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Grape seed procyanidin B2 inhibits adipogenesis of 3T3-L1 cells by targeting peroxisome proliferator-activated receptor γ with miR-483-5p involved mechanism
Biomedicine & Pharmacotherapy 86 (2017) 292–296
Available online at
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Grape seed procyanidin B2 inhibits adipogenesis of 3T3-L1 cells by targeting peroxisome proliferator-activated receptor g with miR-483 -5p involved mechanism Jun Zhanga,b , Yazeng Huangb , Haiyu Shaob , Qing Bib , Jinping Chenb , Zhaoming Yea,* a b
Department of Orthopedics, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang, China Department of Orthopedics, Zhejiang Provincial People’s Hospital, Hangzhou, Zhejiang, China
A R T I C L E I N F O
Article history: Received 11 August 2016 Received in revised form 3 December 2016 Accepted 4 December 2016 Keywords: Procyanidin B2 Adipogenesis PPARg microRNAs 3T3-L1 adipocytes
A B S T R A C T
Procyanidins have lipolysis effect on adipose metabolism, but the underlying mechanism is not fully understood. The aim of present study was to examine the effect of grape seed procyanidin B2 (GSP) on the adipogenic differentiation of 3T3-L1 preadipocyte cell line and investigate the underlying mechanism. The results showed that GSP treatment significantly reduced the intracellular lipid accumulation in induced 3T3-L1 cells by targeting miR-483-5p as well as peroxisome proliferator-activated receptor g (PPARg). In addition, our results revealed that overexpression of miR-483-5p increased adipogenic differentiation, while inhibition of miR-483-5p reduced the lipid accumulation by suppressing the adipogenic differentiation. Moreover, overexpression of miR-483-5p could reverse GSP’s inhibition of adipocyte differentiation as well as increase the level of PPARg. These results demonstrate that GSP inhibits adipogenesis by targeting PPARg and suggest this effect might be mediated by miR-483-5p. © 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Obesity is one of the most common global metabolic disorders. The data from WHO demonstrate that more than 1.3 billion adults worldwide are overweight (body mass index from 25 to 30 kg/m2) and a further 600 million are obese (body mass index 30 kg/m2) [1]. Specifically, the rapid economic development in China since 1980 and the change of living style has resulted in an increased prevalence of overweight and obesity not only in adults but also in children [2–5]. Obesity has been conclusively linked to dyslipidemia, diabetes mellitus, hypertension, some cancers, and osteoarthritis [6,7]. Since obesity is caused by both adipogenesis and adipocyte hypertrophy, inhibition of adipogenesis has been regarded as an effective strategy for preventing obesity. 3T3-L1 preadipocyte cell line is one of the best characterized in vitro systems for unraveling the molecular mechanism during adipogenesis [8]. Procyanidins have been studied heavily due to their benefits for cancer, cardiovascular disease, type 2 diabetes, and ulcerative
* Corresponding author at: Department of Orthopaedics, The Second Affiliated Hospital of Zhejiang University School of Medicine, Jiefang Road #88, Hangzhou, Zhejiang 310009, China. E-mail address: [email protected] (Z. Ye). http://dx.doi.org/10.1016/j.biopha.2016.12.019 0753-3322/© 2016 Elsevier Masson SAS. All rights reserved.
colitis [9–15]. With the limited number of literatures reported so far, the role of procyanidins on adipogenesis, especially its underlying mechanism, is not fully understood [16–21]. MicroRNAs are small noncoding RNAs that associate with the RNA-induced silencing complex (RISC) and bind target mRNAs with partial complementarity. Some microRNAs have been identified as inducers of adipogenesis, including miR-148a, miR125a-3p and miR-483-5p [22,23]. The miRWalk Database identified the down regulation of procyanidins on miR-483’s expression in pancreatic islets [24]. This present study was designed to investigate the putative effect of GSP on the adipogenesis of 3T3-L1 cells and unravel the underlying mechanism by finding some mediated candidate. 2. Material and methods 2.1. Reagents and materials This study protocol was approved by the Institutional Review Boards of Zhejiang Provincial People’s Hospital, Hangzhou. GSP, (CAT No. 29106-49-8, Lot No. HP104375, with purity 98% analyzed using high performance liquid chromatography (HPLC) and LC–MS), was purchased from Hangzhou Neway Chemical Co., Ltd., Hangzhou, China. The miR mimics, mimic-NC, cholesterolconjugated 20 -O-methyl-modified mimics, agomir-NC, antagomir,
J. Zhang et al. / Biomedicine & Pharmacotherapy 86 (2017) 292–296
and antagomir-NC were purchased from RiboBio (RiboBio, Guangzhou, China). The antibodies of PPARg and b-actin were purchased from Cell Signaling Technology (Danvers, MA). Lipofectamine RNAiMAX was purchased from Invitrogen (Carlsbad, CA, USA).
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extraction buffer. An equal volume of dilution buffer supplemented with b-mercaptoethanol was added and the substrate solution was added into each 96-well plate. After incubated at 30 C, 25 ul of diluted sample was added and the absorbance was read at 340 nm at 30 C with a spectrophotometer (Fluostar Optima; BMG Labtech, Offenburg, Germany).
2.2. Cell cultures, treatment and transfection 3T3-L1 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; GIBCO-BRL, Grand Island, NY, USA) containing 10% calf serum (CS; GIOCO-BRL) and antibiotics (100 U/mL penicillin and 100 ug/mL streptomycin; GIBCO-BRL). We seeded 5 105 cells in each well in six-well plate. To induce adipocytic differentiation, two days after reaching 100% confluence (Day 0), the cells were exposed to culture medium containing 0.5 mM 3isobutyl- methylxanthine (IBMX; Sigma), 1 uM dexamethasone (Sigma) and 1 ug/ml insulin (Bovine; Sigma) (MDI hormonal cocktail) for 2 days (Day 2). The medium was replaced with a fresh complete medium containing insulin with 10% FBS and the cells were incubated for another 2 days (Day 4). Thereafter, until the cells were fully differentiated, medium with 10% FBS were changed every other day. We used various concentration of GSP (dissolved in deionized water) (10–200 ug/ml) for screening the optimal concentration for 3T3-L1 cells. Based on its effect on adipogenesis, 150 ug/ml GSP was chosen as the optimum concentration in the following experiment. 3T3-L1 cells were treated with 150 ug/ml GSP for 48 h at day 0. Cells were fed with complete medium without antibiotics 24 h before transfection. miR-483-5p mimics were transfected when the cells were approximately 60–70% confluence and cultured for 72 h before harvesting. Cells were transfected with either agomir or antagomir of miR-483-5p 24 h before they reached 100% confluence. After the treatment and transfection for 72 h, the medium was replaced by the corresponding medium with hormonal cocktail. 2.3. Glycerol-3-phosphate dehydrogenase (G3PDH) assay On day 8 of differentiation, culture medium was removed. 3T3L1 adipocytes were rinsed twice with DPBS and lysed in enzyme
2.4. RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from cells using Trizol (Invitrogen) according to the manufacturer’s instructions. cDNA synthesis was carried out with a Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA). Briefly, 1 ug of RNA was used for reverse transcription into cDNA. Real-time quantitative RT-PCR analysis was performed using the LightCycler (Roche) and SYBR RT-PCR Kits (Takara). The relative expression level of mRNA was normalized to the internal control b-actin while the relative expression level of miRNA was normalized to U6 in each sample using the previous described method [25]. The sequence of primers are as followings: mPPARg sense (50 -TGTCGGTTTCAGAAGTGCCTTG-30 ), antisense (50 TTC AGCTGGTCGATATCACTGGAG-30 ); mC/EBPa sense (50 GTTAGCCATGTGGT- AGGAGACA-30 ), antisense (50 -GTTAGCCATGTGGTAGGAGACA-30 ); mC/EBPb sense (50 -ACGACTTCCTCTCCGACCTCT-30 ), antisense (50 -ACG- ACTTCCTCTCCGACCTCT-30 ); mLPL sense (50 -GGGAGTTTGGCTCCAGAGT- TT-30 ), antisense (50 TGTGTCTTCAGGGGTCCTTAG-30 ); maP2 sense (50 -AGCATCATAACCCTAGATGGCG-30 ), antisense (50 -CATAACACATTCCA- CCACCAGC-30 ) and mb-actin sense (50 -GCATTGTTACCAACTGGGAC-30 ), antisense (50 -CATCACAATGCCTGTGGTAC-30 ). 2.5. Oil red O staining and quantitation Cells were washed twice in DPBS solution, fixed in 4% formaldehyde for 30 min, and washed three times with water. Then, cells were stained with Oil red O (Sigma) for 15 min. Following three washes in water, the lipid droplets were observed and photographed under a microscope (TE2000-E; Nikon, Japan). Oil Red O positive areas were quantified as previously described [26].
Fig. 1. Inhibition of adipogenesis, downregulation of PPARg and differentiation makers and miR-483-5p by GSP. (A) Oil Red O staining was performed 6 days after induction of differentiation with MDI hormonal cocktail. (B) Quantitation of Oil Red O staining. (C) G3PDH activity assay. (D) Western Blotting of indicated proteins and its densitometry and statistical analysis result. (E) The expression level of adipogenic markers was measured by qRT-PCR. (F) qRT-PCR analysis was performed to determine the miR-483-5p level. All results represent mean SD (n = 3). *P < 0.05, **P < 0.01 compared with control group, respectively.
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2.6. Western blot analysis Total proteins were extracted according to the manufacturer’s instructions. Briefly, we left cell pellets on ice for 30 min, with vertex every ten minutes, in radioimmunoprecipitation assay (RIPA) buffer with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA). After that we spin the samples down at 12,000 rpm for 10 min and use the supernatant for the following experiments. BCA protein assay reagent was used for quantification of total protein (Thermo Fisher Scientific, USA). The 10% SDS-PAGE gel and polyvinylidene difluoride (PVDF) membrane (Bio Rad, USA) were used. We loaded 20 ug of protein on the gel and ran for 1 h and a half. We ran the transfer on ice for 1 h and 20 min. After 1 h blocking with 5% fat-free milk in PBST at room temperature, the membrane was incubated with primary antibodies for PPARg (1:1000), C/EBPa (1:1000) and b-actin (1:2000) over night. Signals of PPARg and C/EBPa was detected with Femto Maximum Sensitivity Substrate and b-actin with Pico Chemiluminescent Substrate, respectively (Thermo Fisher Scientific). Image J was used for densitometry analysis. 2.7. Statistical analysis All experiments were repeated at least three times independently. Results were expressed as mean standard deviation (SD). Statistical significance was evaluated by Student’s t-test, with P value of <0.05 considered significant. SPSS (version 16.0 for Windows, SPSS Inc., Chicago, IL, USA) was used for analysis.
(Fig. 1A). Quantification of Oil Red O staining showed that there was significant difference in Oil Red O positive area between GSP and control group (Fig. 1B). In addition to Oil Red O staining, GSP inhibited the G3PDH specific activity, a late marker of differentiation (Fig. 1C). We examined the expression level of PPARg and C/ EBPa. Expression levels of both PPARg and C/EBPa were lower in GSP treated group compared with control group, there was significant difference between them (Fig. 1D). qRT-PCRs showed that mPPARg, mC/EBPa, mC/EBPb, mLPL, maP2 and mus-miR-4835p were significantly decreased in the GSP group compared with control group (Fig. 1E and F). 3.2. MiR-483-5p significantly promoted adipogenesis of 3T3-L1 cells To investigate the effect of miR-483-5p on adipogenesis, we transfected miR-483-5p with agomir into 3T3-L1 cells and induced them with DMI for 6 days. We found that overexpression of miR483-5p significantly promoted adipogenesis, while inhibition of miR-483-5p with the corresponding antagomir remarkably repressed adipogenesis (Fig. 2A and B). The change of G3PDH activity is consistent with results of Oil Red O staining (Fig. 2C). PPARg and C/EBPa were upregulated by the agomir of miR-483-5p, conversely they were downregulated by antagomir of miR-483-5p, the differences were significant (Fig. 2D). To further confirm the changes of PPARg and C/EBPa expressions, significantly increased and decreased level of mPPARg, mC/EBPa and maP2 were detected with transfection of agomir and antagomir of miR-483-5p, respectively (Fig. 2E). Thus miR-483-5p was identified as a promoter of adipogenesis of 3T3-L1 cells.
3. Results 3.1. Adipogenesis was inhibited by GSP by down-regulating the expression of PPARg and miR-483-5p 3T3-L1 cells were induced to differentiate into adipocytes by MDI hormonal cocktail with or without GSP (day 0). Oil Red O was performed on Day 6 to demonstrate triacylglycerol accumulation. As expected, GSP treated cells had significantly less Oil Red O positive adipocytes, indicating that GSP inhibited adipogenesis
3.3. Overexpression of miR-483-5p reversed GSP inhibition of adipogenic differentiation of 3T3-L1 cells The above data indicated that GSP could inhibit adipogenesis by down-regulating miR-483-5p. To further investigate whether the inhibition of adipogenesis by GSP was due to the downregulation of miR-483-5p, a rescue experiment was performed. Intriguingly, GSP inhibition of adipogenesis was reversed by overexpression of miR-483-5p, as assessed by Oil Red O staining and its quantitation
Fig. 2. miR-483-5p significantly promotes adipogenesis of 3T3-L1 cells. (A) Oil Red O staining was performed 6 days after induction of differentiation with MDI hormonal cocktail. (B) Quantitation of Oil Red O staining. (C) G3PDH activity assay. (D) Western Blotting of adipogenic markers and its densitometry and statistical analysis result. (E) qRT-PCR data of adipogenic genes. All results represent mean SD (n = 3). *P < 0.05, **P < 0.01 compared with control group, respectively.
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Fig. 3. Overexpression of miR-483-5p reverses inhibition of GSP on adipogenesis. (A) Oil Red O staining of day 6 revealed that inhibition of GSP can be reversed by overexpression of miR-483-5p. (B) Quantitation of Oil Red O staining. (C) G3PDH activity assay. (D) Western Blotting of adipogenic markers and its densitometry and statistical analysis result. (E) qRT-PCR results of related adipogenic genes. All results represent mean SD (n = 3). *P < 0.05, **P < 0.01 compared with control group, respectively.
(Fig. 3A and B), G3PDH specific activity (Fig. 3C), production of PPARg and C/EBPa (Fig. 3D), and adipogenic gene expression (Fig. 3E). Collectively, these data indicated that GSP could inhibit adipogenesis by allowing lower miR-483-5p, since transfection of miR-483-5p could reverse the adipogenic phenotype. 4. Discussion In present study, we investigated the effect of GSP on differentiation of 3T3-L1 preadipocytes and the underlying mechanism. Our results indicated that GSP could inhibit the differentiation of adipocytes, which is mediated, at least partially, by miR-483-5p. Accumulating data demonstrate that GSP could inhibit adipogenesis [16,20,21,27]. Ardévol et al. first suggested that procyanidins from grape and wine could affect lipid metabolism [16]. Pinent et al. found that grape-seed derived procyanidins could affect adipogenesis and mainly at the induction of differentiation [20]. They also identify a mediation of PPARg on the lipolytic effects of procyanidins on 3T3-L1 adipocytes [27]. Recently, procyanidins from defatted grape seed meal were proved to downregulate lipid metabolism in differentiated murine adipocytes [21]. However, the underlying mechanism that mediated the effect of GSP on PPARg is still not well known. The pharmacokinetic study in human and animals showed that GSP could be detected in plasma and serum within 30 min after a single administration orally or intravenously and reach a peak at 2 h (10 mg/ml). Unfortunately, the level of GSP in plasma is lower than the concentration observed to be effective in various in vitro tests [28,29], due to the low bioavailability in vivo caused by the imperfect administration method, which needs to be improved in future study. To study the underlying mechanism of the effect of GSP on adipogenesis, we used various concentrations of GSP (10– 200 ug/ml) for screening the optimal concentration and 150 ug/ml GSP was chosen as the optimal concentration in the following experiment. Consistent with previous studies, we found decreased Oil Red O staining and G3PDH activity after treating 3T3-L1 cells
with GSP for 48 h. Moreover, we found that the expression of PPARg, a master regulator of adipogenesis, was downregulated, as well as C/EBPa. In line with these results, the mRNA levels of PPARg, C/EBPa, C/EBPb, LPL and aP2 were significantly downregulated. Growing evidences links microRNAs to adipogenesis as therapeutic targets [30]. miR-483-5p is reported to promote adipogenesis of human adipose-derived mesenchymal stem cells by suppressing Erk 1/2 pathway, while transfection of miR-483-5p could increase the expression of PPARg [22]. miR-483-5p is found to be coexpressed with its host gene insulin-like growth factor 2, which is documented to promote adipogenesis in 3T3-L1 cells [31– 33]. Intriguingly, we found that level of miR-483-5p was significantly down-regulated after treatment with GSP. We also found transfection of miR-483-5p could significantly promote adipogenesis of 3T3-L1 cells, while inhibition of miR-483-5p could suppress adipogenesis. mRNA and protein expressions of PPARg were elevated by transfection of Agomir-miR-483-5p and decreased by its Antagomir, which suggested miR-483-5p could mediate the effect of GSP on PPARg. To further investigate the role of miR-483-5p’s in inhibition of adipogenesis by GSP, our rescue experiment showed overexpression of miR-483-5p reversed GSP’s inhibition on adipogenesis of 3T3-L1 cells, as well as G3PDH activity, the expressions of adipogenic markers and related genes. Although miR-483-5p might not directly repress PPARg in 3T3-L1 cells, previous data and our results suggested it could target other genes that negatively regulate PPARg [22]. All of these findings support our hypothesis that GSP could inhibit adipogenesis of 3T3-L1 cells by targeting PPARg with miR-483-5p involved mechanism. In conclusion, we found that miR-483-5p could be significantly downregulated by GSP treatment and could promote adipogenesis of 3T3-L1 cells. Our study also clearly suggested miR-483-5p could mediate the inhibitory effect of GSP on adipogenesis and can be a novel potential therapeutic target for preventing and managing obesity and related metabolic diseases.
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Conflict of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments This study was support by grants from the Zhejiang Provincial Natural Science Foundation of China (LY14H060006), (LY15H060006) and (LY16H060007), Natural Scientific Research Foundation of Zhejiang Medical College (2011XZB01), Zhejiang Provincial Health Bureau Science Foundation of China (2012KYB022) and Zhejiang Provincial Chinese Traditional Medicine Bureau Science Foundation of China (2012ZB015). References [1] WHO. Obesity and overweight. Fact sheet N 311. Geneva: World Health Organization, 2015. [2] C.Y. Ji, Cooperative study on childhood obesity: working group on obesity in China (WGOC), the prevalence of childhood overweight/obesity and the epidemic changes in 1985–2000 for Chinese school-age children and adolescents, Obes. Rev. 9 (Suppl. 1) (2008) 78–81. [3] F. Zhai, H. Wang, S. Du, Y. He, Z. Wang, K. Ge, B.M. Popkin, Prospective study on nutrition transition in China, Nutr. Rev. 67 (Suppl. 1) (2009) S56–61. [4] Z.B. Yu, S.P. Han, J.H. Chu, Z.Y. Xu, C. Zhu, X.R. Guo, Trends in overweight and obesity among children and adolescents in China from 1981 to 2010: a metaanalysis, PLoS One 7 (2012) e51949. [5] Y.Y. Xiao, Y.J. Qiao, L. Pan, J. Liu, T. Zhang, N. Li, E.Q. Liu, Y. Wang, H.Y. Liu, G.S. Liu, G.W. Huang, G. Hu, Trends in the prevalence of overweight and obesity among Chinese preschool children from 2006 to 2014, PLoS One 10 (2015) e0134466. [6] P.G. Kopelman, Obesity as a medical problem, Nature 404 (2000) 635–643. [7] S.Z. Yanovski, J.A. Yanovski, Obesity, N. Engl. J. Med. 346 (2002) 591–602. [8] E.K. Lee, M.J. Lee, K. Abdelmohsen, W. Kim, M.M. Kim, S. Srikantan, J.L. Martindale, E.R. Hutchison, H.H. Kim, B.S. Marasa, R. Selimyan, J.M. Egan, S.R. Smith, S.K. Fried, M. Gorospe, miR-130 suppresses adipogenesis by inhibiting peroxisome proliferator-activated receptor gamma expression, Mol. Cell. Biol. 31 (2011) 626–638. [9] Y. Du, H. Guo, H. Lou, Grape seed polyphenols protect cardiac cells from apoptosis via induction of endogenous antioxidant enzymes, J. Agric. Food Chem. 55 (2007) 1695–1701. [10] Y.H. Wang, B. Ge, X.L. Yang, J. Zhai, L.N. Yang, X.X. Wang, X. Liu, J.C. Shi, Y.J. Wu, Proanthocyanidins from grape seeds modulates the nuclear factor-kappa B signal transduction pathways in rats with TNBS-induced recurrent ulcerative colitis, Int. Immunopharmacol. 11 (2011) 1620–1627. [11] S. Huang, N. Yang, Y. Liu, J. Gao, T. Huang, L. Hu, J. Zhao, Y. Li, C. Li, X. Zhang, Grape seed proanthocyanidins inhibit colon cancer-induced angiogenesis through suppressing the expression of VEGF and Ang1, Int. J. Mol. Med. 30 (2012) 1410–1416. [12] S. Huang, N. Yang, Y. Liu, L. Hu, J. Zhao, J. Gao, Y. Li, C. Li, X. Zhang, T. Huang, Grape seed proanthocyanidins inhibit angiogenesis via the downregulation of both vascular endothelial growth factor and angiopoietin signaling, Nutr. Rev. 32 (2012) 530–536. [13] K.Y. Cheah, S.E. Bastian, T.M. Acott, S.M. Abimosleh, K.A. Lymn, G.S. Howarth, Grape seed extract reduces the severity of selected disease markers in the proximal colon of dextran sulphate sodium-induced colitis in rats, Dig. Dis. Sci. 58 (2013) 970–977.
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Grape seed procyanidin B2 inhibits adipogenesis of 3T3-L1 cells by targeting peroxisome proliferator-activated receptor g with miR-483 -5p involved mechanism Jun Zhanga,b , Yazeng Huangb , Haiyu Shaob , Qing Bib , Jinping Chenb , Zhaoming Yea,* a b
Department of Orthopedics, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang, China Department of Orthopedics, Zhejiang Provincial People’s Hospital, Hangzhou, Zhejiang, China
A R T I C L E I N F O
Article history: Received 11 August 2016 Received in revised form 3 December 2016 Accepted 4 December 2016 Keywords: Procyanidin B2 Adipogenesis PPARg microRNAs 3T3-L1 adipocytes
A B S T R A C T
Procyanidins have lipolysis effect on adipose metabolism, but the underlying mechanism is not fully understood. The aim of present study was to examine the effect of grape seed procyanidin B2 (GSP) on the adipogenic differentiation of 3T3-L1 preadipocyte cell line and investigate the underlying mechanism. The results showed that GSP treatment significantly reduced the intracellular lipid accumulation in induced 3T3-L1 cells by targeting miR-483-5p as well as peroxisome proliferator-activated receptor g (PPARg). In addition, our results revealed that overexpression of miR-483-5p increased adipogenic differentiation, while inhibition of miR-483-5p reduced the lipid accumulation by suppressing the adipogenic differentiation. Moreover, overexpression of miR-483-5p could reverse GSP’s inhibition of adipocyte differentiation as well as increase the level of PPARg. These results demonstrate that GSP inhibits adipogenesis by targeting PPARg and suggest this effect might be mediated by miR-483-5p. © 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Obesity is one of the most common global metabolic disorders. The data from WHO demonstrate that more than 1.3 billion adults worldwide are overweight (body mass index from 25 to 30 kg/m2) and a further 600 million are obese (body mass index 30 kg/m2) [1]. Specifically, the rapid economic development in China since 1980 and the change of living style has resulted in an increased prevalence of overweight and obesity not only in adults but also in children [2–5]. Obesity has been conclusively linked to dyslipidemia, diabetes mellitus, hypertension, some cancers, and osteoarthritis [6,7]. Since obesity is caused by both adipogenesis and adipocyte hypertrophy, inhibition of adipogenesis has been regarded as an effective strategy for preventing obesity. 3T3-L1 preadipocyte cell line is one of the best characterized in vitro systems for unraveling the molecular mechanism during adipogenesis [8]. Procyanidins have been studied heavily due to their benefits for cancer, cardiovascular disease, type 2 diabetes, and ulcerative
* Corresponding author at: Department of Orthopaedics, The Second Affiliated Hospital of Zhejiang University School of Medicine, Jiefang Road #88, Hangzhou, Zhejiang 310009, China. E-mail address: [email protected] (Z. Ye). http://dx.doi.org/10.1016/j.biopha.2016.12.019 0753-3322/© 2016 Elsevier Masson SAS. All rights reserved.
colitis [9–15]. With the limited number of literatures reported so far, the role of procyanidins on adipogenesis, especially its underlying mechanism, is not fully understood [16–21]. MicroRNAs are small noncoding RNAs that associate with the RNA-induced silencing complex (RISC) and bind target mRNAs with partial complementarity. Some microRNAs have been identified as inducers of adipogenesis, including miR-148a, miR125a-3p and miR-483-5p [22,23]. The miRWalk Database identified the down regulation of procyanidins on miR-483’s expression in pancreatic islets [24]. This present study was designed to investigate the putative effect of GSP on the adipogenesis of 3T3-L1 cells and unravel the underlying mechanism by finding some mediated candidate. 2. Material and methods 2.1. Reagents and materials This study protocol was approved by the Institutional Review Boards of Zhejiang Provincial People’s Hospital, Hangzhou. GSP, (CAT No. 29106-49-8, Lot No. HP104375, with purity 98% analyzed using high performance liquid chromatography (HPLC) and LC–MS), was purchased from Hangzhou Neway Chemical Co., Ltd., Hangzhou, China. The miR mimics, mimic-NC, cholesterolconjugated 20 -O-methyl-modified mimics, agomir-NC, antagomir,
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and antagomir-NC were purchased from RiboBio (RiboBio, Guangzhou, China). The antibodies of PPARg and b-actin were purchased from Cell Signaling Technology (Danvers, MA). Lipofectamine RNAiMAX was purchased from Invitrogen (Carlsbad, CA, USA).
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extraction buffer. An equal volume of dilution buffer supplemented with b-mercaptoethanol was added and the substrate solution was added into each 96-well plate. After incubated at 30 C, 25 ul of diluted sample was added and the absorbance was read at 340 nm at 30 C with a spectrophotometer (Fluostar Optima; BMG Labtech, Offenburg, Germany).
2.2. Cell cultures, treatment and transfection 3T3-L1 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; GIBCO-BRL, Grand Island, NY, USA) containing 10% calf serum (CS; GIOCO-BRL) and antibiotics (100 U/mL penicillin and 100 ug/mL streptomycin; GIBCO-BRL). We seeded 5 105 cells in each well in six-well plate. To induce adipocytic differentiation, two days after reaching 100% confluence (Day 0), the cells were exposed to culture medium containing 0.5 mM 3isobutyl- methylxanthine (IBMX; Sigma), 1 uM dexamethasone (Sigma) and 1 ug/ml insulin (Bovine; Sigma) (MDI hormonal cocktail) for 2 days (Day 2). The medium was replaced with a fresh complete medium containing insulin with 10% FBS and the cells were incubated for another 2 days (Day 4). Thereafter, until the cells were fully differentiated, medium with 10% FBS were changed every other day. We used various concentration of GSP (dissolved in deionized water) (10–200 ug/ml) for screening the optimal concentration for 3T3-L1 cells. Based on its effect on adipogenesis, 150 ug/ml GSP was chosen as the optimum concentration in the following experiment. 3T3-L1 cells were treated with 150 ug/ml GSP for 48 h at day 0. Cells were fed with complete medium without antibiotics 24 h before transfection. miR-483-5p mimics were transfected when the cells were approximately 60–70% confluence and cultured for 72 h before harvesting. Cells were transfected with either agomir or antagomir of miR-483-5p 24 h before they reached 100% confluence. After the treatment and transfection for 72 h, the medium was replaced by the corresponding medium with hormonal cocktail. 2.3. Glycerol-3-phosphate dehydrogenase (G3PDH) assay On day 8 of differentiation, culture medium was removed. 3T3L1 adipocytes were rinsed twice with DPBS and lysed in enzyme
2.4. RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from cells using Trizol (Invitrogen) according to the manufacturer’s instructions. cDNA synthesis was carried out with a Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA). Briefly, 1 ug of RNA was used for reverse transcription into cDNA. Real-time quantitative RT-PCR analysis was performed using the LightCycler (Roche) and SYBR RT-PCR Kits (Takara). The relative expression level of mRNA was normalized to the internal control b-actin while the relative expression level of miRNA was normalized to U6 in each sample using the previous described method [25]. The sequence of primers are as followings: mPPARg sense (50 -TGTCGGTTTCAGAAGTGCCTTG-30 ), antisense (50 TTC AGCTGGTCGATATCACTGGAG-30 ); mC/EBPa sense (50 GTTAGCCATGTGGT- AGGAGACA-30 ), antisense (50 -GTTAGCCATGTGGTAGGAGACA-30 ); mC/EBPb sense (50 -ACGACTTCCTCTCCGACCTCT-30 ), antisense (50 -ACG- ACTTCCTCTCCGACCTCT-30 ); mLPL sense (50 -GGGAGTTTGGCTCCAGAGT- TT-30 ), antisense (50 TGTGTCTTCAGGGGTCCTTAG-30 ); maP2 sense (50 -AGCATCATAACCCTAGATGGCG-30 ), antisense (50 -CATAACACATTCCA- CCACCAGC-30 ) and mb-actin sense (50 -GCATTGTTACCAACTGGGAC-30 ), antisense (50 -CATCACAATGCCTGTGGTAC-30 ). 2.5. Oil red O staining and quantitation Cells were washed twice in DPBS solution, fixed in 4% formaldehyde for 30 min, and washed three times with water. Then, cells were stained with Oil red O (Sigma) for 15 min. Following three washes in water, the lipid droplets were observed and photographed under a microscope (TE2000-E; Nikon, Japan). Oil Red O positive areas were quantified as previously described [26].
Fig. 1. Inhibition of adipogenesis, downregulation of PPARg and differentiation makers and miR-483-5p by GSP. (A) Oil Red O staining was performed 6 days after induction of differentiation with MDI hormonal cocktail. (B) Quantitation of Oil Red O staining. (C) G3PDH activity assay. (D) Western Blotting of indicated proteins and its densitometry and statistical analysis result. (E) The expression level of adipogenic markers was measured by qRT-PCR. (F) qRT-PCR analysis was performed to determine the miR-483-5p level. All results represent mean SD (n = 3). *P < 0.05, **P < 0.01 compared with control group, respectively.
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2.6. Western blot analysis Total proteins were extracted according to the manufacturer’s instructions. Briefly, we left cell pellets on ice for 30 min, with vertex every ten minutes, in radioimmunoprecipitation assay (RIPA) buffer with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA). After that we spin the samples down at 12,000 rpm for 10 min and use the supernatant for the following experiments. BCA protein assay reagent was used for quantification of total protein (Thermo Fisher Scientific, USA). The 10% SDS-PAGE gel and polyvinylidene difluoride (PVDF) membrane (Bio Rad, USA) were used. We loaded 20 ug of protein on the gel and ran for 1 h and a half. We ran the transfer on ice for 1 h and 20 min. After 1 h blocking with 5% fat-free milk in PBST at room temperature, the membrane was incubated with primary antibodies for PPARg (1:1000), C/EBPa (1:1000) and b-actin (1:2000) over night. Signals of PPARg and C/EBPa was detected with Femto Maximum Sensitivity Substrate and b-actin with Pico Chemiluminescent Substrate, respectively (Thermo Fisher Scientific). Image J was used for densitometry analysis. 2.7. Statistical analysis All experiments were repeated at least three times independently. Results were expressed as mean standard deviation (SD). Statistical significance was evaluated by Student’s t-test, with P value of <0.05 considered significant. SPSS (version 16.0 for Windows, SPSS Inc., Chicago, IL, USA) was used for analysis.
(Fig. 1A). Quantification of Oil Red O staining showed that there was significant difference in Oil Red O positive area between GSP and control group (Fig. 1B). In addition to Oil Red O staining, GSP inhibited the G3PDH specific activity, a late marker of differentiation (Fig. 1C). We examined the expression level of PPARg and C/ EBPa. Expression levels of both PPARg and C/EBPa were lower in GSP treated group compared with control group, there was significant difference between them (Fig. 1D). qRT-PCRs showed that mPPARg, mC/EBPa, mC/EBPb, mLPL, maP2 and mus-miR-4835p were significantly decreased in the GSP group compared with control group (Fig. 1E and F). 3.2. MiR-483-5p significantly promoted adipogenesis of 3T3-L1 cells To investigate the effect of miR-483-5p on adipogenesis, we transfected miR-483-5p with agomir into 3T3-L1 cells and induced them with DMI for 6 days. We found that overexpression of miR483-5p significantly promoted adipogenesis, while inhibition of miR-483-5p with the corresponding antagomir remarkably repressed adipogenesis (Fig. 2A and B). The change of G3PDH activity is consistent with results of Oil Red O staining (Fig. 2C). PPARg and C/EBPa were upregulated by the agomir of miR-483-5p, conversely they were downregulated by antagomir of miR-483-5p, the differences were significant (Fig. 2D). To further confirm the changes of PPARg and C/EBPa expressions, significantly increased and decreased level of mPPARg, mC/EBPa and maP2 were detected with transfection of agomir and antagomir of miR-483-5p, respectively (Fig. 2E). Thus miR-483-5p was identified as a promoter of adipogenesis of 3T3-L1 cells.
3. Results 3.1. Adipogenesis was inhibited by GSP by down-regulating the expression of PPARg and miR-483-5p 3T3-L1 cells were induced to differentiate into adipocytes by MDI hormonal cocktail with or without GSP (day 0). Oil Red O was performed on Day 6 to demonstrate triacylglycerol accumulation. As expected, GSP treated cells had significantly less Oil Red O positive adipocytes, indicating that GSP inhibited adipogenesis
3.3. Overexpression of miR-483-5p reversed GSP inhibition of adipogenic differentiation of 3T3-L1 cells The above data indicated that GSP could inhibit adipogenesis by down-regulating miR-483-5p. To further investigate whether the inhibition of adipogenesis by GSP was due to the downregulation of miR-483-5p, a rescue experiment was performed. Intriguingly, GSP inhibition of adipogenesis was reversed by overexpression of miR-483-5p, as assessed by Oil Red O staining and its quantitation
Fig. 2. miR-483-5p significantly promotes adipogenesis of 3T3-L1 cells. (A) Oil Red O staining was performed 6 days after induction of differentiation with MDI hormonal cocktail. (B) Quantitation of Oil Red O staining. (C) G3PDH activity assay. (D) Western Blotting of adipogenic markers and its densitometry and statistical analysis result. (E) qRT-PCR data of adipogenic genes. All results represent mean SD (n = 3). *P < 0.05, **P < 0.01 compared with control group, respectively.
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Fig. 3. Overexpression of miR-483-5p reverses inhibition of GSP on adipogenesis. (A) Oil Red O staining of day 6 revealed that inhibition of GSP can be reversed by overexpression of miR-483-5p. (B) Quantitation of Oil Red O staining. (C) G3PDH activity assay. (D) Western Blotting of adipogenic markers and its densitometry and statistical analysis result. (E) qRT-PCR results of related adipogenic genes. All results represent mean SD (n = 3). *P < 0.05, **P < 0.01 compared with control group, respectively.
(Fig. 3A and B), G3PDH specific activity (Fig. 3C), production of PPARg and C/EBPa (Fig. 3D), and adipogenic gene expression (Fig. 3E). Collectively, these data indicated that GSP could inhibit adipogenesis by allowing lower miR-483-5p, since transfection of miR-483-5p could reverse the adipogenic phenotype. 4. Discussion In present study, we investigated the effect of GSP on differentiation of 3T3-L1 preadipocytes and the underlying mechanism. Our results indicated that GSP could inhibit the differentiation of adipocytes, which is mediated, at least partially, by miR-483-5p. Accumulating data demonstrate that GSP could inhibit adipogenesis [16,20,21,27]. Ardévol et al. first suggested that procyanidins from grape and wine could affect lipid metabolism [16]. Pinent et al. found that grape-seed derived procyanidins could affect adipogenesis and mainly at the induction of differentiation [20]. They also identify a mediation of PPARg on the lipolytic effects of procyanidins on 3T3-L1 adipocytes [27]. Recently, procyanidins from defatted grape seed meal were proved to downregulate lipid metabolism in differentiated murine adipocytes [21]. However, the underlying mechanism that mediated the effect of GSP on PPARg is still not well known. The pharmacokinetic study in human and animals showed that GSP could be detected in plasma and serum within 30 min after a single administration orally or intravenously and reach a peak at 2 h (10 mg/ml). Unfortunately, the level of GSP in plasma is lower than the concentration observed to be effective in various in vitro tests [28,29], due to the low bioavailability in vivo caused by the imperfect administration method, which needs to be improved in future study. To study the underlying mechanism of the effect of GSP on adipogenesis, we used various concentrations of GSP (10– 200 ug/ml) for screening the optimal concentration and 150 ug/ml GSP was chosen as the optimal concentration in the following experiment. Consistent with previous studies, we found decreased Oil Red O staining and G3PDH activity after treating 3T3-L1 cells
with GSP for 48 h. Moreover, we found that the expression of PPARg, a master regulator of adipogenesis, was downregulated, as well as C/EBPa. In line with these results, the mRNA levels of PPARg, C/EBPa, C/EBPb, LPL and aP2 were significantly downregulated. Growing evidences links microRNAs to adipogenesis as therapeutic targets [30]. miR-483-5p is reported to promote adipogenesis of human adipose-derived mesenchymal stem cells by suppressing Erk 1/2 pathway, while transfection of miR-483-5p could increase the expression of PPARg [22]. miR-483-5p is found to be coexpressed with its host gene insulin-like growth factor 2, which is documented to promote adipogenesis in 3T3-L1 cells [31– 33]. Intriguingly, we found that level of miR-483-5p was significantly down-regulated after treatment with GSP. We also found transfection of miR-483-5p could significantly promote adipogenesis of 3T3-L1 cells, while inhibition of miR-483-5p could suppress adipogenesis. mRNA and protein expressions of PPARg were elevated by transfection of Agomir-miR-483-5p and decreased by its Antagomir, which suggested miR-483-5p could mediate the effect of GSP on PPARg. To further investigate the role of miR-483-5p’s in inhibition of adipogenesis by GSP, our rescue experiment showed overexpression of miR-483-5p reversed GSP’s inhibition on adipogenesis of 3T3-L1 cells, as well as G3PDH activity, the expressions of adipogenic markers and related genes. Although miR-483-5p might not directly repress PPARg in 3T3-L1 cells, previous data and our results suggested it could target other genes that negatively regulate PPARg [22]. All of these findings support our hypothesis that GSP could inhibit adipogenesis of 3T3-L1 cells by targeting PPARg with miR-483-5p involved mechanism. In conclusion, we found that miR-483-5p could be significantly downregulated by GSP treatment and could promote adipogenesis of 3T3-L1 cells. Our study also clearly suggested miR-483-5p could mediate the inhibitory effect of GSP on adipogenesis and can be a novel potential therapeutic target for preventing and managing obesity and related metabolic diseases.
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