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Article

Soy Protein Peptides Regulate Cholesterol Homeostasis in Hep G2 Cells

Maria R. Lovati¹, Cristina Manzoni, Elisabetta Gianazza, Anna Arnoldi^*, Elzbieta Kurowska, Kenneth K. Carroll^,2 and Cesare R. Sirtori

Institute of Pharmacological Sciences, Faculty of Pharmacy and ^* Department of Agrifood Molecular Sciences, University of Milan, I-20133 Milan, Italy; Centre for Human Nutrition, Department of Biochemistry, The University of Western Ontario, London, N6A 5C1 Canada

¹To whom correspondence should be addressed.

	ABSTRACT

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The activation of LDL receptors was described recently in a human hepatoma cell line (Hep G2) exposed both to + ' subunits from 7S soy globulin and to Croksoy^R70, a commercial isoflavone-poor soy concentrate. To assess the final identity of the peptide(s) putatively responsible for the biochemical effect, experiments were performed in Hep G2 cells, exposed either to synthetic peptides corresponding to specific sequences of 7S soy globulin or to peptides from the in vitro digestion of Croksoy^R70. Moreover, the ability of the whole 7S globulin, its subunits and whole Croksoy^R70 to interfere in the apolipoprotein B (apo B) secretion in the medium as well as in sterol biosynthesis was evaluated in the same model. Increased ¹²⁵I-LDL uptake and degradation vs. controls were shown after Hep G2 incubation with a synthetic peptide (10^-4 mol/L, MW 2271 Da) corresponding to positions 127–150 of the 7S globulin. Cells exposed to Croksoy^R70 enzyme digestion products showed a more marked up-regulation of LDL receptors vs. controls, compared with vs. Hep G2 cells incubated with undigested Croksoy^R70. Among soy-derived products, only the 7S globulin inhibited apo B secretion and ¹⁴C-acetate incorporation when tested in Hep G2 cells at a concentration of 1.0 g/L. These findings support the hypothesis that if one or more peptides can reach the liver after intestinal digestion, they may elicit a cholesterol-lowering effect. Moreover, the protein moiety, devoid of isoflavone components, is likely to be responsible for this major biochemical effect of soy protein.

KEY WORDS: • soy proteins • LDL receptors • apolipoprotein B • cholesterol homeostasis • Hep G2 cells

	INTRODUCTION

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Soy protein has been demonstrated to have cholesterol-lowering properties in various populations, from children (Laurin et al. 1991 ) to renal patients (D’Amico 1992 ). Anderson et al. (1995) summarized the results of these studies in a recent meta-analysis. The final mechanism responsible for the plasma cholesterol reduction remains an open question. Two hypotheses have been the subject of debate, i.e., a direct effect of the major isoflavones in soybeans (particularly daidzein and genistein) or, alternatively, of the protein components (mainly 7S globulin from soybeans or their fragments). The isoflavone hypothesis has been supported by experimental (Anthony et al. 1996 ) and in part by clinical findings (Erdman 1995 ). However, recent reports did not demonstrate an effect of an isoflavone-rich soy extract supplementation on plasma lipid and lipoproteins in ovariectomized cynomolgus monkeys (Greaves et al. 1999 ) or humans (Samman et al. 1999 ). Moreover, Sirtori et al. (1997 and 1998) demonstrated that in major clinical investigations on hypercholesterolemic patients, marked plasma cholesterol reduction was obtained using isoflavone-poor soybean products.

The U.S. Food and Drug Administration (FDA)³ recently approved the health claims concerning the role of soy protein in reducing the risk of coronary hearth disease (FDA 1999 ). This has led to increased interest in determining the identity of the responsible moiety. Studies in humans and in validated animal models have suggested that the mechanism of action of soy protein might be linked to the direct activation of LDL receptors in liver cells (Khosla et al. 1989 , Lovati et al. 1987 , Potter 1998 ), or to a modulation of both synthesis and catabolism of LDL by specific proportions of dietary amino acids corresponding to soy protein (Kurowska and Carroll 1992 and 1996 ). In a human hepatoma cell (Hep G2), high affinity LDL receptors were induced after incubation with 7S globulin from soybeans (Lovati et al. 1992 ), and this was associated with 7S recognition by a specific uptake and degradation system (Lovati et al. 1996 ). In the same cell model, the and ' subunits from 7S soy protein were shown to be more active in LDL receptor up-regulation than either the whole 7S globulin or the ß subunit (Lovati et al. 1998 ). The effect induced by + ' appeared to be due to the ' rather than the subunit because a mutant soy cultivar devoid of ' had no effect on LDL receptor activity (Manzoni et al. 1998 ). Monitoring the LDL receptor mRNA level by Northern blotting clearly established that in Hep G2 cells, 7S or its subunits activated transcription of the LDL receptor gene (Sirtori et al. 1998 ).

Although our previous findings support the hypothesis that specific peptides from soy proteins can modulate cholesterol homeostasis in an in vitro system, this may not be possible in vivo because large undigested peptides are unlikely to cross the intestinal barrier and appear in the circulation. To determine the identity of the smaller size peptide(s) putatively responsible for the biochemical effect, we evaluated LDL receptor activity in Hep G2 cells exposed either to natural peptides from the in vitro digestion (trypsin + pepsin) of Croksoy^R70 or to synthetic peptides, corresponding to specific sequences differing among the + ' and ß subunits of 7S globulin. Experiments were also done to examine LDL receptor activity in Hep G2 cells exposed to a hot ethanol extract of Croksoy^R70. Additionally, the mechanisms of action of 7S globulin, its + ' and ß subunits, and Croksoy^R70 were investigated by examining their effects on the net secretion of apolipoprotein B (apo B), the structural protein of LDL. The most active 7S soy fraction was evaluated subsequently for its ability to alter biosynthesis of cellular lipids. These mechanistic studies were conducted also in view of recent reports demonstrating a coordinate regulation of both apo B synthesis and receptor-mediated LDL catabolism in Hep G2 cells exposed to varying total levels and proportions of mixed amino acids (Kurowska and Carroll 1996 , Twisk et al. 2000 , Zhang et al. 1993 ).

	MATERIALS AND METHODS

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Reagents.

The established human hepatoma cell line (Hep G2) was obtained from American Type Culture Collection (Rockville, MD). Defatted soybean flour was purchased from Cargill BV (Amsterdam, The Netherlands). Croksoy^R70 was a gift from Dr. A. Ferrero, PERFOODS, Milan, Italy. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide, the lactate dehydrogenase (LDH) reagent, bovine serum albumin (BSA), porcine pepsin and bovine trypsin were purchased from Sigma-Aldrich (Milan, Italy) and from Sigma Chemical (St. Louis, MO). The Protein Coomassie Plus Protein Assay kit was purchased from Pierce (Rockford, IL). Antibody against human LDL was obtained from Hoechst (Montreal, Canada). Eagle’s minimum essential medium (MEM), fetal calf serum (FCS), trypsin-EDTA (1X), penicillin (10⁵ U/L), streptomycin (100 g/L), tricine buffer (1 mmol/L, pH 7.4) and nonessential amino acid solutions (100X) were from GIBCO (Madison, WI). Disposable Petri dishes were from Corning Glass Works (Corning, NY). Amicon filters of different cut-off were from Amicon Italia (Milan, Italy). ¹²⁵Iodine, carrier free, in 100 mmol/L NaOH, was from NEN-DuPont de Nemours (Boston, MA). Sephadex G25 columns (PD10), Immobiline II monomers, Ampholine and Pharmalyte carrier ampholytes in the pH ranges 3.5–10, 4–6 and 5–7, GelBond PAG foils, Ultrodex powder, the Electrofocusing Kit for Granulated Gel, a Multiphor II horizontal electrophoresis chamber and an EPS 3500 power supply were from Pharmacia Biotech (Uppsala, Sweden). Acrylamide monomers and catalysts, and 1000/500 Power Supply were from BioRad, Hercules, CA. All other chemicals were of analytical grade from Merck (Darmstadt, Germany).

Preparative extraction of 7S globulin and its subfractions from soybean flour.

The protocol for separation and purification of 7S globulins from defatted soybean flour was described previously (Lovati et al. 1992 ). Fractionation of + ' and ß subunits from purified 7S globulin was done by preparative isoelectric focusing in a granulated bed, as described elsewhere (Lovati et al. 1998 ).

Protein extraction from Croksoy^R70.

Croksoy^R70 (courtesy of Dr. A. Ferrero, PERFOODS, Milan, Italy) is a soy protein concentrate prepared according to a patented procedure (U.S. Patent 4,490,460), making use of rapid heating under high pressure. The total isoflavone concentration of Croksoy^R70 is < 0.15 mg/g dry powder (44 µg/g dry powder, 66 µg/g dry powder and 34 µg/g dry powder, respectively, for daidzein, genistein and glycitein, courtesy of Dr. Patricia Murphy, Iowa State University, Ames, IA) vs. 2.9 mg/g in defatted soy flour (Sirtori et al. 1997 ). Protein extraction from Croksoy^R70 was done using 30 mmol/L Tris, pH 8.0, containing 0.01 mol/L ß-mercaptoethanol. During a subsequent purification, no protein could be precipitated at pH 6.4, due to the absence of 11S globulin; however, >90% protein precipitated at pH 4.8 (Manzoni et al. 1998 ).

Polyacrylamide gel electrophoresis.

Electrophoresis of 7S soy globulin, + ' and ß subunits thereof and Croksoy^R70 was performed on a 10–20% gradient polyacrylamide gel containing 0.1% sodium dodecylsulfate (SDS-PAGE) using a Mini Protean II cell (Bio-Rad), as previously described (Lovati et al. 1992 ).

Separation of Croksoy^R70 enzyme digestion products.

Croksoy^R70 enzyme digestion products were prepared in the following manner: 1 g of Croksoy^R70 was suspended in 100 mL of distilled water, adjusted to pH 2.0 with diluted HCl and incubated at 37°C with 1% porcine pepsin. After 1 h incubation, pepsin (from porcine stomach mucosa; EC 3.4. 23.1) was inactivated by neutralization with 1 mol/L NaOH followed by a further 1 h digestion with 1% trypsin (from bovine pancreas treated with L-1-tosylamide-2-phenylethylchloromethyl ketone; EC 3.4.21.4) under the same experimental conditions. Croksoy^R70 enzyme digests were fractionated further by membrane filtration through Diaflo-Amicon R, with a cut-off of 3000 and 1000 Da at nominal pressure of 55 psi (3.7 atm). Three subfractions were obtained with nominal molecular weights, i.e., >3000 Da, between 1000 and 3000 Da and <1000 Da.

Chemical synthesis of peptides from the sequence of 7S soy globulin.

The + ' subunit differs most from the ß subunit, and from the consensus alignment of all known 7S globulin sequences, in two regions present in + ' but absent in ß. The first of these sequences encompasses positions 10–32, whereas the second begins at position 127 and ends at 150 of the consensus sequence. The two sequences are as follows: QDKESQESEGSESQREPRRHKNK and LRVPAGTTFYVVNPDNDENLRMIA (MW, 2353 and 2271, respectively). These peptides, synthesized on solid phase, as described by Cordopatis et al. (1994) , and purified by reverse-phase HPLC to obtain pure peptides (yield > 98%) suitable for an in vitro test, were kindly provided by Prof. P. Cordopatis (Department of Pharmacy, Laboratory of Pharmacognosy and Chemistry of Natural Products, University of Patras, Greece).

Croksoy^R70 hot ethanol extraction.

To obtain hot ethanol–soluble components, Croksoy^R70 was processed as reported previously (Lovati et al. 1991 ) with a single extraction using 80% ethanol at 60°C for 12 h. The Croksoy^R70 products obtained after hot ethanol processing (bulk, containing mostly proteins; and residue, containing sugars as well as minor soy components, including isoflavones) were solubilized, filtered, evaporated under vacuum, lyophilized and sterilized (Millipore filters, 0.45-µm pore size; Amicon Italia, Milan, Italy) before testing in Hep G2 cells.

Cell culture.

Hep G2 cells were grown in monolayers in 90-mm diameter Petri dishes, and maintained at 37°C in a humidified atmosphere of 95% air, 5% CO₂ in MEM supplemented with 10% FCS, nonessential amino acid solution (1%, v/v), penicillin (10⁵ U/L), streptomycin (0.1 g/L), tricine buffer (20 mmol/L, pH 7.4), NaHCO₃ (24 mmol/L) and sodium pyruvate (0.11 g/L). For experiments designed to evaluate the LDL receptor modulation, cells were seeded in 35-mm plastic dishes (3–5 x 10⁵ cells) and used just before reaching confluence, usually 6 d after plating. For the determination of apo B responses and changes in cellular cholesterol metabolism, cells were seeded in 6- or 24-well plates (6 x 10⁵ cells/plate) and used at confluence (usually 7 d after plating). In all cell culture experiments, the medium was changed every 2–3 d.

Lipoproteins and lipoprotein-deficient serum.

LDL (1.019 < d < 1.063 kg/L) were isolated by sequential preparative ultracentrifugation (Havel et al. 1955 ) from the plasma of clinically healthy normolipidemic volunteers. Lipoproteins were labeled according to the method of McFarlane as modified by Bilheimer et al. (1972) and described previously (Lovati et al. 1998 ). ¹²⁵I-LDL were sterilized by filtration (Millipore filters, 0.45-µm pore size) and stored at 4°C until use (<10 d after preparation). Human lipoprotein-deficient serum (LPDS) was prepared according to Brown et al. (1974) .

Uptake and degradation of ¹²⁵I-LDL.

Monolayers of cells were preincubated for 24 h at 37°C in MEM supplemented with 5% LPDS to up-regulate the LDL receptor (Goldstein et al. 1983 ), in the presence or absence of Croksoy^R70 and the different products thereof (Croksoy^R70 enzyme digests and different MW fractions, Croksoy^R70 products after hot-ethanol extraction) as well as of the synthetic peptides at the concentrations listed in the tables. Cell viability was assessed by LDH and MTT methods (see below). A fixed concentration (7.5 mg/L) of ¹²⁵I-LDL protein was then added to the medium and the incubation continued for a further 4 h at 37°C. Specific uptake (binding + internalization) and degradation of ¹²⁵I-LDL were evaluated as described previously (Lovati et al. 1998 ).

Medium apo B determination.

Cells were seeded in 24-well plates (6 x 10⁵ cells/plate) and used at confluence. Before the experiments, cells were preincubated for 24 h with MEM containing 10 g/L BSA instead of FCS. During the next 24 h, cells were incubated in the same medium in the presence or absence of different concentrations of Croksoy^R70, 7S soy globulin and subunits thereof. The highest nontoxic concentration of each preparation was determined before the experiment by MTT viability assay (see below). Media were collected and cells were washed three times with ice-cold PBS. Medium apo B concentrations were determined subsequently as reported previously (Zhang et al. 1993 ). Briefly, medium was collected and frozen for analysis of apo B, by an ELISA according to Young et al. (1986) , as modified by Ortho Diagnostics (La Jolla, CA). LDL for coating microtiter plates and for preparing a standard curve (range 0.025–0.400 g/L apo B) was isolated from fresh human EDTA-plasma by sequential density gradient centrifugation as reported above. Soluble cellular proteins was extracted with 0.1 mol/L NaOH, measured using the Coomassie Plus Protein Assay, and apo B content was calculated per milligram of total cellular protein before conversion to percentage of control.

Acetate incorporation into cellular lipids.

Confluent HepG2 cells were preincubated for 24 h in MEM containing 10 g/L BSA. Subsequently, they were incubated for another 24 h in the same medium in the presence or absence of 7S globulin (1 g/L). [1-¹⁴C]acetate (20 mmol/L; 18.5 GBq/L) was added for the last 4 h. Cells were washed three times with ice-cold PBS and radiolabeled lipids were extracted using heptane/isopropyl alcohol (3:2, v/v). Free cholesterol, cholesterol esters and triacylglycerols were separated by TLC with a solvent system containing petroleum ether/diethyl ether/acetic acid (70:12:1, v/v/v). The incorporation of [1-¹⁴C]acetate was determined by scintillation counting after collection of lipid fractions visualized with iodine vapor. Radioactivity of samples was expressed as dpm/mg cellular protein.

Viability assays.

The highest nontoxic concentrations of different soybean protein preparations were determined by MTT (Marshall et al. 1995 ) and LDH (Young 1990 ) viability assays. Cells were preincubated for 24 h in a medium containing different preparations at concentrations ranging from 0.05 to 1.0 g/L. After 24 h exposure to different compounds, the culture medium was removed and after washing in PBS, 50 µL of MTT solution was added to each well. After 1 h incubation at 37°C, cells were washed in PBS. Solubilization of formazan crystals formed in viable adherent cells was achieved by adding 50 µL of dimethylsulfoxide, and the spectrophotometric absorbance of the samples was measured at 540 nm. LDH activity was determined using a kinetic, ultraviolet lactate dehydrogenase (LDH/LD) diagnostic kit (Sigma Diagnostics). The LDH leakage was monitored in cell medium after 24 h preincubation with the different soy products

Statistical analyses.

Differences in cell uptake and degradation of LDL after soy protein incubation (Croksoy^R70 and the different products thereof) as well as of the synthetic peptides were determined by ANOVA (SYSTAT, Evanston, IL, run on a Macintosh LC 630) followed by the Dunnett’s test. Differences in apo B cell secretion as well as acetate incorporation into cellular lipids were evaluated by ANOVA followed by Tukey’s t test. Values in the text are means ± SEM Differences were considered significant at P 0.05.

	RESULTS

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Electrophoretic patterns.

The electrophoretic patterns of crude soybean protein, its constituent globulin fractions 7S and 11S, 7S subunits and Croksoy^R70 are presented in Figure 1 . The Croksoy^R70 pattern is characterized by the prevalence of 30-kDa peptide subunits corresponding to 7S soy globulin.

View larger version (65K): [in this window] [in a new window]   Figure 1. SDS-PAGE pattern of crude soybean protein, 7S and 11S globulins from soy, 7S subunits ( + ' and ß) and a commercial soy protein product, CroksoyR70. Fifteen micrograms for soybean proteins and 5 µg for the molecular weight standards (Mr, kDa) were applied to each lane, on a 10–20% gradient polyacrylamide gel containing 0.1% SDS.

The viability assays were carried out on Hep G2 cells preincubated for 24 h in MEM supplemented with 5% LPDS and containing different proteins/peptides at concentrations ranging from 0.05 to 1.0 g/L. At all concentrations tested, none of these (in vitro digested and synthetic peptides) affected Hep G2 cells, i.e., enzyme release beyond that of unexposed cells.

LDL receptor regulation by whole Croksoy^R70, its enzyme digests and subfractions thereof.

Addition of Croksoy^R70 to Hep G2 cells caused a significant dose-dependent rise of LDL receptor–mediated uptake and degradation, with a maximal activity at 0.500 g/L (+35 and + 42%, respectively vs. LPDS), and significantly reduced effectiveness at a concentration of 1.000 g/L (Table 1 ). After enzyme digestion (see Materials and Methods) Croksoy^R70 exerted significantly higher LDL receptor–stimulating activity (vs. undigested compound), which did not appear to be reduced at higher concentrations. Hep G2 cells incubated in the presence of 1.000 g/L of Croksoy^R70 enzyme digests showed, in fact, greater LDL uptake and degradation (+104 and + 122%, respectively) via the LDL receptor, compared with cells exposed to LPDS and to intact Croksoy^R70 (Table 1) .

Evaluation of synthetic peptides corresponding to specific sequences of 7S globulin subunits on the LDL receptor activity in Hep G2 cells.

LDL receptor modulation in Hep G2 cells exposed to peptides corresponding to sequence differences between + ' and ß subunits from 7S globulin is presented in Table 2 . When tested in Hep G2 cells at concentrations of 10^-4 mol/L, a marked increase (P 0.05) in the uptake and degradation of LDL (receptor-mediated) was detected after cell incubation with the LRVPAGTTFYVVNPDNDENLRMIA synthetic peptide. In contrast, Hep G2 cells exposed to the QDKESQESEGSESQREPRRHKNK peptide at concentrations of 10^-5 mol/L had a significantly lower LDL uptake than controls. Although this last result might be due to a toxic effect of the synthetic peptide, cell viability, determined before LDL receptor testing, was unaffected by the peptide addition.

LDL receptor activity in Hep G2 cells exposed to Croksoy^R70 total concentrate, ethanol-extracted bulk and residue containing ethanol-soluble isoflavones is presented in Table 3 and compared with genistein, at concentrations generally occurring in soybean flour and soy globulins (Anderson and Wolf 1995 ). Croksoy^R70 concentrate showed the expected activity on LDL receptor regulation; increased activity was also found in the protein-rich product after ethanol extraction (bulk) vs. Croksoy^R70. A marked dose-related increase in the LDL receptor uptake was, in fact, detected in Hep G2 cells exposed to Croksoy^R70 bulk at concentrations from 0.125 to 0.750 g/L, whereas the LDL degradation, although higher vs. whole Croksoy^R70 concentrate, showed no dose-related response. The hot ethanol extract (residue) did not show any activity; similarly, genistein showed no LDL receptor activation, even at the highest concentrations.

Changes in apo B production by Hep G2 cells induced by incubation with graded non-toxic concentrations of 7S globulin, its + ' and ß subunits and Croksoy^R70 (as determined by MTT viability assay) are presented in Figure 2 . Croksoy^R70 and the soy 7S subfractions only moderately inhibited apo B release into the Hep G2 medium. However, the 7S soy globulin almost totally suppressed (-90%) net apo B secretion (Fig. 2) . In a preliminary experiment (data not shown), we found that the apo B–lowering effect was dose dependent and significant in the presence of a lower 7S globulin concentration (a significant 84 ± 1% reduction for a concentration of 0.25 g/L). 7S globulin at the highest concentration was evaluated for its ability to inhibit ¹⁴C-acetate incorporation into cellular lipids. Hep G2 cells exposed to 7S soy globulin (1.000 g/L) showed a remarkable reduction of ¹⁴C-acetate incorporation mainly into free cholesterol (-73%) but also into cholesterol esters (-48%) and triglycerides (-59%) (P 0.05; Table 4 ).

View larger version (16K): [in this window] [in a new window]   Figure 2. Effect (expressed as percentage change) of soy 7S globulin, its subunits ( + ' and ß) and a commercial soy protein product, CroksoyR70, on apolipoprotein B (apo B) accumulation in the media of HepG2 cell cultures. Confluent HepG2 cells were incubated for 24 h in the absence (control, set to 100%) or presence of soy proteins at the concentrations listed above. Values are means ± SEM, n = 4. Data were evaluated by ANOVA followed by Tukey’s t test. *P 0.05 and **P 0.001, vs. control; oP 0.001 vs. 7S soy globulin at the same concentration.

	DISCUSSION

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The presented data resulted from in vitro experiments; at present, we cannot be sure that they represent what takes place in vivo. On the other hand, by pursuing this in vitro approach, we can obtain clues to the identity of the potentially active peptides. In this way, it might be possible to eventually detect the active components in biological fluids, as was the case for the hypotensive tripeptides, derived from the digestion of sour milk, described recently by Masuda et al. (1996) .

These results confirm that marked LDL receptor up-regulation can be induced in Hep G2 cells exposed to Croksoy^R70. Because Croksoy^R70 in vitro enzyme digestion products of MW > 3000 Da were characterized by the same positive LDL receptor modulation, this suggests that the effect of soy preparations might be due to specific peptides from this fraction. The identity of these active peptide component(s) is being investigated at present. Studies are also being conducted to determine whether peptides with MW > 3000 Da have the ability to cross the intestinal wall. Preliminary results, with rat inverted intestinal sacs, demonstrate that the intact peptides, with MW ranging from 3000 to 20000 Da, can cross the intestinal wall intact at a percentage of 0.20 (unpublished data). In support of these data, Warshaw et al. (1984) showed earlier that as much as 2% of ingested BSA is absorbed and appears in blood intact. Asato et al. (1994) also reported that small quantities of intraluminal peptides can enter the circulation intact and potentially affect lipid metabolism.

Among the objectives of this report was to determine whether synthetic peptides corresponding to sequences differing between + ' and ß subunits of 7S globulin could modulate the activity of LDL receptor in Hep G2 cells. This was done because + ' subunits from 7S soy globulin have been found to be more active than the ß-subunit in LDL receptor stimulation (Manzoni et al. 1998 ). A systematic survey of the protein data bank (Swiss-Prot) of the 7S structure (Wright 1985 ) indicated that + ' subunits differ most from the ß-subunit, based on the consensus alignment of all known 7S globulin sequences, in two regions present in + ' but absent in ß. These results show that the sequence corresponding to a tryptic fragment and devoid of pepsin target sites, with MW 2353 Da, has no LDL receptor–modulating activity in Hep G2 cells. In contrast, a marked up-regulation of LDL receptor–mediated uptake and degradation occurred with the second sequence with MW 2271 Da, added to cells at a concentration of 10^-4 mol/L. These preliminary results clearly suggest a positive modulation of LDL receptor induced by a specific sequence of soy globulin, and indicate a potentially promising area for the development of dietary peptides characterized by important pharmacologic properties.

To determine whether the isoflavone components of soy preparations may play a role in modulation of cholesterol homeostasis, the effect of a hot ethanol extract of Croksoy^R70 on the LDL receptor modulation was also evaluated. High concentrations of genistein (30 mg/L) have been shown to induce a remarkable down-regulation of LDL receptors in Hep G2 cells grown in presence of oncostatin M (Grove et al. 1991 ). However, other data obtained under similar conditions indicate that a threshold concentration of genistein able to activate LDL receptors may be 2 mg/L (Liu et al. 1993 ). Finally, a significant up-regulation of the LDL receptors was reported at concentrations between 10 and 20 mg/L in actively growing liver cells, stimulated by the hepatocyte growth factor (Kanuck and Ellsworth 1995 ). Because the major isoflavones in Croksoy^R70 were present in extremely low concentrations (Sirtori et al. 1997 ), the activity of such low concentrations of genistein was investigated in this experiment. The hot ethanol treatment did not reduce the up-regulating potential of Croksoy^R70 (bulk) on LDL receptors. On the contrary, both the hot ethanol extract (residue) and genistein, at all tested concentrations, did not affect LDL receptor activity (Table 2) . Our observations confirm therefore that the major isoflavone components of soy are unlikely to be responsible for the LDL receptor activation induced by exposure of Hep G2 cells to various soy preparations.

Many authors have speculated that isoflavones, because of a chemical structure similar to that of mammalian estrogens, may bind to estrogen receptors (Shutt and Cox 1972 ), exhibiting a higher affinity for the ß receptor (Kuiper et al. 1997 ) and thus being responsible for the cholesterol-lowering properties of soybeans, as reviewed by Potter (1998) . However, this hypothesis has been followed by conflicting results. Isoflavones, because of their inhibitory action on tyrosine kinase (EC 2.7.1.112), a major regulator of LDL receptor activity in liver cells (Grove et al. 1991 ), might exert a detrimental effect on LDL levels. Data from primates, indicative of a direct cholesterol-lowering effect of ethanol-extractable components of soybean protein (mainly isoflavone) (Anthony et al. 1996 ) were not confirmed by more recent results in gerbils (Tovar-Palacios et al. 1998 ). Furthermore, in apolipoprotein E knockout mice, a soybean protein isolate exerted an impressive antiatherosclerotic activity vs. no effect of the ethanol-extractable components (Ni et al. 1998 ). Finally, Greaves et al. (1999) recently found that the addition to a casein diet of a semipurified ethanol extract of soy, rich in isoflavones, did not improve cholesterolemia in ovariectomized cynomolgus monkeys vs. intact soy protein. Very recent results by Wilcox et al. (1999) show that in Hep G2 cells, very high concentrations of genistein (54 mg/L) can substantially reduce medium content of LDL protein and apo B (70–80% reduction), and at the same time, decrease biosynthesis of cellular lipids, especially cholesteryl esters (40–45% reduction). Because these effects are observed in spite of an impaired receptor-mediated uptake and degradation of LDL (Wilcox et al. 1999 ), they are difficult to interpret.

Because peptides from Croksoy^R70, the 7S globulin and its subunits could also affect the net LDL production by mechanisms other than LDL receptor modulation, these fractions were evaluated for their ability to alter apo B concentrations in the medium of Hep G2 cells. The observed dose-dependent medium apo B reduction induced by exposure of cells to 7S globulins at concentrations of 0.25–1.0 g/L could be due at least in part to a substantial stimulation of uptake and degradation of LDL, as demonstrated in our previous studies (Lovati et al. 1992 ). In the case of + ' subunits and Croksoy^R70, the lack of the expected medium apo B reduction was likely related to the fact that increased uptake and degradation of LDL induced by these preparations was offset by the enhanced secretion of apo B–containing lipoproteins. The substantial apo B–lowering effect produced by 7S globulins was associated with reduced biosynthesis of cellular lipids, especially free cholesterol (73% reduction), and, to a lesser degree, cholesteryl esters and triacylglycerols. These changes differ from those reported for Hep G2 cells incubated with high concentrations of genistein (Wilcox et al. 1999 ). They also contrast with the lack of alterations in cellular cholesterol metabolism in Hep G2 cells, in which variable medium apo B responses were stimulated by a mixture of amino acids (Zhang et al. 1993 ). Very recent data on Ldlr-/- hepatocytes from LDL receptor–deficient mice (Twisk et al. 2000 ) show a direct link between functional LDL receptor expression and post-translational apo B degradation. Adenovirus-mediated overexpression of the LDL receptor in Ldlr-/- cells in this model accounts for an enhanced degradation of apo B, by increasing LDL receptor–mediated reuptake of nascent lipoprotein particles at the cell surface. These results may provide an indirect explanation for our findings in which an LDL receptor up-regulation, promoted by the 7S globulin, is coupled with a significant decrease in apo B secretion by Hep G2 cells. Moreover, because the stimulated uptake of LDL particles by the 7S globulin gives a net flux of lipids into the cell compartment, this may result in a negative feedback on intracellular lipid synthesis, as demonstrated by reduced acetate incorporation into lipids.

In conclusion, the data obtained in this study, although confirming that the protein moiety of soy protein is responsible for the LDL receptor activation, suggest that these cholesterol-lowering properties may be exerted by low-molecular-weight peptides, as the result of a complex mechanism in which different subcellular compartments may cooperate in determining a fine regulation of cell cholesterol homeostasis.

	FOOTNOTES

 
² Deceased.

³ Abbreviations used: apo B, apolipoprotein B; BSA, bovine serum albumin; FCS, fetal calf serum; FDA, Food and Drug Administration; Hep G2, human hepatoma cell line; LDH, lactate dehydrogenase; LPDS, lipoprotein-deficient serum; MEM, minimum essential medium; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.

Manuscript received January 31, 2000. Initial review completed March 15, 2000. Revision accepted May 24, 2000.

	REFERENCES

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