Consumption of Soya Isoflavones Improved Polycystic Ovary Syndrome (PCOS)-Associated Metabolic Disorders in a Rat Model.

Polycystic ovary syndrome (PCOS) is associated with increased risks for certain metabolic disorders such as insulin resistance, non-alcoholic fatty liver disease (NAFLD), and suppressed ovarian follicular development. This study aimed to examine whether soya isoflavones (ISF) mitigate these PCOS-associated metabolic disorders in a rat model. Weanling Sprague-Dawley female rats were randomly divided into 6 groups and were treated with either 0 or 83 µg/day dihydrotestosterone (DHT) to induce PCOS and fed diets containing 0, 0.5, or 1g ISF/kg diet for 8 weeks. DHT treatment increased food intake, body weight gain (BWG, p<0.001), percentage of primordial follicles (60% vs 50.9%, p<0.05), and accumulation of lipid droplets in the livers. It also elevated serum total cholesterol (TC), free cholesterol (FC), triglycerides, non-esterified fatty acids (NEFA), and leptin, and hepatic TC and NEFA. Additionally, DHT treatment reduced the percentage of primary follicles (13.8% vs 30.2%, p<0.05), ovary weight, and length (p<0.001), as well as insulin sensitivity (p<0.01) compared to the Control. ISF intake at 1g/kg reduced BWG, serum TC, FC, NEFA, leptin, and hepatic triglycerides and DHT-induced insulin resistance (p<0.01). ISF intake at both levels decreased DHT-induced lipid droplet accumulation in the livers, and changes in the percentages of primordial and primary follicles. Dietary soya ISF alleviated DHT-induced BWG, insulin resistance, and hepatic lipid droplet accumulation, as well as suppressed ovarian follicular development. This suggests that consumption of soya foods or ISF supplements may be beneficial for the individuals with PCOS, mitigating the associated metabolic disorders such as diabetes and NAFLD.

L-Lysine supplementation affects dietary protein quality and growth and serum amino acid concentrations in rats.

Single amino acid (AA) supplementations in foods are increasing, however their potential nutritional and physiological impacts are not fully understood. This study examined the effects of L-lysine (Lys) supplementation on protein quality of diets, serum AA concentrations and associations between the ratio of supplemental Lys to dietary protein (X) with body weight gain (BWG) in Sprague-Dawley male rats. Rats were fed one of 10 diets containing either 7% or 20% casein and supplemented with 0% (Control), 1.5%, 3%, 6% Lys or 6% Lys + 3% L-arginine (Arg) (8 rats/diet group) for 1 week. Lys supplementation reduced the protein quality of the casein-based diets (p < 0.01). BWG was reduced by supplemental Lys when X > 0.18. Free Lys supplementation dose-dependently increased serum Lys levels (p < 0.01), while increased protein-bound Lys (1.4% vs 0.52%) had little effect on serum Lys (p > 0.05). In the 7% casein diets, ≥ 1.5% supplemental Lys reduced serum alanine, asparagine, glycine, isoleucine, leucine, serine, tyrosine, valine, carnitine, ornithine, and increased urea. Supplementation of ≥ 3% Lys additionally reduced tryptophan and increased histidine, methionine and α-aminoadipic acid (α-AAA) compared to the Control (p < 0.05). In the 20% casein diets, addition of ≥ 1.5% Lys reduced serum asparagine and threonine, and ≥ 3% Lys reduced leucine, proline, tryptophan, valine, and ornithine, and 6% Lys reduced carnitine, and increased histidine, methionine, and α-AAA. Overall, this study showed that free Lys supplementation in a Lys-sufficient diet reduced the protein quality of the diets and modified the serum concentrations of many amino acids. Excess free Lys intake adversely affected growth and utilization of nutrients due to AA imbalance or antagonism. Overall lower protein intake increases susceptibility to the adverse effects of Lys supplementation.

Hypolipidemic Effects of Soy Protein and Isoflavones in the Prevention of Non-Alcoholic Fatty Liver Disease- A Review.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and affects about 25% of the population globally. Obesity and diabetes are the main causes of the disease characterized by excessive accumulation of lipids in the liver. There is currently no direct pharmacological treatments for NAFLD. Dietary intervention and lifestyle modification are the key strategies in the prevention and treatment of the disease. Soy consumption is associated with many health benefits such as decreased incidence of coronary heart disease, type-2 diabetes, atherosclerosis and obesity. The hypolipidemic functions of soy components have been shown in both animal studies and human clinical trials. Dietary soy proteins and associated isoflavones suppressed the formation and accumulation of lipid droplets in the liver and improved NAFLD-associated metabolic syndrome. The molecular mechanism(s) underlying the effects of soy components are mainly through modulation of transcription factors, sterol regulatory element-binding protein-1 and peroxisome proliferator-activated receptor-γ2, and expressions of their target genes involved in lipogenesis and lipolysis as well as lipid droplet-promoting protein, fat-specific protein-27. Inclusion of appropriate amounts of soy protein and isoflavones in the diets might be a useful approach to decrease the prevalence of NAFLD and mitigate disease burden.

Effects of dietary active soybean trypsin inhibitors on pancreatic weights, histology and expression of STAT3 and receptors for androgen and estrogen in different tissues of rats.

Our previous study showed that soy milks could contain high levels of active soybean trypsin inhibitors (SBTI) if they were not properly processed. This study investigated the effects of consuming active SBTI on pancreatic weights, histology, trypsinogen production and expression of STAT3, receptors for androgen (AR) and estrogen (ER) in pancreas, liver and uterus of rats. Weanling Sprague-Dawley rats were randomly divided into 3 groups (8 females and 8 males/group) and fed diets containing either 20% casein protein (Casein) or 20% soy protein (SP) in the presence of high (1.42 BAEE unit/µg, SP + SBTI) or low (0.2 BAEE unit/µg, SP-SBTI) levels of active SBTI for 8 weeks. Ingestion of SP + SBTI diet markedly increased pancreatic weights and trypsinogen content (p < 0.01), and caused acinar cell hypertrophy, and reduced pancreatic STAT3, p-STAT3, AR and ERß content, and increased uterine ERα and ERß compared to the Casein or SP-SBTI diets (p < 0.05). The two SP-containing diets lowered hepatic STAT3, p-STAT3, and pancreatic ERα, and increased hepatic ERα and ERß content in the female rats compared to the Casein diet (p < 0.05). This study demonstrated for the first time that consumption of high level of active SBTI not only increased pancreatic weights and acinar cell secretions, but also attenuated the expression of pancreatic STAT3, p-STAT3, AR, and ERß proteins in both sexes and increased uterine ERα and ERß content, and that dietary soy protein affected hepatic STAT3, p-STAT3, ERα and ERß in a gender-dependent manner.

Dietary supplementation with L-lysine affects body weight and blood hematological and biochemical parameters in rats.

L-Lysine (Lys) is a popular additive in foods, but the physiological effects of excess Lys supplementation are poorly understood and upper limits of safe intake have not been established. The objectives of this study were to examine the effects of dietary supplementation with increasing amounts of Lys on body weight (BW), food intake, and various blood hematological and biochemical parameters in rats. Male Sprague-Dawley rats at 10 weeks of age were assigned to ten diet groups (eight rats/group) and fed diets containing either 7% or 20% casein and supplemented with either 0% (Control), 1.5%, 3%, 6% Lys, or 6% Lys + 3% arginine for 1 week. Rats fed 7% casein with ≥ 1.5% Lys supplementation had lower serum albumin and leptin and higher LDL cholesterol (LDLC), ratios of total cholesterol (TC):HDL cholesterol (HDLC) and LDLC:HDLC than those fed 7% casein Control diet (P < 0.05). Rats fed 7% casein diet supplemented with 3% Lys diet had lower BW gain, food intake, serum alkaline phosphatase activity, and increased mean corpuscular hemoglobin concentration, blood urea nitrogen and serum pancreatic polypeptide compared to rats fed the Control diet (P < 0.05). Addition of 6% Lys in 7% casein caused significant BW loss (P < 0.001) and altered additional parameters. Addition of 6% Lys in a 20% casein diet reduced BW gain and food intake and altered numerous parameters. Arg supplementation normalized many of the endpoints changed by Lys. Collectively, these results show that Lys supplementation affects BW, food intake and a number of hematological and biochemical parameters. These effects of Lys supplementation were confined primarily in diets with lower levels of dietary protein. In the context of a low protein diet (7% casein), levels of Lys supplementation ≥ 1.5% may exert adverse health effects in rats.

Soybean Bioactive Peptides and Their Functional Properties.

Soy consumption has been associated with many potential health benefits in reducing chronic diseases such as obesity, cardiovascular disease, insulin-resistance/type II diabetes, certain type of cancers, and immune disorders. These physiological functions have been attributed to soy proteins either as intact soy protein or more commonly as functional or bioactive peptides derived from soybean processing. These findings have led to the approval of a health claim in the USA regarding the ability of soy proteins in reducing the risk for coronary heart disease and the acceptance of a health claim in Canada that soy protein can help lower cholesterol levels. Using different approaches, many soy bioactive peptides that have a variety of physiological functions such as hypolipidemic, anti-hypertensive, and anti-cancer properties, and anti-inflammatory, antioxidant, and immunomodulatory effects have been identified. Some soy peptides like lunasin and soymorphins possess more than one of these properties and play a role in the prevention of multiple chronic diseases. Overall, progress has been made in understanding the functional and bioactive components of soy. However, more studies are required to further identify their target organs, and elucidate their biological mechanisms of action in order to be potentially used as functional foods or even therapeutics for the prevention or treatment of chronic diseases.

Dietary L-lysine supplementation altered the content of pancreatic polypeptide, enzymes involved in glutamine metabolism, and ß-actin in rats.

This study investigated the effects of Lys supplementation on serum pancreatic polypeptide (PP), glutamine (Gln) levels and the expression of PP, Gln synthetase (GlnS), glutaminase (Gls) and ß-actin in different tissues such as pancreas, skeletal muscle, liver and kidney in rats. Male Sprague-Dawley rats were fed diets containing 7% casein supplemented with either 0% (Control), 1%, 1.5%, 3% Lys or 3% Lys with 1.5% Arg for a week. All rats were necropsied for collection of blood and tissues. Expression of PP, GlnS, Gls, and ß-actin in tissues were determined using Western blotting. The results showed that the rats fed 3% supplemental Lys had significantly lower body weight gain (BWG) and food intake than the ≤ 1.5% Lys groups (P < 0.05). Supplementation with ≥ 1% Lys increased serum PP level (P < 0.05), but had no significant effect on pancreatic PP abundance (P > 0.05). GlnS expression was significantly lowered in skeletal muscle by ≥ 1.5% supplemental Lys compared to the Control (P < 0.05). The expression of Gls in the kidney was increased by the addition of 1.5% Arg to 3% Lys diet (P < 0.05). Liver ß-actin significantly increased with both Lys and Arg supplementation and muscle ß-actin significantly decreased (P < 0.05) with ≥ 1.5% supplemental Lys. Kidney ß-actin significantly increased with Arg supplementation vs 3% Lys alone (P < 0.05). These results showed that dietary supplementation with ≥ 1.5% Lys significantly suppressed GlnS expression in the skeletal muscle, which may contribute to the decreased serum Gln levels, and that increased serum PP by Lys may be due to suppressed catabolism rather than increased synthesis of PP. Lys-induced PP may play a role in reducing food intake and BWG.

The α' subunit of ß-conglycinin and various glycinin subunits of soy are not required to modulate hepatic lipid metabolism in rats.

PURPOSE: This study examined the effect of soy proteins with depletion of different subunits of the two major storage proteins, ß-conglycinin and glycinin, on hepatic lipids and proteins involved in lipid metabolism in rats, since the bioactive component of soy responsible for lipid-lowering is unclear. METHODS: Weanling Sprague Dawley rats were fed diets containing either 20% casein protein in the absence (casein) or presence (casein + ISF) of isoflavones or 20% alcohol-washed soy protein isolate (SPI) or 20% soy protein concentrates derived from a conventional (Haro) or 2 soybean lines lacking the α' subunit of ß-conglycinin and the A1-3 (1TF) or A1-5 (1a) subunits of glycinin. After 8 weeks, the rats were necropsied and liver proteins and lipids were extracted and analysed. RESULTS: The results showed that soy protein diets reduced lipid droplet accumulation and content in the liver compared to casein diets. The soy protein diets also decreased the level of hepatic mature SREBP-1 and FAS in males, with significant decreases in diets 1TF and 1a compared to the casein diets. The effect of the soy protein diets on female hepatic mature SREBP-1, FAS, and HMGCR was confounded since casein + ISF decreased these levels compared to casein alone perhaps muting the decrease by soy protein. A reduction in both phosphorylated and total STAT3 in female livers by ISF may account for the gender difference in mechanism in the regulation and protein expression of the lipid modulators. CONCLUSIONS: Overall, soy protein deficient in the α' subunit of ß-conglycinin and A1-5 subunits of glycinin maintain similar hypolipidemic function compared to the conventional soy protein. The exact bioactive component(s) warrant identification.

l-Lysine supplementation does not affect the bioavailability of copper or iron in rats.

l-lysine (Lys) is an essential amino acid that is added to foods and dietary supplements. Lys may interact with mineral nutrients and affect their metabolism. This study examined the effect of dietary Lys supplementation on the bioavailability of copper (Cu) and iron (Fe). Weanling male Sprague-Dawley rats were fed one of five diets (20% casein) for 4 weeks containing normal Cu and Fe (control) or low Cu or Fe without (LCu, LFe) or with (LCu+Lys, LFe+Lys) addition of 1.5% Lys. Final body weights, body weight gains and food consumption of the rats did not differ (P≥0.05) among diet groups. Rats fed the low Cu or Fe diets showed changes in nutritional biomarkers compared to control rats, demonstrating reduced Cu and Fe status, respectively. Hematological parameters, serum ceruloplasmin activity and Cu and Fe concentrations in serum, liver, kidney and intestinal mucosa were unaffected (P≥0.05) by Lys supplementation. These results indicate that in the context of an adequate protein diet, Lys supplementation at a relatively high level does not affect Cu or Fe bioavailability in rats.

A robust analytical method for measurement of phytoestrogens and related metabolites in serum with liquid chromatography tandem mass spectrometry.

A sensitive and robust method using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed for quantitation of 13 phytoestrogens and related metabolites in rat serum samples. A new type of column, the Kinetex core-shell C18 column, was applied for rapid separation of the target analytes in 10min. Two enzymes, sulfatase H-1 and gulcuronidase H-5 from Helix pomatia were compared on the efficiency of releasing the conjugated forms of the target analytes to their free forms in serum samples. The method detection limit (MDL) defined as three times the signal to noise ratio in spiked serum matrix-based solutions was in the range of 0.1-3.5ng/mL. The linear dynamic calibration was in the broad range of 0.2-500ng/mL for all target compounds. Thirty-two rat serum samples from the rats that were fed with diets containing either casein or soy protein isolates with various amounts of isoflavones for 8 weeks were analyzed for the target analytes with the developed method. Nine target analytes were detected in the serum samples. Those detectable compounds are all the metabolites of the dietary isoflavones, suggesting that the diet isoflavones were mostly metabolized to their metabolites in rat.

Cardio-Metabolic Disease Risks and Their Associations with Circulating 25-Hydroxyvitamin D and Omega-3 Levels in South Asian and White Canadians.

OBJECTIVES: This study compared cardio-metabolic disease risk factors and their associations with serum vitamin D and omega-3 status in South Asian (SAC) and White Canadians (WC) living in Canada's capital region. METHODS: Fasting blood samples were taken from 235 SAC and 279 WC aged 20 to 79 years in Ottawa, and 22 risk factors were measured. RESULTS: SAC men and women had significantly higher fasting glucose, insulin, homeostasis model assessment for insulin resistance (HOMA-IR), apolipoprotein B (ApoB), ratios of total (TC) to HDL cholesterol (HDLC) and ApoB to ApoA1, leptin, E-selectin, P-selectin, ICAM-1 and omega-3 (p < 0.05), but lower HDLC, ApoA1, vitamin D levels than WC (p < 0.05). SAC women had higher CRP and VEGF than WC women. Adequate (50-74.9 nmol/L) or optimal (≥ 75 nmol/L) levels of 25(OH)D were associated with lower BMI, glucose, insulin, HOMA-IR, TG, TC, low density lipoprotein cholesterol (LDLC), ApoB/ApoA1 ratio, CRP, leptin, and higher HDLC, ApoA1, omega-3 index, L-selectin levels in WC, but not in SAC. Intermediate (>4%-<8%) or high (≥ 8%) levels of omega-3 indices were related to lower E-selectin, P-selectin, ICAM-1 and higher HDLC, 25(OH)D levels in WC, but not in SAC. The BMIs of ≤ 25 kg/m2 were related to lower LDLC, ApoB, VEGF, creatinine and higher 25(OH)D in WC, but not in SAC. CONCLUSIONS: The associations of vitamin D, omega-3 status, BMI and risk factors were more profound in the WC than SAC. Compared to WC, vitamin D status and omega-3 index may not be good predictive risk factors for the prevalence of CVD and diabetes in SAC.

Effect of Soyabean Isoflavones Exposure on Onset of Puberty, Serum Hormone Concentration and Gene Expression in Hypothalamus, Pituitary Gland and Ovary of Female Bama Miniature Pigs.

This study was to investigate the effect of soyabean isoflavones (SIF) on onset of puberty, serum hormone concentration, and gene expression in hypothalamus, pituitary and ovary of female Bama miniature pigs. Fifty five, 35-days old pigs were randomly assigned into 5 treatment groups consisting of 11 pigs per treatment. Results showed that dietary supplementation of varying dosage (0, 250, 500, and 1,250 mg/kg) of SIF induced puberty delay of the pigs with the age of puberty of pigs fed basal diet supplemented with 1,250 mg/kg SIF was significantly higher (p<0.05) compared to control. Supplementation of SIF or estradiol valerate (EV) reduced (p<0.05) serum gonadotrophin releasing hormone and luteinizing hormone concentration, but increased follicle-stimulating hormone concentration in pigs at 4 months of age. The expression of KiSS-1 metastasis-suppressor (KISS1), steroidogenic acute regulatory protein (StAR) and 3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase (3ß-HSD) was reduced (p<0.01) in SIF-supplemented groups. Expression of gonadotropin-releasing hormone receptor in the pituitary of miniature pigs was reduced (p<0.05) compared to the control when exposed to 250, 1,250 mg/kg SIF and EV. Pigs on 250 mg/kg SIF and EV also showed reduced (p<0.05) expression of cytochrome P450 19A1 compared to the control. Our results indicated that dietary supplementation of SIF induced puberty delay, which may be due to down-regulation of key genes that play vital roles in the synthesis of steroid hormones.

Dietary soy isoflavones differentially regulate expression of the lipid-metabolic genes in different white adipose tissues of the female Bama mini-pigs.

Soy isoflavones have been shown to affect lipid metabolism, however the underlying molecular mechanism(s) have not yet been fully understood. The present study, using female Bama mini-pig as a model, examined the effects of soy isoflavones on lipid metabolism and involved gene expression in different white adipose tissues. Female Bama Xiang mini-pigs of 35 days old were fed a basal diet (control, Con), or basal diet supplemented with increasing amounts of soy isoflavones (250, 500, or 1250 mg/kg diet) for 120 days. The results showed that soy isoflavones did not affect the body weight, but decreased the dorsal subcutaneous adipose tissue (DSA) mass and increased the mass of abdominal subcutaneous adipose tissue (ASA) and perirenal adipose tissue (PRA). Besides, soy isoflavones decreased the expression of lipogenic genes and increased the expression of lipolytic genes in DSA, while the opposite effects were observed in ASA and PRA. In addition, the expression of lipoprotein lipase was down regulated in DSA while up regulated in ASA and PRA by soy isoflavones. Moreover, the expression of estrogen receptors (ERs) was up regulated in DSA, and down regulated in ASA and PRA by soy isoflavones. Our results suggest that soy isoflavones affected the lipid metabolism in white adipose tissues of Bama mini-pigs in a site-specific manner, which might be mediated through PPARs and ERs regulated gene expression.

Effects of soybean isoflavones on reproductive parameters in Chinese mini-pig boars.

BACKGROUND: Soybean isoflavones are structurally similar to mammalian estrogens and therefore may act as estrogen agonists or antagonists. However, it has not been determined if they have any negative effects on reproductive parameters in male livestock. Therefore, the objective of this study was to evaluate the effects of soybean isoflavones on male reproduction using Chinese mini-pig boars as a model. Fifty Xiang boars were randomly divided into five groups and fed diets containing 0, 125, 250, or 500 ppm soybean isoflavones or 0.5 ppm diethylstilbestrol for 60 days. RESULTS: Dietary supplementation with 250 ppm of soy isoflavones markedly increased the testis index (P < 0.05), fructose content (P < 0.05), and α-glycosidase content in testicular tissue (P < 0.01), as well as increased the number of viable germ cells (P < 0.01) and the level of Bcl-2 protein (P < 0.01). However, 500 ppm of soybean isoflavones significantly reduced both testis and epididymis indexes (P < 0.05) and lactate dehydrogenase levels (P < 0.01), as well as reduced serum LH and testosterone levels (P < 0.05). High levels of soybean isoflavones also increased malondialdehyde levels (P < 0.05), as well as increased the numbers of early and late apoptotic germ cells (P < 0.01) and the level of Bax proteins (P < 0.05) in the testis. CONCLUSIONS: The results of this study indicate that consumption of soy isoflavones at dietary levels up to 250 ppm did not adversely affect reproductive parameters in Chinese mini-pig boars whereas higher levels of soy isoflavones may adversely affect male reproduction.

Increasing Dietary alpha-linolenic acid enhances tissue levels of long-chain n-3 PUFA when linoleic acid intake is low in hamsters.

BACKGROUND/AIMS: We tested whether feeding hamsters diets varying in alpha-linolenic acid (ALA) content and low in linoleic acid (LA) could increase the tissue levels of eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA) to the same extent as a fish oil-supplemented diet. METHODS: For 5 weeks, 60 hamsters were fed 1 of the following 5 diets containing 2% of total dietary energy (TE) as LA and either 0.5% (diet A), 1% (diets B and E), 2% (diet C), or 4% (diet D) ALA of TE, so that the ratio of LA/ALA was 4:1, 2:1, 1:1, or 1:2. Diet E was supplemented with fish oil at the level of 0.2% of total energy intake. At the end of the study, overnight-fasted hamsters were sacrificed, and blood and tissues were collected. RESULTS: Tissue levels of ALA, EPA, DPA, and DHA rose in proportion to the increase in the dietary ALA level (p < 0.01); however, the levels of DHA reached a plateau at ALA intakes above 1% (p < 0.01). These changes were accompanied by decreases in arachidonic acid with or without increases in LA levels (p < 0.01). Hamsters fed diet D had similar or higher EPA, DPA, and DHA tissue levels to those fed diet E (p < 0.01). CONCLUSIONS: In hamsters, diets containing 4% energy as ALA and 2% energy as LA can increase the tissue levels of EPA, DPA, and DHA to the same extent as feeding 0.2% energy as fish oil.

Need for accurate and standardized determination of amino acids and bioactive peptides for evaluating protein quality and potential health effects of foods and dietary supplements.

Accurate standardized methods for the determination of amino acid in foods are required to assess the nutritional safety and compositional adequacy of sole source foods such as infant formulas and enteral nutritionals, and protein and amino acid supplements and their hydrolysates, and to assess protein claims of foods. Protein digestibility-corrected amino acid score (PDCAAS), which requires information on amino acid composition, is the official method for assessing protein claims of foods and supplements sold in the United States. PDCAAS has also been adopted internationally as the most suitable method for routine evaluation of protein quality of foods by the Food and Agriculture Organization/World Health Organization. Standardized methods for analysis of amino acids by ion-exchange chromatography have been developed. However, there is a need to develop validated methods of amino acid analysis in foods using liquid chromatographic techniques, which have replaced ion-exchange methods for quantifying amino acids in most laboratories. Bioactive peptides from animal and plant proteins have been found to potentially impact human health. A wide range of physiological effects, including blood pressure-lowering effects, cholesterol-lowering ability, antithrombotic effects, enhancement of mineral absorption, and immunomodulatory effects have been described for bioactive peptides. There is considerable commercial interest in developing functional foods containing bioactive peptides. There is also a need to develop accurate standardized methods for the characterization (amino acid sequencing) and quantification of bioactive peptides and to carry out dose-response studies in animal models and clinical trials to assess safety, potential allergenicity, potential intolerance, and efficacy of bioactive peptides. Information from these studies is needed for determining the upper safe levels of bioactive peptides and as the basis for developing potential health claims for bioactive peptides. This information is, in turn, needed by regulatory agencies for developing appropriate policy and regulations on adding these substances to foods and for determining if health claims are scientifically substantiated.

Progesterone-modulated induction of apoptosis by interferon-tau in cultured epithelial cells of bovine endometrium.

Interferon-tau (IFN-tau) is produced by the trophoblast prior to implantation in ruminants. It is involved in maternal recognition of pregnancy, and is a pleiotropic molecule that can alter the synthesis of endometrial proteins and inhibit proliferation of some cells. We have observed that IFN-tau reduces the DNA content in cultures of bovine endometrial epithelial cells; therefore, the objective of this study was to determine whether IFN-tau would induce apoptosis in bovine endometrial cells. Epithelial cells were prepared, cultured to confluence, and then incubated for 24 or 48 h in the presence or absence of 10 ng/ml progesterone, 100 ng/ml IFN-tau, or 10 microg/ml cycloheximide (CHX; an apoptosis inducer used as a positive control). Cells undergoing apoptosis exhibit such characteristics as the appearance of apoptotic bodies and DNA fragmentation. The incidence of apoptosis was assessed by using TUNEL, DNA fragmentation analysis, and Western blot analysis of Bax-alpha protein expression. The results showed that IFN-tau and CHX significantly increased the percentage of cells with apoptotic nuclei (33.6% and 44.8%, respectively) compared with controls (11.7%; P < 0.05). Progesterone treatment of the cells significantly inhibited the ability of IFN-tau to induce apoptosis (14.6%) compared with IFN-tau alone (33.6%; P < 0.05). DNA fragmentation analysis showed that INF-tau and CHX treatment resulted in an increase in the appearance of DNA laddering compared with that in untreated control cultures. Western blot analysis showed that IFN-tau and CHX treatment resulted in a greater expression of the proapoptotic protein Bax-alpha compared with that in control cultures. These data demonstrate that IFN-tau can induce apoptosis in bovine uterine epithelial cells and that this effect is modulated by progesterone. We speculate that IFN-tau might play a critical role in the remodeling of the endometrium around the time of implantation.