Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron.

Genetic studies in animal models of microcytic anemia and biochemical studies of transport have implicated the Nramp2 gene in iron transport. Nramp2 generates two alternatively spliced mRNAs that differ at their 3' untranslated region by the presence or absence of an iron-response element (IRE) and that encode two proteins with distinct carboxy termini. Antisera raised against Nramp2 fusion proteins containing either the carboxy or amino termini of Nramp2 and that can help distinguish between the two Nramp2 protein isoforms (IRE: isoform I; non-IRE: isoform II) were generated. These antibodies were used to identify the cellular and subcellular localization of Nramp2 in normal tissues and to study possible regulation by dietary iron deprivation. Immunoblotting experiments with membrane fractions from intact organs show that Nramp2 is expressed at low levels throughout the small intestine and to a higher extent in kidney. Dietary iron starvation results in a dramatic upregulation of the Nramp2 isoform I in the proximal portion of the duodenum only, whereas expression in the rest of the small intestine and in kidney remains largely unchanged in response to the lack of dietary iron. In proximal duodenum, immunostaining studies of tissue sections show that Nramp2 protein expression is abundant under iron deplete condition and limited to the villi and is absent in the crypts. In the villi, staining is limited to the columnar absorptive epithelium of the mucosa (enterocytes), with no expression in mucus-secreting goblet cells or in the lamina propria. Nramp2 expression is strongest in the apical two thirds of the villi and is very intense at the brush border of the apical pole of the enterocytes, whereas the basolateral membrane of these cells is negative for Nramp2. These results strongly suggest that Nramp2 is indeed responsible for transferrin-independent iron uptake in the duodenum. These findings are discussed in the context of overall mechanisms of iron acquisition by the body.     

Characterization of the iron transporter DMT1 (NRAMP2/DCT1) in red blood cells of normal and anemic mk/mk mice.

Divalent metal transporter 1 (DMT1) is the major transferrin-independent iron uptake system at the apical pole of intestinal cells, but it may also transport iron across the membrane of acidified endosomes in peripheral tissues. Iron transport and expression of the 2 isoforms of DMT1 was studied in erythroid cells that consume large quantities of iron for biosynthesis of hemoglobin. In mk/mk mice that express a loss-of-function mutant variant of DMT1, reticulocytes have a decreased cellular iron uptake and iron incorporation into heme. Interestingly, iron release from transferrin inside the endosome is normal in mk/mk reticulocytes, suggesting a subsequent defect in Fe(++) transport across the endosomal membrane. Studies by immunoblotting using membrane fractions from peripheral blood or spleen from normal mice where reticulocytosis was induced by erythropoietin (EPO) or phenylhydrazine (PHZ) treatment suggest that DMT1 is coexpressed with transferrin receptor (TfR) in erythroid cells. Coexpression of DMT1 and TfR in reticulocytes was also detected by double immunofluorescence and confocal microscopy. Experiments with isoform-specific anti-DMT1 antiserum strongly suggest that it is the non-iron-response element containing isoform II of DMT1 that is predominantly expressed by the erythroid cells. As opposed to wild-type reticulocytes, mk/mk reticulocytes express little if any DMT1, despite robust expression of TfR, suggesting a possible effect of the mutation on stability and targeting of DMT1 isoform II in these cells. Together, these results provide further evidence that DMT1 plays a central role in iron acquisition via the transferrin cycle in erythroid cells.     

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

Iron 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 (Trf(hpx)) mice, Hfe knockout mice, and B2m knockout mice. Interestingly, in Trf(hpx) 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.     

Evolutionary matches of enzyme and transporter capacities to dietary substrate loads in the intestinal brush border

Safety factors of enzymes and transporters are defined as the ratio of V_max (maximal reaction rates at high substrate concentrations) to the reaction rate under actual physiological conditions. Although corresponding safety factors have been measured for macroscopic biological structures and for human-engineered structures, safety factors have been little studied at the molecular level. Some evolutionary considerations suggest that safety factors should be modestly in excess of 1.0 (“enough but not too much”) and should tend to be similar for the various steps of a pathway consisting of two or more elements arranged in series. Hence we used a preparation of intact mouse small intestine to measure V_max values (capacities) of brush-border sucrase (yielding glucose plus fructose) and of the brush-border glucose transporter, for comparison with each other and with dietary sucrose loads. Load was manipulated by varying dietary sucrose level or by studying lactating mice with increased energy requirements. Capacities both of sucrase and the glucose transporter increased with sucrose load (i.e., both proteins are inducible) and remained approximately matched to each other except on a carbohydrate-free diet. Their safety factors decreased from ca. 2.7 at low load to 1.0 at high load. Thus, neither sucrase nor the glucose transporter is the rate-limiting step for sucrose digestion; both steps are equally limiting. The modest safety factors and matched capacities must be genetically programmed through natural selection, with benefits of excess capacities being balanced against costs of biosynthetic energy and limited membrane space.     

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.     

Iron transporter Nramp2/DMT-1 is associated with the membrane of phagosomes in macrophages and Sertoli cells

Nramp2 (DMT1) is a pH-dependent divalent cation transporter that acts as the transferrin-independent iron uptake system at the intestinal brush border and also transports iron released from transferrin across the membrane of acidified endosomes. In this study, RAW264.7 macrophages and 2 independently derived murine Sertoli cells lines, TM4 and 15P-1, were used to further study the subcellular localization of Nramp2/DMT1 in phagocytic cells, including possible recruitment to the phagosomal membrane. Nramp2/DMT1 was localized primarily to the EEA1-positive recycling endosome compartment, with some overlapping staining with Lamp1-positive late endosomes. After phagocytosis, immunofluorescence analysis and in vitro biochemical studies using purified latex bead-containing phagosomes indicated Nramp2/DMT1 recruitment to the membrane of Lamp1, cathepsin D, and rab7-positive phagosomes. Nramp2/DMT1 was also found associated with erythrocyte-containing phagosomes in RAW macrophages and with the periphery of sperm-containing phagosomes in Sertoli cells. These results suggest that, as for the macrophage-specific Nramp1 protein, Nramp2/DMT1 may transport divalent metals from the phagosomal space.     

A novel R416C mutation in human DMT1 (SLC11A2) displays pleiotropic effects on function and causes microcytic anemia and hepatic iron overload

A patient suffering from microcytic anemia and hepatic iron overload was found to be compound heterozygote for polymorphisms in the iron transporter DMT1 (Nramp2, SLC11A2), including a 3-bp deletion (DMT1(delCTT)) in intron 4 that partially impairs splicing and an amino acid substitution (DMT1(C1246T), R416C) at a conserved residue in transmembrane domain 9 of the protein. The functional properties and possible contribution to disease of the DMT1 R416C mutation were studied in independent mutants at that position (R416C, R416A, R416K, R416E) expressed in LLC-PK(1) kidney cells. Non-conservative substitutions at R416 (C, A, E) cause multiple functional deficiencies including defective protein processing, loss of transport activity, impaired cell surface targeting, and recycling through endosomes, concomitant with retention of the transporter in the endoplasmic reticulum. Conversely, a conservative isoelectric substitution (R416K) was less vulnerable, resulting in a functional transporter that was properly processed and targeted to the cell surface and to recycling endosomes. We propose that DMT1(C1246T) (R416C) represents a complete loss-of-function, and that a quantitative reduction in DMT1 expression is the cause of the microcytic anemia and iron overload in the patient.     

Liver injury induced by lipopolysaccharide is mediated by TNFR-1 but not by TNFR-2 or Fas in mice.

Endotoxemia causes liver injury in which tumor necrosis factor (TNF)-alpha plays a significant role by inducing hepatic apoptosis. We here examined if such apoptosis is strictly dependent on TNF-alpha and which type of TNF receptor (TNFR) is involved, employing TNFR-1- and -2-knockout mice. Lipopolysaccharide (LPS) dose-dependently induced liver injury in both wild-type (WT) and TNFR-2-knockout mice as indicated by plasma ALT activities, whereas the injury was absent in TNFR-1-knockout mice. Similarly, apoptotic hepatocyte death was observed in WT and TNFR-2-knockout mice after LPS-injection, but not in TNFR-1-knockout mice. Plasma levels of TNF-alpha, interleukin (IL)-6, IL-10 and interferon-gamma as well as hepatic TNF-alpha levels increased equally in mice with either genotype after LPS-injection. LPS also enhanced equally the mRNA expression of Fas but not Fas ligand irrespective of either genotype, as measured by RNase protection assay. These findings suggest that apoptotic liver injury induced by LPS depends on TNF-alpha signaling through TNFR-1 but not via TNFR-2 or Fas-Fas ligand pathway.     

A rapid decrease in the expression of DMT1 and Dcytb but not Ireg1 or hephaestin explains the mucosal block phenomenon of iron absorption

BACKGROUND: A large oral dose of iron will reduce the absorption of a subsequent smaller dose of iron in a phenomenon known as mucosal block. Molecular analysis of this process may provide insights into the regulation of intestinal iron absorption. AIMS: To determine the effect of an oral bolus of iron on duodenal expression of molecules associated with intestinal iron transport in rats and to relate this to changes in iron absorption. METHODS: Rats were given an oral dose of iron and duodenal expression of divalent metal transporter 1 (DMT1), Dcytb, Ireg1, and hephaestin (Hp) was determined using the ribonuclease protection assay, western blotting, and immunofluorescence. Iron absorption was measured using radioactive (59)Fe. RESULTS: A decrease in intestinal iron absorption occurred following an oral dose of iron and this was associated with increased enterocyte iron levels, as assessed by iron regulatory protein activity and immunoblotting for ferritin. Reduced absorption was also accompanied by a rapid decrease in expression of the mRNAs encoding the brush border iron transport molecules Dcytb and the iron responsive element (IRE) containing the splice variant of DMT1. No such change was seen in expression of the non-IRE splice variant of DMT1 or the basolateral iron transport molecules Ireg1 and Hp. Similar changes were observed at the protein level. CONCLUSIONS: These data indicate that brush border, but not basolateral, iron transport components are regulated locally by enterocyte iron levels and support the hypothesis that systemic stimuli exert their primary effect on basolateral transport molecules.     

JNK1 but not JNK2 promotes the development of steatohepatitis in mice

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis and varying degrees of necroinflammation. Although chronic oxidative stress, inflammatory cytokines, and insulin resistance have been implicated in the pathogenesis of NAFLD, the mechanisms that underlie the initiation and progression of this disease remain unknown. c-Jun N-terminal kinase (JNK) is activated by oxidants and cytokines and regulates hepatocellular injury and insulin resistance, suggesting that this kinase may mediate the development of steatohepatitis. The presence and function of JNK activation were therefore examined in the murine methionine- and choline-deficient (MCD) diet model of steatohepatitis. Activation of hepatic JNK, c-Jun, and AP-1 signaling occurred in parallel with the development of steatohepatitis in MCD diet-fed mice. Investigations in jnk1 and jnk2 knockout mice demonstrated that jnk1, but not jnk2, was critical for MCD diet-induced JNK activation. JNK promoted the development of steatohepatitis as MCD diet-fed jnk1 null mice had significantly reduced levels of hepatic triglyceride accumulation, inflammation, lipid peroxidation, liver injury, and apoptosis compared with wild-type and jnk2 -/- mice. Ablation of jnk1 led to an increase in serum adiponectin but had no effect on serum levels of tumor necrosis factor-alpha. In conclusion, JNK1 is responsible for JNK activation that promotes the development of steatohepatitis in the MCD diet model. These findings also provide additional support for the critical mechanistic involvement of JNK1 overactivation in conditions associated with insulin resistance and the metabolic syndrome.     

Cigarette smoking is associated with intestinal barrier dysfunction in the small intestine but not in the large intestine of mice

AimsTo observe the effect of cigarette smoke (CS) on the small bowel and colon in mice and to attempt to explain the potential mechanisms that account for these effects.MethodsMale BALB/c mice age 6–8 weeks were randomly divided into a CS group and a control group (n = 10 per group). CS mice were exposed to CS (five cigarettes each time, four times a day for 5 days a week using Hamburg II smoking machine and CS was diluted with air at a ratio of 1:6) for 10 weeks, and control mice were exposed to room air. After 10 weeks, mice were sacrificed for analysis (colon and small bowel).ResultsCS exposure impaired the intestinal barrier of the small bowel, based on evidence that CS mice exhibited increased intestinal permeability, bacterial translocation, intestinal villi atrophy, damaged tight junctions and abnormal tight junction proteins. These changes were partly mediated through the activated NF-κB (p65) signalling pathway. However, no obvious changes associated with the intestinal barrier were identified in the small bowel of control mice or the colons of control or CS mice.ConclusionsCS is associated with intestinal barrier dysfunction in the small intestine but not in the large intestine of mice.     

The P2Y1 receptor, necessary but not sufficient to support full ADP-induced platelet aggregation, is not the target of the drug clopidogrel

Recently we showed that the P2Y₁ receptor coupled to calcium mobilization is necessary to initiate ADP-induced human platelet aggregation. Since the thienopyridine compound clopidogrel specifically inhibits ADP-induced platelet aggregation, it was of interest to determine whether the P2Y₁ receptor was the target of this drug. Therefore we studied the effects of clopidogrel and of the two specific P2Y₁ antagonists A2P5P and A3P5P on ADP-induced platelet events in rats. Although clopidogrel treatment (50 mg/kg) greatly reduced platelet aggregation in response to ADP as compared to untreated platelets, some residual aggregation was still detectable. In contrast, A2P5P and A3P5P totally abolished ADP-induced shape change and aggregation in platelets from both control and clopidogrel-treated rats. A2P5P and A3P5P (100 μM ) totally inhibited the [Ca²⁺]_i rise induced by ADP (0.1 μM ) in control and clopidogrel-treated platelets, whereas clopidogrel treatment had no effect. Conversely, the inhibition of adenylyl cyclase induced by ADP (5 μM ) was completely blocked by clopidogrel but not modified by A2P5P or A3P5P (100 μM ). A3P5P (1 m M ) reduced the number of [³³P]2MeSADP binding sites on control rat platelets from 907 ± 50 to 611 ± 25 per platelet. After clopidogrel treatment, binding of [³³P]2MeSADP decreased to 505 ± 68 sites per platelet and further decreased to 55 ± 12 sites in the presence of A3P5P (1 m M ). In summary, these results demonstrate that the platelet P2Y₁ receptor responsible for the initiation of aggregation in response to ADP is not the target of clopidogrel. Platelets may express another, as yet unidentified, P2Y receptor, specifically coupled to the inhibition of adenylyl cyclase and necessary to induce full platelet aggregation, which could be the target of this drug.     

Monocyte chemoattractant protein-1-induced tissue inflammation is critical for the development of renal injury but not type 2 diabetes in obese db/db mice

Aims/hypothesis Tissue macrophage accumulation is thought to induce insulin resistance during obesity and stimulate the progression of diabetic nephropathy. Monocyte chemoattractant protein-1 (MCP-1) is a potent stimulator of macrophage recruitment. It is increased in adipose tissue during obesity and in diabetic kidneys, suggesting that inflammation of these tissues may be MCP-1-dependent. Based on these findings, the aim of this study was to examine whether a deficiency in MCP-1 would alter the development of type 2 diabetes and its renal complications. Materials and methods The role of MCP-1 in the progression of type 2 diabetes and its associated renal injury was assessed in obese db/db mice that were deficient in the gene encoding MCP-1 (Ccl2). Results The incidence and development of type 2 diabetes were similar in Ccl2 ^+/+ and Ccl2 ^−/− db/db mice between 8 and 32 weeks of age. Body mass, hyperglycaemia, hyperinsulinaemia, glucose and insulin tolerance, plasma triacylglycerol and serum NEFA were not different between these strains. Pathological changes in epididymal adipose tissue, including increases in macrophage accumulation and Tnfa mRNA and reductions in Adipoq mRNA, were unaffected by the absence of MCP-1. In contrast, kidney macrophage accumulation and the progression of diabetic renal injury (albuminuria, histopathology, renal fibrosis) were substantially reduced in Ccl2 ^−/− compared with Ccl2 ^+/+ db/db mice with equivalent diabetes. Conclusions/interpretation Our study demonstrates that MCP-1 promotes type 2 diabetic renal injury but does not influence the development of obesity, insulin resistance or type 2 diabetes in db/db mice. MCP-1 plays a critical role in inflammation of the kidney, but not adipose tissue, during the progression of type 2 diabetes.     

Blockade of the integrin alphaLbeta2 but not of integrins alpha4 and/or beta7 significantly prolongs intestinal allograft survival in mice

BACKGROUND—Small bowel transplantation remains a difficult therapeutic option endangered by a high rate of rejection and infectious complications. To improve these clinical results, it is mandatory to set up animal models to test alternative immunosuppressive regimens which may lead to immunotolerance.
AIMS—To determine the value of blockade of αLβ2 (LFA-1) and α4 and β7 integrins (α4β1, α4β7, and αEβ7) in the prevention of rejection of fetal small bowel grafts in mice and the effect of the association of calcineurin dependent drugs in anti-LFA-1 treated mice.
METHODS—Adult recipient mice engrafted with allogeneic fetal small bowel received a short course of anti-α4 and/or anti-LFA-1 monoclonal antibodies (mAb) with or without FK506 or cyclosporin A. In addition, in a set of experiment, β7−/− mice were used as recipients. Graft biopsies were performed and processed for standard histology.
RESULTS—Blockade of the pathways of the integrins α4 and β7 had a modest or no effect on intestinal graft survival. In contrast, transitory, short administration of anti-LFA-1 monoclonal antibody alone, when started before engraftment (day −1), allowed long term survival of intestinal grafts, even when associated with calcineurin dependent drugs. However, early withdrawal of FK506 reversed the immunosuppressive effect of anti-LFA-1 treatment.
CONCLUSION—These results suggest that firstly, anti-LFA-1, but not anti-α4 mAb treatment, may be useful in improving the results of intestinal transplantation, and secondly, that this treatment is not incompatible with long term administration of tacrolimus currently used in the prevention of small bowel graft rejection in humans.


Keywords: small bowel transplantation; integrins; calcineurin; tolerance; mouse