Digestion, rumen fermentation and circulating concentrations of insulin, growth hormone and IGF-1 in steers fed diets based on different proportions of maize silage and grass silage

Replacing grass silage with maize silage results in a fundamental change in the ratio of structural to non-structural carbohydrates with commensurate changes in rumen fermentation patterns and nutrient utilisation. This study investigated the effects of feeding four forage mixtures, namely grass silage (G); 67 g/100 g grass silage133 g/100 g maize silage (GGM); 67 g/100 g maize silage133/100 g grass silage (MMG); maize silage (M) to four ruminally and duodenally canulated Holstein Friesian steers. All diets were formulated to be isonitrogenous (22.4 g N/kg DM) using a concentrate mixture. Dietary dry matter (DM) and organic matter (OM) digestibility increased with ascending maize silage inclusion (P,0.1) whereas starch and neutral detergent fibre digestibility declined (P,0.05). Ratio of non-glucogenic to glucogenic precursors in the rumen fluid increased with maize silage inclusion (P,0.01) with a commensurate reduction in rumen pH (P,0.05). Mean circulating concentrations of insulin were greatest and similar in diets MMG and GGM, lower in diet M and lowest in diet G (P,0.01). There were no effects of diet on the mean circulating concentration of growth hormone (GH), or the frequency, amplitude and duration of GH pulses, or the mean circulating concentrations of IGF-1. Increasing levels of DM, OM and starch intakes with the substitution of grass silage with maize silage affected overall digestion, nutrient partitioning and subsequent circulating concentrations of insulin.

Digestion, rumen fermentation and circulating concentrations of insulin, growth hormone and IGF-1 in steers given maize silages harvested at three stages of maturity

Advancing maturity of forage maize is associated with increases in the proportion of dry matter (DM) and starch and decreases in the proportions of structural carbohydrates in the ensiled crop. Three maize silages (286 (low, L), 329 (medium, M) and 379 (high, H) g DM per kg fresh weight) plus a concentrate formulated to give isonitrogenous intakes were offered to Holstein-Friesian steers fitted with a cannula in the dorsal sac of the rumen and a 'T' piece cannula in the proximal duodenum in an experiment with a cross-over design that allowed four collection periods. Nutrient flow to the duodenum was estimated using chromium-EDTA. Steers consumed approximately 0(.)6 kg DM per day less of diet L compared with the other two diets (P=0(.)026), resulting in less DM being digested (P=0(.)005) but digestibility did not differ between diets. Similar results were obtained for organic matter. There were no differences between diets in the intake or digestibility of neutral-detergent fibre. Intake, duodenal flow and faecal output of starch were greater for steers offered diets M and H compared with those given diet L (P < 0(.)05). In all diets rumen digestion contributed to over 90% of total digestion of starch, although rumen digestibility declined significantly with advancing maize maturity (P=0(.)002). Molar proportions of acetic acid were higher in diet H (P < 0(.)05) whilst proportions of propionic acid and n-butyric acid were higher in diets M and L. There were no significant differences between diets in mean rumen pH or ammonia concentrations. Mean circulating concentrations of insulin were higher (P=0(.)009) in cattle given diets L and M compared with diet H. There were no differences between diets in the mean circulating concentration of growth hormone, or the frequency, amplitude and duration of growth hormone pulses, or the mean circulating concentrations of IGF-1. Changes in forage composition that accompany advancing maize maturity affect overall silage digestion and circulating concentrations of insulin.

An intronic growth hormone receptor mutation causing activation of a pseudoexon is associated with a broad spectrum of growth hormone insensitivity phenotypes

Context: Inherited GH insensitivity (GHI) is usually caused by mutations in the GH receptor (GHR). Patients present with short stature associated with high GH and low IGF-I levels and may have midfacial hypoplasia ( typical Laron syndrome facial features). We previously described four mildly affected GHI patients with an intronic mutation in the GHR gene (A.(1) -> G.(1) substitution in intron 6), resulting in the activation of a pseudoexon (6 Psi) and inclusion of 36 amino acids. Objective: The study aimed to analyze the clinical and genetic characteristics of additional GHI patients with the pseudoexon (6 Psi) mutation. Design/Patients: Auxological, biochemical, genetic, and haplotype data from seven patients with severe short stature and biochemical evidence of GHI were assessed. Main Outcome Measures: We assessed genotype-phenotype relationship. Results: One patient belongs to the same extended family, previously reported. She has normal facial features, and her IGF-I levels are in the low-normal range for age. The six unrelated patients, four of whom have typical Laron syndrome facial features, have heights ranging from -3.3 to -6.0 SD and IGF-I levels that vary from normal to undetectable. We hypothesize that the marked difference in biochemical and clinical phenotypes might be caused by variations in the splicing efficiency of the pseudoexon. Conclusions: Activation of the pseudoexon in the GHR gene can lead to a variety of GHI phenotypes. Therefore, screening for the presence of this mutation should be performed in all GHI patients without mutations in the coding exons.

Endocrine regulation of ovarian antral follicle development in cattle

Antral follicle growth in cattle occurs in two distinct phases; the first 'slow' growth phase spans the time from antrum acquisition to a size of approximately 3 mm detectable by transrectal ultrasound, and the second 'fast' phase is gondadotrophin-dependent and includes cohort growth, dominant follicle (DF) selection, and DF growth. This review summarises current concepts of the relative roles FSH and LH, ovarian and metabolic hormones play mainly in the second phase of antral follicle growth in animals of different reproductive and nutritional states. It is proposed that differential FSH response may enable one cohort follicle to become selected, and that follicular secretions, particularly inhibin, suppress FSH and thus are responsible for DF selection and dominance. Acute dependence of the DF on LH pulses will determine DF lifespan, and the LH pulse profile can be influenced by metabolic hormones such as leptin, providing one possible link for nutritional state and reproduction. Direct ovarian effects of acute and chronic changes in growth hormone, insulin and insulin-like growth factor (IGF)-I have been described on cohort follicles, DF oestrogen activity and on DF growth. Influences of metabolic hormones on early antral follicles undergoing their first 'slow' growth phase are less well described, yet metabolic hormones appear to enhance growth into the cohort available for FSH-induced emergence, and may influence subsequent developmental competence of oocytes. (C) 2003 Elsevier Science B.V. All rights reserved.

Pre-pubertal measurements of the somatotrophic axis as predictors of milk production in Holstein-Friesian dairy cows

This study investigated possible relationships between measurements of the somatotrophic axis in pre-pubertal dairy calves and subsequent milk yields. Endogenous growth hormone (GH) release was measured through a fed and fasted period in fifty 6-month-old Holstein-Friesian heifers and they were then challenged with growth hormone-releasing factor (GRF) to assess their GH release pattern. Insulin-like growth factor-I (IGF-I), insulin and glucose concentrations were measured in relation to time of feeding. Cows were subsequently monitored through their first three lactations to record peak and 305-day milk yields. In the first lactation, milk energy output for the first 120 days of lactation was also calculated. The mean 305-day milk yield increased from 7417 +/- 191 kg in the first lactation (n = 37) to 8749 +/- 252 kg in the third (n = 25). There were no significant relationships between any measures of GH secretion and peak or 305-day yield in any lactation. A highly significant positive relationship was established between the GH peak measured 10 min post-GRF challenge and 120-day milk energy values in the first lactation. This relationship was, however, only present in the subpopulation of 12 cows culled after one or two lactations and was absent in the 25 animals remaining for the third lactation. There were no significant relationships between pre-pubertal IGF-I and fed or fasted insulin or glucose concentrations and any subsequent measurement of yield. The usefulness of GH secretagogue challenges in calves as a predictive test for future milk production is thus limited but may have some bearing on nutrient partitioning and longevity. (c) 2005 Elsevier Inc. All rights reserved.

Crosstalk with insulin and dependence on PI3K/Akt/mTOR rather than MAPK pathways in upregulation of basal growth following long-term oestrogen deprivation in three human breast cancer cell lines

Background: MCF-7, T-47-D, ZR-75-1 human breast cancer cell lines are dependent on oestrogen for growth but can adapt to grow during long-term oestrogen deprivation. This serves as a model for identification of therapeutic targets in endocrine-resistant breast cancer. Methods: An overlooked complication of this model is that it involves more than non-addition of oestrogen, and inadequate attention has been given to separating molecular events associated with each of the culture manipulations. Results: Insulin and oestradiol were shown to protect MCF-7 cells against upregulation of basal growth, demonstrating a crosstalk in the growth adaptation process. Increased phosphorylation of p44/42MAPK and c-Raf reflected removal of insulin from the medium and proliferation of all three cell lines was inhibited to a lesser extent by PD98059 and U0126 following long-term oestrogen/insulin withdrawal, demonstrating a reduced dependence on the MAPK pathway. By contrast, long-term oestrogen/insulin deprivation did not alter levels of phosphorylated Akt and did not alter the dose-response of growth inhibition with LY294002 in any of the three cell lines. The IGF1R inhibitor picropodophyllin inhibited growth of all MCF-7 cells but only in the long-term oestrogen/insulin-deprived cells was this paralleled by reduction in phosphorylated p70S6K, a downstream target of mTOR. Long-term oestrogen/insulin-deprived MCF-7 cells had higher levels of phosphorylated p70S6K and developed increased sensitivity to growth inhibition by rapamycin. Conclusions: The greater sensitivity to growth inhibition by rapamycin in all three cell lines following long-term oestrogen/insulin deprivation suggests rapamycin-based therapies might be more effective in breast cancers with acquired oestrogen resistance. Keywords Akt, breast cancer cells, endocrine resistance, insulin, MAPK, MCF-7 cells, mTOR, oestrogen, oestrogen-deprived, PI3K, picropodophyllin, rapamycin, T-47-D cells, ZR-75-1 cells

Molecular mechanisms of oestrogen action on growth of human breast cancer cells in culture

Growth responses to oestrogen can be reproducibly obtained using a selection of oestrogen-receptor-containing human breast cancer cell lines, and molecular mechanisms have been shown to include modulation to growth factor/receptor/signalling pathways, cell-cycle proteins, apoptosis, differentiation, adhesion, motility and migration. Considerable progress has been made in understanding the molecular basis of oestrogen action on gene expression through the ligand-activated transcription factors human oestrogen receptor α (ERα) and ERβ and the resulting effects on global gene expression patterns, but the full profile of coordination of the alterations, which brings about changes in cell growth through genomic and non-genomic mechanisms remain to be fully elucidated. Oestrogen regulation of cell growth involves a complex cross-talk between oestrogen receptor and growth factor signalling pathways such that inhibition of one pathway may lead to stimulation of another, which may explain the remarkable ability of human breast cancer cells to escape from any mode of imposed growth inhibition be it oestrogen deprivation or administration of antioestrogen. Although studies on cell growth have focused to date on the effects of physiological oestrogens, many hundreds of environmental chemicals with oestrogenic properties have now been measured in the human breast. Whether or not the weight of evidence eventually establishes any causal link of complex mixtures of environmental oestrogenic chemicals with breast cancer, the presence of so many oestrogenic chemicals in the breast must influence resulting oestrogenic responses, and the impact of this additional oestrogenic burden needs to be taken into account in future studies on growth regulation of human breast cancer cells.

Numbers of antral follicles during follicular waves in cattle: Evidence for high variation among animals, very high repeatability in individuals, and an inverse association with serum follicle-stimulating hormone concentrations

The extent, causes, and physiological significance of the variation in number of follicles growing during ovarian follicular waves in human beings and cattle are unknown. Therefore, the present study examined the variability and repeatability in numbers of follicles 3 mm or greater in diameter during the follicular waves in bovine estrous cycles, and we determined if the variation in number of follicles during waves was associated with alterations in secretion of FSH, estradiol, inhibin, and insulin-like growth factor I (IGF-I). Dairy cattle were subjected to twice-daily ultrasound analysis to count total number of antral follicles 3 mm or greater in diameter throughout 138 different follicular waves. In another study, blood samples were taken at frequent intervals from cows that consistently had low or very high numbers of follicles during waves and were subjected to immunoassays. Results indicate the following: First, despite an approximately sevenfold variation in number of follicles during waves among animals and marked differences in age, stage of lactation, and season of the year, a very highly repeatable (0.95) number of follicles 3 mm or greater in diameter is maintained during the ovulatory and nonovulatory follicular waves of individuals. Second, variation in number of follicles 3 mm or greater in diameter during waves and the inverse association of number of follicles during waves with FSH are not directly explained by alterations in the patterns of secretion of estradiol, inhibin, or IGF-I. Third, ovarian ultrasound analysis can be used reliably by investigators to identify cattle that consistently have low or high numbers of follicles during waves, thus providing a novel experimental model to determine the causes and physiological significance of the high variation in antral follicle number during follicular waves among single-ovulating species, such as cattle or humans.

Oocyte-mediated suppression of follicle-stimulating hormone- and insulin-like growth factor-induced secretion of steroids and inhibin-related proteins by bovine granulosa cells in vitro: Possible role of transforming growth factor alpha

The objective was to investigate the potential role of the oocyte in modulating proliferation and basal, FSH-induced and insulin-like growth factor (IGF)-induced secretion of inhibin A (inh A), activin A (act A), follistatin (FS), estradiol (E-2), and progesterone (P-4) by mural bovine granulosa cells. Cells from 4- to 6-mm follicles were cultured in serum-free medium containing insulin and androstenedione, and the effects of ovine FSH and IGF analogue (LR3-IGF-1) were tested alone and in the presence of denuded bovine oocytes (2, 8, or 20 per well). Medium was changed every 48 h, cultures were terminated after 144 h, and viable cell number was determined. Results are based on combined data from four independent cultures and are presented for the last time period only when responses were maximal. Both FSH and IGF increased (P < 0.001) secretion of inh A, act A, FS, E-2, and P-4 and raised cell number. In the absence of FSH or IGF, coculture with oocytes had no effect on any of the measured hormones, although cell number was increased up to 1.8-fold (P < 0.0001). Addition of oocytes to FSH-stimulated cells dose-dependently suppressed (P < 0.0001) inh A (6-fold maximum suppression), act A (5.5-fold), FS (3.6-fold), E-2 (4.6-fold), and P-4 (2.4-fold), with suppression increasing with FSH dose. Likewise, oocytes suppressed (P < 0.001) IGF-induced secretion of inh A, act A, FS, and E-2 (P < 0.05) but enhanced IGF-induced P-4 secretion (1.7-fold; P < 0.05). Given the similarity of these oocyte-mediated actions to those we observed previously following epidermal growth factor (EGF) treatment, we used immunocytochemistry to determine whether bovine oocytes express EGF or transforming growth factor (TGF) alpha. Intense staining with TGFalpha antibody (but not with EGF antibody) was detected in oocytes both before and after coculture. Experiments involving addition of TGFalpha to granulosa cells confirmed that the peptide mimicked the effects of oocytes on cell proliferation and on FSH- and IGF-induced hormone secretion. These experiments indicate that bovine oocytes secrete a factor(s) capable of modulating granulosa cell proliferation and responsiveness to FSH and IGF in terms of steroidogenesis and production of inhibin-related peptides, bovine oocytes express TGFalpha but not EGF, and TGFalpha is a prime candidate for mediating the actions of oocytes on bovine granulosa cells.

Formate-dependent growth and homoacetogenic fermentation by a bacterium from human feces: Description of Bryantella formatexigens gen. nov., sp nov.

Formate stimulates growth of a new bacterium from human feces. With high formate, it ferments glucose to acetate via the Wood-Ljungdahl pathway. The original isolate fermented vegetable cellulose and carboxymethylcellulose, but it lost this ability after storage at -76degreesC. 16S rRNA gene sequencing identifies it as a distinct line within the Clostridium coccoides supra-generic rRNA grouping. We propose naming it Bryantella formatexigens gen. nov., sp. nov.

Differential effects of overexpression of ERalpha and ERbeta in MCF10A immortalised, non-transformed human breast epithelail cells

Background: Cellular effects of oestrogen are mediated by two intracellular receptors ERα and ERβ. However, to compare responses mediated through these two receptors, experimental models are needed where ERα and ERβ are individually stably overexpressed in the same cell type. Methods: We compared the effects of stable overexpression of ERα and ERβ in the MCF10A cell line, which is an immortalised but non-transformed breast epithelial cell line without high endogenous ER expression. Results: Clones of MCF10A cells were characterised which stably overexpressed ERα (10A-ERα2, 10A-ERα13) or which stably overexpressed ERβ (10A-ERβ12, 10A-ERβ15). Overexpression of either ERα or ERβ allowed induction of an oestrogen-regulated ERE-LUC reporter gene by oestradiol which was not found in the untransfected cells. Oestradiol also increased proliferation of 10A-ERα13 and 10A-ERβ12 cells, but not untransfected cells, by 1.3-fold over 7 days. The phytoestrogen, genistein, which is reported to bind more strongly to ERβ than to ERα, could induce luciferase gene expression from an ERE-LUC reporter gene at concentrations of 10−6 M and 10−5 M but only in the clones overexpressing ERβ and not in those overexpressing ERα. Clone 10A-ERβ12 also yielded growth stimulation with 10-6 M genistein. Finally, the overexpression of ERα, but not ERβ, gave rise to increased growth in semi-solid methocel suspension culture in the presence of 70 nM oestradiol, suggesting that overexpression of ERα, but not ERβ, produces characteristics of a transformed phenotype. Conclusions: This provides a model system to compare effects of oestradiol with other oestrogenic ligands in cells stably overexpressing individually ERα or ERβ.

Screening human intestinal Bifidobacterium strains for growth, acidification, EPS production and viscosity potential in low-fat milk

Bifidobacterium strains of human origin were screened for their ability to grow in milk and produce exopolysaccharides (EPS). Bifidobacterium strains were grown in low-fat UHT milk and were evaluated for their growth, acidification properties, EPS production and ability to increase the viscosity of fermented milk. The strains that grew well in milk were strains of Bifidobacterium breve and Bifidobacterium longum and B. longum subsp. longum. Among the 22 strains, EPS was produced by Bifidobacterium bifidum ALM 35, B. breve NCIMB 8807 (UCC 2003), B. longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205 at concentrations ranging from 25 to 140 . The molecular mass and the composition varied considerably, depending on the strain. Analysis of the correlation between the apparent viscosity of the fermented milk and pH indicated that the EPS produced during the acidification of milk possibly contributed to the viscosity of the milk products.

Effect of dextransucrase cellobiose acceptor products on the growth of human gut bacteria

The selective fermentation by human gut bacteria of gluco-oligosaccharides obtained from the reaction between the glucosyl group of sucrose and cellobiose, catalyzed by dextransucrases (DSR) from Leuconostoc mesenteroides, has been evaluated. Oligosaccharides were fractionated according to their molecular weight, and their effect on the growth of different bacterial groups was studied. To determine the structure (position and configuration of glycosidic linkages)�function relationship, their properties were compared to those of DSR maltose acceptor products (DSRMal) and of recognized prebiotic carbohydrates (fructo-oligosaccharides, FOS). Cellobiose acceptor products (DSRCel) showed bifidogenic properties similar to those of FOS. However, no significant differences related to molecular weight or isomeric configurations were found for DSRCel and DSRMal products.