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© 2006 American Society for Nutrition J. Nutr. 136:1766-1773, July 2006

Biochemical, Molecular, and Genetic Mechanisms

Red Grape Juice Polyphenols Alter Cholesterol Homeostasis and Increase LDL-Receptor Activity in Human Cells In Vitro1,2

Alberto Dávalos*, Carlos Fernández-Hernando*,3, Francisca Cerrato*, Javier Martínez-Botas*, Diego Gómez-Coronado*, Carmen Gómez-Cordovés{dagger} and Miguel A. Lasunción*,**,4

* Servicio de Bioquímica-Investigación, Hospital Ramón y Cajal, Madrid, Spain; {dagger} Instituto de Fermentaciones Industriales, CSIC, Madrid, Spain; and ** Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá, Madrid, Spain

4 To whom correspondence should be addressed. E-mail: miguel.a.lasuncion{at}hrc.es.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Red grape juice (RGJ) polyphenols have been shown to reduce circulating levels of LDL cholesterol and to increase LDL receptor activity. To explore the effect of RGJ-derived polyphenols on intracellular cholesterol homeostasis, human hepatocarcinoma HepG2 and promyelocytic HL-60 cell lines were incubated in serum-free medium, with or without LDL, in the presence or absence of RGJ. In the presence of LDL, RGJ increased both the activity and cell surface expression of the LDL receptor, and increased the cell total cholesterol content. In cells exposed to LDL, RGJ also increased levels of the active form of sterol regulatory element-binding protein-1 and mRNA expression of the LDL receptor and hydroxymethylglutaryl-CoA reductase. In contrast, RGJ caused a marked reduction in the expression of CYP7A1, apolipoprotein B, ABCA1, and ABCG5. Experiments using the acyl-CoA cholesterol acyltransferase inhibitor S-58035 indicated that no measurable free cholesterol from endocytosed LDL reaches the endoplasmic reticulum in cells treated with RGJ. Finally, fluorescence microscopy revealed that in RGJ-treated cells, DiI-labeled LDL did not colocalize with CD63, a protein localized at steady state in the internal vesicles of late endosomes. These results indicate that RGJ polyphenols disrupt or delay LDL trafficking through the endocytic pathway, thus preventing LDL cholesterol from exerting regulatory effects on intracellular lipid homeostasis.

KEY WORDS: • LDL receptor • red grape juice polyphenols • cholesterol trafficking • LDL cholesterol

Polyphenols, a ubiquitous group of secondary plant metabolites, represent a large group of natural antioxidants abundant in fruits, vegetables, and beverages such as grape juice, wine, and tea. Several studies have linked wine consumption to a reduction in cardiovascular diseases (1–3), attributing some of the cardioprotective effects to the polyphenols present in red wine and grape products (4). Oxidation of LDL was proposed to play a crucial role in atherogenesis (5), and polyphenols were shown to exhibit strong antioxidant properties that could protect LDL from oxidation (6–9).

In addition to their antioxidant activity, polyphenols also possess many different biological properties that may contribute to their cardioprotective effects, including the ability to inhibit platelet activity and thrombosis (10–12) and the potential to reduce plasma lipids. In vivo studies showed that administration of green tea (13), dealcoholized red wine (8), grape seed procyanidins (14), naringin (15), or polymethoxylated flavones (16) lowered plasma cholesterol in laboratory animals with diet-induced hypercholesterolemic. Recently, reduction of cholesterol LDL plasma concentration was reported in women consuming a lyophilized grape powder (17) or red wine (18). The mechanisms underling this hypolipidemic effect may involve the upregulation of LDL receptor expression (19,20), inhibition of hepatic lipid synthesis (21) and lipoprotein secretion (22,23), or increased cholesterol elimination via bile acids (14).

Intracellular cholesterol homeostasis is under the control of the sterol regulatory element-binding protein (SREBP)5 transcription factor (24,25). Once LDL is internalized by the LDL receptor, endosomes fuse with lysosomes and LDL cholesteryl esters are hydrolyzed. Then, free cholesterol is released into the cytoplasm and reaches the different intracellular compartments. Free cholesterol in the endoplasmic reticulum (ER) can be converted to cholesteryl ester by the enzyme acyl-CoA cholesterol acyltransferase (ACAT), transported to the plasma membrane, or used for other metabolic purposes. Free cholesterol in the ER has a negative feedback effect on cholesterol biosynthesis and expression of the LDL receptor via SREBP (24,25). To elucidate the mechanism that accounts for the hypocholesterolemic effect of red grape juice consumption, the present study addresses the effects of red grape juice on intracellular cholesterol homeostasis in human cell lines in vitro.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Materials. DMEM, antibiotics, Trizol reagent, fetal bovine serum (FBS) and normal goat serum were purchased from Gibco-BRL. Lipoprotein-deficient serum (LPDS) was prepared from FBS by ultracentrifugation at a density of 1.21 kg/L. [14C]-acetate (53 mCi/mmol) and anti-LDL receptor monoclonal antibody (IgG C7) were obtained from Amersham Biosciences. Fluorescein isothyocianate-labeled anti-mouse IgG was from Boehringer Mannheim Biochemica. 1,1'-Dioctadecyl-3,3,3,3'-tetramethylindocarbocyanineperchlorate (DiI) was purchased from Molecular Probes. ACAT inhibitor S-58035 was kindly provided by Sandoz and lovastatin was from Merck Sharp & Dohme. Mouse anti-human CD63 monoclonal antibody was purchased from BD Pharmingen and Alexa Fluor® 488 anti-mouse IgG was from Molecular Probes. Concentrated red grape juice (RGJ) from the bobal grape variety was purchased from Dream Fruits.

    Cell culture. Human hepatocarcinoma HepG2 cells (ATCC HB 8065) obtained from the American Type Culture Collection were maintained in DMEM supplemented with 10% FBS and antibiotics, at 37°C in a humidified atmosphere containing 5% CO2. For experiments, cells were incubated in DMEM containing 10% LPDS, supplemented or not with human LDL and/or RGJ. On other occasions, pure antioxidants, lovastatin, and/or compound S-58035 were also added to the incubation medium. For controls, cells were supplemented with glucose and fructose, each at a concentration of 2.1 g/L of medium.

    Analysis of polyphenols in red grape juice. Analysis of flavonoids, phenolic acids, and flavan-3-ols was performed following a previously described method (26). Briefly, 5 mL of RGJ (68° Brix) was diluted with 15 mL of water, separated into 2 aliquots and extracted 3 times each with diethyl ether and ethyl acetate. The organic fractions were mixed and dried with anhydrous Na2SO4 for 30 min, concentrated to dryness and dissolved in 1 mL of methanol/water (50:50). After being filtered through a 0.45-µm membrane, a 100 µL of sample was injected into a reverse-phase Nova-Pak C18 column (300 mm x 3.9 mm, 4µm), using a Waters liquid chromatography system equipped with a 600-MS controller, a 717Plus autosampler, and a 996 photodiode-array detector (26). Detection was performed by scanning from 220 to 380 nm. For anthocyanins, 100 µL of diluted RGJ, previously filtered through a 0.45-µm membrane, was injected into a reverse-phase Nova-Pak C18 column (150 mm x 3.9 mm, 4 µm), and separated by a multistep linear gradient method, as described (27). Detection was performed by scanning from 260 to 600 nm.

    Uptake of RGJ polyphenols by HepG2 cells. HepG2 cells were cultured in 175-cm2 flasks in DMEM containing 10% FBS to reach 80% confluence; then the medium was changed to DMEM containing 10% LPDS, LDL (0.31 mmol cholesterol/L), and RGJ (5 mL/L). At 0, 3, 9, and 20 h of incubation, cells were harvested, washed with PBS, and treated on ice with 300 µL of lysis buffer (Cell Signaling) for 20 min. The cell protein content was determined [bicinchoninic acid (BCA) protein assay kit, Pierce]. Lysates were incubated with 200 µL of 0.1 mol/L ascorbic acid, 100 µL of 0.78 mol/L sodium acetate buffer (pH 4.8), and 100 µL ß-glucuronidase (131100 kU/L ß-glucuronidase and 1070 kU/L sulfatase) from Helix pomatia at 37°C for 8 h. The mixture was extracted twice with 1 mL of ethyl acetate, and pooled supernatants were concentrated to dryness with a SPD121P Speed-Vac system (Thermo Electron). The sample was reconstituted in 300 µL of methanol:water (50:50, v:v), filtered through a 0.45-µm membrane, and 200 µL were subjected to HPLC analysis as described (26).

    LDL receptor activity and cell-surface expression. Human LDL was isolated by ultracentrifugation and labeled with the fluorescent probe DiI as described elsewhere (28). HepG2 cells (4 x 105 cells/well), treated with the test compounds for 20 h in 10% LPDS, were washed twice with PBS and then incubated in duplicate in 10% LPDS medium containing DiI-LDL (0.10 mmol cholesterol/L) for 2 h at 37°C to allow DiI-LDL uptake. Nonspecific DiI-LDL uptake was assessed in a third well containing a 50-fold excess of unlabeled LDL. Cells were washed and analyzed by flow cytometry (FACScalibur, BD Biosciences).

To analyze cell-surface expression of the LDL receptor protein, cells were incubated with anti-LDL receptor monoclonal antibody IgG C7 (15 mg/L) and then FITC-labeled goat anti-mouse IgG, and analyzed by flow cytometry as described previously (29).

    Analysis of cholesterol biosynthesis and lipid determinations. To examine cholesterol biosynthesis, HepG2 cells (6 x 106 cells) were incubated in 10% LPDS medium containing [14C]-acetate (1.5 mCi/L) for 20 h, lysed with 10% KOH, and total cellular de novo cholesterol was analyzed by reverse-phase HPLC as described previously (30). Cell total cholesterol and free cholesterol were measured using an enzymatic method followed by HPLC analysis using cholesterol oxidase and cholesterol esterase as described previously (31). For free cholesterol, cholesterol esterase was not included in the reaction mixture; esterified cholesterol was calculated by subtracting free cholesterol from total cholesterol. Triglycerides were measured using a colorimetric enzymatic kit (A. Menarini Diagnostics).

    Quantitative real-time RT-PCR assay. Total RNA was extracted with Trizol reagent according to the manufacturer's instructions. Total RNA (2 µg) was reverse-transcribed using M-MLV RT enzyme (Promega). Real-time PCR amplification was performed on a LightCycler and data analysis software version 3.5 (Roche Diagnostics GmbH), using FastStart DNA Master SYBR Green I (Roche Diagnostics GmbH). The primers used are listed in supplemental Table S1. Amplification cycles used were 95°C for 10 s, 60°C for 20 s, and 72°C for 20 s, except for ABCG1 and ABCG5, for which the annealing temperature was 62°C. Melting curves were evaluated for each gene, and PCR reaction products were separated on a 2% agarose gel and stained with ethidium bromide to confirm the presence of a single product. All analyses were performed in duplicate, and relative mRNA levels were determined using glyceraldehyde-3-phosphate dehydrogenase as a reference. The relative expression ratio (R) for each target gene was calculated taken into account the reaction efficiency (E) and the crossing point (CP) of the unknown sample vs. the control (Roche Diagnostics, LC relative quantification software, 2001). At least 4 independent experiments were performed in duplicate for each determination.

    ACAT inhibition. ACAT inhibition with S-58035 was used as an indirect measure of free cholesterol reaching the ER. Cells (4 x 105 per well) were incubated in 10% LPDS-containing DMEM supplemented with LDL (0.31 mmol cholesterol/L), in the presence or absence of lovastatin (2.5 µmol/L), S-58035 (5 mg/L), and RGJ (5 mL/L) for 20 h. Cells were washed twice with PBS, and LDL receptor activity was assessed using the DiI-LDL uptake assay described above.

    Western blot analysis of SREBP-1. After incubation, cells were washed with PBS and then treated with lysis buffer. Cell protein content was determined (BCA protein assay kit) and loaded equally (50 µg protein/lane) onto 10% polyacrylamide gels, electrophoresed, and transferred onto a nitrocellulose membrane. Membranes were blocked with 1% defatted milk powder in Tris-buffered saline (TBS; 25 mmol/L Tris pH 7.4, 136 mmol/L NaCl, 2.6 mmol/L KCl) containing 0.1% Tween 20, incubated overnight at 4°C with anti-mouse SREBP-1 (Santa Cruz Biotechnology) and then with horseradish peroxidase-conjugated secondary antibody, and developed with the enhanced chemiluminescence detection system (Amersham Biosciences). Nuclear extracts were obtained as described previously (32) and Western blot was performed as described above, with 100 µg protein loaded per lane. To inhibit rapid degradation of nuclear fragments of SREBPs, cells were treated with N-acetyl-leucinal-leucinal-norleucinal at a final concentration of 25 mg/L 2 h before protein extraction and throughout the extraction procedure.

    Fluorescence microscopy. HepG2 cells were cultured on 12-mm glass coverslips in 24-well dishes for 48 h in medium containing 10% FBS. The medium was then replaced with medium containing 10% LPDS and incubated in the presence of DiI-LDL (0.155 mmol cholesterol/L), RGJ (5 mL/L), or tamoxifen (5 µmol/L) for 20 h. For staining of free cholesterol, cells were washed and fixed in 4% paraformaldehyde for 15 min, washed 3 times with PBS and then stained with filipin (50 mg/L in PBS) for 45 min. Immunostaining was performed as previously described (33), using anti-CD63 (1:100) and Alexa-conjugated anti-mouse IgG (1:400) antibodies. DNA was stained with DAPI. Cells were mounted for microscopy and examined on an Olympus BX51 reflected fluorescence microscope. Merged images were prepared using Adobe Photoshop 6.0.

    Statistical analysis. All values are presented as means ± SEM. Means were compared using Student's t test or ANOVA followed by post hoc multiple comparisons. Comparisons of cholesterol biosynthesis data were made by 1-way repeated measures ANOVA and pairwise comparisons by the Student-Newman-Keuls method. The interactions between RGJ and LDL on LDL receptor activity were analyzed by two-way ANOVA. When the interaction was significant, the subsequent analysis of the effect of RGJ was performed by 1-way ANOVA separately for each group (presence of LDL in the medium). Comparisons of gene expression data were made either by the confidence interval method (for each experimental condition vs. the control) or by 1-way ANOVA and Tukey's HSD post hoc test (for comparisons between the experimental conditions); in the latter case, data from the control condition were excluded from the analyses. Differences with P-values < 0.05 were considered significant. Analyses were performed with Statgraphics Plus v5.0 (Statistical Graphics).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Polyphenol composition of red grape juice and uptake by cells. Analysis of RGJ by RP-HPLC revealed high concentrations of anthocyanins, flavonols, and flavan-3-ols, particularly myricetin, quercetin glycosides, and procyanidin B2 (Table 1). Other polyphenols, such as trans-resveratrol, were detected in trace amounts. RGJ polyphenols were efficiently taken up by human liver HepG2 cells in vitro, in a time-dependent manner, as demonstrated directly for quercetin (Fig. 1).


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  		TABLE 1 Phenolic composition of red grape juice

 

Figure 1
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  		FIGURE 1  Uptake of quercetin from RGJ by HepG2 cells (A) and representative HPLC chromatogram of 20-h incubated cells (B): Q, quercetin. Values are means ± SEM, n = 3. *Different from 0 h, P < 0.05.

 
    Red grape juice upregulates LDL receptor activity. To investigate the potential mechanism(s) involved in the hypolipidemic effect of grape polyphenols, we first addressed the effects of RGJ on LDL receptor activity in HepG2 cells. When cells were incubated in medium lacking LDL, stimulation of LDL receptor activity by RGJ was limited (Fig 2A). The addition of LDL to the incubation medium strongly reduced LDL receptor activity but RGJ counteracted this effect in a concentration-dependent manner; at 5 mL/L RGJ, the LDL receptor activity of cells exposed to LDL was practically indistinguishable from that in unexposed cells. Similar effects occurred in human leukemia HL-60 cells (Fig. 2B).


Figure 2
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  		FIGURE 2  Effect of RGJ on LDL receptor activity in HepG2 (A) and HL60 (B) cells. Values are means ± SEM, n = 3 independent experiments. *Different from the corresponding 0 mL RGJ/L, P < 0.05. Interaction between RGJ and LDL: HepG2, P < 0.001; HL-60, P < 0.01. Main effect of RGJ: HepG2, P < 0.05 for the control, and P < 0.001 for LDL; HL-60, P > 0.05 for the control, and P < 0.01 for LDL.

 
To further confirm the effect of RGJ on LDL receptor activity, cell-surface expression of the LDL receptor was assessed by flow cytometry. In the absence of LDL, RGJ caused a significant increase in the cell-surface expression of the LDL receptor (Table 2). Although supplementing the medium with LDL led to a reduction in this protein expression, this effect was partially blocked by the simultaneous addition of RGJ (Table 2).


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  		TABLE 2 LDL receptor activity and cell-surface expression of LDL receptor protein in HepG2 cells treated with RGJ or quercetin, in the absence or the presence of LDL for 20 h1

 
Similar experiments were performed using the pure flavonoids (+)-catechin, (-)-epicatechin, myricetin, and quercetin, all of which are major components of RGJ. Only the results for the model flavonoid quercetin are shown in Table 2. None of the pure flavonoids studied (1–10 µmol/L) mimicked the effects of RGJ.

To confirm that the effects of RGJ were not mediated by its antioxidant activity, other antioxidants were also evaluated. BHT (1–10 µmol/L) and Trolox (0.1–10 µmol/L) did not stimulate LDL receptor activity in cells exposed to LDL (data not shown), confirming that the effect of RGJ polyphenols cannot be attributed to antioxidant activity.

    Effects of RGJ on cellular lipid mass and cholesterol biosynthesis. To explore whether changes in LDL receptor activity and cell-surface expression are correlated with cell lipid content, both cell cholesterol and triglycerides were quantified. Incubation of HepG2 cells in the presence of LDL significantly increased the cell total cholesterol content, whereas the effect was proportionally much higher for esterified cholesterol (403% of the control) than for free cholesterol (125% of the control) (Table 3). RGJ did not have any detectable effect on cell total cholesterol content in cells incubated in medium lacking LDL. However, in the presence of LDL, RGJ significantly increased total cholesterol. This was confirmed by oil red O staining for neutral lipids (data not shown). Analysis of free and esterified cholesterol revealed significant changes only in esterified cholesterol (149% of the control). Although incubation with LDL did not cause appreciable changes in the triglyceride content of the cells, RGJ caused a significant reduction either in the presence (78.4% of the control) or absence of LDL (81.7% of the control) (Table 3).


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  		TABLE 3 Effect of RGJ on cell lipid content in HepG2 cells incubated with or without LDL for 20 h1

 
Cholesterol biosynthesis was assessed by the incorporation of [14C]-acetate into cholesterol. As expected, supplementing the medium with LDL significantly inhibited de novo biosynthesis of cholesterol (467 ± 150 vs. 779 ± 227 Bq/mg cell protein in the control, P < 0.01). The addition of RGJ increased [14C]-acetate incorporation into cholesterol in cells incubated in the presence of LDL (551 ± 138 Bq/mg cell protein, P < 0.05 compared with the control) but not in the absence of LDL (816 ± 221 Bq/mg cell protein).

    Effect of RGJ on gene expression. We next used quantitative RT-PCR to assess relative changes in the expression of selected genes related to lipid metabolism (Table 4). In cells incubated in the absence of LDL, RGJ significantly reduced the expression of ABCA1, ABCG8, and apoB, whereas the other genes were unaffected. LDL repressed the expression of the LDL receptor, hydroxymethylglutaryl-CoA (HMG-CoA) reductase, and NPC1L1, which are all involved in cholesterol accretion. LDL did not affect the expression of the other genes examined. The simultaneous addition of RGJ abrogated the effect of LDL on the expression of the LDL receptor, partially counteracted the effect on HMG-CoA reductase, and markedly reduced the expression of CYP7A1 and apoB (Table 4).


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  		TABLE 4 Gene expression profile in HepG2 cells incubated with or without RGJ and/or LDL for 20 h1

 
We investigated the processing of SREBP-1 as a major regulator of the transcription of genes involved in lipid metabolism (24,25). As expected, incubation of cells with LDL markedly reduced the nuclear levels of the active, p68-processed form of SREBP-1 (Fig. 3, lane 3). This effect was prevented by simultaneous treatment of the cells with RGJ (Fig. 3, lane 4).


Figure 3
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  		FIGURE 3  Effect of RGJ on SREBP-1 processing in HepG2 cells cultured in control conditions (lane 1), and supplemented with RGJ (5 mL/L) alone (lane 2), LDL (0.31 mmol cholesterol/L) alone (lane 3), or a combination of LDL and RGJ (lane 4). SREBP-1 precursor form (P) was detected in cell lysates and the active form (N) in nuclear extracts, respectively. Actin was used as a loading control.

 
    Effect of ACAT inhibition on RGJ action. To assess the involvement of the free cholesterol content of the ER in the increased uptake of LDL in response to RGJ, we used the ACAT inhibitor S-58035. In the absence of LDL in the medium, S-58035 did not exert any appreciable effect on DiI uptake, either in the presence or absence of RGJ (data not shown). In the presence of LDL, S-58035 significantly reduced DiI-LDL uptake irrespective of the presence or absence of lovastatin added to the medium to avoid interference with endogenously synthesized cholesterol (Fig. 4). These findings indicate that by inhibiting the esterification of cholesterol, S-58035 efficiently increases the free cholesterol content of the ER and thus inhibits LDL receptor expression. Although the addition of S-58035 in the presence of RGJ significantly inhibited DiI-LDL uptake compared with RGJ alone, it was nevertheless much greater than DiI-LDL uptake by cells not exposed to RGJ. Interestingly, when lovastatin was added to inhibit cholesterol biosynthesis in medium containing RGJ and LDL, S-58035 did not decrease LDL receptor activity, indicating that under these conditions, no measurable free cholesterol from endocytosed LDL reaches the ER.


Figure 4
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  		FIGURE 4  Modulation of RGJ effects on LDL receptor activity by inhibition of ACAT with S-58035 in HepG2 cells. Values are means ± SEM, n = 3 independent experiments. Means without a common letter differ, P < 0.05.

 
    Effect of RGJ on intracellular cholesterol distribution. To explore whether RGJ affects the LDL endocytic pathway, HepG2 cells were incubated in the presence of DiI-LDL and stained with filipin. Treatment with tamoxifen, used as a positive control, markedly increased the uptake of DiI-LDL and that increase was accompanied by an accumulation of free cholesterol in membrane-delimited organelles (Fig. 5, E and F), which is in accordance with the inhibition of LDL-derived free cholesterol efflux from the endosome/lysosome compartment that we reported recently (29), In cells treated with RGJ, the filipin staining pattern was quite different, with small fluorescent inclusions in the perinuclear region that were clearly colocalized with DiI (Fig. 5, C and D). The lack of intense accumulation of free cholesterol indicated that RGJ was not acting in the same way as tamoxifen.


Figure 5
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  		FIGURE 5  Effect of RGJ on intracellular accumulation of DiI-LDL (A, C, and E) and free cholesterol (B, D, and F) (filipin staining) in HepG2 cells incubated with DiI-LDL (0.155 mmol cholesterol/L) under control conditions (A and B) or treated with 5 mL/L RGJ (C and D) or 5 µmol/L tamoxifen (E and F).

 
To assess whether the endocytic pathway was affected at other, more proximal steps, cells were immunostained for CD63, a protein localized at steady state in the internal vesicles of late endosomes (34). In the control condition, some degree of colocalization was observed between CD63 and DiI-LDL (Fig. 6 C). In cells treated with tamoxifen, almost all vesicles containing DiI-LDL were also positive for CD63 (Fig. 6 I). In contrast, in cells treated with RGJ, most of the vesicles containing DiI-LDL did not contain CD63 labeling (Fig. 6 F). These results suggest that RGJ may disrupt or delay LDL trafficking through the endocytic pathway—possibly affecting the fusion of primary endosomes with lysosomes—and reduce the availability of LDL-derived cholesterol in the ER.


Figure 6
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  		FIGURE 6  Tracing of the LDL endocytic pathway in HepG2 cells cultured in medium containing 10% LPDS supplemented with DiI-LDL under control conditions (A and B) or treated with 5 mL/L RGJ (D and E) or 5 µmol/L tamoxifen (G and H). A, D and G, DiI staining (red); B, E and H, CD63 staining (green); C, F and I, merged images. Nuclear DNA staining (DAPI) is shown in blue.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Elevated plasma concentrations of LDL and LDL oxidation are recognized as playing an important role in atherogenesis (5), and increasing the efficiency of cholesterol clearance by raising LDL receptor activity is an efficient way to lower plasma LDL (35). Several animal models and human intervention studies showed that consumption of beverages rich in polyphenols, including red wine and dealcoholized red wine, decreases circulating levels of LDL cholesterol (8,18). However, the mechanism underlying this effect and the nature of the active compound have yet to be elucidated. Pal et al. (20) demonstrated that red wine polyphenols increase LDL receptor expression and activity in human cells in vitro. In the present work, we report that grape polyphenols are efficiently taken up by cells, as shown for quercetin, and interfere with the LDL endocytic pathway, counteracting the repression of LDL receptor expression otherwise generated by LDL cholesterol. In both HepG2 and HL-60 cells incubated in the presence of a relatively high concentration of LDL cholesterol, RGJ increased LDL-receptor mRNA levels, the expression of LDL-receptor protein at the cell surface, and the uptake of DiI-LDL by cells. Although these effects were accompanied by an increase in intracellular cholesterol levels, an unexpected finding was that cholesterol biosynthesis was not inhibited. In fact, the expression of HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis, was increased in cells treated with RGJ. In agreement with our findings, Bursill and colleagues reported that tea polyphenols stimulate both the expression of HMG-CoA reductase gene (36) and cholesterol biosynthesis (37) in HepG2 cells.

The LDL receptor and most of the enzymes involved in the cholesterol biosynthesis pathway are regulated at the transcriptional level by a family of membrane-bound transcription factors, the SREBPs (24). In response to a decrease in free cholesterol availability in the ER, SREBP is processed proteolytically, and the active fragment migrates to the nucleus where it interacts with the sterol response elements present in the promotor of those genes and activates their transcription (24). Consistent with this, we observed that incubation of cells with LDL was accompanied by a reduction in SREBP-1 processing. Interestingly, simultaneous treatment with RGJ prevented this effect of LDL. Very recently, Kuhn et al. (19) proposed that the upregulation of LDL receptor expression induced by green tea polyphenols is mediated by inhibition of ubiquitin/proteosome-mediated degradation of active SREBP-2. It remains to be seen whether this mechanism also applies to RGJ polyphenols. Nevertheless, the increased levels of active SREBP are likely to be responsible for the effect of RGJ on the expression of the LDL receptor and HMG-CoA reductase.

In contrast to the LDL receptor and enzymes involved in cholesterol biosynthesis, the expression of genes involved in cholesterol efflux is induced in response to cholesterol loading (38,39). In cells incubated in the presence of LDL, we found that RGJ caused repression of ABCA1 and ABCG5, which are involved in cholesterol efflux, CYP7A1, which directs cholesterol to the synthesis of bile acids, and apoB, which is required for lipoprotein synthesis and secretion. This suggests that RGJ interferes with the intracellular availability of free cholesterol for regulatory purposes. To study how LDL-derived free cholesterol reached the ER, cells were treated with S-58035, a specific inhibitor of ACAT. As expected, LDL inhibited LDL receptor activity, and S-58035 further decreased this activity, a finding that is in agreement with the results of previous studies (40,41). In contrast, S-58035 did not have any effect on RGJ-treated cells, which displayed increased LDL receptor activity. Taken together, these results suggest that RGJ compromises the availability of LDL-derived free cholesterol in the ER, thus explaining the counteracting effect of RGJ in the response to LDL.

Next, we addressed whether RGJ affects the endocytic pathway. After being endocytosed, LDL enters distinct endosomal compartments: first, it enters the early endosome compartments and then the late endosome/lysosome compartments, where LDL cholesteryl ester is hydrolyzed by acid lipase (42,43). It was reported that disruption of intracellular cholesterol trafficking induces upregulation of LDL receptor expression in human hepatocytes (29,44,45). Treatment with tamoxifen, for instance, was shown to exert a strong stimulatory effect on LDL receptor expression in cells exposed to LDL, an effect that was attributed to the retention of LDL-derived free cholesterol in the endosomal/lysosomal compartment (29). In the present study, the filipin staining pattern in cells treated with RGJ was weak and diffuse, quite different from that of tamoxifen-treated cells, which showed intensely stained enlarged vesicles (Fig. 5, F). Because DiI-LDL uptake was similar in the presence of either tamoxifen or RGJ, we conclude that the effect of RGJ was not mediated by the retention of free cholesterol in endosome/lysosome vesicles.

Finally, we analyzed the nature of the LDL-accumulating compartment by examining the expression of CD63, a protein localized to late endosomes (34). Most vesicles containing DiI-LDL also showed CD63 labeling in control and tamoxifen-treated cells, whereas this colocalization was not observed in cells treated with RGJ (Fig. 6, F). These results indicate that RGJ interferes with LDL trafficking through the endocytic pathway, probably by preventing the fusion of lysosomes with endosomes. Based on current understanding of this pathway (43,46), this effect would delay lipolytic processing of LDL and reduce the efflux of lipoprotein cholesterol to the ER, thus explaining the counteracting effect of RGJ on gene expression in cells exposed to LDL.

Epidemiologic and clinical studies suggest that polyphenols are good candidates to explain the protective effects of vegetables, fruits, and red wine against certain cancers and cardiovascular diseases (3,4,12,47). In addition to their antioxidant (6,7), vasorelaxant (48), and anti-inflammatory (17) properties, the effects on cholesterol homeostasis shown here may contribute to the cardioprotective effects of Vitis vinifera polyphenols. Although we were not able to attribute the effects of RGJ to a single polyphenol or family of polyphenols present in RGJ, the variety of polyphenols present in the RGJ concentrate, including flavonols, anthocyanins, flavan-3-ols, procyanidins and phenolic acids, and the limited availability of pure compounds, make the search for the active compound(s) a complex task. The finding that other potent antioxidants do not mimic the effect of RGJ indicates that antioxidant protection is not responsible for the effects of RGJ on LDL receptor activity. Our observation that RGJ interferes with the endocytic pathway offers an alternative mechanism to account for the activity of grape polyphenols.


   ACKNOWLEDGMENTS
 
We acknowledge the contribution of Mercedes García-Villanueva, M.D., Ph.D., and Clara Redondo, M.D., Ph.D. to the microscopy studies. We also thank Rebeca Bustos, Ph.D., for her contribution to laboratory work, Miguel Martín for excellent technical assistance, and Víctor Abraira, Ph.D., for his help in statistical analyses.


   FOOTNOTES
 
1 Supported by grants from Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Ministerio de Educación y Ciencia (VIN03-027 and AGL2004-07075-C02-00). A.D. is a recipient of a fellowship from the Fundación Carolina, Madrid, Spain. Back

2 Supplemental Table 1 and supplemental references are available with the online posting of this paper at www.nutrition.org. Back

3 Present address: Department of Pharmacology, Boyer Center for Molecular Medicine, Yale University, School of Medicine, New Haven, CT 06536-0812. Back

5 Abbreviations used: ACAT, acyl-CoA cholesterol acyltransferase; apo B, apolipoprotein B; BCA, bicinchoninic acid; DiI,1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanineperchlorate; ER, endoplasmic reticulum; FBS, fetal bovine serum; HMG-CoA, hydroxymethylglutaryl-CoA; LPDS, lipoprotein-deficient serum; RGJ, concentrated red grape juice; SREBP, sterol regulatory element-binding protein. Back

Manuscript received 21 December 2005. Initial review completed 8 February 2006. Revision accepted 9 April 2006.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
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