Low and high dietary protein:carbohydrate ratios during pregnancy affect materno-fetal glucose metabolism in pigs.

Inadequate dietary protein during pregnancy causes intrauterine growth retardation. Whether this is related to altered maternal and fetal glucose metabolism was examined in pregnant sows comparing a high-protein:low-carbohydrate diet (HP-LC; 30% protein, 39% carbohydrates) with a moderately low-protein:high-carbohydrate diet (LP-HC; 6.5% protein, 68% carbohydrates) and the isoenergetic standard diet (ST; 12.1% protein, 60% carbohydrates). During late pregnancy, maternal and umbilical glucose metabolism and fetal hepatic mRNA expression of gluconeogenic enzymes were examined. During an i.v. glucose tolerance test (IVGTT), the LP-HC-fed sows had lower insulin concentrations and area under the curve (AUC), and higher glucose:insulin ratios than the ST- and the HP-LC-fed sows (P < 0.05). Insulin sensitivity and glucose clearance were higher in the LP-HC sows compared with ST sows (P < 0.05). Glucagon concentrations during postabsorptive conditions and IVGTT, and glucose AUC during IVGTT, were higher in the HP-LC group compared with the other groups (P < 0.001). (13)C glucose oxidation was lower in the HP-LC sows than in the ST and LP-HC sows (P < 0.05). The HP-LC fetuses were lighter and had a higher brain:liver ratio than the ST group (P < 0.05). The umbilical arterial inositol concentration was greater in the HP-LC group (P < 0.05) and overall small fetuses (230-572 g) had higher values than medium and heavy fetuses (≥573 g) (P < 0.05). Placental lactate release was lower in the LP-HC group than in the ST group (P < 0.05). Fetal glucose extraction tended to be lower in the LP-HC group than in the ST group (P = 0.07). In the HP-LC and LP-HC fetuses, hepatic mRNA expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC) was higher than in the ST fetuses (P < 0.05). In conclusion, the HP-LC and LP-HC sows adapted by reducing glucose turnover and oxidation and having higher glucose utilization, respectively. The HP-LC and LP-HC fetuses adapted via prematurely expressed hepatic gluconeogenic enzymes.

Enhanced sensitivity of skeletal muscle growth in offspring of mice long-term selected for high body mass in response to a maternal high-protein/low-carbohydrate diet during lactation.

AIM: To investigate the effects of a high-protein/low-carbohydrate diet fed to mice of different genotypes during pregnancy and/or lactation on offspring skeletal muscle growth and metabolism. METHODS: Pregnant mice from strains selected for high body mass (DU6) or endurance running performance (DUhLB) and from an unselected control strain (DUK) were fed iso-energetic diets containing 20 % (C) or 40 % protein and low carbohydrate (HP) from mating to weaning at day 21 of age. At birth, offspring were cross-fostered resulting in different exposure to maternal prenatal-preweaning diets (C-C, HP-C, C-HP, HP-HP). Rectus femoris muscle of male mice (n = 291) was examined at day 23, 44, 181 and 396 of age for cellular growth and metabolism. RESULTS: At day 23 of age, body and muscle growth was retarded by 30-40 % (P < 0.0001) in response to the C-HP and HP-HP, but not to the HP-C diet, due to reduced fibre size (P < 0.0001) but not fibre number. DNA was highly reduced in DU6, less in DUhLB, but not in DUK muscle (strain × diet; P < 0.0001). Despite some compensation, muscle growth was still impaired (P < 0.001) in adulthood (day 44; day 181), but at senescence only in DU6 mice (strain × diet; P < 0.05). Only at weaning, isocitrate and lactate dehydrogenase activities were increased or decreased (P < 0.0001), respectively, without influence on fibre type composition. CONCLUSION: A high-protein/low-carbohydrate diet fed to dams during lactation, but not during pregnancy, retards skeletal muscle growth in offspring with greater response of a heavy, obese compared with a physically fit and a control genotype and causes a transient shift towards oxidative versus glycolytic muscle metabolism.

Influence of maternal low protein diet during pregnancy on hepatic gene expression signature in juvenile female porcine offspring.

SCOPE: Epidemiological and experimental evidence indicates that maternal nutrition status contributes to long-term changes in the metabolic phenotype of the offspring, a process known as fetal programming. METHODS AND RESULTS: We have used a swine model (Sus scrofa) to analyze consequences of a maternal low protein diet (about 50% of control) during pregnancy on hepatic lipid metabolism and genome-wide hepatic gene expression profile of juvenile female offspring (mean age 85 days). We found 318 S. scrofa genes to be differentially expressed in the liver at age 85 days. In the low protein offspring group key genes of fatty acid de novo synthesis were downregulated whereas several genes of lipolysis and phospholipid biosynthesis were upregulated. qRT-PCR analysis of selected genes verified microarray data and revealed linear correlations between gene expression levels and slaughter weight. Hepatic cholesterol 7α hydroxylase protein expression tended to be lower in the low protein group. Total lipid and triglyceride content and fatty acid composition of total lipids were not different between groups. CONCLUSION: A maternal low protein diet during pregnancy induces a distinct hepatic gene expression signature in juvenile female pigs which was not translated into phenotypical changes of liver lipid metabolism.

Effects of inadequate maternal dietary protein:carbohydrate ratios during pregnancy on offspring immunity in pigs.

BACKGROUND: Inadequate nutrition in utero may retard foetal growth and alter physiological development of offspring. This study investigated the effects of low and high protein diets fed to primiparous German Landrace sows throughout pregnancy on the immune function of their offspring at different ages. Sows were fed diets with adequate (AP, 12.1%; n = 13), low (LP, 6.5%; n = 15), or high (HP, 30%; n = 14) protein content, made isoenergetic by varying carbohydrate levels. Cortisol, total protein and immunoglobulin (IgG, IgM, IgA) concentrations were measured in the blood of sows over the course of pregnancy. Cortisol, total protein, immunoglobulins, lymphocyte proliferation, immune cell counts, and cytokines were assessed in the blood of offspring at baseline and under challenging conditions (weaning; lipopolysaccharide (LPS) administration). RESULTS: In sows, the LP diet increased cortisol (P < 0.05) and decreased protein levels (P < 0.01) at the end of pregnancy. Immunoglobulin concentrations were decreased in LP (IgA) and HP piglets (IgG, IgM and IgA) on the first day of life (P < 0.05), whereas the number of lymphocytes and mitogen-induced lymphocyte proliferation of the piglets were unaffected by the maternal diet. Mortality during the suckling period was higher in LP piglets compared with AP and HP offspring (P < 0.01). Furthermore, LP piglets showed an elevated cortisol response to weaning, and in HP piglets, the CD4+ cell percentage and the CD4+/CD8+ ratio increased after weaning (P < 0.05). The lipopolysaccharide-induced rise of IL-6 was higher in LP (P = 0.09) and HP (P < 0.01) compared with AP piglets, and LP piglets displayed higher IL-10 levels than AP piglets (P < 0.05). CONCLUSIONS: Our results indicate that both low and high protein:carbohydrate ratios in the diet of pregnant sows can induce short-term as well as long-lasting effects on immune competence in piglets that may have serious consequences for host defence against bacterial pathogens.

Effects of leptin and adiponectin on proliferation and protein metabolism of porcine myoblasts.

The aim of this study was to show the abundance of leptin and adiponectin receptors (LEPR, ADIPOR1, ADIPOR2) and to determine the direct effects of leptin and adiponectin on the in vitro growth of porcine skeletal muscle cells. ADIPOR1 and ADIPOR2 were abundant at mRNA and protein level in proliferating and differentiating myoblast cultures derived from semimembranosus and semitendinosus muscles of newborn piglets, whereas LEPR expression was close to the detection limit. Adiponectin (10, 20, 40 µg/ml) attenuated the proliferation of porcine myoblasts, measured as [(3)H]-thymidine incorporation and real-time monitoring of the cells in response to 24- and 48-h exposure, in a dose-dependent manner. This effect resulted from suppressed basic fibroblast growth factor (bFGF)-mediated stimulation of DNA synthesis in serum-free medium (SFM) containing bFGF. No effects of leptin (5, 10, 20, 40, 80 ng/ml) on myoblast proliferation in SFM were detectable. Neither leptin nor adiponectin altered protein synthesis and degradation in differentiating porcine myoblasts cultured in SFM. The results on receptor abundance suggest that porcine skeletal muscle cells may be sensitive to adiponectin and leptin. However, except via inhibitory interaction of adiponectin with bFGF, these adipokines appear not to affect in vitro proliferation and protein metabolism of porcine muscle cells directly under serum-free culture conditions.

High-protein-low-carbohydrate diet during pregnancy alters maternal plasma amino acid concentration and placental amino acid extraction but not fetal plasma amino acids in pigs.

A high protein-low-carbohydrate diet during pregnancy can cause intra-uterine growth restriction. However, its impact during pregnancy on maternal, umbilical and fetal plasma amino acid (AA) profiles is unknown. A maternal high-protein (30 %)-low-carbohydrate (HP-LC) diet was compared with isoenergetic standard (12·1 % crude protein; ST) and low-protein (6·5 %)-high-carbohydrate (LP-HC) diets fed to nulliparous pregnant sows to examine changes in AA concentrations in maternal, venous and arterial umbilical and fetal plasma in mid and late pregnancy. At 64 and 94 days of pregnancy (dp), sows underwent Caesarean section, and maternal, umbilical and fetal plasma samples were collected. The HP-LC diet mainly affected maternal plasma AA concentrations. Plasma concentrations of Ile and Val were increased and those of Ala, Glu and Gly were decreased (P ≤ 0·05) in HP-LC compared with ST sows at 64 and 94 dp. The LP-HC diet decreased fetal plasma Glu concentration compared with the ST diet at 94 dp. Substantial AA catabolism was reflected by increased (P ≤ 0·05) maternal and fetal plasma urea concentrations with the HP-LC compared with the ST and LP-HC diets at 94 dp. Fractional placental extraction of Val was higher whereas those of Ala, Gln and Glu were lower in the HP-LC compared with the ST sows at 64 and 94 dp (P ≤ 0·05). Reduced fetal mass at 94 dp was accompanied by reduced fetal extraction of Lys and Pro in the HP-LC group (P ≤ 0·05). In conclusion, a maternal HP-LC diet during pregnancy altered maternal plasma composition of many AA and modified placental AA extraction to compensate for imbalanced maternal nutrient intake.

Intrauterine growth retarded progeny of pregnant sows fed high protein:low carbohydrate diet is related to metabolic energy deficit.

High and low protein diets fed to pregnant adolescent sows led to intrauterine growth retardation (IUGR). To explore underlying mechanisms, sow plasma metabolite and hormone concentrations were analyzed during different pregnancy stages and correlated with litter weight (LW) at birth, sow body weight and back fat thickness. Sows were fed diets with low (6.5%, LP), adequate (12.1%, AP), and high (30%, HP) protein levels, made isoenergetic by adjusted carbohydrate content. At -5, 24, 66, and 108 days post coitum (dpc) fasted blood was collected. At 92 dpc, diurnal metabolic profiles were determined. Fasted serum urea and plasma glucagon were higher due to the HP diet. High density lipoprotein cholesterol (HDLC), %HDLC and cortisol were reduced in HP compared with AP sows. Lowest concentrations were observed for serum urea and protein, plasma insulin-like growth factor-I, low density lipoprotein cholesterol, and progesterone in LP compared with AP and HP sows. Fasted plasma glucose, insulin and leptin concentrations were unchanged. Diurnal metabolic profiles showed lower glucose in HP sows whereas non-esterified fatty acids (NEFA) concentrations were higher in HP compared with AP and LP sows. In HP and LP sows, urea concentrations were 300% and 60% of AP sows, respectively. Plasma total cholesterol was higher in LP than in AP and HP sows. In AP sows, LW correlated positively with insulin and insulin/glucose and negatively with glucagon/insulin at 66 dpc, whereas in HP sows LW associated positively with NEFA. In conclusion, IUGR in sows fed high protein:low carbohydrate diet was probably due to glucose and energy deficit whereas in sows with low protein:high carbohydrate diet it was possibly a response to a deficit of indispensable amino acids which impaired lipoprotein metabolism and favored maternal lipid disposal.

Limited and excess protein intake of pregnant gilts differently affects body composition and cellularity of skeletal muscle and subcutaneous adipose tissue of newborn and weanling piglets.

AIM: This study investigated whether dietary protein intake less (50%) or greater (250%) than requirements throughout gestation differently affects offspring body composition and cellular properties of skeletal muscle and subcutaneous adipose tissue (SCAT). METHODS: Primiparous gilts were fed iso-energetic diets containing adequate (22 AP), high (21 HP), or low (19 LP) protein contents. Newborn (n = 166) and weanling piglets cross-fostered to sows fed a standard diet (day 28; n = 83) were examined by morphological, biochemical, histological, and molecular analyses of the body, SCAT, and semitendinosus, longissimus, biceps femoris muscles. RESULTS: Lowered birth weight (BW) in response to the HP and LP diets (p < 0.01) resulted from decreases in all body constituents in LP, and mainly from reduced body fat in HP piglets (p < 0.05). In the light BW class within litters, HP piglets exhibited a greater percentage of muscle tissue (p < 0.05) than LP piglets. Less SCAT mass in HP and LP piglets resulted from reduced (p < 0.05) number, but not the size of adipocytes. The LP diet adversely affected myogenesis and muscular differentiation derived from less (p < 0.01) primary and secondary myofibers, lower creatine kinase activity (p < 0.05), less IGF2 mRNA (p < 0.10), and greater expression of the embryonic myosin heavy chain isoform (p < 0.01). Catch-up growth of LP but not HP pigs until day 28 increased body fat (p = 0.01). Despite compensated muscle growth in LP piglets, the deficit in myofiber number remained. CONCLUSION: Poor intrauterine environment by limited and excess protein supply retards fetal growth, but only limited protein supply impairs myogenesis, persistently restricts muscle growth potential, and favors obesity at infancy.

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.

Potential sources of early-postnatal increase in myofibre number in pig skeletal muscle.

In pigs, myogenesis is a biphasic phenomenon with the formation of primary and secondary fibres. Hyperplasia was reported to be accomplished around 90 days of gestation. However, some studies suggest a substantial increase in the total fibre number (TFN) from birth to weaning by counting fibre number in the muscle cross sections. The aim of this study was to establish in which way TFN increases after birth and whether this increase is imputable to new (tertiary) myofibres and/or fibre elongation. The semitendinosus muscle of 128 piglets was examined at days 1 (n = 63), 7 (n = 12), 21 (n = 12), and 28 (n = 41) of age. TFN was increased at days 7, 21 and 28 of age when compared with day 1 (P < 0.01). From day 1 to 28, TFN increased from 463 × 10(3) to 825 × 10(3). Microscopy of longitudinal and transversal serial sections revealed that at day 7 of age very small fibres expressing the embryonic myosin heavy chain (MyHC) isoform were apparent all over the muscle. In addition, intrafascicular terminations of normal-sized fibres expressed the embryonic MyHC isoform. These data suggest that the TFN in the pig muscle is not fixed at birth and its postnatal increase may be related to both elongation of existing muscle fibres and genesis of tertiary myofibres, mainly between birth and 3 weeks of age.

Hepatic expression of the GH/JAK/STAT/IGF pathway, acute-phase response signalling and complement system are affected in mouse offspring by prenatal and early postnatal exposure to maternal high-protein diet.

PURPOSE: Effects of pre- and early postnatal exposure to maternal high-protein diets are not well understood. Transcription profiling was performed in male mouse offspring exposed to maternal high-protein diet during pregnancy and/or lactation to identify affected hepatic molecular pathways. METHODS: Dams were fed isoenergetic diets with control (20% w/w) or high protein levels (40%). The hepatic expression profiles were evaluated by differential microarray analysis 3 days (d3) and 3 weeks (d21) after birth. Offspring from three different high-protein dietary groups, HP (d3, high-protein diet during pregnancy), HPHP (d21, high-protein diet during pregnancy and lactation) and CHP (d21, control diet during pregnancy and high-protein diet during lactation), were compared with age-matched offspring from dams fed control diet. RESULTS: Offspring body and liver mass of all high-protein groups were decreased. Prenatal high-protein diet affected hepatic expression of genes mapping to the acute response/complement system and the GH/JAK/STAT/IGF signalling pathways. Maternal exposure to high-protein diet during lactation affected hepatic gene expression of the same pathways but additionally affected genes mapping to protein, fatty acid, hexose and pyruvate metabolism. CONCLUSIONS: (1) Genes of the acute response/complement system and GH/JAK/STAT/IGF pathways were down-regulated in offspring of dams exposed to high-protein diets during pregnancy and/or lactation. (2) Genes related to nutrient and energy metabolism, however, were only affected when high-protein diet was administered during lactation. (3) Modulation of the GH/JAK/STAT/IGF pathway might be responsible for reduced body and liver masses by maternal high-protein diet.

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.

Increased fat mass, decreased myofiber size, and a shift to glycolytic muscle metabolism in adolescent male transgenic mice overexpressing IGFBP-2.

To elucidate the functional role of insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) for in vivo skeletal muscle growth and function, skeletal muscle cellularity and metabolism, expression of signal molecules, and body growth and composition were studied in a transgenic mouse model overexpressing IGFBP-2. Postnatal growth rate of transgenic mice was reduced from day 21 of age by 6-8% compared with nontransgenic controls. At 10 wk of age body lean protein and moisture percentages were lower, whereas fat percentage was higher in IGFBP-2 transgenic mice. Muscle weights were reduced (-13% on day 30 of age, -14% on day 72), which resulted from slower growth of myofibers in size but not from decreases in myofiber number. The reduction in muscle mass was associated with lower total DNA, RNA, and protein contents as well as greater DNA/RNA and protein/RNA ratios. The percentage of proliferating (Ki-67-positive) nuclei within myofibers was reduced (3.4 vs. 5.8%) in 30-day-old transgenic mice. These changes were accompanied by slight reductions in specific p44/42 MAPK activity (-18% on day 72) and, surprisingly, by increased levels of phosphorylated Akt (Ser(473)) (+25% on day 30, +66% on day 72). The proportion of white glycolytic fibers (55.9 vs. 53.5%) and the activity of lactate dehydrogenase (+8%) were elevated in 72-day-old transgenic mice. Most of the differences observed between transgenic and nontransgenic mice were more pronounced in males. The results suggest that IGFBP-2 significantly inhibits postnatal skeletal myofiber growth by decreasing myogenic proliferation and protein accretion and enhances glycolytic muscle metabolism.

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.

Establishment and conditions for growth and differentiation of a myoblast cell line derived from the semimembranosus muscle of newborn piglets.

To establish an adequate model to study the proliferation and differentiation of porcine skeletal muscle in response to bioactive compounds, a pool of satellite cells was derived from the semimembranosus muscle (SM) of newborn piglets using a Percoll gradient centrifugation. The final yield amounted to 4.1 x 10(6) cells/g muscle tissue. The percentage of muscle satellite cells has been determined by immunostaining for desmin and subsequent fluorescence analysis by flow cytometry, which revealed 95% of desmin-positive cells. For proliferation studies, satellite cell born myoblasts were seeded in gelatin-coated 96-well microplates at about 5 x 10(3) cells per well. Cells were grown for 1 day in MEMalpha plus 10% fetal bovine serum (FBS) and 10% horse serum (HS), followed by 2 d cultivation in serum-free growth medium. For differentiation studies, myoblasts were cultured in matrigel-coated 24-well plates for 4 d with growth medium containing 10% FBS and 10% HS. At 80% confluence, cells were grown for 24 h in medium plus 10% FBS and 1 microM insulin to initiate differentiation. Subsequently, the cells were cultured in serum-free differentiation medium (SFDM) for 3 d to form myotubes. Cultures reached a maximum fusion rate of approximately 20% after 96 h. By establishing this culture system, we provide an advanced and appropriate in vitro model to study porcine skeletal muscle cell growth and differentiation including the responses to various bioactive compounds.

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.

Muscle energy metabolism: structural and functional features in different types of porcine striated muscles.

Striated muscles exhibit a wide range of metabolic activity levels. Heart and diaphragm are muscles with continuous contractile performance, which requires life-long function. In contrast, skeletal muscles like longissimus muscle can adapt metabolism from resting to different stages of exercise. The aim of this study was to compare the morphological features of these three muscles and the expression of genes that are important for energy metabolism. Therefore, histochemical studies were performed for determination of muscle fibre type composition. Oxidative and glycolytic capacity was assessed by measuring isocitrate dehydrogenase (ICDH) and lactate dehydrogenase (LDH) activities. The mRNA expression of glucose transporter 4 (GLUT 4), growth hormone receptor (GHR) and AMP-activated kinase (AMPK) alpha(1) and alpha(2) subunits was studied by semiquantitative Northern blotting. Heart, and to a slightly lesser extent diaphragm were highly oxidative muscles characterised by high expression of oxidative muscle fibres and ICDH activity. Longissimus muscle exhibited the highest percentage of glycolytic fibres and LDH activity. GLUT 4 mRNA was lowest in heart reflecting the dependency of heart muscle on fatty acids as major energy source. Higher expression of GLUT 4 in diaphragm indicated that glucose is an important energy substrate in this oxidative muscle. Highest GLUT 4 expression in longissimus should be essential for the refilling of glycogen stores after exercise. AMPK subunits, which are important stimulators of GLUT 4 protein insertion into the sarcolemma, are also highest expressed in longissimus muscle indicating the strong capacity to adapt energy metabolism to large changes in energy demand. Interestingly, AMPK alpha(1) subunit expression on protein level is strongly restricted to muscle fibres containing type I myosin in this muscle. GHR mRNA expression was also highest in longissimus muscle indicating that an enhanced effect of growth hormone, which is described to be diabetogenic, could be involved in the lower insulin sensitivity of glycolytic muscles.