Divalent metal transporter I (DMT1) is thought to be involved in transport of iron across the apical cell membrane of villus duodenal cells. To determine its role in hereditary hemochromatosis (HH), we used β2-microglobulin knockout (B2M−/−) mice that accumulate iron as in HH. The B2M−/− and controlC57BL/6 (B2M+/+) mice were fed diets with different iron contents. Increasing the iron availability increased plasma iron levels in both B2M+/+ and B2M−/−mice. Reducing the iron availability decreased the plasma iron concentration in B2M+/+ mice but was without effect on plasma iron in B2M−/− mice. DMT1 was not detectable in mice fed normal or iron-loaded diets when using immunohistochemistry. In Western blots, however, the protein was consistently observed regardless of the dietary regimen. DMT1 expression was increased to the same extent in B2M+/+ and B2M−/− mice when fed an iron-poor diet. In both strains of mice fed an iron-poor diet, DMT1 was evenly distributed in the differentiated enterocytes from the base to the tip of the villi but was absent from the crypts of Lieberkühn. These data suggest that the observed effects were due to the state of iron deficiency in mucosal cells rather than genetic defect.
- gene knockout
- iron deficiency
- Western blotting
several inherited disorders of iron metabolism in humans have been reported. The most common is hereditary hemochromatosis (HH) in which excessive intestinal absorption of iron from a normal diet leads to gradual accumulation in many organs with resultant cellular damage and disturbed function (12). In almost all patients with HH, the disorder can be ascribed to a mutation in a gene related to the major histocompatibility class 1 family (9). The product of this gene is now referred to as HFE. Normally, HFE interacts with β2-microglobulin (B2M) and the complex localizes to the cell membrane. However, in HH the mutated form of HFE (Cys282Tyr) fails to interact withB2M and to move from the endoplasmic reticulum to the plasma membrane (10, 36). How this could lead to increased iron absorption is unknown. One hypothesis is that the HH mutation leads to impaired iron uptake from transferrin by cells of the crypts of Lieberkühn and that when they migrate to the villi, they function as iron-deficient cells with enhanced iron absorption (20).
One mechanism by which this could occur is increased expression of an iron transporter, such as the divalent metal ion transporter I (DMT1), originally called divalent cation transporter I (18). This protein is expressed in duodenal villus enterocytes, and the level of DMT1 expression increases with iron deficiency (5,35). Moreover, a mutation of the protein, Gly∼Arg185, is associated with impaired iron absorption in mice with hereditary microcytic anemia (mk/mk) (14) and homozygous Belgrade rats (b/b) (13). Because the defect affects the uptake phase of iron absorption (8, 28), it is highly likely that DMT1 is the transporter responsible for iron uptake from the intestinal lumen by duodenal enterocytes. Also, one of the two alternatively spliced transcripts of the DMT1 gene contains a putative iron-responsive element (IRE) in its 3′ untranslated region (14,18, 22). This could explain upregulation of DMT1 in iron-deficient enterocytes and increased efficiency of iron absorption in HH.
The B2M knockout mouse (B2M−/−) develops iron overload with a cellular distribution similar to that observed in HH (7, 31) due to increased iron absorption (32,33). In the present investigation, we examined whether DMT1 expression reflects the pool of storage iron (hepatic iron content) and mobile iron (plasma iron) in normal and B2M−/− mice subjected to variations in dietary iron intake. It was the expectation that, despite a reduction in dietary iron, a higher iron uptake would still prevail in the duodenal enterocytes of B2M−/− mouse and affect the expression pattern of DMT1.
MATERIALS AND METHODS
Male C57BL/6 (B2M+/+) (n = 18) and B2M−/− mice raised on an inbred C57BL/6genetic background (n = 18) were obtained at 10 postnatal weeks of age. They were separated into three groups of six mice of each strain and subjected to a normal (170 mg/kg), iron-poor (<10 mg/kg), or iron-high (20 g/kg) diet for 7 wk. The diet containing high iron levels was prepared by mixing the normal diet (model C-1000; Altromin) with carbonyl iron. Before commencing the iron-poor diet (model C-1038; Altromin), the mice were bled to the extent of 1% (wt/vol) from the orbital venous plexus. The animals were housed in cages with free access to food and water and kept at a 12-h light-dark cycle. The animal-handling procedures described in this study were approved by the Danish National Council of Animal Welfare.
Mice were weighed and deeply anesthetized with methohexital at a dose of 0.1 mg/g body wt. Their chests were surgically opened, and blood samples were collected from the hearts in a microcapillary tube and a heparinized syringe. The mice were then transcardially perfused via the left ventricle with heparin (15,000 IE/l) in 0.1 M potassium phosphate-buffered saline for 5 min to remove the blood from the circulation. The duodenum was identified, and a 1.5-cm stump was separated 1 cm from the pylorus. The proximal 0.5 cm was fixed in 3.7% buffered formalin and processed for immunohistochemistry, and the distal 1 cm homogenized 3 ml/g tissue in buffer containing 85 g sucrose, 4.65 g histidine, 0.75 g EDTA, 0.1 g phenylmethylsulfonyl fluoride, 2 mg leupeptin, 1 mg pepstatin, 2 mg apronitin, and 20 ml Triton X-100 in 1:l distilled water. The homogenate was sonicated for 5 s and centrifuged for 30 min at 3,000 g. The supernatant was isolated and measured for its protein concentration using a detergent compatible protein assay kit (Bio-Rad). This colorimetric assay reveals a blue coloring of proteins that was determined at 750 nm using a Beckman spectrophotometer. Samples of the right liver lobule were either weighed and used for nonheme iron measurements or fixed in 3.7% buffered formalin.
Chemical Iron Measurements
The concentration of iron in plasma was measured by atomic absorption spectrometry and expressed as micromoles of iron per milliliter. Liver nonheme iron was measured by the method of Kaldor (19) and expressed as micromoles of iron per gram of liver.
Sections from duodenum and liver were reacted for ferric (nonheme) iron. Sections were incubated for 20 min in 1% H2O2 in 0.1 M PBS to quench endogenous peroxidase activity. They were then immersed in Perl's solution (1:1, 2% HCl and 2% potassium ferrocyanide) at room temperature for 30 min and incubated for 10 min in 0.015% H202 in 3,3-diaminobenzidine tetrahydrochloride (DAB).
Immunohistochemistry was performed as previously described (27). In brief, 1-μm sections of the duodenum mounted on lysine-coated glass slides were incubated with self-made polyclonal rabbit anti-DMT1 (35) diluted 1:200 or polyclonal rabbit anti-DMT1 (NRAMP-22S; Alpha Diagnostics) diluted 1:600. The self-made antibody recognizes a 14-amino acid peptide in the extracellular region of the DMT1 molecule that is present both in the IRE isoform and the non-IRE isoform of the DMT1 molecule (36). The NRAMP-22S antibody (Alpha Diagnostics) recognizes a 17-amino acid peptide sequence located near the COOH terminus of the DMT1 molecule that contains an IRE; hence, this antibody will only detect the IRE isoform of DMT1. Liver sections prepared in an identical manner were incubated with polyclonal rabbit/anti-human ferritin (Dakopatts) diluted 1:50. Binding of primary antibodies was detected using polyclonal swine/anti-rabbit antibody diluted 1:50 (Dakopatts) followed by a polyclonal peroxidase-anti-peroxidase complex diluted 1:50 (Dakopatts). DAB was used as chromogen. The sections were dried and embedded in DePeX (British Drug House).
Immunoblotting was employed to verify the results of the immunohistochemical procedure. Tissue preparations of 10 μg were dissolved in NuPAGE LDS buffer (Invitrogen) containing 0.25% mercaptoethanol (Sigma) and heated for 10 min at 70°C before NuPAGE [4–12% Bis-Tris gel with MOPS running buffer (Invitrogen) containing 0.25% mercaptoethanol] and transferred to a 0.20-μm nitrocellulose membrane (LKB Diagnostics) using NuPAGE transfer buffer (Invitrogen) mixed with 10% methanol to provide optimal protein transfer. Membranes were blocked for nonspecific immunoreactivity by incubation for 30 min with 2% Tween 20 and 3% skim milk powder dissolved in 0.1 M PBS, pH 7.4. The nitrocellulose membranes were then incubated overnight at 4°C with rabbit anti-DMT1 (Alpha Diagnostics) diluted 1:5,000 in 0.1 M PBS, pH 7.4 containing 0.05% Tween 20. Binding of the antibody was detected using peroxidase-coupled swine/anti-rabbit immunoglobulin (Amersham Pharmacia Biotech) diluted 1:3,000 for 60 min at room temperature and visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech). Experiments were repeated in triplicate.
The specificity of staining with the anti-DMT1 serum in the histochemistry and blotting experiments was examined in three ways:1) by omission of the antiserum, 2) by substitution of nonimmune serum for the primary antibody, and3) by performing the incubation with primary antibody in the presence of 50 μg of the peptide used to produce the antibody. Under all such circumstances, immunolabeling was not observed.
Statistical evaluation of iron data (n = 6 mice in all experimental groups) was performed by ANOVA. When differences were detected (P < 0.05), means were tested with the Student-Newman-Keuls test for differences between individual means. Data are presented in means ± SD, unless otherwise noted.
All of the B2M−/− mice and B2M+/+mice weighed between 25 and 30 g, and there were no significant differences between the mean values of the different dietary groups (Table 1). The hematocrits were not significantly different among the three groups ofB2M+/+ mice fed different iron diets (Table 1). For theB2M−/− mice, hematocrits were significantly higher than inB2M+/+ mice. Moreover, with the B2M−/− mice, the mean hematocrit in the group of mice fed an iron-poor diet was significantly lower compared with the B2M−/− mice fed the normal or iron-loaded diet (Table 1).
In mice fed a diet with a normal iron content, the mean plasma iron level was 24.9 ± 3.0 μM in B2M+/+ mice and 33.7 ± 5.1 μM in B2M−/− mice, representing a significant difference (Fig. 1). In B2M+/+mice, varying the diets resulted in significant changes in plasma iron concentrations. Mice fed an iron-poor diet had a concentration of only 16.3 ± 3.4 μM, whereas mice fed an iron-high diet had a plasma iron concentration of 39.0 ± 5.4 μM (Fig. 1). By contrast, reducing iron availability did not significantly reduce plasma iron inB2M−/− mice when the plasma iron concentration was 32.6 ± 3.8 μM. High iron availability significantly increased plasma iron to 42.6 ± 6.0 μM in the B2M−/− mice (Fig. 1). A comparison between the two mouse strains fed the same diet, revealed significant differences between mice fed the normal and iron-poor diets, whereas no significant difference was observed in mice fed the iron-high diet (Fig. 1).
In the liver, changes in dietary iron affected the nonheme iron content in a manner that corresponded to the iron content of the diet (Fig.2). In mice fed the normal diet, nonheme iron levels were 3.02 ± 0.6 (B2M+/+) and 27.5 ± 3.2 μmol/g (B2M−/−). In mice fed the iron-poor diet, nonheme iron levels were 0.9 ± 0.1 (B2M+/+) and 2.1 ± 1.0 μmol/g (B2M−/−). In mice fed the iron-high diet, nonheme iron levels were 69.2 ± 6.7 (B2M+/+) and 115 ± 15.2 μmol/g (B2M−/−). The concentration of nonheme iron was significantly higher in each dietary group of B2M−/− mice compared with the corresponding group of B2M+/+ mice (Fig.2).
Histological investigation of the liver confirmed the presence of iron accumulation in B2M−/− mice subjected to a normal diet and in B2M+/+ and B2M−/− mice subjected to an iron-high diet, whereas the iron content was much lower inB2M+/+ mice fed a normal diet and both types of mice fed the iron-poor diet (not shown). Differences in hepatic iron content were also reflected in a different distribution of ferritin in the liver (not shown). Hence, in both mouse strains, ferritin-immunoreactivity was lower in mice fed an iron-poor diet and higher in mice fed an iron-rich diet. In normal and iron-loaded mice, the ferritin expression decreased with increasing distance from the periportal area. Compared with B2M+/+ mice fed a normal diet, B2M−/− mice fed a normal diet had a higher periportal accumulation of ferritin. InB2M+/+ and B2M−/− mice fed an iron-high diet, ferritin was almost uniformly distributed throughout the liver parenchyma, reflecting the high level of iron-accumulation in the liver. The liver of the B2M−/− mice fed an iron-high diet showed no signs of fibrosis or cirrhosis (not shown).
Iron-content of the different diets was also reflected in the distribution of nonheme iron and ferritin in the duodenal villi. Iron accumulation ranged from average in mice fed a normal diet to low in mice fed an iron-poor diet and high in mice fed an iron-loaded diet (Fig. 3). Accumulation of iron was not higher in B2M−/− mice compared with B2M+/+ mice of each of the dietary groups (Fig. 3). Iron was evenly distributed in differentiated enterocytes from the base to the tip of the villi (Fig.4 A). In the crypts of Lieberkühn, iron was rarely observed in mice fed the normal diet (Fig. 4 A), and always absent in mice fed an iron-poor diet. By contrast, in mice fed the iron-high diet, cells in crypts of Lieberkühn were often labeled with iron (Fig. 4 B). The staining pattern in duodenums stained for ferritin essentially reflected the staining distribution obtained with iron; the only difference between iron and ferritin distribution in the duodenum was that iron, and not ferritin, was found in the crypts of Lieberkühn of both strains of mice fed an iron-high diet (Fig.4 C).
DMT1 immunoreactivity was not detected in B2M+/+ andB2M−/− mice fed normal or iron-high diets (Figs.5 and6 A) but was restricted to mice of each strain fed an iron-poor diet (Fig. 5, B andE). This pattern of immunoreactivity was observed using both anti-DMT1 antibodies, which revealed an undistinguishable immunoreactivity. Both in B2M+/+ and B2M−/−mice fed the iron-poor diet, DMT1 protein staining was observed in enterocytes of the duodenal villi, whereas crypts were unlabeled (Fig.6). The DMT1 immunoreactivity was present along the whole length of the villi from base to tip in the cytoplasm and microvillus membrane, leaving nuclei unstained (Fig. 6, B and C). There was no difference in DMT1 immunoreactivity in the villi of theB2M+/+ and the B2M−/− mice (compare Fig. 6,B and C). Goblet cells were unstained in any strain of mice or dietary condition. Likewise, DMT1 immunoreactivity was not observed in cells of Brunner glands (not shown).
Detection of DMT1 immunoreactivity in Western analyses revealed a consistent band at ∼90 kDa in samples from the different dietary groups of both mouse strains. A band at this molecular mass is compatible with the extensive glycosylation of the native 60-kDa DMT1 protein (17). Samples from B2M+/+ andB2M−/− mice fed an iron-poor diet had a higher DMT1 immunoreactivity, which is compatible with the observation of a high immunoreactivity using immunohistochemistry in enterocytes of this dietary group in mice of both strains (Fig.7). A comparison among samples fromB2M+/+ and B2M−/− mice fed the same diet revealed no differences in DMT1 immunoreactivity (not shown). Immunoreactivity was not observed when the anti-DMT1 antibody was substituted by nonimmune serum or was omitted from the immunoreaction. Likewise, staining was absent if the sections were incubated with the peptide against which the DMT1 antibody was prepared.
The appearance of high levels of plasma iron and iron and ferritin in hepatocytes of the periportal regions of the liver is in keeping with an increased iron absorption in the B2M−/− mice and is comparable to previous reports of hepatic iron levels inB2M−/− mice (7, 33), HFE knockout mice (HFE−/−) (2, 23, 37), and in the early stages of HH (1, 15, 34). The altered iron status ofB2M−/− mice fed a normal diet could not be attributed to redistribution of iron from red blood cells to the liver, because the hematocrit of B2M−/− mice was higher than inB2M+/+ mice.
It is noteworthy that although the iron-poor diet lowered the hepatic iron store in the B2M−/− mice, it left the plasma iron concentration unchanged. There are at least two possibilities for the former observation: 1) as the mucosal transfer of iron is higher in B2M−/− than in B2M+/+ mice (33), B2M−/− mice are probably capable of transporting higher amounts of iron into the blood despite low content of iron in the diet; and 2) the B2M−/− mice had a higher amount of storage iron in the liver when the dietary experiment was initiated. However, the fact that the liver was almost depleted in iron in the B2M−/− mice fed the low-iron diet suggests that an enhanced absorption of iron by the duodenal villi is the quantitatively more important reason for that the plasma iron concentration was almost twice as high in the B2M−/− mice compared with the B2M+/+ mice.
Although the genetic abnormality in HH is different from that inB2M−/− mice, the effect of the abnormalities on iron metabolism are similar in the two conditions. For its normal function, HFE must be translocated from its site of synthesis in the endoplasmic reticulum to the plasma membrane, a process that requires interaction with B2M (10, 36). In HH, this process is impaired due to mutation of the HFE molecule so that it can no longer interact with B2M, and in B2M−/− mice it results from a lack of B2M. Hence, the B2M−/−mouse provides a good animal model of HH (24).
Our findings of DMT1 protein expression in immunoblots in any dietary condition, and not only in samples of mice fed an iron-poor diet, demonstrate that the immunoblotting technique was more sensitive for detection of DMT1 immunoreactivitity than was immunohistochemistry. In mice fed a normal or iron-enriched diet, the level was too low to be detected by PAP immunohistochemistry. The PAP-detection system was chosen instead of the streptavidin-biotin detection system, because the latter caused unacceptable background staining due to endogenous biotin in the intestinal cells (not shown). The finding of a low level of DMT1 protein expression in B2M−/− mice fed a normal diet is compatible with a previous study (6). However, the low level of DMT1 expression is not a rate-limiting factor for absorption in normal or B2M−/− mice. In conditions of dietary iron deficiency, the major function of increased expression of DMT1 protein in the microvillus membrane is probably to maximize the uptake of iron from the small quantity present in the duodenal lumen rather than to increase the total capacity for iron absorption.
HFE−/− mice carrying mutant DMT1 fail to accumulate iron, which indicates that iron is transported through the intestine by DMT1 (23). The detection of DMT1 in the duodenal samples is in agreement with this notion. Impaired intestinal iron absorption in homozygous Belgrade rats (28) also clearly indicates that DMT1 plays a significant role for iron uptake. Recent studies were able to detect more DMT1 protein in enterocytes of HH patients (38) and HFE−/− mice (16), respectively. The latter observations strongly suggest that the DMT1 protein increases in enterocytes in HH, because the cells become iron-deficient due to the lack of a functioning HFE protein. Probably HFE, more than B2M, is responsible for the regulation of iron uptake in enterocytes. As a result, the effects on DMT1 expression and iron absorption are quantitatively greater in HFE−/−mice and HH patients than in B2M−/− mice. However, increased iron absorption does occur in the B2M−/−animals, indicating that the B2M molecule is important for normal iron absorption, probably due to its interaction with HFE.
The fact that DMT1 expression was induced to a similar degree in genetically normal mice and B2M−/− mice when they were fed the iron-poor diet suggests that the observed effects were due to the state of iron deficiency in the mucosal cells rather than being directly due to the genetic defect. The high level of expression and microvillus membrane localization of DMT1 in duodenal enterocytes of iron-depleted B2M−/− mice is a result similar to that previously found in iron-deficient mice and rats (5, 35) in which it was shown to be due to expression of the IRE-containing isoform of DMT1 (5).
In the small intestine, HFE appears to be restricted to the crypts of Lieberkühn where it was observed mainly at intracellular sites (29), probably due to interaction and cotrafficking with the transferrin receptor (3, 11, 21, 36). Recently, we showed that the uptake of transferrin-bound iron from the plasma by the duodenum is inhibited in the HFE−/− mouse compared with the control C57Bl/6 mice. This indicates that HFE regulates duodenal uptake of transferrin-bound iron from the plasma and this iron-sensing mechanism is impaired in HH (35a). This would lead to a decrease in iron levels in the duodenum so that, after migration to the villi, the enterocytes function as in iron deficiency, with increased expression of DMT1 (2). However, in HH, iron transport out of the enterocytes into the blood is accelerated as much or even more than iron uptake from the lumen (4, 25, 26, 30). Possibly, the primary change in HH is exerted on iron transport out of the villus cells as a consequence of impaired HFE function in the crypts, whereas DMT1-mediated iron transport into the cells from the intestinal lumen is not rate limiting when dietary iron levels are normal. Very rapid export of iron from villus cells in HH patients and inHFE−/− mice could lead to cellular iron depletion and increased DMT1 expression despite increased iron uptake from the lumen. In B2M−/− mice, this effect may be smaller and insufficient to cause detectable increase in DMT1.
We thank Grazyna Hahn and Susan Peters for excellent technical assistance.
This work was supported by grants from the Vera and Carl Johan Michaelsens Fund (to T. Moos) and the National Health and Medical Research Council of Australia (to D. Trinder and E. H. Morgan).
Address for reprint requests and other correspondence: T. Moos, Dept. of Medical Anatomy, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark (E-mail:).
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.
April 17, 2002;10.1152/ajpgi.00346.2001
- Copyright © 2002 the American Physiological Society