DMT1 and FPN1 expression during infancy: developmental regulation of iron absorption

Weng-In Leong,1 Christopher L. Bowlus,2 Jonas Tallkvist,3 and Bo Lönnerdal1,2

Departments of 1Nutrition and 2Internal Medicine, University of California, Davis, California 95616; and 3Department of Pharmacology and Toxicology, Uppsala Biomedical Center, SE-75123 Uppsala, Sweden

Submitted 12 March 2003 ; accepted in final form 26 June 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Two iron transporters, divalent metal transporter1 (DMT1) and ferroportin1 (FPN1) have been identified; however, their role during infancy is unknown. We investigated DMT1, FPN1, ferritin, and transferrin receptor expression, iron absorption and tissue iron in iron-deficient rat pups, iron-deficient rat pups given iron supplements, and controls during early (day 10) and late infancy (day 20). With iron deficiency, DMT1 was unchanged and FPN1 was decreased (-80%) at day 10. Body iron uptake, mucosal iron retention, and total iron absorption were unchanged. At day 20, DMT1 increased fourfold and FPN1 increased eightfold in the low-Fe group compared with controls. Body iron uptake and total iron absorption were increased, and mucosal iron retention was decreased with iron deficiency. Iron supplementation normalized expression levels of the transporters, body iron uptake, mucosal iron retention, and total iron absorption of the low-Fe group to those of controls at day 20. In summary, the molecular mechanisms regulating iron absorption during early infancy differ from late infancy when they are similar to adult animals, indicating developmental regulation of iron absorption.

iron deficiency; rat pups; iron transporters; ferritin; ferroportin1


RAPID GROWTH AND EXPANSION of hemoglobin mass make infants particularly susceptible to iron deficiency. The percentage of low-birth-weight infants is high in developing countries, and these infants have an even larger weight gain and lower iron stores. Both of these factors have a negative impact on their iron status, placing them at higher risk for iron deficiency at an early age. Iron deficiency not only results in anemia, but also delayed cognitive and psychomotor development (7, 14, 21, 22). Unlike adults, the main food source for infants is breast milk, which does not contain heme iron. Instead, nonheme iron, which is bound to other proteins or low-molecular-weight ligands in breast milk, is the form of dietary iron for infants. Moreover, iron supplementation, in the form of ferrous sulfate, is the common practice for infants. Hence, the form of dietary iron that infants are exposed to is mainly nonheme iron.

Iron homeostasis is maintained primarily by regulating intestinal iron absorption (2, 3), which is controlled by the enterocyte. The vectorial passage of iron through the enterocyte entails transport of the iron across three cellular barriers: the apical membrane, intracellular translocation across the cytosol, and release of iron across the basolateral membrane into the circulation. The efficiency of iron absorption is normally regulated in accord with iron status. Many of the genes involved in the transport of nonheme iron across enterocytes have been identified, and their regulation has been characterized. These include the apical transporter divalent metal transporter 1 (DMT1) and the basolateral transporter ferroportin 1 (FPN1). DMT1 actively transports reduced dietary iron into the enterocyte (11, 12). A brush-border membrane ferric iron reductase, Dcytb, is believed to reduce dietary ferric iron to ferrous iron before being taken up by DMT1 (18). DMT1 mRNA has been shown to be expressed in villous enterocytes of the duodenum and is up- and downregulated in iron deficiency and iron loading, respectively (5). DMT1 protein staining of the villous enterocytes is also higher in iron deficiency and lower in iron overload (5, 11, 12, 20, 23, 24). Iron traverses the enterocyte and is exported through the basolateral membrane by a process that is incompletely characterized but involves a second transmembrane iron transporter, FPN1 (1, 10, 19). FPN1 localizes to the basolateral membrane of the enterocyte and is similarly induced by iron deficiency (1, 24).

Although the role and regulation of DMT1 and FPN1 have been clearly established in mature tissues, their role in the regulation of iron absorption during early life has not been studied. In contrast to late infancy, iron supplementation during early human infancy does not affect iron absorption, suggesting developmental changes in the regulation of iron absorption (8, 9). We have previously found that intestinal DMT1 and FPN1 are developmentally regulated. In addition, these iron transporters are not downregulated in response to iron supplementation during early infancy in rats (16). Therefore, we investigated the responsiveness of these iron transporters to iron deficiency during early life. Using a rat pup model of infant iron deficiency, we examined (1) DMT1 and FPN1 gene and protein expression, (2) ferritin and transferrin receptor (TfR) protein levels, (3) iron absorption and (4) tissue iron in iron-deficient rat pups, iron-deficient rat pups given iron supplements, and controls during early infancy (day 10) and late infancy (day 20).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Diet. Rats were fed an egg white-based semipurified control diet or a diet low in iron (Table 1). The control diet and the low-iron diet (low Fe) differed only in their iron content; the control diet contained 35 mg iron/kg diet as ferrous sulfate, and the low-Fe diet contained 8 mg iron/kg. Iron concentrations were verified by atomic absorption spectrophotometry (model Smith-Heifjie 4000, Thermo Jarrell Ash, Franklin, MA) before initiating the study. The composition of the mineral and vitamin mixes are shown in Table 1.


View this table:
[in this window]
[in a new window]
 
Table 1. Composition of the semipurified diet

 

Animals. Female virgin Sprague-Dawley rats (~250 g) were purchased (Charles River, Wilmington, MA) and housed individually in stainless steel hanging cages. Animals were kept in a temperature-, humidity-, and light-controlled room (20°C, humidity >60%, and 12:12-h light-dark cycle) and given access to deionized water ad libitum. Animals were fed a control diet for 1 wk and were then randomly fed either the control diet or the low-Fe diet for another 3 wk. Rats were then bred and kept on the same diet throughout pregnancy and lactation. On postnatal day 2, litters were culled to 10 and pups were cross-fostered to dams receiving the same dietary treatment, if needed. Control pups (n = 72) and the low-Fe pups (n = 144) were nursed normally by their dams. The low-Fe group was subdivided into two groups; one group was given daily oral iron supplements, 30 µg iron (as ferrous sulfate)/day in 25 µl 10% sucrose solution (low-Fe + Fe group, n = 72), and the other group received 25 µl 10% sucrose solution only (low-Fe group, n = 72). The control group also received 10% sucrose solution daily. All pups received the daily oral doses from days 2 to 20 after birth. Weight was monitored every other day. Rat pups were killed by asphyxiation with CO2 at day 10 (n = 36/group) or 20 (n = 36/group) after birth. Blood was collected by cardiac puncture, and tissues were dissected. DMT1 and FPN1 gene and protein expression, ferritin and TfR protein levels, 59Fe absorption, and tissue iron were determined. Control and low-Fe rat pups (n = 7 or 8/group) were also killed at birth for tissue iron determination. The study was approved by the Animal Research Services at the University of California Davis, which is accredited by the American Association for the Accreditation of Laboratory Animal Care.

Hemoglobin. Whole blood was collected from animals at days 1, 10, and 20. Hemoglobin was analyzed by the cyanomethemoglobin method using a commercially available kit (Sigma, St. Louis, MO). Blood samples (20 µl) were mixed with 5 ml Drabkin's solution (0.1% sodium bicarbonate, 0.005% potassium cyanide, and 0.02% potassium ferricyanide) for hemoglobin determination.

RNA extraction. Rat pups were fasted for 4 h (day 10) or 6 h (day 20) and killed by CO2 asphyxiation. These fasting times were chosen to allow adequate gastric emptying. Duodenum and liver of the rat pups were dissected and stored in RNA-later (Ambion, Austin, TX) and kept at -20°C until extraction. Tissues (100 mg/ml) were then homogenized in TRIzol reagent (Life Technologies, Rockville, MD), and total RNA was extracted from the whole tissues according to the TRIzol protocol.

Real-time quantitative RT-PCR. The relative expression levels of DMT1 and FPN1 were determined by real-time quantitative RT-PCR using TaqMan EZ RT-PCR core reagents (Applied Biosystems, Foster City, CA). Primers and probes were designed using Primer Express software (Applied Biosystems) to span introns to avoid coamplification of genomic DNA and were purchased from Applied Biosystems. For quantification of DMT1 cDNA, the following primers were used: 5'-GTT TGT CAT GGA GGG ATT CCT-3' and 5'-CAT TCA TCC CTG TCA GAT GCT-3', which recognize both the iron-responsive element (IRE) and the non-IRE forms. For quantification of FPN1 cDNA, the following primers were used: 5'-GTG CCT CCC AGA TCG CAG-3' and 5'-GGG CTG GTT ATA GTA GGA GAC CC-3'. The probes for DMT1 (5'-AAA ATG GTC GCG CTT TGC CCG A-3') and FPN1 (5'-ACC CTT CCG CAC TTT TCG AGA TGG A-3') were 5'-labeled with 6-carboxyfluorescein and 3'-labeled with 6-carboxytetramethylrhodamine.

The RT-PCR reactions were carried out according to the manufacturer's protocol on an ABI Prism 7700 Sequence Detector (Applied Biosystems). RT-PCR conditions were 30 min at 60°C and 10 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Analysis of the data was done using the Sequence Detector 1.7 software. All reactions were performed in duplicate, and difference in fluorescence value ({Delta}Rn) and threshold cycle number (CT) were calculated from fluorescence activity data collected during PCR. A no-template control was included in every reaction. The housekeeping gene, 18S ribosomal RNA, was used for internal normalization using TaqMan ribosomal RNA control reagent (Applied Biosystems). Identical results were obtained by RTPCR analysis of 18S in each group, and therefore 18S was selected as the reference for all experiments.

Membrane and soluble protein preparation. Frozen duodenum (300 mg) samples were homogenized in 5 ml HEPESEDTA buffer [20 mM HEPES, pH 7.4, 1 mM EDTA, 250 mM sucrose, protease inhibitor mixture containing 4-(2-aminoethyl)benzenesulfonyl fluoride, trans-epoxysuccinyl-L-leucylsmido(4-guanidino)butane, bestatin, leupeptin, aprotinin, and sodium EDTA (Sigma)]. The homogenate was centrifuged at 500 g for 5 min at 4°C. The supernatant was then centrifuged at 100,000 g for 30 min at 4°C. The supernatant (soluble protein) was collected for ferritin protein determination, and the crude membrane fraction (pellet) was resuspended in 0.5 ml homogenization buffer for DMT1, FPN1, and TfR protein determination. Samples were stored at -80°C until analysis. Protein concentrations of the membrane fractions and the soluble fractions were quantified by the Lowry method (17).

Production of DMT1 and FPN1 antibodies. Peptide fragments of DMT1 (VKPSQSQVLKGMFV) and FPN1 (KQLTSPKDTEPKPLEGTH) were synthesized with an additional cysteine residue for conjugation to keyhole limpet hemocyanin (KLH) at the COOH-terminal end (Genemed Synthesis, San Francisco, CA). Sequences were verified by amino acid analysis and mass spectroscopy. KLH-conjugated peptides were injected into New Zealand White rabbits (1 mg peptide/rabbit) for polyclonal antibody production. DMT1 peptide was synthesized according to the predicted amino acid sequence obtained from the human DMT1 cDNA corresponding to amino acids 235-248 of the protein in the presumed fourth external loop between putative transmembrane regions five and six. Because this region is common to both the IRE and non-IRE forms of DMT1, it will react with both types of DMT1. The amino acid sequence of this region is highly conserved in mammals with little sequence homology to Nramp1. This peptide is in an extracellular region of the protein away from the glycosylation sites and with only six amino acids in common with human Nramp1. The FPN1 peptide was synthesized according to the predicted amino acid sequence obtained from the rat FPN1 cDNA corresponding to amino acids 253-270 of the protein in the external loop between putative transmembrane regions five and six. Comparison of both peptide sequences with public sequence databases using BLAST identified only DMT1 and FPN1 sequences.

Antibodies specificity was verified by peptide competition analysis. Briefly, membrane protein from duodenum was resolved and transferred as described below. After the blots were blocked, they were incubated with DMT1 (1:1,000) or FPN1 (1:750) antibodies in the presence or absence of the corresponding peptides for 1 h. DMT1 and FPN1 peptides used were in 24- and 18-fold excess over the antibodies used, respectively. The blots were visualized using enhanced chemiluminescence (ECL) after incubation with secondary antibody (Fig. 1).



View larger version (55K):
[in this window]
[in a new window]
 
Fig. 1. Verification of divalent metal transporter1 (DMT1) and ferroportin1 (FPN1) antibodies specificity by peptide competition analysis. Membrane proteins from duodenum were incubated with DMT1 (1:1,000) or FPN1 (1:750) antibodies in the presence or absence of the corresponding peptides, followed by washes and incubation with secondary antibodies. The immunologically detected proteins were visualized by using enhanced chemiluminescence (ECL). Ten microliters of DMT1 and FPN1 peptides (10 µg/ul) were used, which were in 24- and 18-fold excess over the antibodies used, respectively.

 

Purification of antibodies. Immunized rabbit serum was purified by using the immunizing peptides. Two-milliliter volumes of Affi-Gel 10 for DMT1 peptide (pI 9.3) and Affi-Gel 15 for FPN1 peptide (pI 6.3) were transferred to two 1-ml columns (Bio-Rad, Hercules, CA) and washed with coupling buffer (0.1 M NaHCO3, pH 8.3). The immunizing peptides (1 mg/ml) were added to the Affi-Gel columns, which were then incubated overnight on a rotating mixer at 4°C. Coupling buffer was added to the columns and was collected to check coupling efficiency. The columns were then washed with 10 ml coupling buffer, and 0.1 M glycine-HCl, pH 2.5 (elution buffer), was added. The affinity columns were then equilibrated with PBS. A 2-ml volume of the antisera was applied to the affinity columns and was allowed to run through into collection tubes. The columns were washed with 10 ml PBSTween (PBST) containing 0.5 M NaCl. The purified anti-DMT1 IgG and anti-FPN1 IgG were eluted with elution buffer, pH was adjusted to ~7, and Na azide was added.

Western blot analyses. Western blot analyses were performed to determine DMT1, FPN1, ferritin, and TfR protein levels in the duodenum of the animals. Protein samples extracted from the same animals were used for all Western blot analyses. Duodenum membrane protein fractions or the soluble protein fractions (100 µg) were solubilized in Laemmli buffer, boiled for 5 min, and separated by 8 (DMT1, FPN1, and TfR) and 12% (ferritin) SDS-PAGE, respectively. Similar loading and transfer of proteins were verified by staining the blots with Ponceau S (Sigma). Proteins were transferred by electroblotting to nitrocellulose membranes, which were then blocked by 5% nonfat powdered milk in PBST at room temperature for 2 h and at 4°C overnight. The membranes were then washed in several changes of PBST and incubated for 1 h at room temperature in either affinity purified DMT1 (1:1,000), affinity purified FPN1 (1:750), mouse anti-human ferritin antibody (1:2,500) (Alpha Diagnostic, San Antonio, TX), or mouse anti-rat TfR antibody (1:1,000) (Pharmingen, San Diego, CA) in PBST. The membranes were washed again with several changes of PBST, incubated with donkey anti-rabbit Ig, peroxidase-linked species-specific whole antibody (Amersham, Buckinghamshire, England), or peroxidase-conjugated rabbit anti-mouse Ig (DAKO, Copenhagen, Denmark) for1hat room temperature. Membranes were washed again with several changes of PBST. The immunologically detected proteins were visualized by using ECL (Amersham). Processed blots were exposed to X-ray film for the optimum exposure time and quantified using the Chemi-doc Gel Quantification System (Bio-Rad).

Preparation of iron absorption test solutions. The iron absorption test dose contained 5 µg iron (as ferrous sulfate)/ml of PBS with 2.68 mM ascorbic acid giving an ascorbic acid to iron ratio of 30:1 to assure that the iron was in ferrous form. This level of iron is similar to the iron concentration of rat milk during days 10-20 of lactation (14). 59Fe as FeCl3 (Amersham) was then added to the test dose to provide 200,000 counts·min-1·ml-1. This solution was prepared immediately before use and was prewarmed before intubation.

Iron absorption. Animals were fasted for 4 (day 10) or 6 h (day 20) and intubated with 0.5 ml of test dose directly into the stomach using an animal-feeding needle. Animals were killed by CO2 asphyxiation 4-5 h postintubation. The small intestines were then perfused with 2-3 ml saline to remove unabsorbed 59Fe from the lumen. The amount of unabsorbed 59Fe removed from the lumen using saline or saline containing EDTA was not significantly different, and saline alone was used for the perfusion. Radioactivity in the brain, kidney, liver, small intestine, spleen, carcass, cecum-colon, and perfusate were counted in a {gamma}-counter (Beckman, Gamma 8500, Irvine, CA). The results were expressed as percentage of the total radioactivity received.





Tissue iron analysis. Brain, kidney, liver, small intestine, and spleen were dissected and wet ashed in 16 M HNO3 for 7 days (6). Samples were then diluted with appropriate amounts of distilled water and analyzed for iron by atomic absorption spectrophotometry (Model Smith-Heifjie 4000, Thermo Jarrell Ash, Franklin, MA).

Statistical analysis. Values are expressed as means ± SD. Student's unpaired t-test was used to compare two data sets. Comparisons among three groups were tested by one-way ANOVA using Prism GraphPad version 3.02 (GraphPad Software, San Diego, CA). When the P value obtained from ANOVA was significant, Tukey's test was applied to test for differences among groups. Significance was considered to be P < 0.05. When the variances within the group differed significantly according to Bartlett's test, the data were logtransformed before being tested by one-way ANOVA.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of dietary iron deficiency on growth, hemoglobin, and tissue iron. Weight and food intake of dams fed the control and iron-deficient diets did not differ significantly (Table 2). However, weight and hemoglobin of pups born to dams fed the low-Fe diet were significantly lower compared with control pups at day 1 (Table 3). At days 10 and 20, the weights of iron-supplemented pups born to the low-Fe dams (low Fe + Fe) were not significantly different from controls, whereas the weights of unsupplemented low-Fe pups (low Fe) remained significantly lower than controls. Iron supplementation also increased hemoglobin to levels significantly greater than unsupplemented animals at days 10 and 20, but they remained significantly lower than controls.


View this table:
[in this window]
[in a new window]
 
Table 2. Food intake and body weight of dams

 

View this table:
[in this window]
[in a new window]
 
Table 3. Weight and hemoglobin of rat pups at birth and at days 10 and 20 after birth

 

Liver iron was significantly lower at birth in the low-Fe pups. Liver iron concentration was greatest in all groups at birth and subsequently fell to their lowest levels at day 20. This decline was most rapid in the control group so that by day 20, there was no significant difference between groups in liver iron (Table 4). Changes were also noted in spleen iron, with a significantly lower level in both low-Fe groups compared with controls at day 10. At day 20, there was no significant difference between groups, but unlike liver iron, spleen iron increased from days 10 to 20. In contrast to liver and spleen, brain iron concentration in the unsupplemented low-Fe group was significantly lower than controls at both days 10 and 20. However, iron supplementation resulted in brain iron concentrations similar to controls at days 10 and 20 (Table 4). Small intestine iron was lower in both the low-Fe groups at day 10 but was significantly lower only in the low-Fe group compared with controls at day 20. Iron supplementation resulted in small intestine iron similar to controls at day 20 (Table 4).


View this table:
[in this window]
[in a new window]
 
Table 4. Tissue iron of control, low Fe + Fe and low Fe rat pups at day 10 and 20 after birth

 

Effect of iron deficiency on iron absorption. Despite evidence of iron deficiency in the low-Fe groups as reflected by low hemoglobin and liver iron at day 10, there were no significant differences in mucosal iron retention, body iron uptake, or total iron absorption (total unabsorbed iron) among groups (Table 5). However, at day 20, mucosal iron retention was significantly lower and body Fe uptake and total iron absorption (total-unabsorbed Fe) were significantly greater in the unsupplemented low-Fe group. There was no significant difference between the supplemented low-Fe group and controls (Table 5).


View this table:
[in this window]
[in a new window]
 
Table 5. %59Fe uptake in tissues of control, low Fe + Fe, and low-Fe rat pups at days 10 and 20 after birth

 

Effect of iron deficiency on DMT1 and FPN1 gene expression. We have previously shown that intestinal DMT1 and FPN1 expression are developmentally controlled in rats with maximal expression at 40 days of age and that iron supplementation of normal rat pups at 10 days does not affect DMT1 and FPN1 gene expression, whereas it does at 20 days of age (16). In the present study, intestinal DMT1 expression at day 10 did not differ significantly between groups. However, at day 20, DMT1 was nearly fourfold greater in the unsupplemented low-Fe group compared with controls and the supplemented low-Fe group. Intestinal FPN1 gene expression was significantly lower in the unsupplemented low-Fe group at day 10 but increased to eightfold greater than controls at day 20 (Fig. 2).



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2. Quantification of duodenal DMT1 (A) and FPN1 (B) gene expression of the control, low-Fe + Fe, and low-Fe rat pups at days 10 and 20 after birth by real-time RT-PCR. The horizontal lines represent the mean, and each point represents an individual pup. Data are means ± SD for number of rat pups (n) = 8-11/group. Groups without a common letter differ, comparison was only within the same time point.

 

The effect of iron deficiency on liver DMT1 and FPN1 gene expression was also examined. In the liver, DMT1 and FPN1 expression was significantly greater in the unsupplemented low-Fe group compared with the other two groups at day 10 but not at day 20 (Fig. 3).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 3. Quantification of liver DMT1 (A) and FPN1 (B) gene expression of the control, low-Fe + Fe, and low-Fe rat pups at days 10 and 20 after birth by real-time RT-PCR. The horizontal lines represent the mean, and each point represents an individual pup. Data are means ± SD, n = 9 or 10/group. Groups without a common letter differ, comparison was only within the same time point.

 

Effect of iron deficiency on intestinal DMT1, FPN1, ferritin, and TfR protein levels. At day 10, intestinal DMT1 protein level was unchanged but was fivefold greater in the unsupplemented low-Fe group by day 20 compared with controls and the supplemented low-Fe group (Fig. 4). Intestinal FPN1 protein level at day 10 did not differ significantly among groups but increased ~3.3-fold by day 20 in the unsupplemented low-Fe group compared with controls (Fig. 5). The 68-kDa FPN1 protein presented at day 20 was not detectable at day 10, whereas the 130-kDa band observed at day 10 was present in smaller amounts at day 20 (Fig. 5). The change in DMT1 and FPN1 protein levels paralleled the changes in gene expression. Ferritin protein level decreased by 60% in the low-Fe group compared with controls at day 10 but was not affected at day 20 (Fig. 6). TfR protein level was not different among the groups at day 10 (Fig. 6) and was undetectable at day 20 (data not shown).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4. Western blot analysis was used for the detection of DMT1 protein from the duodenum of the control, low-Fe + Fe, and low-Fe rat pups at days 10 (A) and 20 (B) after birth. Data are means ± SD, n = 4/group. Groups without a common letter differ, comparison was only within the same time point.

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5. Western blot analysis was used for the detection of FPN1 protein from the duodenum of the control, low-Fe + Fe, and low-Fe rat pups at days 10 (A) and 20 (B) after birth. C: Western blot analysis of FPN1 protein from the duodenum of the control animals at days 10 and 20. Protein samples used for FPN1 detection were from the same animals used for DMT1 protein detection. Data are means ± SD, n = 4/group. Groups without a common letter differ, comparison was only within the same time point.

 


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6. Western blot analysis was used for the detection of ferritin protein from the duodenum of the control, low-Fe + Fe, and low-Fe rat pups at days 10 (A) and 20 (B) after birth. The immunoreactive bands were the light-chain ferritin. C: Western blot analysis of TfR protein in the duodenum of control, low-Fe + Fe, and low-Fe rat pups at day 10 after birth. Protein samples used for ferritin and TfR detection were from the same animals used for DMT1 and FPN1 protein detection. Data are means ± SD, n = 4/group. Groups without a common letter differ, comparison was only within the same time point.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Regulation of intestinal iron absorption is central to homeostasis of iron stores in adults. DMT1 and FPN1 have both been implicated in this homeostasis, because both are increased in iron deficiency and decreased under iron-replete conditions. Whether DMT1 and FPN1 respond to iron status during early infancy has not been determined. Previously, we have shown that in rat pups born to normal dams, DMT1, and FPN1 transcripts increase during development and are unresponsive to iron supplementation in early infancy. We undertook the current study to determine whether intestinal iron absorption is upregulated in iron-deficient rat pups.

Our model of iron-deficient rat pups has many similarities with human infants. Rat pups born from dams fed a low-iron diet had lower birth weight and hemoglobin. In addition, liver and spleen iron concentrations were reduced. Iron supplementation corrected the low birth weight and eliminated the anemia. Furthermore, brain iron was compromised in the low-Fe group both at days 10 and 20. The difference in brain iron in the low-Fe group was greater at day 10 (-50%) than at day 20 (-27%) compared with the control group. Moreover, an increase in brain iron uptake in the low-Fe group was only observed at day 20, suggesting that mechanisms responsible for regulating brain iron uptake are not present until day 20. This may be functionally important, because iron deficiency during infancy is associated with delayed cognitive and psychomotor development (7, 14, 22).

In the present study, both intestinal DMT1 and FPN1 gene expression increased significantly with iron deficiency at day 20, which corresponded to changes in protein levels. This is in agreement with previous findings that DMT1 and FPN1 gene expression increases in humans with iron deficiency (24) and in iron-deficient rats (4, 5, 23). Giving iron supplements to the low-Fe rat pups (low Fe + Fe) normalized the gene expression and protein levels of the iron transporters to those of controls. With a decrease in intestinal iron in the iron-deficient rat pups, TfR was expected to be increased and ferritin to be decreased. However, ferritin protein level was unchanged, and TfR was undetectable even after increased amounts of antibody and sample were used. It thus appears that the amount of TfR protein present is very low at day 20.

At day 10, ferritin protein level was decreased and TfR protein level was unaffected by iron deficiency. Iron deficiency also had no effect on intestinal DMT1 gene expression and appeared to decrease intestinal FPN1 gene expression. DMT1 and FPN1 protein levels were unaffected by iron deficiency. Thus these intestinal iron transporters are not responsive to iron status at this age. The reason for this is not clear, but it is possible that gut development may be immature during early infancy. In addition, the sensor communicating between body iron stores and the intestinal enterocyte may not be well developed. Rat pups are normally born with sufficient iron stores and do not depend as much on exogenous iron during birth and early infancy (day 10); hence, it is possible that intestinal regulation of iron absorption during early infancy may not be as crucial as during late infancy (day 20) when pups are being weaned and depend on other dietary iron sources than rat milk and their body iron stores.

In our previous study using a similar experimental design (16), DMT1 and FPN1 gene expression were downregulated with iron supplementation at day 20, but there were no changes at day 10. Thus there appears to be a lack of response by both DMT1 and FPN1 to iron deficiency as well as iron supplementation at day 10 (early infancy), whereas by day 20 (late infancy), both DMT1 and FPN1 responded to iron as described in adult animals. This indicates pronounced developmental regulation of iron absorption.

The mechanisms that regulate DMT1 and FPN1 expression are not well known. The changes in DMT1 and FPN1 expression with iron deficiency and iron supplementation in adult animals are believed to be controlled by the interaction of iron regulatory proteins (IRP) and IRE on the DMT1 and FPN1 mRNA (5, 12, 19). However, transcriptional regulation from an alternate start site in the DMT1 promoter suggests other mechanisms are involved (13). In addition, it is not known whether there are differences in the interaction of IRP and IRE between days 10 and 20 or whether the effect of iron deficiency and iron supplementation on IRP formation is different at these developmental stages.

Iron absorption in the rat pups correlated well with the expression of the DMT1 and FPN1. At day 20, as intestinal DMT1 increased with iron deficiency, there was also an increase in iron absorption. At the basolateral side of the enterocyte, FPN1 was also increased with iron deficiency, and there was a decrease in iron retained in the enterocyte. The overall result is an increase in iron influx to the body of the low-Fe rat pups at day 20. In contrast, at day 10, when there was no change in DMT1 and FPN1 protein levels in the iron-deficient group, there was no change in iron absorption. Furthermore, there was no change in iron retained in the enterocyte.

The distribution of iron under these experimental conditions also revealed changes in two important tissues, brain and liver. Although iron-deficient pups had lower brain iron at birth, supplementation reversed this effect. In the liver, which serves as the primary storage site, iron was significantly higher in controls at day 10, but there were no differences among the groups at day 20. Liver iron concentration decreased in all groups from days 10 to 20, most likely the result of increased iron needs for growth. This suggests a sensitive mechanism that maintains liver iron at a certain minimum level during iron deficiency at the expense of other tissues such as the brain.

Liver DMT1 gene expression has been reported to not differ with iron status (23). We also found no significant change in liver DMT1 gene expression level between the low-Fe group and control at day 20, whereas at day 10, there was a significant increase in liver DMT1 gene expression in the low-Fe pups. Similarly, liver FPN1 gene expression was also increased with iron deficiency at day 10, whereas there was no change at day 20. Liver FPN1 has been shown to be upregulated in iron-loaded adult mice using immunohistochemistry and is believed to be regulated by iron in a ferritin-like manner (1). However, we found up-regulation of liver FPN1 in iron-deficient rat pups. These differences may be explained by the difference in iron loading (parental vs. dietary), the age of the animals (adult vs. pups), and methods used (protein vs. gene expression). However, if FPN1 is involved in iron export out of the liver, the iron-deficient rat pups would increase FPN1 to mobilize more iron out of the liver to synthesize more hemoglobin to support the increase in blood volume and the large demand of iron for rapid growth during this period. Apart from the iron-dependent IRE/IRP regulation, whether there is a role for erythropoiesis-mediated regulation of FPN1 is not clear.

In our previous study examining DMT1 gene expression in iron supplemented pups (16), there was a decrease in liver DMT1 gene expression at day 10 in iron-supplemented pups but also no change at day 20. Thus liver DMT1 gene expression increased with iron deficiency and decreased with iron supplementation at day 10, with no change during either deficiency or supplementation at day 20. This leads us to believe that the liver may be playing an important role in regulating iron metabolism at day 10 (during early infancy) when intestinal DMT1 and FPN1 are not responding well to the iron status of the pups.

In conclusion, a lack of response of intestinal DMT1 and FPN1 to iron deficiency during early infancy results in iron-deficient rat pups not being able to increase their iron absorption during early infancy (day 10). However, during late infancy (day 20), intestinal DMT1 and FPN1 are upregulated in iron-deficient rat pups and iron absorption is also upregulated. Thus the molecular mechanisms regulating iron absorption during early infancy differs from late infancy when they are similar to adult animals, indicating developmental regulation of iron absorption.


    FOOTNOTES
 

Address for reprint requests and other correspondence: B. Lönnerdal, Dept. of Nutrition, Univ. of California, One Shields Ave., Davis, CA 95616 (E-mail: bllonnerdal{at}ucdavis.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Abboud S and Haile DJ. A novel mammalian iron-regulated protein involved in intracellular iron metabolism. J Biol Chem 275: 19906-19912, 2000.[Abstract/Free Full Text]
  2. Bothwell TH. The control of iron absorption. Br J Haematol 14: 453-456, 1968.[ISI][Medline]
  3. Bothwell TH and Charlton RW. Absorption of iron. Annu Rev Med 21: 145-156, 1970.[ISI][Medline]
  4. Canonne-Hergaux F, Fleming MD, Levy JE, Gauthier S, Ralph T, Picard V, Andrews NC, and Gros P. The Nramp2/DMT1 iron transporter is induced in the duodenum of microcytic anemia mk mice but is not properly targeted to the intestinal brush border. Blood 96: 3964-3970, 2000.[Abstract/Free Full Text]
  5. Canonne-Hergaux F, Gruenheid S, Ponka P, and Gros P. Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron. Blood 93: 4406-4417, 1999.[Abstract/Free Full Text]
  6. Clegg MS, Keen CL, Lönnerdal B, and Hurley LS. Influence of ashing techniques on the analysis of trace elements in animal tissue. Biol Trace Elem Res 3: 107-115, 1981.[ISI]
  7. De Andraca I, Castillo M, and Walter T. Psychomotor development and behavior in iron-deficient anemic infants. Nutr Rev 55: 125-132, 1997.[ISI][Medline]
  8. Domellöf M, Cohen RJ, Dewey KG, Hernell O, Rivera LL, and Lönnerdal B. Iron supplementation of breast-fed Honduran and Swedish infants from 4 to 9 months of age. J Pediatr 138: 679-687, 2001.[ISI][Medline]
  9. Domellöf M, Lönnerdal B, Abrams SA, and Hernell O. Iron absorption in breast-fed infants: effects of age, iron status, iron supplements, and complementary foods. Am J Clin Nutr 76: 198-204, 2002.[Abstract/Free Full Text]
  10. Donovan A, Brownlie A, Zhou Y, Shepard J, Pratt SJ, Moynihan J, Paw BH, Drejer A, Barut B, Zapata A, Law TC, Brugnara C, Lux SE, Pinkus GS, Pinkus JL, Kingsley PD, Palis J, Fleming MD, Andrews NC, and Zon LI. Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter. Nature 403: 776-781, 2000.[ISI][Medline]
  11. Fleming MD, Trenor C, Su MA, Foernzler D, Beier DR, Dietrich WF, and Andrews NC. Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene. Nat Genet 16: 383-386, 1997.[ISI][Medline]
  12. Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, and Hediger MA. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 388: 482-488, 1997.[ISI][Medline]
  13. Hubert N and Hentze MW. Previously uncharacterized isoforms of divalent metal transporter (DMT)-1: Implications for regulation and cellular function. Proc Natl Acad Sci USA 99: 12345-12350, 2002.[Abstract/Free Full Text]
  14. Hurtado EK, Claussen AH, and Scott KG. Early childhood anemia and mild or moderate mental retardation. Am J Clin Nutr 69: 115-119, 1999.[Abstract/Free Full Text]
  15. Keen CL, Lönnerdal B, Clegg MS, and Hurley LS. Developmental changes in composition of rat milk: trace elements, minerals, protein, carbohydrate and fat. J Nutr 111: 226-236, 1981.[ISI][Medline]
  16. Leong W, Bowlus CL, Tallkvist J, and Lönnerdal B. Intestinal DMT1 gene expression, iron absorption and tissue iron of infant rats given iron supplements (Abstract). FASEB J 15: A60, 2001.
  17. Lowry OH, Rosenbrough NT, Farr AL, and Randall R. Protein measurement with folin phenol reagent. J Biol Chem 193: 265-275, 1951.[Free Full Text]
  18. McKie AT, Barrow D, Latunde-Dada GO, Rolfs A, Sager G, Mudaly M, Richardson C, Barlow D, Bomford A, Peters TJ, Raja KB, Shirali S, Hediger MA, Farzaneth F, and Simpson RJ. An iron-regulated ferric reductase associated with the absorption of dietary iron. Science 291: 1755-1759, 2001.[Abstract/Free Full Text]
  19. McKie AT, Marciani P, Rolfs A, Brennan K, Wehr K, Barrow D, Miret S, Bomford A, Peters TJ, Farzaneh F, Hediger MA, Hentze MW, and Simpson RJ. A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation. Mol Cell 5: 299-309, 2000.[ISI][Medline]
  20. Oates PS, Trinder D, and Morgan EH. Gastrointestinal function, divalent metal transporter-1 expression and intestinal iron absorption. Pflügers Arch 440: 496-502, 2000.[ISI][Medline]
  21. Pollitt E. Iron deficiency and educational deficiency. Nutr Rev 55: 133-141, 1997.[ISI][Medline]
  22. Pollitt E. Early iron deficiency anemia and later mental retardation. Am J Clin Nutr 69: 4-5, 1999.[Free Full Text]
  23. Trinder D, Oates PS, Thomas C, Sadleir J, and Morgan EH. Localisation of divalent metal transporter 1 (DMT1) to the microvillus membrane of rat duodenal enterocytes in iron deficiency, but to hepatocytes in iron overload. Gut 46: 270-276, 2000.[Abstract/Free Full Text]
  24. Zoller H, Koch RO, Theurl I, Obrist P, Pietrangelo A, Montosi G, Haile DJ, Vogel W, and Weiss G. Expression of the duodenal iron transporters divalent-metal transporter 1 and ferroportin 1 in iron deficiency and iron overload. Gastroenterology 120: 1412-1419, 2001.[ISI][Medline]