Differential expression of genes in fetal brain as a consequence of maternal protein deficiency and nematode infection.

Maternal dietary protein deficiency and gastrointestinal nematode infection during early pregnancy have negative impacts on both maternal placental gene expression and fetal growth in the mouse. Here we used next-generation RNA sequencing to test our hypothesis that maternal protein deficiency and/or nematode infection also alter the expression of genes in the developing fetal brain. Outbred pregnant CD1 mice were used in a 2×2 design with two levels of dietary protein (24% versus 6%) and two levels of infection (repeated sham versus Heligmosomoides bakeri beginning at gestation day 5). Pregnant dams were euthanized on gestation day 18 to harvest the whole fetal brain. Four fetal brains from each treatment group were analyzed using RNA Hi-Seq sequencing and the differential expression of genes was determined by the edgeR package using NetworkAnalyst. In response to maternal H. bakeri infection, 96 genes (88 up-regulated and eight down-regulated) were differentially expressed in the fetal brain. Differentially expressed genes were involved in metabolic processes, developmental processes and the immune system according to the PANTHER classification system. Among the important biological functions identified, several up-regulated genes have known neurological functions including neuro-development (Gdf15, Ing4), neural differentiation (miRNA let-7), synaptic plasticity (via suppression of NF-κß), neuro-inflammation (S100A8, S100A9) and glucose metabolism (Tnnt1, Atf3). However, in response to maternal protein deficiency, brain-specific serine protease (Prss22) was the only up-regulated gene and only one gene (Dynlt1a) responded to the interaction of maternal nematode infection and protein deficiency. In conclusion, maternal exposure to GI nematode infection from day 5 to 18 of pregnancy may influence developmental programming of the fetal brain.

Protein kinase C inhibition ameliorates functional endothelial insulin resistance and vascular smooth muscle cell hypersensitivity to insulin in diabetic hypertensive rats.

OBJECTIVE: Insulin resistance, diabetes, and hypertension are considered elements of metabolic syndrome which is associated with vascular dysfunction. We investigated whether inhibition of protein kinase C (PKC) would affect vascular function in diabetic hypertensive (DH) rats. METHODS: A combination of type 2 diabetes and arterial hypertension was produced in male Sprague Dawley rats by intrauterine protein deprivation (IUPD) followed by high salt diet. At the age of 32 weeks, DH rats were treated for 2 weeks with the angiotensin-converting enzyme inhibitor captopril (Capto, 30 mg/kg), PKC inhibitor ruboxistaurin (RBX, 50 mg/kg) or vehicle (n = 8 per group) and blood pressure was monitored using telemetry. At the end of experiments, femoral arteries were dissected, and vascular reactivity was evaluated with isovolumic myography. RESULTS: The IUPD followed by high salt diet resulted in significant elevation of plasma glucose, plasma insulin, and blood pressure. Endothelium-dependent vascular relaxation in response to acetylcholine was blunted while vascular contraction in response to phenylephrine was enhanced in the DH rats. Neither Capto nor RBX restored endothelium-dependent vascular relaxation while both suppressed vascular contraction. Ex-vivo incubation of femoral arteries from control rats with insulin induced dose-response vasorelaxation while insulin failed to induce vasorelaxation in the DH rat arteries. In the control arteries treated with endothelial nitric oxide synthase inhibitor L-NAME, insulin induced vasoconstriction that was exacerbated in DH rats. Capto and RBX partially inhibited insulin-stimulated vascular contraction. CONCLUSION: These findings suggest that PKC inhibition ameliorates functional endothelial insulin resistance and smooth muscle cell hypersensitivity to insulin, but does not restore acetylcholine-activated endothelium-dependent vasodilation in DH rats.

Effects of maternal dietary manipulation during different periods of pregnancy on hepatic glucogenic capacity in fetal and pregnant rats near term.

BACKGROUND AND AIM: Low birth weight is associated with an increased incidence of adult glucose intolerance, type 2 diabetes and cardiovascular disease in humans. In pregnant rats, dietary calorie or protein deprivation results in growth retarded pups, which become glucose intolerant adults with abnormal hepatic glucose metabolism and gluconeogenic enzyme activities. However, whether these abnormalities are present before birth remain unknown. METHODS AND RESULTS: This study examined the effects of manipulating dietary protein and carbohydrate intake during rat pregnancy on the fetal and maternal hepatic activities of the gluconeogenic enzymes, glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK). Wistar rats were fed ad libitum with either standard chow throughout pregnancy (25% protein, 57% carbohydrate, n=6) or an isocaloric, low protein, high carbohydrate diet (LPHC, 8% protein, 81% carbohydrate) for different periods of pregnancy (early, 0-10 days, n=6; late, 10-20 days, n=7; throughout, 0-20 days, n=6) before tissue collection at day 20. The LPHC diet had no effect on fetal or placental weights, or on fetal hepatic activities of G6Pase and PEPCK in the early LPHC group. In contrast, fetuses of dams fed the LPHC diet in late or throughout pregnancy had lower body and placental weights, and higher hepatic G6Pase and PEPCK activities than controls. Maternal hepatic G6Pase activity was elevated in all LPHC groups, while maternal PEPCK activity was only increased significantly in the late LPHC group. CONCLUSIONS: Feeding a LPHC diet, particularly during late pregnancy, therefore, up-regulates fetal and maternal hepatic glucogenic capacity.

Fetal protein loss in gastroschisis as an explanation of associated morbidity.

OBJECTIVE: Our purpose was to examine whether protein deficiency in utero develops in fetuses with gastroschisis. STUDY DESIGN: Twelve infants with prenatally diagnosed gastroschisis were compared with 29 control infants without gastroschisis and 2 infants with exomphalos who were delivered between 35 and 42 weeks of gestation. The groups were compared for birth weight, cord serum total protein and amniotic fluid total protein, and alpha-fetoprotein concentrations. The amniotic fluid samples were collected when the amniotic membranes were ruptured either during cesarean delivery or at artificial rupture of the membranes, and umbilical cord blood was obtained after delivery. RESULTS: In the 10 cases of gastroschisis in which cord serum total protein was measured, the median concentration was 51 g/L (range, 43-61 g/L) and was significantly lower than the median level of 62 g/L (range, 47-78 g/L) in the control group (P <.001). In the 8 cases of gastroschisis in which amniotic fluid total protein and alpha-fetoprotein concentrations were measured, the respective median levels were 5.1 g/L (range, 4.3-18.4 g/L) and 5.0 g/L (range, 2.4-13.2 g/L), which were significantly higher than the median levels of 2.0 g/L (range, 0.5-5.4 g/L) and 0.8 g/L (range, 0.5-1.7 g/L) in the control group (P <.0001). The ratio of amniotic fluid to cord serum total protein was significantly higher than that in the cases of exomphalos and in the control group (P <.001). The median birth weight in the neonates with gastroschisis was 2400 g (range, 1192-3155 g) and was significantly lower than the median value of 3535 g (range, 2520-4680 g) in the control group (P <.0001). CONCLUSIONS: Fetuses with gastroschisis have protein loss that could partly explain associated morbidity. However, whether this is a major contributor to poor fetal outcome remains to be shown.

Maternal dietary protein deficiency decreases amino acid concentrations in fetal plasma and allantoic fluid of pigs.

This study was conducted to test the hypothesis that maternal dietary protein deficiency decreases amino acid availability to the fetus, thereby contributing to retarded fetal growth. Primiparous gilts selected genetically for low or high plasma total cholesterol concentrations (low line and high line, respectively) were mated, and then fed 1.8 kg/d of isocaloric diets containing 13% or 0.5% crude protein. At d 40 or 60 of gestation, they were hysterectomized, and maternal and fetal blood samples as well as amniotic and allantoic fluids were obtained for analyses of amino acids, ammonia and urea. Dietary protein restriction decreased (P < 0.05) the following: 1) maternal plasma concentrations of urea at d 40 and 60 of gestation; 2) fetal plasma concentrations of alanine, arginine, branched-chain amino acids (BCAA), glutamine, glycine, lysine, ornithine, proline, taurine, threonine and urea at d 60 of gestation; 3) amniotic and allantoic fluid concentrations of urea at d 40 and 60 of gestation; and 4) allantoic fluid concentrations of alanine, arginine, BCAA, citrulline, cystine, glycine, histidine, methionine, proline, serine, taurine, threonine and tyrosine at d 40 of gestation, in gilts of both genetic lines. At d 60 of gestation, protein deficiency decreased (P < 0.05) allantoic fluid concentrations of arginine, cystine, glycine, taurine and tyrosine in low line gilts and of cystine, glutamine, ornithine, serine, taurine and tyrosine in high line gilts. Low line and high line gilts also differed remarkably in allantoic fluid concentrations of arginine, glutamine, ornithine and ammonia at d 40 and 60 of gestation. Our results suggest the following: 1) protein-deficient gilts maintain maternal plasma concentrations of amino acids by mobilizing maternal protein stores and decreasing oxidation of amino acids during the first half of gestation; 2) protein deficiency may impair placental transport of amino acids from the maternal to the fetal blood; and 3) low line and high line gilts differ in fetal amino acid metabolism. Decreases in concentrations of the essential and nonessential amino acids in the fetus may be a mechanism whereby maternal dietary protein restriction results in fetal growth retardation.

The effects of protein deprivation on neuronal migration in rats.

Although there is extensive literature documenting the effects of undernutrition on brain development, most studies have been concerned with cell differentiation with little attention given to neuronal migration. In a rat model we have investigated the effect of chronic protein deprivation on cell migration from the anterior lateral ventricle to the olfactory bulb. By using standard autoradiographic techniques and comparing the position of heavily labeled cells within the migratory stream, we estimated the migration rate to be 100 microns/h in 25%-casein-diet rats and between 33 and 70 microns/h in 8%-casein-diet rats. We therefore conclude that migration is slowed in chronically protein-deprived rats and this slowed migration may be related to subsequent abnormalities of cell differentiation seen in protein-deprived rats.

Developmental protein malnutrition in the rat: effects on single-unit activity in the frontal cortex.

This study evaluated the effects of developmental protein malnutrition on the spontaneous electrical activity of frontal cortex neurons in the anesthetized rat. Rats were raised prenatally and postnatally on either an 8% or 6% casein diet until adulthood. Compared to the 25% casein controls, both malnourished groups showed a 30-36% decrease in mean discharge rates and a 100-200% increase in the percentage of cells with very slow (less than 1/s) discharge rates. Most of the diet-related changes were confined to a zone 600-1200 micron below the brain surface, approximately cortical layers III, IV and V. A second set of studies in which diet reversals were introduced at birth or in adulthood found that: (a) restoration of a normal 25% casein diet at birth did not appreciably attenuate the effect of prenatal administration of an 8% casein diet; (b) introduction in adulthood of the 8% casein diet to a normally fed rat had no effect; (c) introduction of the 8% diet at birth, however, produced effects in adulthood comparable to those seen when the protein malnutrition was introduced in the prenatal period. Thus, the rat brain is sensitive to both prenatal and postnatal protein malnutrition (starting at birth). Most importantly, the effects of prenatal protein malnutrition on the activity of frontal cortex neurons do not appear to be reversible by restoration of a normal diet in adulthood or at birth.

Prenatal protein deprivation increases defects of the corpus callosum in BALB/c laboratory mice.

The development of two forebrain fiber tracts, the anterior commissure (CA) and corpus callosum (CC), was examined in BALB/cCF laboratory mice in relation to the maternal dietary protein concentration during gestation. The diets contained either 8 or 27% casein. The BALB/c strain was used because it is prone to having a corpus callosum which is either deficient in size or absent. Female mice were assigned to the low-protein diet either 2 weeks prior to mating (chronic malnutrition) or on the seventh day of gestation (acute malnutrition), and fetal brain development was assessed at 18.5 days after conception. Protein deficiency increased the number of animals which did not have a corpus callosum present in a midsagittal section. In those animals in which the structure was present, it was evident that the cross-sectional area was smaller in the chronically deprived animals than in the control group. In addition to CC area, both CA area and brain weight were reduced by protein deprivation. When brain weight was used as a covariate in an analysis of covariance the effect on CA area was no longer apparent. Part of the effect on the corpus callosum was, however, independent of the effect on brain weight and CA area.

Effect of fetal protein malnutrition on the postnatal structure and function of alveolar macrophages.

To test the hypothesis that intrauterine malnutrition may alter ontogeny of the host defense system, an animal model of fetal protein deprivation was developed. Young adult female rats were fed either a deficient (8% protein) diet or a normal (25% protein) diet for 10 days before insemination and throughout gestation. Offspring of the malnourished animals showed significant growth retardation and were hypoproteinemic. Lavageable pulmonary cells from both groups of neonates were similar with respect to number (2.05 x 10(5) cells per animal), type (95% macrophages), size (approximately 10-micrometer diameter), ultrastructure, and presence of surface receptors for IgG. Despite these similarities, alveolar macrophages from malnourished neonates were significantly impaired in their ability both to ingest and to kill Candida tropicalis. Nutritional supplementation of nursing females reversed these functional macrophage defects in their offspring by the time that weaning occurred. These data indicate that fetal protein malnutrition affects macrophage function but that with postnatal nutritional supplementation these defects are rapidly reversed.