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.
- iron absorption
- mucosal block
- iron deficiency
- excessive iron loading
iron deficiency induces the upregulation of several intestinal iron transporters, including divalent metal transporter 1 (DMT1, formally Slc11a2) and ferroportin (5, 6, 10, 13, 17, 29, 32), which increase iron absorption in the small intestine. However, excess iron loading suppresses intestinal iron absorption, a phenomenon known as “mucosal block” and found to persist for several hours to several days (4, 11, 23, 25). The effect of oral ingestion of 10 mg iron on the duodenal expression of molecules associated with intestinal iron transport was tested in iron-deficient rats and related to the mucosal block phenomenon (8). Three hours after oral iron ingestion, 59Fe uptake (the counts in the carcass and duodenum) and transfer (the counts in the carcass) decreased 60 and 50%, respectively. This inhibition of iron absorption is accompanied by the downregulation of DMT1, duodenal cytochrome b (Dcytb, formally Cybrd1), and ferroportin 1 (FPN1, formally Slc40a1) mRNA and protein (8). Generally, oral administration of 10 mg iron is considered excessive in experiments examining intestinal iron absorption. We found that direct application of 10 mg iron to the loop of the duodenum of iron-deficient rats downregulated Dmt1 mRNA and upregulated ferritin (Ft) mRNA 2 h later, with a longer time (>2 h) required for FT protein synthesis in the duodenal mucosa (1).
Because the mucosal block phenomenon is usually examined in iron-deficient animals, increased intracellular iron concentration in the small intestines following iron loading may be due to the increased expression of DMT1 protein. Regardless of whether mucosal block occurs only in iron-deficient rats, the iron concentration required to induce this phenomenon and the time to onset remain unclear. We therefore administered 10 mg of iron to the ligated duodenum of iron-deficient and iron-sufficient rats and measured their serum iron and duodenal iron uptake. One hour later, arterial serum iron concentration was higher in iron-deficient (928.9 ± 100.3 μg/dl) than in iron-sufficient rats (642.2 ± 30.6 μg), suggesting increased iron absorption in the former, but duodenal iron uptake did not differ significantly (5,658 and 7,533 μg/h, respectively). On the basis of these results, we hypothesized that short-acting mucosal block occurs prior to the regular mucosal block, which is characterized by the downregulation of mRNA and proteins. To determine whether an activating iron load can induce a short-acting mucosal block, we assessed iron uptake from the lumen of the small intestine following duodenal administration of iron loads of 30 μg (low) to 2,000 μg (marginally excessive).
DMT1 was also internalized in the duodenum of these rats several hours after oral loading of a very high concentration of iron (8). Administration of an excess iron load to the stomach of iron-deficient rats reduced duodenal DMT1 protein expression and apical distribution, as well as reducing fluorescence intensity in the cytoplasm (20). Furthermore, DMT1 was internalized by Caco-2 cells within min (20). We therefore assessed whether short acting mucosal block occurs only in iron-deficient rats and also analyzed the time to onset in iron-deficient duodenal mucosa, by comparing the effects of administering FeSO4 to the duodenal loop of iron-deficient and iron-sufficient rats before the decreases in gene and protein expression (i.e., <2 h).
Experimental animals and diets.
Weaned male Wistar rats (4 wk old) were obtained from CLEA Japan (Tokyo, Japan) and fed an AIN-93G iron-sufficient diet (24) or an iron-deficient diet for 3 wk. The AIN-93G diet (CLEA Japan) contained 35 mg/kg iron as FeC6H5O7·nH2O, whereas the iron-deficient diet lacked FeC6H5O7·nH2O. Experimental diets and deionized water were provided ad libitum. The iron contents of the iron-sufficient diet, the iron-deficient diet, and deionized water were 54 mg/kg, 5 mg/kg, and <1 ppm, respectively, as determined by atomic absorption spectrophotometry (AA-7000; Shimadzu, Kyoto, Japan). The rats were housed individually in stainless steel cages maintained at a constant temperature (23 ± 2°C) and humidity (50 ± 10%) with a 12-h light-dark cycle.
Rats were anesthetized by intraperitoneal injection of pentobarbital (0.1 ml/100 mg body wt; Dainippon Pharma, Osaka, Japan), and a midline incision was made in the abdomen. The duodenum was exposed, and two ligatures were tied with disposable microvascular clips (BEAR Medic, Ibaraki, Japan), one ∼0–1 mm from the pylorus and the other at the ligature of Treitz, ∼6–8 cm from the pylorus. Incisions were made proximal to the upper ligature and distal to the lower ligature. The small intestine between the upper and lower ligatures was flushed with prewarmed saline (37°C) from the upper incision. The microclips were moved to seal the pylorus and the distal ends of the duodenum, and the loop was instilled with 10 mM HCl (control) or iron test solution. In experiment 1, the test solution contained 30–2,000 μg iron as FeSO4 (Wako Pure Chemical, Osaka, Japan) in 1 ml of 10 mM HCl. The control and iron solutions contained 0.5% polyethylene glycol (PEG; ICN Biomedicals, Aurora, OH) as a soluble absorption marker. The abdominal incision was sutured with thread and covered with gauze soaked in prewarmed saline (37°C) to prevent the wound from drying. The rats were kept warm for the indicated times to avoid hypothermia. One hour after infusing the test solution, blood was collected from the portal vein and the descending aorta and centrifuged, and serum was separated to measure iron concentration and unsaturated iron binding capacity. The duodenum was then carefully removed from each rat. The duodenal contents were collected, the lumen was rinsed with saline, and the mucosa was scraped off. Mucosal samples were immediately frozen on dry ice and stored at −80°C until use. To estimate iron retention in the duodenal mucosa 1 h after loading, the duodenal loops were collected and washed with 10 mM HCl to remove iron from the mucosal surface.
In experiment 2, the iron solution contained 100 or 1,000 μg of iron as FeSO4. Blood from the portal vein and the descending aorta, as well as the duodenal contents and mucosa, were collected 15 or 30 min after loading. To estimate iron retention and ferritin protein expression in the liver 15 and 30 min after loading, the livers were collected and blood in the liver was replaced with saline. The other experimental procedures were the same as those in experiment 1, unless otherwise noted. All blood/tissue sampling was done between 05:00 and 12:00, when the rat serum iron concentrations were low, to avoid the effects of diurnal variation (2) on serum iron. To prevent iron contamination from the environment, all instruments used to prepare the test solutions and mucosal samples were soaked in 1 M HCl and washed with ultrapure water. The Ethics Committee of Tokyo Metropolitan University approved all animal procedures.
Determination of iron uptake and iron concentrations in the duodenum and the liver.
The concentrations of iron in the test solutions and in the duodenal contents were immediately quantified by atomic absorption spectrophotometry. Precipitates were dissolved by dropwise addition of 1 M HCl. The amount of PEG was estimated by the nephelometric method at 540 nm after deproteinization of the samples (14). Iron uptake was determined by using the formula: Iron uptake (μg) = Fe in iron test solution (μg/5 mg PEG) − Fe in the duodenal content (μg/5 mg PEG). The percentage uptake was calculated as the ratio of iron uptake to iron load. To estimate iron retention in the duodenal mucosa and the liver, the duodenal loops and the liver homogenates were air dried and dried to ash at 450°C. Iron content was then determined by atomic absorption spectrophotometry.
Determination of ferritin protein in the duodenum and liver.
The duodenum and liver were homogenized in bioMasher (Nippi Incorporated Protein Engineering Office, Tokyo, Japan) and FT protein was extracted with RIPA buffer (Thermo Scientific, Vernon Hills, IL). FT proteins were quantified by Rat Ferritin ELISA (Immunology Consultants Laboratory, Portland, OR). Total proteins in duodenum homogenate were quantified by BCA protein assay.
Serum iron concentration, TIBC, and transferrin saturation in portal and arterial blood.
Serum iron concentration and unsaturated iron binding capacity (UIBC) were measured on a 7170 Clinical Analyzer (Hitachi High-Technologies, Tokyo, Japan), by the nitroso-(N-propyl-N-sulfo-propylamino)phenol method. Total iron binding capacity (TIBC; μg/dl) was calculated as the sum of the serum iron concentration and UIBC (the amount of iron able to bind unsaturated part of transferrin.). Transferrin saturation was calculated as serum iron concentration divided by TIBC concentration.
Expression of Dmt1, Ft, and Fpn1 mRNAs.
Mucosal samples were homogenized and total RNA was extracted from 10 mg of each using a QuickGene RNA tissue kit S (RT-S2) and the QuickGene-Mini 800 (Fujifilm, Tokyo, Japan). DNase solution (Nippon Gene, Tokyo, Japan) was added to each sample, and the samples were incubated at room temperature for 5 min. Each total RNA sample had an A260/A280 between 1.7 and 2.0. RNA integrity was assessed with an Agilent 6000 RNA Nano Kit and a Agilent 2100 Bioanalyzer with 2100 expert software. cDNA was synthesized from 5 μg of each total RNA by using a PrimeScript RT reagent Kit according to the manufacturer's instructions (Takara Bio, Shiga, Japan). The relative levels of expression of Dmt1, Ft (heavy chain), and Fpn1 mRNA were determined by real-time PCR (Chromo4 Real-Time PCR Detection System; Bio-Rad Laboratories, Hercules, CA) with TaqMan probes and iQ Supermix (Bio-Rad Laboratories). Relative expression was determined by using calibration curves, with stock cDNA from the duodenal mucosa of iron-deficient rats. Transcript levels were normalized relative to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA in that sample, and divided by the mean level in the control group. Primer sequences and probes are shown in Table 1, and the PCR methods, except for Fpn1, have been described (1). Fpn1-specific primers and probe were designed by use of Universal ProbeLibrary Assay Design Center software (Roche Diagnostics, Basel, Switzerland). The amplification protocol for Fpn1 consisted of an initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 90°C for 10 s, annealing at 55°C for 1 min, and extension at 72°C for 30 s. All reactions were performed in triplicate and the data are presented as means ± SD.
All results are reported as means ± SD for 4–7 rats. Student's t-tests were used to assess differences between rats fed the iron-deficient and iron-sufficient diets at the same iron load in experiment 1. Significant differences among the iron loads in rats fed the iron-deficient and iron-sufficient diets were determined by one-way or two-way ANOVA followed by Tukey's post hoc test. Differences in gene expression between the control and experimental groups were determined by Dunnett's multiple comparison tests. Differences were considered significant at a P value < 0.05.
After 3 wk of feeding, mean ± SD body weights and feed efficiency were significantly lower in rats fed the iron-deficient than the iron-sufficient diet, both in experiments 1 and 2, due to decreased feed intake by iron-deficient rats (Table 2).
Serum iron concentrations and transferrin saturation of portal and arterial blood (experiment 1).
Table 3 shows the serum iron concentrations, UIBC, TIBC, and transferrin saturation of portal blood in iron-deficient and -sufficient rats. Serum iron concentrations were significantly lower in iron-deficient than in iron-sufficient rats administered vehicle (control). Iron saturation markedly decreased as TIBC increased, a phenomenon usually associated with increased iron absorption. We therefore administered various concentrations of nonheme iron (i.e., FeSO4) to the duodenal loops of rats and measured serum iron concentrations in portal blood 1 h later. Iron loading (30–2,000 μg) into the duodenal loops proportionately increased serum iron concentration, with transferrin saturation reaching nearly 100% at iron loads ≥1,000 μg, with no large variations in TIBC. Serum iron concentrations were consistently higher in iron-sufficient than in iron-deficient animals at iron loads ≤30 μg but were lower in iron-sufficient rats at iron loads ≥500 μg. The differences in serum iron contents between rats administered vehicle and 30 μg iron were 26 μg/dl (244.5-218.1 μg/dl) in the iron-sufficient group and 94 μg/dl (170.3-76.4 μg/dl) in the iron-deficient group, indicating that iron absorption was greater in the iron-deficient rats. At iron loads ≥200 μg, the serum iron contents increased 210–302 μg in iron-sufficient and 418–748 μg in iron-deficient rats. At iron loads of 200, 500, 1,000, and 2,000 μg, the increases in iron contents in the iron-deficient group were 418 μg (494.7−76.4 μg/dl), 560 μg (636.5−76.4 μg/dl), 713 μg (789.6−76.4 μg/dl), and 748 μg (824.8−76.4 μg/dl), respectively. Therefore, greater iron loads were associated with greater differences in serum iron contents between iron-sufficient and iron-deficient rats.
Experimental results of arterial blood are shown in Table 4. Hematocrits were significantly lower in iron-deficient (23.5–27.8%) than in iron-sufficient rats (43.9–49.3%). The effects of iron loading on serum iron concentrations, TIBC, and transferrin saturation in arterial blood were consistent with those of portal blood, despite passing through the liver.
Duodenal iron uptake 1 h after luminal administration of a single iron dose (experiment 1).
Iron uptake was higher in iron-deficient than in iron-sufficient rats after loading with 30 and 200 μg iron but did not differ after loading with 500 μg iron (Table 5). Iron uptake after loading with 1,000 and 2,000 μg was significantly lower in iron-deficient rats than in iron-sufficient rats. At 1 h, the percent iron uptake in iron-sufficient rats was 63% (range 60.3–65.5%), regardless of iron load. The percent iron uptake in the iron-deficient group was 76.1% at 30 μg, decreasing approximately 25% to 50.7% at 2,000 μg.
Iron accumulation in the duodenum.
Iron accumulation in the duodenum 1 h after iron loading is shown in Fig. 1. After loading with vehicle, the mean duodenal iron concentrations in the iron-sufficient and iron-deficient groups were 14.4 ± 1.3 and 6.9 ± 0.6 μg/100 mg dry tissue, respectively. Iron accumulation tended to be lower in the iron-deficient than in the iron-sufficient group at any iron load, particularly at loads ≥500 μg.
Serum iron concentration and transferrin saturation in portal and arterial blood (experiment 2).
The duodenal loops of iron-deficient and iron-sufficient rats were administered 100 μg or 1,000 μg of nonheme iron, and portal and arterial blood were obtained 15 or 30 min after loading. Tables 6 and 7 show the serum iron concentration, UIBC, TIBC, and transferrin saturation of portal and arterial blood, respectively. After an iron load of 100 or 1,000 μg, the portal and arterial serum iron concentrations were higher in the iron-sufficient than in the iron-deficient group, except for 30 min after an iron load of 1,000 μg. However, the increments in serum iron concentrations were higher in the iron-deficient group at all iron loads. The increases in serum iron concentration were estimated as differences, by subtracting serum iron concentrations in vehicle-loaded from iron-loaded animals. For example, at an iron load of 100 μg, the incremental iron concentrations at 15 min were 320.9-225.5 μg/dl (control) = 95.4 μg/dl in iron-sufficient and 179.2-23.2 μg/dl = 156.0 μg/dl in iron-deficient rats. At an iron load of 1,000 μg, the incremental iron concentrations at 30 min were 196.6 μg/dl (422.1-225.5 μg/dl) in iron-sufficient and 411.0 μg/dl (434.2-23.2 μg/dl) in iron-deficient animals. Table 7 shows that hematocrits were significantly lower in iron-deficient (20.4–23.1%) than in iron-sufficient (41.3–43.5%) rats. As observed in experiment 1, the effects of iron loading observed in arterial blood were consistent with the effects on portal blood.
Duodenal iron uptake after luminal administration of normal and marginally excessive iron loads (experiment 2).
Iron uptake was assessed 15 or 30 min after loading with 100 or 1,000 μg iron (Fig. 2). Fifteen minutes after a load of 100 μg iron, iron uptake was significantly higher in iron-deficient than in iron-sufficient rats. The percent iron uptake in the iron-deficient group tended to be higher at 15 min than at 30 min but was similar at both times in the iron-sufficient group. After a 1,000 μg load, iron uptake was significantly lower in the iron-deficient than in the iron-sufficient group, particularly at 15 min after loading.
Dmt1, Ft, and Fpn1 mRNA expression.
Following loading with vehicle, the level of Dmt1 mRNA expression in the duodenum was 3.9-fold higher in iron-deficient than in iron-sufficient rats (Fig. 3AI, P < 0.01), whereas the relative levels of ferritin (Fig. 3B) and Fpn1 (Fig. 3C) mRNAs in iron-deficient rats were 0.66 and 0.92, respectively, compared with iron-sufficient rats (not significant). Under these conditions, Dmt1 with iron-responsive element (IRE) and non-IRE were both upregulated in iron-deficient rats, with a greater change in Dmt1 with IRE (Fig. 3AII). As expected, a luminal iron load of 100 μg or 1,000 μg did not significantly alter the levels of expression of any of these genes in either group, relative to vehicle-treated rats.
Iron and ferritin contents in the duodenum and liver.
After loading with vehicle, the mean duodenal iron concentrations in the iron-sufficient and iron-deficient groups were 15.0 ± 2.6 and 7.0 ± 1.2 μg/100 mg dry tissue, respectively, consistent with the results of experiment 1 (Fig. 4). Following an iron load of 1,000 μg, iron accumulation tended to be lower in the iron-deficient than in the iron-sufficient group, although the difference was not significant. Duodenal ferritin, hepatic iron, and ferritin contents were significantly higher in iron-sufficient than in iron-deficient rats.
How does the short-acting mucosal block differ from the regular mucosal block?
Mucosal block is a phenomenon in which iron absorption is suppressed for several days after a large iron load, which was first confirmed in dogs in 1943 (11). Duodenal uptake and transfer of 59Fe after an oral load of 10 mg of nonradioactive iron was found reduced at 3 h and persisted for 12 h (8). Furthermore, DMT1 protein expression was decreased and FT protein increased, suggesting that mucosal block is characterized by suppressed iron uptake coupled with abnormal DMT1 gene and protein expression. This phenomenon has been attributed to excess intracellular iron levels caused by elevated iron uptake capacity resulting from the upregulation of DMT1 expression in the iron-deficient state. Hepatic hepcidin synthesis was enhanced by increased iron storage in the liver, causing FPN degradation on the basolateral surface of epithelial cells (16, 21, 28, 31).
The results presented here showed that the short-acting mucosal block, with no downregulation of mRNA, occurred prior to the induction of the regular mucosal block. The short-acting mucosal block could be induced by much a lower amount of iron (1,000 μg) and much faster (15 min) than previously reported. This transient suppression of iron uptake in response to high iron load was specific to iron-deficient rats, with no inhibition of iron absorption into the portal blood observed. Iron absorption must be enhanced in iron-deficient rats, because the increments in portal and arterial serum iron concentrations were higher in the iron-deficient group at all iron loads. However, mucosal iron accumulation tended to be lower in the iron-deficient than in the iron-sufficient group, particularly at excessive iron load, as a result of transient suppression of iron uptake and increased iron transport to portal blood.
Activating iron load and time to induce the short-acting mucosal block.
In iron-deficient rats, 70% of iron was taken up 1 h after loading with low iron loads, but decreased by 25% to 50% at higher, excessive loads, while being approximately 63% in iron-sufficient rats, regardless of iron load. The iron content in the duodenum was also lower in iron-deficient than in iron-sufficient rats 1 h after loading. Overall 59Fe absorption by jejunal segments from iron-deficient rats was found to be 2.0- to 4.6-fold higher in segments from iron-sufficient rats at luminal concentrations ranging from 1 to 100 μmol/l, much lower than the 0.5–35 mmol/l concentrations in our study (18). We found that duodenal iron uptake was suppressed at iron loads exceeding 500 μg. Because iron uptake was higher in iron-deficient rats than in iron-sufficient rats at iron loads <200 μg, the luminal iron concentration must be monitored in the duodenum, with suppression of iron uptake when iron concentrations exceed a threshold level. In ligated duodenum, the suppression of iron uptake was detected at iron concentrations between 200 and 500 μg/ml. Because the daily intake of iron by rats used in these experiments was approximately 500 μg, not only medical iron preparations but also dietary iron supplements may suppress iron uptake. Generally, a supplemental daily intake of one- to twofold amounts of dietary iron at one time is regarded as not harmful to the small intestine, but lower iron loads may be a burden on the small intestinal mucosa.
We also found that the reduction in iron uptake after loading with 1,000 μg iron was stronger at 15 min than at 30 min, suggesting that this suppressive response occurred very quickly, before changes in gene and protein expression. This is an important difference between the regular and the short-acting mucosal block. As we previously reported (1), significant decreases in Dmt1 mRNA expression were first observed 2 h after the administration of an excess iron load. As the blood vessels and biliary tract are preserved in this experimental model, the pH of the duodenal contents rose from pH 1 to pH 6–7 immediately after administration of iron in 10 mM HCl to the ligated duodenum. Therefore, iron uptake and iron transfer in our model reflect the actual environment of the small intestine.
Although the mechanism responsible for this suppression is not clear, it is important that suppression by marginally excessive iron was induced only in iron-deficient, not in iron-sufficient, rats. The short-acting mucosal block may protect mucosal cells from oxidative stress caused by excess iron, because FT, which chelates iron, has antioxidative properties, and its level of expression was extremely low in enterocytes of iron-deficient rats.
Levels of expression of genes and proteins are not changed by iron loading in the short-acting mucosal block.
The degradation of duodenal DMT1 protein was reported to require >1 h after oral administration of 10 mg iron (8). The findings reported here are unlikely to be due to the degradation of DMT1 protein because we observed suppression of iron uptake as early as 15 min after loading with 1,000 μg iron. Although iron uptake was strongly suppressed within 15 min after loading, the level of suppression declined over time, resulting in comparable rates of iron uptake by iron-sufficient and iron-deficient rats 30 min after loading. These findings suggest that the short-acting mucosal block involves a suppressive response mediated by DMT1 protein (e.g., internalization) that persists until the regular mucosal block (i.e., mucosal block accompanied by decreases in DMT1 gene and protein expression). The IRE in Dmt1 mRNA (5, 7, 27) may be involved in this short-acting suppression, because the level of Dmt1-IRE mRNA is upregulated 18.5-fold in the duodenum of iron-deficient rats. The IRE in Dmt1 mRNA has been reported induced by dietary iron deprivation (5). DMT1 proteins with distinct COOH-termini (15) have been reported translated from distinct mRNAs by alternative splicing of 3′ exons encoding different 3′ untranslated regions. During the transferrin cycle, DMT1 not containing IRE is recycled with transferrin receptor between the plasma membrane and endosome, whereas DMT1 containing IRE is not (26). However, this mechanism has not been confirmed in duodenal enterocytes. Iron has been reported to induce apical-basal movement of DMT1 in Caco-2 cell monolayers (20) and in rat intestines (20, 22, 30). Cellular internalization of DMT1 in the duodenum of Belgrade and Sprague-Dawley rats was observed 1–2.5 h after dietary iron intake and within 30 min in Caco-2 cells. As predicted by the membrane distribution of DMT1, high extracellular iron levels caused a robust decrease in apical iron uptake, suggesting that mucosal block may occur earlier in iron-deficient rats than previously thought. Therefore, the suppression of iron uptake 15–30 min after loading is likely due to a change in function of DMT1 protein, possibly due to the internalization of DMT1 in an apical-basal manner (19, 20). Possible differences in DMT1 localization, especially focused on DMT1 with and without IRE, should be assessed immunohistochemically, to determine whether DMT1 internalization is accompanied by transient suppression of iron uptake in vivo. The transport of iron to the portal blood after iron load was consistently higher in iron-deficient than in iron-sufficient rats, regardless of the iron load. Transient suppression of iron uptake was observed only at the apical surface (i.e., transport from the lumen into the mucosal cells), not at the basolateral surface (i.e., from the mucosal cells to the portal blood). Serum hepcidin levels in rats were observed to increase within 1 h after loading excess iron through a nasogastric tube, resulting in acute intoxication levels of 20 or 40 mg/100 g body wt, and were found to remain elevated for an additional 6 h (3). Therefore, the transient mucosal block would not involve FPN1 degradation by upregulated serum hepcidin.
Iron regulation of DMT1 has been reported to involve the tissue-specific expression of an upstream 5′ exon (exon 1A), with higher expression in the duodenum and kidneys than in other tissues (12). This exon 1A contains a conserved sequence of 29–31 amino acids, which may be required to induce the transient mucosal block. The cytoplasmic tail domain of IRE-less DMT1 was also found to determine the isoform-specific localization of DMT1 (26). Clearly, additional studies are needed to assess the roles of these sequences in the function of duodenal DMT1.
The luminal iron concentration must be monitored in the duodenum if iron concentrations exceed a threshold level, to induce the short-acting mucosal block. Thus our finding that the short-acting mucosal block was induced only in iron-deficient, not in iron-sufficient, rats was likely important. Dmt1-IRE mRNA is induced by iron deficiency, with the level increased 18.5-fold in iron-deficient duodenum. Moreover, DMT1 proteins with distinct COOH-termini containing IRE (15) have been reported. The iron-regulatory protein (IRP) is also expected to play key role in the mucosal block, since IRP deficiency impairs iron absorption and promotes mucosal iron retention via a ferritin-mediated mucosal block (9). Thus experiments are currently underway to quantitatively determine whether DMT1-IRE protein undergoes in internalization or functional changes in vivo.
This work was supported by a Grant-in-Aid (no. 22650182) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).
No conflicts of interest, financial or otherwise, are declared by the author(s).
S.S., S.Y., E.N., and A.A. conception and design of research; S.S., S.Y., E.N., K.T., and A.A. performed experiments; S.S., S.Y., E.N., and A.A. analyzed data; S.S. interpreted results of experiments; S.S. prepared figures; S.S. and A.A. drafted manuscript; S.S. edited and revised manuscript; S.S. approved final version of manuscript.
Present address for E. Nozaki: Department of Laboratory Medicine, Kyorin University, 6-20-2 Shinkawa Mitaka, Tokyo 181-8611, Japan.
Present address for K. Tadai: Department of Nutrition, Shubun University, 6 Nikko-cho, Ichinomiya, Nagoya 491-0938, Japan.
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