RESUMO
In order to supply adequate iron during pregnancy, the levels of the iron regulatory hormone hepcidin in the maternal circulation are suppressed, thereby increasing dietary iron absorption and storage iron release. Whether this decrease in maternal hepcidin is caused by changes in factors known to regulate hepcidin expression, or by other unidentified pregnancy factors, is not known. To investigate this, we examined iron parameters during pregnancy in mice. We observed that hepatic iron stores and transferrin saturation, both established regulators of hepcidin production, were decreased in mid and late pregnancy in normal and iron loaded dams, indicating an increase in iron utilization. This can be explained by a significant increase in maternal erythropoiesis, a known suppressor of hepcidin production, by mid-pregnancy, as indicated by an elevation in circulating erythropoietin and an increase in spleen size and splenic iron uptake. Iron utilization increased further in late pregnancy due to elevated fetal iron demand. By increasing maternal iron levels in late gestation, we were able to stimulate the expression of the gene encoding hepcidin, suggesting that the iron status of the mother is the predominant factor influencing hepcidin levels during pregnancy. Our data indicate that pregnancy-induced hepcidin suppression likely occurs because of reductions in maternal iron reserves due to increased iron requirements, which predominantly reflect stimulated erythropoiesis in mid-gestation and increased fetal iron requirements in late gestation, and that there is no need to invoke other factors, including novel pregnancy factor(s), to explain these changes.
Assuntos
Hepcidinas , Deficiências de Ferro , Feminino , Gravidez , Camundongos , Animais , Hepcidinas/genética , Hepcidinas/metabolismo , Ferro/metabolismo , Ferro da Dieta , Feto/metabolismo , EritropoeseRESUMO
BACKGROUND: Many women enter pregnancy with iron stores that are insufficient to maintain maternal iron balance and support fetal development and consequently, often require iron supplements. However, the side effects associated with many currently available iron supplements can limit compliance. OBJECTIVE: This study aimed to test the safety and efficacy of a novel nanoparticulate iron supplement, a dietary ferritin analog termed iron hydroxide adipate tartrate (IHAT), in pregnant mice. METHODS: Female C57BL/6 mice were maintained on either an iron-deficient or a control diet for 2 wk prior to timed mating to develop iron-deficient or iron-sufficient pregnancy models, respectively. Mice from each model were then gavaged daily with 10 mg iron/kg body weight as either IHAT or ferrous sulfate, or with water only, beginning on embryonic day (E) 4.5. Mice were killed on E18.5 and maternal iron and hematological parameters were measured. The expression of genes encoding iron transporters and oxidative stress markers in the duodenum and placenta were determined, along with hepatic expression of the gene encoding the iron regulatory hormone hepcidin and fetal iron. RESULTS: Oral IHAT and ferrous sulfate were equally effective at increasing maternal hemoglobin (20.2% and 16.9%, respectively) and hepatic iron (30.2% and 29.3%, respectively), as well as total fetal iron (99.7% and 83.8%, respectively), in iron-deficient pregnant mice compared with those gavaged with water only, with no change in oxidative stress markers seen with either treatment. However, there was a significant increase in the placental expression of the oxidative stress marker heme oxygenase 1 in iron-replete pregnant mice treated with ferrous sulfate when compared with iron-replete pregnant mice gavaged with IHAT (96.9%, P <0.05). CONCLUSIONS: IHAT has proved a safe and effective alternative to oral ferrous sulfate in mice, and it has potential for treating iron deficiency in human pregnancy.
Assuntos
Anemia Ferropriva , Deficiências de Ferro , Anemia Ferropriva/tratamento farmacológico , Animais , Feminino , Ferritinas/uso terapêutico , Compostos Ferrosos/uso terapêutico , Hemoglobinas/análise , Humanos , Ferro , Camundongos , Camundongos Endogâmicos C57BL , Placenta/química , Gravidez , ÁguaRESUMO
Iron deficiency is one of the most common nutritional deficiencies worldwide and is often treated with oral iron supplements. However, commonly used supplements, including those based on ferrous iron salts, are associated with gastrointestinal side effects and unfavorable changes in the intestinal microbiome. Sucrosomial® iron is a novel iron formulation that is effective at treating iron deficiency, and with fewer gastrointestinal side effects, yet its effect on the gut microbiome has not been examined previously. Thus, we treated mice for two weeks with diets containing either Sucrosomial® iron or ferrous sulfate as the sole iron source and examined bacterial communities in the intestine using 16S Microbial Profiling of DNA extracted from feces collected both prior to and following dietary treatment. Mice treated with Sucrosomial® iron showed an increase in Shannon diversity over the course of the study. This was associated with a decrease in the abundance of the phylum Proteobacteria, which contains many pathogenic species, and an increase in short chain fatty acid producing bacteria such as Lachnospiraceae, Oscillibacter and Faecalibaculum. None of these changes were observed in mice treated with ferrous sulfate. These results suggest that Sucrosomial® iron may have a beneficial effect on the intestinal microbiome when compared to ferrous sulfate and that this form of iron is a promising alternative to ferrous iron salts for the treatment of iron deficiency.
Assuntos
Anemia Ferropriva , Microbioma Gastrointestinal , Deficiências de Ferro , Anemia Ferropriva/tratamento farmacológico , Animais , Suplementos Nutricionais , Compostos Ferrosos/farmacologia , Ferro , Camundongos , Sais/uso terapêuticoRESUMO
BACKGROUND: The ferroxidase zyklopen (Zp) has been implicated in the placental transfer of iron to the fetus. However, the evidence for this is largely circumstantial. OBJECTIVES: This study aimed to determine whether Zp is essential for placental iron transfer. METHODS: A model was established using 8- to 12-wk-old pregnant C57BL/6 mice on standard rodent chow in which Zp was knocked out in the fetus and fetal components of the placenta. Zp was also disrupted in the entire placenta using global Zp knockout mice. Inductively coupled plasma MS was used to measure total fetal iron, an indicator of the amount of iron transferred by the placenta to the fetus, at embryonic day 18.5 of gestation. Iron transporter expression in the placenta was measured by Western blotting, and the expression of Hamp1, the gene encoding the iron regulatory hormone hepcidin, was determined in fetal liver by real-time PCR. RESULTS: There was no change in the amount of iron transferred to the fetus when Zp was disrupted in either the fetal component of the placenta or the entire placenta. No compensatory changes in the expression of the iron transport proteins transferrin receptor 1 or ferroportin were observed, nor was there any change in fetal liver Hamp1 mRNA. Hephl1, the gene encoding Zp, was expressed mainly in the maternal decidua of the placenta and not in the nutrient-transporting syncytiotrophoblast. Disruption of Zp in the whole placenta resulted in a 26% increase in placental size (P < 0.01). CONCLUSIONS: Our data indicate that Zp is not essential for the efficient transfer of iron to the fetus in mice and is localized predominantly in the maternal decidua. The increase in placental size observed when Zp is knocked out in the entire placenta suggests that this protein may play a role in placental development.
Assuntos
Ceruloplasmina , Placenta , Animais , Ceruloplasmina/genética , Feminino , Feto/metabolismo , Ferro/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Placenta/metabolismo , Placentação , GravidezRESUMO
Iron-loading disorders, such as hereditary hemochromatosis, are associated with inappropriately low expression of the iron regulatory hormone, hepcidin. A recent study has demonstrated that food deprivation can increase hepcidin production in mice. We have examined this effect in more detail to determine whether the pathway(s) that are responsible might provide novel targets for pharmaceutical intervention in disorders of iron homeostasis. C57BL/6 mice were deprived of food for 5, 10, 16, or 24 h before euthanasia, then blood and tissue samples were collected for analysis. The effect of food deprivation was also examined in Hfe-/- mice, a model of hereditary hemochromatosis, as well as mice that were maintained on an iron-deficient diet or injected with erythropoietin. Food deprivation increased the hepatic expression of the gene that encodes hepcidin, hepcidin antimicrobial peptide 1 ( Hamp1), with maximal expression observed after 16 h, and was able to overcome the reduction in Hamp1 expression associated with Hfe deficiency. Food deprivation also increased Hamp1 expression in response to stimuli that more strongly suppress the gene, such as iron deficiency and erythropoietin treatment, but the effects were not significant. These results indicate that Hamp1 induction by food deprivation is independent of HFE and suggest that targeting the pathway regulated by food deprivation could have clinical benefit in iron-loading conditions.-Mirciov, C. S. G., Wilkins, S. J., Anderson, G. J., Frazer, D. M. Food deprivation increases hepatic hepcidin expression and can overcome the effect of Hfe deletion in male mice.
RESUMO
The stimulation of erythrocyte formation increases the demand for iron by the bone marrow and this in turn may affect the levels of circulating diferric transferrin. As this molecule influences the production of the iron regulatory hormone hepcidin, we hypothesized that erythropoiesis-driven changes in diferric transferrin levels could contribute to the decrease in hepcidin observed following the administration of erythropoietin. To examine this, we treated mice with erythropoietin and examined diferric transferrin at various time points up to 18 hours. We also investigated the effect of altering diferric transferrin levels on erythropoietin-induced inhibition of Hamp1, the gene encoding hepcidin. We detected a decrease in diferric transferrin levels 5 hours after erythropoietin injection and prior to any inhibition of the hepatic Hamp1 message. Diferric transferrin returned to control levels 12 hours after erythropoietin injection and had increased beyond control levels by 18 hours. Increasing diferric transferrin levels via intravenous iron injection prevented the inhibition of Hamp1 expression by erythropoietin without altering hepatic iron concentration or the expression of Erfe, the gene encoding erythroferrone. These results suggest that diferric transferrin likely contributes to the inhibition of hepcidin production in the period shortly after injection of erythropoietin and that, under the conditions examined, increasing diferric transferrin levels can overcome the inhibitory effect of erythroferrone on hepcidin production. They also imply that the decrease in Hamp1 expression in response to an erythropoietic stimulus is likely to be mediated by multiple signals.
Assuntos
Eritropoese/efeitos dos fármacos , Eritropoetina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hepcidinas/sangue , Transferrina/farmacologia , Animais , Masculino , Camundongos , Fatores de TempoRESUMO
In conditions such as ß-thalassaemia, stimulated erythropoiesis can reduce the expression of the iron regulatory hormone hepcidin, increasing both macrophage iron release and intestinal iron absorption and leading to iron loading. However, in certain conditions, sustained elevation of erythropoiesis can occur without an increase in body iron load. To investigate this in more detail, we made use of a novel mouse strain (RBC14), which exhibits mild ß-thalassaemia intermedia with minimal iron loading. We compared iron homeostasis in RBC14 mice to that of Hbbth3/+ mice, a more severe model of ß-thalassaemia intermedia. Both mouse strains showed a decrease in plasma iron half-life, although the changes were less severe in RBC14 mice. Despite this, intestinal ferroportin and serum hepcidin levels were unaltered in RBC14 mice. In contrast, Hbbth3/+ mice exhibited reduced serum hepcidin and increased intestinal ferroportin. However, splenic ferroportin levels were increased in both mouse strains. These data suggest that in low-grade chronic haemolytic anaemia, such as that seen in RBC14 mice, the increased erythroid iron requirements can be met through enhanced macrophage iron release without the need to increase iron absorption, implying that hepcidin is not the sole regulator of macrophage iron release in vivo.
Assuntos
Hepcidinas/metabolismo , Ferro/metabolismo , Talassemia beta/metabolismo , Animais , Biomarcadores , Proteínas de Transporte de Cátions/metabolismo , Modelos Animais de Doenças , Células Precursoras Eritroides/metabolismo , Eritropoese , Feminino , Hepcidinas/sangue , Ferro/sangue , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , alfa-Globinas/metabolismo , Talassemia beta/sangueRESUMO
The ferritin core is composed of fine nanoparticulate Fe(3+) oxohydroxide, and we have developed a synthetic mimetic, nanoparticulate Fe(3+) polyoxohydroxide (nanoFe(3+)). The aim of this study was to determine how dietary iron derived in this fashion is absorbed in the duodenum. Following a 4 wk run-in on an Fe-deficient diet, mice with intestinal-specific disruption of the Fpn-1 gene (Fpn-KO), or littermate wild-type (WT) controls, were supplemented with Fe(2+) sulfate (FeSO4), nanoFe(3+), or no added Fe for a further 4 wk. A control group was Fe sufficient throughout. Direct intestinal absorption of nanoFe(3+) was investigated using isolated duodenal loops. Our data show that FeSO4 and nanoFe(3+) are equally bioavailable in WT mice, and at wk 8 the mean ± SEM hemoglobin increase was 18 ± 7 g/L in the FeSO4 group and 30 ± 5 g/L in the nanoFe(3+) group. Oral iron failed to be utilized by Fpn-KO mice and was retained in enterocytes, irrespective of the iron source. In summary, although nanoFe(3+) is taken up directly by the duodenum its homeostasis is under the normal regulatory control of dietary iron absorption, namely via ferroportin-dependent efflux from enterocytes, and thus offers potential as a novel oral iron supplement.
Assuntos
Proteínas de Transporte de Cátions/fisiologia , Duodeno/metabolismo , Enterócitos/metabolismo , Compostos Férricos/farmacocinética , Absorção Intestinal/fisiologia , Ferro da Dieta/farmacocinética , Nanopartículas , Administração Oral , Anemia Ferropriva/metabolismo , Animais , Disponibilidade Biológica , Proteínas de Transporte de Cátions/biossíntese , Proteínas de Transporte de Cátions/deficiência , Proteínas de Transporte de Cátions/genética , Compostos Ferrosos/farmacocinética , Regulação da Expressão Gênica , Hemoglobinas/análise , Hepcidinas/biossíntese , Hepcidinas/genética , Homeostase , Deficiências de Ferro , Camundongos , Camundongos Knockout , Baço/metabolismoRESUMO
The BMP/SMAD signalling pathway plays an important role in iron homeostasis, regulating hepcidin expression in response to body iron levels. However, the role of this pathway in the reduction in hepcidin associated with increased erythropoiesis (and secondary iron loading) is unclear. To investigate this, we established a mouse model of chronic stimulated erythropoiesis with secondary iron loading using the haemolytic agent phenylhydrazine. We then examined the expression of components of the BMP6/SMAD signalling pathway in these animals. We also examined this pathway in the Hbb(th3/+) mouse, a model of the iron loading anaemia ß-thalassaemia intermedia. Increasing doses of phenylhydrazine led to a progressive increase in both liver iron levels and Bmp6 mRNA expression, but, in contrast, hepatic Hamp expression declined. The increase in Bmp6 expression was not associated with a corresponding change in the phosphorylation of hepatic SMAD1/5/8, indicating that stimulated erythropoiesis decreases the ability of BMP6 to alter SMAD phosphorylation. Increased erythropoiesis also reduces the capacity of phosphorylated SMAD (pSMAD) to induce hepcidin, as Hamp levels declined despite no changes in pSMAD1/5/8. Similar results were seen in Hbb(th3/+) mice. Thus the erythroid signal probably affects some components of BMP/SMAD signalling, but also may exert some independent effects.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteína Morfogenética Óssea 6/metabolismo , Eritropoese/efeitos dos fármacos , Sobrecarga de Ferro/metabolismo , Anemia Hemolítica/induzido quimicamente , Anemia Hemolítica/metabolismo , Animais , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fator 15 de Diferenciação de Crescimento/metabolismo , Hemólise/efeitos dos fármacos , Hepcidinas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenil-Hidrazinas/efeitos adversos , Fenil-Hidrazinas/farmacologia , Fosforilação , Receptores da Transferrina/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Baço/metabolismo , Transferrina/metabolismo , Talassemia beta/metabolismoRESUMO
BACKGROUND & AIMS: Suckling mammals absorb high levels of iron to support their rapid growth. In adults, iron absorption is controlled by systemic signals that alter expression of the iron-regulatory hormone hepcidin. We investigated whether hepcidin and absorption respond appropriately to systemic stimuli during suckling. METHODS: In Sprague-Dawley rats, iron levels increased following administration of iron dextran, and inflammation was induced with lipopolysaccharide. Gene expression was measured by quantitative reverse-transcription polymerase chain reaction; protein levels were measured by immunoblot analyses. Iron absorption was determined based on retention of an oral dose of 59Fe. RESULTS: Iron absorption was high during suckling and reduced to adult levels upon weaning. In response to iron dextran or lipopolysaccharide, iron absorption in adults decreased substantially, but, in suckling animals, the changes were minimal. Despite this, expression of hepcidin messenger RNA was strongly induced by each agent, before and after weaning. The hyporesponsiveness of iron absorption to increased levels of hepcidin during suckling correlated with reduced or absent duodenal expression of ferroportin 1 (Fpn1), normally a hepcidin target. Fpn1 expression was robust in adults. Predominance of the Fpn1A splice variant, which is under iron-dependent translational control, accounts for the low level of Fpn1 in the iron-deficient intestine of suckling rats. CONCLUSIONS: Iron absorption during suckling is largely refractory to changes in expression of the systemic iron regulator hepcidin, and this in turn reflects limited expression of Fpn1 protein in the small intestine. Iron absorption is therefore not always controlled by hepcidin.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Duodeno/metabolismo , Absorção Intestinal , Ferro da Dieta/metabolismo , Complexo Ferro-Dextran/metabolismo , Lactação , Fatores Etários , Envelhecimento , Animais , Animais Recém-Nascidos , Animais Lactentes , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Transporte Biológico , Western Blotting , Proteínas de Transporte de Cátions/genética , Modelos Animais de Doenças , Regulação para Baixo , Retroalimentação Fisiológica , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hepcidinas , Inflamação/induzido quimicamente , Inflamação/metabolismo , Lipopolissacarídeos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , DesmameRESUMO
BACKGROUND: Expression of the key iron regulatory hormone hepcidin is increased by some stimuli (iron loading, inflammation) but decreased by others (increased erythropoiesis, iron deficiency). We investigated the response of hepcidin to increased erythropoiesis and iron deficiency in the presence of an acute inflammation to assess the relative strengths of these stimuli. DESIGN AND METHODS: Sprague-Dawley rats were maintained on control or iron-deficient diets and treated with lipopolysaccharide to induce inflammation or phenylhydrazine to stimulate erythropoiesis. The levels of Hamp, IL-6 and α2m mRNA were determined by qualitative real-time polymerase chain reaction and those of serum interleukin-6 and tumor necrosis factor-α were measured by enzyme-linked immunosorbent assay. Cultured RAW264.7 and HuH7 cells were used in associated studies. RESULTS: The increase in hepatic hepcidin levels induced by lipopolysaccharide was not affected by phenylhydrazine treatment but was blunted by iron deficiency. Lipopolysaccharide-treated iron-deficient animals also showed lower liver α2m mRNA and reduced serum interleukin-6 and tumor necrosis factor-α, suggesting a more generalized effect of iron deficiency. Similarly, RAW 264.7 cells treated with iron chelators and then stimulated with lipopolysaccharide showed lower IL-6 mRNA than cells treated with lipopolysaccharide alone. Huh7 cells treated with an iron chelator showed a blunted hepcidin response to interleukin-6, suggesting that the response of hepatic parenchymal cells to inflammatory cytokines may also be iron-dependent. CONCLUSIONS: In any one physiological situation, net hepcidin levels are determined by the relative strengths of competing stimuli. The ability of severe iron deficiency to blunt the response to lipopolysaccharide of both hepcidin and other markers of inflammation suggests that adequate iron levels are necessary for a full acute phase response.
Assuntos
Anemia Ferropriva/imunologia , Peptídeos Catiônicos Antimicrobianos/genética , Citocinas/genética , Lipopolissacarídeos/farmacologia , Anemia Ferropriva/genética , Animais , Peptídeos Catiônicos Antimicrobianos/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Hepcidinas , Inflamação/imunologia , Ferro/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Growth restriction, craniofacial dysmorphology, and central nervous system defects are the main diagnostic features of fetal alcohol syndrome. Studies in humans and mice have reported that the growth restriction can be prenatal or postnatal, but the underlying mechanisms remain unknown.We recently described a mouse model of moderate gestational ethanol exposure that produces measurable phenotypes in line with fetal alcohol syndrome (e.g., craniofacial changes and growth restriction in adolescent mice). In this study, we characterize in detail the growth restriction phenotype by measuring body weight at gestational day 16.5, cross-fostering from birth to weaning, and by extending our observations into adulthood. Furthermore, in an attempt to unravel the molecular events contributing to the growth phenotype, we have compared gene expression patterns in the liver and kidney of nonfostered, ethanol-exposed and control mice at postnatal day 28.We find that the ethanol-induced growth phenotype is not detectable prior to birth, but is present at weaning, even in mice that have been cross-fostered to unexposed dams. This finding suggests a postnatal growth restriction phenotype that is not due to deficient postpartum care by dams that drank ethanol, but rather a physiologic result of ethanol exposure in utero. We also find that, despite some catch-up growth after 5 weeks of age, the effect extends into adulthood, which is consistent with longitudinal studies in humans.Genome-wide gene expression analysis revealed interesting ethanol-induced changes in the liver, including genes involved in the metabolism of exogenous and endogenous compounds, iron homeostasis, and lipid metabolism.
Assuntos
Etanol/efeitos adversos , Transtornos do Espectro Alcoólico Fetal/genética , Transtornos do Espectro Alcoólico Fetal/metabolismo , Retardo do Crescimento Fetal/genética , Expressão Gênica , Animais , Peso Corporal , Anormalidades Craniofaciais/metabolismo , Modelos Animais de Doenças , Feminino , Retardo do Crescimento Fetal/metabolismo , Humanos , Estudos Longitudinais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , GravidezRESUMO
Manganese is an essential metal that is required for a wide range of biological functions. Ferroportin (FPN), the only known cellular exporter of iron, has also been proposed to play a role in manganese export, but this relationship is incompletely understood. To investigate this in more detail in vivo, we examined the relative distributions of manganese and iron in TMPRSS6 deficient mice, which are characterized by constitutively high expression of the iron regulatory hormone hepcidin and, consequently, very low FPN levels in their tissues. Tmprss6-/- mice showed frank iron deficiency and reduced iron levels in most tissues, consistent with FPN playing an important role in the distribution of this metal, but manganese levels were largely unaffected. Associated studies using intestine-specific FPN knockout mice showed that loss of FPN significantly reduced the dietary absorption of iron, but had no effect on manganese intake. Taken together, our data suggest that FPN does not play a major role in Mn transport in vivo. They do not exclude a minor role for FPN in manganese homeostasis, nor the possibility that the transporter may be relevant at high Mn levels, but at physiological levels of this metal, other transport proteins appear to be more important.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Hepcidinas/metabolismo , Homeostase , Manganês/metabolismo , Animais , Regulação da Expressão Gênica , Hepcidinas/genética , Ferro/metabolismo , Manganês/sangue , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Serina Endopeptidases/deficiência , Serina Endopeptidases/metabolismoRESUMO
Background & Aims: Multicopper ferroxidases (MCFs) facilitate intestinal iron absorption and systemic iron recycling, likely by a mechanism involving the oxidization of Fe2+ from the iron exporter ferroportin 1 for delivery to the circulating Fe3+ carrier transferrin. Hephaestin (HEPH), the only MCF known to be expressed in enterocytes, aids in the basolateral transfer of dietary iron to the blood. Mice lacking HEPH in the whole body (Heph-/- ) or intestine alone (Hephint/int ) exhibit defects in dietary iron absorption but still survive and grow. Circulating ceruloplasmin (CP) is the only other known MCF likely to interact with enterocytes. Our aim was to assess the effects of combined deletion of HEPH and CP on intestinal iron absorption and homeostasis in mice. Methods: Mice lacking both HEPH and CP (Heph-/-Cp-/- ) and mice with whole-body knockout of CP and intestine-specific deletion of HEPH (Hephint/intCp-/- ) were generated and phenotyped. Results: Heph-/-Cp-/- mice were severely anemic and had low serum iron, but they exhibited marked iron loading in duodenal enterocytes, the liver, heart, pancreas, and other tissues. Hephint/intCp-/- mice were moderately anemic (similar to Cp-/- mice) but were iron loaded only in the duodenum and liver, as in Hephint/int and Cp-/- mice, respectively. Both double knockout models absorbed iron in radiolabeled intestinal iron absorption studies, but the iron was inappropriately distributed, with an abnormally high percentage retained in the liver. Conclusions: These studies indicate that HEPH and CP, and likely MCFs in general, are not essential for intestinal iron absorption but are required for proper systemic iron distribution. They also point to important extra-intestinal roles for HEPH in maintaining whole-body iron homeostasis.
Assuntos
Ceruloplasmina/deficiência , Ferro/metabolismo , Proteínas de Membrana/deficiência , Absorção Fisiológica , Anemia/patologia , Animais , Animais Lactentes , Tamanho Corporal , Peso Corporal , Proteínas de Transporte de Cátions/metabolismo , Ceruloplasmina/metabolismo , Modelos Animais de Doenças , Duodeno/metabolismo , Enterócitos/metabolismo , Feminino , Ligadura , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , FenótipoRESUMO
Iron is crucial for many biological functions, but quantitatively the most important use of iron is in the production of hemoglobin in red blood cell precursors. The amount of iron in the plasma, and hence its availability for hemoglobin synthesis, is determined by the liver-derived iron regulatory hormone hepcidin. When the iron supply to erythroid precursors is limited, as often occurs during stimulated erythropoiesis, these cells produce signals to inhibit hepatic hepcidin production, thereby increasing the amount of iron that enters the plasma. How stimulated erythropoiesis suppresses hepcidin production is incompletely understood, but erythroferrone, Gdf15 and Twsg1 have emerged as candidate regulatory molecules. To further examine the relationship between erythropoiesis and the candidate erythroid regulators, we have studied five mouse models of anemia, including two models of ß-thalassemia (Hbbth3/+ and RBC14), the hemoglobin deficit mouse (hbd), dietary iron deficient mice and mice treated with phenylhydrazine to induce acute hemolysis. Hematological parameters, iron status and the expression of Erfe (the gene encoding erythroferrone), Gdf15 and Twsg1 in the bone marrow and spleen were examined. Erfe expression was the most consistently upregulated of the candidate erythroid regulators in all of the mouse models examined. Gene expression was particularly high in the bone marrow and spleen of iron deficient animals, making erythroferrone an ideal candidate erythroid regulator, as its influence is strongest when iron supply to developing erythroid cells is limited. Gdf15 expression was also upregulated in most of the anemia models studied although the magnitude of the increase was generally less than that of Erfe. In contrast, very little regulation of Twsg1 was observed. These results support the prevailing hypothesis that erythroferrone is a promising erythroid regulator and demonstrate that Erfe expression is stimulated most strongly when the iron supply to developing erythroid cells is compromised.
Assuntos
Anemia/metabolismo , Anemia/patologia , Células Precursoras Eritroides/metabolismo , Hepcidinas/metabolismo , Anemia/sangue , Animais , Modelos Animais de Doenças , Eritropoetina/sangue , Ferro/metabolismo , Deficiências de Ferro , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Modelos Genéticos , Fenil-Hidrazinas , Receptores da Transferrina/metabolismoRESUMO
BACKGROUND & AIMS: Previous studies have suggested that iron absorption in suckling mammals is refractory to stimuli that normally would decrease absorption in adults. To better understand the regulation of iron absorption during suckling, we have characterized the relationship between hepcidin, ferroportin, and iron absorption at this crucial stage of life. METHODS: To determine whether ferroportin is involved in iron absorption during suckling, absorption was measured in intestine-specific ferroportin knockout mice. The effect of constitutive hepcidin overexpression on intestinal iron absorption also was investigated in suckling transmembrane serine protease 6 knockout mice. Finally, suckling mice were injected with lipopolysaccharide to induce hepcidin expression. Blood was collected for serum iron analysis, and liver tissue and duodenal enterocytes were collected for gene and protein expression profiles. RESULTS: Iron absorption was very low in suckling ferroportin knockout mice, indicating that ferroportin is responsible for the majority of the iron absorbed at this time. However, increases in hepcidin during suckling, as seen in transmembrane serine protease 6 knockout mice and in mice injected with lipopolysaccharide, did not affect enterocyte ferroportin levels. Immunofluorescent localization of ferroportin showed that the protein localized to the basolateral membrane of duodenal enterocytes in both suckling and weaned mice. CONCLUSIONS: These data show that the high iron absorption occurring during suckling is mediated by ferroportin. However, enterocyte ferroportin is hyporesponsive to hepcidin at this time, despite being expressed on the basolateral membrane. Alterations to ferroportin that prevent hepcidin binding during suckling may allow iron absorption to remain high regardless of hepcidin expression levels, reducing the likelihood of iron deficiency during development.
RESUMO
The amount of iron in the body is controlled at the point of absorption in the proximal small intestine. Dietary iron enters the intestinal epithelium via the brush-border transporter DMT1 and exits through the basolateral membranes. The basolateral transfer of iron requires two components: a copper-containing iron oxidase known as hephaestin and a membrane transport protein IREG1. The amount of iron traversing the enterocytes is directly related to body iron requirements and inversely related to the iron content of the intestinal epithelium. We propose that body signals control iron absorption by first acting on crypt enterocytes to determine the expression of basolateral transport components. This, in turn, modulates the intracellular iron content of mature epithelial cells, which ultimately determines the activity of the brush-border transporter DMT1.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Proteínas de Membrana/metabolismo , Animais , Duodeno/metabolismo , Enterócitos/enzimologia , Enterócitos/metabolismo , Regulação da Expressão Gênica , Humanos , Absorção Intestinal/fisiologia , Proteínas de Ligação ao Ferro/metabolismo , Modelos BiológicosRESUMO
Hephaestin is a vertebrate multicopper ferroxidase important for the transfer of dietary iron from intestinal cells to the blood. Hephaestin is mutated in the sex-linked anemia mouse, resulting in iron deficiency. However, sex-linked anemia mice still retain some hephaestin ferroxidase activity. They survive, breed, and their anemia improves with age. To gain a better understanding of the role of hephaestin in iron homeostasis, we used the Cre-lox system to generate knockout mouse models with whole body or intestine-specific (Villin promoter) ablation of hephaestin. Both types of mice were viable, indicating that hephaestin is not essential and that other mechanisms, multicopper ferroxidase-dependent or not, must compensate for hephaestin deficiency. The knockout strains, however, both developed a microcytic, hypochromic anemia, suggesting severe iron deficiency and confirming that hephaestin plays an important role in body iron acquisition. Consistent with this, the knockout mice accumulated iron in duodenal enterocytes and had reduced intestinal iron absorption. In addition, the similarities of the phenotypes of the whole body and intestine-specific hephaestin knockout mice clarify the important role of hephaestin specifically in intestinal enterocytes in maintaining whole body iron homeostasis. These mouse models will serve as valuable tools to study the role of hephaestin and associated proteins in iron transport in the small intestine and other tissues.
Assuntos
Absorção Intestinal , Mucosa Intestinal/metabolismo , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Animais , Peso Corporal , Feminino , Genótipo , Absorção Intestinal/genética , Deficiências de Ferro , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , FenótipoRESUMO
BACKGROUND: Some years ago we established an N-ethyl-N-nitrosourea screen for modifiers of transgene variegation in the mouse and a preliminary description of the first six mutant lines, named MommeD1-D6, has been published. We have reported the underlying genes in three cases: MommeD1 is a mutation in SMC hinge domain containing 1 (Smchd1), a novel modifier of epigenetic gene silencing; MommeD2 is a mutation in DNA methyltransferase 1 (Dnmt1); and MommeD4 is a mutation in Smarca 5 (Snf2h), a known chromatin remodeler. The identification of Dnmt1 and Smarca5 attest to the effectiveness of the screen design. RESULTS: We have now extended the screen and have identified four new modifiers, MommeD7-D10. Here we show that all ten MommeDs link to unique sites in the genome, that homozygosity for the mutations is associated with severe developmental abnormalities and that heterozygosity results in phenotypic abnormalities and reduced reproductive fitness in some cases. In addition, we have now identified the underlying genes for MommeD5 and MommeD10. MommeD5 is a mutation in Hdac1, which encodes histone deacetylase 1, and MommeD10 is a mutation in Baz1b (also known as Williams syndrome transcription factor), which encodes a transcription factor containing a PHD-type zinc finger and a bromodomain. We show that reduction in the level of Baz1b in the mouse results in craniofacial features reminiscent of Williams syndrome. CONCLUSIONS: These results demonstrate the importance of dosage-dependent epigenetic reprogramming in the development of the embryo and the power of the screen to provide mouse models to study this process.
Assuntos
Desenvolvimento Embrionário , Epigênese Genética , Animais , Feminino , Genes Letais , Genoma , Heterozigoto , Histona Desacetilase 1 , Histona Desacetilases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Transgênicos , Fatores de Transcrição/metabolismo , Síndrome de Williams/fisiopatologiaRESUMO
Intestinal iron absorption is extremely high in neonatal mammals but falls rapidly to adult levels following weaning. The aim of this study was to investigate the molecular basis of this elevated neonatal absorption using the rat as an experimental model. RNA was extracted from various sections of the intestine of 10-, 15-, 20-, 25-, and 300-day-old rats and the expression of the genes encoding DMT1 (Slc11a2), ferroportin (Slc40a1), Cybrd1 (Cybrd1), and hephaestin (heph) determined by ribonuclease protection assay. The hepatic expression of Hamp was studied at the same ages. Iron absorption was examined by following (59)Fe uptake in both whole animals and in isolated intestinal loops. Slc11a2, Slc40a1, and Cybrd1 mRNAs were highly expressed in all regions of the small intestine and colon studied in suckling rats. However, after weaning, when iron absorption declined significantly, strong expression was retained only in the duodenum. No change in hephaestin mRNA occurred in any part of the digestive tract. In the distal small intestine and colon, Slc40a1 expression most closely followed the change in absorption that occurred after weaning. Hamp expression was low during the neonatal period and increased to adult levels following weaning. Our results suggest that the distal small intestine and colon contribute significantly to the high intestinal iron absorption seen in neonatal animals and that this reflects increased expression of the iron transporters, particularly Slc40a1.