Tissue Nonheme Iron Research Articles

Establishing Dietary Iron Requirements for Mouse and Rat Models of Hereditary Hemochromatosis

The established iron requirement for laboratory rodents is 35‐50 ppm, but standard rodent chows contain up to 350 ppm iron. Excess dietary iron exacerbates iron accumulation in hepcidin (Hamp) knockout (KO) mice and rats, which model the iron‐overload disorder hereditary hemochromatosis (HH) in humans. Establishing iron requirements is important to allow for proper experimental design when studying Hamp KOs since WT littermates are not iron‐loaded (and thus not an appropriate control group for iron‐loaded Hamp KOs). We hypothesized that Hamp KOs have iron requirements at least 50% below those of WT animals, given that intestinal iron absorption is inappropriately elevated (by 2‐3‐fold) in the KOs. In the present study, WT and Hamp KO mice and rats of both sexes were weaned onto one of four AIN‐93G purified rodent diets, with the following iron concentrations (in ppm): 5‐7 (low), 17‐20 (low marginal), 24‐27 (high marginal), or 45‐49 (adequate). After 6 weeks on the diets, bioindicators of iron status were assessed, including blood hemoglobin content and hematocrit, serum ferritin and nonheme iron content, tissue nonheme iron concentration, and blood transferrin saturation. Outcomes showed that significant iron loading occurred in Hamp KO animals when fed the high‐marginal and adequate iron diets, as indicated by higher serum ferritin and nonheme iron content, elevated hepatic iron levels and increased TSAT. Iron loading, however, did not occur when the KO rats and mice were fed the low‐marginal iron diets. Animals fed the low‐iron diet, however, developed IDA. We thus conclude that the optimal amount of dietary iron that will not result in iron loading post‐weaning in Hamp KOs, while also not causing iron depletion, is ~22 ppm. Consistent with our hypothesis then, Hamp KO rodents require about half as much iron as WT controls.

Dietary Iron Increases Expression of Liver Hepcidin Relative to BMP6 By a Mechanism Involving Transferrin Receptor 2 and Specificity of Transferrin Lobe Iron Occupancy

Introduction: Prior studies have identified two iron signals regulating liver hepcidin expression (PMID 21488083, 21480335). One is mediated by liver sinusoidal BMP6 (and BMP2) and is reflective of liver iron concentration (LIC). The other is independent of BMP expression and reflective of transferrin saturation. The mechanism by which TF regulates hepcidin is unclear but appears to involve hepatocellular transferrin receptor 2 (TFR2). We previously generated mice with TF mutations that block iron binding to either N-lobe (Tf N-bl) or C-lobe (Tf C-bl). These mice demonstrated differences in Epo sensitivity and hepcidin regulation (PMID 31434707). To characterize the differential regulation of Hamp1 in these mice we analyzed the effect of dietary iron in juvenile mice. This model advantages a physiologic low iron state with very low basal Hamp1 and Bmp6 expression.Methods: Twelve-day old WT, Tf N-bl, Tf C-bland Tfr2 Y245X (functional knockout) mice were gavaged with 4 mg/kg/d FeSO 4 (or saline) in 2 divided doses for 3 days and sacrificed on the fourth day. The primary outcome was liver Hamp1 relative to Bmp6 expression by RT-PCR. Other iron-related parameters included liver Bmp2, serum and tissue non-heme iron, and TIBC. Erythroid parameters included splenic Fam132b (Erfe gene) expression, kidney Epo, blood packed cell volume (PCV), splenic weight index (SWI).Results: Iron-treated mice demonstrated increased serum iron, TF saturation, LIC, liver Bmp6 in all strains compared to untreated. Liver Hamp1 was increased in all strains with iron-treatment; however, the magnitude was least in the Tf N-bland greatest in the Tf C-bl mice. Splenic Fam132b was unchanged. The iron-treated Tf C-bl had ~16 fold greater Hamp1 compared with iron-treated Tf N-bl mice (P=0.0005), despite similar Bmp6, Bmp2, and splenic Fam132b expression. The iron-treated Tf C-bl mice also had modestly higher splenic index and PCV compared with iron-treated Tf N-bl mice, despite similar kidney Epo. In Tfr2 Y245X mice Hamp1 and Hamp1 indexed to Bmp6 expression was intermediate between Tf N-bland Tf C-bl mice (figure).Conclusion: Iron treatment increased liver Bmp6 expression as well as Bmp6 sensitivity (Hamp:Bmp6), in association with increases in LIC and in TF saturation. The effect of iron on Bmp6 sensitivity is markedly influenced by which TF lobe iron occupies, as well as the presence of TFR2. These observations support a model in which TF modulates the hepcidin response to BMP6 in either direction, via interaction with TFR2. We speculate that iron occupancy of the N-lobe of transferrin confers an upregulatory signal to hepcidin expression in the hepatocyte, and that its absence confers a downregulatory signal to BMP6 sensitivity in the hepatocyte. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Study of liver fat and iron deposition quantification based on magnetic resonance imaging in rats with nonalcoholic fatty liver disease

Objective: To investigate the accuracy of magnetic resonance imaging (MRI) for quantitative determination of liver fat and iron content through a rat model of non-alcoholic fatty liver disease (NAFLD) induced by methionine-choline deficient (MCD) diet. Methods: Sixty SD rats were randomly divided into experimental (MCD-diet group, n = 30) and normal control group (normal diet, n = 30). Rats were subjected to special MRI examinations at the ends of 2, 4, and 8 weeks. Proton density fat fraction (PDFF) and R2* value were obtained, and then the rats were sacrificed. The liver tissues were stained with HE, Prussian blue, etc. Liver tissue non-heme iron (NHI) homogenate was determined by flame atomic absorption spectrometry. According to different data, one-way analysis of variance, t-test or χ (2) test was used for statistical analysis. Results: PDFF and R2 * values in the MCD diet group at 2, 4 and 8 weeks were 23.37% ± 9.20%, 28.07% ± 6.84%, 25.40% ± 7.04% (P < 0.01) and 90.58 ± 15.92, 104.12 ± 13.47, 106.35 ± 15.76 (P < 0.05), respectively, which were significantly higher than the normal control group PDFF (2.39% ± 0.50%, 2.45% ± 0.45%, 3.26% ± 0.80%) and R2* (48.93 ± 7.90, 54.71 ± 5.91, 64.25 ± 15.76). Additionally, with the disease progression, R2 * had gradually increased, which was consistent with the NHI trend in liver tissue homogenates of each group. Conclusion: MRI, as a non-invasive quantitative method, can accurately assess liver fat and iron content in fatty liver disease, and with the degree of severity of fat changes, iron deposits tend to increase.

Iron and zinc homeostases in female rats with physically active and sedentary lifestyles.

To determine the effects of repeated physical activity on iron and zinc homeostases in a living system, we quantified blood and tissue levels of these two metals in sedentary and physically active Long-Evans rats. At post-natal day (PND) 22, female rats were assigned to either a sedentary or an active treatment group (n = 10/group). The physically active rats increased their use of a commercially-constructed stainless steel wire wheel so that, by the end of the study (PND 101), they were running an average of 512.8 ± 31.9 (mean ± standard error) min/night. After euthanization, plasma and aliquots of liver, lung, heart, and gastrocnemius muscle were obtained. Following digestion, non-heme iron and zinc concentrations in plasma and tissues were measured using inductively coupled plasma optical emission spectroscopy. Concentrations of both non-heme iron and zinc in plasma and liver were significantly decreased among the physically active rats relative to the sedentary animals. In the lung, both metals were increased in concentration among the physically active animals but the change in zinc did not reach significance. Similarly, tissue non-heme iron and zinc levels were both increased in heart and muscle from the physically active group. It is concluded that repeated physical activity in an animal model can be associated with a translocation of both iron and zinc from sites of storage (e.g. liver) to tissues with increased metabolism (e.g. the lung, heart, and skeletal muscle).

Supplementation of Fructooligosaccharide Mildly Improves the Iron Status of Anemic Rats Fed a Low-Iron Diet

Also known as a prebiotic, fructooligosaccharide (FOS) resists digestion by gastric acid and pancreatic enzymes in vivo, but is preferentially fermented by beneficial intestinal bacteria once it reaches the colon. While some studies suggest that FOS and its fermentation products may influence the iron absorption process, the effects of prolonged FOS supplementation on iron status remain unclear. The objective of this study was therefore to determine the enhancing effects of FOS supplementation on the iron status of anemic rats. Male Sprague-Dawley rats receiving a low-iron diet (12 μg/g) for 14 days showed significantly lower hemoglobin concentration, as well as lower tissue non-heme iron levels than rats receiving a regular diet (45 μg/g), confirming iron-deficiency anemia. On the first day of the feeding trial, two groups of anemic rats (n = 6) were fed the same low-iron diet with or without FOS supplementation, while two other groups of anemic rats were switched to the regular diet with or without FOS supplementation to allow recovery. FOS was provided to the rats by dissolving in water at 5% (w/v). Anemic rats fed the low-iron diet showed a mild increase (p < 0.05) in hemoglobin level after 21 days of FOS supplementation when compared to rats without FOS. For anemic rats switched to the regular diet, hemoglobin level returned to normal after 14 days and FOS supplementation showed no additional effects. Our results suggest that FOS supplementation has a mild enhancing effect on the iron status of anemic subjects on a low-iron diet.

Effects of Testosterone on Erythropoiesis in a Female Mouse Model of Anemia of Inflammation.

The anemia of inflammation is a common problem in inflammatory and autoimmune diseases. We characterized a mouse model of anemia of chronic inflammation induced by repeated injections of low doses of heat-killed Brucella abortus (HKBA), and determined the effects of T administration on erythropoiesis in this model. Female C57BL/6NCrl mice were injected weekly with HKBA for 10 wk. Weekly injections of T or vehicle oil were started 4 wk later. Control mice were injected with saline and vehicle oil in parallel. HKBA-injected mice had significantly lower hemoglobin, hematocrit, mean corpuscular volume, reticulocyte hemoglobin, transferrin saturation (TSAT), and tissue nonheme iron in liver and spleen, enlarged spleen, and up-regulated hepatic expression of inflammatory markers, serum amyloid A1, and TNFα, but down-regulated IL-6, bone morphogenic protein 6, and hepcidin compared with saline controls. HKBA also reduced serum hepcidin and increased serum erythropoietin. Bone marrow erythroid precursors were substantially reduced in HKBA-injected mice. Cotreatment with T increased the percentage of late-stage erythroid precursors in the bone marrow relative to HKBA-injected and saline controls and reversed HKBA-induced suppression of hemoglobin and hematocrit. T also normalized serum erythropoietin, TSAT, and reticulocyte hemoglobin without correcting the expression of the hepatic inflammation markers. Conclusions are that low-dose HKBA induces moderate anemia characterized by chronic inflammation, decreased iron stores, and suppression of erythroid precursors in the bone marrow. T administration reverses HKBA-induced anemia by stimulating erythropoiesis, which is associated with a shift toward accelerated maturation of erythroid precursors in the bone marrow.

Age-dependent expression of duodenal cytochrome b, divalent metal transporter 1, ferroportin 1, and hephaestin in the duodenum of rats.

The body's requirement for iron is different at different developmental stages. However, the molecular mechanisms of age-dependent iron metabolism are poorly understood. In the present study, we investigated the expression of iron transport proteins in the duodenum of Sprague-Dawley rats at five different age stages. Male Sprague-Dawley rats at postnatal week (PNW) 1, 3, 12, 44, and 88 were employed in the study. Serum iron status and tissue non-heme iron concentrations in the spleen, liver, bone marrow, heart, kidney, duodenal epithelium, and gastrocnemius were examined at each age stage. The expression of duodenal cytochrome b (DcytB), divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), hephaestin, and hepcidin were measured by real-time polymerase chain reaction or Western blot. The levels of serum iron and transferrin saturation were higher in the rats at PNW1 and 3 than in those at PNW12, 44, and 88. Non-heme iron contents decreased from PNW1 to PNW3 and then increased thereafter. Duodenal DcytB, DMT1, and FPN1 increased to the highest level at PNW3 and then decreased from PNW12 to 88. The hepatic hepcidin mRNA level decreased to the lowest level at PNW3 and then increased with age. Our findings showed that age had a significant effect on body iron status. The increased duodenal DcytB, DMT1, and FPN1 expression can enhance intestinal iron absorption to meet the high iron requirements in infants. Hepcidin or enterocyte iron levels may be involved in the regulation of age-dependent FPN1, DMT1, and DcytB expression in the duodenum.

Sex Differences in Iron Status and Hepcidin Expression in Rats

Studies have shown that men and women exhibit significant differences regarding iron status. However, the effects of sex on iron accumulation and distribution are not well established. In this study, female and male Sprague-Dawley rats were killed at 4 months of age. Blood samples were analyzed to determine the red blood cell (RBC) count, hemoglobin (Hb) concentration, hematocrit (Hct), and mean red blood cell volume (MCV). The serum samples were analyzed to determine the concentrations of serum iron (SI), transferrin saturation (TS), ferritin, soluble transferrin receptor (sTfR), and erythropoietin (EPO). The tissue nonheme iron concentrations were measured in the liver, spleen, bone marrow, kidney, heart, gastrocnemius, duodenal epithelium, lung, pallium, cerebellum, hippocampus, and striatum. Hepatic hepcidin expression was detected by real-time PCR analysis. The synthesis of ferroportin 1 (FPN1) in the liver, spleen, kidney, and bone marrow was determined by Western blot analysis. The synthesis of duodenal cytochrome B561 (DcytB), divalent metal transporter 1 (DMT1), FPN1, hephaestin (HP) in the duodenal epithelium was also measured by Western blot analysis. The results showed that the RBC, Hb, and Hct in male rats were higher than those in female rats. The SI and plasma TS levels were lower in male rats than in female rats. The levels of serum ferritin and sTfR were higher in male rats than in female rats. The EPO levels in male rats were lower than that in female rats. The nonheme iron contents in the liver, spleen, bone marrow, and kidney in male rats were also lower (56.7, 73.2, 60.6, and 61.4 % of female rats, respectively). Nonheme iron concentrations in the heart, gastrocnemius, duodenal epithelium, lung, and brain were similar in rats of both sexes. A moderate decrease in hepatic hepcidin mRNA content was also observed in male rats (to 56.0 % of female rats). The levels of FPN1 protein in the liver, spleen, and kidney were higher in male rats than in female rats. There was no significant change in FPN1 expression in bone marrow. Significant difference was also not found in DcytB, DMT1, FPN1, and HP protein levels in the duodenal epithelium between male and female rats. These data suggest that iron is distributed differently in male and female rats. This difference in iron distribution may be associated with the difference in the hepcidin level.

Duodenal Reductase Activity and Spleen Iron Stores Are Reduced and Erythropoiesis Is Abnormal in Dcytb Knockout Mice Exposed to Hypoxic Conditions1–3

Duodenal cytochrome b (Dcytb, Cybrd1) is a ferric reductase localized in the duodenum that is highly upregulated in circumstances of increased iron absorption. To address the contribution of Dcytb to total duodenal ferric reductase activity as well as its wider role in iron metabolism, we first measured duodenal ferric reductase activity in wild-type (WT) and Dcytb knockout (Dcytb−/−) mice under 3 conditions known to induce gut ferric reductase: dietary iron deficiency, hypoxia, and pregnancy. Dcytb−/− and WT mice were randomly assigned to control (iron deficiency experiment, 48 mg/kg dietary iron; hypoxia experiment, normal atmospheric pressure; pregnancy experiment, nonpregnant animals) or treatment (iron deficiency experiment, 2–3 mg/kg dietary iron; hypoxia experiment, 53.3 kPa pressure; pregnancy experiment, d 20 of pregnancy) groups and duodenal reductase activity measured. We found no induction of ferric reductase activity in Dcytb−/− mice under any of these conditions, indicating there are no other inducible ferric reductases present in the duodenum. To test whether Dcytb was required for iron absorption in conditions with increased erythropoietic demand, we also measured tissue nonheme iron levels and hematological indices in WT and Dcytb−/− mice exposed to hypoxia. There was no evidence of gross alterations in iron absorption, hemoglobin, or total liver nonheme iron in Dcytb−/− mice exposed to hypoxia compared with WT mice. However, spleen nonheme iron was significantly less (6.7 ± 1.0 vs. 12.7 ± 0.9 nmol · mg tissue-1; P < 0.01, n = 7–8) in hypoxic Dcytb−/− compared with hypoxic WT mice and there was evidence of impaired reticulocyte hemoglobinization with a lower reticulocyte mean corpuscular hemoglobin (276 ± 1 vs. 283 ± 2 g · L-1; P < 0.05, n = 7–8) in normoxic Dcytb−/− compared with normoxic WT mice. We therefore conclude that DCYTB is the primary iron-regulated duodenal ferric reductase in the gut and that Dcytb is necessary for optimal iron metabolism.

Iron metabolism in hepcidin1 knockout mice in response to phenylhydrazine-induced hemolysis

Hepcidin, an iron regulatory peptide, plays a central role in the maintenance of systemic iron homeostasis by inducing the internalization and degradation of the iron exporter, ferroportin. Hepcidin expression in the liver is regulated in response to several stimuli including iron status, erythropoietic activity, hypoxia and inflammation. Hepcidin expression has been shown to be reduced in phenylhydrazine-treated mice, a mouse model of acute hemolysis. In this mouse model, hepcidin suppression was associated with increased expression of molecules involved in iron transport and recycling. The present study aims to explore whether the response to phenylhydrazine treatment is affected by hepcidin deficiency and/or the subsequently altered iron metabolism. Hepcidin1 knockout (Hamp(-/-)) and wild type mice were treated with phenylhydrazine or saline and parameters of iron homeostasis were determined 3 days after the treatment. In wild type mice, phenylhydrazine administration resulted in significantly reduced serum iron, increased tissue non-heme iron levels and suppressed hepcidin expression. The treatment was also associated with increases in membrane ferroportin protein levels and spleen heme oxygenase 1 mRNA expression. In addition, trends toward increased mRNA expression of duodenal iron transporters were also observed. In contrast, serum iron and tissue non-heme iron levels in Hamp(-/-) mice were unaffected by the treatment. Moreover, the effects of phenylhydrazine on the expression of ferroportin and duodenal iron transporters were not observed in Hamp(-/-) mice. Interestingly, mRNA levels of molecules involved in splenic heme uptake and degradation were significantly induced by Hamp disruption. In summary, our study demonstrates that the response to phenylhydrazine-induced hemolysis differs between wild type and Hamp(-/-) mice. This observation may be caused by the absence of hepcidin per se or the altered iron homeostasis induced by the lack of hepcidin in these mice.

Role of Iron in Brain Injury After Intraventricular Hemorrhage

Intraventricular extension of hemorrhage is a predictor of poor outcome in intracerebral hemorrhage, and iron overload contributes to brain injury after intracerebral hemorrhage. The current study investigated the role of iron in ventricular dilatation and neuronal death in a rat model of intraventricular hemorrhage (IVH). There were 2 parts in this study. First, male Sprague-Dawley rats had a 200-μL injection of either autologous blood or saline into the right lateral ventricle and were euthanized at different time points. Rats had MRI and brains were used for Western blot analysis, immunohistochemistry, histology, and brain tissue nonheme iron measurements. Second, rats had IVH and were treated with deferoxamine or vehicle, and rats were euthanized 4 weeks later for brain tissue loss and lateral ventricle size measurements. IVH resulted in brain iron accumulation, bilateral enlargement of the lateral ventricles, and hippocampal brain tissue loss. Iron accumulation was associated with upregulation of heme oxygenase-1 and ferritin. Systemic deferoxamine treatment reduced IVH-induced ventricular enlargement (eg, day 28: 32.7±10.6 vs 43.8±9.7 mm(3) in vehicle-treated group, n=8 to 9; P<0.05) and hippocampal brain tissue loss (hippocampal volume: 89.0±2.7 vs 85.2±4.1 mm(3) in the vehicle-treated group; P<0.05). Iron has a role in brain injury after IVH. Deferoxamine may be a therapy for patients with IVH or intraventricular extension after intracerebral hemorrhage.

Tmprss6, An Inhibitor of Hepatic Bmp/Smad Signaling, Is Required for Hepcidin Suppression and Iron Loading In a Mouse Model of β-Thalassemia

AbstractAbstract 164TMPRSS6, a transmembrane protease produced by the liver, is an essential regulator of mammalian iron homeostasis. TMPRSS6 inhibits the expression of hepcidin, a circulating peptide that decreases intestinal iron absorption and macrophage iron release, by down-regulating hepatic BMP/SMAD signaling for hepcidin production. Accordingly, TMPRSS6 mutations result in elevated hepcidin levels, impaired absorption of dietary iron, and systemic iron deficiency. Interestingly, in congenital iron loading anemias such as β-thalassemia, hepcidin levels are inappropriately low relative to body iron stores, a finding that has been postulated to result from the production of a hepcidin-repressing factor in the setting of ineffective erythropoiesis. Here we asked if Tmprss6 is required to achieve the hepcidin suppression present in Hbbth3/+ mice, a model of β-thalassemia intermedia. To test this, we bred Hbbth3/+ mice to mice harboring a targeted disruption of the Tmprss6 serine protease domain. We generated mice of various Hbb-Tmprss6 genotype combinations and compared parameters of systemic iron homeostasis at 8 weeks of age. Consistent with prior studies of Hbbth3/+ mice, Hbbth3/+ mice harboring 2 wild-type Tmprss6 alleles (Hbbth3/+Tmprss6+/+ mice) showed non-heme iron concentrations of liver, spleen, and kidney that were significantly elevated compared to wild-type controls. Homozygosity for Tmprss6 mutation, however, ameliorated the iron overload phenotype of Hbbth3/+ mice and led to systemic iron deficiency. Tissue non-heme iron concentrations were markedly reduced in Hbbth3/+Tmprss6−/− mice as compared to Hbbth3/+Tmprss6+/+ mice and were similar to levels observed in Tmprss6−/− mice harboring 2 wild-type Hbb alleles. Hbbth3/+Tmprss6−/− mice had hemoglobin levels similar to the thalassemic levels present in Hbbth3/+Tmprss6+/+ mice. However, compared to Hbbth3/+Tmprss6+/+ mice, Hbbth3/+Tmprss6−/− mice showed markedly reduced erythrocyte mean corpuscular volume and serum transferrin saturation, as well as increased red blood cell count. Interestingly, homozygous loss of Tmprss6 in Hbbth3/+ mice also led to marked reduction in splenomegaly and improvement in peripheral red blood cell morphology. Consistent with prior studies of Hbbth3/+ mice, Hbbth3/+Tmprss6+/+ mice displayed hepatic hepcidin mRNA levels that were similar to wild-type and were, therefore, inappropriately decreased relative to their increased hepatic iron stores. Hepatic mRNA levels of Bmp6, encoding a Bmp ligand that is transcriptionally regulated by iron and acts as a key regulator of hepcidin expression in vivo, were significantly elevated in Hbbth3/+Tmprss6+/+ mice, suggesting that their relative hepcidin deficiency does not result from impaired Bmp6 transcription. While Hbbth3/+Tmprss6+/+ mice showed suppressed hepcidin levels, homozygous loss of Tmprss6 alleviated their hepcidin suppression and led to elevated hepcidin mRNA levels similar to Tmprss6−/− mice harboring 2 wild-type Hbb alleles. Hbbth3/+Tmprss6−/− mice also showed elevated hepatic mRNA encoding Id1, another transcriptional target of Bmp/Smad signaling. These findings indicate that Tmprss6 is required to achieve the suppression of hepatic hepcidin expression that underlies systemic iron overload in Hbbth3/+ mice. Furthermore, our results demonstrate that, by up-regulating hepatic Bmp/Smad signaling for hepcidin production, genetic loss of Tmprss6 induces profound changes in systemic iron homeostasis in this thalassemia model. Disclosures:No relevant conflicts of interest to declare.

Hemochromatosis and pregnancy: iron stores in the Hfe−/− mouse are not reduced by multiple pregnancies

Hereditary hemochromatosis (HH), a widespread hereditary iron metabolism disorder, is characterized by an excessive absorption of dietary iron, resulting in increased body iron stores. Some studies indicate a sex difference in disease expression, with women showing a slower disease progression and a less severe clinical profile. This is usually attributed to iron loss during menstruation and pregnancy. However, this link has not been clearly demonstrated. The Hfe-/- mouse model recapitulates key aspects of HH, including an iron overload phenotype similar to that observed in human patients. In this study, we use it to test the impact of multiple pregnancies in the iron stores. One-year-old nulliparous and pluriparous (averaging 29 weaned pups per female) C57BL/6 (B6) and Hfe-/- mice were euthanized, and blood and tissues were collected. Several serological and erythroid parameters were evaluated, as well as tissue nonheme iron content and serum ferritin. Hepcidin 1, hepcidin 2, and bone morphogenetic protein 6 (BMP6) expressions in the liver were determined by real-time PCR. No significant differences were observed for many serological and erythroid parameters although differences occurred in transferrin saturation and mean corpuscular volume in Hfe-/- mice and total iron-binding capacity in B6 mice. Hepatic iron concentration was similar for nulliparous and pluriparous mice of both genotypes, but total iron per organ (liver, spleen, heart, and pancreas) was higher overall in pluriparous females than nulliparous. Hepcidin 1 and 2 and BMP6 expressions were significantly decreased in pluriparous females, when compared with nulliparous, in both genotypes. In conclusion, multiple pregnancies do not reduce body iron stores in Hfe-/- mice.

DMT1 expression is increased in the lungs of hypotransferrinemic mice.

Despite a lack of transferrin, hypotransferrinemic (Hp) mice demonstrate an accumulation of iron in peripheral organs including the lungs. One potential candidate for such transferrin-independent uptake of iron is divalent metal transporter-1 (DMT1), an established iron transporter. We tested the hypothesis that increased concentrations of iron in the lungs of Hp mice are associated with elevations in DMT1 expression. With the use of inductively coupled plasma emission spectroscopy, measurements of nonheme iron confirmed significantly elevated concentrations in the lung tissue of Hp mice relative to the wild-type mice. Western blot analyses for the expression of two isoforms of DMT1 in the Hp mice relative to the wild-type animals demonstrated an elevation for the isoform that lacks an iron-responsive element (IRE) with significant decrements in the expression of +IRE DMT1. With the use of immunohistochemistry, -IRE DMT1 was localized to both airway epithelial cells and alveolar macrophages in wild-type mice. Staining appeared increased in both types of cells in the Hp mice. Elevated concentrations of both tissue nonheme iron and expression of -IRE DMT1 in the Hp mice were associated with increased quantities of -IRE mRNA. There was no difference between wild-type and homozygotic Hp mice in the amount of mRNA for DMT1 +IRE. We conclude that differences between Hp and wild-type mice in nonheme iron concentrations were accompanied by increases in the expression of -IRE DMT1. Increased expression of -IRE DMT1 in the lungs of the Hp mice could be responsible for elevated concentrations of the metal in these tissues.

Effect of different durations of exercise on transferrin-bound iron uptake by rat erythroblast

This study evaluated effects of different durations of exercise on transferrin receptor (TfR) expression on the membrane of rat erythroblasts. Female rats were assigned to six groups: 3, 6 and 12 months of strenuous exercise (swimming 2 h/day, 5 days/wk) groups and their corresponding controls. At the end of experiments, the erythroblasts were isolated for Tf binding assay and transferrin-bound iron (Tf-Fe) uptake. Tissue non-heme iron and hematological iron indices were also measured. The TfR number on the cells was about 603,189 ± 107,562, 890,150 ± 164,849 and 384,695 ± 46,295 molecules/cell in three control groups (3, 6, 12 months) respectively. Exercise groups had significantly higher levels of TfR than those of the control groups, being 1,374,137 ± 243,677, 2,175,360 ± 462,737 and 1,012,759 ± 249,423 molecules/cell in 3, 6 and 12 months of exercise groups respectively (p < 0.05). After 30 min of incubation, cellular Tf approached to levels of 8.28 ± 1.94, 10.73 ± 3.30 and 6.60 ± 0.93 fmole/10 6 cells in 3, 6 and 12 months of exercise groups, while the corresponding control values were 3.09 ± 0.36, 5.03 ± 1.01 and 2.51 ± 0.88 fmole/10 6 cells respectively (all P < 0.05). The rates of cellular iron accumulation were 7.07 (3), 8.79 (6) and 5.96 (12 month) fmole/10 6 cells/min in the exercised rats and 2.91, 3.85, and 2.03 fmole/10 6 cells/min in their corresponding controls (all p < 0.05). However, no significant difference was observed in the ratios (Exercise/Corresponding control) of the increased TfR expression, Tf-Fe uptake and Tf endocytosis as well as of the decreased plasma iron and tissue non-heme iron levels induced by different periods of exercise. Furthermore, the increase in the length of exercise (6 or 12 month) did not induce a remarkable decrease in plasma hemoglobin and hematocrit. These results indicate that a true iron deficiency or ‘sport anemia’ can not develop even if under longer periods (6 or 12 month) of strenuous exercise.

Increased expression of transferrin receptor on membrane of erythroblasts in strenuously exercised rats.

This study investigated the effects of strenuous exercise on transferrin (Tf)-receptor (TfR) expression and Tf-bound iron (Tf-Fe) uptake in erythroblasts of rat bone marrow. Female Sprague-Dawley rats were randomly assigned to either an exercise or sedentary group. Animals in the exercise group swam 2 h/day for 3 mo in a glass swimming basin. Both groups received the same amount of handling. At the end of 3 mo, the bone marrow erythroblasts were freshly isolated for Tf-binding assay and determination of Tf-Fe uptake in vitro. Tissue nonheme iron and hematological iron indexes were measured. The number of Tf-binding sites found in erythroblasts was approximately 674,500 +/- 132,766 and 1,270,011 +/- 235,321 molecules/cell in control and exercised rats, respectively (P < 0. 05). Total Fe and Tf uptake by the cells was also significantly increased in the exercised rats after 30 min of incubation. Rates of cellular Fe accumulation were 5.68 and 2.58 fmol. 10(6) cells(-1). min(-1) in the exercised and control rats, respectively (P < 0.05). Tf recycling time and TfR affinity were not different in exercised and control rats. Increased cellular Fe was mainly located in the stromal fraction, suggesting that most of accumulated Fe was transported to the mitochondria for heme synthesis. The findings demonstrated that the increased cellular Fe uptake in exercised rats was a consequence of the increased TfR expression rather than the changes in TfR affinity and Tf recycling time. The increase in TfR expression and cellular Fe accumulation, as well as the decreased serum Fe concentration and nonheme Fe in the liver and the spleen induced by exercise, probably represented the early signs of Fe deficiency.

Effect of dietary aluminum on tissue nonheme iron and ferritin levels in the chick

Aluminum toxicity is well documented but the mechanism of action is poorly understood. In renal failure patients with aluminum overload, disturbances in iron metabolism leading to anemia are apparent. Few animal models, however, have been used to study the effects of dietary aluminum on iron metabolism. The purpose of this study was to determine if dietary aluminum exposure alters tissue iron and ferritin concentrations in the chick, as has been found in cultured human cells exposed to aluminum. Groups of day-old chicks were fed purified diets containing one of two levels of iron (control or high iron), and one of three levels of aluminum chloride in a 2×3 factorial design. Diets were consumed ad libitum for 1 week, then pair-feeding was initiated for 2 more weeks. A seventh group consumed a low iron diet ad libitum for comparative purposes. After the 3-week feeding period, samples of kidney, liver, and intestinal mucosa were analyzed for nonheme iron and ferritin concentrations by a colorimetric assay and SDS-PAGE, respectively. Results showed that dietary aluminum intake reduced iron stores in liver and intestine, but had no effect on nonheme iron levels in the kidney. Ferritin levels were reduced by aluminum intake in all tissues studied. The decreases in tissue ferritin levels were proportionately more than the decreases in tissue nonheme iron levels. This resulted in increased nonheme iron to ferritin ratios that amounted to as much as 140 and 525% in kidney and intestine, respectively. These findings are consistent with the interpretation that, in the growing chick, dietary aluminum can inhibit iron absorption, disrupt the regulation of tissue ferritin levels by iron, and potentially alter the compartmentalization and protective sequestration of iron within cells.

Accumulation of Iron in the Rat Lung after Intratracheal Instillation of Coal Dust

Abstract Inhalation of coal dust is associated with an accumulation of iron in the human lung. Collagen deposition in the lungs of coal miners can correlate with the concentration of tissue nonheme iron. Associations between the accumulation of this metal and fibrotic injury could result through either (1) catalysis of free radicals by iron associated with the dust and subsequent oxidant dependent mediator release, or (2) metal dependent enzyme activation. To better quantify and characterize the accumulation of the metal in the lung, 60-day old, male Sprague Dawley rats were intratracheally instilled with 1.0 mg coal dust (Dust 1508; Pennsylvania State University repository) twice a week for 6 months. Every 4 weeks, animals were euthanized and the lungs excised. Tissue nonheme iron was measured and lungs, fixed with 10 percent formalin, were stained for iron (Perls' Prussian blue), ferritin using a polyclonal antibody, and collagen (Masson trichrome). Nonheme iron accumulated during the 6 months of study....

Effects of experimental hemosiderosis on intestinal morphology, permeability, and tissue iron content

Effects of iron overload on intestinal function and structure are unknown and were, therefore, investigated. Sprague-Dawley rats were randomized into an iron-overloaded group, which received a single subcutaneous injection of 1.2 g/kg elemental iron-dextran complex, and placebo-treated pair-fed controls. Animals were studied after a 10-month observation period. Intestinal permeability was assessed by measuring the urinary excretion of lactulose, rhamnose, and mannitol after oral administration. In addition, tissue nonheme iron content was measured, and histologic examination and morphometric measurements were carried out. The chronic iron-overloaded group showed a significant increase in intestine tissue iron content and stainable iron in the submucosa and muscularis propria and adipose tissue of the small intestine and lamina propria and muscularis mucosa of the large intestine. There was a significant decrease in the crypt depths without discernible change in the intestine permeability to any of the markers used. In addition, the iron-overloaded animals showed a significant number of iron-laden cells, which primarily consisted of macrophages, fibroblasts, myocytes, and adipocytes. In contrast, no iron-laden cells were present in tissues obtained from the normal control group. Thus, chronic experimental iron overload in rats leads to significant morphologic, but no permeability, alterations of the alimentary tract.

The relationship between serum ferritin concentration and tissue non-heme iron or tissue ferritin in dairy cattle.

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