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Article

A Cooperative Interaction between Soy Protein and Its Isoflavone-Enriched Fraction Lowers Hepatic Lipids in Male Obese Zucker Rats and Reduces Blood Platelet Sensitivity in Male Sprague-Dawley Rats^{1 ,2}

Michael R. Peluso^*,, Todd A. Winters^*,, Michael F. Shanahan and William J. Banz^*,3

Departments of ^* Animal Science, Food and Nutrition and Physiology, Southern Illinois University, Carbondale, IL 62901

³To whom correspondence should be addressed.

	ABSTRACT

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Soy protein diets lower plasma cholesterol in hyperlipoproteinemic human subjects, as well as in animal models. We fed 7-wk-old male obese (fa/fa) and lean Zucker rats a modified AIN-76 diet (20 g protein/kg diet) containing casein (C), low isoflavone soy protein (38 mg isoflavones/kg diet; LI), or high isoflavone soy protein (578 mg isoflavones/kg diet; HI) for 70 d. In obese rats, plasma total cholesterol was 21 and 29% lower in the LI and HI groups, respectively, than in the C group (P 0.004). Liver weight and liver triglyceride and cholesteryl ester concentrations were 27, 33 and 46% lower, respectively, in the LI group than in the C group (P < 0.003). These liver measurements were 23, 24 and 57% lower, respectively, in the HI group than in the LI group (P < 0.05). In a complementary study, 5-wk-old male Sprague-Dawley rats were fed the same C, LI and HI diets for 42 d. Thrombin-mediated platelet serotonin release in vitro was 13% lower in the HI group than in the C group (P = 0.003). In a third study, 7-wk-old male Sprague-Dawley rats were fed either a modified AIN-76 control diet or a high fat casein-based atherogenic diet (140 g fat, 12 g cholesterol, and 2 g cholic acid/kg diet) with or without a soy isoflavones extract (983 mg isoflavones/kg diet) for 63 d. Addition of the isoflavones extract to the atherogenic diet lowered the liver triglyceride concentration by 33% relative to the atherogenic diet without isoflavones (P = 0.0001). Our studies suggest that the hypocholesterolemic mechanism of dietary soy protein involves a cooperative interaction between the protein and isoflavone-enriched fraction that lowers hepatic lipid concentrations. We speculate that modulation of liver and plasma lipid homeostasis can also lower blood platelet sensitivity.

KEY WORDS: • soy protein • soy isoflavones • rats • hepatic lipids • platelets

	INTRODUCTION

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Dietary soy protein has been shown to be hypocholesterolemic in human and animal studies (Carroll and Kurowska 1995 ). In humans, the cholesterol-lowering effect was observed primarily in persons who are hypercholesterolemic before dietary intervention (Anderson et al. 1995 ). Soy protein diets have been particularly beneficial in the treatment of type II hyperlipoproteinemia (Sirtori et al. 1977 and 1995 ), which is characterized by elevated plasma LDL cholesterol (type IIa) or plasma LDL and VLDL cholesterol and triglyceride (type IIb). Hyperlipidemia is associated with the development of atherosclerosis, cardiovascular disease (CVD),⁴ and noninsulin-dependent diabetes mellitus (NIDDM) (Despres et al. 1990 ). Therefore, a hypocholesterolemic effect of soy protein may lower CVD and NIDDM risk.

Hypotheses have been proposed for mechanisms responsible for the cholesterol-lowering effect of soy protein (Anthony et al. 1998 , Potter 1998 , Sirtori et al. 1998 ). The soy protein amino acid composition, specific soy peptides and globulins, and the isoflavones and saponins associated with soy protein have all been suggested as factors participating in the hypocholesterolemic response. The liver centrally regulates whole-body cholesterol excretion through the production and secretion of bile. Therefore, a mechanism responsible for the hypocholesterolemic effect of soy protein likely includes normalization of aberrant hepatic cholesterol and bile acid metabolism. The liver also centrally regulates plasma cholesterol and triglyceride concentrations through production, secretion and catabolism of apolipoprotein B (apoB). Furthermore, visceral obesity and elevated portal-hepatic free fatty acid flux induce hepatic steatosis and elevate the production of triglyceride-rich apoB-lipoproteins (Despres et al. 1990 ). Therefore, a mechanism responsible for the hypocholesterolemic effect of soy protein may also include normalization of aberrant hepatic fatty acid and triglyceride metabolism.

Obese (fa/fa) Zucker rats are hyperinsulinemic, hyperlipoproteinemic and develop hepatic steatosis within a few weeks after birth (Krief and Bazin 1991 ). These rats can be used as a model system for symptoms associated with the development of CVD and NIDDM (Kasiske et al. 1992 , St. John and Bell 1991 ). Markedly elevated pancreatic insulin secretion suppresses hepatic fatty acid catabolism and stimulates hepatic lipogenesis and fatty acid esterification. Elevated triglyceride and cholesteryl ester availability up-regulates secretion of apoB-lipoproteins and induces lipid storage in cytosolic droplets (Fukuda et al. 1982 ). Furthermore, there is an absence of the feeding-induced diurnal rise and fall of hepatic cholesterogenesis in adult male obese rats (Lin 1985 ), and fecal neutral sterols are 50% lower in obese rats than in lean rats (McNamara 1985 ). Expression of the hepatic LDL receptor is 60% lower in obese rats than in lean rats, without a difference in LDL receptor mRNA (Liao et al. 1997 ). The diurnal rhythm of hepatic cholesterol 7-hydroxylase has also been shown to be absent in obese rats (Tang et al. 1988 ). Hepatic steatosis, hepatic overproduction of VLDL, and abnormal hepatic cholesterol and bile acid metabolism are characteristics that make the obese Zucker rat a model system for studying mechanisms responsible for the hypocholesterolemic effect of soy protein.

Blood platelets also play an integral role in the development of CVD (Ross 1986 ). Arterial cholesterol deposition and blood platelet sensitivity are elevated by plasma LDL and lowered by plasma HDL (Miller et al. 1981 , Surya and Akkerman 1993 ). The variation in platelet sensitivity found among species has been shown to correlate directly with susceptibility of the species to CVD (Hayes and Pronczuk 1996 ). Furthermore, the plasma LDL cholesterol:HDL cholesterol ratio has been found to vary directly with platelet sensitivity both across and within species. Platelet activation is accompanied by release of compounds from intraplatelet granules that promote atherosclerotic lesion formation (Ross et al. 1984 ). For example, platelet-derived growth factor stimulates vascular smooth muscle cell migration and proliferation in the arterial intima. Activated platelets also release 5-hydroxytryptamine (5HT), commonly known as serotonin, which plays a role in the pathophysiology of essential hypertension (Nityanand et al. 1990 ). Dietary soy protein, rich in isoflavones, has been shown to reduce atherosclerotic lesion development in male cynomolgus monkeys fed an atherogenic diet (Anthony et al. 1997 ). An inhibitory effect of isoflavone-rich soy protein on platelet aggregability has been reported in female rhesus monkeys (Williams and Clarkson 1998 ). These effects of isoflavone-rich soy protein may result in part from a reduction in the plasma LDL cholesterol:HDL cholesterol ratio. Another study showed rapid inhibition of vasoconstriction in stenotic arteries of female macaques after intravenous infusion of the soy isoflavone genistein (Honore et al. 1997 ). Improved systemic arterial compliance has also been shown after dietary isoflavone supplementation in menopausal women (Nestel et al. 1997 ). These studies suggest that plasma isoflavones may interact directly with blood platelets and cells of the arterial wall.

Mechanisms responsible for the effects of soy protein and its isoflavone-enriched fraction on CVD and NIDDM risk certainly involve beneficial changes in liver and plasma lipid metabolism, as well as in the function of blood platelets and cells of the vascular wall. Our primary objective was to discriminate between the effects of soy protein and the isoflavone-enriched fraction on liver and blood risk factors associated with CVD and NIDDM. The following project used male lean and obese Zucker rats and male Sprague-Dawley rats to examine the effects of soy protein and its isoflavone-enriched fraction on liver and plasma lipid concentrations and on blood platelet sensitivity. Our studies with these animal models suggest that soy protein and the isoflavone-enriched fraction act cooperatively to lower hepatic lipid concentrations and reduce blood platelet sensitivity.

	MATERIALS AND METHODS

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Animals, diets, and experimental design.

Rats were obtained from Harlan Sprague Dawley, (Indianapolis, IN) and housed individually in stainless steel wire-mesh cages at 21°C in a room with an automatically controlled 12-h light:dark cycle. Upon arrival, rats were fed a nonpurified diet (Purina Formulab Rodent Chow; El-Mel, Florissant, MO) and acclimated to the facility for 10 d before administration of experimental diets. Rats had free access to deionized water and the assigned diets for the length of each study. Growth and food intake were measured weekly. At the end of each experimental period, rats were deprived of food for 12 h and then anesthetized with an intraperitoneal injection of sodium pentobarbitol (5 mg/100 g body). Blood was drawn by cardiac puncture, and platelets were isolated from platelet-rich plasma for measurement of platelet sensitivity as described below. Remaining plasma was stored at -80°C for lipid analysis. Livers were removed, weighed, frozen in liquid N₂ and stored at -80°C for lipid analysis.

Three studies were designed to examine the effects of soy protein and the isoflavone-enriched fraction of soy protein on liver and plasma lipid concentrations and on blood platelet sensitivity as follows: Study 1: male lean and obese Zucker rats; Study 2: male Sprague-Dawley rats; and Study 3: male Sprague-Dawley rats fed an atherogenic diet. High nitrogen casein (ICN Biomedicals, Costa Mesa, CA) was used as the control protein in each study. All experimental protocols for animal care and use were approved by the Animal Care and Use Committee at Southern Illinois University, Carbondale, IL.

Study 1.

Seven-week-old male obese Zucker rats (fa/fa, n = 5/diet group) and lean Zucker rats (Fa/?, n = 3/diet group) were fed one of three diets containing casein (C diet group), low isoflavone soy protein isolate (LI diet group) or high isoflavone soy protein isolate (HI diet group) as the protein source for 70 d (Table 1 ). The control (C) diet represented a modified AIN-76 semipurified diet for laboratory rodents (AIN 1977 ). To elevate the ratio of complex:simple carbohydrates, sucrose was lowered from 500 g/kg diet (AIN-76 diet) to 300 g/kg diet, and cornstarch was raised from 150 g/kg diet (AIN-76 diet) to 350 g/kg diet. Net protein concentration of all three diets was 174 g/kg diet, and protein source (casein, low isoflavone soy protein or high isoflavone soy protein) was the only dietary variable. High isoflavone soy protein was alcohol-washed to lower the isoflavone concentration and produce low isoflavone soy protein (both soy protein isolates were donated by Protein Technologies International, St. Louis, MO). Total soy isoflavone concentration in the LI and HI diets was 38 and 578 mg/kg diet, respectively. Individual isoflavone concentrations as genistein-, daidzein- and glycitein-containing compounds (aglycones + glycosides + glycoside esters) were 24, 12 and 2 mg/kg diet (LI diet) and 370, 178 and 30 mg/kg diet (HI diet), respectively (values calculated from isoflavone concentrations of the soy protein isolates as provided by Protein Technologies International).

Five-week-old male Sprague-Dawley rats (n = 10/diet group) were fed one of three diets containing casein (C diet group), low isoflavone soy protein isolate (LI diet group) or high isoflavone soy protein isolate (HI diet group) as the protein source for 42 d. Diets were identical to those used in Study 1 (Table 1) .

Study 3.

Seven-week-old male Sprague-Dawley rats (n = 10/diet group) were fed one of five diets for 63 d (Table 2 ). Two diet groups were administered a control (C) diet, and three diet groups were administered an atherogenic (A) diet. One group of rats fed the control diet (C + I diet group) and one group of rats fed the atherogenic diet (A + I diet group) were fed the respective diets supplemented with a powdered soy isoflavones extract (donated by Archer Daniels Midland, Decatur, IL). The remaining group of rats was fed the atherogenic diet containing high isoflavone soy protein isolate (identical to that used in Studies 1 and 2) in place of casein (A + HI diet group). The control diet represented a modified AIN-76 semipurified diet for laboratory rodents (AIN 1977 ). To elevate the ratio of complex:simple carbohydrates, sucrose was lowered from 500 g/kg diet (AIN-76 diet) to 200 g/kg diet, and cornstarch was raised from 150 g/kg diet (AIN-76 diet) to 450 g/kg diet. The control diet was converted to an atherogenic diet by raising the sucrose concentration from 200 to 400 g/kg diet, lowering the cornstarch concentration from 450 to 145 g/kg diet, lowering the corn oil concentration from 50 to 20 g/kg diet and by the addition of coconut oil (70 g/kg diet), lard (50 g/kg diet), cholesterol (12 g/kg diet) and cholic acid (2 g/kg diet). The atherogenic diet was expected to elevate plasma and liver total cholesterol and triglyceride concentrations. However, the efficacy of the atherogenic diet to actually promote atherosclerotic lesion development was not examined. Net protein concentration of all five diets was 174 g/kg diet. Total soy isoflavone concentration in the C + I and A + I diets was 983 mg/kg diet, and total soy isoflavone concentration in the A + HI diet was 578 mg/kg diet. Individual isoflavone concentrations as genistein-, daidzein- and glycitein-containing compounds (aglycones + glycosides + glycoside esters) were 535, 411 and 37 mg/kg diet (C + I and A + I diets) and 370, 178 and 30 mg/kg diet (A + HI diet), respectively (values calculated from isoflavone concentrations of the powdered soy isoflavones extract and the high isoflavone soy protein isolate as provided by Archer Daniels Midland Company and by Protein Technologies International, respectively).

	Analytical methods

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Frozen livers were thawed on ice to 4°C, and total lipids were extracted from 0.5-g liver portions (Folch et al. 1957 ). For cholesterol analysis, aliquots of lipid extracts (5–50 µL) were mixed with 50 µL of Triton X-100 in acetone (150 g/L), and solvents were evaporated under vacuum. Total and unesterified cholesterol concentrations were determined by an enzymatic colorimetric method (Allain et al. 1974 ). The molar concentration of cholesteryl ester was calculated by difference. Liver total triglycerides were quantified colorimetrically (Fletcher 1968 ). Plasma total cholesterol was measured using an enzymatic colorimetric method (Allain et al. 1974 ). Plasma total triglycerides were quantified with a kit (Sigma Diagnostics no. 336, St. Louis, MO) using an adaptation of an enzymatic procedure (Bucolo and David 1973 ).

Blood platelet sensitivity.

Blood (9 mL) was drawn by cardiac puncture into 10-mL syringes preloaded with 1 mL sodium citrate (40 g/L). Anticoagulated blood from each rat was mixed with 1.5 mL of pH 7.4 buffered saline glucose-citrate (84 mmol/L NaCl, 8.5 mmol/L Na₂HPO₄, 1.5 mmol/L KH₂PO₄, 13.6 mmol/L sodium citrate and 11.1 mmol/L D-glucose). All procedures were performed at 22°C. Platelet-rich plasma was obtained by sedimentation of blood cells at 850 x g for 5 min. An equivalent aliquot of platelet-rich plasma from each rat was layered on top of a single-step CellSep Platelets density gradient (Cardinal, Santa Fe, NM). Platelets were sedimented through the upper layer of the gradient at 1450 x g for 20 min. Isolated platelets were removed from the gradient interface, washed with 8 mL of buffered saline glucose-citrate, pelleted at 600 x g for 8 min, and resuspended at a concentration of 2–4 x 10¹¹ cells/L in pH 7.6 Tyrode’s buffer (137 mmol/L NaCl, 0.4 mmol/L NaH₂PO₄, 2.6 mmol/L KCl, 12.1 mmol/L NaHCO₃ and 5.5 mmol/L D-glucose). The expected purity of this washed platelet population is 99%.

Four 300-µL aliquots of suspended platelets from each rat were equilibrated at 22°C for 3 h. Duplicate platelet aliquots were then incubated for 3 min either without (unstimulated) or with thrombin (150 U/L) in Tyrode’s buffer (pH 7.6) containing 1 mmol/L CaCl₂, 2.5 µmol/L imipramine and 250 µmol/L ascorbic acid in a final volume of 400 µL. Thrombin-stimulated and unstimulated "resting" platelets were pelleted at 2200 x g for 1 min. Supernatants were immediately removed and frozen at -80°C for serotonin (5HT) analysis. Platelet pellets were covered with 125 µL of ice-cold lysis buffer (pH 7.5; 20 mmol/L Tris, 150 mmol/L NaCl, 3.5 mmol/L SDS, 10 g/L Triton X-100, 5 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 400 mg/L 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mg/L leupeptin and 1 mg/L pepstatin), and platelets were solubilized with sonication for 5 s. Protein was precipitated from 30-µL aliquots with trichloroacetic acid (120 g/L). Platelet protein was quantified using a modification of the method of Lowry et al. (1951) and Sigma Assay Kit no. P5656 with bovine serum albumin as a standard. Remaining platelet lysates were stored at -80°C for 5HT analysis.

Platelet lysate and supernatant samples (from above) were diluted with ice-cold 0.32 mol/L perchloric acid containing 400 µmol/L Na₂S₂O₅ as an antioxidant and 100 µg/L methyl 5-hydroxytryptamine (methylserotonin) as an internal standard. Samples were centrifuged briefly at 10,000 x g to sediment precipitated protein, and 20-µL aliquots were loaded onto a C18 reverse-phase HPLC column (5-µm diameter silica, Beckman Instruments, Fullerton, CA). Serotonin and methylserotonin were eluted with a mobile phase (pH 4.5; 109 mmol/L citric acid, 167 mmol/L sodium acetate and 82 µmol/L EDTA) containing 10% (v/v) methanol. Peaks were detected electrochemically, and 5HT was quantified by the method of internal standards (Sasa et al. 1978 ). The initial platelet 5HT concentration was computed by adding the amount of 5HT in each platelet lysate to the amount of 5HT released (secreted) from each platelet sample. Release of 5HT was expressed as a percentage of the initial platelet 5HT concentration (% initial platelet 5HT).

	Statistical methods

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ANOVA was from StatView 4.02 (Abacus Concepts, Berkeley, CA). Analysis of covariance (ANCOVA) was from Systat 7.0 (SPSS, Chicago, IL). Values reported in results, tables, and figures are group means ± SEM

Study 1.

Animal observations, liver and plasma lipid concentrations, and platelet measurements were analyzed for the main effect of phenotype (lean rats vs. obese rats) with two-way ANOVA (significance at P 0.05). Within each phenotype, the effect of dietary treatment (C, LI, HI) was analyzed with one-way ANOVA [Fisher’s least significant difference (LSD) test, significance at P 0.05]. Platelet data sets were also analyzed with one-way ANOVA for the effect of dietary treatment independent of phenotype (n = 3 lean rats plus 5 obese rats per diet group). Unstimulated and thrombin-stimulated 5HT release were analyzed with platelet protein as a covariate (one-way ANCOVA, Fisher’s LSD test, significance at P 0.05). Values for 5HT release are reported as least-squares means.

Study 2.

Data sets were analyzed for the effect of dietary treatment (C, LI, HI) with either one-way ANOVA or one-way ANCOVA (Fisher’s LSD test, significance at P 0.05).

Study 3.

Data sets were analyzed for the main and interaction effects of diet (control vs. atherogenic) and the isoflavone-enriched fraction of soy protein (C, A vs. C + I, A + I, A + HI) with either two-way ANOVA or two-way ANCOVA (significance at P 0.05). Liver weight and liver and plasma lipid concentrations were also analyzed for the effect of dietary treatment with one-way ANOVA (Fisher’s LSD test, significance at P 0.05). Liver total cholesterol and cholesteryl ester concentrations were transformed (log₁₀) before ANOVA.

	RESULTS

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Animal observations

    Study 1. Initial body weight of male obese Zucker rats (242 ± 4 g) was higher than that of male lean Zucker rats (183 ± 3 g) (P < 0.0001). The effect of phenotype was significant for all of the following animal observations (two-way ANOVA, P < 0.0001). Final body weights were 408 ± 10 and 584 ± 8 g in lean and obese rats, respectively, without a significant effect of dietary treatment (P > 0.05). This corresponded to a weight gain of 3.2 ± 0.1 g/d in lean rats and 4.9 ± 0.1 g/d in obese rats. Energy intake and energy efficiency ratio were 360 ± 14 kJ/d and 9.0 ± 0.3 g/MJ, respectively, in lean rats, and 441 ± 9 kJ/d and 11.1 ± 0.3 g/MJ, respectively, in obese rats. In lean plus obese rats fed the HI diet, energy intake was 10% lower (P = 0.03) and there was a trend for energy efficiency ratio to be 8% higher (P = 0.1) than in lean plus obese rats fed the C diet.

    Study 2. Initial body weight of all 30 male Sprague-Dawley rats was 124 ± 1 g, and final body weights were 337 ± 8, 341 ± 6 and 344 ± 6 g in the C, LI, and HI groups, respectively, without a significant effect of dietary treatment (P > 0.05). Weight gain was 5.1 ± 0.1 g/d for all 30 rats. Energy intake was lower in rats fed the LI diet (310 ± 7 kJ/d) and in rats fed the HI diet (324 ± 6 kJ/d) than in rats fed the C diet (352 ± 13 kJ/d) (P < 0.04). Therefore, the energy efficiency ratio rose from 14.5 ± 0.5 g/MJ in the C group to 16.6 ± 0.3 g/MJ in the LI group and 16.0 ± 0.4 g/MJ in the HI group (P < 0.008).

    Study 3. Initial body weight of all 50 male Sprague-Dawley rats was 183 ± 1 g. The energy density of the atherogenic diets (A, A + I, A + HI) was 10% higher than the energy density of the control diets (C, C + I) (see Table 2 ). However, weight gain was 3.8 ± 0.1 g/d and final body weight was 420 ± 4 g for all 50 rats, without a significant effect of dietary treatment (P > 0.05). Although growth rates were similar, the energy intake was higher in rats fed the control diets (355 ± 8 kJ/d) than in rats fed the atherogenic diets (302 ± 4 kJ/d) (P < 0.0001). Therefore, the energy efficiency ratio was higher in rats fed the atherogenic diets (12.7 ± 0.2 g/MJ) than in rats fed the control diets (10.4 ± 0.4 g/MJ) (P < 0.0001). Energy intake was 4.5% higher and the energy efficiency ratio was 7.2% lower in rats fed diets containing the isoflavone-enriched fraction of soy protein (C + I, A + I, A + HI) than in rats fed diets not containing soy isoflavones (C, A) (P < 0.05).

Liver weight and liver and plasma lipid concentrations

    Study 1 (Fig. 1 ). Obese (fa/fa) Zucker rats develop an enlarged fatty liver within a few weeks of birth (Krief and Bazin 1991 ). Study 1 examined the effects of replacing casein in a modified AIN-76 control diet (C diet) with either high isoflavone soy protein isolate (HI diet) or with the same soy protein isolate that was alcohol-washed to remove most of the isoflavone-enriched fraction (LI diet). The relative liver weight (Fig. 1A ) was 27 and 44% lower in obese rats fed the LI and HI diets, respectively, than in obese rats fed the C diet (P < 0.003). The relative liver weight was 23% lower in obese rats fed the HI diet than in obese rats fed the LI diet (P < 0.05). Corresponding to reductions in liver weight, the liver triglyceride concentration (Fig. 1B ) was 33 and 49% lower in obese rats fed the LI and HI diets, respectively, than in obese rats fed the C diet (P < 0.001), and the liver triglyceride concentration was 24% lower in obese rats fed the HI diet than in obese rats fed the LI diet (P < 0.05). The liver total cholesterol concentration (Fig. 1C ) was 34 and 48% lower in obese rats fed the LI and HI diets, respectively, than in obese rats fed the C diet (P 0.0001). The liver cholesteryl ester concentration (Fig. 1D ) was 46 and 77% lower in obese rats fed the LI and HI diets, respectively, than in obese rats fed the C diet (P 0.0006). Furthermore, the liver cholesteryl ester concentration was 57% lower in obese rats fed the HI diet than in obese rats fed the LI diet (P < 0.02). In lean rats, the liver total cholesterol concentration was 35% lower in the HI group than in the C group (P = 0.007), and the liver cholesteryl ester concentration was 51 and 88% lower in the LI and HI groups, respectively, than in the C group (P < 0.03). The plasma total cholesterol concentration (Fig. 1E ) was 21 and 29% lower in obese rats fed the LI and HI diets, respectively, than in obese rats fed the C diet (P 0.004). The plasma total triglyceride concentration (Fig. 1F ) was not significantly affected by dietary treatment in lean or obese rats (P > 0.05).

View larger version (37K): [in this window] [in a new window]   Figure 1. Liver and plasma lipid concentrations in male lean and obese Zucker rats fed casein (C)-, low isoflavone soy protein (LI)-, or high isoflavone soy protein (HI)-based diets for 70 d (Study 1). Values are diet group means ± SEM, n = 3 lean rats or 5 obese rats/diet group. Main effect of phenotype (lean vs. obese, two-way ANOVA): (A) liver weight, P = 0.005; (B) liver total triglyceride concentration, P < 0.0001; (C) liver total cholesterol concentration, P = 0.02; (D) liver cholesteryl ester concentration, P = 0.003; (E) plasma total cholesterol concentration, P < 0.0001; (F) plasma total triglyceride concentration, P < 0.0001. Within each phenotype (panels A–F), values without a common letter denotation are significantly different [one-way ANOVA, Fisher’s least significant difference (LSD) test, P 0.05].

    Study 2 (Table 3 ).

    Study 3 (Table 4 ).

    Study 1 (Table 5 ). In our studies, serotonin (5-hydroxytryptamine, 5HT) release from "resting" unstimulated platelets and from thrombin-stimulated platelets was measured to quantify platelet sensitivity. In Study 1 with male Zucker rats, to assist delineation of any effects of dietary treatment, one-way ANCOVA was applied to data from lean plus obese rats combined (n = 8/diet group). Unstimulated 5HT release was 27% lower in rats fed the HI diet than in rats fed the C diet (P < 0.04). There was a trend for unstimulated 5HT release to be 23% lower in rats fed the LI diet than in rats fed the C diet (P = 0.06). Neither soy protein–based diet had a significant effect on thrombin-stimulated 5HT release (P > 0.05).

    Study 2 (Fig. 2 ).

View larger version (20K): [in this window] [in a new window]   Figure 2. Serotonin (5HT) release from platelets isolated from male Sprague-Dawley rats fed casein (C)-, low isoflavone soy protein (LI)-, or high isoflavone soy protein (HI)-based diets for 42 d (Study 2). Values are diet group means ± SEM, n = 10 rats/diet group. Values without a common letter denotation are significantly different [one-way analysis of covariance (ANCOVA), Fisher’s least significant difference (LSD) test, P 0.003].

    Study 3 (Table 6 ).

	DISCUSSION

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Obese Zucker rats are hyperinsulinemic, and obese rats fed a casein-based control diet in our study developed an enlarged lipid-infiltrated liver. In animal models of obesity, chronic hyperinsulinemia stimulates hepatic lipogenesis and the assembly and secretion of apoB-lipoproteins (Jeanrenaud 1978 ). Obesity in humans is associated with hepatic steatosis, hyperlipoproteinemia, CVD and NIDDM (Sheth et al. 1997 ). Visceral (abdominal) obesity elevates the flux of free fatty acids to the liver through the portal vein (Bjorntorp 1990 ), and hepatic steatosis in obese Zucker rats is a result of elevated fatty acid synthesis and esterification, and reduced fatty acid oxidation (Fukuda et al. 1982 ). Dietary soy protein relative to casein has been shown to suppress hepatic fatty acid synthesis in male Wistar rats (Iritani et al. 1986 ) and lower hepatic lipogenic enzyme gene expression and stimulate triglyceride degradation in female Wistar fatty rats (Iritani et al. 1996 ). The liver triglyceride concentration was 33 and 49% lower in male obese Zucker rats fed the LI and HI diets, respectively, than in obese rats fed the C diet (Fig. 1B ). Speculation from one of our complementary studies with male Sprague-Dawley rats suggests that soy isoflavones could stimulate hepatic triglyceride degradation. Indeed, a powdered isoflavone-enriched extract of soy protein added to a casein-based high fat atherogenic diet (140 g fat/kg diet) lowered the liver triglyceride concentration by 33% (Table 4) . The soy isoflavone genistein is metabolized in rat liver by microsomal cytochrome P-450 3A (CYP3A) (Jager et al. 1998 ), and there is an inverse relationship between the concentration of CYP3A protein and the severity of fatty liver in animal models of hepatic steatosis (Leclercq et al. 1998 ). A triglyceride-lowering effect of isoflavones in the liver could be related to isoflavone metabolism and microsomal CYP3A.

The liver cholesteryl ester concentration was 46 and 77% lower in obese Zucker rats fed the LI and HI diets, respectively, than in obese rats fed the C diet (Fig. 1D ). The plasma total cholesterol concentration was 21–29% lower in obese rats fed the two soy protein–based diets than in obese rats fed casein (Fig. 1E ). Enhanced fecal steroid elimination and induction of the hepatic LDL receptor are considered integral components of the mechanism responsible for the cholesterol-lowering effects of soy protein (Potter 1995 , Sirtori et al. 1995 ). Interactions in the intestinal tract that involve mixed micelles, soy protein and components of the isoflavone-enriched fraction such as saponins likely modulate cholesterol and bile acid absorption. Fundamentally, modulation of lipid digestion and absorption could also be associated with the higher energy efficiency ratio observed in rats fed high isoflavone soy protein than in rats fed casein (see Results, Studies 1 and 2). Furthermore, in cultured liver cells, genistein (7–70 µmol/L) has been shown to potentiate induction of LDL receptor mRNA by hepatocyte growth factor (Kanuck and Ellsworth 1995 ). The citrus flavanone naringenin, which like genistein is hydroxylated at the 5, 7, and 4' positions, was recently shown (at 100–200 µmol/L) to inhibit cholesteryl ester synthesis and apoB secretion in HepG2 cells (Borradaile et al. 1999 ). Genistein undergoes enterohepatic cycling in rats (Sfakianos et al. 1997 ), and speculation from these last-mentioned studies suggests that hepatic isoflavone accumulation could affect cholesterol metabolism independently. However, the addition of a powdered soy isoflavones extract (983 mg isoflavones/kg diet) to either a casein-based control diet or to a casein-based atherogenic diet did not lower plasma total cholesterol or liver cholesteryl ester concentrations in male Sprague-Dawley rats (Table 4) . This is consistent with other studies showing no effect of dietary isoflavones on plasma lipid concentrations in normocholesterolemic human subjects (55 mg isoflavones/d for 8 wk) (Hodgson et al. 1998 ) and in hypercholesterolemic cynomolgus monkeys (386 mg isoflavones/kg diet for 12 wk) (Greaves et al. 1999 ). Perhaps the cooperative interaction between soy protein and its isoflavone-enriched fraction is related to hepatic isoflavone metabolism. Conjugation of genistein with glucuronic acid occurs in rat hepatocytes before biliary secretion (Sfakianos et al. 1997 ), and hepatic canalicular transport of conjugated isoflavones has been shown to stimulate bile flow (Jager et al. 1997 ). A choleretic effect of isoflavones may occur in tandem with soy protein-micellar interactions in the intestinal tract to regulate hepatic cholesterol distribution and flux. Elevation of hepatic unesterified cholesterol by the isoflavone-enriched fraction of soy protein is indicative of an enlarged metabolically active cholesterol pool (Tables 3 and 4) .

In addition to plasma lipoprotein concentrations, blood platelets play an important role in the development of atherosclerosis (Ross 1986 ), and the release of vasoactive compounds such as serotonin (5-hydroxytryptamine, 5HT) from activated platelets into atherosclerotic artery walls aggravates the development of hypertension (Nityanand et al. 1990 ). Intravenous genistein administration (140 mg) improved arterial elasticity in female rhesus monkeys (Honore et al. 1997 ), and dietary isoflavone supplementation (80 mg/d for 10 wk) improved arterial compliance in menopausal women (Nestel et al. 1997 ). In female rhesus monkeys, isolated soy protein (350 mg isoflavones/kg diet for 6 mo) relative to isoflavone-depleted soy protein was reported to lower thrombin-stimulated platelet aggregation in vitro (Williams and Clarkson 1998 ). Our studies measured 5HT release from unstimulated "resting" platelets and from thrombin-stimulated "activated" platelets in vitro as indices of platelet sensitivity. Serotonin release from unstimulated platelets was significantly lower in normocholesterolemic male Sprague-Dawley rats fed both low and high isoflavone soy protein than in rats fed casein (Fig. 2A ). Soy protein relative to casein has occasionally been shown to elevate the plasma HDL cholesterol concentration. For example, a study of male cynomolgus monkeys fed either low or high isoflavone soy protein (200 mg/kg diet for 14 mo) containing either 34 or 300 mg isoflavones/kg diet, respectively, showed both soy protein diets to raise the plasma HDL cholesterol concentration and lower the plasma total cholesterol:HDL cholesterol ratio (Anthony et al. 1997 ). LDL has been shown to hypersensitize platelets and HDL has been shown to desensitize platelets in vitro (Surya and Akkerman 1993 ). However, the platelets used to measure 5HT release in our studies were isolated and washed, with an expected purity of 99%. Therefore, a direct effect of LDL or HDL in our in vitro assay was unlikely. Nevertheless, lipid constituents of plasma lipoproteins are exchanged with lipids in the platelet membrane, and platelet membrane cholesterol and phospholipid play an integral role in platelet function (Boesze-Battaglia and Schimmel 1997 ). We speculate that the reduction in resting platelet sensitivity observed in rats fed soy protein resulted at least in part from a change in the plasma lipoprotein lipid distribution that affected platelet function.

Platelet activation by thrombin is initiated with the binding of thrombin to its receptor in the platelet membrane, and thrombin binding is followed by G-protein–mediated activation of phospholipase C. Subsequent activation of phospholipase A₂ catalyzes hydrolytic release of membrane arachidonic acid, which is metabolized to thromboxane A₂, a potentiator of platelet activation (Thomas and Holub 1992 ). Both genistein and daidzein have been shown to exert a dose-dependent inhibition (0.4–110 µmol/L) of thromboxane receptor binding in human platelets in vitro (Nakashima et al. 1991 ). In our studies, the plasma isoflavone concentrations were likely chronically higher in rats fed high isoflavone soy protein than in rats fed either low isoflavone soy protein or casein. For example, high isoflavone soy protein (386 mg isoflavones/kg diet for 12 wk) was shown to produce plasma genistein and daidzein concentrations of 110 and 92 nmol/L, respectively, in cynomolgus monkeys (Greaves et al. 1999 ). Additionally, the plasma genistein and daidzein concentrations in Japanese men consuming traditional diets have been reported to be as high as 2.4 and 0.9 µmol/L, respectively (Adlercreutz et al. 1993 ). Therefore, platelet isoflavone uptake and accumulation could lower platelet sensitivity through antagonism at the thromboxane receptor.

We did not find an effect of the isoflavone-enriched fraction of soy protein (administered without soy protein) on platelet sensitivity in normocholesterolemic or hypercholesterolemic male Sprague-Dawley rats (Table 6) . However, the platelet 5HT concentration and unstimulated 5HT release were lower in platelets isolated from hypercholesterolemic rats than in platelets isolated from normocholesterolemic rats. This is consistent with a reduction in platelet 5HT found in human patients with familial hypercholesterolemia (Smith and Betteridge 1997 ). In contrast, thrombin-mediated 5HT release was higher in platelets isolated from hypercholesterolemic rats than in platelets isolated from normocholesterolemic rats (Table 6) . Hyperreactive platelets obtained from human subjects with type II hyperlipoproteinemia have a higher cholesterol concentration than platelets obtained from normocholesterolemic subjects (Shastri et al. 1980 ), and platelet hypersensitivity has been observed in vitro after incubation with cholesterol-rich liposomes (Shattil et al. 1975 ). The inability of soy protein or the isoflavone-enriched fraction to lower platelet 5HT release in our diet-induced hypercholesterolemic model may be related to an inability to lower platelet cholesterol.

In conclusion, the protein and isoflavone-enriched fractions of isolated soy protein cooperatively reduced the development of an enlarged fatty liver in young male obese Zucker rats. High isoflavone soy protein also lowered thrombin-stimulated platelet 5HT release in male Sprague-Dawley rats. Our studies support further investigation into the therapeutic use of soy protein and components of its isoflavone-enriched fraction as a part of dietary treatment for hepatic steatosis and atherosclerosis to lower CVD and NIDDM risk.

	ACKNOWLEDGMENTS

 
The authors wish to gratefully acknowledge Richard W. Steger and Clare Fadden for the use of and assistance with the HPLC, and Jennifer Cameron, Brian Carroll, Stephanie Ellis, Brian Gename, Matt Hagemayer, Melissa McDearmon, Theodora Perseli, Shannon Read and Mark Williams for valuable contributions to the project. We also thank Andrzej Bartke for kindly providing critical review of the manuscript, and Protein Technologies International and Archer Daniels Midland Company for donating isolated soy protein and powdered soy isoflavones products, respectively.

	FOOTNOTES

 
¹ Presented in part in poster form at Experimental Biology 99, April 1999, Washington, DC [Banz, W., Peluso, M., Winters, T. & Shanahan, M. (1999) The effects of soy protein and isoflavones on platelet, lipid and liver measurements in Zucker rats. FASEB J. 13: A885 (abs.)] and at the World Soybean Research Conference VI, August 1999, Chicago, IL [Peluso, M., Williams, M., Hagemayer, M., Steger, R., Winters, T., Shanahan, M. & Banz, W. (1999) Soy protein rich in isoflavones modulates hepatic lipids and platelet sensitivity in rats. Proceedings, p. 716 (abs.)].

² Supported by the Illinois Council on Food and Agricultural Research, the Illinois Soybean Program Operating Board and the Southern Illinois University Office of Research and Development.

⁴ Abbreviations used: ANCOVA, analysis of covariance; apoB, apolipoprotein B; CVD, cardiovascular disease; CYP3A, cytochrome P-450 3A; HI, high isoflavone soy protein isolate; 5HT, 5-hydroxytryptamine, serotonin; LI, low isoflavone soy protein isolate; LSD, least significant difference; NIDDM, noninsulin-dependent diabetes mellitus.

Manuscript received December 28, 1999. Initial review completed February 11, 2000. Revision accepted April 7, 2000.

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