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Changes in the Expression of Intestinal Iron Transport and Hepatic 655 SMALL INTESTINE Changes in the expression of intestinal iron transport and Gut: first published as 10.1136/gut.2003.031153 on 13 April 2004. Downloaded from hepatic regulatory molecules explain the enhanced iron absorption associated with pregnancy in the rat K N Millard, D M Frazer, S J Wilkins, G J Anderson ............................................................................................................................... Gut 2004;53:655–660. doi: 10.1136/gut.2003.031153 Background: Iron absorption increases during pregnancy to cater for the increased iron requirements of the growing fetus. Aims: To investigate the role of the duodenal iron transport molecules and hepatic regulatory molecules in coordinating the changes in iron absorption observed during pregnancy. Methods: Rats at various days of gestation and 24–48 hours post-partum were examined for hepatic expression of hepcidin, transferrin receptors 1 and 2, and HFE (the gene mutated in the most prevalent See end of article for authors’ affiliations form of hereditary haemochromatosis), and duodenal expression of divalent metal transporter 1 (DMT1), ....................... duodenal cytochrome b (Dcytb), iron regulated mRNA (Ireg1), and hephaestin (Hp) by ribonuclease protection assay, western blotting, and immunohistochemistry. Correspondence to: Dr G J Anderson, Iron Results: Decreased hepatic non-haem iron and transferrin saturation and increased expression of Metabolism Laboratory, transferrin receptor 1 in the liver indicated a progressive reduction in maternal body iron stores during Queensland Institute of pregnancy. Duodenal expression of the iron transport molecules DMT1, Dcytb, and Ireg1 increased during Medical Research, PO pregnancy, and this corresponded with a reduction in hepcidin, HFE, and transferrin receptor 2 Royal Brisbane Hospital, Brisbane, Queensland expression in the liver. Expression of all molecules returned towards control values by 24–48 hours post- 4029, Australia; gregA@ partum. qimr.edu.au Conclusions: These data indicate that increased expression of key iron transport molecules is responsible Accepted for publication for the elevated iron absorption associated with pregnancy, and implicate hepcidin, HFE, and transferrin 14 November 2003 receptor 2 in determining how the maternal iron homeostatic machinery responds to the increased iron ....................... demands accompanying gestation. ron requirements increase during pregnancy due to Recent advances in our understanding of the mechanism http://gut.bmj.com/ expansion of the maternal erythrocyte mass and high iron and regulation of intestinal iron transport have come from Idemand of the growing fetus. These requirements are met the discovery of a number of key molecules involved in the initially through mobilisation of maternal iron stores process. These include the iron transporters divalent metal (principally from the liver) but as iron stores become transporter 1 (DMT1) and iron regulated mRNA (Ireg1; also depleted, intestinal iron absorption increases to maintain known as ferroportin 1), and the enzymes duodenal an adequate iron supply for both the mother and her cytochrome b (Dcytb) and hephaestin (Hp). The brush border 1–3 offspring. The fetus obtains its iron via the placenta which transporter DMT1, putative basolateral transporter Ireg1, and on October 1, 2021 by guest. Protected copyright. sequesters transferrin bound iron from the maternal circula- the ferrireductase Dcytb are all upregulated under iron tion. The rate of materno-fetal transfer increases with the deficient conditions9 and these changes underlie the increase increasing size of the fetus and placenta, and is maximal just in iron absorption accompanying iron depletion. Disruption prior to parturition.45Iron absorption is also maximal at this of the ceruloplasmin homologue Hp, a putative iron oxidase, time. For example, in humans, there is a ninefold increase in leads to microcytic hypochromic anaemia in mice10 but this iron absorption between weeks 12 and 36 of gestation6 while protein does not appear to be modulated significantly by in rats absorption increases substantially from day 16 variations in iron levels.11 Several other molecules have been 1 through to term at day 21. Absorption rapidly returns to implicated in determining how the body responds to changes control levels post-partum (for example, within 18 hours in in iron requirements, including HFE (the gene mutated in the 7 rats), indicating the strength of the absorption stimulus most prevalent form of hereditary haemochromatosis), provided by pregnancy. transferrin receptor 2 (TfR2), and the antimicrobial peptide The ability of the small intestine to respond to the iron hepcidin. These are all highly expressed in the liver and demands of pregnancy is indicative of a highly regulated mutations in any of these genes can result in iron overload.12– process. Under normal physiological conditions the two main 14 Indeed, HFE is the gene most commonly mutated in the factors that regulate iron absorption are the level of body iron prevalent iron loading disorder hereditary haemochromato- 8 stores and rate of erythropoiesis. Iron absorption increases sis. Hepcidin is secreted into the circulation and has been when stores are low and decreases when stores are high, so it is not surprising that the depleted body iron levels associated with pregnancy are associated with enhanced iron absorp- Abbreviations: Dcytb, duodenal cytochrome b; DMT1, divalent metal tion. However, one study has found that iron loaded rats have transporter 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; increased absorption during pregnancy, in spite of their high HFE, the gene mutated in the most prevalent form of hereditary 1 haemochromatosis; Hp, hephaestin; IRE, iron responsive element; Ireg1, iron stores. This observation suggests that the level of iron regulated mRNA (also known as ferroportin 1); NP, non-pregnant; maternal iron stores may not be the sole regulator of iron PP, post-partum; RPA, ribonuclease protection assay; TfR, transferrin absorption during pregnancy. receptor www.gutjnl.com 656 Millard, Frazer, Wilkins, et al proposed to be the messenger between body iron stores and Ribonuclease protection assays iron absorption by duodenal enterocytes.15 Gene expression in liver and enterocytes was analysed by There has been no comprehensive analysis of the molecular ribonuclease protection assay (RPA) using 5 mg of total RNA mechanisms underlying the enhanced iron absorption isolated with TRIzol reagent (Invitrogen, Melbourne, Gut: first published as 10.1136/gut.2003.031153 on 13 April 2004. Downloaded from associated with pregnancy. We thus sought to investigate Australia) as per the manufacturer’s instructions. the role of the duodenal iron transport molecules and hepatic Expression of the housekeeping gene glyceraldehyde 3-phos- regulatory molecules in coordinating the changes in iron phate dehydrogenase (GAPDH), and the iron metabolism genes absorption observed as gestation progresses. These studies Hp, HFE, hepcidin, Dcytb, Ireg1, transferrin receptor 1 (TfR1), TfR2, demonstrated a progressive increase in expression of iron and DMT1 (both the iron responsive element (IRE) contain- transport molecules in the duodenum during pregnancy that ing splice variant and the non-IRE form) were examined corresponded with decreasing body iron stores and a using riboprobes corresponding to the complementary DNA reduction in liver hepcidin expression. Interestingly, expres- sequences of these genes, as previously described.911 The sion of HFE and TfR2 also declined as pregnancy proceeded. riboprobe for TfR2 corresponded to nucleotides 1859 to 2039 After birth, all parameters returned towards control levels of the cDNA sequence listed in GenBank (accession No XM within two days. 22022). Band intensities were quantified by densitometry, and iron metabolism gene expression was reported as a METHODS proportion of GAPDH gene expression. Animals Female Sprague-Dawley rats fed a standard rodent pellet diet Western blot analysis (370 mg iron/kg) and time mated at 10–12 weeks of age were Protein was extracted from isolated duodenal enterocytes and obtained from the Herston Medical Research Centre, DMT1 and Ireg1 protein expression examined by western Brisbane, Australia. Rats were sacrificed at 9, 15, 18, and blotting using polyclonal antibodies raised as previously 21 days of gestation, and 24–48 hours post-partum. Those described.91118 Band intensities were quantified by densito- that failed to fall pregnant were used as non-pregnant metry, and expression of DMT1 and Ireg1 were reported as a controls. Rats were anaesthetised (44 mg/kg ketamine and proportion of actin protein expression to control for any 8 mg/kg xylazine intraperitoneally) and exsanguinated from discrepancies in gel loading. the abdominal aorta to provide blood for serum iron determination. Samples of liver and duodenum were Immunohistochemistry removed, and duodenal enterocytes were isolated as Sections cut from duodenal tissue embedded and snap frozen described previously.16 Tissues and pellets of isolated cells in OCT embedding compound (Sakura Finetek, Torrance, were snap frozen in liquid nitrogen. This study was approved California, USA) were used to localise DMT1 and Dcytb by the Queensland Institute of Medical Research Animal proteins by immunofluorescence microscopy, as previously Ethics Committee. described.9 Serum and tissue iron analyses Statistical analysis Serum transferrin saturation
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