Original Papers
Phillyrin, a Natural Lignan, Attenuates Tumor Necrosis Factor α-Mediated Insulin Resistance and Lipolytic Acceleration in 3T3-L1 Adipocytes
Authors
Poren Kong 1, Linlin Zhang 2, Yuyu Guo 1, Yingli Lu 1, Dongping Lin 1
Affiliations
1
2
Key words " phillyrin l " 3T3‑L1 adipocytes l " TNFα l " obesity l " insulin resistance l
received revised accepted
January 5, 2014 May 9, 2014 May 24, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368614 Published online July 4, 2014 Planta Med 2014; 80: 880–886 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. Poren Kong (Bo-ren Jiang in Chinese) Department of Endocrine and Metabolic Diseases Laboratory of Endocrine and Metabolic Diseases, Ninth Peopleʼs Hospital Shanghai Jiaotong University, School of Medicine No. 639, Zhizhaoju Road 200211 Shanghai China Phone: + 86 21 23 27 16 99 51 39 Fax: + 86 21 23 27 16 99 [email protected] Correspondence Dr. Dongping Lin Department of Endocrine and Metabolic Diseases Laboratory of Endocrine and Metabolic Diseases, Ninth Peopleʼs Hospital Shanghai Jiaotong University, School of Medicine No. 639, Zhizhaoju Road 200211 Shanghai China Phone: + 86 21 23 27 16 99 51 39 Fax: + 86 21 23 27 16 99 [email protected]
Kong P et al. Phillyrin, a Natural …
Department of Endocrine and Metabolic Diseases, Laboratory of Endocrine and Metabolic Diseases, Ninth Peopleʼs Hospital, Shanghai Jiaotong University, School of Medicine, Shanghai, China Yantai Traditional Chinese Medicine Hospital, Affiliated to Shandong University of Traditional Chinese Medicine, Yantai, China
Abstract !
In obese adipose tissue, tumor necrosis factor-α secreted from macrophages plays an important role in the adipocyte dysfunctions, including insulin resistance, lipolytic acceleration, and changes of adipokines, which promote the development of obesity-related complications. Phillyrin, an active ingredient found in many medicinal plants and certain functional foods, elicits antiobesity and anti-inflammatory properties in vivo. The aim of the current study was to investigate the role of phillyrin in preventing tumor necrosis factor α-induced insulin resistance or lipolytic acceleration in 3T3-L1 adipocytes. Our results showed that phillyrin partially restored insulinstimulated 2-DOG uptake, which was reduced by tumor necrosis factor-α, with concomitant restoration in serine phosphorylation of insulin receptor substrate-1 and insulin-stimulated Glut4 translocation to plasma membrane. Phillyrin also dose-dependently prevented tumor necrosis factor α-stimulated adipocyte lipolysis with preserved downregulation of perilipin. The mitogen-activated protein kinases and I kappaB kinase activation was promoted in tumor necrosis factor α-stimulated adipocytes, but pretreatment with 40 µM phillyrin inhibited the phosphorylation of extracellular signal-regulated kinases1/2, stressactivated protein kinase/Jun N-terminal kinase and I kappaB kinase (p < 0.05). Moreover, phillyrin could inhibit the expressions of interleukin-6 and monocyte chemoattractant protein-1 induced by tumor necrosis factor-α. Using transwell coculture method with 3T3-L1 adipocytes and RAW 264.7 macrophages, the enhanced productions of tumor necrosis factor-α and free fatty acids in the medium were significantly reduced by phillyrin (p < 0.05). These results indicate that phillyrin exerts a beneficial effect on adipocyte dysfunctions induced by tumor necrosis factor-α through sup-
Planta Med 2014; 80: 880–886
pression of the activation of I kappaB kinase and N-terminal kinase. Phillyrin may have the potential to ameliorate the inflammatory changes and insulin resistance in obese adipose tissue.
Abbreviations !
BSA: DMEM:
bovine serum albumin Dulbeccoʼs modified Eagleʼs medium 2-DOG: 2-deoxy-D-[1-3H] glucose DPM: disintegrations per minute ERK: extracellular signal-regulated kinases FFAs: free fatty acids Glut: glucose transporter HDM: high density microsomes IRS-1: insulin receptor substrate-1 IKK: I kappaB kinase IL-6: interleukin-6 IRS-1: insulin receptor substrate-1 JNK: Jun N-terminal kinase KRP: Krebs-Ringerʼs phosphate LDM: low density microsomes MAP kinases:mitogen-activated protein kinases MCP-1: monocyte chemoattractant protein-1 MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide NFκB nuclear factor-κB OD: optical density PM: plasma membranes PMSF: phenylmethanesulfonyl fluoride RIPA: radioimmunoprecipitation assay SAPK: stress-activated protein kinase TNFα: tumor necrosis factor-α Supporting information available online at http://www.thieme-connect.de/products
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Introduction Obesity is strongly associated with the development of insulin resistance, non-alcoholic fatty liver disease, and cardiovascular diseases. Obese adipose tissue is markedly infiltrated by macrophages, with subsequently increased production of proinflammatory cytokines such as TNFα and IL-6, which contribute to chronic, low-grade inflammation [1–4]. It has been shown that TNFα secreted in adipose tissue has pleiotropic effects on adipocytes physiology including an induction of lipolysis to increase the mobilization of FFAs, promoting insulin resistance and activating proinflammatory cytokine expression [5–7], which plays an important role in the development of obesity-related complications. In the absence of TNFα (TNFα−/−), mice had lower levels of circulating FFAs and were protected from obesity-related reduction in the insulin receptor signaling in adipose tissues, resulting in significantly improved insulin sensitivity in both dietinduced obesity and ob/ob model of obesity [8]. Therefore, agents that can disrupt the action of TNFα on adipocytes may have the potential to prevent or delay the onset of or ameliorate obesityrelated diseases. Phillyrin (C27H34O11), one of the main natural lignans from Forsythia suspensa (Thunb.) Vahl (Oleaceae), is commonly used as an important ingredient in the food, beverage, and cosmetic industries [9]. Previous studies showed that phillyrin exerted anti-obesity and triglyceride-lowering effects in nutritive obese mice [10, 11], and Forsythia suspensa leaves could decrease the fasting glucose level in streptozotocin-induced diabetic mice [12]. A recent research has found that phillyrin inhibits lipid accumulation inside HepG2 cells through the stimulation of AMP-activated protein kinase-dependent signaling, which may partially explain its anti-obesity effects [13]. However, the detailed mechanisms of phillyrin on other tissues, especially the key adipose tissue in obesity, are not clear. Some in vitro studies have found that phillyrin could disrupt the production of various inflammatory factors (i.e., nitric oxide, prostaglandin E2, and thromboxane B2) from macrophages [14, 15], suggesting its anti-inflammatory properties. We hypothesize that the inhibition of enhanced inflammatory responses and insulin resistance in adipose tissue may contribute to phillyrinʼs actions on glucose and lipid metabolic disorder in obesity. Therefore, in the present study, the effects of phillyrin on the TNFα-induced insulin resistance and lipolytic acceleration in 3T3-L1 adipocytes were investigated. We hoped to obtain in vitro evidences of the action mechanisms of phillyrin on the improvement of insulin resistance and low-grade inflammation, which might be beneficial to the further understanding of its potential role in the treatment of obesity and type 2 diabetes.
Results and Discussion !
To determine the cytotoxic effect of phillyrin and TNFα on adipo" Fig. 1, phillyrin at cytes, we performed a MTT test. As shown in l concentrations of 5 to 80 µM, in the presence or absence of 10 ng/ mL TNFα, did not show significant cytotoxicity to 3T3-L1 adipocytes. The relative viability values of the adipocytes were all greater than 95.3 % when measured at 96 h after treatment. The effect of phillyrin on glucose transportation in adipocytes was evaluated using 2-DOG uptake. Treatment with phillyrin for 24 h increased glucose uptake of 3T3-L1 adipocytes at concentrations of 40 and 80 µM (17% and 20 %, respectively, p < 0.05)
Fig. 1 Cytotoxicity of phillyrin and 10 ng/mL tumor necrosis factor-α to 3T3-L1 adipocytes. 3T3-L1 adipocytes were exposed to increasing concentrations of phillyrin (from 5 to 80 µM, closed circles) or with TNF-α (10 ng/ mL, closed triangles) for 96 h; the cell viability was then determined by the MTT assay. The values are normalized with those of untreated control (DMSO solvent control) and are shown as percentages of relative cell viability. The results are mean ± SE of six independent experiments.
" Fig. 2 A). The time course with 40 µM phillyrin showed that (l glucose uptake was increased after 24 to 96 h of incubation " Fig. 2 B). Treatment with 10 ng/mL TNFα for 96 h decreased (l the insulin-stimulated 2-DOG uptake by 45 %. Co-treatment with phillyrin antagonized the effect of TNFα and partially reversed TNFα–induced decrease in 2-DOG uptake in a dose-dependent " Fig. 2 C). It is well-known that TNFα impairs insulin manner (l signaling pathway via MAPKs [16] and transcription factors such as NF-κB [17, 18]. Our results also showed that TNFα-induced insulin sensitivity was ameliorated by SP600125 (JNK inhibitor) " Fig. 2 C). and BAY11–7085 (NF-κB inhibitor) (l Serine phosphorylation of IRS-1 is tightly involved in the suppression of IRS-1 activity and insulin resistance by TNFα [19]. From our data, serine phosphorylation of IRS-1 was dramatically enhanced by TNFα, but treatment with phillyrin significantly re" Fig. 2 D). To determine the Glut4 translocaduced this effect (l tion, PM and LDM fractions were separately investigated. The immunoblotting showed that TNFα treatment for 96 h decreased the total Glut4 protein level and insulin-stimulated Glut4 translocation to PM in adipocytes, and phillyrin could attenuate the ef" Fig. 2 E and Fig. 1S, Supporting Information). These data fects (l indicate that phillyrin prevents TNFα–mediated insulin resistance possibly by inhibiting upstream mediators of inflammation and their downstream negative regulators of insulin signaling. TNFα plays an important role in elevated circulating FFAs concentrations in obesity [20, 21]. Triacylglycerol hydrolysis proportionally releases glycerol and FFAs from adipocytes, thus glycerol release in the culture medium was assayed as an index of lipolysis. When 3T3-L1 adipocytes were incubated with 10 µM to 80 µM phillyrin for 24 h, the glycerol levels in the medium were not " Fig. 3 A). The addition of 10 ng/mL TNFα elevated changed (l glycerol release by threefold compared with that under basal conditions, but co-treatment with phillyrin attenuated this effect " Fig. 3 B). One most imin a concentration-dependent manner (l portant molecular event in TNFα-mediated lipolysis is the downregulation of perilipin [22]. We further examined whether phil" Fig. 3 C, phillyrin atlyrin affected perilipin levels. As shown in l tenuated the reduction of perilipin protein expressions in TNFαtreated adipocytes (p < 0.05).
Kong P et al. Phillyrin, a Natural …
Planta Med 2014; 80: 880–886
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Original Papers
Fig. 2 Effects of phillyrin and tumor necrosis factor-α on glucose uptake in 3T3-L1 adipocytes (A to C). 2-DOG uptake change is represented as fold of the control (no treatment). A Change of adipocytes [3H]2-DOG uptake with phillyrin at the indicated concentration for 24 h. B Time course of glucose uptake with or without 40 µM phillyrin. C 3T3-L1 adipocytes were treated with phillyrin at the indicated concentrations, 20 μΜ PD98059 (PD), 10 µM SP600125 (SP), 10 µM BAY11–7085 (BAY), together with TNFα (10 ng/mL) for 96 h. [3H]2-DOG uptake was measured as described under Materials and Methods. Means ± SE for 6 independent experiments are shown. * p < 0.05 compared with the control (A and B), * p < 0.05 compared with TNFα plus insulin (C). D and E Effects of phillyrin and TNFα on phosphorylation of
In 3T3-L1 adipocytes or differentiated human adipocytes, the phosphorylation of ERK1/2 was shown to play a central role in the regulation of TNFα-stimulated lipolysis [23], and the activation of JNK and NF-κB may also participate in this process [6, 24]. It is also well-known that TNFα impairs insulin signaling pathway via MAPKs [16] and IKK/NF-κB activation [17]. To investigate the mechanisms by which phillyrin inhibits TNFα-induced lipolytic acceleration and insulin resistance, we performed immunoblot analysis to examine the phosphorylations of three MAPKs and IKK/NF-κB pathways, including ERK1/2, P38, SAPK/JNK, and IKK" Fig. 4 A, MAPKs phosphorylations were proIκBα. As shown in l moted by TNFα in 3T3-L1 adipocytes, but pretreatment with 40 µM phillyrin significantly inhibited the activation of ERK1/2 and SAPK/JNK. IKK phosphorylation and IκBα degradation were also induced by TNFα, and phillyrin partially blocked this re" Fig. 4 B, p < 0.05). sponse (l In the lipolysis assay, our results showed that PD98059 (ERK inhibitor), but not SP600125 or BAY11–7085, suppressed TNFα-in" Fig. 3 B). However, TNFαduced lipolysis in 3T3-L1 adipocytes (l induced insulin resistance was ameliorated by SP600125 and " Fig. 2 C). These results imply BAY11–7085, but not PD98059 (l that phillyrin exerts its action on lipolysis and insulin resistance through different signaling pathways; further studies are needed to elucidate the detailed mechanism of this action. TNFα can up-regulate the production of several important cytokines and chemokines from adipocytes, such as IL-6 and MCP-1, which are negative regulators of insulin resistance [25, 26]. Here
Kong P et al. Phillyrin, a Natural …
Planta Med 2014; 80: 880–886
Ser307-IRS-1 and Glut4 translocation in 3T3-L1 adipocytes. D 3T3-L1 adipocytes were preincubated with indicated concentrations of phillyrin for 2 h and were then treated with or without TNFα (2 ng/mL) for 30 min to determine the protein concentration of serine residue 307 phosphorylation of IRS1 (p-IRS-1-Ser307) by immunoblotting. Results are representative of three individual experiments. E 3T3-L1 adipocytes were treated with TNFα (10 ng/ mL) in the absence or presence of 40 µM phillyrin for 96 h followed by 10 nM insulin treatment for 30 min. Total Glut1, Glut4, Glut4 in PM and LDM were prepared. Representative blots from three independent experiments are shown. * p < 0.05 compared with TNFα alone.
we investigated whether phillyrin also modulated the expressions of MCP-1 and IL-6 induced by TNFα. Results from quantitative-PCR analysis showed that the enhanced expressions of MCP1 and IL-6 mRNA by TNF-α-stimulation were effectively inhibited " Fig. 5). Activation of the transcription by phillyrin treatment (l factor NF-κB is considered to play a major role in TNFα-induced inflammatory responses [27, 28]. NF-κB is activated by TNF-α via phosphorylation and proteasomal degradation of IκBα, resulting in its translocation into the nucleus, where it promotes the expression of numerous target genes whose products induce further insulin resistance, such as MCP-1 and IL-6 [27]. According to these mechanisms, the anti-inflammatory properties of phillyrin, such as attenuation of MCP-1 and IL-6 expressions, may be attributed to the suppression of NF-κB activation in adipocytes. As outlined above, phillyrin attenuated TNFα-mediated lipolytic acceleration and inflammatory factor release from adipocytes. TNFα is mainly secreted from macrophages infiltrated in adipose tissue. The paracrine loop involving saturated FFAs and TNFα derived from adipocytes and macrophages, respectively, aggravates obesity-induced adipose tissue inflammation [3, 29]. Since Suganami et al. firstly developed an in vitro coculture system composed of adipocytes and macrophages in 2005 [29], the method was widely adopted to mimic the inflammatory status in obese adipose tissue in vivo. We investigated whether phillyrin suppressed the inflammatory interaction between 3T3-L1 adipocytes and RAW 264.7 macrophages using this transwell coculture " Fig. 6 A, TNFα concentrations in the coculmethod. As shown in l
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Fig. 4 Effects of phillyrin on the phosphorylation of mitogen-activated protein kinases and I kappaB kinase in adipocytes. 3T3-L1 adipocytes were pretreated with 40 µM phillyrin for 2 h, then stimulated with 10 ng/ mL TNFα, phillyrin, or both for another 5– 15 min. Change of ERK1/2, SAPK/JNK, P38 phosphorylations (A) and IKK phosphorylation, IκBα degradation (B) were analyzed by Western blotting. Representative blots are shown from at least three independent experiments.
Fig. 3 Effect of phillyrin on tumor necrosis factor α-induced lipolysis in adipocyte. A 3T3-L1 adipocytes were incubated with phillyrin at the indicated concentration for 24 h. B 3T3-L1 adipocytes were preincubated with indicated concentrations of phillyrin, 20 μΜ PD98059 (PD), 10 µM SP600125 (SP), 10 µM BAY11–7085 (BAY) for 2 h; the cells were then treated for 24 h after the addition of 10 ng/mL TNFα. Glycerol releases in the culture media were assayed. Data are expressed as means ± SE of 6 tests. * p < 0.05 compared with 10 ng/mL TNFα alone. C Adipocytes were preincubated for 2 h with 40 µM phillyrin and then treated for 24 h in the presence of 10 ng/mL TNFα. The cells were lysed, and perilipin protein expression was analyzed by immunoblotting. Results are representative of three individual experiments. * p < 0.05 compared with TNFα alone.
ture medium were three times higher than those for macrophages alone, and the secretions were significantly suppressed by phillyrin in a dose-dependent manner. Similar effects of phillyrin on FFAs secretions were also observed. FFAs levels in coculture medium were markedly increased compared with those from adipocytes alone, but treatment with phillyrin significantly
Fig. 5 Effects of phillyrin on the mRNA expression of interleukin-6 and monocyte chemoattractant protein-1 induced by tumor necrosis factor-α in 3T3-L1 adipocytes. Adipocytes were preincubated for 2 h with 40 µM phillyrin and then treated for 24 h in the presence of 10 ng/mL TNFα before total mRNA was extracted using Trizol reagent. Real-time PCR for IL-6 (A) and MCP-1 (B) expressions was performed as described in Materials and Methods. Data are expressed as means ± SE (n = 4). * p < 0.05 compared with TNFα alone.
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Original Papers
Original Papers
Materials and Methods !
Materials Phillyrin was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing), purity ≥ 98% (HPLC). Recombinant murine TNFα, PD98059 (purity ≥ 98%, HPLC), SP600125 (purity ≥ 98 %, HPLC), and BAY11–7085 (purity ≥ 98 %, HPLC) were obtained from Sigma-Aldrich. Antibodies against p-ERK1/2, p-P38, p-SAPK/JNK, p-IKKα/IKKβ, IκBα, p-IRS-1(Ser307), and IRS-1 were from Cell Signaling Technology Inc.; antibody against perilipin A was from Affinity Bioreagents; antibodies against Glut-1, Glut-4, and HRP-linked anti-goat IgG were from Santa Cruz Biotechnology Inc. 2-DOG was purchased from Amersham. Unless otherwise specified, all other reagents were purchased from Sigma.
Cell culture and cytotoxicity test
Fig. 6 Effects of phillyrin on the secretion of inflammatory mediators in coculture system. Differentiated 3T3-L1 adipocytes were cocultured with RAW 264.7 macrophages for 24 h with or without various concentrations of phillyrin. Released TNFα (A) levels in the coculture medium were measured as described in Materials and Methods, and those from macrophages alone were used as a negative control. FFAs (B) concentrations were also measured in the coculture medium, and FFAs from adipocytes alone were used as a negative control. The values are means ± SE of 6 tests. * p < 0.05 compared with non-treated coculture.
inhibited the secretion even at the lower concentration (10 µM) " Fig. 6 B). These results indicate that phillyrin suppresses the (l production of pro-inflammatory factors induced in the co-culture, thus suggesting that phillyrin might have the potential to improve chronic inflammatory responses in obese adipose tissue. In summary, these data demonstrate that phillyrin 1) blocks TNFα-induced adipocytes insulin resistance and lipolytic acceleration through inhibiting MAP kinases and IKK phosphorylations, 2) inhibits the expressions of IL-6 and MCP-1 induced by TNFα, 3) suppresses the production of proinflammatory mediators in the coculture system composed of adipocytes and macrophages. The improvement of phillyrin on glucose and lipid metabolic disorder in obesity may be attributed to the attenuation of chronic inflammatory conditions and the improvement in insulin sensitivity in obese adipose tissue.
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3T3-L1 preadipocytes (American Type Culture Collection) were maintained in DMEM (Gibco) containing 10% fetal bovine serum. Differentiation of 3T3-L1 preadipocytes to mature adipocytes was performed as previously described [30]. Phillyrin was dissolved in DMSO as a 1000-fold stock and added to the medium at various concentrations as shown in each figure. DMSO was present in the control culture at a concentration of 0.1 % (v/v). Possible cytotoxicity of phillyrin was assessed by the MTT staining method. Briefly, the mature adipocytes were treated with various concentrations of phillyrin or with 10 ng/mL TNF-α for 96 h. Subsequent incubation of the cells with 100 µl MTT solution (5.0 mg/ml) dissolved in serum-free DMEM for 4 h at 37 °C allowed formation of a violet precipitate, formazan. All liquid in each well was removed, and 100 µl of DMSO was added. The OD at 570 nm was determined by ELISA spectrometry.
Induction of insulin resistance and glucose uptake assay To induce insulin resistance, differentiated 3T3-L1 adipocytes were exposed to 10 ng/mL TNFα for 96 h. During the treatment period, fresh TNFα or phillyrin was changed every 24 h. Transport assay was initiated by washing the cells twice in KRP buffer (1.32 mM NaCl, 4.71 mM KCl, 47 mM CaCl2, 1.24 mM MgSO4, 2.48 mM Na3PO4, 10 mM HEPES, pH 7.4). Cells were then incubated in the KRP solution for another 30 min with or without 100 nM insulin at 37 °C. Then 0.5 µCi/mL 2-DOG (final concentration) was added to the cells. After incubation for 10 min, the medium was aspirated and the cells were washed 3 times with icecold KRP containing 10 mM glucose. The cells were lysed with 0.1 N NaOH and subsequently solubilized in scintillation fluids (Triton X-100/methylbenzene, 1 : 2.5) overnight. The radioactivity taken up by the cells was determined using a scintillation counter (Beckman Instruments). DPM value was corrected by protein content in each well, which was measured with BCA protein assay kit (Pierce).
Lipolysis assay Differentiated 3T3-L1 adipocytes were incubated overnight in DMEM with 0.5 % BSA in the absence of serum. The following morning, cells were treated as described in Results. The culture supernatant was transferred to another set of tubes and heated at 70 °C for 10 min to inactivate any enzymes released by the cells. Samples (25 µL) were then used for glycerol assay with 175 µL of glycerol reagent (GPO Trinder Reagent A, Sigma-Aldrich) in a flat-bottom 96-well plate. Absorption was measured
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Extract of total cell protein and subcellular membrane fractions Whole cell lysates from adipocytes grown on 10 cm dishes were prepared using cell lysis RIPA buffer, containing 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 % Triton x-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, complete protease inhibitor cocktail, and complete phosphatase inhibitors. The lysates were centrifuged at 12 000 g for 10 min at 4 °C to remove the insoluble materials. After washing with PBS, adipocytes were scraped in icecold HES buffer (20 mM HEPES, 250 mM sucrose, 1 mM EDTA (pH 7.4)) containing a protease inhibitor cocktail and subsequently homogenized with 30 strokes in a glass Dounce homogenizer at 4 °C. The homogenate was centrifuged at 16 000 g for 20 min. The supernatant was further centrifuged at 36 000 g for 5 min for HDM fraction and at 200 000 g for 24 min for LDM fraction. The PM fraction was collected from the interface of a 1.12 M sucrose cushion following centrifugation of the 16 000 g pellet at 70 000 g for 10 min. PM were then resuspended in homogenization buffer and pelleted at 200 000 g for 4 min. The obtained fractions were then resuspended in HES buffer. Protein concentrations of PM and LDM were determined with BCA protein assay kit (Pierce) and subjected to Western blotting analysis.
Immunoblotting Equal amounts of protein from each sample were boiled in SDSloading buffer for 5 min and loaded and separated by SDS-polyacrylamide gel electrophoresis. Gels were then blotted onto PVDF membranes (Amersham). The membranes were blocked and incubated with different antibodies, followed by incubation with secondary antibodies conjugated to horseradish peroxidase. Antigen-antibody complexes were detected by chemiluminescence and exposed to high-performance Kodak film. Films were scanned, and specific bands were quantified using the Qualityone Image software.
Coculture of adipocytes and macrophages Differentiated 3T3-L1 adipocytes were cocultured with RAW 264.7 in the medium containing 2 % fatty acid-free BSA for 24 hours in a transwell system. Briefly, differentiated 3T3-L1 adipocytes were incubated overnight in DMEM with 0.5 % BSA in the absence of serum. RAW 264.7 cells (2 × 105 cells/mL) were plated onto dishes with serum-starved and hypertrophied 3T3-L1 cells by using transwell inserts with a 0.4-mm porous membrane (Corning), and the coculture was incubated in serum-free DMEM for 24 h. RAW264.7 and 3T3-L1 cells of equal numbers to those in the coculture were cultured separately as control cultures. Phillyrin was added to the coculture at various concentrations as shown in each figure. After 24 h of treatment, culture supernatants were collected and stored at − 20 °C until measurement.
Measurement of tumor necrosis factor-α and free fatty acids concentrations in the coculture medium The concentrations of TNFα in the culture medium were determined by commercially available enzyme-linked immunosorbent assay (R&D Systems), according to the manufacturerʼs protocol. The concentrations of FFAs in the medium were measured by an acyl-coenzyme A oxidase-based colorimetric assay kit (NEFA‑C, Wako Pure Chemicals).
Statistical analysis Statistical analysis of the data were performed by using one-factor analysis of variance (ANOVA) or by testing the main effects of TNF-α alone, phillyrin alone, and their interaction by using 2-factor ANOVA (SPSS). Tukeyʼs HSD tests were used to compute individual pairwise comparisons of means. All results were expressed as mean ± SE. A p value < 0.05 was considered to be statistically significant.
Suppporting information The relative quantities of total Glut4 and Glut4 in the PM fraction " Fig. 2 E are available as Supporting Information. shown in l
Real time quantitative reverse transcription PCR
Acknowledgments
Total RNA from 3T3-L1 cells was isolated with Trizol reagent, and reverse transcription of 1 µg RNA was carried out with the SuperScript III reverse transcriptase following Invitrogenʼs protocol with oligo dT primer. Quantitative PCR amplification and detection were performed with SYBR Premix Ex Taq (TaKaRa), according to the manufacturerʼs protocol on ABI Prism 7300 sequence detection system (Applied Biosystems). Cycling parameters were 95 °C for 10 s, then 40 cycles of 95 °C for 5 s and 60 °C for 34 s. After PCR, a melting curve analysis was performed to demonstrate the specificity of the PCR product, which was displayed as a single peak (data not shown). Quantifications were performed in duplicate, and the experiments were repeated independently three times. As a control, the mRNA level of 36B4 was determined in the real time PCR assay for each RNA sample and was used to correct for experimental variations. The following primer sequences were used: " IL-6, 5′-tagtccttcctaccccaatttcc-3′ (forward), 5′-ttggtccttagccactccttc-3′ (reverse); " MCP-1, 5′-TTCCTCCACCACCATGCAG‑3′ (forward), 5′-CCAGCCGGCAACTGTGA‑3′ (reverse); " 36B4, 5′-AAGCGCGTCCTGGCATTGTCT‑3′ (forward), 5′-CCGCAGGGGCAGCAGTGGT‑3′ (reverse).
!
This work was supported by the National Natural Science Foundation of China (grant No. 81000345) and by the Shanghai Natural Science Foundation (grant No. 09ZR1417100).
Conflict of Interest !
The authors declare that they have no competing interests.
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19 Zhang Z, Li X, Lv W, Yang Y, Gao H, Yang J, Shen Y, Ning G. Ginsenoside Re reduces insulin resistance through inhibition of c-Jun NH2-terminal kinase and nuclear factor-kappaB. Mol Endocrinol 2008; 22: 186–195 20 Orban Z, Remaley AT, Sampson M, Trajanoski Z, Chrousos GP. The differential effect of food intake and beta-adrenergic stimulation on adipose-derived hormones and cytokines in man. J Clin Endocrinol Metab 1999; 84: 2126–2133 21 Doerrler W, Feingold KR, Grunfeld C. Cytokines induce catabolic effects in cultured adipocytes by multiple mechanisms. Cytokine 1994; 6: 478–484 22 Mottagui-Tabar S, Ryden M, Lofgren P, Faulds G, Hoffstedt J, Brookes AJ, Andersson I, Arner P. Evidence for an important role of perilipin in the regulation of human adipocyte lipolysis. Diabetologia 2003; 46: 789– 797 23 Souza SC, Palmer HJ, Kang YH, Yamamoto MT, Muliro KV, Paulson KE, Greenberg AS. TNF-alpha induction of lipolysis is mediated through activation of the extracellular signal related kinase pathway in 3T3-L1 adipocytes. J Cell Biochem 2003; 89: 1077–1086 24 Laurencikiene J, van Harmelen V, Arvidsson Nordstrom E, Dicker A, Blomqvist L, Naslund E, Langin D, Arner P, Ryden M. NF-kappaB is important for TNF-alpha-induced lipolysis in human adipocytes. J Lipid Res 2007; 48: 1069–1077 25 Fasshauer M, Kralisch S, Klier M, Lossner U, Bluher M, Klein J, Paschke R. Adiponectin gene expression and secretion is inhibited by interleukin6 in 3T3-L1 adipocytes. Biochem Biophys Res Commun 2003; 301: 1045–1050 26 Kim KY, Kim JK, Jeon JH, Yoon SR, Choi I, Yang Y. c-Jun N-terminal kinase is involved in the suppression of adiponectin expression by TNF-alpha in 3T3-L1 adipocytes. Biochem Biophys Res Commun 2005; 327: 460– 467 27 Yoshida H, Takamura N, Shuto T, Ogata K, Tokunaga J, Kawai K, Kai H. The citrus flavonoids hesperetin and naringenin block the lipolytic actions of TNF-alpha in mouse adipocytes. Biochem Biophys Res Commun 2010; 394: 728–732 28 Teferedegne B, Green MR, Guo Z, Boss JM. Mechanism of action of a distal NF-kappaB-dependent enhancer. Mol Cell Biol 2006; 26: 5759–5770 29 Suganami T, Nishida J, Ogawa Y. A paracrine loop between adipocytes and macrophages aggravates inflammatory changes: role of free fatty acids and tumor necrosis factor alpha. Arterioscler Thromb Vasc Biol 2005; 25: 2062–2068 30 Jiang B, Qiao J, Yang Y, Lu Y. Inhibitory effect of paeoniflorin on the inflammatory vicious cycle between adipocytes and macrophages. J Cell Biochem 2012; 113: 2560–2566
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Phillyrin, a Natural Lignan, Attenuates Tumor Necrosis Factor α-Mediated Insulin Resistance and Lipolytic Acceleration in 3T3-L1 Adipocytes
Authors
Poren Kong 1, Linlin Zhang 2, Yuyu Guo 1, Yingli Lu 1, Dongping Lin 1
Affiliations
1
2
Key words " phillyrin l " 3T3‑L1 adipocytes l " TNFα l " obesity l " insulin resistance l
received revised accepted
January 5, 2014 May 9, 2014 May 24, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368614 Published online July 4, 2014 Planta Med 2014; 80: 880–886 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. Poren Kong (Bo-ren Jiang in Chinese) Department of Endocrine and Metabolic Diseases Laboratory of Endocrine and Metabolic Diseases, Ninth Peopleʼs Hospital Shanghai Jiaotong University, School of Medicine No. 639, Zhizhaoju Road 200211 Shanghai China Phone: + 86 21 23 27 16 99 51 39 Fax: + 86 21 23 27 16 99 [email protected] Correspondence Dr. Dongping Lin Department of Endocrine and Metabolic Diseases Laboratory of Endocrine and Metabolic Diseases, Ninth Peopleʼs Hospital Shanghai Jiaotong University, School of Medicine No. 639, Zhizhaoju Road 200211 Shanghai China Phone: + 86 21 23 27 16 99 51 39 Fax: + 86 21 23 27 16 99 [email protected]
Kong P et al. Phillyrin, a Natural …
Department of Endocrine and Metabolic Diseases, Laboratory of Endocrine and Metabolic Diseases, Ninth Peopleʼs Hospital, Shanghai Jiaotong University, School of Medicine, Shanghai, China Yantai Traditional Chinese Medicine Hospital, Affiliated to Shandong University of Traditional Chinese Medicine, Yantai, China
Abstract !
In obese adipose tissue, tumor necrosis factor-α secreted from macrophages plays an important role in the adipocyte dysfunctions, including insulin resistance, lipolytic acceleration, and changes of adipokines, which promote the development of obesity-related complications. Phillyrin, an active ingredient found in many medicinal plants and certain functional foods, elicits antiobesity and anti-inflammatory properties in vivo. The aim of the current study was to investigate the role of phillyrin in preventing tumor necrosis factor α-induced insulin resistance or lipolytic acceleration in 3T3-L1 adipocytes. Our results showed that phillyrin partially restored insulinstimulated 2-DOG uptake, which was reduced by tumor necrosis factor-α, with concomitant restoration in serine phosphorylation of insulin receptor substrate-1 and insulin-stimulated Glut4 translocation to plasma membrane. Phillyrin also dose-dependently prevented tumor necrosis factor α-stimulated adipocyte lipolysis with preserved downregulation of perilipin. The mitogen-activated protein kinases and I kappaB kinase activation was promoted in tumor necrosis factor α-stimulated adipocytes, but pretreatment with 40 µM phillyrin inhibited the phosphorylation of extracellular signal-regulated kinases1/2, stressactivated protein kinase/Jun N-terminal kinase and I kappaB kinase (p < 0.05). Moreover, phillyrin could inhibit the expressions of interleukin-6 and monocyte chemoattractant protein-1 induced by tumor necrosis factor-α. Using transwell coculture method with 3T3-L1 adipocytes and RAW 264.7 macrophages, the enhanced productions of tumor necrosis factor-α and free fatty acids in the medium were significantly reduced by phillyrin (p < 0.05). These results indicate that phillyrin exerts a beneficial effect on adipocyte dysfunctions induced by tumor necrosis factor-α through sup-
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pression of the activation of I kappaB kinase and N-terminal kinase. Phillyrin may have the potential to ameliorate the inflammatory changes and insulin resistance in obese adipose tissue.
Abbreviations !
BSA: DMEM:
bovine serum albumin Dulbeccoʼs modified Eagleʼs medium 2-DOG: 2-deoxy-D-[1-3H] glucose DPM: disintegrations per minute ERK: extracellular signal-regulated kinases FFAs: free fatty acids Glut: glucose transporter HDM: high density microsomes IRS-1: insulin receptor substrate-1 IKK: I kappaB kinase IL-6: interleukin-6 IRS-1: insulin receptor substrate-1 JNK: Jun N-terminal kinase KRP: Krebs-Ringerʼs phosphate LDM: low density microsomes MAP kinases:mitogen-activated protein kinases MCP-1: monocyte chemoattractant protein-1 MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide NFκB nuclear factor-κB OD: optical density PM: plasma membranes PMSF: phenylmethanesulfonyl fluoride RIPA: radioimmunoprecipitation assay SAPK: stress-activated protein kinase TNFα: tumor necrosis factor-α Supporting information available online at http://www.thieme-connect.de/products
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Introduction Obesity is strongly associated with the development of insulin resistance, non-alcoholic fatty liver disease, and cardiovascular diseases. Obese adipose tissue is markedly infiltrated by macrophages, with subsequently increased production of proinflammatory cytokines such as TNFα and IL-6, which contribute to chronic, low-grade inflammation [1–4]. It has been shown that TNFα secreted in adipose tissue has pleiotropic effects on adipocytes physiology including an induction of lipolysis to increase the mobilization of FFAs, promoting insulin resistance and activating proinflammatory cytokine expression [5–7], which plays an important role in the development of obesity-related complications. In the absence of TNFα (TNFα−/−), mice had lower levels of circulating FFAs and were protected from obesity-related reduction in the insulin receptor signaling in adipose tissues, resulting in significantly improved insulin sensitivity in both dietinduced obesity and ob/ob model of obesity [8]. Therefore, agents that can disrupt the action of TNFα on adipocytes may have the potential to prevent or delay the onset of or ameliorate obesityrelated diseases. Phillyrin (C27H34O11), one of the main natural lignans from Forsythia suspensa (Thunb.) Vahl (Oleaceae), is commonly used as an important ingredient in the food, beverage, and cosmetic industries [9]. Previous studies showed that phillyrin exerted anti-obesity and triglyceride-lowering effects in nutritive obese mice [10, 11], and Forsythia suspensa leaves could decrease the fasting glucose level in streptozotocin-induced diabetic mice [12]. A recent research has found that phillyrin inhibits lipid accumulation inside HepG2 cells through the stimulation of AMP-activated protein kinase-dependent signaling, which may partially explain its anti-obesity effects [13]. However, the detailed mechanisms of phillyrin on other tissues, especially the key adipose tissue in obesity, are not clear. Some in vitro studies have found that phillyrin could disrupt the production of various inflammatory factors (i.e., nitric oxide, prostaglandin E2, and thromboxane B2) from macrophages [14, 15], suggesting its anti-inflammatory properties. We hypothesize that the inhibition of enhanced inflammatory responses and insulin resistance in adipose tissue may contribute to phillyrinʼs actions on glucose and lipid metabolic disorder in obesity. Therefore, in the present study, the effects of phillyrin on the TNFα-induced insulin resistance and lipolytic acceleration in 3T3-L1 adipocytes were investigated. We hoped to obtain in vitro evidences of the action mechanisms of phillyrin on the improvement of insulin resistance and low-grade inflammation, which might be beneficial to the further understanding of its potential role in the treatment of obesity and type 2 diabetes.
Results and Discussion !
To determine the cytotoxic effect of phillyrin and TNFα on adipo" Fig. 1, phillyrin at cytes, we performed a MTT test. As shown in l concentrations of 5 to 80 µM, in the presence or absence of 10 ng/ mL TNFα, did not show significant cytotoxicity to 3T3-L1 adipocytes. The relative viability values of the adipocytes were all greater than 95.3 % when measured at 96 h after treatment. The effect of phillyrin on glucose transportation in adipocytes was evaluated using 2-DOG uptake. Treatment with phillyrin for 24 h increased glucose uptake of 3T3-L1 adipocytes at concentrations of 40 and 80 µM (17% and 20 %, respectively, p < 0.05)
Fig. 1 Cytotoxicity of phillyrin and 10 ng/mL tumor necrosis factor-α to 3T3-L1 adipocytes. 3T3-L1 adipocytes were exposed to increasing concentrations of phillyrin (from 5 to 80 µM, closed circles) or with TNF-α (10 ng/ mL, closed triangles) for 96 h; the cell viability was then determined by the MTT assay. The values are normalized with those of untreated control (DMSO solvent control) and are shown as percentages of relative cell viability. The results are mean ± SE of six independent experiments.
" Fig. 2 A). The time course with 40 µM phillyrin showed that (l glucose uptake was increased after 24 to 96 h of incubation " Fig. 2 B). Treatment with 10 ng/mL TNFα for 96 h decreased (l the insulin-stimulated 2-DOG uptake by 45 %. Co-treatment with phillyrin antagonized the effect of TNFα and partially reversed TNFα–induced decrease in 2-DOG uptake in a dose-dependent " Fig. 2 C). It is well-known that TNFα impairs insulin manner (l signaling pathway via MAPKs [16] and transcription factors such as NF-κB [17, 18]. Our results also showed that TNFα-induced insulin sensitivity was ameliorated by SP600125 (JNK inhibitor) " Fig. 2 C). and BAY11–7085 (NF-κB inhibitor) (l Serine phosphorylation of IRS-1 is tightly involved in the suppression of IRS-1 activity and insulin resistance by TNFα [19]. From our data, serine phosphorylation of IRS-1 was dramatically enhanced by TNFα, but treatment with phillyrin significantly re" Fig. 2 D). To determine the Glut4 translocaduced this effect (l tion, PM and LDM fractions were separately investigated. The immunoblotting showed that TNFα treatment for 96 h decreased the total Glut4 protein level and insulin-stimulated Glut4 translocation to PM in adipocytes, and phillyrin could attenuate the ef" Fig. 2 E and Fig. 1S, Supporting Information). These data fects (l indicate that phillyrin prevents TNFα–mediated insulin resistance possibly by inhibiting upstream mediators of inflammation and their downstream negative regulators of insulin signaling. TNFα plays an important role in elevated circulating FFAs concentrations in obesity [20, 21]. Triacylglycerol hydrolysis proportionally releases glycerol and FFAs from adipocytes, thus glycerol release in the culture medium was assayed as an index of lipolysis. When 3T3-L1 adipocytes were incubated with 10 µM to 80 µM phillyrin for 24 h, the glycerol levels in the medium were not " Fig. 3 A). The addition of 10 ng/mL TNFα elevated changed (l glycerol release by threefold compared with that under basal conditions, but co-treatment with phillyrin attenuated this effect " Fig. 3 B). One most imin a concentration-dependent manner (l portant molecular event in TNFα-mediated lipolysis is the downregulation of perilipin [22]. We further examined whether phil" Fig. 3 C, phillyrin atlyrin affected perilipin levels. As shown in l tenuated the reduction of perilipin protein expressions in TNFαtreated adipocytes (p < 0.05).
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Fig. 2 Effects of phillyrin and tumor necrosis factor-α on glucose uptake in 3T3-L1 adipocytes (A to C). 2-DOG uptake change is represented as fold of the control (no treatment). A Change of adipocytes [3H]2-DOG uptake with phillyrin at the indicated concentration for 24 h. B Time course of glucose uptake with or without 40 µM phillyrin. C 3T3-L1 adipocytes were treated with phillyrin at the indicated concentrations, 20 μΜ PD98059 (PD), 10 µM SP600125 (SP), 10 µM BAY11–7085 (BAY), together with TNFα (10 ng/mL) for 96 h. [3H]2-DOG uptake was measured as described under Materials and Methods. Means ± SE for 6 independent experiments are shown. * p < 0.05 compared with the control (A and B), * p < 0.05 compared with TNFα plus insulin (C). D and E Effects of phillyrin and TNFα on phosphorylation of
In 3T3-L1 adipocytes or differentiated human adipocytes, the phosphorylation of ERK1/2 was shown to play a central role in the regulation of TNFα-stimulated lipolysis [23], and the activation of JNK and NF-κB may also participate in this process [6, 24]. It is also well-known that TNFα impairs insulin signaling pathway via MAPKs [16] and IKK/NF-κB activation [17]. To investigate the mechanisms by which phillyrin inhibits TNFα-induced lipolytic acceleration and insulin resistance, we performed immunoblot analysis to examine the phosphorylations of three MAPKs and IKK/NF-κB pathways, including ERK1/2, P38, SAPK/JNK, and IKK" Fig. 4 A, MAPKs phosphorylations were proIκBα. As shown in l moted by TNFα in 3T3-L1 adipocytes, but pretreatment with 40 µM phillyrin significantly inhibited the activation of ERK1/2 and SAPK/JNK. IKK phosphorylation and IκBα degradation were also induced by TNFα, and phillyrin partially blocked this re" Fig. 4 B, p < 0.05). sponse (l In the lipolysis assay, our results showed that PD98059 (ERK inhibitor), but not SP600125 or BAY11–7085, suppressed TNFα-in" Fig. 3 B). However, TNFαduced lipolysis in 3T3-L1 adipocytes (l induced insulin resistance was ameliorated by SP600125 and " Fig. 2 C). These results imply BAY11–7085, but not PD98059 (l that phillyrin exerts its action on lipolysis and insulin resistance through different signaling pathways; further studies are needed to elucidate the detailed mechanism of this action. TNFα can up-regulate the production of several important cytokines and chemokines from adipocytes, such as IL-6 and MCP-1, which are negative regulators of insulin resistance [25, 26]. Here
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Ser307-IRS-1 and Glut4 translocation in 3T3-L1 adipocytes. D 3T3-L1 adipocytes were preincubated with indicated concentrations of phillyrin for 2 h and were then treated with or without TNFα (2 ng/mL) for 30 min to determine the protein concentration of serine residue 307 phosphorylation of IRS1 (p-IRS-1-Ser307) by immunoblotting. Results are representative of three individual experiments. E 3T3-L1 adipocytes were treated with TNFα (10 ng/ mL) in the absence or presence of 40 µM phillyrin for 96 h followed by 10 nM insulin treatment for 30 min. Total Glut1, Glut4, Glut4 in PM and LDM were prepared. Representative blots from three independent experiments are shown. * p < 0.05 compared with TNFα alone.
we investigated whether phillyrin also modulated the expressions of MCP-1 and IL-6 induced by TNFα. Results from quantitative-PCR analysis showed that the enhanced expressions of MCP1 and IL-6 mRNA by TNF-α-stimulation were effectively inhibited " Fig. 5). Activation of the transcription by phillyrin treatment (l factor NF-κB is considered to play a major role in TNFα-induced inflammatory responses [27, 28]. NF-κB is activated by TNF-α via phosphorylation and proteasomal degradation of IκBα, resulting in its translocation into the nucleus, where it promotes the expression of numerous target genes whose products induce further insulin resistance, such as MCP-1 and IL-6 [27]. According to these mechanisms, the anti-inflammatory properties of phillyrin, such as attenuation of MCP-1 and IL-6 expressions, may be attributed to the suppression of NF-κB activation in adipocytes. As outlined above, phillyrin attenuated TNFα-mediated lipolytic acceleration and inflammatory factor release from adipocytes. TNFα is mainly secreted from macrophages infiltrated in adipose tissue. The paracrine loop involving saturated FFAs and TNFα derived from adipocytes and macrophages, respectively, aggravates obesity-induced adipose tissue inflammation [3, 29]. Since Suganami et al. firstly developed an in vitro coculture system composed of adipocytes and macrophages in 2005 [29], the method was widely adopted to mimic the inflammatory status in obese adipose tissue in vivo. We investigated whether phillyrin suppressed the inflammatory interaction between 3T3-L1 adipocytes and RAW 264.7 macrophages using this transwell coculture " Fig. 6 A, TNFα concentrations in the coculmethod. As shown in l
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Fig. 4 Effects of phillyrin on the phosphorylation of mitogen-activated protein kinases and I kappaB kinase in adipocytes. 3T3-L1 adipocytes were pretreated with 40 µM phillyrin for 2 h, then stimulated with 10 ng/ mL TNFα, phillyrin, or both for another 5– 15 min. Change of ERK1/2, SAPK/JNK, P38 phosphorylations (A) and IKK phosphorylation, IκBα degradation (B) were analyzed by Western blotting. Representative blots are shown from at least three independent experiments.
Fig. 3 Effect of phillyrin on tumor necrosis factor α-induced lipolysis in adipocyte. A 3T3-L1 adipocytes were incubated with phillyrin at the indicated concentration for 24 h. B 3T3-L1 adipocytes were preincubated with indicated concentrations of phillyrin, 20 μΜ PD98059 (PD), 10 µM SP600125 (SP), 10 µM BAY11–7085 (BAY) for 2 h; the cells were then treated for 24 h after the addition of 10 ng/mL TNFα. Glycerol releases in the culture media were assayed. Data are expressed as means ± SE of 6 tests. * p < 0.05 compared with 10 ng/mL TNFα alone. C Adipocytes were preincubated for 2 h with 40 µM phillyrin and then treated for 24 h in the presence of 10 ng/mL TNFα. The cells were lysed, and perilipin protein expression was analyzed by immunoblotting. Results are representative of three individual experiments. * p < 0.05 compared with TNFα alone.
ture medium were three times higher than those for macrophages alone, and the secretions were significantly suppressed by phillyrin in a dose-dependent manner. Similar effects of phillyrin on FFAs secretions were also observed. FFAs levels in coculture medium were markedly increased compared with those from adipocytes alone, but treatment with phillyrin significantly
Fig. 5 Effects of phillyrin on the mRNA expression of interleukin-6 and monocyte chemoattractant protein-1 induced by tumor necrosis factor-α in 3T3-L1 adipocytes. Adipocytes were preincubated for 2 h with 40 µM phillyrin and then treated for 24 h in the presence of 10 ng/mL TNFα before total mRNA was extracted using Trizol reagent. Real-time PCR for IL-6 (A) and MCP-1 (B) expressions was performed as described in Materials and Methods. Data are expressed as means ± SE (n = 4). * p < 0.05 compared with TNFα alone.
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Materials and Methods !
Materials Phillyrin was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing), purity ≥ 98% (HPLC). Recombinant murine TNFα, PD98059 (purity ≥ 98%, HPLC), SP600125 (purity ≥ 98 %, HPLC), and BAY11–7085 (purity ≥ 98 %, HPLC) were obtained from Sigma-Aldrich. Antibodies against p-ERK1/2, p-P38, p-SAPK/JNK, p-IKKα/IKKβ, IκBα, p-IRS-1(Ser307), and IRS-1 were from Cell Signaling Technology Inc.; antibody against perilipin A was from Affinity Bioreagents; antibodies against Glut-1, Glut-4, and HRP-linked anti-goat IgG were from Santa Cruz Biotechnology Inc. 2-DOG was purchased from Amersham. Unless otherwise specified, all other reagents were purchased from Sigma.
Cell culture and cytotoxicity test
Fig. 6 Effects of phillyrin on the secretion of inflammatory mediators in coculture system. Differentiated 3T3-L1 adipocytes were cocultured with RAW 264.7 macrophages for 24 h with or without various concentrations of phillyrin. Released TNFα (A) levels in the coculture medium were measured as described in Materials and Methods, and those from macrophages alone were used as a negative control. FFAs (B) concentrations were also measured in the coculture medium, and FFAs from adipocytes alone were used as a negative control. The values are means ± SE of 6 tests. * p < 0.05 compared with non-treated coculture.
inhibited the secretion even at the lower concentration (10 µM) " Fig. 6 B). These results indicate that phillyrin suppresses the (l production of pro-inflammatory factors induced in the co-culture, thus suggesting that phillyrin might have the potential to improve chronic inflammatory responses in obese adipose tissue. In summary, these data demonstrate that phillyrin 1) blocks TNFα-induced adipocytes insulin resistance and lipolytic acceleration through inhibiting MAP kinases and IKK phosphorylations, 2) inhibits the expressions of IL-6 and MCP-1 induced by TNFα, 3) suppresses the production of proinflammatory mediators in the coculture system composed of adipocytes and macrophages. The improvement of phillyrin on glucose and lipid metabolic disorder in obesity may be attributed to the attenuation of chronic inflammatory conditions and the improvement in insulin sensitivity in obese adipose tissue.
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3T3-L1 preadipocytes (American Type Culture Collection) were maintained in DMEM (Gibco) containing 10% fetal bovine serum. Differentiation of 3T3-L1 preadipocytes to mature adipocytes was performed as previously described [30]. Phillyrin was dissolved in DMSO as a 1000-fold stock and added to the medium at various concentrations as shown in each figure. DMSO was present in the control culture at a concentration of 0.1 % (v/v). Possible cytotoxicity of phillyrin was assessed by the MTT staining method. Briefly, the mature adipocytes were treated with various concentrations of phillyrin or with 10 ng/mL TNF-α for 96 h. Subsequent incubation of the cells with 100 µl MTT solution (5.0 mg/ml) dissolved in serum-free DMEM for 4 h at 37 °C allowed formation of a violet precipitate, formazan. All liquid in each well was removed, and 100 µl of DMSO was added. The OD at 570 nm was determined by ELISA spectrometry.
Induction of insulin resistance and glucose uptake assay To induce insulin resistance, differentiated 3T3-L1 adipocytes were exposed to 10 ng/mL TNFα for 96 h. During the treatment period, fresh TNFα or phillyrin was changed every 24 h. Transport assay was initiated by washing the cells twice in KRP buffer (1.32 mM NaCl, 4.71 mM KCl, 47 mM CaCl2, 1.24 mM MgSO4, 2.48 mM Na3PO4, 10 mM HEPES, pH 7.4). Cells were then incubated in the KRP solution for another 30 min with or without 100 nM insulin at 37 °C. Then 0.5 µCi/mL 2-DOG (final concentration) was added to the cells. After incubation for 10 min, the medium was aspirated and the cells were washed 3 times with icecold KRP containing 10 mM glucose. The cells were lysed with 0.1 N NaOH and subsequently solubilized in scintillation fluids (Triton X-100/methylbenzene, 1 : 2.5) overnight. The radioactivity taken up by the cells was determined using a scintillation counter (Beckman Instruments). DPM value was corrected by protein content in each well, which was measured with BCA protein assay kit (Pierce).
Lipolysis assay Differentiated 3T3-L1 adipocytes were incubated overnight in DMEM with 0.5 % BSA in the absence of serum. The following morning, cells were treated as described in Results. The culture supernatant was transferred to another set of tubes and heated at 70 °C for 10 min to inactivate any enzymes released by the cells. Samples (25 µL) were then used for glycerol assay with 175 µL of glycerol reagent (GPO Trinder Reagent A, Sigma-Aldrich) in a flat-bottom 96-well plate. Absorption was measured
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Extract of total cell protein and subcellular membrane fractions Whole cell lysates from adipocytes grown on 10 cm dishes were prepared using cell lysis RIPA buffer, containing 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 % Triton x-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, complete protease inhibitor cocktail, and complete phosphatase inhibitors. The lysates were centrifuged at 12 000 g for 10 min at 4 °C to remove the insoluble materials. After washing with PBS, adipocytes were scraped in icecold HES buffer (20 mM HEPES, 250 mM sucrose, 1 mM EDTA (pH 7.4)) containing a protease inhibitor cocktail and subsequently homogenized with 30 strokes in a glass Dounce homogenizer at 4 °C. The homogenate was centrifuged at 16 000 g for 20 min. The supernatant was further centrifuged at 36 000 g for 5 min for HDM fraction and at 200 000 g for 24 min for LDM fraction. The PM fraction was collected from the interface of a 1.12 M sucrose cushion following centrifugation of the 16 000 g pellet at 70 000 g for 10 min. PM were then resuspended in homogenization buffer and pelleted at 200 000 g for 4 min. The obtained fractions were then resuspended in HES buffer. Protein concentrations of PM and LDM were determined with BCA protein assay kit (Pierce) and subjected to Western blotting analysis.
Immunoblotting Equal amounts of protein from each sample were boiled in SDSloading buffer for 5 min and loaded and separated by SDS-polyacrylamide gel electrophoresis. Gels were then blotted onto PVDF membranes (Amersham). The membranes were blocked and incubated with different antibodies, followed by incubation with secondary antibodies conjugated to horseradish peroxidase. Antigen-antibody complexes were detected by chemiluminescence and exposed to high-performance Kodak film. Films were scanned, and specific bands were quantified using the Qualityone Image software.
Coculture of adipocytes and macrophages Differentiated 3T3-L1 adipocytes were cocultured with RAW 264.7 in the medium containing 2 % fatty acid-free BSA for 24 hours in a transwell system. Briefly, differentiated 3T3-L1 adipocytes were incubated overnight in DMEM with 0.5 % BSA in the absence of serum. RAW 264.7 cells (2 × 105 cells/mL) were plated onto dishes with serum-starved and hypertrophied 3T3-L1 cells by using transwell inserts with a 0.4-mm porous membrane (Corning), and the coculture was incubated in serum-free DMEM for 24 h. RAW264.7 and 3T3-L1 cells of equal numbers to those in the coculture were cultured separately as control cultures. Phillyrin was added to the coculture at various concentrations as shown in each figure. After 24 h of treatment, culture supernatants were collected and stored at − 20 °C until measurement.
Measurement of tumor necrosis factor-α and free fatty acids concentrations in the coculture medium The concentrations of TNFα in the culture medium were determined by commercially available enzyme-linked immunosorbent assay (R&D Systems), according to the manufacturerʼs protocol. The concentrations of FFAs in the medium were measured by an acyl-coenzyme A oxidase-based colorimetric assay kit (NEFA‑C, Wako Pure Chemicals).
Statistical analysis Statistical analysis of the data were performed by using one-factor analysis of variance (ANOVA) or by testing the main effects of TNF-α alone, phillyrin alone, and their interaction by using 2-factor ANOVA (SPSS). Tukeyʼs HSD tests were used to compute individual pairwise comparisons of means. All results were expressed as mean ± SE. A p value < 0.05 was considered to be statistically significant.
Suppporting information The relative quantities of total Glut4 and Glut4 in the PM fraction " Fig. 2 E are available as Supporting Information. shown in l
Real time quantitative reverse transcription PCR
Acknowledgments
Total RNA from 3T3-L1 cells was isolated with Trizol reagent, and reverse transcription of 1 µg RNA was carried out with the SuperScript III reverse transcriptase following Invitrogenʼs protocol with oligo dT primer. Quantitative PCR amplification and detection were performed with SYBR Premix Ex Taq (TaKaRa), according to the manufacturerʼs protocol on ABI Prism 7300 sequence detection system (Applied Biosystems). Cycling parameters were 95 °C for 10 s, then 40 cycles of 95 °C for 5 s and 60 °C for 34 s. After PCR, a melting curve analysis was performed to demonstrate the specificity of the PCR product, which was displayed as a single peak (data not shown). Quantifications were performed in duplicate, and the experiments were repeated independently three times. As a control, the mRNA level of 36B4 was determined in the real time PCR assay for each RNA sample and was used to correct for experimental variations. The following primer sequences were used: " IL-6, 5′-tagtccttcctaccccaatttcc-3′ (forward), 5′-ttggtccttagccactccttc-3′ (reverse); " MCP-1, 5′-TTCCTCCACCACCATGCAG‑3′ (forward), 5′-CCAGCCGGCAACTGTGA‑3′ (reverse); " 36B4, 5′-AAGCGCGTCCTGGCATTGTCT‑3′ (forward), 5′-CCGCAGGGGCAGCAGTGGT‑3′ (reverse).
!
This work was supported by the National Natural Science Foundation of China (grant No. 81000345) and by the Shanghai Natural Science Foundation (grant No. 09ZR1417100).
Conflict of Interest !
The authors declare that they have no competing interests.
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at 540 nm on a BioRad plate reader. Protein content was determined using the BCA protein assay (Pierce).
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