated with WSF-7, rosiglitazone or insulin for 1h to examine the effect on Akt phosphorylation.
Western Blot AnalysisCell lysates of 3T3-L1 adipocytes were subjected to 2–15% gradient gel electrophoresis under non-reducing and non-heat-denaturing conditions. Adiponectin oligomers were detected by antibodies specific for the C-terminal globular domain.13) To examine the total amount of adiponectin, the cell lysates were incubated with sample buffer containing 10 mM dithiothreitol (DTT) for 1h at room temperature, followed by heat denaturation at 95°C for 10 min. Adiponectin monomer was detected using antibody raised against the N-terminal peptide.10) PPARγ was detected by antibodies for Ser273 pPPARγ or total PPARγ. Akt was detected using antibodies specific for Ser473 pAkt or total Akt. The amount of each protein was quantified by analyzing the western blots using the NIH ImageJ software. All experiments were performed at least three times and the representative results were presented.
PPARγ Ligand Binding AssayIn vitro binding of WSF-7 to PPARγ was analyzed by a LanthaScreen™ time-resolved fluorescent energy transfer (TR-FRET) PPARγ competitive binding assay kit (Invitrogen, Shanghai, China), in accordance with the manufacturer’s instructions. WSF-7 or rosiglitazone was mixed with Fluormone Pan-PPAR Green, GST-PPARγ-LBD, and Tb-GST antibody in a black 384-well assay plate and incubated at room temperature for 2 h. The fluorescent emission signal was measured by PerkinElmer, Inc. EnSpire® Multimode Microplate Reader at 490 and 520 nm, using a 340 nm excitation filter.
Luciferase Reporter Assay293T cells were transfected with PPRE-TK-Luciferase reporter along with PPARγ and retinoid X receptor α (RXRα) expression vectors. 24 h after transfection, the cells were treated with WSF-7 or rosiglitazone in the presence or absence of GW9662. The cells were harvested for the luciferase assay after treatment for 24 h. Luciferase activities were normalized to Renilla activities cotransfected as an internal control.14)
Glucose Uptake Assay3T3-L1 adipocytes were treated with WSF-7 or rosiglitazone in the presence or absence of 25 µM GW9662 or 2.5 µM wortmanin for 24 h, then incubated in glucose-free DMEM for 2 h, followed by stimulation with 5 mg/mL insulin for 10 min. Glucose uptake was measured by a bioluminescent assay,15) using the Glucose Uptake-Glo™ Assay Kit (Promega, Beijing, China).
Membrane Isolation3T3-L1 adipocytes were harvested for membrane isolation after treatment with WSF-7 or rosiglitazone for 24h. Total membranes were prepared using Membrane and Cytosol Protein Extraction Kit (Beyotime, Shanghai, China),15) in accordance with the manufacturer’s instructions. Membrane-bound and cytoplasmic GLUT4 were detected by anti-GLUT4 antibody.
Site-Directed MutagenesisSite-directed mutants were generated in PPARγ expression vector using Fast Mutagenesis System (TransGen Biotech, Beijing, China). DNA sequencing was used to confirm the presence of the desired mutations in the LBD of mouse PPARγ1, including K288A, F310A, and H489A.
Statistical AnalysisAll data were expressed as the means ± standard deviation (S.D.) Student’s t-test was used to compare two samples. Statistical comparisons among three samples of the same treatment group were performed using one-way ANOVA with Dunnett’s test as post-hoc test. In all cases, a p-value less than 0.05 was considered to be statistically significant.
RESULTS
WSF-7 Is a Novel Partial PPARγ AgonistWSF-7, derived from natural monoterpene α-pinene (Fig. 1A), was identified as a potential PPARγ agonist in our screen for synthetic chemicals that inhibit Ser273 phosphorylation of PPARγ. To confirm that WSF-7 is a ligand for PPARγ, we examined in vitro binding of WSF-7 to PPARγ-LBD in a TR-FRET PPARγ competitive binding assay. Similar to rosiglitazone, WSF-7 binds directly to the LBD of PPARγ with an IC50 value of 99 nM (Fig. 1B).
To further demonstrate the PPARγ-agonistic activity of WSF-7, we performed luciferase reporter assay to investigate the effect of WSF-7 on PPARγ transcriptional activity. WSF-7 dose-dependently activates PPARγ transcription. The effect of WSF-7 was inhibited when GW9662, a specific PPARγ antagonist, was present (Fig. 1C). WSF-7 is less potent than rosiglitazone in activating PPARγ transcription, demonstrating that WSF-7 is a weak PPARγ agonist.
To study the mechanism of PPARγ activation by WSF-7, we generated site-directed mutants in the LBD of mouse PPARγ1, including K288A, F310A and H489A, which correspond to K265A, F287A, and H466A of human PPARγ1, respectively.16) The K288A mutation did not have any substantial effect on PPARγ transcriptional activity activated by rosiglitazone, WSF-7, or luteolin, another PPARγ agonist,17) whereas the H489A mutation significantly abolished the transactivation activity of luteolin or WSF-7. Different response was seen for the F310A mutant between luteolin and WSF-7 (Fig. 1D). These results suggest that WSF-7, unlike rosiglitazone or luteolin, might activate PPARγ transcription by a novel mechanism.
WSF-7 Promotes Preadipocyte DifferentiationPPARγ agonists were reported to promote adipogenesis.11,18,19) As shown in Figs. 2A and B, WSF-7 also promoted the differentiation of 3T3-L1 preadipocytes. Expression of PPARγ and aP2, a marker of adipocyte differentiation, was increased by WSF-7 (Fig. 2C). The effect of WSF-7 on adipogenesis was abolished by GW9662 (Fig. 2). These results demonstrate that WSF-7 promotes adipogenesis by activating PPARγ. However, 20 µM of WSF-7 induced differentiation to a lesser extent than 0.5 µM rosiglitazone (Fig. 2), suggesting that WSF-7 is weaker than rosiglitazone in promoting preadipocyte differentiation. Therefore, WSF-7 is a novel PPARγ agonist with weak adipogenic activity.
WSF-7 Promotes Adiponectin Oligomerization in 3T3-L1 AdipocytesAdiponectin, an insulin-sensitizing adipokine, circulates in the serum as low molecular weight (LMW) trimer, medium molecular weight (MMW) hexamer and high molecular weight (HMW) oligomers.13) Lower level of HMW is closely associated with insulin resistance, therefore, HMW adiponectin possesses the most potent insulin-sensitizing effect.20) PPARγ agonists were reported to elevate the level of adiponectin mRNA via a PPRE (PPARγ-responsive regulatory element) present in promotor. PPARγ agonists also upregulate expression of DsbA-L and Ero1-Lα, which have been demonstrated to be involved in promoting adiponectin oligomerization.21)
After treatment for 48 h, the level of adiponectin monomer (Total Ad) was elevated by WSF-7 (Fig. 3A), confirming that WSF-7 upregulates adiponectin expression. Adiponectin oligomerization was also promoted by WSF-7, as can be seen by increased level of LMW, MMW and HMW oligomers. Rosiglitazone was reported to enhance insulin sensitivity by increasing the ratio of HMW/total adiponectin.20) WSF-7 treatment also increased this ratio (Fig. 3B). The effects of WSF-7 on adiponectin were abolished by GW9662 (Fig. 3). Therefore, WSF-7 promotes adiponectin oligomerization by PPARγ activation in 3T3-L1 adipocytes.
WSF-7 Enhances Insulin-Induced Glucose Uptake in 3T3-L1 AdipocytesInsulin-induced glucose uptake, an important function of adipocytes, can be activated by PPARγ agonists.22,23) To investigate the effect of WSF-7, 3T3-L1 adipocytes were treated with WSF-7 for 24h, then induced with insulin for ten minutes. We found that WSF-7 promoted insulin-stimulated glucose uptake and that WSF-7-mediated effect was abrogated by GW9662 (Fig. 4A).
GLUT4 plays a key role in facilitating insulin-induced glucose uptake in adipocytes.24) As a PPARγ target gene, GLUT4 expression was increased by WSF-7, which was inhibited in the presence of GW9662 (Fig. 4B), consistent with previous reports that PPARγ agonists increase the expression of GLUT4 by activating PPARγ.25,26) The level of membrane-bound GLUT4 was also increased by WSF-7 (Fig. 4B).
To investigate whether WSF-7 activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, 3T3-L1 adipocytes were treated with WSF-7 together with wortmanin, a PI3K inhibitor. Insulin-induced glucose uptake was inhibited by wortmanin, however, the level of membrane-bound GLUT4 was still increased by WSF-7 in the presence of wortmanin (Fig. 4C). No WSF-7-induced Akt phosphorylation was detected (Fig. 4D). Therefore, WSF-7 does not activate the PI3K/Akt pathway directly. The inhibition of insulin-induced glucose uptake by wortmanin might be caused by the indirect inhibitory effect on the insulin signaling pathway, independent of WSF-7. Our results demonstrate that WSF-7 enhances insulin-induced glucose uptake via GLUT4 upregulation in 3T3-L1 adipocytes.
WSF-7 Inhibits PPARγ Ser273 Phosphorylation in 3T3-L1 AdipocytesPPARγ agonists were reported to block obesity-mediated PPARγ phosphorylation at Ser273 by Cdk5.7,8,11) To examine whether WSF-7 has the similar effect, 3T3-L1 adipocytes were treated with WSF-7 after TNF-α pretreatment. WSF-7 dose-dependently decreased PPARγ Ser273 phosphorylation (Fig. 5). Approximately 60% inhibition was seen with 20 µM WSF-7. Therefore, WSF-7 inhibits Ser273 phosphorylation of PPARγ in 3T3-L1 adipocytes.
DISCUSSION
In this paper, we found that WSF-7 binds directly to PPARγ and activates the transcriptional activity of PPARγ (Fig. 1). We demonstrated that WSF-7 stimulates the endogenous PPARγ activity of mature 3T3-L1 adipocytes by the following results: (1) WSF-7 increases PPARγ expression (Fig. 4B); (2) PPARγ-responsive genes, such as adiponectin and GLUT4, are also upregulated by WSF-7 (Figs. 3A, 4B); (3) GW9662 suppresses WSF-7-mediated effect on gene expression. In addition, we found that WSF-7 increases the expression of PPARγ and aP2 (another PPARγ target gene) during preadipocyte differentiation (Fig. 2C). Therefore, WSF-7 stimulates the endogenous PPARγ activity both during and after adipogenesis. However, WSF-7 exhibits much weaker PPARγ-agonistic effects than rosiglitazone (Figs. 1, 2). Therefore, WSF-7 is a partial PPARγ agonist.
PPARγ activities are selectively regulated by the binding of specific ligands at LBD. A small four-stranded β-sheet and 12 α-helices in a three-layer sandwich are present in PPARγ LBD, which provides a large binding pocket and multiple points to interact with ligands. Full agonists, like TZDs, stabilize C-terminal helix 12, which is part of a binding cleft for coactivator in AF2, leading to efficient recruitment of transcriptional coactivator. There is an alternate site for ligand binding in LBD, which is adjacent to a flexible loop with about 15 amino acid residues between helices H2′ and H3.27) When luteolin binds to PPARγ, the amino-terminal portion of AF2 is incompletely folded into α-helical conformation.28) Our results suggest that WSF-7 might activate PPARγ by a novel mechanism (Fig. 1D). We are currently in the process of determining how WSF-7 binds to the LBD of PPARγ.
PPARγ phosphorylation at Ser273 has been linked to insulin resistance.7) PPARγ agonists, both full and partial, specifically inhibit this phosphorylation.6–8,11) Moreover, non-agonist PPARγ ligands also improve insulin sensitivity by inhibiting this phosphorylation.5) The adverse side effects of TZDs have been reported to be reduced significantly by PPARγ partial agonists or even non-agonist without loss of insulin sensitization. Therefore, blocking Ser273 phosphorylation without classical agonism is a powerful strategy for developing novel antidiabetic drugs targeting PPARγ. We have previously identified several natural products from traditional Chinese medicine as partial PPARγ agonists.11,14) WSF-7, the first synthetic PPARγ agonist we have identified in our screen, also inhibits phosphorylation of PPARγ at Ser273 (Fig. 5). It is possible that the binding of WSF-7 at the LBD causes the conformational change of PPARγ, blocking the accessibility of Cdk5 to Ser273. The effect of WSF-7 on the insulin sensitivity of diabetic rats is currently under investigation.
Insulin-induced glucose uptake, with GLUT4 as the major glucose transporter, is very important for maintaining glucose homeostasis in adipocytes.29) The level of GLUT4 correlates highly with insulin sensitivity.30) When blood glucose level is low, GLUT4 is sequestered in the membranes of small internal vesicles. Insulin, secreted from pancreatic β-cells in response to high glucose, induces these vesicles to move to the plasma membrane, with which they fuse, increasing the number of membrane-bound GLUT4. As a result, glucose uptake is stimulated. Our results demonstrate that WSF-7 promotes insulin-induced glucose uptake through activating the transcriptional activity of PPARγ, leading to increased expression and translocation of GLUT4 (Fig. 4), as reported previously for other PPARγ agonists.8,25,26) We failed to detect WSF-7-induced Akt phosphorylation (Fig. 4D). Therefore, WSF-7 does not activate the PI3K/Akt pathway directly. We also demonstrate that WSF-7 promotes adiponectin oligomerization by increasing the level of HMW adiponectin (Fig. 3), which possesses the most potent insulin-sensitizing effect. Therefore, in addition to inhibiting PPARγ Ser273 phosphorylation, WSF-7 also improves insulin sensitivity of adipocytes by increasing the HMW/total Ad ratio and promoting insulin-stimulated glucose uptake. WSF-7 can be easily synthesized from natural monoterpene α-pinene, which is found in the oils of many coniferous trees. These characteristics, combined with its weak PPARγ-agonistic but effective insulin-sensitizing activities, make WSF-7 a potential safer drug for T2DM treatment.
