Phillip S. Oates

Localisation of divalent metal transporter 1 (DMT1) to the microvillus membrane of rat duodenal enterocytes in iron deficiency, but to hepatocytes in iron overload

BACKGROUND The mechanism of iron absorption by the intestine and its transfer to the main iron storage site, the liver, is poorly understood. Recently an iron carrier was cloned and named DMT1 (divalent metal transporter 1). AIMS To determine the level of DMT1 gene expression and protein distribution in duodenum and liver. METHODS A DMT1 cRNA and antibody were produced and used in in situ hybridisation and immunohistochemistry, respectively, in rats in which the iron stores were altered by feeding diets with normal, low, and high iron content. RESULTS Duodenal DMT1 mRNA was low in crypts and increased at the crypt-villus junction in iron deficient and control rats; it fell in the iron loaded state. Staining for DMT1 protein was not detected in crypts. In villus enterocytes, protein staining was localised to the microvillus membrane in iron deficiency, in the cytoplasm and to a lesser extent in the membrane in controls, and entirely in the cytoplasm of iron loaded animals. Liver DMT1 mRNA was distributed evenly across hepatocytes. DMT1 protein staining was observed on hepatocyte plasma membranes, with highest values in the iron loaded state, lower values in control animals, and none after iron depletion. CONCLUSIONS Results are consistent with a role for DMT1 in the transmembrane transport of non-transferrin bound iron from the intestinal lumen and from the portal blood.

Expression of the neuronal transferrin receptor is age dependent and susceptible to iron deficiency.

In order to characterize the mechanism by which Iron (Fe) is taken up by neurons, we examined the neuronal expression of transferrin receptor (TR) in rats during development and iron (Fe) deficiency by using immunohistochemistry, in vitro receptor autoradiography and in situ hybridization. In contrast to the continuous expression of TR in brain capillary endothelial cells regardless of the age of the animals studied, the expression of neuronal TR was almost absent at late embryonic and early postnatal ages but increased with increasing age to reach a plateau from postnatal (P) 21 through adulthood as verified by immunohistochemical staining. Reducing the Fe stores potentiated the expression of TR immunoreactivity in neurons of both young and adult rats in several grey matter regions. Increased TR immunoreactivity was also observed in neuronal extensions of neurons of the medial habenular nucleus, reticular neurons of the brainstem, and fibers projecting to the area postrema. TR immunoreactivity was never observed in white matter regions, except for that recorded in brain capillaries. In vitro receptor autoradiography verified the increased capacity for transferrin binding during Fe deficiency. By contrast, TR mRNA expression was not affected by Fe deficiency. These findings demonstrate that the expression of the neuronal TR protein is age dependent and susceptible to Fe deficiency. J. Comp. Neurol. 398:420–430, 1998.

Ferroportin/IREG-1/MTP-1/SLC40A1 modulates the uptake of iron at the apical membrane of enterocytes

Background: Absorption of non-haeme iron occurs mainly in the duodenum. It involves the divalent metal transporter 1 (DMT1) in the uptake of ferrous Fe(II) iron and the basolateral transporter ferroportin/IREG-1/MTP-1/SLC40A1 in its release. Whether ferroportin functions in this process at other sites in the enterocyte is unknown. In this study the effect of a blocking antibody to ferroportin on the uptake and release of iron was evaluated in enterocyte-like cells (IEC-6 and Caco-2) and in freshly isolated duodenal enterocytes from rats. Methods: Uptake of 1 μM Fe(II) and its release by cells was studied in the presence of the antibody. Ferroportin expression was determined by western blot analysis of duodenal mucosa enriched microvillus membranes, Caco-2 cells, IEC-6 cells, and freshly isolated enterocytes. Immunofluorescent detection of ferroporitn was performed on frozen sections of duodenum from rats with variations in body iron stores. Results: Ferroportin was expressed in all cell types. In these cells, the antibody significantly reduced (p<0.05) uptake of Fe(II) by 40–50% but had no effect on the release of iron. In Caco-2 cells, Fe(II) uptake was reduced only when the antibody was in contact with the apical membrane. Ferroportin protein was enriched in microvillus membrane preparations. In enterocytes from iron deficient rats, ferroportin was expressed along the brush border where it colocalised with lactase. Ferroportin was seen in the basal cytoplasm and along the basolateral membranes. Iron loading markedly reduced intracellular expression of ferroportin. In Caco-2 cells, ferroportin also localised to the microvillus and lateral and basal membranes. Conclusions: In addition to release, ferroportin functions in the uptake of iron at the apical membrane, possibly by modulating the activity of DMT1.

Effect of dietary fat to produce non‐alcoholic fatty liver in the rat

Background and Aim:  Non‐alcoholic steatohepatitis (NASH) belongs to a spectrum of non‐alcoholic fatty liver disease (NAFLD). Oxidative stress is hypothesized to play an important role in the progression of the disease. We used the Lieber/DeCarli model for NASH to investigate the mechanisms involved in its progression.

Iron-independent neuronal expression of transferrin receptor mRNA in the rat.

Neuronal transferrin receptor protein expression is highly upregulated widely in CNS following iron deficiency. Using the medial habenular nucleus as a model of neuronal transferrin receptor mRNA expression, the present study examined 17-day-old rats subjected to variations in dietary iron. Changing the iron availability resulted in alterations in plasma and cerebrospinal fluid (CSF) levels of transferrin and iron. The iron-binding capacity of transferrin in CSF was exceeded in normal and iron-overloaded rats. In spite of a lowering of the concentration of brain iron by approximately 22% in iron-deficient rats, neuronal transferrin receptor mRNA was not affected when measured by quantitative densitometry. Brain iron and neuronal transferrin receptor mRNA expression was unaltered in iron overloaded rats. The absence of a rise in transferrin receptor mRNA during iron deficiency suggests that neuronal transferrin receptor mRNA expression is regulated by another mechanism than the post-transcriptional regulation mechanism, which has been attributed to cells of non-neural tissue.

Effects of dietary iron loading with carbonyl iron and of iron depletion on intestinal growth, morphology, and expression of transferrin receptor in the rat

The intestine has one of the highest cell turnovers of the body, which is characterised by cell proliferation and differentiation occurring at specified locations along the crypt to the villus axis. These processes require iron for the synthesis of iron‐dependent proteins, the supply of which is mediated through the transferrin receptor. In this study, we varied dietary iron intake to determine whether this affected the pattern of transferrin receptor expression and activity on intestinal cell turnover and cell differentiation.

Transferrin receptor activity and localisation in the rat duodenum

Abstract. It is not known how the efficiency of intestinal iron absorption is regulated. One hypothesis suggests that an interaction between the transferrin receptor (TfR) and the haemochromatosis protein (HFE) regulates the level of iron loading in crypt cells. The hypothesis goes on to suggest that this determines the amount of transport protein, expressed in villus enterocytes, that is involved in iron absorption. Mice with haploinsufficiency for TfR are iron deficient and this is thought to be caused by reduced iron absorption. This suggests that TfR may play a role in the regulation and/or mechanism of iron absorption. We investigated TfR function and distribution by measuring iron uptake from plasma transferrin and by immunohistochemistry. The uptake of transferrin-bound iron (Tf–Fe2) into mucosal cells subsequently separated along the crypt–villus axis was compared to the presence of TfR, determined by immunohistochemistry using frozen and wax sections. Frozen sections showed TfR staining in crypt and villus epithelial cells. In wax sections TfR was only identified in a supranuclear region commencing in enterocytes at the crypt–villus junction and attaining greatest levels at the villus tip. This indicates that the processing of wax tissue exposes a TfR epitope that otherwise remains undetectable when studied in frozen sections. This appearance in paraffin sections was inversely related to the uptake of Tf–Fe2. Supranuclear TfR was not associated with lysosomes, since there was no difference in the uptake of normal Tf–Fe2 and that of the non-digestible cellobiose Tf–Fe2, and Western blot analysis revealed similar amounts of TfR in crypt and villus cells. Also the uptake of Tf–Fe2 increased linearly with time, albeit less in villus than crypt cells, suggesting that maturation of an efflux system in villus cells is not responsible for this difference. We hypothesise that TfR in the supranuclear region of villus enterocytes may play a role in iron absorption.

Molecular regulation of hepatic expression of iron regulatory hormone hepcidin

Iron is a micronutrient that is an essential component that drives many metabolic reactions. Too little iron leads to anemia and too much iron increases the oxidative stress of body tissues leading to inflammation, cell death, and system organ dysfunction, including cancer. Maintaining normal iron balance is achieved by rigorous control of the amount absorbed by the intestine, that released from macrophages following erythrophagocytosis of effete red cells and by either release or uptake from hepatocytes. Hepcidin is a recently characterized molecule that appears to play a key role in the regulation of iron efflux from enterocytes, macrophages, and hepatocytes. It is produced by hepatocytes under basal conditions, in response to alterations in increased iron stores or reduced requirement for erythropoiesis and by inflammation. The proteins that regulate hepcidin expression are presently being defined, albeit that our present understanding is still far from complete. This review focuses on the molecules which regulate hepcidin expression. The subsequent characterization of these proteins using molecular, cellular, and physiological approaches also is discussed along with inflammatory signals and receptors involved in hepcidin expression.

Subcellular location of heme oxygenase 1 and 2 and divalent metal transporter 1 in relation to endocytotic markers during heme iron absorption

Background and Aim:  Heme is an important dietary micronutrient, although its absorptive mechanisms are poorly understood. One hypothesis suggests enterocytes take up heme by receptor‐mediated endocytosis (RME) which then undergoes catabolism by heme oxygenase (HO) inside internalized vesicles. This would require the translocation of HO‐1 or HO‐2 to endosomes and/or lysosomes and the presence of a transporter, possibly divalent metal transporter 1 (DMT1), to transfer released iron to the cytoplasm. Currently, the location of HO‐1 and HO‐2 in enterocytes is unknown.

The relevance of the intestinal crypt and enterocyte in regulating iron absorption.

Rigorous regulation of iron absorption is required to meet the requirements of the body and to limit excess iron accumulation that can produce oxidative stress. Regulation of iron absorption is controlled by hepcidin and probably by the crypt program. Hepcidin is a humoral mediator of iron absorption that interacts with the basolateral transporter, ferroportin. High levels of hepcidin reduce iron absorption by targeting ferroportin to lysosomes for destruction. It is also proposed that ferroportin is expressed on the apical membrane and coordinates with ferroportin-hepcidin derived from the basal surface to modulate the uptake phase of iron absorption. The crypt program suggests that as crypt cells differentiate and migrate into the absorptive zone they absorb iron from the diet at levels inverse to the amount of iron taken up from transferrin. Under most circumstances, intestinal iron absorption is controlled at multiple levels that lead to hepcidin/ferroportin modulation of the enterocyte labile iron pool (LIP). It is likely that transcription of iron transport proteins involved in the apical and basolateral transport of iron are differentially regulated by separate LIPs. Iron-responsive protein (IRP) 1 and IRP2 do not appear to play a significant role in the expression of iron transport proteins, although IRP2 regulates L- and H-ferritin expression. Despite the importance of hepcidin, there is evidence of hepcidin-independent regulation of iron absorption possibly involving haemojuvelin (HJV) and neogenin, which may be up-regulated during ineffective erythropoiesis.