Iron-deficient Rats Research Articles

Involvement of Sp1 in Transcriptional Regulation of Atp7a During Hypoxia

Genes with GC‐rich promoters were preferentially induced in the rodent intestine during iron deficiency. Moreover, promoters of iron homeostasis related genes (e.g. Dmt1, Dcytb) contain evolutionarily conserved Sp1 binding sites (GC‐rich sequences). The Menkes copper ATPase (Atp7a) gene was strongly induced during iron deficiency, and we previously demonstrated that Atp7a was co‐regulated with iron transport related genes by HIF2α. In this study, we sought to test the role of Sp‐like trans‐acting factors in transcriptional regulation of Atp7a. Phylogenetic footprinting revealed several conserved Sp‐like binding sites in the Atp7a promoter. These sites were mutated in a −224 bp promoter construct driving the luciferase reporter gene. Several of these sites participated in the induction of promoter activity in response to hypoxia in transfected rat intestinal epithelial (IEC‐6) cells. Moreover, phosphorylation of Sp1 increased in IEC‐6 cells exposed to hypoxia; Sp1 is known to be regulated by phosphorylation, which increases DNA binding and trans‐activation properties. Further studies will explore Sp factor interaction with the Atp7a promoter by chromatin immunoprecipitation (ChIP) assay in IEC‐6 cells and in intestine of iron deficient rats. Current data suggest that synergistic interactions between Sp1 (or a related factor) and HIF2α mediate induction of Atp7a gene expression during hypoxia.Grant Funding Source : 1R01 DK074867

Iron-Responsive Olfactory Uptake of Manganese Improves Motor Function Deficits Associated with Iron Deficiency

Iron-responsive manganese uptake is increased in iron-deficient rats, suggesting that toxicity related to manganese exposure could be modified by iron status. To explore possible interactions, the distribution of intranasally-instilled manganese in control and iron-deficient rat brain was characterized by quantitative image analysis using T1-weighted magnetic resonance imaging (MRI). Manganese accumulation in the brain of iron-deficient rats was doubled after intranasal administration of MnCl2 for 1- or 3-week. Enhanced manganese level was observed in specific brain regions of iron-deficient rats, including the striatum, hippocampus, and prefrontal cortex. Iron-deficient rats spent reduced time on a standard accelerating rotarod bar before falling and with lower peak speed compared to controls; unexpectedly, these measures of motor function significantly improved in iron-deficient rats intranasally-instilled with MnCl2. Although tissue dopamine concentrations were similar in the striatum, dopamine transporter (DAT) and dopamine receptor D1 (D1R) levels were reduced and dopamine receptor D2 (D2R) levels were increased in manganese-instilled rats, suggesting that manganese-induced changes in post-synaptic dopaminergic signaling contribute to the compensatory effect. Enhanced olfactory manganese uptake during iron deficiency appears to be a programmed “rescue response” with beneficial influence on motor impairment due to low iron status.

Discovery of a cytosolic/soluble ferroxidase in rodent enterocytes

Hephaestin (Heph), a membrane-bound multicopper ferroxidase (FOX) expressed in duodenal enterocytes, is required for optimal iron absorption. However, sex-linked anemia (sla) mice harboring a 194-amino acid deletion in the Heph protein are able to absorb dietary iron despite reduced expression and mislocalization of the mutant protein. Thus Heph may not be essential, and mice are able to compensate for the loss of its activity. The current studies were undertaken to search for undiscovered FOXs in rodent enterocytes. An experimental approach was developed to investigate intestinal FOXs in which separate membrane and cytosolic fractions were prepared and FOX activity was measured by a spectrophotometric transferrin-coupled assay. Unexpectedly, FOX activity was noted in membrane and cytosolic fractions of rat enterocytes. Different experimental approaches demonstrated that cytosolic FOX activity was not caused by contamination with membrane Heph or a method-induced artifact. Cytosolic FOX activity was abolished by SDS and heat (78 °C), suggesting protein-mediated iron oxidation, and was also sensitive to Triton X-100. Furthermore, cytosolic FOX activity increased ∼30% in iron-deficient rats (compared with controls) but was unchanged in copper-deficient rats (in contrast to the reported dramatic reduction of Heph expression and activity during copper deficiency). Additional studies done in sla, Heph-knockout, and ceruloplasmin-knockout mice proved that cytosolic FOX activity could not be fully explained by Heph or ceruloplasmin. Therefore rodent enterocytes contain a previously undescribed soluble cytosolic FOX that may function in transepithelial iron transport and complement membrane-bound Heph.

Effects of Citric Acid and Lemon Juice on Iron Absorption and Improvement of Anemia in Iron-Deficient Rats

Effects of Citric Acid and Lemon Juice on Iron Absorption and Improvement of Anemia in Iron-Deficient Rats

Influence of Iron Deficiency on Olfactory Behavior in Weanling Rats

Chronically high occupational exposure to airborne metals like iron can impair olfactory function, but little is known about how low iron status modifies olfactory behavior. To investigate the influence of body iron status, weanling rats were fed a diet with low iron content (4 - 7 ppm) to induce iron deficiency anemia and olfactory behavior was compared to control rats fed an isocaloric diet sufficient in iron (210 - 220 ppm). Iron-deficient rats had prolonged exploratory time for attractive odorants in behavioral olfactory habituation/dis-habituation tests, olfactory preference tests and olfactory sensitivity tests compared with control rats. No significant differences were observed for aversive odorants between the two groups. These findings suggest that iron-dependent functions may be involved in controlling and processing of olfactory signal transduction via self and lateral inhibition such that odorant signal remains stronger for longer times prolonging exploratory activity on attractive odorants in the behavioral tests. These findings establish that iron deficiency can modify olfactory behavior.

아스파르트산 킬레이트 철분의 철분 결핍쥐에서의 생물학적 유용성

본 연구에서는 아스파르트산, 탄산칼슘 및 황산철을 반응 시켜 아스파르트산 킬레이트 철분(aspartic acid chelated iron, Asp-Fe)을 제조하고 Asp-Fe의 철분결핍 쥐(iron-deficient rat, ID)에서의 생물학적 유용성을 확인하였다. 시험군은 철분이 함유된 식이를 섭취한 정상군(NC), 철분 결핍 식이를 1개월간 투여하여 철분 결핍 상태를 유도한 쥐(ID)에 생리식염수를 공급한 결핍 대조군(ID＋C), 철분 결핍 쥐에 햄철(heme-Fe) 투여군(ID＋heme-Fe) 및 Asp-Fe 투여군 (ID＋Asp-Fe)으로 나누어 실시하였다. 그 결과 식이섭취량, 장기무게, 체중증가 정도에서 각 군에 따른 차이가 없는 것으로 나타났다. 7일간 투여 후 혈액 중 철분의 함량을 측정한 결과 결핍쥐에 Asp-Fe 투여군(175.2 μg/dL)과 heme-Fe 투여군(140.8 μg/dL)은 결핍 대조군(96.1 μg/dL)보다 유의적인 수준으로 증가하였다. 총 철분 결합능(total iron binding capacity, TIBC)를 측정한 결과 Asp-Fe 투여군(735.4 μg/ dL)은 결핍 대조군(841.9 μg/dL)보다 유의적 수준으로 정상화되었다. 헤마토크리트(HCT) 수치를 측정한 결과에서 Asp- Fe 및 heme-Fe 모두 결핍 대조군보다 증가하는 경향은 보였지만 유의적인 차이는 없었다. 흡수율에서는 heme-Fe의 경우 21.3%인 반면에 Asp-Fe의 경우 50.2%로 약 2.3배 높은 것으로 나타났으며, 혈청에서의 철분농도 및 transferrin saturation( TS)는 heme-Fe 투여군 및 결핍 대조군에 비하여 Asp-Fe 투여군이 유의하게 높은 수준을 유지하였다. 이상의 결과로 미루어 볼 때 아미노산 킬레이트 철분은 heme-Fe과 유사한 수준의 생체 이용율을 가지고 있으며, 철분 결핍을 회복시키는데 매우 효과적인 보충제로서 이용될 수 있을 것으로 사료된다.

Severe Postnatal Iron Deficiency Alters Emotional Behavior and Dopamine Levels in the Prefrontal Cortex of Young Male Rats

Iron deficiency in early human life is associated with abnormal neurological development. The objective of this study was to evaluate the effect of postnatal iron deficiency on emotional behavior and dopaminergic metabolism in the prefrontal cortex in a young male rodent model. Weanling, male, Sprague-Dawley rats were fed standard nonpurified diet (220 mg/kg iron) or an iron-deficient diet (26 mg/kg iron). After 1 mo, hematocrits were 0.42 ± 0.0043 and 0.16 ± 0.0068 (mean ± SEM;P< 0.05;n= 8), liver nonheme iron concentrations were 2.3 ± 0.24 and 0.21 ± 0.010mol/g liver (P< 0.05;n= 8), and serum iron concentrations were 47 ± 5.4 and 23 ± 7.1mol/L (P< 0.05;n= 8), respectively. An elevated plus maze was used to study emotional behavior. Iron-deficient rats displayed anxious behavior with fewer entries and less time spent in open arms compared to control rats (0.25 ± 0.25 vs. 1.8 ± 0.62 entries; 0.88 ± 0.88 vs. 13 ± 4.6 s;P< 0.05;n= 8). Iron-deficient rats also traveled with a lower velocity in the elevated plus maze (1.2 ± 0.15 vs. 1.7 ± 0.12 cm/s;P< 0.05;n= 8), behavior that reflected reduced motor function as measured on a standard accelerating rotarod device. Both the time on the rotarod bar before falling and the peak speed attained on rotarod by iron-deficient rats were lower than control rats (156 ± 12 vs. 194 ± 12 s; 23 ± 1.5 vs. 28 ± 1.6 rpm;P< 0.05;n= 78). Microdialysis experiments showed that these behavioral effects were associated with reduced concentrations of extracellular dopamine in the prefrontal cortex of the iron-deficient rats (79 ± 7.0 vs. 110 ± 14 ng/L;P< 0.05;n= 4). Altered dopaminergic signaling in the prefrontal cortex most likely contributes to the anxious behavior observed in young male rats with severe iron deficiency.

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 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 hemoglobin synthesis are unclear. The current 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 hemoglobin 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/day, 3 days/week) 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 4 h after exercise (L). A single bout of exercise performed after the 3-week training period increased the bone marrow ALAD activity, plasma iron concentration, and transferrin saturation. Although the plasma iron concentration and transferrin saturation were lower in the E group than the L group after a single bout of exercise, the basal hematocrit, hemoglobin, and TIBC 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 hemoglobin concentration in iron-deficient rats.

Serum ceruloplasmin protein expression and activity increases in iron-deficient rats and is further enhanced by higher dietary copper intake

AbstractIncreases in serum and liver copper content are noted during iron deficiency in mammals, suggesting that copper-dependent processes participate during iron deprivation. One point of intersection between the 2 metals is the liver-derived, multicopper ferroxidase ceruloplasmin (Cp) that is important for iron release from certain tissues. The current study sought to explore Cp expression and activity during physiologic states in which hepatic copper loading occurs (eg, iron deficiency). Weanling rats were fed control or low iron diets containing low, normal, or high copper for ∼ 5 weeks, and parameters of iron homeostasis were measured. Liver copper increased in control and iron-deficient rats fed extra copper. Hepatic Cp mRNA levels did not change; however, serum Cp protein was higher during iron deprivation and with higher copper consumption. In-gel and spectrophotometric ferroxidase and amine oxidase assays demonstrated that Cp activity was enhanced when hepatic copper loading occurred. Interestingly, liver copper levels strongly correlated with Cp protein expression and activity. These observations support the possibility that liver copper loading increases metallation of the Cp protein, leading to increased production of the holo enzyme. Moreover, this phenomenon may play an important role in the compensatory response to maintain iron homeostasis during iron deficiency.

Rapid alteration in rat red blood cell copper chaperone for superoxide dismutase after marginal copper deficiency and repletion

There is increased incidence of human copper deficiency (CuD). A sensitive and reliable blood biomarker may reveal additional cases of marginal deficiency. Two experiments were designed to test the hypothesis that the copper chaperone for superoxide dismutase (CCS) would be a robust marker after marginal CuD. Experiment 1 used weanling male Sprague-Dawley rats that were offered a CuD diet for 4 weeks, and samples were evaluated after 1, 2, and 4 weeks and compared with copper-adequate (CuA) controls. Furthermore, iron-deficient rats were included for comparison after 2 weeks of depletion. Red blood cell and plasma cuproenzymes were evaluated through Western blot analysis. Superoxide dismutase (Sod1) and ceruloplasmin protein were found to be altered by both iron and CuD, whereas CCS and CCS/Sod1 ratio were found to only be altered only in CuD rats and, importantly, after only 1 week of treatment. Two weeks on CuA diet restored cuproenzyme levels to control values after 4 weeks of CuD depletion. In experiment 2, marginal CuD (CuM) rats were compared with CuA and CuD rats after 2 weeks of treatment. Superoxide dismutase, ceruloplasmin, and CCS/Sod1 abundances were lower in CuM and CuD groups compared with CuA rats, but there was no statistical difference between CuM and CuD rats. However, CCS was statistically different between all groups, and abundance highly correlated with liver copper concentration. Results suggest that red blood cell CCS may be an excellent biomarker for diagnosis of rapid and marginal CuD.

Exploration of the copper-related compensatory response in the Belgrade rat model of genetic iron deficiency

The Menkes copper ATPase (Atp7a) and metallothionein (Mt1a) are induced in the duodenum of iron-deficient rats, and serum and hepatic copper levels increase. Induction of a multi-copper ferroxidase (ceruloplasmin; Cp) has also been documented. These findings hint at an important role for Cu during iron deficiency. The intestinal divalent metal transporter 1 (Dmt1) is also induced during iron deficiency. The hypothesis that Dmt1 is involved in the copper-related compensatory response during iron deficiency was tested, utilizing a mutant Dmt1 rat model, namely the Belgrade (b/b) rat. Data from b/b rats were compared with phenotypically normal, heterozygous +/b rats. Intestinal Atp7a and Dmt1 expression was increased in b/b rats, whereas Mt1a expression was unchanged. Serum and liver copper levels did not increase in the Belgrades nor did Cp protein or activity. The lack of fully functional Dmt1 may thus partially blunt the compensatory response to iron deficiency by 1) decreasing copper levels in enterocytes, as exemplified by a lack of Mt1a induction and a lesser induction of Atp7a, 2) abolishing the frequently described increase in liver and serum copper, and 3) attenuating the documented increase in Cp expression and activity.

Responses of HSC70 expression in diencephalon to iron deficiency anemia in rats

A powdered diet containing 100 or 3ppm Fe was fed to rats starting at the age of 3weeks. The voluntary activity level was checked using a wheel in the cage during the 17th week after the beginning of supplementation. Significantly less activity was seen in the 3ppm Fe group during both light and dark periods. After 20weeks, the blood and diencephalon were sampled from both groups. Lower hematocrit and blood hemoglobin content was observed in the 3ppm Fe group. The level of 70kDa heat shock cognate (HSC70) expression was greater in the diencephalon of the 3ppm Fe group. In addition, the distribution of HSC70 was determined by proximity ligation assay. More HSC70-positive as well as total cells were noted in several areas of the diencephalon of the iron-deficient rats. The altered expression and distribution of HSC70 might play some role in the neurological changes.

Early iron deficiency enhances stimulus-response learning of adult rats in the context of competing spatial information

Iron deficiency early in life results in neurocognitive deficits that persist into adulthood despite iron treatment. The hippocampus is particularly vulnerable to iron deficiency during the fetal and neonatal periods as evidenced by poorer hippocampus-mediated spatial recognition learning. However, the extent to which early iron deficiency alters interactions between hippocampus-based and extra-hippocampus based learning systems remains undetermined. The present study used an ambiguous maze-learning task to examine the learning process in iron sufficient young adult rats that had recovered from iron deficiency in the fetal and neonatal period. Animals were presented with a stimulus response-learning task in the context of spatial information; a procedure designed to elicit competition between dorsal striatum- and hippocampus-based systems respectively. Formerly iron deficient adult rats showed enhanced stimulus-response learning in the context of competing spatial/distal cue information, a finding suggestive of reduced hippocampal functional influence. The study provides evidence that early iron deficiency alters how different learning systems develop and ultimately interact in adulthood. The potential unbalancing of activity among major memory systems during early life has been postulated by others as a relevant factor underlying the developmental origins of certain psychiatric disorders.

Effect of Resistance Exercise on Iron Status in Moderately Iron-Deficient Rats

Resistance exercise increases heme synthesis in the bone marrow, but it does not improve the hemoglobin status in severe iron-deficient rats on a diet containing less than 5mg iron/kg. The current study investigated whether resistance exercise could mitigate hemoglobin status via increasing heme synthesis in moderately iron-deficient rats. Male 4-week-old Sprague-Dawley rats were fed an iron-deficient diet containing 12mg iron/kg for 3weeks. The rats were divided into two groups: a sedentary (S) group (n = 7) or an exercise (E) group (n = 7). The rats in the E group performed a climbing exercise (5min × 6sets/day, 3days/week). The aminolevulinic acid dehydratase activity, hematocrit, and hemoglobin tended to be higher in group E than S. The iron content in the flexor hallucis longus muscle was significantly higher in E than S, whereas the content in the liver, spleen, kidney, and heart did not significantly differ between the groups. Therefore, resistance exercise appears to improve hemoglobin via increasing heme synthesis in the bone marrow in moderately iron-deficient rats.

Lack of iron-related phenotype in Sp6 intestinal knockout mice

Based on a microarray study, which demonstrated the upregulation of Sp6 transcriptional factor in iron deficient rat duodenum, we reasoned that SP6 could regulate iron absorption by controlling the expression of iron absorption genes (Collins,2006). For that, we generated Sp6 specific intestinal knockout mice. Our data suggest a lack of transcriptional upregulation of Sp6 in mice in conditions where iron absorption is promoted (phlebotomy, iron deficiency, hpx mouse), and an absence of iron-related phenotype in Sp6 intestinal knockout model. We propose that other Sp6 orthologues may be involved in the genetic response to increase iron absorption, possibly in co-operation with hypoxia inducible factor 2 alpha (HIF-2α)—a newly discovered regulator of iron absorption.

Exploration of Iron and Copper Homeostasis in the Belgrade Rat Model of Iron Deficiency

Dmt1 and Atp7a are strongly upregulated in the duodenum of iron deficient rats. Induction of a circulating, multi-copper ferroxidase (ceruloplasmin; Cp) was also documented. These findings exemplify an important interaction between Fe and Cu during iron deficiency. The hypothesis that Dmt1 is necessary for alterations in the expression/activity of copper-related proteins during iron deficiency was tested. Experiments were performed in the Belgrade (b) rat model of genetic iron deficiency. Dmt1 and Atp7a mRNA expression, along with other metal homeostasis related genes (e.g. Tfrc) was increased in enterocytes from b/b rats (as compared to +/b rats), while hephaestin (Heph) did not change. In the liver, Cp mRNA expression was unaltered. Immunoblots demonstrated that Atp7a protein was increased in the b/b rats, along with duodenal Heph protein expression (< 3.0-fold). Cp protein expression in the liver did not change nor did serum ferroxidase activity. Lastly, enterocyte membrane ferroxidase activity was also unchanged in the mutants. Thus, Dmt1 activity is not necessary for the induction of Atp7a mRNA and protein expression, but it is necessary for the induction of serum ferroxidase activity documented in rats deprived of dietary iron. It is thus concluded that Dmt1 is at least partially necessary for the copper-related compensatory response during iron deficiency. Grant Funding Source: 1R01 DK074867

Transcriptional regulation of the Menkes copper ATPase (Atp7a) gene by hypoxia-inducible factor (HIF2α) in intestinal epithelial cells

Iron homeostasis-related genes (e.g., Dmt1 and Dcytb) are upregulated by hypoxia-inducible factor 2α (HIF2α) during iron deficiency in the mammalian intestine. Menkes copper ATPase (Atp7a) gene expression is also strongly induced in the duodenum of iron-deficient rats. The current study was thus designed to test the hypothesis that Atp7a is regulated by HIF2α. Rat intestinal epithelial (IEC-6) cells were utilized to model the intestinal epithelium, and CoCl(2) and 1% O(2) were applied to mimic hypoxia in vitro. Both treatments significantly increased endogenous Atp7a mRNA levels; mRNA induction with CoCl(2) treatment was blunted by a transcriptional inhibitor. The rat Atp7a promoter was thus cloned and studied. Various sized promoter constructs were inserted into a luciferase reporter vector and transfected into cells. A -224/+88 bp construct had full activity and was induced by CoCl(2); this promoter fragment was thus utilized for subsequent analyses. Interestingly, this region contains three phylogenetically conserved, putative hypoxia response elements (HRE; 5'-NCGTGN-3'). It was further noted that HIF2α overexpression caused a significant upregulation of promoter activity while HIF1α overexpression had little effect. To determine whether Atp7a is a direct HIF target, three putative HREs were deleted individually or in combination; all were shown to be essential for transcriptional induction. Chromatin immunoprecipitation studies also demonstrated that HIF2α binds to the Atp7a promoter region. Lastly, Atp7a and HIF2α protein levels were shown to be increased by both treatments. In conclusion, the Atp7a gene is upregulated by direct interaction with HIF2α, demonstrating coordinate regulation with genes related to intestinal iron homeostasis.

Effect of calcium-fortified milk-rich diets (either goat’s or cow’s milk) on copper bioavailability in iron-deficient anemia

As Cu is a mineral involved in the hematopoietic system whose deficiency is associated with anemia due to its requirement for efficient Fe utilization, the objective of the present study was to assess the effect of fortifying Ca in goat’s milk, in comparison to similarly fortified cow’s milk. This was performed to check whether Ca-fortified goat’s milk minimizes Ca–Cu interactions which would favor Cu bioavailability in experimentally induced iron-deficient (ID) rats. Currently, Ca-enriched dairy products are consumed despite the possibility of mineral interactions such as Ca–Cu. Previous studies have shown that consuming goat’s milk improves Cu bioavailability by minimizing Cu–Fe interactions. In the present study, Ca-fortified goat’s milk (2× Ca requirement), compared to fortified cow’s milk, increased the digestive and metabolic utilization of Cu (P < 0.001) and Cu content in target organs involved in erythropoiesis (sternum) in ID rats (P < 0.001). We conclude that goat’s milk, even fortified with Ca, could be beneficial for the recovery from iron-deficient anemia by increasing the Cu bioavailability, an essential mineral for erythropoiesis.

Suppression of hepatic hepcidin expression in response to acute iron deprivation is associated with an increase of matriptase-2 protein

AbstractRecent studies demonstrate a pivotal role for bone morphogenic protein-6 (BMP6) and matriptase-2, a protein encoded by the TMPRSS6 gene, in the induction and suppression of hepatic hepcidin expression, respectively. We examined their expression profiles in the liver and showed a predominant localization of BMP6 mRNA in nonparenchymal cells and exclusive expression of TMPRSS6 mRNA in hepatocytes. In rats fed an iron-deficient (ID) diet for 24 hours, the rapid decrease of transferrin saturation from 71% to 24% (control vs ID diet) was associated with a 100-fold decrease in hepcidin mRNA compared with the corresponding controls. These results indicated a close correlation of low transferrin saturation with decreased hepcidin mRNA. The lower phosphorylated Smad1/5/8 detected in the ID rat livers suggests that the suppressed hepcidin expression results from the inhibition of BMP signaling. Quantitative real-time reverse transcription polymerase chain reaction analysis revealed no significant change in either BMP6 or TMPRSS6 mRNA in the liver. However, an increase in matriptase-2 protein in the liver from ID rats was detected, suggesting that suppression of hepcidin expression in response to acute iron deprivation is mediated by an increase in matriptase-2 protein levels.

Interrelationships between tissue iron status and erythropoiesis during postweaning development following neonatal iron deficiency in rats

Dietary iron is particularly critical during periods of rapid growth such as in neonatal development. Human and rodent studies have indicated that iron deficiency or excess during this critical stage of development can have significant long- and short-term consequences. Since the requirement for iron changes during development, the availability of adequate iron is critical for the differentiation and maturation of individual organs participating in iron homeostasis. We have examined in rats the effects of dietary iron supplement following neonatal iron deficiency on tissue iron status in relation to erythropoietic ability during 16 wk of postweaning development. This physiological model indicates that postweaning iron-adequate diet following neonatal iron deficiency adversely affects erythroid differentiation in the bone marrow and promotes splenic erythropoiesis leading to splenomegaly and erythrocytosis. This altered physiology of iron homeostasis during postweaning development is also reflected in the inability to maintain liver and spleen iron concentrations and the altered expression of iron regulatory proteins in the liver. These studies provide critical insights into the consequences of neonatal iron deficiency and the dietary iron-induced cellular signals affecting iron homeostasis during early development.