Iron-deficient Rats Research Articles

Dietary iron supplements and Moringa oleifera leaves influence the liver hepcidin messenger RNA expression and biochemical indices of iron status in rats

In this study, the effects of iron depletion and repletion on biochemical and molecular indices of iron status were investigated in growing male Wistar rats. We hypothesized that iron from Moringa leaves could overcome the effects of iron deficiency and modulate the expression of iron-responsive genes better than conventional iron supplements. Iron deficiency was induced by feeding rats an iron-deficient diet for 10 weeks, whereas control rats were maintained on an iron-sufficient diet (35.0-mg Fe/kg diet). After the depletion period, animals were repleted with different source of iron, in combination with ascorbic acid. Iron deficiency caused a significant (P < .05) decrease in serum iron and ferritin levels by 57% and 40%, respectively, as compared with nondepleted control animals. Significant changes in the expression (0.5- to100-fold) of liver hepcidin (HAMP), transferrin, transferrin receptor-2, hemochromatosis type 2, ferroportin 1, ceruloplasmin, and ferritin-H were recorded in iron-depleted and iron-repleted rats, as compared with nondepleted rats (P < .05). Dietary iron from Moringa leaf was found to be superior compared with ferric citrate in overcoming the effects of iron deficiency in rats. These results suggest that changes in the relative expression of liver hepcidin messenger RNA can be used as a sensitive molecular marker for iron deficiency.

Marginally excessive iron loading transiently blocks mucosal iron uptake in iron-deficient rats

Regular "mucosal block" is characterized by decreased uptake of a normal iron load 3-72 h after the administration of excess iron (generally 10 mg) to iron-deficient animals. We found that short-acting mucosal block could be induced by much lower iron concentration and much shorter induction time than previously reported, without affecting levels of gene expression. A rapid endocytic mechanism was reported to decrease intestinal iron absorption after a high iron load, but the activating iron load and the time to decreased absorption were undetermined. We assessed the effects of 30-2,000 μg iron load on iron uptake in the duodenal loop of iron-deficient and iron-sufficient rats under anesthesia. One hour later, mucosal cellular iron uptake in iron-deficient rats administered 30 μg iron was 76.1%, decreasing 25% to 50.7% in rats administered 2,000 μg iron. In contrast, iron uptake by iron-sufficient rats was 63% (range 60.3-65.5%) regardless of iron load. Duodenal mucosal iron concentration was significantly lower in iron-deficient than in iron-sufficient rats. Iron levels in portal blood were consistently higher in iron-deficient rats regardless of iron load, in contrast to the decreased iron uptake on the luminal side. Iron loading blocked mucosal uptake of marginally excess iron (1,000 μg), with a greater effect at 15 min than at 30 min. The rapid induction of short-acting mucosal block only in iron-deficient rats suggests DMT1 internalization.

Effect of iron and zinc supplementation and its discontinuation on lipid profile in rats

The aim of this research was to investigate whether combined iron/zinc supplementation is more beneficial than iron supplementation alone from the perspective of the lipid profile in rats. The study was conducted on 6-week male Wistar rats in 3 stages: (1) 4-week adaptation to the diets: C (AIN-93M) and D (mineral mix without iron); (2) 4-week supplementation: 10-times more iron or iron and zinc compared to C; (3) 2-week post-supplementation period (the same diets as in the first stage). The iron and zinc content in serum was measured using ASA. Total cholesterol (TC), HDL cholesterol (HDL-C), non-HDL cholesterol (non-HDL-C) and triglycerides (TG) were determined. After 4-week supplementation (stage II) and post-supplementation (stage III) periods combined iron/zinc supplementation decreased HDL-C and increased non-HDL-C concentrations in control rats, and in contrast to iron supplementation alone TG concentration decreased. After stage II combined iron/zinc supplementation did not result in increased non-HDL-C and TG concentrations in iron deficient rats in contrast to iron supplementation alone. After stage III both iron and simultaneous iron/zinc supplementation were the cause of TC increase which was the result of the increase of non-HDL-C but not HDL-C concentration in iron deficient rats. In conclusion, there were no beneficial effects of simultaneous iron and zinc supplementation on the lipid profile of rats fed control and iron deficient diets. Combined iron and zinc supplementation may contribute to lower HDL-C and higher non-HDL-C concentrations.

Effect of iron status in rats on the absorption of metal ions from plant ferritin.

An isolate of lead-ferritin obtained from soybean seeds sprouted in 25 mM of PbNO3 was introduced into the diet of both iron-deficient and iron non-deficient male rats. After a 21-day administration period, statistical differences in the lead accumulation in the femurs of the rats were noted. Iron-deficient rats accumulated more than four times the amount of lead in their bones than rats without iron-deficiency. No further decrease was observed in haemoglobin concentrations in the groups of animals fed with lead isolates, either iron-deficient or iron non-deficient. Also, no differences in the mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) were observed at the end of the experiment in the group of iron non-deficient rats fed with lead-ferritin isolate compared to the control group of iron non-deficient rats. In the iron-deficient group fed with lead-ferritin isolate, a small increase in haemoglobin concentrations, MCH, MCV and mean corpuscular haemoglobin concentrations (MCHC) was recorded. The results presented in this paper confirm that lead from the tested preparation—lead ferritin isolate—was better absorbed by those rats with induced iron deficiency anaemia. Additionally, we may also suspect based on the obtained results that absorption of ferritin-iron depends on iron status in the body.Electronic supplementary materialThe online version of this article (doi:10.1007/s11130-014-0413-1) contains supplementary material, which is available to authorized users.

Resistance exercise improves the iron status without increasing the iron absorption in iron‐deficient rats (907.1)

We have previously reported that resistance exercise improved the iron status in iron‐deficient rats (Fujii 2011). The current study investigated the mechanisms underlying this exercise‐related effect. Male four‐week‐old rats were divided into a sedentary (S) or an exercise (E) group, and all rats were fed an iron‐deficient diet (12 mg iron/kg) for eight weeks. The rats in the E group performed climbing exercise (5 min x 6 sets/day, 3 days/week). The hematocrit and hemoglobin levels were significantly higher in the E group than in the S group. The plasma iron and transferrin saturation and total iron content in the body measured using the whole body homogenates of the rats tended to be higher in the E group. The mRNA expression of hepcidin tended to be higher in the E group, while the mRNA expression of duodenal cytochrome b, divalent metal transporter 1 and ferroportin 1 did not differ between the groups. The apparent absorption of iron was significantly lower in the E group than in the S group. Therefore, the resistance exercise‐induced improvement of the iron status does not appear to be due to an enhancement of iron absorption.

Management of experimental hypochlorhydria with iron deficiency by the composite extract of Fumaria vaillantii L. and Benincasa hispida T. in rat

The aim of the present study was to search the effective ratio of whole plant of Fumaria vaillantii Loisel (Fumaria vaillantii L.) and fruit of Benincasa hispida Thunb. (Benincasa hispida T.) in composite form, namely “FVBH” for the management of hypochlorhydria along with iron deficiency in male albino rats. Hypochlorhydria refers to suppression of hydrochloric acid secretion by the stomach. Hypochlorhydria was induced by ranitidine in this study. We used four composite extracts of the mentioned plant and fruit with different ratios (1:1, 1:2, 2:1, and 3:2) for searching the most effective composite extract for the correction of hypochlorhydria. Gastric acidity is an important factor for iron absorption. Thus, hypochlorhydria causes iron deficiency in rat and it was prevented significantly by the extract treatment at the ratio of 1:1 of the said plant and fruit. The correction of iron deficiency by the composite extract was compared with iron supplementation to hypochlorhydric rat. It was found that preadministration followed by coadministration of FVBH-1 (1:1) able to prevent the ranitidine-induced hypochlorhydria and iron deficiency. The composite extract, FVBH-1 (1:1) significantly (P<0.05) increased the pepsin concentration, chloride level in gastric juice, iron levels in serum and liver along with blood hemoglobin level than other ratios used here. Hence, it can be concluded that FVBH-1 (1:1) is an effective herbal formulation for the management of hypochlorhydria and related iron deficiency.

Divalent metal transporter 1 (Dmt1) Mediates Copper Transport in the Duodenum of Iron-Deficient Rats and When Overexpressed in Iron-Deprived HEK-293 Cells

Intracellular copper-binding proteins (metallothionein I/II) and a copper exporter (Menkes copper-transporting ATPase) are upregulated in duodenal enterocytes from iron-deficient rats, consistent with copper accumulation in the intestinal mucosa. How copper enters enterocytes during iron deficiency is, however, not clear. Divalent metal transporter 1 (Dmt1), the predominant iron importer in the mammalian duodenum, also transports other metal ions, possibly including copper. Given this possibility and that Dmt1 expression is upregulated by iron deprivation, we sought to test the hypothesis that Dmt1 transports copper during iron deficiency. Two model systems were utilized: the Belgrade (b) rat, expressing mutant Dmt1, and an inducible Dmt1-overexpression cell culture system. Mutant rats (b/b) were fed a semipurified, AIN93G-based control diet and phenotypically normal littermates (+/b) were fed control or iron-deficient diets for ∼14 wk. An everted gut sleeve technique and a colorimetric copper quantification assay were utilized to assess duodenal copper transport. The control diet-fed +/b rats had normal hematological parameters, whereas iron-deprived +/b and b/b rats were iron deficient and Dmt1 mRNA and protein levels increased. Importantly, duodenal copper transport was similar in the control +/b and b/b rats; however, it significantly increased (∼4-fold) in the iron-deprived +/b rats. Additional experiments in Dmt1 overexpressing HEK-293 cells showed that copper (64Cu) uptake was stimulated (∼3-fold) in the presence of an iron chelator. Dmt1 transcript stabilization due to a 3′ iron-responsive element was also documented, likely contributing to increased transport activity. In summary, these studies suggest that Dmt1 enhances copper uptake into duodenal enterocytes during iron deprivation.

Severe Iron Deficiency Is Associated with a Reduced Conception Rate in Female Rats

Background/Aims: Iron deficiency is a global nutritional disorder, especially for pregnant women. There is a close relationship between deficiency in trace elements and unexplained infertility in females. However, the relationship between iron deficiency and unexplained infertility has not been determined. This study was designed to determine the effect of iron deficiency on conception in a rat model. Methods: Female rats were randomly divided into two groups (n = 15 each): an iron-deficiency group fed a low iron diet and a normal control group. Both groups of female rats were mated with healthy male rats after the iron-deficiency model was established. Results: Iron-deficient rats developed white skin and eyes, hair loss, and weight loss. Hemoglobin levels and red blood cell count were significantly lower than in controls, showing successful establishment of the iron-deficiency model. There was a significantly lower conception rate in the iron-deficiency group; there also appeared to be a disruption of estrus and a delay in conception in the iron-deficiency group. Conclusions: Severe iron deficiency has a significant influence on fertility, and may be an important factor in unexplained infertility. Further research on the role of iron in conception is warranted.

Multigenerational effects of fetal-neonatal iron deficiency on hippocampal BDNF signaling.

Fetal-neonatal iron deficiency induces adult learning impairments concomitant with changes in expression of key genes underlying hippocampal learning and memory in spite of neonatal iron replenishment. Notably, expression of brain-derived neurotrophic factor (BDNF), a gene critical for neuronal maturation and synaptic plasticity, is lowered both acutely and in adulthood following early-life iron deficiency. Although the mechanism behind its long-term downregulation remains unclear, epigenetic modification in BDNF, as seen in other models of early-life adversity, may play a role. Given that early iron deficiency occurs during critical periods in both hippocampal and gonadal development, we hypothesized that the iron-sufficient offspring (F2 IS) of formerly iron-deficient (F1 FID) rats would show a similar suppression of the BDNF gene as their parents. We compared hippocampal mRNA levels of BDNF and functionally related genes among F1 IS, F1 ID, and F2 IS male rats at postnatal day (P) 15 and P65 using RT-qPCR. As expected, the F1 ID group showed a downregulation of BDNF and associated genes acutely at P15 and chronically at P65. However, the F2 IS group showed an upregulation of these genes at P15, returning to control levels at P65. These results demonstrate that adverse effects of early iron deficiency on hippocampal gene expression observed in the F1 are not present in the F2 generation, suggesting differential effects of nutritionally induced epigenetic programing during the critical periods of hippocampal and gonadal development.

Early-Life Iron Deficiency Anemia Alters the Development and Long-Term Expression of Parvalbumin and Perineuronal Nets in the Rat Hippocampus

Early-life iron deficiency anemia (IDA) alters the expression of critical genes involved in neuronal dendritic structural plasticity of the hippocampus, thus contributing to delayed maturation of electrophysiology, and learning and memory behavior in rats. Structural maturity in multiple cortical regions is characterized by the appearance of parvalbumin-positive (PV⁺) GABAergic interneurons and perineuronal nets (PNNs). Appearance of PV⁺ interneurons and PNNs can serve as cellular markers for the beginning and end of a critical developmental period, respectively. During this period, the system progresses from an immature yet highly plastic condition, to a more mature and efficient state that is however less flexible and may exhibit poorer potential for recovery from injury. To test if fetal-neonatal IDA alters parvalbumin (PV) mRNA expression, protein levels, and the number of PV⁺ interneurons and PNNs in the male rat hippocampus, pregnant dams were given an iron-deficient (ID) diet (3 mg iron/kg chow) from gestational day 2 to postnatal day (P) 7 and then placed on an iron-sufficient (IS) diet (198 mg/kg) for the remainder of the experiment. On this regimen, formerly ID animals become fully iron-replete by P56. Minimal levels of PV (mRNA and protein), PV⁺ interneurons, and PNNs were found in IS and ID P7 rats. By P15, and continuing through P30 and P65, ID rats had reduced PV mRNA expression and protein levels compared to IS controls. While there were no differences in the number of PV⁺ neurons at either P30 or P65, the percentage of PV⁺ cells surrounded by PNNs was slightly greater in ID rats as compared to IS controls. The lower levels of these acknowledged critical period biomarkers in the ID group are consistent with studies that demonstrate later maturation of the acutely ID hippocampus and lower plasticity in the adult formerly ID hippocampus. The findings provide additional potential cellular bases for previously described electrophysiologic and behavioral abnormalities found during and following early-life IDA.

Transcription Factors Sp1 and Hif2α Mediate Induction of the Copper-transporting ATPase (Atp7a) Gene in Intestinal Epithelial Cells during Hypoxia

Genes with G/C-rich promoters were up-regulated in the duodenal epithelium of iron-deficient rats including those encoding iron (e.g. Dmt1 and Dcytb) and copper (e.g. Atp7a and Mt1) metabolism-related proteins. It was shown previously that an intestinal copper transporter (Atp7a) was co-regulated with iron transport-related genes by a hypoxia-inducible transcription factor, Hif2α. In the current study, we sought to test the role of Sp1 in transcriptional regulation of Atp7a expression during iron deprivation/hypoxia. Initial studies in IEC-6 cells showed that mithramycin, an Sp1 inhibitor, reduced expression of Atp7a and iron transport-related genes (Dmt1, Dcytb, and Fpn1) and blocked their induction by CoCl2, a hypoxia mimetic. Consistent with this, overexpression of Sp1 increased endogenous Atp7a mRNA and protein expression and stimulated Atp7a, Dmt1, and Dcytb promoter activity. Site-directed mutagenesis and functional analysis of a basal Atp7a promoter construct revealed four functional Sp1 binding sites that were necessary for Hif2α-mediated induction of promoter activity. Furthermore, chromatin immunoprecipitation (ChIP) assays confirmed that Sp1 specifically interacts with the Atp7a promoter in IEC-6 cells and in rat duodenal enterocytes. This investigation has thus revealed a novel aspect of Atp7a gene regulation in which Sp1 may be necessary for the HIF-mediated induction of gene transcription during iron deficiency/hypoxia. Understanding regulation of Atp7a expression may help further clarify the physiological role of copper in the maintenance of iron homeostasis. Furthermore, this Sp1/Hif2α regulatory mechanism may have broader implications for understanding the genetic response of the intestinal epithelium to maintain whole-body iron homeostasis during states of deficiency.

Effects of Dietary Factors on the Pharmacokinetics of 58Fe-labeled Hemin After Oral Administration in Normal Rats and the Iron-deficient Rats

Hemin, iron (III) protoporphyrin chloride (IX), as a stable form of heme iron, has been used in iron absorption studies. The aim of the present study was to elucidate the influences of body iron status and three dietary factors (green tea extract, ascorbic acid, and calcium) on the pharmacokinetics of hemin using stable isotope labeling methods followed by ICP-MS measurement. In this study, a rapid, sensitive, and specific ICP-MS method for the determination of (58)Fe originating from hemin in rat plasma was developed and a rat model of iron deficiency anemia was established. It was found that hemin iron absorption increased significantly under iron deficiency anemia status, with AUC0-t and AUC0-∞ showing significant increase in anemic rats compared to normal ones. Green tea extract strongly inhibited hemin iron absorption in both normal rats and iron-deficient rats. In normal rats administered with green tea extract, C max resulted significantly reduced, whereas in anemic rats administered with green tea extract both AUC0-t and AUC0-∞ were reduced. On the other hand, ascorbic acid significantly affected hemin iron absorption only in iron-deficient rats, in which C max showed a significant increase. Interestingly, calcium slowed down the hemin iron absorption rate in normal rats, MRT0-t being significantly different in calcium-treated animals compared to untreated ones. This trend also appeared in the iron-deficient group but it did not reach statistical significance. Our data suggest that the mechanism of hemin iron absorption is regulated by body iron status and dietary factors can influence hemin iron absorption to varying degrees. Moreover, these results may also have general implication in the iron deficiency treatment with iron supplements and fortification of foods.

Post‐exercise meal timing does not influence hemoglobin synthesis in iron deficient rats

Resistance exercise increases heme synthesis in the bone marrow and the hemoglobin (Hb) concentration in iron‐deficient rats. Post‐exercise early nutrient provision facilitated exercise‐induced skeletal muscle protein hypertrophy compared to late provision in a rat study conducted for 8 weeks. However, ingesting meals early after exercise did not increase the Hb concentration in a 3‐week study using iron‐deficient rats (Fujii, T., et al. EB2012). There is a possibility that the latter study period might be too short, as no significant muscle hypertrophy was observed. Therefore, in this study, the study period was prolonged to 8 weeks to investigate the effect of post‐exercise meal timing on the Hb concentration and other blood parameters in iron‐deficient rats. Male 4‐week‐old rats were fed an iron‐deficient diet (12 mg iron/kg) and performed climbing exercise (3 d/w) for 8 weeks. The rats were divided into a group fed a post‐exercise meal early after exercise (E) or a group fed the meal 4 h after exercise (L). The skeletal muscle weights were significantly higher in the E group than in the L group. However, the Hb concentration did not differ between the groups. In addition, the bone marrow ALAD activity and plasma iron concentration did not differ. Therefore, post‐exercise meal timing that enhances exercise‐induced muscle hypertrophy has no effect on either the biosynthesis or concentration of Hb.

Study on iron availability from prepared soybean sprouts using an iron-deficient rat model

During soya seeds germination in FeSO4 solutions their phytoferritin content is multiplied. Prepared soybean sprouts have been proposed as a safe and easily available source of iron supplementation. The preparation was compared with FeSO4 and ferritin isolates, using rats with induced iron deficiency anaemia. After the end of the 2-week supplementation experiment, it was observed that no statistically significant differences in haemoglobin concentration, mean corpuscular volume, mean corpuscular haemoglobin, and mean corpuscular haemoglobin concentration existed between those animals supplemented with sprouts enriched in ferritin, ferritin isolate and FeSO4 and healthy animals forming the control group. Moreover, the examined preparation had a beneficial influence on the recreation of ferritin reserves in both the liver and the blood serum, and also did not induce negative alterations in general growth parameters of animals. Use of an easily obtainable ferritin iron source may be a profitable alternative in supplementation due to its wide availability and food preservative properties.

Effect of iron overload and iron deficiency on liver hemojuvelin protein.

IntroductionHemojuvelin (Hjv) is a key component of the signaling cascade that regulates liver hepcidin (Hamp) expression. The purpose of this study was to determine Hjv protein levels in mice and rats subjected to iron overload and iron deficiency.MethodsC57BL/6 mice were injected with iron (200 mg/kg); iron deficiency was induced by feeding of an iron-deficient diet, or by repeated phlebotomies. Erythropoietin (EPO)-treated mice were administered recombinant EPO at 50 U/mouse. Wistar rats were injected with iron (1200 mg/kg), or fed an iron-deficient diet. Hjv protein was determined by immunoblotting, liver samples from Hjv−/− mice were used as negative controls. Mouse plasma Hjv content was determined by a commercial ELISA kit.ResultsLiver crude membrane fraction from both mice and rats displayed a major Hjv-specific band at 35 kDa, and a weaker band of 20 kDa. In mice, the intensity of these bands was not changed following iron injection, repeated bleeding, low iron diet or EPO administration. No change in liver crude membrane Hjv protein was observed in iron-treated or iron-deficient rats. ELISA assay for mouse plasma Hjv did not show significant difference between Hjv+/+ and Hjv−/− mice. Liver Hamp mRNA, Bmp6 mRNA and Id1 mRNA displayed the expected response to iron overload and iron deficiency. EPO treatment decreased Id1 mRNA, suggesting possible participation of the bone morphogenetic protein pathway in EPO-mediated downregulation of Hamp mRNA.DiscussionSince no differences between Hjv protein levels were found following various experimental manipulations of body iron status, the results indicate that, in vivo, substantial changes in Hamp mRNA can occur without noticeable changes of membrane hemojuvelin content. Therefore, modulation of hemojuvelin protein content apparently does not represent the limiting step in the control of Hamp gene expression.

Multiple Menkes copper ATPase (Atp7a) transcript and protein variants are induced by iron deficiency in rat duodenal enterocytes

The Menkes copper ATPase (Atp7a) pumps copper into the trans-Golgi for cuproenzyme synthesis, and translocates to the basolateral membrane of enterocytes for copper export. Recent studies demonstrated that three 5′ end splice variants of the Atp7a transcript exist in rat duodenum, all of which are strongly induced during iron deprivation. To explore a possible role for Atp7a (and copper) in intestinal iron absorption, the current studies were undertaken to test the hypothesis that multiple Atp7a transcript and protein variants exist in intestinal epithelial cells. Northern blot analyses using probes generated from the full-length Atp7a cDNA revealed several specific hybridization bands, all of which were more intense in RNA samples extracted from duodenal enterocytes isolated from iron-deficient rats. A PCR-based approach, using forward primers specific for the alternative 5′ end splice variants and a reverse primer in exon 23, demonstrated that 3 full-length transcripts exist in rat IEC-6 cells. To identify possible Atp7a protein variants, three distinct polyclonal antisera were utilized. The specificity of the antisera was first established by western blotting and immunoprecipitation studies using samples derived from isolated rat enterocytes and Atp7a knockdown IEC-6 cells. Several specific immunoreactive bands were documented, and a unique Atp7a protein distribution in cytosolic vesicle-like structures was noted. In conclusion, multiple Atp7a transcript and protein variants exist in rodent intestinal epithelial cells and are induced by dietary iron deprivation. Further studies will be designed to determine the subcellular distribution of Atp7a protein variants and possible unique functions of each.

Olfactory ferric and ferrous iron absorption in iron-deficient rats

The absorption of metals from the nasal cavity to the blood and the brain initiates an important route of occupational exposures leading to health risks. Divalent metal transporter-1 (DMT1) plays a significant role in the absorption of intranasally instilled manganese, but whether iron uptake would be mediated by the same pathway is unknown. In iron-deficient rats, blood (59)Fe levels after intranasal administration of the radioisotope in the ferrous form were significantly higher than those observed for iron-sufficient control rats. Similar results were obtained when ferric iron was instilled intranasally, and blood levels of (59)Fe were even greater in the iron-deficient rats compared with the amount of ferrous iron absorbed. Experiments with Belgrade (b/b) rats showed that DMT1 deficiency limited ferric iron uptake from the nasal cavity to the blood compared with +/b controls matched for iron deficiency. These results indicate that olfactory uptake of ferric iron by iron-deficient rats involves DMT1. Western blot experiments confirmed that DMT1 levels are significantly higher in iron-deficient rats compared with iron-sufficient controls in olfactory tissue. Thus the molecular mechanism of olfactory iron absorption is regulated by body iron status and involves DMT1.

The effects of resistance exercise and post‐exercise meal timing on the iron status in iron deficient rats

Resistance exercise increases heme synthesis in the bone marrow and the hemoglobin (Hb) in iron deficient rats. Post‐exercise early nutrient provision facilitates skeletal muscle protein synthesis compared to late provision. However, the effects of post‐exercise nutrition timing on Hb synthesis are unclear. This study investigated the effect of post‐exercise meal timing on the activity of the key enzyme involved in hemoglobin synthesis, δ‐aminolevulinic acid dehydratase (ALAD), in the bone marrow and examined the Hb concentration in iron deficient rats. Male 4‐week‐old Sprague‐Dawley rats were fed an iron deficient diet containing 12 mg iron/kg and performed climbing exercise (5 min × 6 sets/d, 3 d/w) for 3 weeks. The rats were divided into a group fed a post‐exercise meal early after exercise (E) or a group fed the meal 4h after exercise (L). A single bout of exercise after resistance exercise training for 3 weeks increased the bone marrow ALAD activity, plasma iron concentration and transferrin saturation (TfS). Although the plasma iron concentration and TfS were lower in the E group than in the L group after a single bout of exercise, the basal hematocrit, Hb and total iron binding capacity after 3 weeks did not differ between the groups. Therefore, resistance exercise increases the bone marrow ALAD activity, while the post‐exercise meal timing has no effect on the Hb concentration in iron deficient rats.

Discovery of a novel cytosolic ferroxidase in rodent enterocytes

During iron absorption in the doudenum, iron that exits enterocytes must be oxidized by a membrane bound multi‐copper ferroxidase, hephaestin. We designed experiments to assess Heph activity during iron deficiency and made the unexpected observation that ferroxidase (FOX) activity (by a transferrin‐coupled assay) was present in the cytosol of isolated rodent enterocytes. Several different experimental approaches allowed us to conclude that cytosolic FOX (cytoFOX) activity was not due to contamination with membrane Heph. cytoFOX activity was inhibited by sodium azide (a known FOX inhibitor), pPD (a competing substrate), D‐penicillamine (a copper chelator), and by SDS and heat (showing that it is mediated by a protein). Moreover, activity was increased (~30%) in iron deficient rats but was unchanged in copper deficient rats (in contrast to the reported dramatic reduction of Heph during copper deficiency). Finally, studies done in Heph KO, sla (Heph mutant) and ceruloplasmin (Cp) KO mice proved that cytoFOX activity was not contributed by Heph (or Cp) as activity in enterocyte cytosol was not different between KO/mutant mice and wt littermates. In conclusion, rodent enterocytes contain a cytosolic FOX that may function in transepithelial iron transport, perhaps acting in acidified intracellular vesicles containing the iron exporter ferroportin 1 (Fpn1) and complementing membrane Heph.Grant Funding Source: NIH

Physiologic Role of the Menkes Copper ATPase (Atp7a) in Intestinal Iron Transport

Atp7a is a copper transporting ATPase involved in intestinal copper export that is strongly induced in the intestine of iron deficient rats. We evaluated the possible role of Atp7a in iron homeostasis in Atp7a knock down (KD) rat intestinal epithelial (IEC‐6) cells and Brindle mice that express a mutant, inactive form of Atp7a. In KD cells with &gt;90% decrease in Atp7a protein levels, copper loading led to significantly increased intracellular Cu levels, demonstrating a reduction in the copper export function of Atp7a. Iron (59Fe) transport studies in IEC‐6 cells grown in bicameral cell culture inserts revealed significant transepithelial iron flux when cells were deprived of iron. Transport in Atp7a KD cells showed that 59Fe transported into cells and transferred to the basolateral chamber was reduced. Next, Brindled mice were rescued by perinatal copper injection and studies were done to consider the in vivo role of Atp7a in iron absorption. Brindled mice were anemic compared to wild type littermates, and Dmt1, TfR1 and Atp7a expression was induced by dietary iron deprivation. Furthermore, serum ferroxidase activity, reflecting ceruloplasmin, was decreased in Brindled mice as was activity of a novel cytosolic ferroxidase in enterocytes. These data demonstrate a physiologic role for Atp7a in intestinal iron transport possibly related to copper delivery to multi‐copper ferroxidases involved in iron homeostasis.Grant Funding Source : NIH