Maternal protein restriction induce skeletal muscle changes without altering the MRFs MyoD and myogenin expression in offspring

Stimuli during pregnancy, such as protein restriction, can affect morphophysiological parameters in the offspring with consequences in adulthood. The phenomenon known as fetal programming can cause short- and long-term changes in the skeletal muscle phenotype. We investigated the morphology and the myogenic regulatory factors (MRFs) MyoD and myogenin expression in soleus, SOL; oxidative and slow twitching and in extensor digitorum longus, EDL; glycolytic and fast twitching muscles in the offspring of dams subjected to protein restriction during pregnancy. Four groups of male Wistar offspring rats were studied. Offspring from dams fed a low-protein diet (6?% protein, LP) and normal protein diet (17?% protein, NP) were euthanized at 30 and 112?days old, and their muscles were removed and kept at ?80?°C. Muscles histological sections (8?μm) were submitted to a myofibrillar adenosine triphosphatase histochemistry reaction for morphometric analysis. Gene and protein expression levels of MyoD and myogenin were determined by RT-qPCR and western blotting. The major findings observed were distinct patterns of morphological changes in SOL and EDL muscles in LP offspring at 30 and 112?days old without changes in MRFs MyoD and myogenin expression. Our results indicate that maternal protein restriction followed by normal diet after birth induced morphological changes in muscles with distinct morphofunctional characteristics over the long term, but did not alter the MRFs MyoD and myogenin expression. Further studies are necessary to better understand the mechanisms underlying the maternal protein restriction response on skeletal muscle.     

Dietary protein deficiency in pregnant mice and offspring

Previous studies suggest an association between dermal contact hypersensitivity and preterm delivery. We hypothesized that dietary protein deficiency produces cell-mediated immune hypersensitivity in pregnant animals and their offspring akin to those known to produce tissue damage. We compared the effects of feeding a 20% protein diet (controls) to those of feeding a 10% protein (deficient) diet ad libitum to pregnant BALB/c mice. We measured dermal contact sensitivity to 2,4-dinitrofluorobenzene (DNFB) by the increment in ear skin thickness (swelling) 72 h after immunization and parity by the number of viable pups delivered. Dams fed the protein-deficient diet ingested less food, gained less weight and delivered fewer viable pups than the dams fed the control diet. Greater DNFB-stimulated increment in ear skin thickness was found in the protein-deficient mothers and in their offspring than in the control mothers and their offspring. We conclude that dietary protein deficiency limits parity and induces immune hypersensitivity. These findings suggest the potential for dietary protein deficiency to activate a T-cell-mediated branch of the immune response that may put pregnant animals at risk for preterm delivery.     

Magnesium deficiency in pregnant rats alters methylation of specific cytosines in the hepatic hydroxysteroid dehydrogenase-2 promoter of the offspring

Prenatal under-nutrition involves changes in the epigenetic regulation of specific genes. Maternal magnesium (Mg) deficiency affects maternal glucocorticoid metabolism, but the mechanisms underlying changes in glucocorticoid homeostasis of offspring are not well understood. In this study, we investigated the effects of feeding pregnant rats a Mg-deficient diet (0.003% magnesium) on the methylation of cytosine-guanine (CpG) dinucleotides in hepatic glucocorticoid genes of neonatal offspring, compared with controls (0.082% magnesium). Methylation of CpG dinucleotides in the peroxisome proliferator-activated receptor α (Ppara), glucocorticoid receptor (Nr3c1) and 11β-hydroxysteroid dehydrogenase-2 (Hsd11b2) promoters in the liver were measured by pyrosequencing. Quantitative real-time PCR was used to assess hepatic mRNA expression of each gene. Mean methylation of the Hsd11b2 promoter in the Mg-deficient offspring (33.2%) was higher than in controls (10.4%). This was due to a specific increase at CpG dinucleotides 1 (20.0% vs. control 10.1%), 2 (58.8% vs. 17.0%), 3 (29.7% vs. 6.2%) and 4 (38.7% vs. 8.8%) (p < 0.05). Ppara and Nr3c1 methylation status and expression did not differ between the groups. No significant difference was noted between male and female pups, which were equally represented. Therefore, a Mg-deficient diet alters glucocorticoid metabolism, predicting higher hepatic intracellular glucocorticoid concentrations, and is possibly a key mechanism that induces the metabolic complications of Mg deficiency.     

Gestational low protein diet in the rat mediates Igf2 gene expression in male offspring via altered hepatic DNA methylation

Biological responses to environmental stress, including nutrient limitation are mediated in part by epigenetic modifications including DNA methylation. Insulin-like growth factor II (Igf2) and H19 are subject to epigenetic modifications leading to genomic imprinting. The present study was designed to test the effect of maternal low protein diet on the Igf2/H19 locus in offspring. Pregnant Sprague-Dawley rats were fed diets containing 180 g/kg casein (control) or 90 g/kg (LP) casein with either 1 mg/kg (LP) or 3 mg/kg folic acid (LPF). LP diet increased Igf2 and H19 gene expression in the liver of day 0 male offspring and the addition of folic acid reduced the mRNA level in LPF rats to that of the control group. DNA methylation in Imprinting Control Region (ICR) of Igf2/H19 locus increased significantly following maternal LP diet but rats fed the LPF diet did not exhibit the hypermethylation. The Differential Methylation Region 2 (DMR2) did not show any change in methylation in either LP or LPF rats. The expression of Dnmt1 and Dnmt3a, the members of DNA methyltransferase family, and methyl CpG-binding domain 2 (Mbd2) was significantly increased following the maternal LP diet but did not differ between the control and LPF group. There is a strong correlation between methylation of ICR with the expression of Igf2 and H19. These results suggested that maternal exposure to a low protein diet and folic acid during gestation alters gene expression of Igf2 and H19 in the liver by regulating the DNA methylation of these genes. The DNA methyltransferase machinery may be involved into the programming of imprinted genes through the imprinted control region.     

Effect of protein or energy restriction during late gestation on hormonal and metabolic status in pregnant goats and postnatal male offspring

The objective of this study was to investigate the effects of maternal protein or energy restriction on hormonal and metabolic status of pregnant goats during late gestation and their postnatal male kids. Forty-five pregnant goats were fed a control (CON), 40% protein-restricted (PR) or 40% energy-restricted (ER) diet from 90 days of gestation until parturition. Plasma of mothers (90, 125 and 145 days of gestation) and kids (6 weeks of age) were sampled to determine metabolites and hormones. Glucose concentration for pregnant goats subjected to PR or ER was less (P<0.001) than that of CON goats at 125 and 145 days of gestation. However, plasma nonesterified fatty acids concentration was greater (P<0.01) at 125 and 145 days for PR and ER than CON. Protein restriction increased (P<0.01) maternal cortisol concentration by 145 days of gestation, and ER decreased (P<0.01) maternal insulin concentration at 125 days of gestation. Moreover, maternal amino acid (AA) concentrations were affected by nutritional restriction, with greater (P<0.05) total AA (TAA) and nonessential AA (NEAA) for PR goats but less (P<0.05) TAA and NEAA for ER goats at 125 days of gestation. After 6 weeks of nutritional recovery, plasma concentrations of most metabolic and hormonal parameters in restricted kids were similar to CON kids, except for reduced (P<0.05) insulin concentration in ER, and reduced (P<0.05) Asp concentration in PR and ER kids. These results provide information on potential metabolic mechanisms responsible for fetal programming.     

Differential hepatic lobar gene expression in offspring exposed to altered maternal dietary protein intake

Increased plasma fibrinogen concentrations are a recognized risk factor for coronary heart disease, and increased fibrinogen levels in adults are associated with parameters of reduced early growth. We studied fibrinogen gene expression in adult offspring of dams fed either a 20% (control) or an 8% protein diet [maternal low-protein (MLP) rats] during pregnancy and lactation and determined whether any effects were consistent between left and right liver lobes, since the fetal liver has a unique blood supply that produces differential stimuli to the left and right lobes. In MLP offspring, there was a reduction in all three fibrinogen mRNA copy numbers in the left liver lobe [left vs. right lobes for alpha-, beta-, and gamma-fibrinogen (x10(6) copies/ng total RNA): 8.04 vs. 23.16, P<0.001; 4.74 vs. 13.07, P<0.001; and 4.61 vs. 16.38, P = 0.007, respectively], with a parallel reduction in fibrinogen concentration in the left liver lobe (8.53+/-0.33 vs. 10.41 +/-0.65 arbitrary units, P = 0.014, left and right lobes, respectively). No such effect was observed in offspring of control dams. To investigate the underlying mechanism, glucocorticoid receptor function and mRNA levels were studied, since expression of fibrinogen genes is regulated by glucocorticoid hormones. The binding affinity of the high-affinity glucocorticoid receptor was reduced only in the left liver lobe of the MLP offspring (P = 0.02, left. vs. right), with a parallel reduction in this lobe in glucocorticoid receptor mRNA level (P = 0.006, left vs. right). In conclusion, maternal dietary protein restriction reduces fibrinogen gene expression, fibrinogen protein, and mRNA level and binding affinity of glucocorticoid receptors only in the left liver lobe of the adult offspring.     

Disruption of the acyl-CoA-binding protein gene delays hepatic adaptation to metabolic changes at weaning

The acyl-CoA-binding protein (ACBP)/diazepam binding inhibitor is an intracellular protein that binds C(14)-C(22) acyl-CoA esters and is thought to act as an acyl-CoA transporter. In vitro analyses have indicated that ACBP can transport acyl-CoA esters between different enzymatic systems; however, little is known about the in vivo function in mammalian cells. We have generated mice with targeted disruption of ACBP (ACBP(-/-)). These mice are viable and fertile and develop normally. However, around weaning, the ACBP(-/-) mice go through a crisis with overall weakness and a slightly decreased growth rate. Using microarray analysis, we show that the liver of ACBP(-/-) mice displays a significantly delayed adaptation to weaning with late induction of target genes of the sterol regulatory element-binding protein (SREBP) family. As a result, hepatic de novo cholesterogenesis is decreased at weaning. The delayed induction of SREBP target genes around weaning is caused by a compromised processing and decreased expression of SREBP precursors, leading to reduced binding of SREBP to target sites in chromatin. In conclusion, lack of ACBP interferes with the normal metabolic adaptation to weaning and leads to delayed induction of the lipogenic gene program in the liver.     

Maternal protein restriction leads to early life alterations in the expression of key molecules involved in the aging process in rat offspring

Recent findings demonstrate that nutrition during the fetal and neonatal periods can affect the life span of an organism. Our previous studies in rodents using a maternal low protein diet have shown that limiting protein and growth during lactation [postnatal low protein (PLP group)] increases longevity, while in utero growth restriction (IUGR) followed by "catch up growth" (recuperated group) shortens life span. The aim of this study was to investigate mechanisms in early postnatal life that could underlie these substantial differences in longevity. At weaning, PLP animals had improved insulin sensitivity as suggested by lower concentrations of insulin required to maintain concentrations of glucose similar to those of the control group and significant upregulation of insulin receptor-beta, IGF-1 receptor, Akt1, Akt2, and Akt phosphorylated at Ser 473 in the kidney. These animals also had significantly increased SIRT1 (mammalian sirtuin) expression. Expression of the antioxidant enzymes catalase, CuZnSOD, and glutathione peroxidase-1 was elevated in these animals. In contrast, recuperated animals had a significantly increased fasting glucose concentration, while insulin levels remained comparable to those of the control group suggesting relative insulin resistance. MnSOD expression was increased in these animals. These data suggest that early nutrition can lead to alterations in insulin sensitivity and antioxidant capacity very early in life, which may influence life span.     

Maternal protein restriction elevates cholesterol in adult rat offspring due to repressive changes in histone modifications at the cholesterol 7alpha-hydroxylase promoter

Adverse events in utero, such as intrauterine growth restriction (IUGR), can permanently alter epigenetic mechanisms leading to the metabolic syndrome, which encompasses a variety of symptoms including augmented cholesterol. The major site for cholesterol homeostasis occurs via the actions of hepatic cholesterol 7α-hydroxylase (Cyp7a1), which catabolizes cholesterol to bile acids. To determine whether posttranslational histone modifications influence the long-term expression of Cyp7a1 in IUGR, we used a protein restriction model in rats. This diet during pregnancy and lactation led to IUGR offspring with decreased liver to body weight ratios, followed by increased circulating and hepatic cholesterol levels in both sexes at d 21 and exclusively in the male offspring at d 130. The augmented cholesterol was associated with decreases in the expression of Cyp7a1. Chromatin immunoprecipitation revealed that this was concomitant with diminished acetylation and enhanced methylation of histone H3 lysine 9 [K9,14], markers of chromatin silencing, surrounding the promoter region of Cyp7a1. These epigenetic modifications originate in part due to dietary-induced decreases in fetal hepatic Jmjd2a expression, a histone H3 [K9] demethylase. Collectively, these findings suggest that the augmented cholesterol observed in low-protein diet-derived offspring is due to permanent repressive posttranslational histone modifications at the promoter of Cyp7a1. Moreover, this is the first study to demonstrate that maternal undernutrition leads to long-term cholesterol dysregulation in the offspring via epigenetic mechanisms.     

Maternal consumption of low-isoflavone soy protein isolate alters hepatic gene expression and liver development in rat offspring

In utero environment is known to affect fetal development. Especially, the distinct fetal programming of carcinogenesis was reported in offspring exposed to maternal diets containing soy protein isolate (SPI) or genistein. Therefore, we investigated whether maternal consumption of low-isoflavone SPI or genistein alters hepatic gene expression and liver development in rat offspring. Female Sprague–Dawley rats were fed a casein diet, a low-isoflavone SPI diet or a casein diet supplemented with genistein (250 mg/kg diet) for 2 weeks before mating and throughout pregnancy and lactation. Male offspring were studied on postnatal day 21 (CAS, SPI and GEN groups). Among 965 differentially expressed hepatic genes related to maternal diet (P<.05), the expression of 590 was significantly different between CAS and SPI groups. Conversely, the expression of 88 genes was significantly different between CAS and GEN groups. Especially, genes involved in drug metabolism were significantly affected by the maternal diet. SPI group showed increased cell proliferation, reduced apoptosis and activation of the mTOR pathway, which may contribute to a higher relative liver weight compared to other groups. We observed higher serum homocysteine levels and lower global and CpG site-specific DNA methylation of Gadd45b, a gene involved in cell proliferation and apoptosis, in SPI group compared to CAS group. Maternal SPI diet also reduced histone H3-Lysine 9 (H3K9) trimethylation and increased H3K9 acetylation in offspring. These results demonstrate that maternal consumption of a low-isoflavone SPI diet alters the hepatic gene expression profile and liver development in offspring possibly by epigenetic processes.     

Effects of feed restriction during pregnancy on maternal reproductive outcome,foetal hepatic IGF gene expression and offspring performance in the rabbit

Primiparous female rabbits have high nutritional requirements and, while it is recommended that they are subjected to an extensive reproductive rhythm, this could lead to overweight, affecting reproductive outcomes. We hypothesised that restricting food intake during the less energetic period of gestation could improve reproductive outcome without impairing offspring viability. This study compares two groups of primiparous rabbit does in an extensive reproductive programme, one in which feed was restricted from Day 0 to Day 21 of gestation (R021), and another in which does were fed ad libitum (control) throughout pregnancy. The mother and offspring variables compared were (1) mother reproductive outcomes at the time points pre-implantation (Day 3 postartificial insemination [AI]), preterm (Day 28 post-AI) and birth; and (2) the prenatal offspring characteristic IGF system gene expression in foetal liver, liver fibrosis and foetus sex ratio, and postnatal factor viability and growth at birth, and survival and growth until weaning. Feed restriction did not affect the conception rate, embryo survival, or the number of morulae and blastocysts recovered at Day 3 post-AI. Preterm placenta size and efficiency were similar in the two groups. However, both implantation rate (P < 0.001) and the number of foetuses (P = 0.05) were higher in the R021 mothers than controls, while there was no difference in foetal viability. Foetal size and weight, the weights of most organs, organ weight/BW ratios and sex ratio were unaffected by feed restriction; these variables were only affected by uterine position (P < 0.05). Conversely, in the R021 does, foetal liver IGBP1 and IGF2 gene expression were dysregulated despite no liver fibrosis and a normal liver structure. No effects of restricted feed intake were produced on maternal fertility, prolificacy, or offspring birth weight, but control females weaned more kits. Litter weight and mortality rate during the lactation period were also unaffected. In conclusion, pre-implantation events and foetal development were unaffected by feed restriction. While some genes of the foetal hepatic IGF system were dysregulated during pregnancy, liver morphology appeared normal, and the growth of foetuses and kits until weaning was unmodified. This strategy of feed restriction in extensive reproductive rhythms seems to have no significant adverse effects on dam reproductive outcome or offspring growth and viability until weaning.     

Dietary protein restriction during lactation in primiparous sows with different live weights at farrowing: I. Consequences on sow metabolic status and litter growth

The hypothesis that the restriction of dietary protein during lactation has different impacts on sow metabolic status and milk production according to body weight was evaluated. From 5-months of age until farrowing, the gilts were fed to achieve body weights of 180 or 240 kg at farrowing. At this time, 38 sows were assigned to one of three groups: " 180 kg" sows not restricted in dietary protein during lactation (180CP); "180 kg" restricted in protein (180LP), or "240 kg" sows restricted in protein (240LP). Catheters were fitted in the jugular vein of 24 sows and serial blood samples were collected 1 d before and 1 d after weaning. Amongst the protein-restricted animals, heavy sows (240LP) had a smaller appetite than light sows in early lactation, resulting in lower energy and protein intakes in the 240LP than in the 180LP sows. Body protein losses were 8, 11 and 13.5% of calculated body protein mass at farrowing in the 180CP, 180LP and 240LP sows, respectively. At the end of lactation, IGF-I concentrations were lower in the 180LP than in the sows from the other groups, probably because of the uncoupling between GH and IGF-I secretions. Low IGF-I concentrations likely promote lean tissue mobilization. Glucose and insulin profiles suggested an insulin resistance state in the 240LP sows compared with the 180LP sows, which may explain, at least in part, the lower feed intake and body reserve mobilization in these sows. Plasma pre- and post-prandial concentrations of amino acids in late lactation differed among the three treatment groups. Throughout lactation, litters from the 180LP and 240LP sows had a slower growth rate than litters from sows which were not restricted, suggesting that endogenous protein mobilization throughout lactation does not completely compensate for a low protein intake.     

Dietary protein restriction during lactation in primiparous sows with different live weights at farrowing: II. Consequences on reproductive performance and interactions with metabolic status

The hypothesis that the restriction of dietary protein during lactation has different impacts on reproductive performance in light and heavy sows at farrowing was investigated, as well as the relationships between reproductive parameters and sow metabolic data. At farrowing, 38 primiparous sows were assigned to one of three groups: sows weighing 180 kg not restricted in dietary protein during lactation (180CP); sows weighing 180 or 240 kg restricted in protein (180LP and 240LP). Twenty-four sows were catheterized and serial blood samples were collected 1 d before and 1 d after weaning. The sows were inseminated at the first estrus after weaning and slaughtered at d 30 of gestation. Protein restriction reduced the proportion of sows that returned to estrus within 8 d after weaning in the 180LP sows (P < 0.03), but not in the 240LP sows. It also induced a reduction in ovulation rate in the 180LP sows (P < 0.05) and, to a lesser extent, in the 240LP sows (P = 0.12). When the sows were categorized according to return to estrus (WOI < or = 8 or > 8 d), basal and mean concentrations of LH increased after weaning only in sows with a short WOI. Sows with a delayed estrus exhibited a higher ratio of plasma tyrosine to large neutral amino acids (AA, P < 0.01). In conclusion, large body reserves at farrowing buffer, at least in part, the detrimental effect of a strongly negative nitrogen balance on reproduction. We suggest that the alteration of AA profiles induced by dietary protein restriction and body protein loss alters LH secretion via modifications of the neurotransmitters involved in GnRH secretion.     

DNA methylation and gene expression changes in monozygotic twins discordant for psoriasis: identification of epigenetically dysregulated genes

Monozygotic (MZ) twins do not show complete concordance for many complex diseases; for example, discordance rates for autoimmune diseases are 20%-80%. MZ discordance indicates a role for epigenetic or environmental factors in disease. We used MZ twins discordant for psoriasis to search for genome-wide differences in DNA methylation and gene expression in CD4(+) and CD8(+) cells using Illumina's HumanMethylation27 and HT-12 expression assays, respectively. Analysis of these data revealed no differentially methylated or expressed genes between co-twins when analyzed separately, although we observed a substantial amount of small differences. However, combined analysis of DNA methylation and gene expression identified genes where differences in DNA methylation between unaffected and affected twins were correlated with differences in gene expression. Several of the top-ranked genes according to significance of the correlation in CD4(+) cells are known to be associated with psoriasis. Further, gene ontology (GO) analysis revealed enrichment of biological processes associated with the immune response and clustering of genes in a biological pathway comprising cytokines and chemokines. These data suggest that DNA methylation is involved in an epigenetic dysregulation of biological pathways involved in the pathogenesis of psoriasis. This is the first study based on data from MZ twins discordant for psoriasis to detect epigenetic alterations that potentially contribute to development of the disease.