Alcohol-washed Soy Protein Isolate Research Articles

Dietary soy isoflavones increased hepatic protein disulfide isomerase content and suppressed its enzymatic activity in rats

Protein disulfide isomerase (PDI) is a multifunctional protein and plays important roles in protein folding, triglyceride transfer, insulin degradation, and thyroid hormone transportation. This study examined the modulation of PDI expression by soy consumption using rat as a model. Sprague-Dawley male and female rats at 50 days (d) of age were fed diets containing either 20% casein or alcohol-washed soy protein isolate (SPI, containing 50 mg isoflavones (ISFs)/kg diet) or SPI plus ISF (250 mg/kg diet) and mated at age of 120 d. The offspring (F1) were fed the same diets as their parents. Addition of ISF to SPI diet markedly increased PDI protein content in the liver and testis of the adult rats compared with the casein or SPI diet. PDI mRNA abundance in the liver and protein content in the brain, thyroid, heart, and uterus were unchanged by the diets. Two-dimensional Western blot showed that the rats fed diets containing SPI had a diminished hepatic PDI protein with an isoelectric point (pI) of 6.12, a dephosphorylated form, compared with the rats fed diets containing either casein or SPI with supplemental ISF. Soy ISF added into SPI diet remarkably suppressed hepatic PDI activity of the rats compared with the casein diet. Moreover, soy ISF dose-dependently increased PDI and thyroid hormone receptor (TR) β protein content, whereas reduced TR DNA binding ability in human hepatocytes. Overall, this study shows that soy ISF increased hepatic PDI protein content, but addition of ISF into SPI diet inhibited its enzymatic activities and this effect may be mediated through a post-transcriptional mechanism.

Dietary supplementation with soy isoflavones or replacement with soy proteins prevents hepatic lipid droplet accumulation and alters expression of genes involved in lipid metabolism in rats

Accumulation of hepatic lipid droplet (HLD) is the hallmark pathology of non-alcoholic fatty liver disease (NAFLD). This study examined the effects of soy isoflavones (ISF) and different amounts of soy proteins on the accumulation of HLD, lipid metabolism and related gene expression in rats. Weanling Sprague-Dawley rats were fed diets containing either 20% casein protein without (D1) or with (D2) supplemental ISF (50mg/kg diet) or substitution of casein with increasing amounts of alcohol-washed soy protein isolate (SPI, 5, 10, and 20%; D3, D4, D5) for 90 days. Dietary casein (20%) induced accumulation of HLD in female, but not in male rats. Both soy proteins and ISF remarkably prevented the formation of HLD. Soy proteins lowered hepatic total cholesterol and triglyceride in a dose-dependent manner. Interestingly, soy proteins but not ISF significantly increased free fatty acids in the liver of the female rats compared to D1. Proteomic analysis showed that at least 3 enzymes involved in lipogenesis were down-regulated and 7 proteins related to fatty acid β-oxidation or lipolysis were up-regulated by soy protein over D1. Additionally, 9 differentially expressed proteins identified were related to amino acid metabolism, 5 to glycolysis and 2 to cholesterol metabolism. Dietary ISF and SPI markedly reduced hepatic-peroxisome-proliferator-activated receptor γ2 (PPARγ2) and fat-specific protein 27 (FSP27) in female rats. Overall, this study has shown that partial or full replacement of dietary casein by soy protein or supplementation with soy ISF can effectively prevent the accumulation of HLD. The potential molecular mechanism(s) involved might be due to suppression of lipogenesis and stimulation of lipolysis and down-regulation of PPARγ2 and FSP27. This suggests that consumption of soy foods or supplements might be a useful strategy for the prevention or treatment of fatty liver diseases.

The α′ subunit of β-conglycinin and the A1–5 subunits of glycinin are not essential for many hypolipidemic actions of dietary soy proteins in rats

This study examined the effects of dietary soy protein (SP) lacking different storage protein subunits and isoflavones (ISF) on the abdominal fat, blood lipids, thyroid hormones, and enzymatic activities in rats. Weanling Sprague-Dawley rats (8 males and 8 females/group) were fed diets containing either 20 % casein without or with supplemental isoflavones or alcohol-washed SP isolate or SP concentrates (SPC) prepared from 6 different soy bean lines for 8 weeks. Feeding of diets containing SPC regardless of their subunit compositions significantly lowered relative liver weights, blood total, free, and LDL cholesterol in both genders (P < 0.05) and also reduced serum free fatty acids (FFA) and abdominal fat in females (P < 0.05) compared to the casein or casein + ISF diets. Dietary SPC significantly elevated the plasma free triiodothyronine (T3) in both genders and total T3 in females compared to the casein diet (P < 0.05). The SPC lacking β-conglycinin α' and either the glycinin A1-3 or A1-5 subunits increased total T3 in males and reduced plasma enzymatic activities of creatine kinase and lactate dehydrogenase compared to casein or casein + ISF diet (P < 0.05). Soy isoflavones were mainly responsible for the hypocholesterolemic effects and increased plasma free T3, whereas reduction in FFA, abdominal fat, liver weight and increased plasma total T3 were the effects of the soy proteins. Neither the α' subunit of β-conglycinin nor the A1-5 subunits of glycinin are essential for the hypolipidemic properties of soy proteins.

Distribution of Isoflavones in Samples of Serum, Liver and Mammary Glands of Rats or Pigs Fed Dietary Isoflavones

Background/Aims: There has been great interest in the potential beneficial and adverse health effects of dietary isoflavones. Determination of tissue concentrations of isoflavone metabolites provides an insight into the potential bioactivity of dietary isoflavones. However, data on the distribution of isoflavones in animal models fed dietary isoflavones are limited. In this study, additional data on the distribution of isoflavones in serum and/or tissues of rats and pigs fed dietary isoflavones were generated. Methods: Rats (male and female) were fed a casein control diet (containing no isoflavones) and an isoflavone-supplemented diet (containing an alcohol-washed soy protein isolate plus NOVASOY, providing a total of 1,047 mg/kg of total isoflavones). Female pigs were fed a control diet (without soy) containing 17.5 mg/kg of isoflavones, a soy diet containing 582.8 mg/kg of isoflavones or a soy diet supplemented with a daily dose of 2.3 g (equivalent to 42.0 and 14.5 mg/kg of body weight at the onset and end of treatment, respectively) of crystalline genistein. The concentrations of isoflavones in serum and tissues (liver and mammary gland) and in tissues (liver and mammary gland) of pigs were determined via a sensitive and rapid method using liquid chromatography/mass spectrometry. Results: Rats fed the control diet containing no isoflavones had nondetectable levels of isoflavone metabolites in serum, liver and mammary gland samples. Rats fed the isoflavone-supplemented diet had the greatest levels of equol, followed by genistein, daidzein and glycitein, respectively, in their serum, livers and mammary glands. The concentrations of total isoflavones (daidzein, equol and genistein plus glycitein) in serum were significantly (p < 0.05) greater in male rats vs. female rats, but the reverse was true in the case of livers. Concentrations of daidzein, equol, genistein and glycitein were lowest (p < 0.05) in the livers of pigs fed the control diet, and in the mammary glands of female pigs there was only an effect of feeding soy plus genistein on the concentrations of daidzein and equol (p <0.05). Conclusions: The current data therefore show gender as well as species differences in the tissue distribution of isoflavones.

Soy Protein Isolate Increases Urinary Estrogens and the Ratio of 2:16α-Hydroxyestrone in Men at High Risk of Prostate Cancer ,

Specific estrogen metabolites may initiate and promote hormone-related cancers. In epidemiological studies, significantly lower excretion of urinary estradiol (E2) and lower ratio of urinary 2-hydroxy estrogens to 16α-hydroxyestrone (2:16 OH-E1) have been reported in prostate cancer cases compared to controls. Although soy supplementation has been shown to increase the ratio 2:16 OH-E1 in women, no studies to our knowledge have investigated the effects of soy supplementation on estrogen metabolism in men. The objective of this randomized controlled trial was to determine the effects of soy protein isolate consumption on estrogen metabolism in men at high risk for developing advanced prostate cancer. Fifty-eight men supplemented their habitual diets with 1 of 3 protein isolates: 1) isoflavone-rich soy protein isolate (SPI+) (107 mg isoflavones/d); 2) alcohol-washed soy protein isolate (SPI−) (<6 mg isoflavones/d); or 3) milk protein isolate (MPI), each providing 40 g protein/d. At 0, 3, and 6 mo of supplementation, the urinary estrogen metabolite profile was measured by GC-MS. Both soy groups had higher E2 excretion than the MPI group at 3 and 6 mo. After 6 mo of supplementation, the SPI+ group had a significantly higher urinary 2:16 OH-E1 ratio than the MPI group. Increased urinary E2 excretion and 2:16 OH-E1 ratio in men consuming soy protein isolate are consistent with studies in postmenopausal women and suggest that soy consumption may be beneficial in men at high risk of progressing to advanced prostate cancer as a result of effects on endogenous estrogen metabolism.

Consumption of Soy Protein Isolate Modulates the Phosphorylation Status of Hepatic ATPase/ATP Synthase β Protein and Increases ATPase Activity in Rats

ATPase/ATP synthase plays important roles in the regulation of carbohydrate, protein, and lipid metabolism through modulating energy homeostasis. The purpose of this study was to examine the effects of feeding soy proteins and isoflavones (ISF) on the enzymatic activity and protein modification of hepatic mitochondrial ATPase/ATP synthase. In Expt. 1, Sprague-Dawley rats aged 50 d were fed diets containing either 20% casein or 20% alcohol-washed soy protein isolate (SPI) with or without supplemental ISF (770.7 μmol/kg diet) for 70 d. In Expt. 2, weanling Sprague-Dawley rats were fed diets containing 20% casein with or without added ISF (154.1 μmol/kg diet) or 20% SPI for 90 d. Hepatic mitochondrial ATPase activity was significantly higher in the rats fed SPI than in those fed casein. Addition of ISF to SPI eliminated the action of SPI. ATPase/ATP synthase β protein contents in the liver were unchanged; however, its patterns measured by 2-dimensional Western blot were different among dietary groups. The rats fed SPI or SPI plus ISF had 3 more major protein spots with the same molecular weights (80 kDa and 55 kDa) as those presented in the rats fed casein but with different isoelectric points. Pretreatment of hepatic mitochondrial proteins from the rats fed casein with alkaline phosphatase produced the same ATPase/ATP synthase β patterns as observed in the SPI-fed rats and significantly elevated the ATPase activity. These results suggest that consumption of soy proteins increases hepatic ATPase activity, which might be a consequence of increased dephosphorylation or decreased phosphorylation of the mitochondrial ATPase/ATP synthase β protein.

Isoflavone-Rich Soy Protein Isolate Suppresses Androgen Receptor Expression without Altering Estrogen Receptor-β Expression or Serum Hormonal Profiles in Men at High Risk of Prostate Cancer

The purpose of this study was to determine the effects of soy protein isolate consumption on circulating hormone profiles and hormone receptor expression patterns in men at high risk for developing advanced prostate cancer. Fifty-eight men were randomly assigned to consume 1 of 3 protein isolates containing 40 g/d protein: 1) soy protein isolate (SPI+) (107 mg/d isoflavones); 2) alcohol-washed soy protein isolate (SPI−) (<6 mg/d isoflavones); or 3) milk protein isolate (0 mg/d isoflavones). For 6 mo, the men consumed the protein isolates in divided doses twice daily as a partial meal replacement. Serum samples collected at 0, 3, and 6 mo were analyzed for circulating estradiol, estrone, sex hormone-binding globulin, androstenedione, androstanediol glucuronide, dehydroepiandrosterone sulfate, dihydrotestosterone, testosterone, and free testosterone concentrations by RIA. Prostate biopsy samples obtained pre- and postintervention were analyzed for androgen receptor (AR) and estrogen receptor-β expression by immunohistochemistry. At 6 mo, consumption of SPI+ significantly suppressed AR expression but did not alter estrogen receptor-β expression or circulating hormones. Consumption of SPI− significantly increased estradiol and androstenedione concentrations, and tended to suppress AR expression (P = 0.09). Although the effects of SPI− consumption on estradiol and androstenedione are difficult to interpret and the clinical relevance is uncertain, these data show that AR expression in the prostate is suppressed by soy protein isolate consumption, which may be beneficial in preventing prostate cancer.

Dietary soy protein isolate modifies hepatic retinoic acid receptor-beta proteins and inhibits their DNA binding activity in rats.

Retinoic acid receptors (RAR) belong to the same nuclear receptor superfamily as thyroid hormone receptors (TR) that were previously shown to be modulated by dietary soy protein isolate (SPI). This study has examined the effect of dietary SPI and isoflavones (ISF) on hepatic RAR gene expression and DNA binding activity. In Expt. 1, Sprague-Dawley rats were fed diets containing 20% casein or 20% alcohol-washed SPI in the absence or presence of increasing amounts of ISF (5-1250 mg/kg diet) for 70, 190, or 310 d. In Expt. 2, weanling Sprague-Dawley rats were fed diets containing 20% casein with or without supplemental ISF (50 mg/kg diet) or increasing amounts of alcohol-washed SPI (5, 10, and 20%) for 90 d. Intake of soy proteins significantly elevated hepatic RARbeta2 protein content dose-dependently compared with a casein diet, whereas supplemental ISF had no consistent effect. Neither RARbeta protein in the other tissues measured nor the other RAR (RARalpha and RARgamma) in the liver were affected by dietary SPI, indicating a tissue and isoform-specific effect of SPI. RARbeta2 mRNA abundances were not different between dietary groups except that its expression was markedly suppressed in male rats fed SPI for 310 d. DNA binding activity of nuclear RARbeta was significantly attenuated and the isoelectric points of RARbeta2 were shifted by dietary SPI. Overall, these results show for the first time, to our knowledge, that dietary soy proteins affect hepatic RARbeta2 protein content and RARbeta DNA binding activity, which may contribute to the suppression of retinoid-induced hypertriglyceridemia by SPI as reported.

Soy Protein Isolate Increases Hepatic Thyroid Hormone Receptor Content and Inhibits Its Binding to Target Genes in Rats1-3

Our previous studies showed that intake of 20% alcohol-washed soy protein isolate (SPI) significantly increased hepatic thyroid hormone receptor (TR) beta1 protein content in rats. However, whether SPI influences the binding ability of TR to its target genes is unknown. The purpose of this study was to examine the effect of increasing amounts of dietary SPI on hepatic TRbeta1 content and the binding of TR to thyroid hormone response element (TRE) in rats. Sprague-Dawley rats (28 d old) were fed diets containing casein (20%) with or without isoflavone supplementation (50 mg/kg diet) or alcohol-washed SPI (5, 10, or 20%) for 90 d. The hepatic TRbeta1 protein content was measured by Western blot, and the binding ability of TR to DNA was examined by electrophoretic mobility shift assay. Consumption of the 20% SPI diet increased pancreatic relative weight and decreased spleen relative weight. Intake of SPI markedly elevated TRbeta1 content in both male and female rats compared with a casein-based control diet. The increase in TRbeta1 in females was much higher than that in males. Interestingly, the binding abilities of TR to DNA were significantly inhibited by increasing amounts of dietary SPI in female rats. In conclusion, this study shows for the first time that dietary SPI increases hepatic TRbeta1 protein content and inhibits the binding of TR to target genes. Modulation of hepatic TRbeta1, a key regulator of gene expression involved in lipid metabolism, by SPI may be a novel mechanism by which soy components lower blood lipid level and exert their hypocholesterolemic actions.

Dietary Soy Protein Isolate and Isoflavones Modulate Hepatic Thyroid Hormone Receptors in Rats

Thyroid hormone receptors (TRs) are regulators of many genes involved in cholesterol and lipid metabolism. The purpose of this study was to examine the effect of soy protein isolate (SPI) and isoflavones on hepatic TRs in rats. In Expt. 1, Sprague-Dawley rats were fed diets containing either casein or alcohol-washed SPI with or without isoflavone supplementation (5-1250 mg/kg diet) for 70, 190, and 310 d. The offspring (F1) were fed the same diets as their parents (F0). In Expt. 2, Sprague-Dawley rats were fed diets containing casein or casein plus isoflavones (50-400 mg/kg diet) for 120 d. The mRNA and protein contents of the hepatic TRs were measured by semiquantitative RT-PCR and Western blot, respectively. TRalpha1, TRalpha2, and TRbeta2 contents were not affected by SPI. However, the content of the 52-kDa TRbeta1 protein, the major isoform present in the liver, was markedly increased by dietary SPI in both sexes of F0 and F1 compared with casein. The supplemental isoflavones had no effect on TRbeta1, whereas the high doses of isoflavones (250 and 1250 mg/kg diet) reduced the hepatic TRalpha1 protein content in F1 male rats on d 28. SPI had no effect on total T3 and T4 levels. However, higher dose of supplemental isoflavones markedly increased T4 level in female rats. Overall, this study demonstrates for the first time that SPI upregulates hepatic TRbeta1 expression, and that isoflavones reduce the hepatic TRalpha1 level in young male rats. The SPI-induced TRbeta1 may play a role in mediating the hypocholesterolemic and lipid-lowering actions of soy protein.

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.