Divalent Metal Ion Transporter Research Articles

Effect of berberine on copper and zinc levels in chickens infected with Eimeria tenella

Berberine, a traditional Chinese medicine, was found to exhibit anticoccidial activity. However, its mechanism is unclear. Trace metals such as copper and zinc are extremely low (less than 0.01% of the total weight of the body) but play a vital role in organisms. In the present study, we investigated the effect of berberine on copper and zinc levels in chickens infected with Eimeria tenella. Firstly, our data confirmed that infected chickens with E. tenella exhibited classic impairment on the 8th day of post infection, such as weight loss and increased feed conversion. Further study showed that E. tenella infection decreased the contents of copper and zinc in the liver and serum of chickens. Berberine was similar to amprolium and significantly improved the pathogenic conditions. Berberine could restore copper and zinc imbalance caused by E. tenella in chickens to a large extent. Studies on the development of cecum lesions demonstrated that the protective effect of berberine on the intestinal cecum was similar to that of the Cu/Zn mixture. Additionally, the mRNA expression of several metal transport related genes of the chick small intestine, including zinc transporter 1, copper transporter 1 and divalent metal ion transporter 1, was elevated by the treatment with berberine. Taken together, we speculate that the anticoccidial activity of berberine may be related to the maintenance of certain metals (Cu/Zn) homeostasis by affecting mRNA expression of their transport genes. However, the mode of action of BBR on these vital metals in the chicks infected with E. tenella still needs to be further studied.

A comparation between different iron sources on growth performance, iron utilization, antioxidant capacity and non-specific immunity in Eriocheir sinensis

This study was to determine the effects of different iron sources on the growth performance, iron utilization, antioxidant capacity and non-specific immunity in Eriocheir sinensis. Juvenile crabs (3.29 ± 0.06 g) were fed with six basic diets supplemented with 100 mg/kg iron in the form of ferrous sulfate (S-100), ferrous fumarate (F-100), ferrous glycine (G-100), ferrous sulfate + ferrous fumarate (S-50 + F-50), ferrous sulfate + ferrous glycine (S-50 + G-50) and ferrous fumarate + ferrous glycine (F-50 + G-50) for 10 weeks. The crab in the G-100 group and the S-50 + G-50 group had higher weight gain, specific growth rate and lower whole-body total lipid than those in the S-100 group. The expression levels of divalent metal ion transporter 1 and ferroportin in the intestine and hepatopancreas were significantly enhanced in the G-100 group and the S-50 + G-50 group compared with the S-100 group. Crabs fed the G-100 diet possessed higher hemolymph iron, transferrin concentration and iron contents in the whole body, hepatopancreas and muscle than in other treatments. In addition, the total antioxidant capacity in the G-100 group and the activities of acid phosphatase and phenoloxidase in the G-100 group and the S-50 + G-50 group significantly improved compared to the S-100 group. This study demonstrates that ferrous glycine might be the proper iron source for improving growth performance, iron utilization, antioxidant capacity and non-specific immunity of E. sinensis, and can be a source of effective iron supplementation in aquafeed.

Four AAs increase DMT1 abundance in duodenal brush-border membrane vesicles and enhance iron absorption in iron-deprived mice

AbstractIron-deficiency anemia is common worldwide and typically treated by oral iron supplementation. Excess enteral iron, however, may cause pathological outcomes. Developing new repletion approaches is thus warranted. Previous experimentation revealed that select amino acids (AAs) induce trafficking of transporters onto the enterocyte brush-border membrane (BBM) and enhance electrolyte absorption/secretion. Here, we hypothesized that certain AAs would increase the abundance of the main intestinal iron importer, divalent metal-ion transporter 1 (DMT1), on the BBM of duodenal enterocytes, thus stimulating iron absorption. Accordingly, all 20 AAs were screened using an ex vivo duodenal loop/DMT1 western blotting approach. Four AAs (Asp, Gln, Glu, and Gly) were selected for further experimentation and combined into a new formulation. The 4 AAs stimulated 59Fe transport in mouse duodenal epithelial sheets in Ussing chambers (∼4-fold; P < .05). In iron-deprived mice, oral intragastric administration of the 4 AA formulation increased DMT1 protein abundance on the enterocyte BBM by ∼1.5-fold (P < .05). The 4 AAs also enhanced in vivo 59Fe absorption by ∼2-fold (P < .05), even when ∼26 µg of cold iron was included in the transport solution (equal to a human dose of ∼73 mg). Further experimentation using DMT1int/int mice showed that intestinal DMT1 was required for induction of iron transport by the 4 AAs. Select AAs thus enhance iron absorption by inducing DMT1 trafficking onto the apical membrane of duodenal enterocytes. We speculate that further refinement of this new 4 AA formulation will ultimately allow iron repletion at lower effective doses (thus mitigating negative side effects of excess enteral iron).

Relative Bioavailability of Broiler Chickens Fed with Zinc Hydroxychloride and Sulfate Sources for Corn-Soybean Meal.

This study was conducted to determine relative bioavailability (RBV) of basic zinc chloride (BZC) compared to zinc sulfate monohydrate (ZSM) for broilers. A randomized design involving a 2 × 3 factorial arrangement of the different treatment regimens plus one negative control was set up for this study. A total of 630 newly hatched male AA broiler chicks were randomly allocated to 42 different pens (15 chickens/pen) and assigned to 7 dietary treatments in a completely randomized design. The diet was supplemented with 0, 20, 40, or 80mg of Zn mg/kg of feed in the form of ZSM or BZC. The results showed that zinc supplementation altered average daily gain (ADG) and feed conversion ratio (FCR) (P < 0.05) for both zinc sources. It was observed that the weight gain increased linearly (P < 0.01) and FCR decreased linearly as dietary BZC and ZSM concentration increased. Moreover, compared with chickens fed with ZSM, chickens fed with BZC had higher ADG and lower FCR from days 0 to 14 (P < 0.05), and higher activity of plasma alkaline phosphatase (ALP) (P < 0.05), total superoxide dismutase (T-SOD), and CuZn superoxide dismutase (CuZn-SOD) (P < 0.01) in the plasma of chickens fed with BZC at zinc level 80mg/kg at day 14. The pancreas divalent metal-ion transporter-1 (DMT1) mRNA expression of chickens fed with BZC was found to be significantly enhanced at day 28, and the pancreas metallothionein (MT) mRNA expression for BZC fed group was also markedly increased at Zn levels of 20 and 40mg/kg respectively. The relative bioavailability (RBV) of BZC (Zn sulfate 100%) based on ADG in the starter phase was 110.82%, whereas the tibia zinc content, as well as the activities of plasma ALP and CuZn-SOD, and the pancreas MT mRNA level were in the range between 108 and 119%. It was thus concluded that BZC was more efficacious than Zn sulfate and could serve as a potentially novel zinc source in the broilers.

Effect of zinc depletion/repletion on intestinal iron absorption and iron status in rats

Iron and zinc deficiencies likely coexist in general population. We have previously demonstrated that zinc treatment induces while zinc deficiency inhibits iron absorption in intestinal cell culture models, but this needs to be tested in vivo. In the present study we assessed intestinal iron absorption, iron status (haemoglobin), red blood cell number, plasma ferritin, transferrin receptor, hepcidin) and tissue iron levels in zinc depleted, replete and pair fed control rats. Zinc depletion led to reduction in body weight, tissue zinc levels, intestinal iron absorption, protein and mRNA expression of iron transporters, the divalent metal ion transporter-1, hephaestin and ferroportin, but elevated the intestinal and liver tissue iron levels compared with the pair fed control rats. Zinc repletion led to a significant weight gain compared to zinc deficient rats and normalized the iron absorption, iron transporter expression, tissue iron levels to that of pair fed control rats. Surprisingly, haemoglobin levels and red blood cell number reduced significantly in zinc repleted rats, which could be due to rapid weight gain. Together, these results indicate that whole body zinc status has profound influence on growth, intestinal absorption and systemic utilization of iron, mediated via modulation of iron transporter expression.

Iron induces two distinct Ca2+ signalling cascades in astrocytes

Iron is the fundamental element for numerous physiological functions. Plasmalemmal divalent metal ion transporter 1 (DMT1) is responsible for cellular uptake of ferrous (Fe2+), whereas transferrin receptors (TFR) carry transferrin (TF)-bound ferric (Fe3+). In this study we performed detailed analysis of the action of Fe ions on cytoplasmic free calcium ion concentration ([Ca2+]i) in astrocytes. Administration of Fe2+ or Fe3+ in μM concentrations evoked [Ca2+]i in astrocytes in vitro and in vivo. Iron ions trigger increase in [Ca2+]i through two distinct molecular cascades. Uptake of Fe2+ by DMT1 inhibits astroglial Na+-K+-ATPase, which leads to elevation in cytoplasmic Na+ concentration, thus reversing Na+/Ca2+ exchanger and thereby generating Ca2+ influx. Uptake of Fe3+ by TF-TFR stimulates phospholipase C to produce inositol 1,4,5-trisphosphate (InsP3), thus triggering InsP3 receptor-mediated Ca2+ release from endoplasmic reticulum. In summary, these findings reveal the mechanisms of iron-induced astrocytic signalling operational in conditions of iron overload.

Knocking out alpha-synuclein in melanoma cells dysregulates cellular iron metabolism and suppresses tumor growth

The protein alpha-synuclein (α-syn) is unusual because, depending on its conformation and the type of cell in which it is expressed, it is pro-death or pro-survival, triggering neurodegeneration in Parkinson’s disease and enhancing cell survival of some melanomas. To probe the function of α-syn in melanoma, we used CRISPR/Cas9 to knockout SNCA, the gene that codes for α-syn, in SK-Mel-28 melanoma cells. The SNCA-knockout clones in culture exhibited a decrease in the transferrin receptor 1 (TfR1), an increase in ferritin, an increase of reactive oxygen species and proliferated slower than control cells. These SNCA-knockout clones grafted into SCID mice grew significantly slower than the SK-Mel-28 control cells that expressed α-syn. In the excised SNCA-knockout xenografts, TfR1 decreased 3.3-fold, ferritin increased 6.2-fold, the divalent metal ion transporter 1 (DMT1) increased threefold, and the iron exporter ferroportin (FPN1) decreased twofold relative to control xenografts. The excised SNCA-KO tumors exhibited significantly more ferric iron and TUNEL staining relative to the control melanoma xenografts. Collectively, depletion of α-syn in SK-Mel-28 cells dysregulates cellular iron metabolism, especially in xenografts, yielding melanoma cells that are deficient in TfR1 and FPN1, that accumulate ferric iron and ferritin, and that undergo apoptosis relative to control cells expressing α-syn.

Efficient iron utilization compensates for loss of extracellular matrix of ovarian cancer spheroids

Metastasis is the major cause of death in women with advanced ovarian cancer. Epithelial ovarian cancer cells can dissociate directly from extracellular matrix (ECM) and form spheroids to spread through the peritoneal cavity. Loss of ECM hinders the survival of ECM-detached epithelial cells. It is still largely unknown how ovarian cancer spheroids maintain their viability after loss of ECM. We find that spheroids derived either from ovarian cancer ascites or cell lines are iron-replete. In accordance with iron-replete condition, proteins involved in iron uptake, transport and storage including divalent metal ion transporter 1 (DMT1), transferrin receptor 1 (TFR1), ferritin, poly(rC)-binding proteins 1 and 2 (PCBP1 and 2) and nuclear factor E2-related factor 2 (NRF2) all increase in ovarian cancer spheroids. Genes linking iron homeostasis and lipid metabolism including stearoyl coenzyme A desaturase 1 (SCD1) are up-regulated in ovarian cancer spheroids. The product of SCD1 oleic acid can restore the viability of ovarian cancer spheroids inhibited by deprivation of iron. Extracellular signal-regulated kinase (ERK) activation contributes to autophagy activation in ovarian cancer spheroids. Impairment of autophagy by U0126 or Olaparib results in lysosomal iron accumulation and decrease of the cytosolic labile iron pool, leading to reduction of SCD1, lipid level and cell viability. Combination of U0126 and Olaparib has synergistic cytotoxicity toward ovarian cancer spheroids. Our findings reveal that ovarian cancer spheroids develop efficient iron utilization system to survive. Targeting iron utilization in ovarian cancer spheroids may have the potential to become new treatment strategies for ovarian cancer metastasis.

Use of Dual Electron Probes Reveals Role of Ferritin in &lt;i&gt;Ex Vivo&lt;/i&gt; Erythropoiesis

In the finely regulated process of mammalian erythropoiesis, the path of the labile iron pool and its transport to mitochondria for heme production is not well understood1. Existing models for erythropoiesis do not involve a central role for the ubiquitous iron storage protein ferritin, but instead indicate that incoming endosomal Fe3+ attached to transferrin enters the cytoplasm through divalent metal ion transporters after reduction to Fe2+, and is immediately taken up into mitochondria through the mitoferrin-1 transporter. Here, we apply a dual 3D imaging and spectroscopic technique, based on scanned electron probes, to measure distributions of Fe3+ in ex vivo human erythropoietic stem cells. After seven days in culture, we find that the cells display a highly specialized architecture of organelles with anchored clustering of mitochondria and massive accumulation of nanoparticles containing high iron concentrations localized to lysosomal storage depots, which we identify as ferritin. Our results support the hypothesis that lysosomal ferritin iron depots are utilized by developing reticulocytes to continue heme production after much of the cellular machinery has been expelled.

A holistic view of mammalian (vertebrate) cellular iron uptake.

Cell iron uptake in mammals is commonly distinguished by whether the iron is presented to the cell as transferrin-bound or not: TBI or NTBI. This generic perspective conflates TBI with canonical transferrin receptor, endosomal iron uptake, and NTBI with uptake supported by a plasma membrane-localized divalent metal ion transporter, most often identified as DMT1. In fact, iron uptake by mammalian cells is far more nuanced than this somewhat proscribed view suggests. This view fails to accommodate the substantial role that ZIP8 and ZIP14 play in iron uptake, while adhering to the traditional premise that a relatively high endosomal [H+] is thermodynamically required for release of iron from holo-Tf. The canonical view of iron uptake also does not encompass the fact that plasma membrane electron transport - PMET - has long been linked to cell iron uptake. In fact, the known mammalian metallo-reductases - Dcytb and the STEAP proteins - are members of this cohort of cytochrome-dependent oxido-reductases that shuttle reducing equivalents across the plasma membrane. A not commonly appreciated fact is the reduction potential of ferric iron in holo-Tf is accessible to cytoplasmic reducing equivalents - reduced pyridine and flavin mono- and di-nucleotides and dihydroascorbic acid. This allows for the reductive release of Fe2+ at the extracellular surface of the PM and subsequent transport into the cytoplasm by a neutral pH transporter - a ZIP protein. What this perspective emphasizes is that there are two TfR-dependent uptake pathways, one which does and one which does not involve clathrin-dependent, endolysosomal trafficking. This raises the question as to the selective advantage of having two Tf, TfR-dependent routes of iron accumulation. This review of canonical and non-canonical iron uptake uses cerebral iron trafficking as a point of discussion, a focus that encourages inclusion also of the importance of ferritin as a circulating 'chaperone' of ferric iron.

Iron and copper transport activities of the mammalian metal‐ion transporters DMT1 and CTR1

Divalent metal‐ion transporter‐1 (DMT1) and the high‐affinity copper transporter‐1 (CTR1) play essential roles in the intestinal absorption of iron and copper respectively. Both transporters are potential therapeutic targets in disorders of metal metabolism. Disagreement remains over whether the metal‐ion specificities of the two transporters overlap. For example, others have concluded that DMT1 transports Cu+ and CTR1 transports Fe2+ based on the results of shRNA knockdown of gene expression in Caco‐2 cells [Espinoza A et al (2012) Biol Trace Elem Res 146, 281]. Since these observations contradicted our published data regarding the substrate profile of DMT1 in vitro [Illing AC et al (2012) J Biol Chem 287, 30485] and in vivo [Shawki A et al (2015) Am J Physiol Gastrointest Physiol 309, G635] or of CTR1 in vitro [Lee J et al (2002) J Biol Chem 277, 4380], we have re‐examined metal‐ion transporter substrate profiles. We tested the hypothesis that DMT1 can transport copper and that CTR1 can transport iron by using radiotracer assays and the voltage clamp in RNA‐injected oocytes expressing human DMT1 or CTR1. Expression of CTR1 in oocytes stimulated up to 20‐fold the uptake of 2 μM 64Cu+ (pH 5.5) but did not stimulate the uptake of 64Cu2+ or 55Fe2+. Expression of DMT1 in oocytes stimulated 80‐fold the uptake of 2 μM 55Fe2+ (pH 6.0) but transport of 64Cu+ or 64Cu2+ did not differ from that in control oocytes. We found that Cu+ inhibited DMT1‐mediated 55Fe2+ uptake in a competitive fashion (Ki ≈ 58 μM). In DMT1‐expressing oocytes, Cu+ evoked inward currents; however, such currents were also observed in control oocytes and their I/V relationships did not resemble those for DMT1 substrates Fe2+ and Cd2+. Addition of Fe2+ or Cd2+ abolished the presteady‐state (transient) currents observed in the absence of metal ion whereas addition of Cu+ or Cu2+ did not. We found, however, that Cu2+ accelerated the decay of the transient currents. We conclude that (1) whereas Cu+ and Cu2+ are capable of interacting with DMT1, neither are transported substrates; and (2) CTR1 mediates transport of Cu+ but not of Cu2+ or Fe2+. We therefore find no evidence of overlapping metal‐ion specificities in DMT1 and CTR1.Support or Funding InformationPHS grants R01‐DK080047, R01‐DK107309, R01‐DK074192, and R25‐HL115473; and the American Physiological Society

Role of Heme metabolism in the oxidative state of NO‐receptor soluble guanylyl cyclase (sGC) in aging mouse brain

IntroductionAging is an important risk factor for cardiovascular diseases and neurodegeneration. Heme synthesis and metabolism decline with age. Heme moiety is essential for proper function of the Nitric Oxide receptor soluble guanylyl cyclase (sGC). Evidence suggests the involvement of NO/cGMP signaling in human neurological disorders. sGC also an important role in maintenance of cerebral blood flow, vascular homeostasis and platelet function. NO binds to the ferrous heme moiety of sGC and activates cGMP‐forming activity hundred fold. Therefore, the integrity of reduced sGC heme moiety is essential for proper transduction of NO signal.Methods and ResultsWe investigated if the oxidative state and the relative content of sGC heme moiety changes with age in mouse brain tissue. Three groups of animals have been used in our studies: young, 3.5 months old, and mature, 15 months old, and old, 24 months, C57Bl6 mice. sGC‐dependent synthesis of cGMP in protein homogenates from mouse brains in response to sGC regulators was evaluated by quantifying the amount P32‐labeled cGMP using thin layer chromatography. To estimate the changes in the amount of heme‐deficient sGC, protoporphyrin IX (PPIX) was used as a heme‐independent sGC activator. NO‐independent activator BAY 58‐2667, which has a higher affinity for sGC with ferric heme, was used to evaluate the changes in the amount of sGC with oxidized heme. NO donor DEA‐NO was used to evaluate the level of sGC with ferrous heme. For each individual sample the ratios of sGC‐[HemeFe3+] vs sGC‐[HemeFe2+] activities (BAY 58‐2667/DEA‐NO) and heme‐deficient vs sGC‐[HemeFe2+] activities (PPIX/DEA‐NO) were determined. Our analysis demonstrated that the relative amount of heme‐deficient and heme‐oxidized sGC increased in both males and females of older age. Decline in sGC protein expression in oldest animal groups was detected by Western blot analysis. We also investigated if the accumulation of heme‐deficient sGC or sGC with ferric heme in ageing brain correlates with changes in the level of proteins involved in iron and heme metabolism. We found no difference in the level of HSP90 chaperone needed for sGC heme insertion and unchanged level in the iron chaperone PCBP2, which is essential for iron trafficking. The expression of divalent metal ion transporter (DMT1) was also not affected by age. However, we observe a decrease in the expression of Heme Oxygenase 1 (HO1) and Aconitase 2 proteins involved in maintenance of cellular oxidative balance in old animals of both genders.ConclusionOur data indicate that changes in heme homeostasis might contribute to the decline of NO/sGC in brain with aging.

Mechanistic basis of the inhibition of SLC11/NRAMP-mediated metal ion transport by bis-isothiourea substituted compounds.

In humans, the divalent metal ion transporter-1 (DMT1) mediates the transport of ferrous iron across the apical membrane of enterocytes. Hence, its inhibition could be beneficial for the treatment of iron overload disorders. Here we characterize the interaction of aromatic bis-isothiourea-substituted compounds with human DMT1 and its prokaryotic homologue EcoDMT. Both transporters are inhibited by a common competitive mechanism with potencies in the low micromolar range. The crystal structure of EcoDMT in complex with a brominated derivative defines the binding of the inhibitor to an extracellular pocket of the transporter in direct contact with residues of the metal ion coordination site, thereby interfering with substrate loading and locking the transporter in its outward-facing state. Mutagenesis and structure-activity relationships further support the observed interaction mode and reveal species-dependent differences between pro- and eukaryotic transporters. Together, our data provide the first detailed mechanistic insight into the pharmacology of SLC11/NRAMP transporters.

The iron chelator desferrioxamine synergizes with chemotherapy for cancer treatment

BackgroundCisplatin (CDDP) resistance remains a major obstacle for treatment of ovarian cancer. Iron contributes to the growth and reproduction of malignant cells, thus iron chalators can inhibit the growth of tumor cells by depleting the intracellular iron pool. The iron chelator, desferrioxamine (DFO), has performed anticancer in previous study. The aim of our study is to determine the correlation between iron-deprivation and tumor chemosensitivity in ovarian cancer. MethodsTo investigate the prognostic value of ferritin light (FTL), ferroportin (FPN), hepcidin (HAMP) and divalent metal-ion transporter-1 (DMT1) in ovarian cancer, the Kaplan–Meier analysis and the Gene Expression Profiling Interactive Analysis (GEPIA) were used. The ovarian cancer cell lines (SKOV-3 and OVCAR-3) were exposed to a gradient concentration of DFO (10, 20, 50, 100, 200 μM) and CDDP (1, 5, 10, 50,100 μM) for 24 h. The protein expression of FTL was tested. The expression of cancer stem cell (CSC) markers, including Sox2, Nanog and C-myc, were downregulated with treatment of DFO. Also, the mamosphere formation and the plation of CD44+/high/CD133+/high and Aldehyde dehydrogenase (ALDH)+/high SKOV-3 cells were reduced after treatment for 7d. Furthermore, we detected the expression of p53, BCL-2, BAX, and caspase-8. ResultsThe survival analysis revealed that high expression of FTL, DMT1, HAMP, showed poor overall survival (OS) in ovarian cancer patients. Our combined data found that DFO could effectively inhibit CSCs, improve the resistance to chemotherapy, and significantly enhanced the efficacy of CDDP therapy in vitro in promoting apoptosis. Besides, targeting molecular targets, including BAX, BCL-2, p53 and caspase-8 could serve as the clinical biomarkers to evaluate the effects of ovarian cancer. It is reasonable to believe that DFO adjuvant therapy in combination with CDDP chemotherapy can promote the improvement of treatment response in ovarian cancer patients. ConclusionOur research suggests the experimental evidence for DFO and CDDP as a new effective combination therapy to enhance the efficacy of chemical therapy in ovarian cancer.

A direct comparison of divalent metal-ion transporter (DMT1) and hinokitiol, a potential small molecule replacement

Hinokitiol, a natural lipophilic chelator, appears capable of replacing several iron transporters after they have been genetically ablated. Divalent metal-ion transporter (DMT1) is the major iron importer in enterocytes and erythroblasts. We have compared DMT1 and hinokitiol in multiple fashions to learn if the smaller molecule is a suitable substitute using two HEK293 cell lines engineered to overexpress different isoforms of DMT1. Both the macromolecule and the lipophilic chelator enable import of ferrous ions into HEK293 cells. Hinokitiol also mediates ferric ion import but DMT1 cannot do so. While DMT1 can also import Mn2+ ions, hinokitiol lacks this ability. The Michaelis–Menten analysis for kinetics of macromolecular catalysis is also suitable for hinokitiol-supported iron import. To compare hinokitiol to DMT1 relative to other metal ions that DMT1 can transport, we employed an organic extraction procedure with which we initially matched the results obtained for Fe2+, Fe3+ and Mn2+, and then showed that multiple other cations were unlikely to enter via hinokitiol. The small chelator thus shares some functional properties with DMT1, but distinct difference were also noted.

Zinc induces iron uptake and DMT1 expression in Caco-2 cells via a PI3K/IRP2 dependent mechanism.

The absorption of iron is influenced by numerous dietary and physiological factors. We have previously demonstrated that zinc treatment of intestinal cells increases iron absorption via induction of the apical membrane iron transporter divalent metal ion transporter-1 (DMT1). To better understand the mechanisms of zinc-induced iron absorption, we have studied the effect of zinc on iron uptake, iron transporter and iron regulatory protein (IRP 1 and 2) expression and the impact of the PI3K pathway in differentiated Caco-2 cells, an intestinal cell culture model. We found that zinc induces DMT1 protein and mRNA expression. Zinc-induced DMT1 expression and iron absorption were inhibited by siRNA silencing of DMT1. Furthermore, zinc treatment led to increased abundance of IRP2 protein in cell lysates and in polysomal fractions, implying its binding to target mRNAs. Zinc treatment induced Akt phosphorylation, indicating the activation of the PI3K pathway. LY294002, a specific inhibitor of PI3K inhibited zinc-induced Akt phosphorylation, iron uptake, DMT1 and IRP2 expression. Furthermore, LY294002 also decreased the basal level of DMT1 mRNA but not protein expression. siRNA silencing of IRP2 led to down-regulation of both basal and zinc-induced DMT1 protein expression, implying possible involvement of post-transcriptional regulatory mechanisms. In agreement with these findings, zinc treatment stabilized DMT1 mRNA levels in actinomycin D-treated cells. Based on these findings, we conclude that zinc-induced iron absorption involves elevation of DMT1 expression by stabilization of its mRNA, by a PI3K/IRP2-dependent mechanism.

Improving Zinc and Iron Accumulation in Maize Grains Using the Zinc and Iron Transporter ZmZIP5.

Zinc (Zn) and iron (Fe) are essential micronutrients for plant growth. Thus, it is important to understand the mechanisms of uptake, transport and accumulation of these micronutrients in maize to improve crop nutritional quality. Members of the zinc-regulated transporters, iron-regulated transporter-like protein (ZIP) family are responsible for the uptake and transport of divalent metal ions in plant. Previously, we showed that ZmZIP5 functionally complemented the Zn uptake double mutant zrt1zrt2, Fe-uptake double mutant fet3fet4 in yeast. In our β-glucuronidase (GUS) assay, the germinated seeds, young sheaths, and stems of ZmZIP5-promoter-GUS transgenic plants were stained. We generated and compared two maize lines for this study: Ubi-ZmZIP5, in which ZmZIP5 was constitutively overexpressed, and ZmZIP5i, a RNAi line. At the seedling stage, high levels of Zn and Fe were found in the roots and shoots of Ubi-ZmZIP5 plants, whereas low levels were found in the ZmZIP5i plants. Zn and Fe contents decreased in the seeds of Ubi-ZmZIP5 plants and remained unchanged in the seeds of ZmZIP5i plants. The seeds of Leg-ZmZIP5 plants, in which ZmZIP5 overexpression is specific to the endosperm, had higher levels of Zn and Fe. Our results imply that ZmZIP5 may play a role in Zn and Fe uptake and root-to-shoot translocation. Endosperm-specific ZmZIP5 overexpression could be useful for Zn and Fe biofortification of cereal grains.

Select Amino Acids Alter DMT1 Abundance on the BBM of Intestinal Enterocytes and Enhance or Reduce Iron Flux in Duodenal Organ Cultures

Approximately 1.6 billion people worldwide are anemic, with iron deficiency (ID) as the most common cause. Moreover, iron overload (IO), most commonly associated with the genetic disease Hereditary Hemochromatosis (HH), affects some 1 million individuals in the US. Both conditions warrant better treatment options, as iron supplementation is not always effective for individuals with ID, and current treatments for IO (phlebotomy and Fe chelation) are non‐specific and have untoward side effects. Our previous studies demonstrated that select amino acids (AA) can influence trafficking of membrane transport proteins in enterocytes and thus influence electrolyte absorption in the gut. We thus hypothesized that certain AAs could also influence trafficking of the predominant intestinal iron transporter, divalent metal‐ion transporter 1 (DMT1). We further surmised, that if true, this could represent a novel therapeutic approach to modulate intestinal iron absorption. This is particularly important since overall body iron levels are determined by assimilation of dietary iron in the intestine since humans and other mammals cannot excrete excess iron (and iron imbalance is common in many diseases). For an initial screen of the effect of AAs on DMT1 trafficking, ex vivo duodenal loops excised from adult mice were filled with a buffer containing individual AAs and incubated at 37° C in an oxygenated bath for 45 min. Subsequently, brush‐border membrane vesicles (BBMVs) were purified from mucosal scrapes and DMT1 protein expression levels were determined by immunoblotting. Amino acids were grouped based on how they influenced DMT1 protein expression on the BBM. Based upon the Western blotting data, two formulations were made that contained mixtures of AAs that either increased or decreased DMT1 protein expression on the BBM. To confirm these observations at a functional level, 59Fe flux studies were next performed with mouse duodenal organ cultures using Ussing chambers after exposure to the AA mixtures. These experiments initially showed that one AA formulation increased 59Fe flux by ≈4‐fold (p&lt;0.05), which was in agreement with the Western blotting data. Interestingly, this same AA formulation decreased conductance (p&lt;0.0001), which indicates a tightening of the epithelial barrier. To test the other AA formulation (which decreased DMT1 BBM protein expression), we utilized a mouse model of HH, hepcidin (Hamp) KO mice, in which intestinal iron absorption is inappropriately elevated. Importantly, 59Fe flux studies with Hamp−/− mice showed an approximate 7‐fold reduction in iron transport (p&lt;0.05) after exposure to the AA mixture, again confirming the immunoblotting data. We thus conclude that select AAs can influence DMT1 trafficking to and from the enterocyte BBM and thus alter vectorial iron flux. Although additional in vivo testing is warranted, these initial findings could lead to the development of specific AA formulations that could be used to mitigate pathological changes in intestinal iron transport associated with common disease states.Support or Funding InformationThis work was supported by NIH grants R01 DK074867 and R01 DK109717 (to JFC).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ablation of Na+/H+ exchanger‐3 prevents iron loading in the Hfe mouse model of hereditary hemochromatosis

Iron deficiency is the most prevalent micronutrient deficiency worldwide. Meanwhile, several common hereditary disorders result in iron overload (hemochromatosis) and subsequently liver disease, cardiomyopathy, and endocrine disorders. Since there exists no regulated mechanism for the excretion of iron, systemic iron homeostasis is achieved by tightly regulating iron absorption. We have demonstrated that the H+‐coupled divalent metal‐ion transporter‐1 (DMT1) is essential for the uptake of nonheme iron at the apical membrane of intestinal enterocytes [Shawki A et al (2015) Am J Physiol Gastrointest Liver Physiol 309, G635–G647]. DMT1 is a validated therapeutic target in hereditary hemochromatosis. More recently, we identified a role for the intestinal brush‐border Na+/H+ exchanger‐3 (NHE3) in driving iron absorption by generating the H+ gradient to energize DMT1 [Shawki A et al (2016) Am J Physiol Gastrointest Liver Physiol 311, G423–G430]. Here we tested the hypothesis that pathological iron loading depends on the activity of NHE3. We examined the effects of ablating NHE3 on the iron‐overload phenotype of the Hfe mouse model of hereditary hemochromatosis. We crossed the NHE3 and Hfe knockout lines (on the FVB/N background) and examined (at age ≈ 120 d) hematological variables, and tissue and blood iron status in male and female offspring fed a standard diet (NIH‐07). The increased nonheme iron observed in the livers of Hfe‐null mice relative to wildtype mice was absent in the double null (Hfe−/− | NHE3−/−) (n = 89, 2‐way interaction P &lt; 0.001), an effect that did not differ by sex (no 3‐way interaction, P = 0.54). Liver histology, using Perls' Prussian blue stain to detect iron deposits, supported this finding. NHE3 knockout also normalized serum iron levels in the Hfe null. Our data reveal that ablation of NHE3 prevents iron loading in the Hfe mouse model of hereditary hemochromatosis, and present the possibility of targeting intestinal NHE3 in the treatment of iron overload.Support or Funding InformationNIH grants R01 DK080047, R01 DK107309, P30 DK078392, and R25 HL115473, and the American Physiological SocietyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oral Gavage of Ginger Nanoparticle-Derived Lipid Vectors Carrying Dmt1 siRNA Blunts Iron Loading in Murine Hereditary Hemochromatosis.

Nanoparticles (NPs) have been utilized to deliver drugs to the intestinal epithelium in vivo. Moreover, NPs derived from edible plants are less toxic than synthetic NPs. Here, we utilized ginger NP-derived lipid vectors (GDLVs) in a proof-of-concept investigation to test the hypothesis that inhibiting expression of divalent metal-ion transporter 1 (Dmt1) would attenuate iron loading in a mouse model of hereditary hemochromatosis (HH). Initial experiments using duodenal epithelial organ cultures from intestine-specific Dmt1 knockout (KO) (Dmt1int/int) mice in the Ussing chamber established that Dmt1 is the only active iron importer during iron-deficiency anemia. Further, when Dmt1int/int mice were crossed with mice lacking the iron-regulatory hormone, hepcidin (Hepc−/−), iron loading was abolished. Hence, intestinal Dmt1 is required for the excessive iron absorption that typifies HH. Additional experiments established a protocol to produce GDLVs carrying functional Dmt1 small interfering RNAs (siRNAs) and to target these gene delivery vehicles to the duodenal epithelium in vivo (by incorporating folic acid [FA]). When FA-GDLVs carrying Dmt1 siRNA were administered to weanling Hepc−/− mice for 16 days, intestinal Dmt1 mRNA expression was attenuated and tissue iron accumulation was blunted. Oral delivery of functional siRNAs by FA-GDLVs is a suitable therapeutic approach to mitigate iron loading in murine HH.