Helen Hayes

Cell cycle regulation and cytoskeletal remodelling are critical processes in the nutritional programming of embryonic development.

Many mechanisms purport to explain how nutritional signals during early development are manifested as disease in the adult offspring. While these describe processes leading from nutritional insult to development of the actual pathology, the initial underlying cause of the programming effect remains elusive. To establish the primary drivers of programming, this study aimed to capture embryonic gene and protein changes in the whole embryo at the time of nutritional insult rather than downstream phenotypic effects. By using a cross-over design of two well established models of maternal protein and iron restriction we aimed to identify putative common “gatekeepers” which may drive nutritional programming. Both protein and iron deficiency in utero reduced the nephron complement in adult male Wistar and Rowett Hooded Lister rats (P<0.05). This occurred in the absence of damage to the glomerular ultrastructure. Microarray, proteomic and pathway analyses identified diet-specific and strain-specific gatekeeper genes, proteins and processes which shared a common association with the regulation of the cell cycle, especially the G1/S and G2/M checkpoints, and cytoskeletal remodelling. A cell cycle-specific PCR array confirmed the down-regulation of cyclins with protein restriction and the up-regulation of apoptotic genes with iron deficiency. The timing and experimental design of this study have been carefully controlled to isolate the common molecular mechanisms which may initiate the sequelae of events involved in nutritional programming of embryonic development. We propose that despite differences in the individual genes and proteins affected in each strain and with each diet, the general response to nutrient deficiency in utero is perturbation of the cell cycle, at the level of interaction with the cytoskeleton and the mitotic checkpoints, thereby diminishing control over the integrity of DNA which is allowed to replicate. These findings offer novel insight into the primary causes and mechanisms leading to the pathologies which have been identified by previous programming studies.

Role of the placenta in regulation of fetal iron status

During pregnancy, the developing fetus is entirely dependent on its mother for nutrition and waste disposal, which take place almost exclusively across the placenta. The placenta is, however, not just a passive mediator; it can also regulate the rates and amounts of nutrient transported. This is particularly true for iron transport. This review examines how the fetus, and not the mother, regulates iron uptake and transfer across the placenta, how these processes interact, and how the fetus can modulate iron metabolism in the mother.

Maternal iron status in early pregnancy and birth outcomes: insights from the Baby's Vascular health and Iron in Pregnancy study.

Fe deficiency anaemia during early pregnancy has been linked with low birth weight and preterm birth. However, this evidence comes mostly from studies measuring Hb levels rather than specific measures of Fe deficiency. The present study aimed to examine the association between maternal Fe status during the first trimester of pregnancy, as assessed by serum ferritin, transferrin receptor and their ratio, with size at birth and preterm birth. In the Baby VIP (Babys Vascular health and Iron in Pregnancy) study, we recruited 362 infants and their mothers after delivery in Leeds, UK. Biomarkers were measured in maternal serum samples previously obtained in the first trimester of pregnancy. The cohort included sixty-four (18 %) small for gestational age (SGA) babies. Thirty-three babies were born preterm (9 %; between 34 and 37 weeks). First trimester maternal Fe depletion was associated with a higher risk of SGA (adjusted OR 2·2, 95 % CI 1·1, 4·1). This relationship was attenuated when including early pregnancy Hb in the model, suggesting it as a mediator (adjusted OR 1·6, 95 % CI 0·8, 3·2). For every 10 g/l increase in maternal Hb level in the first half of pregnancy the risk of SGA was reduced by 30 % (adjusted 95 % CI 0, 40 %); levels below 110 g/l were associated with a 3-fold increase in the risk of SGA (95 % CI 1·0, 9·0). There was no evidence of association between maternal Fe depletion and preterm birth (adjusted OR 1·5, 95 % 0·6, 3·8). The present study shows that depleted Fe stores in early pregnancy are associated with higher risk of SGA.

Processes Underlying the Nutritional Programming of Embryonic Development by Iron Deficiency in the Rat

Poor iron status is a global health issue, affecting two thirds of the world population to some degree. It is a particular problem among pregnant women, in both developed and developing countries. Feeding pregnant rats a diet deficient in iron is associated with both hypertension and reduced nephron endowment in adult male offspring. However, the mechanistic pathway leading from iron deficiency to fetal kidney development remains elusive. This study aimed to establish the underlying processes associated with iron deficiency by assessing gene and protein expression changes in the rat embryo, focussing on the responses occurring at the time of the nutritional insult. Analysis of microarray data showed that iron deficiency in utero resulted in the significant up-regulation of 979 genes and down-regulation of 1545 genes in male rat embryos (d13). Affected processes associated with these genes included the initiation of mitosis, BAD-mediated apoptosis, the assembly of RNA polymerase II preinitiation complexes and WNT signalling. Proteomic analyses highlighted 7 proteins demonstrating significant up-regulation with iron deficiency and the down-regulation of 11 proteins. The main functions of these key proteins included cell proliferation, protein transport and folding, cytoskeletal remodelling and the proteasome complex. In line with our recent work, which identified the perturbation of the proteasome complex as a generalised response to in utero malnutrition, we propose that iron deficiency alone leads to a more specific failure in correct protein folding and transport. Such an imbalance in this delicate quality-control system can lead to cellular dysfunction and apoptosis. Therefore these findings offer an insight into the underlying mechanisms associated with the development of the embryo during conditions of poor iron status, and its health in adult life.

An exploratory study of the associations between maternal iron status in pregnancy and childhood wheeze and atopy

Maternal nutritional status during pregnancy has been reported to be associated with childhood asthma and atopic disease. The Avon Longitudinal Study of Parents and Children has reported associations between reduced umbilical cord Fe status and childhood wheeze and eczema; however, follow-up was short and lung function was not measured. In the present study, the associations between maternal Fe status during pregnancy and childhood outcomes in the first 10 years of life were investigated in a subgroup of 157 mother-child pairs from a birth cohort with complete maternal, fetal ultrasound, blood and child follow-up data. Maternal Fe intake was assessed using FFQ at 32 weeks of gestation and Hb concentrations and serum Fe status (ferritin, soluble transferrin receptor and TfR-F (transferrin receptor:ferritin) index) were measured at 11 weeks of gestation and at delivery. Maternal Fe intake, Hb concentrations and serum Fe status were found to be not associated with fetal or birth measurements. Unit increases in first-trimester maternal serum TfR concentrations (OR 1.44, 95% CI 1.05, 1.99) and TfR-F index (OR 1.42, 95% CI 1.10, 1.82) (i.e. decreasing Fe status) were found to be associated with an increased risk of wheeze, while unit increases in serum ferritin concentrations (i.e., increasing Fe status) were found to be associated with increases in standardised mean peak expiratory flow (PEF) (β 0.25, 95% CI 0.09, 0.42) and forced expiratory volume in the first second (FEV1) (β 0.20, 95% CI 0.08, 0.32) up to 10 years of age. Increasing maternal serum TfR-F index at delivery was found to be associated with an increased risk of atopic sensitisation (OR 1.35, 95% CI 1.02, 1.79). The results of the present study suggest that reduced maternal Fe status during pregnancy is adversely associated with childhood wheeze, lung function and atopic sensitisation, justifying further studies on maternal Fe status and childhood asthma and atopic disease.

Pregnancy and maternal iron deficiency stimulate hepatic CRBPII expression in rats

Iron deficiency impairs vitamin A (VA) metabolism in the rat but the mechanisms involved are unknown and the effect during development has not been investigated. We investigated the effect of pregnancy and maternal iron deficiency on VA metabolism in the mother and fetus. 54 rats were fed either a control or iron deficient diet for 2weeks prior to mating and throughout pregnancy. Another 15 female rats followed the same diet and were used as non-pregnant controls. Maternal liver, placenta and fetal liver were collected at d21 for total VA, retinol and retinyl ester (RE) measurement and VA metabolic gene expression analysis. Iron deficiency increased maternal hepatic RE (P<.05) and total VA (P<.0001), fetal liver RE (P<.05), and decreased placenta total VA (P<.05). Pregnancy increased Cellular Retinol Binding Protein (CRBP)-II gene expression by 7 fold (P=.001), decreased VA levels (P=.0004) and VA metabolic gene expression (P<.0001) in the liver. Iron deficiency increased hepatic CRBPII expression by a further 2 fold (P=.044) and RBP4 by~20% (P=.005), increased RBPR2 and decreased CRBPII, LRAT, and TTR in fetal liver, while it had no effect on VA metabolic gene expression in the placenta. Hepatic CRBPII expression is increased by pregnancy and further increased by iron deficiency, which may play an important role in VA metabolism and homeostasis. Maternal iron deficiency also alters VA metabolism in the fetus, which is likely to have consequences for development.

Transcriptional regulation of copper metabolism genes in the liver of fetal and neonatal control and iron-deficient rats

Copper and iron metabolism have been known to interact for many years. We have previously shown, during pregnancy, that copper levels in the maternal liver rise as a consequence of iron deficiency, but that levels in the fetal liver decrease. In this paper, we measure expression of genes involved in copper metabolism in fetal and postnatal liver, to test whether alterations can explain this observation. Additionally, we study the extent to which gene expression changes in the latter stages of pregnancy and in the perinatal period. Ctr1 expression levels dropped to term, rising again thereafter. There was no difference in gene expression between control and iron deficient animals. Atox1 expression remained approximately stable until term, and then there was a rise to a maximum at about Day 8. Atp7a expression levels remained constant, except for a brief drop at term. Atp7b levels, in contrast, decreased from a maximum early in gestation to low levels in the term and post-natal livers. Ceruloplasmin expression appeared to be diametrically opposite to Atp7b. The other two metallochaperones showed the same pattern of expression as Atox1, with a decrease to term, a rise at Day 1, or a rise after birth followed by a brief decrease at about Day 3. None of the genes were significantly affected by iron deficiency, suggesting that changes in expression cannot explain the altered copper levels in the fetal and neonatal liver.

The effect of iron deficiency on the temporal changes in the expression of genes associated with fat metabolism in the pregnant rat

Iron is essential for the oxidative metabolism of lipids. Lipid metabolism changes during gestation to meet the requirements of the growing fetus and to prepare for lactation. The temporal effects of iron deficiency during gestation were studied in female rats fed complete or iron‐deficient diets. Plasma triglycerides were elevated in the iron‐deficient group throughout gestation. There were time‐dependent changes in the triglyceride content of the maternal liver, falling at the midpoint of gestation and then increasing on d21.5. Compared to the control, triglycerides in the maternal liver were not different in the iron‐deficient group prior to pregnancy and on d12.5, but were markedly reduced by d21.5. The abundance of mRNAs in the maternal liver suggests that lipogenesis is unchanged and beta‐oxidation is reduced on d21.5 by iron deficiency. On d21.5 of gestation, the expression of placental lipase was unchanged by iron deficiency, however, the abundance of mRNAs for SREBP‐1c, FABP4 were reduced, suggesting that there were changes in fatty acid handling. In the fetal liver, iron deficiency produced a marked decrease in the abundance of the L‐CPT‐1 mRNA, suggesting that beta‐oxidation is reduced. This study shows that the major effect of iron deficiency on maternal lipid metabolism occurs late in gestation and that perturbed lipid metabolism may be a common feature of models of fetal programming.

Use of β-glucan from spent brewer's yeast as a thickener in skimmed yogurt: Physicochemical, textural, and structural properties related to sensory perception

Powdered β-glucan extracted from brewers yeast (Yestimun, Leiber GmbH, Bramsche, Germany) was incorporated into skimmed-milk yogurt at varying concentrations (0.2-0.8% wt/wt) to investigate its potential application as a thickener. The effect of β-glucan fortification on the nutritional profile, microstructure, physicochemical properties, and texture of freshly prepared yogurts was investigated. Sensory evaluation was also conducted and was correlated with instrumental analysis. The addition of Yestimun significantly reduced the fermentation time of the yogurt mix from 4 h to 3 h. Scanning electron microscopy revealed that β-glucan particles formed small spherical clusters within the yogurt matrix. The majority of the physicochemical properties (syneresis, viscosity, color, and titratable acidity) remained unaffected by the incorporation of Yestimun in the recipe. Textural properties showed a gradual increment with increasing β-glucan concentration. Hardness, total work done, adhesive force, and adhesiveness increased by 19.27, 23.3, 21.53, and 20.76%, respectively, when using the highest amount of Yestimun powder. Sensory analysis (n = 40) indicated that fortifying yogurt with Yestimun at 0.8% (wt/wt) concentration may affect overall acceptance ratings, which was attributed to adverse flavor and aftertaste effects. However, the overall liking score of the yogurt (5.0/9.0) shows potential for commercialization of the product.

Prenatal iron exposure and childhood type 1 diabetes

Iron overload due to environmental or genetic causes have been associated diabetes. We hypothesized that prenatal iron exposure is associated with higher risk of childhood type 1 diabetes. In the Norwegian Mother and Child cohort study (n = 94,209 pregnancies, n = 373 developed type 1 diabetes) the incidence of type 1 diabetes was higher in children exposed to maternal iron supplementation than unexposed (36.8/100,000/year compared to 28.6/100,000/year, adjusted hazard ratio 1.33, 95%CI: 1.06–1.67). Cord plasma biomarkers of high iron status were non-significantly associated with higher risk of type 1 diabetes (ferritin OR = 1.05 [95%CI: 0.99–1.13] per 50 mg/L increase; soluble transferrin receptor: OR = 0.91 [95%CI: 0.81–1.01] per 0.5 mg/L increase). Maternal but not fetal HFE genotypes causing high/intermediate iron stores were associated with offspring diabetes (odds ratio: 1.45, 95%CI: 1.04, 2.02). Maternal anaemia or non-iron dietary supplements did not significantly predict type 1 diabetes. Perinatal iron exposures were not associated with cord blood DNA genome-wide methylation, but fetal HFE genotype was associated with differential fetal methylation near HFE. Maternal cytokines in mid-pregnancy of the pro-inflammatory M1 pathway differed by maternal iron supplements and HFE genotype. Our results suggest that exposure to iron during pregnancy may be a risk factor for type 1 diabetes in the offspring.