Hypotransferrinemic Mice Research Articles

Abnormal iron accumulation in the brain of neonatal hypotransferrinemic mice

Transferrin is a plasma protein involved in iron delivery to tissues. To study iron transport into the brain under a transferrin deficiency, iron concentration and 59Fe uptake in the brain were measured in neonatal hypotransferrinemic (HP) mice at 7 days of age. Brain iron concentration of the HP mice, in which iron concentration was relatively high in the cerebral cortex and cerebellum, was approximately three times higher than that of non-mutant mice, whereas serum iron concentration of HP mice was significantly lower than that of non-mutant mice. When 59FeCl 3 was subcutaneously injected into HP and non-mutant mice, 59Fe was distributed highly in the choroid plexus in the ventricles of HP mice 24 h after injection. The 59Fe distribution in the brain was different between HP and non-mutant mice. On the other hand, the clearance of 59Fe from the blood was very high in HP mice and the hepatic 59Fe concentration of HP mice was more than ten times of that of non-mutant mice. The present findings demonstrate that iron distribution in the brain is changed by transferrin deficiency and that iron abnormally accumulates in the brain of HP mice. It is likely that the management of iron is different in the brain of HP mice.

Expression of the DMT1 (NRAMP2/DCT1) iron transporter in mice with genetic iron overload disorders

AbstractIron overload is highly prevalent, but its molecular pathogenesis is poorly understood. Recently, DMT1 was shown to be a major apical iron transporter in absorptive cells of the duodenum. In vivo, it is the only transporter known to be important for the uptake of dietary non-heme iron from the gut lumen. The expression and subcellular localization of DMT1 protein in 3 mouse models of iron overload were examined: hypotransferrinemic (Trfhpx) mice, Hfeknockout mice, and B2m knockout mice. Interestingly, in Trfhpx homozygotes, DMT1 expression was strongly induced in the villus brush border when compared to control animals. This suggests that DMT1 expression is increased in response to iron deficiency in the erythron, even in the setting of systemic iron overload. In contrast, no increase was seen in DMT1 expression in animals with iron overload resembling human hemochromatosis. Therefore, it does not appear that changes in DMT1 levels are primarily responsible for iron loading in hemochromatosis.

The molecular defect in hypotransferrinemic mice

Hypotransferrinemic (Trfhpx/hpx) mice have a severe deficiency in serum transferrin (Trf) as the result of a spontaneous mutation linked to the murine Trf locus. They are born alive, but before weaning, die from severe anemia if they are not treated with exogenous Trf or red blood cell transfusions. We have determined the molecular basis of the hpx mutation. It results from a single point mutation, which alters an invariable nucleotide in the splice donor site after exon 16 of the Trf gene. No normalTrf messenger RNA (mRNA) is made from the hpx allele. A small amount of mRNA results from the usage of cryptic splice sites within exon 16. The predominant cryptic splice site produces aTrf mRNA carrying a 27-base pair (bp), in-frame deletion. Less than 1% of normal levels of a Trf-like protein is found in the serum of Trfhpx/hpx mice, most likely resulting from translation of the internally deleted mRNA. Despite their severe Trf deficiency, however,Trfhpx/hpx mice initially treated with transferrin injections can survive after weaning without any further treatment. They have massive tissue iron overload develop in all nonhematopoietic tissues, while they continue to have severe iron deficiency anemia. Their liver iron burden is 100-fold greater than that of wild-type mice and 15- to 20-fold more than that of mice lacking the hemochromatosis gene, Hfe. Trfhpx/hpx mice thus provide an additional model with a defined molecular defect for the study of genetic iron disorders.

The molecular defect in hypotransferrinemic mice

AbstractHypotransferrinemic (Trfhpx/hpx) mice have a severe deficiency in serum transferrin (Trf) as the result of a spontaneous mutation linked to the murine Trf locus. They are born alive, but before weaning, die from severe anemia if they are not treated with exogenous Trf or red blood cell transfusions. We have determined the molecular basis of the hpx mutation. It results from a single point mutation, which alters an invariable nucleotide in the splice donor site after exon 16 of the Trf gene. No normalTrf messenger RNA (mRNA) is made from the hpx allele. A small amount of mRNA results from the usage of cryptic splice sites within exon 16. The predominant cryptic splice site produces aTrf mRNA carrying a 27-base pair (bp), in-frame deletion. Less than 1% of normal levels of a Trf-like protein is found in the serum of Trfhpx/hpx mice, most likely resulting from translation of the internally deleted mRNA. Despite their severe Trf deficiency, however,Trfhpx/hpx mice initially treated with transferrin injections can survive after weaning without any further treatment. They have massive tissue iron overload develop in all nonhematopoietic tissues, while they continue to have severe iron deficiency anemia. Their liver iron burden is 100-fold greater than that of wild-type mice and 15- to 20-fold more than that of mice lacking the hemochromatosis gene, Hfe. Trfhpx/hpx mice thus provide an additional model with a defined molecular defect for the study of genetic iron disorders.

Diminished injury in hypotransferrinemic mice after exposure to a metal-rich particle.

Using the hypotransferrinemic (Hp) mouse model, we studied the effect of altered iron homeostasis on the defense of the lung against a catalytically active metal. The homozygotic (hpx/hpx) Hp mice had greatly diminished concentrations of both serum and lavage fluid transferrin relative to wild-type mice and heterozygotes. Fifty micrograms of a particle containing abundant concentrations of metals (a residual oil fly ash) was instilled into wild-type mice and heterozygotic and homozygotic Hp animals. There was an oxidative stress associated with particle exposure as manifested by decreased lavage fluid concentrations of ascorbate. However, rather than an increase in lung injury, diminished transferrin concentrations in homozygotic Hp mice were associated with decreased indexes of damage, including concentrations of relevant cytokines, inflammatory cell influx, lavage fluid protein, and lavage fluid lactate dehydrogenase. Comparable to other organs in the homozygotic Hp mouse, siderosis of the lung was evident, with elevated concentrations of lavage fluid and tissue iron. Consequent to these increased concentrations of iron, proteins to store and transport iron, ferritin, and lactoferrin, respectively, were increased when assayed by immunoprecipitation and immunohistochemistry. We conclude that the lack of transferrin in Hp mice did not predispose the animals to lung injury after exposure to a particle abundant in metals. Rather, these mice demonstrated a diminished injury that was associated with an increase in the metal storage and transport proteins.

Resistance of hypotransferrinemic mice to hyperoxia-induced lung injury.

Oxidative stress plays a central role in the pathogenesis of acute and chronic pulmonary diseases. Safe sequestration of iron, which participates in the formation of the hydroxyl radical, is crucial in the lung's defense. We used a mouse line defective in the major iron transport protein transferrin to investigate the effect of aberrant iron metabolism on the lung's defense against oxidative injury. The tolerance to hyperoxic lung injury was greater in the hypotransferrinemic than in wild-type mice as documented by histopathology and biochemical indexes for lung damage. There was no increase in the levels of intracellular antioxidants, inflammatory cytokines, and heme oxygenase-1 in the hypotransferrinemic mouse lung compared with those in wild-type mice. However, there were elevated expressions of ferritin and lactoferrin in the lung of hypotransferrinemic mice, especially in the alveolar macrophages. Our results suggest that pulmonary lactoferrin and ferritin protect animals against oxidative stress, most likely via their capacity to sequester iron, and that alveolar macrophages are the key participants in iron detoxification in the lower respiratory tract.

Importance of Anemia and Transferrin Levels in the Regulation of Intestinal Iron Absorption in Hypotransferrinemic Mice

AbstractThe hypotransferrinemic mouse (trf hpx) is a mutant strain exhibiting transferrin deficiency, marked anemia, hyperabsorption of iron, and elevated hepatic iron stores. We set out to investigate the relative roles of anemia and of transferrin in the malregulation of intestinal iron absorption in these animals. Transfusion of erythrocytes obtained from littermate controls increased hemoglobin levels and reduced reticulocyte counts in recipient animals. Although mucosal to carcass 59Fe transfer was reduced, total duodenal iron uptake was not significantly affected. Iron absorption in homozygotes, in contrast to littermate controls, was not reduced by hyperoxia. Mouse transferrin injections, in the short term, increased delivery of iron to the marrow and raised hemoglobin levels. Although mucosal transfer and total iron uptake were reduced at the higher transferrin doses, total uptake was still higher than in controls. Daily injections of mouse/human transferrin for 3 weeks from weaning, normalized hemoglobin values, and markedly reduced liver iron and intestinal iron absorption values in trf hpxanimals. When such daily-injected mice were left for a week to allow transferrin clearance, iron absorption values were significantly enhanced; hemoglobin or hepatic iron levels were, however, not significantly altered. These data indicate that hyperabsorption of iron in trf hpx mice is not solely because of the anemia; transferrin levels per se do affect iron absorption, possibly via a direct effect on the intestinal mucosa.

Importance of Anemia and Transferrin Levels in the Regulation of Intestinal Iron Absorption in Hypotransferrinemic Mice

The hypotransferrinemic mouse (trf hpx) is a mutant strain exhibiting transferrin deficiency, marked anemia, hyperabsorption of iron, and elevated hepatic iron stores. We set out to investigate the relative roles of anemia and of transferrin in the malregulation of intestinal iron absorption in these animals. Transfusion of erythrocytes obtained from littermate controls increased hemoglobin levels and reduced reticulocyte counts in recipient animals. Although mucosal to carcass 59Fe transfer was reduced, total duodenal iron uptake was not significantly affected. Iron absorption in homozygotes, in contrast to littermate controls, was not reduced by hyperoxia. Mouse transferrin injections, in the short term, increased delivery of iron to the marrow and raised hemoglobin levels. Although mucosal transfer and total iron uptake were reduced at the higher transferrin doses, total uptake was still higher than in controls. Daily injections of mouse/human transferrin for 3 weeks from weaning, normalized hemoglobin values, and markedly reduced liver iron and intestinal iron absorption values in trf hpxanimals. When such daily-injected mice were left for a week to allow transferrin clearance, iron absorption values were significantly enhanced; hemoglobin or hepatic iron levels were, however, not significantly altered. These data indicate that hyperabsorption of iron in trf hpx mice is not solely because of the anemia; transferrin levels per se do affect iron absorption, possibly via a direct effect on the intestinal mucosa.

Metal ion transporters in mammals: structure, function and pathological implications.

Despite the importance of metal ions in several catalytic functions, there has been, until recently, little molecular information available on the mechanisms whereby metal ions are actively taken up by mammalian cells. The classical concept for iron uptake into mammalian cells has been the endocytosis of transferrin-bound Fe3+ by the transferrin receptor. Studies with hypotransferrinaemic mice revealed that in the intestine mucosal transferrin is derived from the plasma and that its presence is not required in the intestinal lumen for dietary iron absorption. This suggests that, at least in the intestine, other non-receptor-mediated uptake systems exist. The molecular identification of metal ion transporters is of great importance, in particular since an increasing number of human diseases are thought to be related to disturbances in metal ion homeostasis, including metal ion overload and deficiency disorders (i.e. anaemia, haemochromatosis, Menkes disease, Wilson's disease), and neurodegenerative diseases (i.e. Alzheimer's, Friedreich's ataxia and Parkinson's diseases). Furthermore, susceptibilities to mycobacterial infections are caused by metal ion transporter defects. The pathological implications of disturbed metal ion homeostasis confirm the vital roles these metal ions play in the catalytic function of many enzymes, in gene regulation (zinc-finger proteins), and in free radical homeostasis. Recent insights have significantly advanced our knowledge of how metal ions are taken up or released by mammalian cells. The purpose of this review is to summarize these advances and to give an overview on the growing number of mammalian metal ion transporters.

Iron proteins of duodenal enterocytes isolated from mice with genetically and experimentally altered iron metabolism.

The molecular basis for the control of iron absorption by the duodenum remains unknown: however, ferritin (Ft) and the iron status of enterocytes have been suggested as regulatory factors. We determined the iron and Ft status of duodenal enterocytes from mice with hypotransferrinaemia, a genetic defect leading to greatly enhanced iron absorption, and for comparison we also investigated mice with experimentally-altered iron absorption. Duodenal enterocytes were isolated and analysed for Ft and non-haem iron content and for transferrin binding (as a measure of transferrin receptor activity). RNA was extracted from the duodenal mucosa and examined for transferrin receptor and H- and L-Ft mRNA levels by Northern hybridization analysis. Ft levels were elevated in enterocytes of hypotransferrinaemic mice, similar to that seen in iron dextran-injected mice of the CD1-strain. Enterocyte Ft levels were reduced in mice fed a diet diminished in iron, but unchanged in hypoxic mice enterocytes. Enterocytes of hypotransferrinaemic mice had normal non-haem iron levels and transferrin binding; however, enterocytes from CD-1 mice fed a low iron diet had increased transferrin binding and a decreased non-haem iron content. Duodenal mRNA levels for transferrin receptor and H-Ft were unchanged in hypotransferrinaemic mice, whereas L-Ft was increased. We conclude from the Ft and non-haem iron contents and transferrin binding that duodenal enterocytes from hypotransferrinaemic mice are not simply iron deficient, leading to increased expression of iron carriers proteins. Duodenal iron absorption can be enhanced in mice even when enterocyte Ft levels are raised or unchanged, suggesting that iron absorption is regulated by developmentally programmed expression of iron transporters by enterocytes.

Immunohistochemical analysis of transferrin receptor: regional and cellular distribution in the hypotransferrinemic (hpx) mouse brain

The hypotransferrinemic (hpx) mouse mutant produces <1% of the normal circulating level of transferrin (Tf). Heterozygote animals of this strain (hpx/+) have approximately 50% of normal plasma Tf levels. In this study we examine the cellular and regional distribution of Tf receptor (Tf-R) in the brain of wild type, hpx/+ and mutant (hpx/hpx) mice. Also, using slot-blot (immunoblot) analysis, we describe the relative amount of Tf-R in brain microvessels of hpx/+ animals compared with wild type. Tf-R was seen primarily in neurons throughout the brains of wild type, hpx/+ and hpx/hpx animals. Gray matter areas immunoreacted more robustly than white matter areas. Oligodendrocytes and third ventricle tanycytes, both of which we have previously described as iron-positive, did not immunoreact for Tf-R. Tf-R immunohistochemical reaction in wild type, hpx/+ and hpx/hpx brains appeared similar. Immunoblot analysis of isolated cortical microvessels from wild type and hpx/+ animals revealed no upregulation of Tf-R expression in hpx/+ (relative to normal) despite a 50% decrease in circulating Tf levels. These results indicate that Tf-R is primarily expressed by neurons and that half normal levels of Tf (hpx/+) or transferrin supplementation (hpx/hpx) are apparently sufficient for normal expression and distribution of Tf-R. Because of the lack of circulating Tf, but unaltered Tf-R expression, hpx mice could serve as a model for delivery of therapeutic agents via the Tf/Tf-R system.

Evidence for non-transferrin-mediated uptake and release of iron and manganese in glial cell cultures from hypotransferrinemic mice.

Transferrin (Tf) is accepted as the iron mobilization protein, but its role in transport of other metals is controversial. In this study, we used mixed glial cultures from hypotransferrinemic (Hp) mice to determine the dependence of these cells on transferrin for iron and manganese delivery and release. Hp mice have a splicing defect in the transferrin (Tf) gene, resulting in < 1% of the normal plasma levels of Tf. Cellular iron and manganese uptake increases over 24 hr in cultures of normal and Hp glial cells in the presence of standard concentrations of Tf in the media; although total 59iron uptake in the Hp mouse cultures was 2X greater than normal, 54Mn uptake was similar between the two groups. The absence of Tf in the media resulted in a significant increase in 59iron uptake in both normal and Hp glial but did not affect Mn uptake. Elevated Tf (10X normal) in the media reduced both 59iron and 54Mn uptake. Efflux of 59Iron and 54Mn occurred in normal and Hp cultures, indicating the existence of a dynamic exchange of metals, and that intracellular Tf is not necessary for metal release. However, in the absence of Tf in the media, significantly more iron was retained in the cells than if Tf were present in both normal and Hp glial cultures. 54Mn release was minimally affected by extracellular Tf. The data demonstrate that Tf is not required for iron and Mn uptake into glial cells. These data further demonstrate a dynamic metal exchange system for glial cells which is not dependent on intracellular Tf.

Time-course of iron overload and biochemical, histopathological and ultrastructural evidence of pancreatic damage in hypotransferrinaemic mice.

1. The time course of iron overload of the pancreas in hypotransferrinaemic mice maintained on a standard rodent diet was compared with biochemical and histological markers of tissue damage. 2. Pancreatic iron levels increased linearly from weaning till 9 months of age [73.3 nmol/mg of tissue (SEM 9.9; n = 5) compared with 0.9 nmol/mg of tissue (SEM 0.1; n = 4) in age-matched controls] then decreased linearly till at least 18 months of age. 3. Investigation of tissue distribution of newly absorbed radioiron suggested that significant redistribution of iron from liver to pancreas (rather than direct dietary iron sources) must be invoked to explain the rate of pancreatic iron loading in hypotransferrinaemic mice. 4. Pancreatic epithelial cells first showed altered morphology at 9 months of age. At 12 months of age, the pancreatic epithelium had developed a micronodular appearance, with large numbers of acini replaced by atrophic, degenerated acinar cells. Increased collagen fibre deposition was evident by trichrome staining and by electron microscopy. Biochemical markers of pancreatitis (serum lipase, tissue pancreatitis-associated protein mRNA) were elevated before 9 months of age, whereas the levels of pancreatic amylase mRNA declined from 9 months of age. 5. The data suggest that iron loading of hypotransferrinaemic mouse pancreas proceeds up to a threshold level at 9 months of age followed by a progressive atrophy of secretory epithelium. The hypotransferrinaemic mouse pancreas is a useful model system for investigation of parenchymal cell damage by iron.

AMS measurements to study uptake and distribution of 26Al in mice and the role of the transferrin receptor in aluminium absorption mechanisms

We report the use of a 20 MV tandem Van de Graaff accelerator and accelerator mass spectrometry (AMS) to measure the distribution and uptake mechanisms of 26Al in vivo in mice. The influence of antibodies against the transferrin receptor and of hypotransferrinaemia (mice with virtual absence of circulating transferrin) were studied with regard to the uptake of 26Al from infusion with aluminium citrate. In both hypotransferrinaemic and antibody-treated mice, uptake of 26Al was similar to control values in all tissues except spleen and muscle for the antibody-treated mice and bone for the hypotransferrinaemic mice. It appears that non-transferrin mediated transport of aluminium can occur into tissues and that transport of the metal bound to citrate and other low molecular weight complexes may be important. Generally, the uptake of aluminium into control tissue follows the order bone (femur) > renal cortex > skeletal muscle ≈ liver > spleen > brain. Comparisons with the uptake and distribution of the radiotracer analogue of aluminium, (Ga-67) were made.

Distribution of injected iron 59 and manganese 54 in hypotransferrinemic mice

Transferrin has been proposed as the mobilization protein for iron and manganese. To better understand the role of transferrin in the transport of these metals, we studied the tissue distribution of injected iron 59 and manganese 54 in the hypotransferrinemic (Hp) mouse mutant. The Hp mouse has a mutation in the transferrin gene and produces <1% of normal transferrin levels. The tissue distribution of 59Fe and 54Mn in Hp mice was compared with that in animals heterozygous for the Hp mutation (50% transferrin levels) and wild-type animals. Formed elements in the brain, liver, spleen, heart, sternum/rib, plasma, and blood were analyzed for isotope incorporation at 24 hours, 7 days, and 4 weeks after injection. Tissue distribution of both 59Fe and 54Mn was similar in wild-type and heterozygote animals, indicating that decreased transferrin concentration and increased saturation did not influence the tissue distribution of the injected metals. The absence of transferrin in the Hp mutant was associated with abnormal tissue distribution of radiolabeled iron; there was 4 times more 59Fe than normal in the Hp liver and 10 times less 59Fe in the spleen and blood formed elements than normal. Injected manganese also accumulated at abnormally high levels in the Hp mouse liver. Distribution of either metal to the brain, heart, and sternum/rib was not affected by the absence of plasma transferrin. These results reveal that transferrin is required for proper targeting of manganese and iron, especially from the liver to other organs, but further indicate that nontransferrin transport mechanisms for iron and manganese must exist.

Control of Intestinal Iron Absorption in Hypotransferrinaemic (HPX/HPX) Mice

Control of Intestinal Iron Absorption in Hypotransferrinaemic (HPX/HPX) Mice

Duodenal Enterocytes in Genetic Hypotransferrinaemic Mice Are Not Iron Deficient

Duodenal Enterocytes in Genetic Hypotransferrinaemic Mice Are Not Iron Deficient

Role of transferrin in the tissue delivery of injected 59Fe 2+ and 54Mn 2+ in the hypotransferrinemic mouse : [formula omitted]. Penn State University College of Medicine, Milton S Hershey Medical Center, Hershey, PA 17033

Role of transferrin in the tissue delivery of injected 59Fe 2+ and 54Mn 2+ in the hypotransferrinemic mouse : [formula omitted]. Penn State University College of Medicine, Milton S Hershey Medical Center, Hershey, PA 17033

The hypotransferrinaemic mouse: ultrastructural and laser microprobe analysis observations.

Homozygote hypotransferrinaemic mice (hpx/hpx) have cytopathological features similar to those of human congenital atransferrinaemia, genetic haemochromatosis, and neonatal haemochromatosis. These conditions all have in common high levels of cytotoxic non-transferrin-bound serum iron. This study describes the ultrastructural features of iron overload in liver, pancreas, heart, and small intestine of 2- and 12-month-old hypotransferrinaemic mice. Electron microscopic studies of unstained sections showed early parenchymal cell siderosis, with accumulation of numerous ferritin particles and clusters in the cytosol, as well as ferritin and haemosiderin in lysosomes (siderosomes). In the 12-month-old animals, iron was also found in Kupffer cells and macrophages in other tissues. In addition, there were conspicuous iron-containing compounds in the bile canaliculi, and marked iron deposition in the pancreas and heart. Laser microprobe mass analysis (LAMMA) enabled localization and relative quantitation of iron deposition in subcellular compartments providing in situ documentation of iron accumulation in siderosomes and contributed in assessing total cytosolic iron in various cell types. Moreover, it demonstrated the importance and magnitude of the biliary route for iron excretion in these animals.

Cellular distribution of iron, transferrin, and ferritin in the hypotransferrinemic (Hp) mouse brain

Hypotransferrinemic (Hp) mice have a point mutation or small deletion in the transferrin (Tf) gene, resulting in defective splicing of precursor Tf mRNA. Hp animals produce < 1% of normal Tf levels and require supplemental serum or purified Tf for survival. Because of the lack of endogenous brain Tf, we examined regional and cellular distributions of iron and iron regulatory proteins (Tf and ferritin) in selected brain regions of Hp mice. The regional distribution of iron, Tf, and ferritin in Hp brain was similar to normal except for the pattern of iron staining in hippocampus. The cellular distribution of iron, ferritin, and Tf was similar between Hp and normal animals. The predominant cell type staining for Tf and iron was oligodendrocytes. Qualitative observations suggest that the number of cells staining for iron was similar between Hp and normal mice, whereas the number of Hp Tf-positive cells was reduced. Ferritin immunostaining was similar in both cases. However, ferritin-positive cells were predominantly astrocytes, an observation unique to mice among species studied previously. Western blot analysis revealed that Tf present in Hp brain was of exogenous origin (from supplemental injections). Presumably, Tf transports the iron found in Hp oligodendrocytes. These data demonstrate that, despite reduced endogenous Hp brain Tf, iron and plasma Tf migrate or are transported to the appropriate cells (oligodendrocytes), bringing into question the role of endogenous brain Tf in extracellular iron transport.