Divalent Metal Transporter 1 Expression Research Articles

Lower Expression of Ndfip1 Is Associated With Alzheimer Disease Pathogenesis Through Decreasing DMT1 Degradation and Increasing Iron Influx.

We have previously reported that high expression of divalent metal transporter 1 (DMT1) plays a crucial role in iron dyshomeostasis and β-amyloid (Aβ) peptide generation in the brain of Alzheimer’s disease (AD). Recent studies have shown that Nedd4 family interacting protein 1 (Ndfip1) can degrade DMT1 through ubiquitination pathway and reduce the accumulation of intracellular iron. The present study aims to evaluate whether Ndfip1 is involved in AD pathogenesis through mediating DMT1 degradation and iron metabolism. β-amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mouse and Ndfip1 transfected SH-SY5Y cells were used in this study. Immunohistochemistry and Western blot were performed to examine the distribution and expression levels of Ndfip1 and DMT1. In addition, ELISA and calcein fluorescence were carried out for analyzing the levels of Aβ peptide and iron influx, respectively. The results showed that Ndfip1 immunoreactivity was decreased in the cortex and hippocampus of APP/PS1 mice, compared with wild type (WT) controls. Colocalization of Ndfip1 and Aβ within senile plaques could be observed. Immunoblot analyses showed that low expression of Ndfip1 and high expression of DMT1 proteins were detected in APP/PS1 mouse brain, compared with age-matched WT animals. Overexpression of Ndfip1 down-regulated DMT1 expression, and reduced iron influx and Aβ secretion in SH-SY5Y cells. Further, overexpressed Ndfip1 significantly attenuated iron-induced cell damage in Ndfip1 transfected cells. The present study suggests that lower expression of Ndfip1 might be associated with the pathogenesis of AD, through decreasing DMT1 degradation and increasing iron accumulation in the brain.

Bacteria-Carried Iron Oxide Nanoparticles for Treatment of Anemia.

The efficiency of maghemite nanoparticles for the treatment of anemia was sensibly higher when nanoparticles were incorporated onto the probiotic bacterium Lactobacillus fermentum (MNP-bacteria) than when administrated as uncoated nanoparticles (MNP). Plasma iron and hemoglobin, intestine expression of divalent metal transporter 1 (DMT1) and duodenal Cytochrome b (DcytB), as well as hepatic expression of the hormone hepcidin were fully restored to healthy levels after administration of MNP-bacteria but not of MNP. A magnetic study on biodistribution and biodegradation showed accumulation of maghemite nanoparticles in intestine lumen when MNP-bacteria were administrated. In contrast, MNP barely reached intestine. In vivo MRI studies suggested the internalization of MNP-bacteria into enterocytes, which did not occur with MNP. Transmission electronic microscopy confirmed this internalization. The collective analysis of results point out that L. fermentum is an excellent carrier to overcome the stomach medium and drive maghemite nanoparticles to intestine, where iron absorption occurs. Due the probiotic ability to adhere to the gut wall, MNP-bacteria internalize into the enterocyte, where maghemite nanoparticles are delivered, providing an adequate iron level into enterocyte. This paper advances a new route for effective iron absorption in the treatment of anemia.

Expression of Duodenal Iron Transporter Proteins in Diabetic Patients with and without Iron Deficiency Anemia.

The role of iron transport proteins in the pathogenesis of anemia in patients with diabetes mellitus (T2DM) is still unclear. We investigated the expression of duodenal transporter proteins in diabetic patients with and without iron deficiency anemia (IDA). Methods. Overall, 39 patients were included: 16 with T2DM and IDA (group A), 11 with T2DM without IDA (group B), and 12 controls (group C). Duodenal mucosal expression of divalent metal transporter 1 (DMT1), ferroportin 1 (FPN), hephaestin (HEPH), and transferrin receptor 1 (TfR) was evaluated by Western blotting. Chronic disease activity markers were measured as well. Results. FPN expression was increased in group A compared to group B and controls: 1.17 (0.72–1.46), 0.76 (0.53–1.04), and 0.71 (0.64–0.86), respectively (p = 0.011). TfR levels were over expressed in groups A and B compared to controls: 0.39 (0.26–0.61), 0.36 (0.24–0.43), and 0.18 (0.16–0.24), respectively, (p = 0.004). The three groups did not differ significantly with regard to cellular HEPH and DMT1 expression. The normal CRP and serum ferritin levels, accompanied with normal FPN among diabetic patients without IDA, do not support the association of IDA with chronic inflammatory state. Conclusion. In patients with T2DM and IDA, duodenal iron transport protein expression might be dependent on body iron stores rather than by chronic inflammation or diabetes per se.

METABOLIC CHANGES INDUCED BY BUSHENHUOXUE GRANULES ON STRIATUM AND SUBSTANTIA NIGRA IN A RAT MODEL OF PARKINSON’S DISEASE

Background: Parkinson’s disease is a neurodegenerative disease, while its mechanism is still unclear. Long-term levodopa-based treatment leads to decreased response or loss of response, as well as severe side effects. Our previous study has proved that Bushenhuoxue Granules have effects on Parkinson’s disease, but the underlying mechanism is still need to be explored. Our research is to investigate the mechanisms of Bushenhuoxue Granules on Parkinson’s disease (PD) by examining changes in the expression of the adenosine A2A receptor、vesicular monoamine transporter 2 (VMAT2)、divalent metal transporter 1(DMT1) and nuclear factor E2 related (Nrf2) in a rat model of Parkinson’s disease (PD) . 
 Materials and Methods: Changes in the apomorphine (APO)-induced rotational behavior of rats were observed after treatment. Immunofluorescence and immunohistochemistry were performed to investigate changes in adenosine A2A receptor 、VMAT2、DMT1 and Nrf2 expression in the rat striatum and substantia nigra. 
 Results: Rotations after treatment were199.11 ± 27.16, which significantly decreased compared with that before treatment ( 273.0 ± 44.61, p < 0.01). Adenosine A2A receptor expression in the striatum was 3.10 ± 0.34 significantly increased in the model group and decreased in the normal control group, whereas the expression level in the Bushenhuoxue group was 1.13 ± 0.23,p < 0.05 between the two control groups. No adenosine A2A receptor expression was observed in the substantia nigra. VMAT2 expression in the rat striatum was 23.20 ± 2.68 and substantia nigra was 15.98 ± 0.70 increased in the normal control group. They were 8.99 ± 0.48 in the rat striatum and 8.45 ± 0.59 substantia nigra significantly decreased in the model control group, whereas the expression level in the Bushenhuoxue group was 15.36 ± 0.89 in the rat striatum and 11.69 ± 1.17 in the rat substantia nigra (p < 0.05), also between the two control groups. DMT1 expression in the rat striatum was 3.30 ± 0.30 and substantia nigra was 6.56 ± 0.64 decreased in the normal control group. They were 7.92 ± 0.52 in the rat striatum and 12.76 ± 0.86 substantia nigra significantly increased in the model control group, whereas the expression level in the Bushenhuoxue group was 6.17 ± 0.27 in the rat striatum and 9.13 ± 0.44 in the rat substantia nigra (p < 0.05), also between the two control groups. Nrf2 expression in the rat striatum was 7.90 ± 0.29 and substantia nigra was 15.22 ± 1.22 increased in the normal control group. They were 3.09 ± 0.43 in the rat striatum and 8.57 ± 0.54 substantia nigra significantly decreased in the model control group, whereas the expression level in the Bushenhuoxue group was 5.00 ± 0.34 in the rat striatum and 12.46 ± 0.62 in the rat substantia nigra (p< 0.05), also between the two control groups. Conclusion: Bushenhuoxue Granules significantly improved the rotational behavior of PD’s rats, decreased adenosine A2A receptor expression, and increased VMAT2 expression; decreased DMT1 expression, and increased Nfr2 expression.

Interleukin-6 regulates iron-related proteins through c-Jun N-terminal kinase activation in BV2 microglial cell lines.

Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra (SN) and subsequent DA depletion in the striatum. Microglia activation and nigral iron accumulation play important roles in the pathogenesis of PD. Activated microglia show increased iron deposits. However, the relationship between microglia activation and iron accumulation remains unclear. In the present study, we aimed to determine how iron levels affect interleukin-6 (IL-6) synthesis, and the effect of IL-6 on cellular iron metabolism in BV2 microglial cells.IL-6 mRNA was up-regulated after FAC treatment for 12 h in BV2 cells. Iron regulatory protein 1 (IRP1) and divalent metal transporter 1 (DMT1) were up-regulated and iron exporter ferroportin 1 (FPN1) was down-regulated in BV2 cells after 24 h of IL-6 treatment. Phosphorylated JNK increased significantly compared to the control after BV2 cells were treated with IL-6 for 1 h. Pretreatment with SP600125 attenuated the up-regulation of IRP1 and DMT1 and down-regulation of FPN1 (compared to IL-6-treated group). These results suggest that iron load could increase IL-6 mRNA expression in BV2 cells. Further, IL-6 likely up-regulates IRP1 and DMT1 expression and down-regulates FPN1 expression in BV2 microglial cells through JNK signaling pathways.

Altered expression of intestinal duodenal cytochrome b and divalent metal transporter 1 might be associated with cardio-renal anemia syndrome.

The interaction among heart failure (HF), chronic kidney disease (CKD), and anemia is called cardio-renal anemia syndrome. The mechanism of anemia in cardio-renal anemia syndrome is complex and remains completely unknown. We have previously reported that impaired intestinal iron transporters may contribute to the mechanism of anemia in HF using in vivo HF model rats. In this study, we assessed intestinal iron transporters in CKD model rats to investigate the association of intestinal iron transporters in the mechanism of cardio-renal anemia syndrome. CKD was induced by 5/6 nephrectomy in Sprague-Dawley rats. Sham-operated rats served as a control. After 24-week surgery, CKD rats exhibited normocytic normochromic anemia and normal serum erythropoietin levels despite of anemia. Serum iron levels were decreased in CKD rats compared with the controls. Of interest, intestinal expression of critical iron importers, such as duodenal cytochrome b (Dcyt-b) and divalent metal transporter 1 (DMT-1), was decreased in CKD rats compared with the controls. On the other hand, intestinal expression of ferroportin, an intestinal iron exporter, was not different in the control and CKD groups. Moreover, hepatic expression of hepcidin, a regulator of iron homeostasis, did not differ between the control and CKD groups. These results suggest that impaired intestinal expression of Dcyt-b and DMT-1 might be associated with the reduction of an iron uptake in CKD. Taken together, impaired these intestinal iron transporters may become a novel therapeutic target for cardio-renal anemia syndrome.

Effect of iron source on iron absorption and gene expression of iron transporters in the ligated duodenal loops of broilers.

This experiment was conducted to investigate the effect of iron source on Fe absorption and the gene expression of divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) in the ligated duodenal loops of broilers. The in situ ligated duodenal loops from Fe-deficient broiler chicks (28-d-old) were perfused with Fe solutions containing 0 to 14.33 mmol Fe/L from 1 of the following: Fe sulfate (FeSO∙7HO), Fe methionine with weak chelation strength (Fe-Met W; chelation strength is expressed as quotient of formation [Q] value, Q = 1.37), Fe proteinate with moderate chelation strength (Fe-Prot M; Q = 43.6), and Fe proteinate with extremely strong chelation strength (Fe-Prot ES; Q = 8,590) for up to 30 min. The gene expression of DMT1 and FPN1 in the duodenal loops from the control group and the groups treated with 3.58 mmol Fe/L from 1 of 4 Fe sources was analyzed. The absorption kinetics of Fe from different Fe sources in the duodenum followed a saturated carrier-dependent transport process. The maximum transport rate (J) values in the duodenum were greater ( < 0.03) for Fe-Prot ES and Fe-Prot M than for Fe-Met W and FeSO∙7HO. The Fe perfusion inhibited ( < 0.05) the mRNA expression of but enhanced ( < 0.0008) the mRNA expression of in the duodenum and had no effect ( > 0.14) on the protein expression levels of the 2 transporters. These results indicated that organic Fe sources with greater Q values showed higher Fe absorption; however, all Fe sources followed the same saturated carrier-dependent transport process in the duodenum, and DMT1 and FPN1 might participate in Fe absorption in the duodenum of broilers regardless of Fe source.

Protoporphyrin IX regulates peripheral benzodiazepine receptor associated protein 7 (PAP7) and divalent metal transporter 1 (DMT1) in K562 cells

BackgroundProtoporphyrin IX (PP IX), the immediate precursor to heme, combines with ferrous iron to make this product. The effects of exogenous PP IX on iron metabolism remain to be elucidated. Peripheral-type benzodiazepine receptor (PBR) is implicated in the transport of coproporphyrinogen into the mitochondria for conversion to PP IX. We have demonstrated that PBR-Associated Protein 7 (PAP7) bound to the Iron Responsive Element (IRE) isoform of divalent metal transporter 1 (DMT1). PP IX and PAP7 are ligands for PBR, thus, we hypothesized that PAP7 interact with PP IX via PBR. MethodsWe have examined in K562 cells, which can be induced to undergo erythroid differentiation by PP IX and hemin, the effects of PP IX on the expression of PAP7 and other proteins involved in cellular iron metabolism, transferrin receptor 1 (TfR1), DMT1, ferritin heavy chain (FTH), c-Myc and C/EBPα by western blot and quantitative real time PCR analyses. ResultsPP IX significantly decreased mRNA levels of DMT1 (IRE) and (non-IRE) from 4h. PP IX markedly decreased protein levels of C/EBPα, PAP7 and DMT1. In contrast, hemin, which like PP IX also induces K562 cell differentiation, had no effect on PAP7 or DMT1 expression. ConclusionWe hypothesize that PP IX binds to PBR displacing PAP7 protein, which is then degraded, decreasing the interaction of PAP7 with DMT1 (IRE) and resulting in increased turnover of DMT1. General significanceThese results suggest that exogenous PP IX disrupts iron metabolism by decreasing the protein expression levels of PAP7, DMT1 and C/EBPα.

Cancer cell-specific mitochondrial reactive oxygen species promote non-heme iron uptake and enhance the proliferation of gastric epithelial cancer cell.

Iron is an essential nutrient for life and is involved in many important processes such as oxygen transport and DNA synthesis. However, excess amounts of iron can cause carcinogenesis by producing reactive oxygen species. Thus, the cellular transport of iron must be tightly regulated. In the human body, iron may be present as heme or non-heme iron. The mechanisms governing the cellular transport of these forms have not been clearly elucidated. We previously reported that the expression of an important heme transporter, heme carrier protein 1 was regulated by cancer-specific reactive oxygen species derived from mitochondria. In this study, we have asked if mitochondrial reactive oxygen species may also be related with non-heme iron transport. In order to address this question, we have investigated the relationship between mitochondrial reactive oxygen species and accumulation of cellular non-heme iron in a rat gastric normal, cancer and manganese superoxide dismutase-overexpressing cancer cell line, in which reactive oxygen species from mitochondria are specifically scavenged. We have also analyzed the expression of divalent metal transporter 1 and ferroprotin, involved in the incorporation and excretion of non-heme iron, respectively, as well as a hypoxia-related transcription factor HIF-1α, to elucidate the molecular mechanism of non-heme iron accumulation.

Regulation of DMT1 on autophagy and apoptosis in osteoblast.

Iron overload has recently been associated with the changes in the bone microstructure that occur in osteoporosis. However, the effect of iron overload on osteoblasts is unclear. The purpose of this study was to explore the function of divalent metal transporter 1 (DMT1) in the pathological processes of osteoporosis. Osteoblast hFOB1.19 cells were cultured in medium supplemented with different concentrations (0, 50, 100, 200, 300, 400, 500 μmol/L) of ferric ammonium citrate (FAC) as a donor of ferric ions. We used western blotting and immunofluorescence to determine the levels of DMT1 after treatment with FAC. Apoptosis was evaluated by detecting the levels of cleaved caspase 3, BCL2, and BAX with western blotting. Autophagy was evaluated by detecting the levels of LC3 with western blotting and immunofluorescence. Beclin-1 expression was also assessed with western blotting. The autophagy inhibitor 3-methyladenine was used to determine whether autophagy affects the apoptosis induced by FAC. Our results show that FAC increased the levels of DMT1, upregulated the expression of BCL2, and downregulated the apoptosis-related proteins cleaved caspase 3 and BAX. Both LC3I/LC3II levels and beclin-1 were also increased, indicating that FAC increases the accumulation of autophagosomes in hFOB1.19 cells. FAC-induced autophagy was increased by the apoptosis inhibitor 3-MA but was reduced in DMT1 shRNA hFOB1.19 cells. These results suggest that the increased expression of DMT1 induces iron overload and iron overload induces osteoblast autophagy and apoptosis, thus affecting the pathological processes of osteoporosis. Clarifying the mechanisms underlying the effects of DMT1 will allow the identification of novel targets for the prevention and treatment of osteoporosis.

Effects of alpha-lipoic acid on expression of iron transport and storage proteins in BV-2 microglia cells

The antioxidant properties of alpha-lipoic acid (ALA) are associated with its ability to reduce iron in cells and tissues, which is partly due to its inhibiting effect on iron uptake from transferrin and its promoting effect on iron deposition into ferritin. However, the relevant mechanisms are unknown. We therefore investigated the effects of ALA on the expression of transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (Fpn1) and ferritin in BV-2 microglia cells. We demonstrated that ALA significantly inhibited DMT1 expression, lowered ferritin-light-chain (Ft-L) and ferritin-heavy-chain (Ft-H) content, and had no effect on TfR1 and Fpn1 in BV-2 microglia cells. This indicated that the inhibiting effect of ALA on DMT1 might be one of the causes of the ALA-induced reduction in cellular transferrin-bound-iron uptake. We also demonstrated that ALA enhanced DMT1 and TfR1 expression in ferric ammonium citrate (FAC)-treated cells. FAC treatment led to a significant increase in Ft-L, Ft-H and Fpn1, and pre-treatment with ALA resulted in a further increase in the contents of Ft-L and Ft-H but not Fpn1 in cells. ALA could up-regulate TfR1, DMT1 and ferritin expression when iron is increased outside of the cell, promoting iron deposition into ferritin by increasing cell iron uptake, and then reducing free iron both inside and outside of the cell.

Supplementation of infant formulas with recombinant human lactoferrin and/or galactooligosaccharides increases iron bioaccessibility as measured by ferritin formed in Caco-2 cell model

Recently, lactoferrin (Lf), a glycoprotein belonging to transferrin family, has received more attention for the discovery of its wide spectrum of functionalities. Among its important functionalities is its ability to bind Fe and its high stability against in vitro digestion. Galactooligosaccharides (GOS) also has functionality related to Fe bioaccessibility and it also positively affect Lf stability. This study aimed to evaluate the functionality of recombinant human Lf (rhLf) and GOS as factors that improve iron bioaccessibility in human milk. In this study, rhLf and GOS were added to First Infant Formula (FIF) in three concentrations (0.10, 0.15 and 0.20 and 3.3, 5 and 10g 100mL−1 of the reconstituted formula, respectively), whether alone or in combination. In vitro digestion model was applied to different infant formulas to evaluate Fe solubility and the obtained digests were added to Caco-2 cell line to determine Fe bioaccessibility through the measurement of synthesized ferritin. The obtained results revealed that supplementation of formulas with rhLf and GOS enhanced Fe solubility to 66–97% which turns in an improvement of Fe bioaccessibility. Caco-2 cell ferritin content ranged from 6 to 46ng per mg cell protein with 5–7.5 fold increases in cells incubated with digested samples containing both rhLF and GOS. Although mineral solubility is the most important factor, which affects on Fe bioaccessibility, other factors may also interfere with it, such as the expression of divalent metal transporter 1 (DMT1). The findings also revealed that rhLf is a highly stable protein, especially in the presence of GOS and more researches are needed to clarify this hidden effect of GOS. Thus, rhLf and GOS together are prominent components of human milk could improve Fe bioaccessibility and it is noteworthy that these finding must be taken into account during infant formula evolution to prevent some disease related to Fe deficiency.

Endometrial expression and in vitro modulation of the iron transporter divalent metal transporter-1: implications for endometriosis

To evaluate divalent metal transporter-1 (DMT1) expression in healthy women's and endometriosis patients' endometrium and to analyze DMT1 and ferritin light chain (Fn-L) expression modulation by iron overload and IL-1β in endometrial stromal cells (ESCs). Observational and experimental study. University hospital research laboratory. Thirty-one healthy women and 24 endometriosis patients. Menstrual, proliferative, and secretory endometrial biopsies. Isolated ESCs from seven endometrial biopsies incubated with IL-1β or FeSO4 overload for 24hours. Divalent metal transporter-1 endometrial protein expression assessed by immunohistochemistry and Western blot. Divalent metal transporter-1 and Fn-L proteins expression in stimulated ESCs evaluated by Western blot. Divalent metal transporter-1 is expressed throughout the menstrual cycle in human endometrium. Four endometrial DMT1 variants were identified accordingly to their molecular weight: DMT-80, -65, -55, and -50. Endometrial expression of DMT-80 and -55 is higher in endometriosis patients than in healthy women. In ESCs, iron overload induces an overexpression of DMT-80, DMT-50, and Fn-L, whereas IL-1β increases DMT-80 and -50 expressions and decreases Fn-L expression. Divalent metal transporter-1 overexpression in endometriosis patients' endometrium can increase iron influx toendometrial cells, inducing oxidative stress-mediated proinflammatory signaling. In turn, endometriosis-related conditions, as iron overload and inflammation (IL-1β), enhance endometriosis patients endometrial DMT1 expression, creating a vicious circle on DMT-1-modulated pathways.

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.

Fermented Goat's Milk Consumption Improves Duodenal Expression of Iron Homeostasis Genes during Anemia Recovery.

Despite the crucial roles of duodenal cytochrome b (Dcytb), divalent metal transporter 1 (DMT1), ferritin light chain (Ftl1), ferroportin 1 (FPN1), transferrin receptor 1 (TfR1), and hepcidin antimicrobial peptide (Hamp) in Fe metabolism, no studies have investigated the modulations of these genes during Fe repletion with fermented milks. Analysis included Fe status markers and gene and protein expression in enterocytes of control and anemic animals fed fermented milks. Fermented goat's milk up-regulated enterocyte Dcytb, DMT1, FPN1, and Ftl1 and down-regulated TfR1 and Hamp gene expression in control and anemic animals. Anemia decreased Dcytb, DMT1, and Ftl1 in animals fed fermented cow's milk and up-regulated TfR1 and Hamp expression. Fe overload down-regulated Dcytb and TfR1 in animals fed fermented cow's milk and up-regulated DMT1 and FPN1 gene expression. Fermented goat's milk increased expression of duodenal Dcytb, DMT1, and FPN1 and decreased Hamp and TfR1, improving Fe metabolism during anemia recovery.

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.

Expression of Iron-Related Proteins at the Neurovascular Unit Supports Reduction and Reoxidation of Iron for Transport Through the Blood-Brain Barrier.

The mechanisms for iron transport through the blood-brain barrier (BBB) remain a controversy. We analyzed for expression of mRNA and proteins involved in oxidation and transport of iron in isolated brain capillaries from dietary normal, iron-deficient, and iron-reverted rats. The expression was also investigated in isolated rat brain endothelial cells (RBECs) and in immortalized rat brain endothelial (RBE4) cells grown as monoculture or in hanging culture inserts with defined BBB properties. Transferrin receptor 1, ferrireductases Steap 2 and 3, divalent metal transporter 1 (DMT1), ferroportin, soluble and glycosylphosphatidylinositol (GPI)-anchored ceruloplasmin, and hephaestin were all expressed in brain capillaries in vivo and in isolated RBECs and RBE4 cells. Gene expression of DMT1, ferroportin, and soluble and GPI-anchored ceruloplasmin were significantly higher in isolated RBECs with induced BBB properties. Primary pericytes and astrocytes both expressed ceruloplasmin and hephaestin, and RBECs, pericytes, and astrocytes all exhibited ferrous oxidase activity. The coherent protein expression of these genes was demonstrated by immunocytochemistry. The data show that brain endothelial cells provide the machinery for receptor-mediated uptake of ferric iron-containing transferrin. Ferric iron can then undergo reduction to ferrous iron by ferrireductases inside endosomes followed by DMT1-mediated pumping into the cytosol and subsequently cellular export by ferroportin. The expression of soluble ceruloplasmin by brain endothelial cells, pericytes, and astrocytes that together form the neurovascular unit (NVU) provides the ferroxidase activity necessary to reoxidize ferrous iron once released inside the brain.

Hyperglycemia Does Not Affect Iron Mediated Toxicity of Cultured Endothelial and Renal Tubular Epithelial Cells: Influence of L-Carnosine.

Iron has been suggested to affect the clinical course of type 2 diabetes (T2DM) as accompanying increased intracellular iron accumulation may provide an alternative source for reactive oxygen species (ROS). Although carnosine has proven its therapeutic efficacy in rodent models of T2DM, little is known about its efficacy to protect cells from iron toxicity. We sought to assess if high glucose (HG) exposure makes cultured human umbilical vein endothelial cells (HUVECs) and renal proximal tubular epithelial cells (PTECs) more susceptible to metal induced toxicity and if this is ameliorated by L-carnosine. HUVECs and PTECs, cultured under normal glucose (5 mM, NG) or HG (30 mM), were challenged for 24 h with FeCl3. Cell viability was not impaired under HG conditions nor did HG increase susceptibility to FeCl3. HG did not change the expression of divalent metal transporter 1 (DMT1), ferroportin (IREG), and transferrin receptor protein 1 (TFRC). Irrespective of glucose concentrations L-carnosine prevented toxicity in a dose-dependent manner, only if it was present during the FeCl3 challenge. Hence our study indicates that iron induced cytotoxicity is not enhanced under HG conditions. L-Carnosine displayed a strong protective effect, most likely by chelation of iron mediated toxicity.

Mechanisms of divalent metal toxicity in affective disorders.

Metals are required for proper brain development and play an important role in a number of neurobiological functions. The divalent metal transporter 1 (DMT1) is a major metal transporter involved in the absorption and metabolism of several essential metals like iron and manganese. However, non-essential divalent metals are also transported through this transporter. Therefore, altered expression of DMT1 can modify the absorption of toxic metals and metal-induced toxicity. An accumulating body of evidence has suggested that increased metal stores in the brain are associated with elevated oxidative stress promoted by the ability of metals to catalyze redox reactions, resulting in abnormal neurobehavioral function and the progression of neurodegenerative diseases. Metal overload has also been implicated in impaired emotional behavior, although the underlying mechanisms are not well understood with limited information. The current review focuses on psychiatric dysfunction associated with imbalanced metabolism of metals that are transported by DMT1. The investigations with respect to the toxic effects of metal overload on behavior and their underlying mechanisms of toxicity could provide several new therapeutic targets to treat metal-associated affective disorders.

Iron Regulatory Protein 1 Suppresses Hypoxia-Induced Iron Uptake Proteins Expression and Decreases Iron Levels in HepG2 Cells.

Transferrin receptor (TfR1) and divalent metal transporter 1 (DMT1) are important proteins for cellular iron uptake, and both are regulated transcriptionally through the binding of hypoxia-inducible factor 1 (HIF-1) to hypoxia-responsive elements (HREs) under hypoxic conditions. These proteins are also regulated post-transcriptionally through the binding of iron regulatory protein 1 (IRP1) to iron-responsive elements (IREs) located in the mRNA untranslated region (UTR) to control cellular iron homeostasis. In iron-deficient cells, IRP1-IRE interactions stabilize TfR1 and DMT1 mRNAs, enhancing iron uptake. However, little is known about the impact of IRP1 on the regulation of cellular iron homeostasis under hypoxia. Thus, to investigate the role of IRP1 in hypoxic condition, overexpression and knockdown assays were performed using HepG2 cells. The overexpression of IRP1 suppressed the hypoxia-induced increase in TfR1 and DMT1 (+IRE) expression and reduced the stability of TfR1 and DMT1 (+IRE) mRNAs under hypoxia, whereas IRP1 knockdown further increased the hypoxia-induced expression of both proteins, preventing the decrease in IRE-dependent luciferase activity induced by hypoxia. Under hypoxic conditions, ferrous iron uptake, the labile iron pool (LIP), and total intracellular iron reduced when IRP1 was overexpressed and further increased when IRP1 was knocked down. IRP1 expression declined and TfR1/DMT1 (+IRE) expression increased with the time of hypoxia prolonged, whereas the binding of IRP1 to the IRE of TfR1/DMT1 mRNA maintained. In summary, IRP1 suppressed TfR1/DMT1 (+IRE) expression, limited the cellular iron content and decreased lactate dehydrogenase (LDH) release induced by hypoxia.