International Journal of Obesity (2005) 29, 934–941. doi:10.1038/sj.ijo.0802988; published online 10 May 2005

Grape-seed derived procyanidins interfere with adipogenesis of 3T3-L1 cells at the onset of differentiation

M Pinent1, M C Bladé1, M J Salvadó1, L Arola1, H Hackl2, J Quackenbush3, Z Trajanoski2 and A Ardévol1

  1. 1Department of Biochemistry and Biotechnology, Rovira i Virgili University, Tarragona, Spain
  2. 2Institute for Genomics and Bioinformatics, Graz University of Technology, Austria
  3. 3The Institute for Genomic Research, Rockville, MD, USA

Correspondence: Dr A Ardévol, Departament de Bioquímica i Biotecnologia, Universitat Rovira i Virgili, c/ Marcel.lí Domingo s/n, 43007, Tarragona, Spain. E-mail:

Received 3 November 2004; Revised 14 February 2005; Accepted 17 March 2005; Published online 10 May 2005.





Our group's previous results on the effects of a grape seed procyanidin extract (GSPE) on adipose metabolism showed that peroxisome proliferator-activated receptor-γ (PPARγ) plays a central role in the lipolytic effects of GSPE on adipocytes. Since PPARγ2 is a main regulator of the differentiation process of adipocytes, we investigated whether GSPE affects the adipogenesis of 3T3-L1 cells.



We performed a time point screening by treating 3T3-L1 cells with GSPE during the differentiation process for 24h.



Differentiation markers and differential gene expression due to GSPE treatment (using the microarray technique).



Twenty four hour-GSPE treatment at the onset of differentiation reduces adipose-specific markers and maintains the expression of preadipocyte marker preadipocyte factor-1 (Pref-1) significantly elevated. These effects were not found in other time points. Microarray analysis of gene expression after GSPE treatment at the early stage of differentiation showed a modified gene expression profile in which cell cycle and growth-related genes were downregulated by GSPE.



These results suggest that GSPE affects adipogenesis, mainly at the induction of differentiation, and that procyanidins may have a new role in which they impede the formation of adipose cells.


procyanidin, differentiation, 3T3-L1 adipocytes, PPARγ, microarray



Flavonoids are phenolic compounds that are ubiquitously found in plants, fruits and beverages. They have been widely studied and shown to have beneficial effects on human health.1 Flavonoids have traditionally been studied due to their properties as antioxidants, though more recently it has been suggested that they act through the modulation of intracellular signalling cascades.2 Among other properties, flavonoids are antigenotoxic,3 antiatherogenic1, 4 and ameliorators of diabetes.5 Their effects on proliferation and differentiation have been studied both in cancerous and noncancerous cell lines.1, 6, 7 However, there is little information about their effects on adipocytes.6, 8, 9 Few studies have focused on the action of procyanidins, an oligomeric class of flavonoids, in the adipose cell10 or on the relationship between procyanidins and the adipose differentiation process.

The study of the adipocyte metabolism is important because obesity is a growing problem in industrialized societies. A key step to determine the possible mechanisms of potential ameliorators of obesity is to define their role in the process of differentiation from preadipocytes to adipose cells. A good model to study differentiation is the 3T3-L1 cell line, provided it can differentiate from preadipocytes to adipocytes under certain stimulating conditions.11 Adipose differentiation is a complex process that is highly regulated by hormones, cytokines and growth factors. Insulin, insulin-like growth factor-1, glucocorticoids (as dexamethasone), cAMP generating agents as isobutyl-methylxanthine,12 as well as other molecules such as thiazolidinediones,13 have been shown to trigger the differentiation of preadipocytes to adipocytes. Before starting the differentiation program, 3T3-L1 may re-enter the cell cycle. Intracellular signals promote the phosphorylation of the retinoblastoma (Rb) protein, which results in the release of the E2F family of transcription factors. This class of transcription factors initiates subsequent events needed for transition through the cell cycle.14 A highly regulated expression of cyclins, CDKs (cyclin-dependent kinases) and CIKs (cyclin-dependent kinase inhibitors) drives the cell through cell cycle progression. This event, called mitotic clonal expansion, ceases coincident with the expression of the key transcription factors PPARγ and CCAAT/enhancer binding protein-α (C/EBPα).15 PPARγ is the regulator of many genes involved in adipose phenotype and lipid metabolism, such as fatty acid synthase and adipocyte-specific fatty acid binding protein (aP2).15, 16 After 6 days postinduction of differentiation, cells acquire adipose morphology and accumulate triglyceride droplets. Although the transcriptional regulation of adipose differentiation has been widely studied, the intracellular signalling cascades that control this process are not fully understood. Mitogen-activated protein kinase (MAPK)17 and STAT18 among others are intracellular mediators shown to be involved in the process of adipogenesis.

In addition to the induction of differentiation factors, inhibitors of adipogenesis, such as myostatin,19 tumor necrosis factor-α,20 retinoic acid21 and rapamycin,22 have also been described. Genistein, a class of flavonoid, has also been shown to inhibit 3T3-L1 proliferation and differentiation.6

Previous results from our group showed that GSPE modified the adipose metabolism.10 Long-term GSPE treatment increased the lipolytic rate in 3T3-L1 adipocytes and downregulated some mature adipocyte markers, such as glycerol-3-phosphate dehydrogenase (G3PDH) activity, PPARγ and hormone-sensitive lipase (HSL) mRNA.23 Owing to the central role of PPARγ2 in the control of the differentiation process of adipose cells, we hypothesized that GSPE affects the adipogenesis of 3T3-L1 cells.

This study was designed to determine whether GSPE affects the differentiation of 3T3-L1 into adipocytes. We studied differentiation cell markers at various time points and performed a more in-depth screening of GSPE effects on gene expression profile in order to clarify its involvement in adipose cell differentiation.



Cells, reagents and materials

GSPE was kindly provided by Les Dérives Résiniques et Terpéniques (Dax, France). According to the manufacturer, this procyanidin extract contained essentially monomeric (21.3%), dimeric (17.4%), trimeric (16.3%), tetrameric (13.3%) and oligomeric (5–13 units) (31.7%) procyanidins.

Cell culture reagents were obtained from BioWhittaker (Verviers, Belgium). Bradford protein reagent was obtained from Bio-Rad Laboratories (Life Sciences Group, Hercules, CA, USA). Reagents for G3PDH activity assay were obtained from SIGMA (St Louis, MO, USA), as were amino allyl dUTP for microarrays. RNA isolation was carried out with the High Pure RNA Isolation Kit (Roche). The reagents for reverse transcription were purchased from Invitrogen. The probes for PCR and the reagents for microarray verification were obtained from Applied Biosystems. N-Hydroxysuccinimide esters of Cy3 and Cy5 were from Amersham.

Cell culture and treatment

3T3-L1 preadipocytes were cultured and induced to differentiate as previously described.10 Briefly, postconfluent cells were induced to adipocyte differentiation (day 0) with 0.25μmol/l dexamethasone, 0.5mmol/l 3-isobutyl-methylxanthine, and 5μg/ml insulin in 10% FBS/supplemented DMEM; after 48h (day 2) cells were switched to 10% FBS/supplemented DMEM containing only insulin, and at day 4 insulin was removed.

3T3-L1 postconfluent preadipocytes were treated at various time points with 140mg/l GSPE (dissolved in water) for 24h at day 0, 2, or 4. After treatment, the medium was replaced for the corresponding following differentiation protocol. At day 10, the triglyceride content, G3PDH activity and gene expression were measured. For the microarray assays, RNA was extracted just immediately after the 24-h GSPE treatment at day 0. Water treatments were used as controls.

Measurement of glycerol-3-phoshate dehydrogenase (G3PDH) activity

After treatment, differentiated 3T3-L1 adipocytes were rinsed twice with PBS, scraped into 750μl of 50mmol/l Tris-HCl, 1mmol/l EDTA, 1mmol/l β-mercaptoethanol and sonicated. The resulting extract was used to measure G3PDH, in accordance with the Wise and Green method.24

Triglyceride assay and lipid content

Remaining cell lysates, as described above, were used to determine the total triacylglyceride content, measured using the enzymatic test, glycerol-phosphate oxidase method (QCA), following the manufacturer's instructions. Values were corrected by their protein content, which was measured by the Bradford method.25 We determined the lipid content by staining the cells with Oil Red O.26

Microarray assay and data analysis

Total RNA was isolated and its quality was checked by Agilent 2100 Bioanalyzer RNA assays. The labelling and hybridization procedures used were based on those developed at The Institute for Genomic Research.27 The mouse slides, also obtained from The Institute for Genomic Research, consisted of 27648 elements that can be found on the web page
. Briefly, cDNA was prepared from 25μg total RNA with Random Hexamers and Superscript Reverse Transcriptase II, in the presence of amino allyl dUTP. cDNA sample was purified (QIAquick kit, QIAGEN) according to the manufacturer's instructions, but using potassium phosphate wash and elution buffer instead of supplied buffers. N-Hydroxysuccinimide esters of Cy3 and Cy5 were coupled to the aadUTP incorporated in the cDNA. Coupling reactions were quenched by 0.1M sodium acetate (pH=5.2) and unincorporated dyes were removed using QIAquick columns (QIAGEN). Fluorescent samples were dried, resuspended in hybridization buffer (50% formamide, 5 × SSC, 0.1% SDS) and combined. In all, 20μg mouse Cot1 DNA and 20μg poly(A) DNA were added and denatured at 95°C for 3min. The sample was applied to the prehybridized slide (incubation at 42°C for 45min in 5 × SSC, 0.1% SDS, 1% BSA) and hybridized in a humidified chamber overnight at 42°C in the dark. The slides were washed at room temperature twice for 2min in a1 × SSC, 0.2% SDS solution, for 4min in 0.1 × SSC, 0.2% SDS, for 4min in 0.1 × SSC; for 2min more in 0.1 × SSC and dipped twice in MQ water. The slides were dried and scanned with a GenePix 4000B microarray scanner (Axon Instruments) and the resulting TIFF images were analysed with GenePix Pro 4.1 (Axon instruments).

Microarray assay was performed by triplicate with dye swap, corresponding to three independent experiments (biological replicates). Features were filtered for low-quality spots and arrays were global mean normalized using ArrayNorm.28 We chose a two-fold change cutoff to the mean of the ratios to find the most variable genes. Differentially expressed genes were classified according to the Gene Ontology™ (GO) Consortium,29 considering the biological process description for each gene.

Quantitative RT-PCR

Microarray results were verified by quantitative real-time PCR. cDNA corresponding to each RNA experiment was generated using TaqManR Reverse Transcription Reagents (Applied Biosystems) and quantitative PCR amplification and detection were performed using specific TaqMan Assay-on-Demand probes (Mm00438064_m1, for Cyclin A2; Mm00772471_m1 for Cell division control protein 2 homologue; Mm00495703_m1 for DNA topoisomerase II alpha isozyme) and the TaqMan PCR Core Reagent Kit as recommended by the manufacturers. Quantifications were performed in triplicate. mRNA 18S was used as the reference gene (HS99999901_sl, human mRNA 18S).

PPARγ2, HSL and Pref-1 were analyzed as previously described.23 Briefly, 1μg of total RNA was reverse transcribed and mRNA levels were measured by real-time RT-PCR analyses in a fluorescent thermal cycler (GeneAmp 5700 Sequence Detection System, Applied Biosystems) according to the manufacturer's instructions. The level of mRNA for each gene was normalized to the level of glyceraldehyde-3-phosphate dehydrogenase mRNA detected in each sample. Amplification was performed with the probes already described plus Pref-1, TGC GCC AAC AAT GGA ACT T (forward), TGG CAG TCC TTT CCA GAG AAC (reverse).

Calculations and statistical analysis

Results are expressed as the mean±s.e.m. Effects were assessed using Student's t test. All calculations were performed using SPSS software.



Effects of GSPE on differentiation markers

To determine the effect of GSPE on the differentiation program of 3T3-L1 cells, we checked some differentiation markers after a 24-h GSPE treatment at different time points and subsequent differentiation of cells. Figure 1a and b show that after 10 days postinduction of differentiation, the triglyceride content on cells treated at day 0 (day of induction of differentiation) was significantly reduced (32%), whereas when treatment was performed at day 2 or 4, triglyceride content remained unchanged (Figure 1a). G3PDH activity was also reduced when GSPE treatment was performed at day 0 or 2 (~26% reduction), but there were no changes when the treatment was carried out at day 4 (Figure 2). Gene expression markers were also analyzed at the end of differentiation (10 days) after the three time-point treatments. mRNA of preadipocytes corresponding to each biological replicate was isolated and analysed in order to check the behaviour of each marker during differentiation. Figure 3a shows that PPARγ2 mRNA levels increased after GSPE treatment at day 0. On the other hand, GSPE treatments at day 2 or 4 reduced PPARγ2 gene expression (31 and 43%, respectively). A late differentiation marker of adiposity is HSL, a key enzyme in the lipid metabolism. The mRNA levels of this enzyme did not change when treatment was performed at day 0 or 2, but in day 4 treatments mRNA levels showed a 44% reduction (Figure 3b). Pref-1 was chosen as a preadipocyte marker, as its levels have been shown to be downregulated during adipogenesis.30 Pref-1 mRNA levels due to GSPE treatment after induction of differentiation remained five-fold upregulated (Figure 3c) but did not reach preadipocyte levels (10-fold increase). When treatments were performed at day 2 or 4 Pref-1 showed the normal downregulation in the process of differentiation.

Figure 1.
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Effect of GSPE on triglyceride cell content. 3T3-L1 preadipocytes were induced to differentiate as described in Methods. Cells were incubated 24h with 140mg/l of GSPE at different time points: day 0 (induction of differentiation), day 2 or 4. (a) Triglyceride (TAG) content was assayed after 10 days of differentiation for all treatments. Data reflect the means±s.e.m. of at least three independent experiments; values were corrected to controls in each experiment. (a, b) indicate statistically significant differences between means with P<0.05. (b) Oil red staining for lipid accumulation was assayed after 10 days of differentiation for cells treated at day 0.

Full figure and legend (90K)

Figure 2.
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Effect of GSPE on G3PDH activity of 3T3-L1 adipocytes. Postconfluent 3T3-L1 preadipocytes were induced to differentiate. At several time points (day 0, 2, or 4), cells were incubated with 140mg/l GSPE for 24h. After 10 days postinduction of differentiation, cells were collected and G3PDH activity was assayed. Data reflect the means±s.e.m. of at least three independent experiments; values were corrected to controls in each experiment. (a, b) indicate statistically significant differences between means with P<0.05.

Full figure and legend (13K)

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the author

Procyanidin effects on mRNA levels (a, PPARγ2; b, HSL; c, Pref-1). 3T3-L1 postconfluent preadipocytes were induced to differentiate and 24h GSPE treatment (140mg/l) was performed at day 0 (induction of differentiation), day 2 or 4. At day 10, total RNA was extracted and gene expression was quantified by real-time RT-PCR. Gene expression, normalized by glyceraldehyde-3-phosphate dehydrogenase mRNA levels, is expressed relative to control cells. Control: untreated fully differentiated cells; preadipocyte: untreated postconfluent fibroblasts. Values represent mean±s.e.m. a, b, c indicate significantly different groups with P<0.05.

Full figure and legend (68K)

Gene profile is highly modified due to GSPE treatment during induction of differentiation

The time-point screening showed that GSPE had stronger effects when added, together with the hormonal cocktail, at the onset of differentiation. We therefore decided to analyse the gene profile after 24-h GSPE treatment of postconfluent preadipocytes (day 0).

We found 398 differentially expressed transcripts (more than two-fold change). Of these, 38.3% were upregulated and 61.7 % were downregulated. In all, 206 genes could be categorized based on the biological processes in which they are involved. We did this classification according to the GO consortium, obtaining a view of the biological processes in which the differentially expressed genes are involved. The products of the genes are usually involved in several biological processes, so the results of the classification were redundant because some EST were classified into several categories. This redundancy helped us to get a more complete idea of the biological processes affected by GSPE.

Table 1 summarizes the number of differentially expressed EST classified according to their biological function (complete list of the data files are available on the web site:
). Most differentially expressed transcripts fell into the categories related to cell growth and maintenance, including cell cycle, cytokinesis, cell organization and biogenesis. The protein metabolism category also contained a large number of differentially expressed genes, despite the fact that many of them were also contained in the cell growth group. Table 2 lists the genebank accession numbers of these growth and cell cycle-related differentially expressed genes. These include cyclins (A2, B2, F, etc), cell division control protein 2, histone H2A.X. and other cell cycle regulators. Almost all these genes were downregulated, with mRNA expression differences that achieved more than four-fold change. We also checked the expression of p21, a cell cycle-related gene involved in the effects of flavonoids on cell proliferation.31, 32, 33, 34, 35 We found that p21 (GeneBank ID BE282021), despite not being classified in the first screening of two-fold change cutoff, was 1.41-fold upregulated.



In this paper, we studied the effect of a GSPE on the differentiation process of 3T3-L1 adipocytes. First we performed a time-point screening of 24h 140mg/l GSPE, analysing several adipose differentiation markers after 10 days postinduction of differentiation. Simultaneous GSPE treatment with the hormonal cocktail at the onset of differentiation reduced triglyceride accumulation and decreased G3PDH activity. This shows that GSPE interferes negatively with the differentiation process, prohibiting cells from acquiring complete adipose phenotype. Moreover, at the molecular level Pref-1, a preadipose marker and inhibitor of adipogenesis,30 remained upregulated in day 0 treated cells. Surprisingly, at this time point of treatment, PPARγ2 mRNA increased. It has been reported that this nuclear transcription factor (PPARγ2) is upregulated during differentiation, with mRNA levels starting to rise at 24h and remaining stable until the end of the differentiation regimens.36 Therefore, a higher expression than the controls is a rare feature that suggests that GSPE not only partially inhibits differentiation but also produces other gene expression changes that are not characteristic of the differentiation process. When GSPE treatment was performed 2 or 4 days after induction, cell triglyceride content remained unchanged and morphology and G3PDH activity remained unaffected after GSPE treatment at day 4. On the other hand, PPARγ2 levels were significantly downregulated. The induction of PPARγ mRNA at the early stages of differentiation and its action as a stimulator of the transcription of adipogenic genes has been well described (reviewed in Rosen et al,15 Fajas et al,37 and Tong and Hotamisligil38). Despite the well-known role of PPARγ in the induction of adipose maturation, however, its role in the maintenance of adipocyte phenotype is still unclear. Both the upregulation of PPARγ when treatment was carried out at the beginning of differentiation and its downregulation in later time-point treatments point to PPARγ as a target for GSPE irrespective of its actions on differentiation. This hypothesis is reinforced by previous results from our group that pointed to PPARγ as a target for GSPE in fully differentiated adipocytes, since BRL 49653, a high-affinity PPARγ ligand, removed the GSPE lipolytic effects on 3T3-L1.23

As GSPE had inhibitory effects at the early stage of differentiation, we took our study further. We studied the gene expression profile of day 0 GSPE-treated cells after 24h. We used microarray technology to obtain a wide view of the intracellular dynamics after GSPE treatment. This paper shows that GSPE modifies the gene expression profile of differentiating cells, since 398 transcripts were found to be, at least, two-fold up/downregulated. Their functional classification showed that many of the differentially expressed genes were cell cycle-related genes. This was not unexpected because within 24–36h of induction, cells re-enter the cell cycle and undergo mitotic clonal expansion. Also, microarray-based studies of adipose differentiation have shown that mRNAs maximally expressed at 16–24h correspond to many genes associated with the cell cycle.36, 39, 40, 41 Most of the cell cycle related genes in our microarray were downregulated, which suggests that GSPE-treated cells were not in the mitotic clonal expansion phase. Opinions on whether mitotic clonal expansion is necessary to allow adipose differentiation are controversial,42 but recent studies support the idea that mitotic clonal expansion is required for adipogenesis.43 In fact, Tang et al44 demonstrated that treatment with PD98059, an inhibitor of MEK (MAPK/extracellular signal-regulated kinase kinase), delayed mitotic clonal expansion and that this delay was proportional to decreases in differentiation markers and triacylglyceride accumulation. As 24-h GSPE treatment is followed by a 24-h normal induction cocktail, it is likely that GSPE acts delaying mitotic clonal expansion and therefore partially inhibiting differentiation. Although in this study we did not assay the potential inhibition of proliferation by GSPE, in breast carcinoma cells 24h procyanidin treatment up to 75mg/l—a time and dose quite similar to the one we used—has been shown to have antiproliferative properties that correlated to an increase in p2145 (a CIK). CIKs are also key regulators of cell cycle re-entry because they inhibit cyclin-dependent kinase actions.

As far as we know, there are no studies on effects of procyanidins on adipose differentiation. Although most studies of the antiproliferative effects of flavonoids have been carried out in cancerous cells,46, 47, 48, 49 there is also evidence that proliferation is inhibited by flavonoids in 3T3-L1. Genistein, a soy flavonoid, was shown to inhibit both proliferation and differentiation in 3T3-L1 when added at the induction of differentiation,6 and this induced growth arrest was accompanied by an increase in p21 expression and subsequent cyclin E/CDK 2 supression.31 In fact, several flavonoids have been shown to exert growth-inhibitory effects through upregulation of p21 in different cell lines.32, 33, 34, 35

We also found an upregulation of p21 by GSPE treatment at day 0. P21 upregulation favours its binding to cyclin E/CDK2 and the inhibition of its kinase activity, so cyclin E/CDK2 cannot contribute to pRb phosphorylation. Unphosphorylated pRb maintains its ability to repress E2F transcriptional activity.50 The E2F family represents transcription factors that regulate several genes necessary for progression through the cell cycle (recently reviewed in Bracken et al51). Concomitantly to the GSPE upregulation of p21 mRNA, in our microarrays we also observed some of the E2F target genes (and therefore downstream of p21) downregulated due to GSPE treatment, such as cyclin A, cell division control protein 2, DNA topoisomerase II, polo-like kinase (PLK) and other cell cycle-related genes. This agrees with the hypothesis that, in 3T3-L1, GSPE induce growth arrest by an upregulation of p21. However, more studies are needed to confirm this hypothesis.

In conclusion, this paper shows for the first time that a GSPE interferes with the differentiation process of 3T3-L1 adipocytes mainly at the onset of differentiation. At this time point the significantly modified gene expression profile of 3T3-L1 due to GSPE treatment suggests that GSPE can interfere with the progression of adipocytes through the cell cycle necessary to fully differentiate. These effects support the hypothesis that grape-seed procyanidins help to prevent the development of obesity and obesity-related pathologies by reducing the formation of new fat cells.



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This study was supported by Grant number AGL2002-00078 from the Comisión Interministerial de Ciencia y Tecnología (CICYT) of the Spanish Government and by the Austrian Science Fund (SBF Biomembranes). M Pinent is the recipient of a fellowship from the autonomous government of Catalonia.



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