High and low protein∶ carbohydrate dietary ratios during gestation alter maternal-fetal cortisol regulation in pigs.

Imbalanced maternal nutrition during gestation can cause alterations of the hypothalamic-pituitary-adrenal (HPA) system in offspring. The present study investigated the effects of maternal low- and high-protein diets during gestation in pigs on the maternal-fetal HPA regulation and expression of the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11ß-hydroxysteroid dehydrogenase 1 and 2 (11ß-HSD1 and 11ß-HSD2) and c-fos mRNAs in the placenta and fetal brain. Twenty-seven German Landrace sows were fed diets with high (HP, 30%), low (LP, 6.5%) or adequate (AP, 12.1%) protein levels made isoenergetic by varying the carbohydrate levels. On gestational day 94, fetuses were recovered under general anesthesia for the collection of blood, brain and placenta samples. The LP diet in sows increased salivary cortisol levels during gestation compared to the HP and AP sows and caused an increase of placental GR and c-fos mRNA expression. However, the diurnal rhythm of plasma cortisol was disturbed in both LP and HP sows. Total plasma cortisol concentrations in the umbilical cord vessels were elevated in fetuses from HP sows, whereas corticosteroid-binding globulin levels were decreased in LP fetuses. In the hypothalamus, LP fetuses displayed an enhanced mRNA expression of 11ß-HSD1 and a reduced expression of c-fos. Additionally, the 11ß-HSD2 mRNA expression was decreased in both LP and HP fetuses. The present results suggest that both low and high protein∶carbohydrate dietary ratios during gestation may alter the expression of genes encoding key determinants of glucocorticoid hormone action in the fetus with potential long-lasting consequences for stress adaptation and health.

Adiponectin action from head to toe.

Adiponectin, the most abundant protein secreted by white adipose tissue, is known for its involvement in obesity-related disorders such as insulin resistance, type 2 diabetes mellitus and atherosclerosis. Moreover, modulation of the circulating adiponectin concentration is observed in pathologies that are more or less obesity-related, such as cancer and rheumatoid arthritis. The wide distribution of adiponectin receptors in various organs and tissues suggests that adiponectin has pleiotropic effects on numerous physiological processes. Besides its well-known insulin-sensitizing, anti-inflammatory and antiatherosclerotic properties, accumulating evidence suggests that adiponectin may also have anticancer properties and be cardioprotective. A beneficial effect of adiponectin on female reproductive function was also suggested. Since adiponectin has numerous beneficial biological functions, its use as a therapeutic agent has been suggested. However, the use of adiponectin or its receptors as therapeutic targets is complicated by the presence of different adiponectin oligomeric isoforms and production sites, by multiple receptors with differing affinities for adiponectin isoforms, and by cell-type-specific effects in different tissues. In this review, we discuss the known and potential roles of adiponectin in various tissues and pathologies. The therapeutic promise of administration of adiponectin and the use of its circulating levels as a diagnostic biomarker are further discussed based on the latest experimental studies.

Dose-dependent effects of genistein and daidzein on protein metabolism in porcine myotube cultures.

This study was conducted to investigate whether the isoflavones genistein and daidzein, which are components of soy-based diets, and the estrogen 17beta-estradiol affect differentiation and protein metabolism of porcine skeletal muscle cells in vitro. Serum-free porcine myotube cultures expressing the estrogen receptors ERalpha and ERbeta were treated with various concentrations of genistein, daidzein, or 17beta-estradiol for 26 h. The degree of differentiation by creatine phosphokinase activity was not altered by treatment. At 100 micromol/L both genistein and daidzein caused decreases in protein amount due to cell loss. In addition, 100 micromol/L genistein reduced protein synthesis rate of the surviving cells (P < 0.05) measured as [3H]-phenylalanine incorporation. Interestingly, genistein (0.1 micromol/L), daidzein (10, 100 micromol/L), and 17beta-estradiol (0.1, 1 nmol/L) slightly reduced protein degradation (P < 0.05). The results suggest that both genistein and daidzein affect protein metabolism in a dose-dependent manner and that estrogenic actions may play a role in decreasing protein degradation in porcine skeletal muscle.

IGF-I- and EGF-dependent DNA synthesis of porcine myoblasts is influenced by the dietary isoflavones genistein and daidzein.

Soy-derived isoflavones have been reported to be specific inhibitors of protein tyrosine kinases like the type 1 insulin-like growth factor receptor (IGF-1R) and the epidermal growth factor receptor (EGFR). This study was conducted to investigate, whether IGF-I and EGF stimulate porcine myoblast growth and whether the responses are influenced by isoflavones. Satellite cell-born myoblasts derived from the semimembranosus muscle of newborn piglets were treated for 26 h with IGF-I or EGF alone and in combination with genistein or daidzein. The DNA amount was measured and DNA synthesis was recorded as 6 h-[(3)H]thymidine incorporation during exponential growth in serum-free basal medium. IGF-I and EGF synergistically stimulated DNA synthesis of porcine myoblast with EGF causing a greater response. Genistein (100 micromol/l) effectively reduced the growth factor-mediated DNA synthesis, which was associated with an inhibition of growth factor receptor protein expression. In response to daidzein no reduction in growth factor-mediated DNA synthesis was found. Daidzein (1; 10 micromol/l) combined with IGF-I caused even a slight increase in DNA amount compared with the untreated control. The expression of the IGF-1R precursor protein was reduced with 10 and 100 micromol/l daidzein, whereas the EGFR expression remained unchanged with daidzein. The results suggest that dietary isoflavones may interact with growth factor-induced stimulation of pig skeletal muscle growth.

Developmental changes and the impact of isoflavones on mRNA expression of IGF-I receptor, EGF receptor and related growth factors in porcine skeletal muscle cell cultures.

OBJECTIVE: Soy that is widely used in human nutrition and in livestock production is a rich source of isoflavones. In addition to the estrogenic or antiestrogenic effects, isoflavones are suggested to affect cell growth via inhibition of protein tyrosine kinases (e.g. growth factor receptors). Therefore, the present in vitro-study was undertaken to determine, whether genistein and daidzein affect the mRNA expression of growth factor receptors (IGF-I receptor and EGF receptor) and their related growth factors in porcine skeletal muscle cell cultures. DESIGN: First, we investigated the basal mRNA expression of IGF-I, IGF-II, EGF, IGF-I receptor, and EGF receptor in proliferating and differentiating porcine skeletal muscle cell cultures using real-time PCR. Secondly, we measured the changes in the mRNA expression in these cell cultures treated with 0 (control), 1, 10, 100 microM genistein or daidzein over 26 h in serum-free medium (n=3). RESULTS: The mRNA expression of IGF-I was slightly decreased, whereas transcript concentrations of IGF-II and EGF were increased during differentiation compared with the proliferating stage of porcine muscle cell cultures. IGF-I receptor transcripts tended to be increased, whereas EGF receptor mRNA expression remained unchanged from proliferation to differentiation. Genistein and daidzein at 1 microM and 10 microM showed no effects on the mRNA expression of these genes, neither in proliferating nor in differentiating cells. However, high-concentrated isoflavones (100 microM) decreased the mRNA expression of IGF-I receptor and of the growth factors examined. CONCLUSIONS: The present study confirms the role of the IGF and EGF system in proliferation and differentiation of skeletal muscle cell culture especially under serum-free culture conditions. Furthermore, the results of this in vitro-study suggest that there is no effect of isoflavones at concentrations resulting from dietary consumption (1 and 10 microM) on IGF- and EGF-associated gene expression in porcine skeletal muscle tissue. Genistein and daidzein at high concentration (100 microM) reduced the mRNA expression of the IGF-I receptor and the growth factors examined, and therefore, may modify their autocrine and paracrine actions in skeletal muscle tissue.

Effects of dietary isoflavones on proliferation and DNA integrity of myoblasts derived from newborn piglets.

Soy-based formulas are consumed by growing numbers of infants and used as regular food supplements in livestock production. Moreover, constituent dietary phytoestrogens may compete with endogenous estrogens and affect individual growth. This study aimed to investigate the in vitro effects of isoflavones in comparison with estrogens on the proliferation of porcine satellite cells derived from neonatal muscle. After 7 h of exposure in serum-free medium, 17beta-estradiol (1 nM, 1 microM), estrone (1 microM), and daidzein (1, 100 microM) slightly decreased whereas 100 microM genistein substantially lowered DNA synthesis. Declines in DNA amount were observed with genistein (1, 100 microM) and daidzein (100 microM). After 26 h of exposure, 100 microM genistein reduced DNA synthesis, whereas it was increased by 10 microM genistein and 10 and 100 microM daidzein. In the case of 10 microM genistein and 100 microM daidzein, these increases apparently resulted from the repair of damaged DNA. Genistein and daidzein (100 microM) reduced protein synthesis, caused a G2/M phase block, and decreased DNA amount in association with higher rates of cell death partially resulting from apoptosis. Conclusively, isoflavones at concentrations of greater than 1 muM act as inhibitors of porcine skeletal muscle cell proliferation.

Evidence for estrogen receptor alpha and beta expression in skeletal muscle of pigs.

Recent research suggests that estrogen receptors (ERs) are of significance in skeletal muscle function. The aim of the present study was to investigate, whether ERalpha and ERbeta are expressed in different porcine skeletal muscles and in satellite cells derived from semimembranosus muscle (SM) at the protein and mRNA level. Immunohistochemistry demonstrated positive staining for ERalpha in the nuclei of skeletal muscle cells, while the ERbeta stain showed positive signals in nuclei and cytoplasm of skeletal myofibers and myoblasts derived from satellite cells. Additionally, a weak expression of both ER subtypes was seen in skeletal muscle tissue and SM satellite cells with Western blot analysis. A clear expression of the ERalpha mRNA and a weak expression of the ERbeta mRNA was seen in skeletal muscle tissue and SM satellite cell cultures, as determined by reverse transcription (RT)-PCR. The present study shows for the first time that both ERalpha and ERbeta are expressed in porcine skeletal muscle, which, consequently, could be considered as a target tissue for estrogens or estrogen-like compounds. However, more detailed studies on the functional impact of both receptor subtypes in skeletal muscle are necessary. The porcine SM satellite cell culture provides a suitable in vitro model to investigate estrogenic effects on pig skeletal muscle.

Long-term growth selection of mice changes the intrinsic susceptibility of myogenic cells to apoptosis.

Myogenic cells were derived from mice long-term selected over 70 generations for high 6-weeks body weight (DU-6) and from unselected control mice (DU-Ks). The cells were grown in medium with 10% foetal bovine serum (FBS) for 8 days or transferred to low serum conditions (1% FBS) at days 4 and 6 of cultivation, respectively, and maintained for two further days. In both cell lines, serum reduction induced decreases in DNA and protein contents, and in DNA synthesis rate. It also triggered apoptotic cell death as demonstrated by increased DNA strand breaks and expression of active caspase-3. Concomitantly, the anti-apoptotic protein bcl-2 was enhanced. The basal frequency of apoptotic cells decreased with time of cultivation in both lines and was lower in DU-6 than in DU-Ks cells. However, the increase in apoptosis induced by serum reduction was more pronounced in DU-6 than in DU-Ks cells and did not differ between the time points of serum reduction. The results suggest that growth selection decreases the basal apoptosis frequency of muscle satellite cells under normal supply, but enhances the intrinsic susceptibility to growth factor withdrawal by serum deprivation as a severe apoptotic stimulus. Furthermore, the apoptotic response to growth factor withdrawal seems to be largely independent of the stage of myogenic development.

Susceptibility to apoptosis in different murine muscle cell lines.

Objective of the study was to investigate growth characteristics and susceptibility to apoptosis in different murine muscle cell lines (selected for high body weight, DU-6; randomly mated control, DU-Ks; immortalized myoblast cell line, C2C12). Apoptosis was induced by serum deprivation. At days 4, 5, and 6 of cultivation, protein, DNA and the frequency of apoptotic cells were determined. Until day 4, C2C12 accumulated more DNA and protein compared with DU-Ks and DU-6, while exhibiting a lower percentage of apoptotic cells. Serum deprivation impaired the growth of each cell line. C2C12 continued to accumulate DNA and protein after serum deprivation, whereas reductions, indicative of cell death, were apparent in DU-Ks and DU-6. Serum deprivation did not enhance apoptosis in C2C12. Higher percentages of apoptosis were observed in DU-Ks and DU-6 after 2 days of serum deprivation with greater responsiveness of DU-6 to serum deprivation. The results suggest that cell loss in response to serum deprivation is in part due to induction of apoptosis. C2C12 are less sensitive to sub-optimal culture conditions compared with DU-Ks and DU-6 which are at a closer distance to the in vivo status. Moreover, long-term selection for growth decreases the basic frequency of apoptosis of muscle satellite cells, but increases their susceptibility to apoptosis induction.