Endogenous and exogenous equol are antiestrogenic in reproductive tissues of apolipoprotein e-null mice.

Equol is an isoflavone (IF) metabolite produced by intestinal microbiota in a subset of people consuming dietary soy. Equol producers may show different responses to soy foods and phenotypes related to cancer risk. Here, we assessed the effects of soy IF, endogenous microbial equol production, and dietary racemic equol in a 3 × 2 × 2 factorial experiment using gnotobiotic apoE-null mice (n = 9-11/group/sex). At age 3-6 wk, equol-producing microbiota were introduced to one-half of the colony (n = 122). At age 6 wk, mice were randomized to receive a diet that contained 1 of 3 protein sources: casein and lactalbumin, alcohol-washed soy protein (low IF), and intact soy protein (high IF), with total IF amounts of 0, 42, and 566 mg/kg diet, respectively. One-half of each diet group also received racemic equol (291 mg/kg diet). After 16 wk of dietary treatment, serum isoflavonoid profiles varied with sex, soy IF amount, and intestinal microbiota status. There were no treatment effects on tissues of male mice. In females, reproductive tissue phenotypes differed by equol-producing ability (i.e., microbiota status) but not dietary equol or IF content. Equol producers had lower uterine weight, vaginal epithelial thickness, total uterine area, endometrial area, and endometrial luminal epithelial height compared with nonproducers (P < 0.05 for all), with an association between microbiota status and estrous cycle (P > chi-square = 0.03). Exogenous equol reduced expression of progesterone receptor (PGR) and the proliferation marker Ki67 (P < 0.0001) in vaginal epithelium and endometrium; for endogenous equol, only PGR was reduced (P < 0.0005). Our findings indicate that equol diminishes estrogen-dependent tissue responses in apoE-null mice.

The atheroprotective effect of dietary soy isoflavones in apolipoprotein E-/- mice requires the presence of estrogen receptor-alpha.

OBJECTIVE: Although the mechanisms by which dietary soy inhibits atherosclerosis are unclear, one line of evidence implicates an important role for its phytoestrogenic isoflavones. We sought to determine whether soy isoflavones exert atheroprotective effects through estrogen receptor-dependent processes and, if so, which estrogen receptor subtype (ie, alpha or beta) is involved. METHODS AND RESULTS: We compared the effects of diets rich in soy protein that were either isoflavone depleted (0.04 mg/g protein isolate) or isoflavone-replete, or Soy(+IF) (1.72 mg/g protein isolate) in apolipoprotein E-deficient (ee) mice that had been crossed with estrogen receptor-alpha- and -beta-deficient mice to produce double-knockout alphaalphaee and betabetaee mice and (estrogen receptor) wild-type controls (AAee and BBee). Both male and ovariectomized female mice were studied (n=10 to 17 per treatment group; total n=201). After 16 weeks, atherosclerosis was assessed by quantifying the aortic content of esterified cholesterol. Atherosclerosis was reduced 20% to 27% (P<0.05) by Soy(+IF) in betabetaee, BBee, and AAee mice but was unaffected in alphaalphaee mice. The inhibitory effect of Soy(+IF) was unrelated to sex, total plasma cholesterol, VLDL, LDL, and HDL cholesterol. CONCLUSIONS: The results indicate a necessary role for estrogen receptor-alpha-dependent processes in mediating the atheroprotective effects of dietary soy isoflavones.

A diet rich in green and yellow vegetables inhibits atherosclerosis in mice.

Although dietary patterns characterized by a high intake of fruits and vegetables are associated with reduced risk of coronary heart disease, the mechanisms involved are uncertain. We determined the effects of a diet rich in green and yellow vegetables on the development of atherosclerosis, the underlying cause of coronary heart disease, in a mouse model of atherosclerosis, the LDL receptor -/-, apolipoprotein B transgenic mouse. The mice were randomized into 2 diet groups: 1) a vegetable-free control diet (n = 53) and 2) the same diet with 30% (w:w) replaced by an equal-parts mixture of freeze-dried peas, green beans, broccoli, corn, and carrots (n = 54). Mice were fed these diets for 16 wk. Aortic atherosclerosis, as estimated by cholesteryl ester content, was reduced 38% (P < 0.001) in mice fed the vegetable-rich diet. Plasma total cholesterol (-12%), VLDL + ILDL cholesterol (-32%), serum amyloid A (-37%), and body weight (-7%) (all P < 0.01) were also lower in these mice at the end of the treatment period. In a regression model, antiatherogenic effects of the vegetable diet remained largely unexplained by the variation in plasma lipoproteins or body weight. Although the pathway(s) involved remain uncertain, the results indicate that a diet rich in green and yellow vegetables inhibits the development of atherosclerosis and may therefore lead to a reduction in the risk of coronary heart disease.

Soy protein containing isoflavones reduces the size of atherosclerotic plaques without affecting coronary artery reactivity in adult male monkeys.

The cardiovascular effects of dietary soy on men or adult male experimental animals have received little attention. We determined the effects of long-term (31 mo) consumption of a commercially available soy protein concentrate containing experimentally varied concentrations of isoflavones on the development of atherosclerosis and vascular reactivity in adult male monkeys. The monkeys were fed atherogenic diets that differed only in the source of protein: Control (n = 30), casein and lactalbumin; low-isoflavone soy (n = 30), a mixture of unmodified soy protein isolate and isoflavone-depleted soy protein isolate containing 0.94 mg of isoflavones/g protein; and high-isoflavone soy (n = 31), unmodified soy protein isolate containing 1.88 mg of isoflavone/g protein. Plasma LDL cholesterol was reduced, whereas HDL cholesterol and apolipoprotein A-1 (P < 0.05) were increased in both groups that consumed soy protein. Atherosclerosis (mean plaque size in the coronary arteries) was reduced by approximately 34% (P < 0.05) in both groups fed soy protein. There were no effects of dietary soy on endothelium-dependent or -independent reactivity of coronary arteries. The results indicate that long-term consumption of soy protein containing a modest amount of isoflavones inhibits the early progression of coronary artery atherosclerosis without affecting endothelium-dependent or -independent arterial function.

Effects of dietary isoflavone aglycones on the reproductive tract of male and female mice.

We assessed the effects of dietary consumption of soy isoflavone aglycones on the reproductive tract of sexually mature male and female mice. Isoflavone concentrates with a ratio of 10:1, 2:1 or 1:10 genistein:daidzein (G:D) were added to provide 120 mg total isoflavones/1800 Calories (approximately 40 mg/kg body weight) to diets having either casein/lactalbumin or soy protein isolate as the source of protein. After 16 weeks, mice were necropsied and gross and histopathologic assessments of uterus, vagina, testes and accessory sex glands were completed. Effects of the 10G:1D isoflavone concentrates were absent or minimal in females but in males included atrophy of accessory sex glands. In contrast, the 2G:1D and 1G:10D concentrates caused dramatic estrogenic effects in both male and female mice. Effects in females included endometritis and effects typical of estrogenic stimulation (i.e., uterine enlargement, keratinization of vaginal epithelium, increased height of endometrial surface epithelial cells, and uterine squamous metaplasia). Effects in males included reduced plasma testosterone concentrations, atrophy of seminiferous epithelium, atrophy of accessory sex glands, and squamous metaplasia of seminal vesicles. Some effects varied with protein source. We conclude that a diet containing approximately 40 mg/kg soy isoflavone aglycones with a genistein:daidzein ratio of 2:1 or less has marked estrogenic effects on the reproductive system of male and female mice.

Dietary soy beta-conglycinin (7S globulin) inhibits atherosclerosis in mice.

Although beta-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 beta-conglycinin on the cardiovascular system are unknown. We assessed the effects of dietary beta-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) beta-conglycinin, 4) glycinin (11S globulin, another major soy storage protein), 5) beta-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 beta-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 beta-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.

The inhibitory effect of soy protein isolate on atherosclerosis in mice does not require the presence of LDL receptors or alteration of plasma lipoproteins.

The mechanisms by which dietary soy favorably influences lipoprotein metabolism and inhibits atherosclerosis are uncertain. Studies of blood mononuclear cells and cultured hepatocytes have indicated that certain soy peptides (i.e., 7S globulins) stimulate expression of LDL receptors. This pathway represents a hypothetical mechanism by which soy's hypocholesterolemic and antiatherosclerotic effects may be mediated. However, direct evidence supporting this hypothesis is lacking. To address this, we compared effects of dietary soy protein isolate in two genetically engineered mouse models of atherosclerosis. One mouse [LDL receptor -/- + apolipoprotein (apo) B transgenic] is devoid of LDL receptors and overproduces apolipoprotein B, whereas the other (apoE -/-) has a normal complement of LDL receptors but does not produce apolipoprotein E. Male (n = 10-12/group) and ovariectomized female (n = 10-12/group) mice were studied. There were three treatment groups, which differed principally by the source of the protein component of the diet: 1) casein/lactalbumin (no isoflavones), 2) alcohol-washed soy protein isolate (total isoflavones = 0.04 mg/g), and 3) intact soy protein isolate (total isoflavones = 1.72 mg/g). Atherosclerosis was assessed by quantifying the aortic content of esterified cholesterol. Atherosclerosis was inhibited (relative to the casein/lactalbumin group) by both alcohol-washed (45 and 31%) (P < 0.05) and intact (65 and 41%) (P < 0.05) soy protein isolate in LDL receptor -/- and apoE -/- mice, respectively. There was no sex difference. In a two-way analysis, there were significant effects of type of soy isolate and type of mouse. The antiatherosclerosis effect was enhanced in LDL receptor -/- mice (P < 0.001) and diminished in mice fed alcohol-washed soy protein isolate (P < 0.001). Furthermore, inhibitory effects of soy on atherosclerosis were unrelated to plasma LDL, VLDL or HDL cholesterol concentrations. The results represent direct evidence for the existence of LDL receptor- and plasma lipoprotein-independent pathways by which dietary soy protein isolate inhibits atherosclerosis.