Divalent Metal Transporter 1 mRNA Research Articles

Genetic Determined Iron Starvation Signature in Friedreich's Ataxia.

Early studies in cellular models suggested an iron accumulation in Friedreich's ataxia (FA), yet findings from patients are lacking. The objective is to characterize systemic iron metabolism, body iron storages, and intracellular iron regulation in FA patients. In FA patients and matched healthy controls, we assessed serum iron parameters, regulatory hormones as well as the expression of regulatory proteins and iron distribution in peripheral blood mononuclear cells (PBMCs). We applied magnetic resonance imaging with R2*-relaxometry to quantify iron storages in the liver, spleen, and pancreas. Across all evaluations, we assessed the influence of the genetic severity as expressed by the length of the shorter GAA-expansion (GAA1). We recruited 40 FA patients (19 women). Compared to controls, FA patients displayed lower serum iron and transferrin saturation. Serum ferritin, hepcidin, mean corpuscular hemoglobin and mean corpuscular volume in FA inversely correlated with the GAA1-repeat length, indicating iron deficiency and restricted availability for erythropoiesis with increasing genetic severity. R2*-relaxometry revealed a reduction of splenic and hepatic iron stores in FA. Liver and spleen R2* values inversely correlated with the GAA1-repeat length. FA PBMCs displayed downregulation of ferritin and upregulation of transferrin receptor and divalent metal transporter-1 mRNA, particularly in patients with >500 GAA1-repeats. In FA PBMCs, intracellular iron was not increased, but shifted toward mitochondria. We provide evidence for a previously unrecognized iron starvation signature at systemic and cellular levels in FA patients, which is related to the underlying genetic severity. These findings challenge the use of systemic iron lowering therapies in FA. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Ferroportin Q248H Mutation Leads to a Moderate Iron Load in Mutant Slc40a1Q248H/Q248H Mice

BACKGROUND: In Africans, ferroportin (Scl40a1 or FPN) Q248H mutation is associated with increased iron load. FPN Q248H has high allele frequency (2.2-13.4%) and is linked to reduction of inflammation and reduced anemia in Africa. We previously showed that FPN Q248H resists degradation by physiologic hepcidin concentrations and thus may facilitate higher iron uptake. OBJECTIVES: to analyze the effect of FPN Q248H mutation in vivo in a knock-in Slc40a1Q248H mouse model on normal and high iron diet. METHODS: The study was approved by Howard University IACUC committee. The Slc40a1Q248H/WT mice were generated by Flp- FRT recombination on the C57BL/6 background and homozygous Slc40a1Q248H/Q248H mutant mice were produced in Howard University animal facility by breeding. Twenty-three weeks old homozygous and wild type C57BL/6 (WT) females were fed normal or 2% carbonyl iron diet. Total iron concentration and ferritin were measured in serum by ELISA. Expression of FPN, transferrin receptor (TFR), ferritin heavy chain (FHC), divalent metal transporter 1 (DMT1), duodenal cytochrome B (Dcytb), hepcidin, and IL-1β, IL-6, and IL-18 cytokines were measured by real-time RT-PCR. Iron deposition was determined by Perl's staining. Hepatic oxidative stress was measured by malondialdehyde ELISA. RESULTS: On normal iron diet, Slc40a1Q248H/Q248H mice grew normally and showed a tendency to increased serum ferritin, decreased serum iron and reduced hematocrit compared to WT mice. Levels of FPN,Dcytb and DMT1 mRNA were increased in duodenum of mutant mice. In the spleen of Slc40a1Q248H/Q248H mice, FPN,FTH1, TFR and DMT1 mRNAs were also elevated. In contrast, in the liver of Slc40a1Q248H/Q248H mice, levels of FPN and TFR mRNA were decreased, but hepcidin levels were increased compared to WT mice. There was a trend towards hepatic FTH1 level increase in the mutant mice. Hepatic expression of pro-inflammatory IL-1β, IL-6, and IL-18 cytokines mRNA was similar between mutant and WT mice. No iron accumulation was found in the liver, and iron accumulation in the spleen was comparable between WT and Slc40a1Q248H/Q248H mice. On high iron diet, Slc40a1Q248H/Q248H mice showed significant increase in serum ferritin compared to WT mice on normal diet. Levels of FPN mRNA remained increased whereas Dcytb and DMT1 mRNA levels were decreased in duodenum of the mutant mice. In the liver, FPN mRNA levels were further decreased in the mutant mice, and FTH1 levels were equally increased in WT and Slc40a1Q248H/Q248H mice. In the spleen, FPN mRNA levels were equally increased for WT and Slc40a1Q248H/Q248H mice. Spleen FTH1 mRNA levels were further elevated in mutant mice compared to WT mice. Higher iron accumulation was found in the liver and spleen of Slc40a1Q248H/Q248H mice compared to WT mice. Elevated levels of hepatic oxidative stress were observed in the liver of Slc40a1Q248H/Q248H mice. Expression of pro-inflammatory IL-1β, IL-6, and IL-18 cytokines mRNA remained low in the liver of Slc40a1Q248H/Q248H mice. CONCLUSION: Upregulation of duodenal FPN, Dcytb and DMT1 and splenic FPN,FTH1, TFR and DMT1 expression indicated increased iron absorption and recycling in female Slc40a1Q248H/Q248H mice. The mutantmice showed tendency to mild iron load in tissues as indicated by increased serum ferritin and higher ferritin levels in the spleen and liver. High iron diet led to significant iron accumulation in the liver and increased hepatic stress compared to WT mice. Surprisingly, hepatic levels of pro-inflammatory cytokines were reduced in the mutant mice. Taken together, our study of the first in vivo model of FPNQ248H mutation indicates that it alters iron metabolism and might reduce liver inflammation. ACKNOWLEDGEMENTS: This work was supported by NIH grants 1R01HL125005, 5U54MD007597, 1P30AI117970-06 and 1SC1HL150685.

Effect of Bis(maltolato)oxovanadium(IV) on Zinc, Copper, and Manganese Homeostasis and DMT1 mRNA Expression in Streptozotocin-Induced Hyperglycemic Rats.

Simple SummaryDiabetes mellitus is a disease that affects some metabolic processes, altering the metabolism of trace elements associated with the antioxidant defense system. The insulin-mimetic activity of vanadium has received attention in recent years. However, the use of vanadium as an antidiabetic agent is limited due to its pro-oxidant properties, which are probably caused by interactions with other trace elements. There is currently little information on the interactions between vanadium and zinc, copper and manganese in diabetic rats. For these reasons, the objective of this study was to examine whether diabetic rats treated with bis(maltolato)oxovanadium(IV) (BMOV(IV)) undergo alterations in zinc, copper and manganese homeostasis, and whether such changes are related to the gene expression of the divalent metal transporter 1 (DMT1). The results obtained from this study showed that the metabolism of Zn, Cu and, to a lesser extent, Mn is altered by diabetes and that BMOV(IV) administered to diabetic rats at a dose of 3 mg V/day reverted the alterations in zinc and copper homeostasis produced by diabetes. The effect of this supplementation was possibly mediated by a decrease in food intake and DMT1 gene expression.Our aim was to examine whether vanadium (IV) corrects alterations in zinc, copper and manganese homeostasis, observed in streptozotocin-induced hyperglycemic rats, and whether such changes are related to divalent metal transporter 1 (DMT1) mRNA expression, and antioxidant and proinflammatory parameters. Four groups of Wistar rats were examined: control; hyperglycemic (H); hyperglycemic treated with 1 mg V/day (HV); and hyperglycemic treated with 3 mg V/day (HVH). Vanadium was supplied in drinking water as bis(maltolato)oxovanadium(IV) for five weeks. Zinc, copper and manganese were measured in food, excreta, serum and tissues. DMT1 mRNA expression was quantified in the liver. Hyperglycemic rats showed increased Zn and Cu absorption and content in the liver, serum, kidneys and femurs; DMT1 expression also increased (p < 0.05 in all cases). HV rats showed no changes compared to H rats other than decreased DMT1 expression (p < 0.05). In the HVH group, decreased absorption and tissular content of studied elements (p < 0.05 in all cases) and DMT1 expression compared to H (p < 0.05) were observed. Liver zinc, copper and manganese content correlated positively with glutathione peroxidase activity and negatively with catalase activity (p < 0.05 in both cases). In conclusion, treatment with 3 mg V/d reverted the alterations in zinc and copper homeostasis caused by hyperglycemia, possibly facilitated by decreased DMT1 expression.

Increased DMT1 and FPN1 expression with enhanced iron absorption in ulcerative colitis human colon.

Iron deficiency anemia is a common complication of ulcerative colitis (UC) that can profoundly impact quality of life. Most iron absorption occurs in the duodenum via divalent metal transporter 1 (DMT1)-mediated uptake and ferroportin-1 (FPN1)-mediated export across the apical and basolateral membranes, respectively. However, the colon also contains iron transporters and can participate in iron absorption. Studies have shown increased duodenal DMT1 and FPN1 in patients with UC, but there is conflicting evidence about whether expression is altered in UC colon. We hypothesized that expression of colonic DMT1 and FPN1 will also increase to compensate for iron deficiency. Quantitative RT-PCR and Western blot analyses were performed on duodenal and colonic segmental (right colon, transverse colon, left colon, and rectum) biopsies obtained during colonoscopy. DMT1 mRNA and protein abundances in colonic segments were approximately equal to those in the duodenum, whereas colonic FPN1 mRNA and protein abundances of colonic segments were about one-quarter of those of the duodenum. DMT1 specific mRNA and protein abundances were increased twofold, whereas FPN1 mRNA and protein expressions were increased fivefold in UC distal colon. Immunofluorescence studies revealed enhanced expression of apical membrane- and basolateral membrane-localized DMT1 and FPN1 in UC human colon, respectively. Increased DMT1 expression was associated with enhanced 2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea (CISMBI, DMT1 specific inhibitor)-sensitive 59Fe uptake in UC human colon. We conclude from these results that patients with active UC have increased expression of colonic iron transporters and increased iron absorption, which may be targeted in the treatment of UC-related anemia.

Site-specific intestinal DMT1 silencing to mitigate iron absorption using pH-sensitive multi-compartmental nanoparticulate oral delivery system

Iron is a nutrient metal, but excess iron promotes tissue damage. Since iron chelation therapies exhibit multiple off-target toxicities, there is a substantial demand for more specific approaches to decrease iron burden in iron overload. While the divalent metal transporter 1 (DMT1) plays a well-established role in the absorption of dietary iron, up-regulation of intestinal DMT1 is associated with iron overload in both humans and rodents. Hence, we developed a novel pH-sensitive multi-compartmental particulate (MCP) oral delivery system that encapsulates DMT1 siRNA and validated its efficacy in mice. Using the gelatin NPs coated with Eudragit® L100-55, we demonstrated that DMT1 siRNA-loaded MCPs down-regulated DMT1 mRNA levels in the duodenum, which was consistent with decreased intestinal absorption of orally-administered 59Fe. Together, the Eudragit® L100-55-based oral siRNA delivery system could provide an effective strategy to specifically down-regulate duodenal DMT1 and mitigate iron absorption.

Broiler intestine DMT1 gene expression and bone characteristics, as affected by in ovo injection of different forms of manganese

This study was conducted to compare the effects of in ovo injection of different sources of manganese (Mn) on Divalent Metal Transporter 1 (DMT1) gene expression in the small intestine of broilers embryo and hatchling, and its subsequent effects on bone development. A total of 480 fertile eggs (ROSS 308) on embryonic day 17 were divided into 6 treatments with 3 replicates of 20 fertile eggs per replicate. Treatments were included: non-injected (NC) and diluent-injected controls (PC), manganese sulphate (MnS), manganese-methionine (Mn-Met), nano manganese (NMn) and nano manganese-methionine (NMn-Met) groups. Results showed that in ovo injection of different sources of Mn could change the DMT1 mRNA level of intestinal cells (p < .05). DMT1 mRNA level was up-regulated significantly (p < .05) by both nano forms of Mn on embryonic day 18 (E18) and post-hatch day 1 (P1) in all intestinal segments. On post-hatch day 7 (P7), bone length was higher in NMn treatment (p < .05) and tibia ash were affected by NMn and NMn-Met groups (p < .05). On (E18), the percentage of fresh and dry tibia weights were affected by Mn-Met, while on P14, the same characteristics were affected by NMn and MnS treatments, respectively (p < .05). The percentage of Mn in the tibiae ash content significantly (p < .05) enhanced by nano forms of Mn. It is concluded that in ovo injection of nano forms of Mn, either NMn or NMn-Met can be considered as a new strategy to improve Mn absorption in broilers intestine.HighlightsIn ovo injection of nanoparticles led to positive effects on DMT1 mRNA level in different intestinal segment of broilers embryo and hatchling.In ovo injection of nanoparticles enhance the percentage of Mn in the tibiae ash content of broilers embryo and hatchling.In ovo injection of Mn sources improved bone development.

Kinetics of iron absorption by in situ ligated small intestinal loops of broilers involved in iron transporters.

Two experiments were conducted with 28-d-old commercial male broilers to study the kinetics of iron (Fe) absorption and the effect of Fe treatment on divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) mRNA levels in in situ ligated segments from different small intestinal regions of broilers. In Exp. 1, we compared Fe absorption in 3 small intestinal segments at different post-perfusion time points after perfusion with 0.45 m of Fe as Fe sulfate (FeSO ∙ 7HO), and found that the Fe absorption in the duodenum at 30, 45, and 60 min was greater ( < 0.006) than that in the jejunum, and at 60 min, the Fe absorption in the duodenum was greater ( = 0.034) than that in the ileum. In addition, the Fe absorption at 30 min was more than 85.0% of the maximum absorption in each segment. In Exp. 2, a kinetic study of Fe absorption was performed with the duodenal, jejunal, and ileal loops perfused with solutions containing 0 (control), 0.11, 0.22, 0.45, 0.80, 1.79, or 3.58 m of Fe as FeSO 7HO. The Fe concentrations in perfusates were measured at 30 min after perfusion, and in the control group and the group treated with 0.45 m Fe as FeSO 7HO, the DMT1 and FPN1 mRNA levels in the ligated duodenum, jejunum, and ileum were analyzed. The kinetic curves of Fe absorption showed that Fe absorption in the duodenum and jejunum depended on a saturated carrier-mediated process. The maximum absorption rate in the duodenal segment was greater ( < 0.0001) than that in the jejunum (42.75 vs. 8.16 nmol × cm × min), and the Michaelis-Menten constant value was higher ( < 0.0001) in the duodenum than in the jejunum (6.16 vs. 1.31 m). In the ileum, however, the Fe absorption was a non-saturated diffusion process, and the diffusive constant was 3.54 × 10 cm × min. The DMT1 and FPN1 mRNA levels in the duodenum were greater ( < 0.0001) than those in the jejunum and ileum, and greater ( < 0.009) in the jejunum than in the ileum. No differences ( > 0.25) were detected in the DMT1 and FPN1 mRNA levels of the duodenum or jejunum and the DMT1 mRNA level of the ileum between the control and the 0.45 m Fe group, but Fe perfusion increased ( < 0.03) FPN1 mRNA level in the ileum. The above results indicate that the duodenum is the main site of Fe absorption in the small intestine of broilers, and Fe absorption in the duodenum and jejunum is a saturated carrier-mediated process, but a non-saturated diffusion process in the ileum.

Hematopoietic effect of fermented deer antler extract in iron deficient diet-induced anemic rats.

To assess the hematopoietic effects of fermented deer antler extract using a dietinduced anemic animal model to facilitate the utilization of fermented deer antler extract and its derived products. Thirty 3-week-old female Sprague-Dawley rats were treated for 5 weeks. The rats were randomly divided into 6 groups and treated as follows: control, saline; NFA200, non-fermented deer antler extract 200 mg/kg; NFA500, non-fermented deer antler extract 500 mg/kg; FAB200, fermented deer antler extract 200 mg/kg; FAB500, fermented deer antler extract 500 mg/kg; and PC, heme iron 0.2 mg/kg. Blood parameters, iron content in the liver and spleen, hepatic δ-aminolevulinic acid dehydrogenase (ALAD) activity and divalent metal transporter 1 (DMT1) mRNA expression were analyzed. No detectable significant differences were observed in blood parameters among groups. The decrease in the hepatic ALAD activity in anemic rats was significantly improved by fermented deer antler extract supplementation (P<0.05); however, non-fermented deer antler extract supplementation did not result in a significant improvement (P>0.05). The hepatic DMT1 mRNA expression level was increased significantly by supplementation with both the fermented deer antler extract and the non-fermented deer antler extract in a dose-dependent manner compared with nontreatment in anemic rats (P<0.05). The hematopoietic activity induced by deer antler extract in dietinduced anemic rats might be increased through the fermentation process.

Evidence there are at least three ways for copper to be taken up by enterocytes across the brush border, as investigated in the Caco2 cell model

The specifics of intestinal copper absorption by enterocytes and the effects of copper and iron status on this process are still not completely understood. The Caco2 polarized cell culture models for enterocytes was used to determine whether copper transporter 1 (CTR1) participates in Cu uptake across the brush border membrane and is located on that side of the cell monolayer; whether divalent metal transporter 1 (DMT1), a chloride dependent transporter, and another as yet unknown transporter are also involved; and whether the status of Cu and Fe of the cells has any effect. Quantitative PCR analysis of Caco2 cells grown on six‐well tissue culture plates showed that CTR1 and DMT1 mRNA were expressed in about equal proportions relative to 18S rRNA. Both DMT1 and CTR1 expression increased overall when the cell culture medium was supplemented with 1 uM Cu(II) histidine for 24 hours. Potential effects of changes in iron status are under investigation. To test if increased mRNA expression resulted in a greater rate of copper uptake, cells were seeded on bicameral chambers and allowed to polarize. Transepithelial electrical resistance was measured to determine the formation of tight junctions. Cells were then untreated or pretreated with 5uM copper to induce copper excess or triethylenetetramine (TETA) to induce copper deficiency. Rates of uptake of Cu(I) (5uM) were measured by following uptake of 67Cu into cells and the basolateral fluid over 1h, after application to the apical (brush border) side of the monolayer, in serum‐free HEPES buffered medium. Copper supplementation did not enhance the rate of copper uptake, but copper deficiency significantly reduced it by 20–35%. Supplementation with 5uM Fe(II) (as the nitrilotriacetate complex) to induce iron excess, or 10 uM desferrioxamine (to induce deficiency) had little to no effect on the ability of the monolayers to take up copper across the brush border. To check for activity of a chloride dependent transporter, uptake of 67Cu(I) across the apical portion of the monolayer was measured in HEPES buffered medium in which Na2SO4, MgSO4, and K2SO4 was substituted for NaCl, MgCl2, and KCl. Cells were also incubated with and without silver in order to also determine the importance of CTR1 to dietary copper uptake. Generally, copper uptake was reduced by a small amount by 10–20% when chloride was replaced with sulfate. Addition of Ag(I) (50 uM) reduced uptake 20–50%, the largest effect being in the presence of silver and sulfate, thus leaving at least half of the uptake occurring through other means. The assumed role of a reductase in copper uptake was also examined. By qPCR, we found that Caco2 cells expressed STEAPs 2 and 3 but not 4 mRNA, and did not express duodenal cytochrome B. Measurements of Cu(II) reductase activity by the method of Wyman et al, using 50 uM Cu(II)‐NTA and 200 uM bathocuproine disulfonate, indicated the presence of cell surface reductase activity. Efforts to knock out DMT1 and STEAP2 by CRISPR/Cas9 are currently underway to see their impact on copper absorption, as is confocal microscopy to definitively locate CTR1. We conclude that copper absorption by enterocytes is more complex than generally assumed, and there may be an as yet unidentified copper uptake transporter in the brush border membrane of enterocytes.

Different Zinc Sources Have Diverse Impacts on Gene Expression of Zinc Absorption Related Transporters in Intestinal Porcine Epithelial Cells.

This study was conducted to investigate the effects of zinc sources on gene expression of zinc-related transporters in intestinal porcine epithelial cells (IPEC-1). IPEC-1 cells were treated with zinc glycine chelate (Zn-Gly), zinc methionine (Zn-Met), and zinc sulfate (ZnSO4), respectively, for measurement of cell viability. Then, the relative expression of zinc-related transporters in IPEC-1 in response to different zinc sources (50μmol/L zinc) was measured. Zinc transporter SLC39A4 (ZIP4) expression was selectively silenced to assess the function of ZIP4 in inorganic and organic zinc absorption. The result showed that Zn-Gly and Zn-Met had lower cell damage compared with ZnSO4 on the same zinc levels. Different zinc sources improved the expression of metallothionein1 (MT1) and zinc transporter SLC30A1 (ZnT1) messenger RNA (mRNA) compared with the control (P<0.05), while ZIP4 decreased (P<0.05) in response to zinc addition. MT1 and ZnT1 mRNA expressions in Zn-Gly and Zn-Met were higher than those in ZnSO4, and ZIP4 mRNA expression in Zn-Met was the lowest among three kinds of zinc sources (P<0.05). Expression of divalent metal transporter 1 (DMT1) mRNA in control was significantly higher (P<0.05) than added different zinc sources groups. Silencing of ZIP4 significantly decreased MT1 mRNA expression in ZnSO4 and Zn-Gly treatments, reduced zinc absorption rate, and increased DMT1 mRNA expression in ZnSO4 compared with negative control. In summary, different zinc sources could improve zinc status on IPEC-1 cells and organic zinc had lower cell damage compared with ZnSO4. Moreover, Zn-Gly and Zn-Met are more efficient on zinc absorption according to the expression of various zinc-related transporters MT1, ZIP4, ZnT1, and DMT1. ZIP4 played a direct role in inorganic zinc uptake, and the absorption of zinc in Zn-Gly depends on ZIP4 partly, while absorption of Zn-Met is less dependent on ZIP4.

Increased duodenal iron absorption through up-regulation of divalent metal transporter 1 from enhancement of iron regulatory protein 1 activity in patients with nonalcoholic steatohepatitis.

Increased hepatic iron accumulation is thought to be involved in the pathogenesis of nonalcoholic steatohepatitis (NASH). Hepatic iron accumulation, as well as oxidative DNA damage, is significantly increased in NASH livers. However, the precise mechanism of iron accumulation in the NASH liver remains unclear. In this study, 40 cases with a diagnosis of NASH (n = 25) or simple steatosis (SS; n = 15) by liver biopsy were enrolled. An oral iron absorption test (OIAT) was used, in which 100 mg of sodium ferrous citrate was administered to each individual. The OIAT showed that absorption of iron from the gastrointestinal (GI) tract was increased significantly in NASH patients, compared to SS and control subjects. Iron reduction therapy was effective in patients with NASH, who exhibited iron deposition in the liver and no alanine aminotransferase improvement after other therapies (n = 9). Serum hepcidin concentration and messenger RNA (mRNA) levels of divalent metal transporter 1 (DMT1) also were significantly elevated in patients with NASH. OIAT results were correlated with grade of liver iron accumulation and DMT1 mRNA levels. Then, we demonstrated that DMT1 mRNA levels increased significantly in Caco-2/TC7 cell monolayers cultured in transwells with serum from NASH patients. An electrophoresis mobility shift assay showed activation of iron regulatory protein (IRP) in those cells, and IRP1 small interfering RNA clearly inhibited the increase of DMT1 mRNA levels. In spite of elevation of serum hepcidin, iron absorption from the GI tract increased through up-regulation of DMT1 by IRP1 activation by humoral factor(s) in sera of patients with NASH.

Participation of divalent cation transporter DMT1 in the uptake of inorganic mercury.

Mercury (Hg) is found in food in various chemical forms, which differ in terms of accumulation, transport, and toxicity. Although methylmercury (CH3Hg) is the predominant mercury species in the diet, contributed mostly by seafood products, there is also a contribution of inorganic mercury [Hg(II)] from vegetables, cereals, and seafood products. The main pathway for exposure to mercury is oral, and therefore the gastrointestinal mucosa is the first barrier that the contaminant meets when it enters the systemic circulation. However, the transport mechanisms responsible for the process of mercury absorption are not known. The aim of this study is to evaluate the possible participation of divalent metal transporter 1 (DMT1) in Hg(II) intestinal uptake. For this purpose, we have used various complementary approaches. We have studied mercury acquisition in a Saccharomyces cerevisiae strain expressing murine DMT1. Moreover, we have evaluated the effect of a reduction of DMT1 expression in Caco-2 cells, by means of small interfering RNA and of treatment with hepcidin, on mercury uptake and transport. The results show that expression of the transporter DMT1 in yeast produces an increase in Hg(II) accumulation. Furthermore, a decrease in the levels of DMT1 mRNA in Caco-2 cells in various stages of differentiation leads to a reduction in cellular accumulation and apical-basolateral transport of Hg(II). These data point clearly to the mediation of the divalent cation transporter DMT1 in the entry of Hg(II) into the intestinal epithelium.

Kinetics of manganese transport and gene expressions of manganese transport carriers in Caco-2 cell monolayers

Two experiments were conducted to investigate the kinetics of manganese (Mn) transport in Caco-2 cell monolayers and the gene expressions of Mn transport carriers in apical (AP) and basolateral (BL) membranes. In experiment 1, the cells were treated with the medium containing 146 μmol/L of Mn (MnSO4·H2O). Both the uptake and transport of Mn from AP-BL or from BL-AP at different time-points were assessed to determine the optimal time for kinetics of Mn transport. The transport of Mn increased linearly with higher efficiency values in AP-BL than in BL-AP direction, however, the uptake of Mn revealed an asymptotic pattern within 120 min. In experiment 2, the kinetics of Mn transport in AP-BL was determined with media containing Mn concentrations from 0 to 2,500 μmol/L at 40 and 120 min, respectively, and mRNA levels of divalent metal transporter 1 (DMT1) and ferroportin (FPN1) were determined in Caco-2 cells treated with the medium containing 0 or 800 μmol/L of Mn for 120 min. The kinetics of Mn transport showed a carrier-mediated process when Mn concentrations were lower than 1,000 μmol/L and a linear increment when Mn concentrations exceeded 1,000 μmol/L at either 40 or 120 min. Mn treatment decreased (P < 0.01) DMT1 mRNA level and increased (P < 0.01) FPN1 mRNA level. The results from the present study suggested that Mn transport in AP-BL fit both carrier-mediated saturable and non-saturable diffusion processes, and Mn transport carriers DMT1 and FPN1 mediate the apical uptake and basolateral exit of Mn in Caco-2 cells.

Increasing dietary protein selectively upregulates the DMT1 isoform that contains an iron responsive element

Intestinal iron absorption is a critical regulator of whole body iron homeostasis. The Divalent Metal Transporter 1 (DMT1) is a nonheme iron importer found on the brush border membrane of enterocytes. Its genetic absence in experimental animals leads to markedly impaired intestinal iron absorption. Recent data from our laboratory demonstrate that increasing dietary protein in rats enhances intestinal iron absorption by 50%, and that this is associated with a 3.8‐fold induction in DMT1 mRNA expression. To determine whether dietary protein affects the expression of the isoform of DMT1 that includes an iron responsive element (IRE), isoform‐specific qPCR was performed using RNA isolated from the duodenum of rats consuming either a 20% (control) or 40% (high) protein diet for 1 wk. Rats on the 40% protein diet exhibited significantly greater DMT1A‐IRE mRNA expression compared to animals consuming the 20% protein diet (fold change: 2.8; p&lt;0.01), while no significant change was observed in the level of expression of the DMT1A isoform that does not include an IRE. These findings indicate that duodenal DMT1A‐IRE is selectively and significantly upregulated in response to increasing dietary protein.

Release of iron from cellular iron stores in lysosomes: Potential involvement of divalent metal transporter 1 (DMT1) and common metabolites

Excess cellular Fe is stored in ferritin (Ft), and this is drawn upon when cells or organisms become Fe deficient. In several cell types, lysosomal protease inhibitors prevent release of Fe from Ft, indicating that degradation of the protein “shell” surrounding its Fe is required. We have begun to address several questions on the details of this process. Confocal microscopic studies with hepatoma cells showed that lysosomal protease inhibitors caused the accumulation of Ft in lysosomes, confirmed by ELISA and Fe assays of organelles separated in density gradients. The shift of Ft into lysosomes induced by incubation in a low Fe medium was accompanied by increased co‐localization of DMT1. Solubilization of the Ft ferrihydrate crystallites by common metabolites was demonstrated. Incubation of purified (insoluble) crystallites with ascorbate, glutathione (GSH) or citrate (pH 5.5 or 7) markedly increased levels of soluble Fe. Treatment of whole cells with ascorbate, GSH or other reducing agents, and manipulation of intracellular GSH concentrations, did not induce ferritin Fe release. Knockdown (90%) of DMT1 mRNA by exposure to specific siRNA for 72 h did not affect the rate of Ft Fe release. However, 72 h was not sufficient to appreciatively knock down expression of DMT1 protein. Our findings suggest that after degradation, Ft Fe is easily solubilized and returned to the cytosol via DMT1.

Relative contribution of CTR1 and DMT1 in copper transport by the blood–CSF barrier: Implication in manganese-induced neurotoxicity

The homeostasis of copper (Cu) in the cerebrospinal fluid (CSF) is partially regulated by the Cu transporter-1 (CTR1) and divalent metal transporter-1 (DMT1) at the blood–CSF barrier (BCB) in the choroid plexus. Data from human and animal studies suggest an increased Cu concentration in blood, CSF, and brains following in vivo manganese (Mn) exposure. This study was designed to investigate the relative role of CTR1 and DMT1 in Cu transport under normal or Mn-exposed conditions using an immortalized choroidal Z310 cell line. Mn exposure in vitro resulted in an increased cellular 64Cu uptake and the up-regulation of both CTR1 and DMT1. Knocking down CTR1 by siRNA counteracted the Mn-induced increase of 64Cu uptake, while knocking down DMT1 siRNA resulted in an increased cellular 64Cu uptake in Mn-exposed cells. To distinguish the roles of CTR1 and DMT1 in Cu transport, the Z310 cell-based tetracycline (Tet)-inducible CTR1 and DMT1 expression cell lines were developed, namely iZCTR1 and iZDMT1 cells, respectively. In iZCTR1 cells, Tet induction led to a robust increase (25 fold) of 64Cu uptake with the time course corresponding to the increased CTR1. Induction of DMT1 by Tet in iZDMT1 cells, however, resulted in only a slight increase of 64Cu uptake in contrast to a substantial increase in DMT1 mRNA and protein expression. These data indicate that CTR1, but not DMT1, plays an essential role in transporting Cu by the BCB in the choroid plexus. Mn-induced cellular overload of Cu at the BCB is due, primarily, to Mn-induced over-expression of CTR1.

Manganese source affects manganese transport and gene expression of divalent metal transporter 1 in the small intestine of broilers

In the present study, two experiments were conducted to investigate the effect of Mn source on Mn transport and the expression of a Mn transporter, divalent metal transporter 1 (DMT1), in the small intestine of broilers. In Expt 1, in situ ligated duodenal loops from Mn-deficient chicks (29-d-old) were perfused with solutions containing 0-8.74 mmol Mn/l from either MnSO4, or one of two organic chelates of Mn and amino acids with moderate (OM) or strong (OS) chelation strength (Q(f)) up to 30 min. In Expt 2, Mn-deficient intact broilers (14-d-old) were fed a control diet (12.45 mg Mn/kg) or the control diet supplemented with 100 mg Mn/kg as one of all Mn sources for 14 d. The uptake kinetics of Mn from different Mn sources in the ligated duodenal loops followed a saturable process as determined by regression analysis of concentration-dependent uptake rates. The maximum transport rate (Jmax) and K(m) values, and DMT1 mRNA levels in the ligated duodenal loops were higher (P < 0.01) for OM and OS than for MnSO4. DMT1 mRNA levels were much higher (P < 0.01) in the duodenum than in the jejunum and ileum. Both DMT1 mRNA levels in the duodenum and plasma Mn contents from the hepatic portal vein of intact chicks on day 14 post-feeding increased (P < 0.05) in the following order: control < MnSO4 < OM < OS. These results indicated that organic Mn sources with stronger Q(f) showed higher Mn transport and absorption, and DMT1 might be involved in the regulation of organic Mn transport in the proximal small intestine of broilers.

Divalent Metal Transporter 1 Expression and Regulation in Human Placenta

Divalent metal transporter 1 (DMT1) is likely responsible for the release of iron from endosomes to the cytoplasm in placental syncytiotrophoblasts (STB). To determine the localization and the regulation of DMT1 expression by iron directly in placenta, the expression of DMT1 in human term placental tissues and BeWo cells (human placental choriocarcinoma cell line) was detected and the change in expression in response to different iron treatments on BeWo cells was observed. DMT1 was shown to be most prominent near the maternal side in human term placenta and predominantly in the cytoplasm of BeWo cells. BeWo cells were treated with desferrioxamine (DFO) and human holotransferrin (hTf-2Fe) and it was found that both DMT1 mRNA and protein increased significantly with DFO treatment and decreased with hTf-2Fe treatment. Further, DMT1 mRNA responded more significantly to treatments if it possessed an iron-responsive element than mRNA without this element. This study indicated that DMT1 is likely involved in endosomal iron transport in placental STB and placental DMT1 + IRE expression was primarily regulated by the IRE/IRP mechanism.

Hepcidin Treatment Modulates the Expression of Divalent Metal Transporter-1, Ceruloplasmin, and Ferroportin-1 in the Rat Cerebral Cortex and Hippocampus

Elevated iron levels are considered to play a role in the neurodegenerative mechanisms that underlie Alzheimer's and Parkinson's disease. The linkage between hepcidin (Hepc) and ferroportin-1 (FPN1), the divalent metal transporter 1 (DMT1), and ceruloplasmin (CP) in the brain is unknown. To discern the role of Hepc in regulating the expression of these proteins, we investigated FPN1, DMT1, and CP protein and mRNA expression in the brain after the intracerebroventricular injection of Hepc. Our results show that after Hepc injection, expression of FPN1 mRNA and FPN1 protein was inhibited in the cerebral cortex and hippocampus. Furthermore, we showed a clear change of DMT1 and CP protein and mRNA levels in the brain. The immunohistochemical analysis revealed an increase of DMT1 and a decrease of CP levels. Semi-quantitative analysis using PCR methods showed an increase of DMT1(+IRE) mRNA, and a decrease of DMT1(-IRE) mRNA and CP mRNA levels. Since alterations in iron levels in the brain are causally linked to degenerative conditions such as Alzheimer's disease, an improved understanding of the regulation of iron transport protein expression such as FPN1, DMT1, and CP could lead to novel strategies for treatments.

DMT1 is not essential for Cu uptake from blood by internal organs

Divalent metal transporter 1 (DMT1) is the main mechanism by which Fe(II) is imported into enterocytes from the intestinal lumen. This uptake is inhibited by Cu(I) and some divalent metal ions; knockdown of expression is accompanied by a reduction in Cu as well as Fe uptake. In contrast, cells of internal organs mainly obtain their Fe from blood plasma transferrin by receptor mediated endocytosis, and DMT1 is mainly localized in endosomes, implying little would be at the cell surface to mediate Cu uptake. To verify this, we compared initial (1h) rates of uptake of tracer 64Cu(II) by organs of virgin and lactating Belgrade rats with those in the background (Fisher) strain. Lactation greatly increased 64Cu uptake by the mammary gland and reduced uptake by liver; but no significant differences ascribable to lack of DMT1 were detected in any of the organs tested. HepG2 cells were used to further explore potential involvement of DMT1 by measuring initial (30 min) rates of 64Cu uptake from purified plasma proteins. No inhibition by Fe(II) was observed. Conversely, uptake of 59Fe(II) (± ascorbate) was not inhibited by excess Cu(I) or Cu(II). Pretreatment of cells with Fe for 24h enhanced rates of 59Fe(II) uptake but did not alter those for Cu. Fe pretreatment did not alter DMT1 mRNA or protein expression. We conclude that DMT1 is unlikely to be important for uptake of Cu by cells of internal organs. Supported in part by USPHS Grant HD 46949.