Characterization of folate-dependent enzymes and indices of folate status in laying hens supplemented with folic acid or 5-methyltetrahydrofolate.

The conversion of folic acid (FA) to the biologically active 5-methyltetrahydrofolate (5-MTHF) is necessary for the deposition of folate in the egg. A study was conducted to compare egg folate concentrations, indices of folate status, and activities of folate-dependent enzymes in response to equimolar intake of either FA or 5-MTHF in laying hens. Forty-eight laying hens, 24 wk of age, from 2 different strains (Shaver White and Shaver Brown) were randomly assigned to receive 1 of 3 (n = 8 per strain) dietary treatments: 1) basal diet with no supplemental folate, 2) basal diet + 10 mg/kg of FA, or 3) basal diet + 11.3 mg/kg of 5-MTHF for 3 wk. A completely randomized design with 3 dietary treatments and 2 laying hen strains in a 3 x 2 factorial arrangement was used. Data were subjected to ANOVA, using the PROC GLM procedure of SAS. Plasma homocysteine, serum, and egg folate concentrations; hepatic serine hydroxymethyltransferase; and methionine synthase activity were affected by dietary folate supplementation but not by its form (FA and 5-MTHF). Relative to control hens, plasma homocysteine was decreased (P < 0.05) by 14.2%, whereas serum and egg folate were increased (P < 0.05) by 78.3 and 61.8%, respectively, in hens consuming either folate compound. Hepatic serine hydroxymethyltransferase and methionine synthase activity were increased and decreased (P < 0.05), respectively, in folate-fed birds compared with control-fed birds. Hepatic dihydrofolate reductase was influenced by both the addition and form of dietary folate, being higher (P < 0.05) in FA-fed birds than in 5-MTHF and control-fed birds. Feed efficiency was improved (P < 0.05) in 5-MTHF-fed birds relative to FA-fed birds. Strain of hen influenced serum folate and plasma homocysteine concentrations but not other indices of folate metabolism. Overall, FA and 5-MTHF have equivalent effects in enhancing egg folate concentrations and improving folate status in laying hens. Also, supplementation and form of folate may modulate the activity of folate-dependent enzymes.

Effect of plant sterol-enriched diets on plasma and egg yolk cholesterol concentrations and cholesterol metabolism in laying hens.

Egg exists as a major dietary source of cholesterol in Western diets. In North America, laying hen diets are usually devoid of cholesterol when diets are formulated to exclude animal-based products. Hence, laying hens meet their physiological cholesterol requirement through de novo synthesis. Plant sterols exert a cholesterol-lowering effect in humans by interfering with intestinal sterol absorption. However, it is unknown whether plant sterol supplementation could be effective in reducing intestinal reabsorption of biliary cholesterol in laying hens, thus modulating whole body cholesterol in favor of lower plasma and yolk cholesterol content. The current study was designed to investigate the effect of diets enriched with 0, 0.5, 1, and 2% plant sterols on cholesterol absorption, synthesis, as well as plasma, liver, and egg yolk cholesterol concentrations in laying hens. After 8 wk of plant sterol intervention (first 2 wk were acclimatization), feed intake, BW, egg weight, egg yolk weight, egg production, Haugh units, liver mass, plasma, and hepatic cholesterol concentrations did not differ as a function of plant sterol supplementation. Egg cholesterol concentrations (mg/g) fluctuated during the 6-wk experimental period. At wk 6, a minor reduction in egg yolk cholesterol concentration (mg per g of yolk, P<0.05, vs. control) was observed in hens fed 1 and 2% cholesterol-enriched diets, respectively. However, such result failed to affect total egg cholesterol content. No statistical difference was observed across treatments over 6 wk. Neither cholesterol absorption rates nor synthesis differed as a function of treatment. Results suggested that overall cholesterol content in egg yolk was not affected by feeding hens plant sterol-enriched diets over 6 wk.

IL-1alpha, IL-1beta, and IFN-gamma mark beta cells for Fas-dependent destruction by diabetogenic CD4(+) T lymphocytes.

Cytokines such as IL-1alpha, IL-1beta, and IFN-gamma have long been implicated in the pathogenesis of autoimmune diabetes, but the mechanisms through which they promote diabetogenesis remain unclear. Here we show that CD4(+) T lymphocytes propagated from transgenic nonobese diabetic (NOD) mice expressing the highly diabetogenic, beta cell-specific 4.1-T-cell receptor (4.1-TCR) can kill IL-1alpha-, IL-1beta-, and IFN-gamma-treated beta cells from NOD mice. Untreated NOD beta cells and cytokine-treated beta cells from Fas-deficient NOD.lpr mice are not targeted by these T cells. Killing of islet cells in vitro was associated with cytokine-induced upregulation of Fas on islet cells and was independent of MHC class II expression. Abrogation of Fas expression in 4.1-TCR-transgenic NOD mice afforded nearly complete protection from diabetes and did not interfere with the development of the transgenic CD4(+) T cells or with their ability to cause insulitis. In contrast, abrogation of perforin expression did not affect beta cell-specific cytotoxicity or the diabetogenic potential of these T cells. These data demonstrate a novel mechanism of action of IL-1alpha, IL-1beta, and IFN-gamma in autoimmune diabetes, whereby these cytokines mark beta cells for Fas-dependent lysis by autoreactive CD4(+) T cells.

Effects of estradiol and some antiestrogens (clomiphene, tamoxifen, and hydroxytamoxifen) on luteinizing hormone secretion by rat pituitary cells in culture.

Primary pituitary cell cultures from adult female rats were incubated for 4 or 24 h with various concentrations of estradiol (E2) or the antiestrogens (AE) tamoxifen (TMX), 4-hydroxytamoxifen (OH-TMX), and clomiphene citrate (CC). The luteinizing hormone (LH)-response of these cultures to gonadotrophin releasing hormone (GnRH) was monitored. Treatment for 4 or 24 h with high concentrations (10(-5) M) of the AE significantly decreased the GnRH-induced LH-release by the gonadotrophs. The negative E2-effect, which is observed in this model after 4 h was enhanced and the positive E2-effect, which occurs after 24 h, was completely reversed into a negative effect by these high AE-concentrations. Treatment of pituitary cells with increasing concentrations of E2 (10(-13)-10(-6) M) or AE (10(-12)-10(-5) M) for 24 h led first to a dose dependent increase of the LH-response to 5 X 10(-10) M GnRH. At higher E2- or AE-concentrations this positive effect was lost, resulting in bell shaped dose-response curves. The following maximal effective concentrations (EDmax) were found: E2 = 10(-10) M, OH-TMX = 10(-9) M, CC = 10(-7) M, TMX = 10(-6) M. Incubation of pituitary cells for 24 h with concentrations of AE near their EDmax and stimulation with increasing concentrations (10(-11)-10(-7) M) of GnRH resulted in significant increases of LH-secretion over a wide range of GnRH-concentrations. It is concluded that AE possess marked intrinsic activities on pituitary LH-secretion: at extremely high concentrations they suppress the GnRH-induced release of the gonadotrophin. At lower concentrations they increase the pituitary LH-response to GnRH in a manner which is qualitatively indistinguishable from that of E2.

Prostaglandins have no effect on spontaneous and gonadotrophin-releasing hormone stimulated luteinizing hormone release in cultured rat pituitary cells.

Primary cultures of pituitary cells from adult female rats were exposed to increasing concentrations (10(-13) to 10(-6)M) of selected prostaglandins (PGE2, PGF2 alpha, PGI2, 6-keto-PGF1 alpha, and sulproston). The release of luteinizing hormone (LH) by the pituicytes was monitored. In a second series of experiments, pituitary cells were treated with prostaglandins as described above and additionally challenged with a submaximal stimulus of gonadotrophin-releasing hormone (GnRH; 10(-9) M). The spontaneous LH-release in all prostaglandin treated cultures did not differ from the controls. When stimulated with 10(-9) M GnRH, the LH-release increased approx. six-fold in all cultures, with no difference between prostaglandin treated cells and the respective controls. Thus, the prostaglandins tested - at least at concentrations less than or equal to 10(-6) M - have neither a direct positive or negative effect on pituitary LH-release, nor do they enhance or decrease the LH-releasing effect of GnRH. These prostaglandins are probably not involved in the cellular regulation of LH-release by the pituicyte.