Beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation.

The Wnt/beta-catenin/TCF4 pathway plays critical roles in the maintenance of small intestinal epithelium; however, downstream targets of the beta-catenin/TCF4 complex are not extensively characterized. We identified miR-30e as an immediate target activated by the beta-catenin/TCF4 complex. miR-30e was detected in the peri-nuclear region of the intestinal crypt IEC-6 cells. Bioinformatics analysis revealed clustered beta-catenin/TCF4 binding sites within the miR-30e promoter region. This promoter region was cloned into pGL3-control luciferase reporter vector, with the enhancer region removed. Transfection of pCMV-SPORT6-beta-catenin expression vector dose-dependently increased luciferase activity, and co-transfection of pCMV-SPORT6-TCF4 expression vector further enhanced the promoter activity. Dexamethasone-induced IEC-6 cells differentiation caused a 2.5-fold increase in miR-30e expression, and upon beta-catenin siRNA transfection, miR-30e increased 1.3-fold. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed the binding between beta-catenin/TCF4 complexes from IEC-6 nuclear extracts and the putative sequences in the miR-30e promoter. These results demonstrate that beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation.

miR-584 mediates post-transcriptional expression of lactoferrin receptor in Caco-2 cells and in mouse small intestine during the perinatal period.

MicroRNAs function as gene expression modulators that are critical for mammalian development. Lactoferrin receptor on the apical membrane of enterocytes has been suggested to play key roles in the absorption of lactoferrin-bound iron from breast milk. The objective of this study was to identify mechanisms of microRNA mediated post-transcriptional regulation of the lactoferrin receptor. Sequence analyses revealed that the miR-584 sequence is identical in human, mouse and rat, and there is a conserved region complementary to the seed region (5' nucleotides 2-8) of miR-584 within the lactoferrin receptor mRNA-3'-untranslated region. miR-584 was further found to co-localize with lactoferrin receptor mRNA in mouse small intestine. The 3'-untranslated region of human lactoferrin receptor mRNA was cloned into pGL3-control luciferase reporter vector. By luciferase reporter assays in HEK293 cells, miR-584 mimic specifically repressed the reporter activity in a dose-dependent manner. miR-584 mimic reduced endogenous lactoferrin receptor protein expression in Caco-2 cells, without significantly affecting the mRNA level. We also determined that miR-584 expression is inversely correlated with lactoferrin receptor mRNA and protein expression. Taken together, we propose that miR-584 contributes to the post-transcriptional expression of lactoferrin receptor during the perinatal period. These findings demonstrate a novel example of how microRNAs may be involved in regulation of nutrient metabolism in the newborn.

Factors influencing intestinal cadmium uptake in pregnant Bangladeshi women--a prospective cohort study.

Experimental studies indicate that zinc (Zn) and calcium (Ca) status, in addition to iron (Fe) status, affect gastrointestinal absorption of cadmium (Cd), an environmental pollutant that is toxic to kidneys, bone and endocrine systems. The aim of this study was to evaluate how various nutritional factors influence the uptake of Cd in women, particularly during pregnancy. The study was carried out in a rural area of Bangladesh, where malnutrition is prevalent and exposure to Cd via food appears elevated. The uptake of Cd was evaluated by associations between erythrocyte Cd concentrations (Ery-Cd), a marker of ongoing Cd exposure, and concentrations of nutritional markers. Blood samples, collected in early pregnancy and 6 months postpartum, were analyzed by inductively coupled plasma mass spectrometry (ICPMS). Ery-Cd varied considerably (range: 0.31-5.4microg/kg) with a median of 1.1microg/kg (approximately 0.5microg/L in whole blood) in early pregnancy. Ery-Cd was associated with erythrocyte manganese (Ery-Mn; positively), plasma ferritin (p-Ft; negatively), and erythrocyte Ca (Ery-Ca; negatively) in decreasing order, indicating common transporters for Cd, Fe and Mn. There was no evidence of Cd uptake via Zn transporters, but the association between Ery-Cd and p-Ft seemed to be dependent on adequate Zn status. On average, Ery-Cd increased significantly by 0.2microg/kg from early pregnancy to 6 months postpartum, apparently due to up-regulated divalent metal transporter 1 (DMT1). In conclusion, intestinal uptake of Cd appears to be influenced either directly or indirectly by several micronutrients, in particular Fe, Mn and Zn. The negative association with Ca may suggest that Cd inhibits the transport of Ca to blood.

[Conclusions and recommendations of a WHO expert consultation meeting on iron supplementation for infants and young children in malaria endemic areas]. / Conclusions et recommandations à l'issue de la consultation de l'OMS sur la lutte contre la carence martiale chez le nourrisson et le jeune enfant dans les pays d'endémie palustre.

This article presents the results of an expert consultation meeting aimed at evaluating the safety and public health implications of administering supplemental iron to infants and young children in malaria-endemic areas. Participants at this meeting that took place in Lyon, France on June 12-14, 2006 reached consensus on several important issues related to iron supplementation for infants and young children in malaria-endemic areas. The conclusions in this report apply specifically to regions where malaria is endemic.

Iron at the center of ferritin, metal/oxygen homeostasis and novel dietary strategies.

Bioiron - central to respiration, photosynthesis and DNA synthesis and complicated by radical chemistry with oxygen - depends on ferritin, the super family of protein nanocages (maxi-ferritins in humans, animals, plant, and bacteria, and mini-ferritins, also called DPS proteins, in bacteria) for iron and oxygen control. Regulation of ferritin synthesis, best studied in animals, uses DNA transcription and mRNA translation check points. Ferritin is a member of both the "oxidant stress response" gene family that includes thioredoxin reductase and quinine reductase, and a member of the iron responsive gene family that includes ferroportin and mt-aconitase ferritin DNA regulation responds preferentially to oxidant response inducers and ferritin mRNA to iron inducers: heme confers regulator synergy. Ferritin proteins manage iron and oxygen, with ferroxidase sites and iron + oxygen substrates to form mineral of both Fe and O atoms; maxi-ferritins contribute more to cellular iron metabolism and mini-ferritins to stress responses. Iron recovery from ferritin is controlled by gated protein pores, possibly contributing to iron absorption from ferritin, a significant dietary iron source. Ferritin gene regulation is a model for integrating DNA/mRNA controls, while ferritin protein function is central to molecular nutrition cellular metabolism at the crossroads of iron and oxygen in biology.

Iron at the center of ferritin, metal/oxygen homeostasis and novel dietary strategies

Bioiron _ central to respiration, photosynthesis and DNA synthesis and complicated by radical chemistry with oxygen _ depends on ferritin, the super family of protein nanocages (maxi-ferritins in humans, animals, plants and bacteria, and mini-ferritins, also called DPS proteins, in bacteria) for iron and oxygen control. Regulation of ferritin synthesis, best studied in animals, uses DNA transcription and mRNA translation check points. Ferritin is a member of both the "oxidant stress response" gene family that includes thioredoxin reductase and quinine reductase, and a member of the iron responsive gene family that includes ferroportin and mt-aconitase ferritin DNA regulation responds preferentially to oxidant response inducers and ferritin mRNA to iron inducers; heme confers regulator synergy. Ferritin proteins manage iron and oxygen, with ferroxidase sites and iron + oxygen substrates to form mineral of both Fe and O atoms; maxi-ferritins contribute more to cellular iron metabolism and mini-ferritins to stress responses. Iron recovery from ferritin is controlled by gated protein pores, possibly contributing to iron absorption from ferritin, a significant dietary iron source. Ferritin gene regulation is a model for integrating DNA/mRNA controls, while ferritin protein function is central to molecular nutrition cellular metabolism at the crossroads of iron and oxygen in biology.

Mammalian lactoferrin receptors: structure and function.

Mammalian lactoferrin (Lf) receptors are suggested to have pivotal roles for mediating multiple functions of Lf. In this review, we focus on current knowledge of the structure and function of mammalian Lf receptors, mainly the first cloned Lf receptor that has been shown to be expressed in the infant small intestine at high levels but also in virtually all other tissues. The small intestinal Lf receptor takes up iron from Lf into cells and presumably exerts other physiological functions. Other Lf receptors in various tissues have also been reported to mediate some functions of Lf, such as modulating immune function, inhibiting platelet aggregation and enhancing collagen gel contractile strength. The detailed mechanisms behind the receptor-Lf interactions still need to be elucidated.

Anaemia and iron deficiency during pregnancy in rural Bangladesh.

OBJECTIVE: To study the prevalence of anaemia and its association with measures of iron deficiency (ID) among a group of pregnant women. DESIGN: Cross-sectional survey. SETTING: Pregnant women identified through house-to-house visits and participating in community-based antenatal care activities in a rural location of Mymensingh, Bangladesh. SUBJECTS: The estimates are based on 214 reportedly healthy pregnant women in their second trimester. Information on socio-economic status and reproductive history were obtained through home visits and venous blood samples were collected at antenatal care centres. Haemoglobin concentration (Hb) was measured by HemoCue, serum ferritin (sFt) by radioimmunoassay and serum transferrin receptor (sTfR) by enzyme-linked immunosorbent assay methods. ID was defined as presence of either low sFt (<12 microg l(-1)) or high sTfR (>8.5 mg l(-1)). RESULTS: The prevalence of anaemia (Hb <110 g l(-1)) was 50%, but severe anaemia (Hb <70 g l(-1)) was absent. Low sFt was observed in 42%, high sTfR in 25%, either low sFt or high TfR in 54% and both low sFt and high TfR in 13% of the pregnant women. Two out of three anaemic women had an indication of ID, which was present in 80% of women with moderate (Hb 70-99 g l(-1)) and 50% with mild (Hb 100-109 g l(-1)) anaemia. Four out of 10 non-anaemic women (Hb >/=110 g l(-1)) also had ID, but the prevalence was significantly lower than that observed in anaemic women (P=0.001). CONCLUSIONS: Despite the high prevalence of anaemia, severe cases were absent. The prevalence of ID increased at lower Hb. However, an increased prevalence was also found among women in the highest category of Hb.

Is iron deficiency in infants and young children common in Scandinavia and is there a need for enforced primary prevention?

A significant problem in diagnosing infants and young children with iron deficiency (ID) and iron deficiency anemia (IDA) is that it is not clear how the biochemical markers used correspond to physiological outcomes such as growth, neurodevelopment and morbidity. Thus, the prevalence of iron deficiency and iron deficiency anemia in infants and young children is difficult to assess and therefore not really known. Recent studies have shown that iron metabolism is subjected to developmental changes during infancy, which affect indicators of iron status. Hence, better defined cut-off values for the indicators used are needed and, in particular, how they are correlated to functional outcomes.

Effect of iron supplementation on cognition in Greek preschoolers.

OBJECTIVE: To examine effects of iron supplementation on vigilance, attention and conceptual learning in preschool children in Greece. DESIGN: Randomized Double-Blind Placebo Controlled trial of iron. Randomization stratified by iron status and day care center (DCC). SETTING: Nine public DCCs in Athens, Greece. SUBJECTS: In all, 49 3-4-y olds (21 anemic, 28 good iron status) with birth weight not less than 2500 g, currently healthy; benign past medical history, IQ > or =1 s.d. below the age-adjusted mean, serum Pb < or =200 ppb (none exceeded 50 ppb), and height, weight and head circumference for age > or =10th percentile. Anemia defined as: (1) pretreatment Hgb <112 g/l and TS <16% and ferritin <12 microg/L OR (2) Hgb rise of >10 g/l (T2-T0) with iron supplementation. Good iron status was defined as baseline levels of Hgb >120 g/l and either TS >20% or serum ferritin >12 microg/l. INTERVENTION: The intervention consisted of a 2-month supplementation of 15 mg iron (and MV) vs placebo (MV alone). RESULTS: After iron treatment, the anemic subjects made significantly fewer errors of commission (14% higher specificity, P<0.05), exhibited 8% higher accuracy (P<0.05) and were significantly more efficient (mean difference=1.09, P<0.05) than those given placebo. These effects of iron were not found among preschoolers with good iron status. No effects of iron treatment were found on the Oddity Learning task. CONCLUSIONS: This study demonstrated that iron supplementation of iron-deficient anemic preschoolers results in an improvement in discrimination, specifically selective attention.

Effects of weaning cereals with different phytate content on growth, development and morbidity: a randomized intervention trial in infants from 6 to 12 months of age.

BACKGROUND: Phytate decreases iron and zinc bioavailability and contributes to deficiencies of iron and zinc, potentially causing anaemia, poor psychomotor development, impaired growth and increased risk of diarrhoea and respiratory infections. AIM: To investigate whether a reduced dietary intake of phytate, either via extensively phytate-reduced infant cereals [milk cereal drinks (MCDs) and porridge] or a milk-based infant formula, would improve growth and development and reduce morbidity in infants. DESIGN: Infants (n = 300) were, in a double-blind design, randomized to three diet intervention groups from 6 to 12 mo of age-commercial MCD and porridge (CC group), phytate-reduced MCD and phytate-reduced porridge (PR group), or milkbased infant formula and porridge with regular phytate content (IF group)-then followed until 18 mo. Dietary intake, anthropometry, development (Bayley Scales of Infant Development) and episodes of infectious diseases were registered. RESULTS: There were no significant differences between study groups in growth, development or morbidity until 12 mo of age. The IF group had a 77% higher risk (95% CI: 1.05-2.97) of diarrhoea compared to the PR group during the 12-17-mo period. Infants with haemoglobin concentration (Hb) < 110 g/l at 12 mo had lower attained weight at 18 mo (11.14 kg vs 11.73 kg, p = 0.012). Infants with serum zinc (S-Zn) <10.7 pmol/l at 12 mo had higher risk of respiratory infections (RR = 1.74, 95% CI: 1.19-2.56) compared to controls. CONCLUSION: Phytate reduction had no effect on growth, development or incidence of diarrhoeal or respiratory infections. Infants with low Hb or low S-Zn may be at higher risk of poor growth and respiratory infections, even in this high-income population.

Serum ferritin and transferrin receptor concentrations during the transition from adolescence to adulthood in a healthy Swedish population.

AIM: As part of a longitudinal nutrition study of adolescents, changes in serum ferritin (s-Ft), serum transferrin receptors (s-TfR) and the TfR/Ft ratio from 15 to 21 y of age were studied in randomly selected Swedish adolescents. METHODS: Blood samples from 60 males and 66 females were drawn at 15, 17 and 21 y of age. RESULTS: In males, median s-Ft increased significantly from 33 microg l(-1) at 15 y to 96 microg l(-1) at 21 y, and in females, after a non-significant decrease at 17 y, from 27 at 15 y to 34 microg l(-1) at 21 y. Between 15 and 17 y, s-TfR increased significantly, in males from 5.9 to 7.7 microg l(-1), and in females from 5.2 to 7.6 microg l(-1), whereas s-TfR decreased significantly in both genders between 17 and 21 y. The log (TfR/Ft) ratio did not change in males between 15 and 17 y, but decreased significantly from 2.2 +/- 0.3 to 1.8 +/- 0.3 by the age of 21 y. In females, the TfR/Ft ratio increased significantly between 15 and 17 y, whereas a significant decrease was found by the age of 21 y. S-Ft <12 microg l(-1) at 15, 17 and 21 y was 3, 2 and 2% in males, and in females 18, 26 and 21%, respectively. CONCLUSION: This study shows an increase in s-Ft and a decline in s-TfR in males, indicating increased iron stores. In females, s-Ft did not increase significantly, whereas s-TfR and the TfR/Ft ratio decreased between 17 and 21 y, indicating that cellular iron needs were met.

Molecular cloning and functional expression of a human intestinal lactoferrin receptor.

Lactoferrin (Lf), a major iron-binding protein in human milk, has been suggested to have multiple biological roles such as facilitating iron absorption, modulating the immune system, embryonic development, and cell proliferation. Our previous binding studies suggested the presence of a specific receptor for Lf (LfR) in the small intestine of newborn infants, which may facilitate iron absorption. We here report the cloning and the functional expression of the human intestinal LfR and the evidence of its involvement in iron metabolism. The entire coding region of the LfR cDNA was cloned by PCR based on amino acid sequences of the purified native LfR (nLfR). The recombinant LfR (rLfR) was then expressed in a baculovirus-insect cell system and purified by immobilized human Lf (hLf) affinity chromatography where binding of hLf to the rLfR was partially Ca(2+) dependent. The apparent molecular mass was 136 kDa under nonreducing conditions and 34 kDa under reducing conditions. 125I-hLf bound to the rLfR with an apparent K(d) of approximately 360 nM. These biochemical properties of the rLfR are similar to those of the nLfR. RT-PCR revealed that the gene was expressed at high levels in fetal small intestine and in adult heart and at lower levels in Caco-2 cells. PI-PLC treatment of Caco-2 cells indicated that the LfR is GPI anchored. In Caco-2 cells transfected with the LfR gene, 125I-hLf binding and 59Fe-hLf uptake were increased by 1.7 and 3.4 times, respectively, compared to those in mock-transfected cells. Our findings demonstrate the presence of a unique receptor-mediated mechanism for nutrient uptake by the newborn.

Long-term marginal intakes of zinc and retinol affect retinol homeostasis without compromising circulating levels during lactation in rats.

Marginal zinc or vitamin A intake is more common than previously thought in industrialized and developing countries, with pregnant and lactating women believed to be particularly at risk. However, the lack of sensitive indicators of zinc and vitamin A status precludes accurate assessment of marginal nutriture. Concurrent deficiencies in zinc and vitamin A intake often coexist, and the interaction between zinc deficiency and vitamin A metabolism may confound results from epidemiologic or intervention studies. To investigate effects of a maternal diet chronically restricted in zinc or vitamin A intake on indices of vitamin A metabolism, we fed rats a control diet (C) or a diet marginal in zinc (ZD), marginal in vitamin A (AD), marginal in both (DD) or pair-fed to DD (PF), preconception through lactation. Plasma retinol (ROH) was greater and retinol binding protein (RBP) was lower in rats fed ZD, AD and DD compared with those fed C. Hepatic cellular retinol binding protein (CRBP) expression was greater than controls in rats fed ZD and AD and lower in those fed DD, whereas RBP expression was greater in the DD- and PF-fed groups compared with rats C. Mammary gland CRBP and RBP expression were not affected by the diets. Milk ROH was lower in rats fed AD, and milk RBP was lower in those fed ZD and DD compared with rats fed C. In summary, chronic, marginal intake of zinc or vitamin A resulted in alterations in tissue retinol metabolism and milk retinol levels without decreasing plasma zinc, retinol or ROH:RBP during lactation. These observations are of concern because these parameters, which are commonly used to assess zinc and vitamin A status, may lead to misassessment of marginal zinc or vitamin A nutriture in some human populations.

Frataxin expression rescues mitochondrial dysfunctions in FRDA cells.

Friedreich's ataxia (FRDA) is the result of mutations in the nuclear-encoded frataxin gene, which is expressed in mitochondria. Several lines of evidence have suggested that frataxin is involved in mitochondrial iron homeostasis. We have transfected the frataxin gene into lymphoblasts of FRDA compound heterozygotes (FRDA-CH) with deficient frataxin expression to produce FRDA-CH-t cells in which message and protein are rescued to near-physiological levels. FRDA-CH cells were more sensitive to oxidative stress by challenge with free iron, hydrogen peroxide and the combination, consistent with a Fenton chemical mechanism of pathophysiology, and this sensitivity was rescued to control levels in FRDA-CH-t cells. Iron challenge caused increased mitochondrial iron levels in FRDA-CH cells, and a decreased mitochondrial membrane potential (MMP), both of which were rescued in FRDA-CH-t cells. The rescue of the low MMP, and high mitochondrial iron concentration by frataxin overexpression suggests that these cellular phenotypes are relevant to the central pathophysiological process in FRDA which is aggravated by exposure to free iron. However, even at physiological iron concentrations, FRDA-CH cells had decreased MMP as well as lower activities of aconitase and ICDH (two enzymes supporting MMP), and twice the level of filtrable mitochondrial iron (but no increase in total mitochondrial iron), and the observed phenotypes were either fully or partially rescued in FRDA-CH-t cells. Free iron is known to be toxic. The observation that frataxin deficiency (either directly or indirectly) causes an increase in filtrable mitochondrial iron provides a new hypothesis for the mechanism of cell death in this disease, and could be a target for therapy.

Effects of type of fat in the diet on iron bioavailability assessed in suckling and weanling rats.

Differences in iron bioavailability from human milk and milk formulas may in part be due to differences in lipid composition. We investigated the short and long term effects of diets based on different fats [corn, coconut, olive, or soy oil, human milk fat (HMF) and a formula fat blend (FF)] on iron absorption in rats. Suckling rat pups dosed with 59Fe-labeled diets containing different fat sources were killed after 6 h, and blood and individual tissues were counted. Iron availability was estimated by % 59Fe in blood. Pups dosed with a more saturated fat (coconut oil) had a higher % 59Fe in blood than those fed other fat sources. Weanling rats were used to determine iron bioavailability from fat sources using both the hemoglobin repletion method and whole body counting. Hemoglobin regeneration was significantly higher for rats fed the HMF diet (8.4 +/- 0.5 g/dl) than from the FF diet (6.5+/-0.6 g/dl) or the corn oil diet (less saturated) (6.4 +/- 0.3 g/dl). Rats fed diets based on coconut oil (more saturated) had significantly higher % 59Fe retention (61.6 +/- 1.4) than rats fed diets based on FF (49.8 +/- 3.4). There was a significant positive association between oleic acid in the diet and oleic acid in the intestinal mucosa (r = 0.95, p < 0.05) and between linoleic acid in the diet and linoleic acid in the intestinal mucosa (r = 0.97, p < 0.05) suggesting that the dietary treatment altered the fatty acid composition of the brush border membrane. Our results suggest that saturated fats may increase iron absorption and that part of this may be achieved by changes in the fatty acid composition of the intestinal mucosa. Hemoglobin regeneration and % 59Fe retention data suggest that differences in iron absorption from infant diets may in part be due to differences in fat composition. Therefore, lipid composition of infant formulas should also be taken into consideration as a factor influencing iron bioavailability.

High affinity binding of the transcobalamin II-cobalamin complex and mRNA expression of haptocorrin by human mammary epithelial cells.

Little is known about the acquisition of cobalamin by the mammary gland and its secretion into milk. Human milk and plasma contain at least two types of cobalamin binding proteins: transcobalamin II (TC) and haptocorrin (HC). In plasma, TC is responsible for the transport of cobalamin to tissues and cells; however, cobalamin in milk is present exclusively bound to HC. We show that human mammary epithelial cells (HMEC) exhibit high affinity for TC; Scatchard analysis revealed a single class of binding sites for the TC-[(57)Co]cyanocobalamin complex with a dissociation constant (K(d)) of 4.9 x 10(-11) M. Uptake of the TC-[(57)Co]cyanocobalamin complex at 37 degrees C was saturable by 24 h. Binding of free [(57)Co]cyanocobalamin to HMEC was not saturable and very limited binding of the HC-[(57)Co]cyanocobalamin complex was observed. Expression of the haptocorrin gene by HMEC was confirmed by Northern blot and PCR analysis. Thus, a specific cell surface receptor for the TC-cobalamin complex exists in the mammary gland and once cobalamin is internalized, it may be transferred to HC and subsequently secreted into milk as a HC-cobalamin complex.