Mechanisms Regulating Iron Absorption over a Wide Range of Dietary Iron Content in the Mouse.

Abstract

Dietary iron absorption by enterocytes is mediated by a ferrous transporter (DMT1) and possibly a ferric reductase (Cbyrd1). The role of Cbyrd1 is uncertain, as a knockout mouse has no defect in absorption (Gunshin et al. Blood, 2005, June 16, Epub). Transfer of iron to plasma is mediated by ferroportin (FPN). FPN's residence on the basolateral membrane is regulated by hepcidin (Nemeth et al. Science 2004, 306:2090–3). Iron absorption responds to erythropoiesis, hypoxia and iron stores. A dietary iron content of 120 mg/kg consumed will maintain hepatic iron stores near that of mice found in the wild. Mice will grow and breed given diets containing 35 mg/kg. Commercial mouse chow iron content ranges from 200–350 mg/kg. We studied the effects of diets containing 2, 35, 120, 350 and 2000 mg iron/kg on iron absorption, liver and spleen iron content and transcriptional levels of DMT1, FPN and hepcidin in A/J mice. Mice were weaned at 3 weeks of age and groups of 8 animals were placed on one of the 5 diets for 4 weeks. No differences between groups were noted in hematocrit, hemoglobin and MCV. Mean hepatic iron content was 52.7 ±3.7 ug/g (wet wt) in mice fed the 2 mg/kg diet. Mean hepatic iron content was 560 ±23.7 ug/g in mice fed the 2000 mg/kg diet. There was no difference in hepatic iron content in mice fed intermediate iron diets (35–350 mg/kg). Mean hepatic iron concentration in these groups was 110 ±3 ug/g. Mean spleen iron content was 132 ±12.2 ug/g (wet wt) in mice maintained on 2 mg/kg. Mean spleen iron content was 598 ±49 ug/g in mice fed the 2000 mg/kg diet. There was no difference in spleen iron content in mice maintained on intermediate iron diets (mean 359 ±10 ug/g). These data indicate that mice maintain constant levels of hepatic and splenic iron over a ten-fold range in dietary iron content. Iron absorption was measured as percent of a measured dose of 59Fe (5 ug total) remaining in the carcass (minus the GI tract) 24 h after administration by gavage. Absorption was inversely proportional to dietary iron content. Mean absorption was 86% ±4 in the group on the 2 mg/kg diet, 42% ±3 on the 35 mg/kg diet, 26% ±7 on the 120 mg/kg diet, 19% ±4 on the 350 mg/kg diet and 6% ±1 on the 2000 mg/kg diet. Transcript levels of hepcidin, DMT1 and FPN were measured by real-time PCR and normalized to beta actin mRNA. Liver hepcidin expression was 20-fold greater in mice on the 2000 mg/kg diet than in mice on the 2 mg/kg diet (3900 ±1021 copies/actin copy vs. 198 ±47). Hepcidin expression did not differ in mice on intermediate diets (745 ±147 copies). Enterocytes were isolated from everted gut explants by elution in EDTA. Transcript levels of enterocyte DMT1 and FPN were 4566 ±SEM and 236 ±SEM copies respectively in mice on the 2 mg/kg diet. No detectable transcripts were found in mice on the 2000 mg/kg diet. Enterocyte transcript levels for DMT1 and FPN were no different in groups on intermediate iron diets (17 ±2 copies and 20 ±9 copies respectively). These data indicate that tissue iron content, hepcidin, DMT1 and FPN remain constant over a ten-fold range in dietary iron and only vary at extremes, while iron absorption is inversely proportional to dietary iron. The data also suggest that dietary iron, within defined limits, regulates iron absorption by a mechanism intrinsic to the enterocyte.