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Biochemical and Molecular Actions of Nutrients

Dietary Soy ß-Conglycinin (7S Globulin) Inhibits Atherosclerosis in Mice^1,2

Michael R. Adams³, Deborah L. Golden, Adrian A. Franke^*, Susan M. Potter, Houston S. Smith and Mary S. Anthony

Department of Pathology, Section of Comparative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157 ^* Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, HI 96813; and the Solae Company, St. Louis, MO 63188

³To whom correspondence should be addressed. E-mail: madams{at}wfubmc.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Although ß-conglycinin (7S globulin), a major soy storage protein, stimulates the expression of LDL receptors and the degradation of LDL by hepatocytes in vitro, the in vivo effects of dietary ß-conglycinin on the cardiovascular system are unknown. We assessed the effects of dietary ß-conglycinin and other soy peptide fractions on the development of atherosclerosis in atherosclerosis-susceptible mice. At 6 wk of age, male and ovariectomized female apolipoprotein (apo) E-null mice and LDL receptor-null, apoB transgenic mice were assigned randomly to treatment groups that differed only in the source of dietary protein: 1) casein/lactalbumin, 2) isoflavone-containing soy protein isolate, 3) ß-conglycinin, 4) glycinin (11S globulin, another major soy storage protein), 5) ß-conglycinin-devoid soy protein, and 6) W008 (a peptide fraction produced by hydrolysis and precipitation of soy protein isolate). After 4 mo, aortic atherosclerosis (cholesteryl ester content) and plasma lipoprotein cholesterol concentrations were quantified using GLC. Relative to mice fed casein/lactalbumin–based diets, the extent of atherosclerosis was reduced in ovariectomized female mice fed all soy protein–containing diets. Relative to mice fed isoflavone-containing soy protein isolate, atherosclerosis was reduced only in mice fed the ß-conglycinin–containing diet. Mean reductions were 39 and 67% (all P <0.05) in male and ovariectomized female apoE null mice and 66% (P < 0.05) in male LDL receptor null mice. These effects were unrelated to variation in isoflavone content of the protein source and only minimally related to plasma lipoprotein cholesterol concentrations. We conclude that a diet rich in ß-conglycinin has atheroprotective effects that greatly exceed those of isoflavone-containing soy protein isolate and do not depend on LDL receptors or influences on plasma lipoproteins.

KEY WORDS: • atherosclerosis • mice • soy protein • 7S globulin • lipoproteins

Diet-induced atherosclerosis is reduced in animals fed soy protein–based diets relative to those fed animal protein–based diets. However, the components of soy protein responsible for this effect and the mechanisms involved remain uncertain. Although some evidence implicates the isoflavones contained in soy beans and many soy-based products, other evidence implicates certain peptides or peptide fractions. Some of the evidence supporting a role for peptides comes from the studies of Sugano et al. (1,2) who employed extensive enzymatic digestion of soy protein followed by separation of the peptide products into soluble and insoluble fractions. These authors determined that there was an undigested insoluble high-molecular-weight fraction (HMF)⁴ that bound bile acids, increased the excretion of acidic and neutral steroids, and caused a marked reduction in plasma and hepatic cholesterol concentrations in rats. It has also been shown that increased consumption of HMF by human subjects increased bile acid secretion, raised plasma HDL cholesterol, and decreased plasma LDL cholesterol concentrations relative to subjects consuming casein or a soy protein isolate (3).

Soy peptides can also be separated on the basis of molecular size. The major storage proteins of soy beans are ß-conglycinin, or 7S globulins, and glycinin, or 11S globulins. The studies of Lovati et al. (4,5) demonstrated that 7S globulins stimulate the expression of LDL receptors and the degradation of LDL in cultured hepatocytes (HepG2 cells). The study of Sirtori et al. (6) showed that, when fed to rats, 7S globulin reduced plasma cholesterol concentration by 35%. More recently, these authors (7) showed that small peptides (3–20 kDa) produced by enzymatic digestion of soy protein and a small synthetic peptide (2271 Da), which corresponds to certain sequences of the complete 7S globulin, have the same effects on cultured hepatocytes as the complete 7S globulin. This led to speculation that there are bioactive small peptide fractions produced by the digestion of soy protein that are absorbed from the intestinal tract and have favorable effects on lipoprotein metabolism and cardiovascular health (7,8). However, we are unaware of evidence from studies of whole animals that supports this speculation.

For this reason, we studied the effects of ß-conglycinin and a peptide fraction (termed W008) similar to the HMF of Sugano et al. (1,2) on plasma lipoproteins and atherosclerosis in apoE null and LDL receptor null mice. The effects of glycinin (which is not believed to affect lipoprotein metabolism) and soy protein isolate produced from a cultivar of soy bean that does not produce ß-conglycinin were also studied.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Mice and diets. The mice used in these studies were bred and reared in our animal facilities which are fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Wake Forest University School of Medicine. The original breeding pair of LDL receptor -/-, human apoB transgenic mice (9) was provided by Dr. Helen Hobbs, Departments of Internal Medicine and Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX. This mouse is a hybrid cross between the LDL receptor -/- mouse (10) which is itself a hybrid of 129sv and C57BL/6 strains and the human apolipoprotein (apo)B transgenic mouse (11) a hybrid of SJL and C57BL/6B strains. ApoE -/- mice (12), backcrossed >99% to C57BL6/J were provided by Dr. Nobuyo Maeda, Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC.

At 6 wk of age, male and ovariectomized female mice of each genotype were assigned randomly to 10 diet groups. There were 10–16 mice of each sex and genotype in each diet group (total n = 416).

The diets are described in Table 1. The principal difference between the diets was the source of the protein component. Casein/lactalbumin was used in an isoflavone-free, soy protein-free control diet. The soy protein sources varied in their isoflavone content. Therefore, to control for the possibility that differences in isoflavone content might contribute to differences in the effects of protein source, the protein sources were fed to separate groups of animals 1) in the form they were produced and 2) with isoflavone concentrate added in amounts sufficient to equalize isoflavone intake across soy protein groups. The total isoflavone content of these diets was equivalent to the amount (0.4 mg/kJ) consumed by mice fed the diet with isoflavone-containing soy protein isolate. Isoflavone concentrate was produced by ethanol extraction of defatted soy flake. The ß-conglycinin and glycinin protein sources were made from defatted soy flake using the typical isolate process with the exception of precipitation pH. This soy protein isolate was then fractionated on the basis of the isoelectric points of the various protein types and the fractions were spray-dried. W008 was produced using the typical isolate process with an added hydrolysis step and the hydrolyzed precipitated fraction was spray-dried for incorporation into the experimental diets. The ß-conglycinin–free protein source was made using the typical isolate process with a cultivar of soy bean that produces no ß-conglycinin. Soy protein isolates and isoflavone concentrates were provided by the Solae Company. The isoflavone content of these products was determined by HPLC-MS (13) in the Nestle Purina Analytical Laboratory, St. Louis, MO.

    Atherosclerosis and plasma lipoproteins. Plasma lipoproteins were separated by HPLC (14), and aliquots of isolated lipoprotein fractions were used for enzymatic determination of cholesterol (15).

Analysis for aortic free and esterified cholesterol content was conducted as described previously (16). The aorta was placed on the platform of a dissecting microscope and the adventitia was carefully and completely dissected away from the intima/media and removed. The intima/media was then placed in 3 mL of chloroform:methanol (2:1, v:v) containing 5 -cholestane as an internal standard and the lipids were extracted. The lipid extract was separated by filtration and extracts were dried under N₂ at 60°C and then dissolved in hexane. Analysis of free and total cholesterol was done with two injections per sample on a DB 17 (0.53 mm i.d. x 15 m x 1 µm) GLC column at 250°C and installed in a Hewlett -Packard 5890 GC equipped with an HP 7673A automatic injector using on-column injection and a flame ionization detector. Cholesteryl ester was calculated as the difference between free and total cholesterol, as measured before and after saponification and reextraction of the nonsaponifiable sterol into hexane. The delipidated tissue protein was then digested and dissolved in 1 mol/L NaOH and total protein was determined (17).

    Data analysis. To reduce skewness and equalize group variances, all data sets underwent logarithmic transformation before analysis. Three-way (protein type x sex x isoflavone content), 2-way (protein type x sex) and 1-way (protein type within each sex) ANOVA were used for detecting effects of protein type, isoflavone content, and sex on atherosclerosis and plasma lipoproteins. Pairwise comparisons were made using Duncan’s New Multiple Range Test. Multiple linear regression was used to assess the relationship between effects of treatment on plasma lipoproteins and effects on atherosclerosis and to select covariates for analysis of covariance. Analyses were done using BMDP Statistical Software. Results are presented as mean ± SEM. Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Atherosclerosis. As we observed previously, the extent of atherosclerosis was 4–5 times greater in apoE -/- mice than LDL receptor -/-, apoB transgenic mice (Figs. 1234). For this reason, data were analyzed separately for the two mouse types. Three-way ANOVA revealed main effects of protein type (P < 0.001) and sex (P < 0.02) but not isoflavone content (P > 0.2) and a significant sex x protein interaction term (P < 0.001) in both mouse types. Because there was no effect of isoflavone content, groups of mice fed diets with and without added isoflavones were combined for further analysis. Patterns of response differed greatly between males and ovariectomized females. Among male mice, atherosclerosis was reduced only in mice fed ß-conglycinin (both mouse types) (all P <0.05) relative to those fed casein/lactalbumin (Figs. 1and 2). Among females, the extent of atherosclerosis was reduced in both mouse types in all soy protein groups (all P <0.05) relative to those fed casein/lactalbumin (Figs. 3 and 4). Relative to mice fed isoflavone-containing soy protein, the extent of atherosclerosis was reduced only in mice fed ß-conglycinin. Among males, the extent of atherosclerosis was reduced 66% (P < 0.05) (LDL receptor -/-) and 39% (P < 0.05) (apoE -/-) (Figs. 1and 2), whereas among ovariectomized females, the extent of atherosclerosis was reduced 67% (P < 0.05) in apoE -/- mice (Figs. 3and 4).

View larger version (14K): [in this window] [in a new window]   FIGURE 1 Aortic cholesteryl ester levels of male apoE -/- mice fed diets with different sources of protein. The molecular weight of the cholesteryl ester standard is 650. C/L, casein/lactalbumin; soy+, isoflavone-containing soy protein; W008, a precipitated peptide fraction of hydrolyzed soy protein. Values are means ± SEM, n = 11–24. Bars labeled with different letters differ (P < 0.05).

View larger version (12K): [in this window] [in a new window]   FIGURE 2 Aortic cholesteryl ester levels of male LDL receptor -/- mice fed diets with different sources of protein. The molecular weight of the cholesteryl ester standard is 650. C/L, casein/lactalbumin; soy+, isoflavone-containing soy protein; W008, a precipitated peptide fraction of hydrolyzed soy protein. Values are means ± SEM, n = 10–24. Bars labeled with different letters differ (P < 0.05).

View larger version (12K): [in this window] [in a new window]   FIGURE 3 Aortic cholesteryl ester levels of female apoE -/- mice fed diets with different sources of protein. The molecular weight of the cholesteryl ester standard is 650. C/L, casein/lactalbumin; soy+, isoflavone-containing soy protein; W008, a precipitated peptide fraction of hydrolyzed soy protein. Values are means ± SEM, n = 11–24. Bars labeled with different letters differ (P < 0.05).

View larger version (12K): [in this window] [in a new window]   FIGURE 4 Aortic cholesteryl ester levels of female LDL receptor -/- mice fed diets with different sources of protein. The molecular weight of the cholesteryl ester standard is 650. C/L, casein/lactalbumin; soy+, isoflavone-containing soy protein; W008, a precipitated peptide fraction of hydrolyzed soy protein. Values are means ± SEM, n = 10–23. Bars labeled with different letters differ (P < 0.05).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The principle finding was that ß-conglycinin, one of the major soy bean storage proteins, had a pronounced inhibitory effect on the development of atherosclerosis that greatly exceeded the effect of isoflavone-containing soy protein isolate. To our knowledge, the results represent the first direct in vivo evidence for atheroprotective effects of soy peptides or peptide fractions that were found previously to have hypolipoproteinemic effects in human beings (3) or experimental animals (1,2,6) and the ability to upregulate LDL receptor activity in vitro (4,5,7). Interestingly, however, neither of these mechanisms seemed to play a substantial role. Because the effects were of similar magnitude in mice with and without LDL receptors, it does not appear that there is a necessary role for LDL receptors. Furthermore, the effects were unrelated or only minimally related to plasma lipoprotein concentrations in both mouse types. As in previous studies (18–20), plasma total cholesterol and lipoprotein cholesterol concentrations were unaffected by dietary soy protein isolate in apoE -/- mice. In the present study, there was also no effect of dietary ß-conglycinin on total or lipoprotein cholesterol concentrations in these mice. Also consistent with a previous study (18), female, but not male LDL receptor -/- apoB transgenic mice had reduced total, LDL, and VLDL cholesterol concentrations in response to dietary soy protein isolate. In the present study, there was a similar response to ß-conglycinin. However, analysis of covariance indicated that the atheroprotective effects of soy protein isolate and ß-conglycinin were not explained by effects on total, VLDL, or LDL cholesterol concentration. Therefore, it seems that the atheroprotective effects of ß-conglycinin cannot be accounted for by effects on these atherosclerosis risk factors in either type of mouse.

Isoflavone concentrates were added in relatively small amounts (<1 mg/g diet) to some diets to equalize isoflavone intake across diet groups. These concentrates were produced by ethanol extraction of defatted soy flakes; therefore, they contained other ethanol-soluble substances that could influence the development of atherosclerosis. Perhaps most notable in this regard are the soy saponins. Some saponins have hypolipoproteinemic effects (21), thereby having the potential to influence the development of atherosclerosis. The saponin content of this concentrate was not determined. However, the saponin content of a similar concentrate used by Yamakoshi et al. (22) was 11%. In that study (22), there was no effect of the isoflavone concentrate or another saponin-rich concentrate on plasma lipoprotein cholesterol concentrations in rabbits. Furthermore, in the present study, there was no difference in the extent of atherosclerosis between mice fed the diets with isoflavone concentrate and those fed diets without added isoflavone concentrate. Also, the effects of ß-conglycinin on atherosclerosis were unexplained by plasma lipoprotein concentrations. Therefore, although saponins may influence lipoprotein metabolism and atherosclerosis under some circumstances, it seems unlikely that saponins had an influence on the outcome of this study.

How, then, might these atheroprotective effects have been mediated? Because large peptides like 7S globulin are hydrolyzed in the gastrointestinal tract and probably not absorbed intact in meaningful amounts, it seems unlikely that these effects are attributable to the intact peptide. However, it is possible that smaller peptide digestion products escape further digestion, are absorbed, and reach the circulation. Supporting this possibility are the findings of Masuda et al. (23) who administered sour milk containing small antihypertensive peptides to spontaneously hypertensive rats and found a reduction in angiotensin I-converting enzyme in association with a reduction in blood pressure. Furthermore, the intact peptides were detected in the aorta. Although there are no published data demonstrating the absorption and transport of soy protein digestion products to the liver or cardiovascular system, Lovati et al. (7) described the in vitro effects of small peptides produced by the digestion of a soy protein concentrate and a small synthetic peptide (MW 2271 Da) corresponding to specific sequences of 7S soy globulin. In these studies, both peptide products had significant effects on LDL metabolism in cultured Hep G2 cells. This indicates that, if such products do survive digestion and reach the circulation, they may have in vivo biological activity within the cardiovascular system. We can only speculate on the processes that might be involved. Biological effects that have been attributed to bioactive peptides include antioxidation, antiproliferation, and anti-inflammation [reviewed in (24)]. Because the initiation and progression of atherosclerosis are known to involve the oxidation of plasma lipoproteins in the arterial intima, cell proliferation, and a localized inflammatory reaction, these represent potential pathways by which soy peptides could directly inhibit atherosclerosis.

It was surprising to observe that although isoflavone-containing soy protein isolate had a potent atheroinhibitory effect in female mice, it had no effect in male mice. We speculate that this has to do with the isoflavone content of the isolate used. This isolate contained 0.4 mg/kJ of total isoflavones/Cal of diet, an amount 50% higher than we used in previous studies (18,19) in which atheroinhibitory effects were observed in both male and female mice. We hypothesize that there is a range of effective doses above and below which isoflavones are not atheroprotective. This is supported by the findings of Clarkson et al. (25) who found that among monkeys fed an isoflavone-rich diet, there was no inhibition of atherosclerosis in animals with the highest plasma isoflavone concentrations. We suspect that there was no atheroinhibition in male mice because they have plasma isoflavone concentrations 2–3 times those of females (18). This meant that plasma isoflavone concentrations were in an optimal range for inhibition of atherosclerosis in the females but were above that range in the males fed this high-isoflavone isolate.

Nonetheless, the atheroinhibitory effects of ß-conglycinin were pronounced in both males (a mean reduction of 53%) and females (a mean reduction of 56%) relative to mice fed isoflavone-containing soy protein isolate. Among female mice, the extent of atherosclerosis had a mean reduction of 85% relative to female mice fed casein/lactalbumin. The magnitude of the atheroprotective effect of isoflavone-containing ß-conglycinin in female mice substantially exceeds that achieved by the feeding of isoflavone-containing soy protein isolate in the current study and a previous study (18). These findings suggest that favorable cardiovascular effects of soy may be enhanced by the consumption of products rich in ß-conglycinin.

Although the pathways by which these effects are mediated remain unclear, we conclude that the consumption of diets rich in ß-conglycinin has an inhibitory effect on the development of atherosclerosis that greatly exceeds the effect of whole isoflavone-containing soy protein isolate and does not depend on LDL receptors or effects on plasma lipoproteins.

	FOOTNOTES

 
¹ Presented in abstract form at the International Symposium on the Role of Soy in Prevention and Treatment of Chronic Disease, Orlando, FL, September 21–24 [Adams, M. R., Golden, D. L., Franke, A. A., Potter, S. M., Smith, H. S. & Anthony, M. S. (2003) Dietary 7S globulin (ß-conglycinin) inhibits atherosclerosis in mice. (abs.)].

² Supported by grant RO1 HL 64746 from the National Institutes of Health.

⁴ Abbreviations used: apo, apolipoprotein; HMF, high-molecular-weight fraction; ILDL, intermediate-sized LDL.

Manuscript received 30 September 2003. Initial review completed 1 December 2003. Revision accepted 15 December 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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