Divalent Metal Ion Transporter Research Articles

Naodesheng Pills Ameliorate Cerebral Ischemia Reperfusion-Induced Ferroptosis via Inhibition of the ERK1/2 Signaling Pathway.

Ferroptosis plays a crucial role in the occurrence and development of cerebral ischemia-reperfusion (I/R) injury and is regulated by mitogen-activated protein kinase 1/2 (ERK1/2). In China, Naodesheng Pills (NDSP) are prescribed to prevent and treat cerebrosclerosis and stroke. However, the protective effects and mechanism of action of NDSP against cerebral I/R-induced ferroptosis remain unclear. We investigated whether NDSP exerts its protective effects against I/R injury by regulating ferroptosis and aimed to elucidate the underlying mechanisms. The efficacy of NDSP was evaluated using a Sprague-Dawley rat model of middle cerebral artery occlusion and an in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) model. Brain injury was assessed using 2,3,5-triphenyltetrazolium chloride (TTC), hematoxylin and eosin staining, Nissl staining, and neurological scoring. Western blotting was performed to determine the expression levels of glutathione peroxidase 4 (GPX4), divalent metal-ion transporter-1 (DMT1), solute carrier family 7 member 11 (SLC7A11), and transferrin receptor 1 (TFR1). Iron levels, oxidative stress, and mitochondrial morphology were also evaluated. Network pharmacology was used to assess the associated mechanisms. NDSP (1.08 g/kg) significantly improved cerebral infarct area, cerebral water content, neurological scores, and cerebral tissue damage. Furthermore, NDSP inhibited I/R- and OGD/R-induced ferroptosis, as evidenced by the increased protein expression of GPX4 and SLC7A11, suppression of TFR1 and DMT1, and an overall reduction in oxidative stress and Fe2+ levels. The protective effects of NDSP in vitro were abolished by the GPX4 inhibitor RSL3. Network pharmacology analysis revealed that ERK1/2 was the core target gene and that NDSP reduced the amount of phosphorylated ERK1/2. NDSP exerts its protective effects against I/R by inhibiting cerebral I/R-induced ferroptosis, and this mechanism is associated with the regulation of ferroptosis via the ERK1/2 signaling pathway.

Intracellular Regulation Limits the Response of Intestinal Ferroportin to Iron Status in Suckling Rats.

Iron status is regulated via iron absorption as there is no active iron excretion. Divalent metal-ion transporter-1 (DMT1) and ferroportin (FPN) are two key proteins vital for iron absorption, but the regulation of them in suckling mammals differs from that in adults. This study aims to explore regulation of iron transporters under different iron conditions during suckling. This study developed suckling rats under different iron conditions. Unexpectedly, unchanged FPN at different iron status are detected. Since FPN is the only known iron exporter for mammals, unchanged FPN limits iron exported into blood during suckling. Thus, factors regulating FPN at transcriptional, post-transcriptional, and post-translational levels are detected. Results showed that Fpn mRNA is upregulated, while micro RNA-485(miR-485) which could silence Fpn mRNA is upregulated at low iron status limiting translation of Fpn mRNA. Besides, serum hepcidin and liver Hamp mRNA are upregulated, but ring finger protein 217(Rnf217) mRNA remained unchanged at high iron status leading to FPN not downregulated as adults. Overall, this study indicates that translational regulation limits intestinal FPN protein response to iron deficiency and Rnf217 cannot effectively mediate the degradation of FPN at high iron status, which provides a reference for maintaining iron homeostasis during suckling.

Protein S-palmitoylation enhances profibrotic signaling in response to cadmium

Cadmium (Cd) is a naturally occurring, toxic environmental metal found in foods. Humans do not have an efficient mechanism for Cd elimination; thus, Cd burden in humans increases with age. Cell and mouse studies show that Cd burden from low environmental levels of exposure impacts lung cell metabolism, proliferation signaling and cell growth as part of disease-promoting profibrotic responses in the lungs. Prior integrative analysis of metabolomics and transcriptomics identified the zDHHC11 transcript as a central functional hub in response to Cd dose. zDHHC11 encodes a protein S-palmitoyltransferase, but no evidence is available for effects of Cd on protein S-palmitoylation. In the present research, we studied palmitoylation changes in response to Cd and found increased protein S-palmitoylation in human lung fibroblasts that was inhibited by 2-bromopalmitate (2-BP), an irreversible palmitoyltransferase inhibitor. Mass spectrometry-based proteomics showed palmitoylation of proteins involved in divalent metal transport and in fibrotic signaling. Mechanistic studies showed that 2-BP inhibited palmitoylation of divalent metal ion transporter ZIP14 and also inhibited cellular Cd uptake. Transcription analyses showed that Cd stimulated transforming growth factor (TGF)-β1 and β3 expression within 8 h and lung fibrotic markers α-smooth muscle actin, matrix metalloproteinase-2, and collagen 1α1 gene expression and that these effects were blocked by 2-BP. Because 2-BP also blocked palmitoylation of proteins controlled by TGFβ1, these results show that palmitoylation impacts Cd-dependent fibrotic signaling both by enhancing cellular Cd accumulation and by supporting post-translational processing of TGFβ1-dependent proteins.

Effect of electroacupuncture on oxidative stress and cell apoptosis induced by ferroptosis in mice with Parkinson's disease.

To observe the effect of electroacupuncture (EA) on ferroptosis and apoptosis-related proteins in the substantia nigra of midbrain in mice with Parkinson's disease (PD), so as to explore its possible mechanisms in the treatment of PD. Twenty-four C57BL/6 mice were randomly divided into blank, model and EA groups, with 8 mice in each group. The PD model was established by continuous gavage of rotenone for 4 weeks. EA was applied at "Baihui" (GV20), "Quchi" (LI11) and "Zusanli" (ST36) for 20 min, once a day for 14 days, with 2-day rest after every 5-day treatment. The open field test was used to evaluate the residence time in the central area, ave-rage movement speed, and total distance of the open field. Western blot was used to detect the protein expression le-vels of divalent metal ion transporter 1 (DMT1), membrane ferroportin 1 (FPN1), glutathione peroxidase 4 (GPX4), proapoptotic protein Bax, and anti apoptotic protein Bcl-2 in the substantia nigra. Immunohistochemical method was used to detect the morphological changes of neurons and the positive expression of tyrosine hydroxylase (TH) in the substantia nigra of mice. After 4 weeks of modeling, compared with the blank group, the residence time in the central area, average speed and total distance of open field were significantly lower (P<0.000 1, P<0.01, P<0.001)；the protein expression levels of DMT1 and Bax in the substantia nigra were increased (P<0.001, P<0.000 1), while the protein expression levels of FPN1, GPX4 and Bcl-2, and the optical density of TH+ cells in the substantia nigra were decreased (P<0.000 1, P<0.001) in the model group. In comparison with the model group, the residence time in the central area, average speed, and total distance of the EA group were increased (P<0.01, P<0.05)；the protein expression levels of DMT1 and Bax in the substantia nigra were decreased (P<0.01, P<0.001), while the protein expression levels of FPN1, GPX4, and Bcl-2, and the optical density of TH+ cells in the substantia nigra were increased (P<0.000 1, P<0.01, P<0.001, P<0.05). EA has a protective effect on dopaminergic neurons in the substantia nigra of midbrain in PD model mice, which may be related with its effect in regulating oxidative stress and cell apoptosis induced by ferroptosis.

Prion Protein Octarepeat Domain Forms Transient β-Sheet Structures upon Residue-Specific Binding to Cu(II) and Zn(II) Ions.

Misfolding of the cellular prion protein (PrPC) is associated with the development of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). Metal ions appear to play a crucial role in PrPC misfolding. PrPC is a combined Cu(II) and Zn(II) metal-binding protein, where the main metal-binding site is located in the octarepeat (OR) region. Thus, the biological function of PrPC may involve the transport of divalent metal ions across membranes or buffering concentrations of divalent metal ions in the synaptic cleft. Recent studies have shown that an excess of Cu(II) ions can result in PrPC instability, oligomerization, and/or neuroinflammation. Here, we have used biophysical methods to characterize Cu(II) and Zn(II) binding to the isolated OR region of PrPC. Circular dichroism (CD) spectroscopy data suggest that the OR domain binds up to four Cu(II) ions or two Zn(II) ions. Binding of the first metal ion results in a structural transition from the polyproline II helix to the β-turn structure, while the binding of additional metal ions induces the formation of β-sheet structures. Fluorescence spectroscopy data indicate that the OR region can bind both Cu(II) and Zn(II) ions at neutral pH, but under acidic conditions, it binds only Cu(II) ions. Molecular dynamics simulations suggest that binding of either metal ion to the OR region results in the formation of β-hairpin structures. As the formation of β-sheet structures can be a first step toward amyloid formation, we propose that high concentrations of either Cu(II) or Zn(II) ions may have a pro-amyloid effect in TSE diseases.

Evaluating the role of intestinal divalent metal-ion transporter 1 (DMT1) in high dietary iron induced copper deficiency in mice

Iron (Fe) and Copper (Cu) are essential trace minerals that are important in various biological processes within the body. High iron intake has been associated with perturbations in copper metabolism leading to copper deficiency in rats and mice. We hypothesized that high dietary iron blocks intestinal copper transport via the divalent metal transporter 1 (DMT1), which is essential for iron absorption but can also transport other divalent cations, including possibly copper. Prior to conducting experiments, to evaluate the effect of high iron intake on copper status, pilot studies were conducted in male and female intestine-specific DMT1 KO (DMT1int/int) mice and controls (DMT1fl/fl) to make both genotypes similarly iron deficient/anemic. At weanling, mice were divided into groups and administered IP iron dextran at doses of 0.5, 0.75, or 1.0 mg (DMT1int/int), or PBS (DMT1fl/fl), to raise the Hb levels in the DMT1int/int mice since they are severely anemic. DMT1int/int mice were then fed a 50-ppm control iron AIN-93G diet, while DMT1fl/fl mice were fed a 2-3 ppm (iron-deficient) diet, for 4 weeks. Weekly hemoglobin (Hb) measurements were taken via tail vein bleeds. For copper studies, iron status in weanling mice of both genotypes and sexes was modified based on pilot studies, and subsequently, mice were switched to experimental diets containing 1x iron (50 ppm Fe) with marginal copper (2-3 ppm Cu) or 20x iron (1000 ppm Fe) with marginal copper for 3 weeks. Biomarkers of iron and copper status were assessed including hemoglobin content and hematocrit (Hct), serum, and tissue nonheme iron concentration, blood transferrin saturation, liver hepcidin and copper transporter (Ctr1) expression and serum ceruloplasmin (Cp) activity. Outcomes showed that administration of 0.75 mg iron dextran at weanling combined with dietary intervention resulted in lowered and comparable iron status in mice of both genotypes and sexes as indicated by Hb, Hct and hepatic nonheme iron and liver hepcidin expression. In dietary copper studies, male and female DMT1fl/fl mice fed a high iron / marginal copper diet loaded iron in the liver and spleen, while DMT1int/int mice had significantly lower liver and spleen nonheme iron when fed the same diet. Liver hepcidin expression was elevated in both genotypes with high iron / marginal copper feeding while no changes were observed in Ctr1 expression between genotypes or diets. Serum Cp activity was decreased by 48% in female DMT1int/int mice as compared to a 23% decrease in DMT1fl/fl mice fed the high iron / marginal copper diet. These findings suggest that high dietary iron feeding in DMT1int/int mice does not cause severe iron loading but leads to lower copper content when compared to DMT1fl/fl mice. This investigation was funded by grant DK074867 from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (JFC, PI), and grant DK109717 from NIDDK and the Office of Dietary Supplements (to JFC, PI). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Whole genome DNA methylation and mutational profiles identify novel changes in proliferative verrucous leukoplakia

Proliferative verrucous leukoplakia (PVL) is a rare form of oral leukoplakia with a relatively high transformation rate resulting in oral squamous cell carcinoma (OSCC). Molecular analysis of PVL at the genome level is limited and has only identified molecular similarities between PVL and OSCC. However, the clinical profile of PVL suggests that molecular differences may be more important. Whole exome sequencing of 5 PVL-associated OSCC (PVL-OSCC) and paired blood samples was used to identify somatic mutations common to the tumors. Whole methylome analysis of samples from 4 PVL-associated OSCC and 3 OSCC of non-PVL origin samples was conducted to explore differential methylation. In contrast to conventional OSCC, PVL-associated OSCC showed infrequent TP53 mutation and altered spectra of PIK3CA and NOTCH1 mutations. Unsupervised hierarchical clustering identified 63 probes that discriminated between PVL-associated OSCC and OSCC of non-PVL origin. Differences in methylation were most significant for divalent metal ion transport, particularly calcium movement. Specific differences in mutation and methylation profiles between PVL-derived OSCC and OSCC of non-PVL origin suggest differences in their transformation pathways. Further studies of early PVL lesions may identify markers of transformation that are also applicable to more common oral premalignant disorders such as oral epithelial dysplasia.

Slc11a1 gene polymorphism influences dextran sulfate sodium (DSS)-induced colitis in a murine model of acute inflammation

Ulcerative Colitis (UC) is an inflammatory disease characterized by colonic mucosal lesions associated with an increased risk of carcinogenesis. UC pathogenesis involves environmental and genetic factors. Genetic studies have indicated the association of gene variants coding for the divalent metal ion transporter SLC11A1 protein (formerly NRAMP1) with UC susceptibility in several animal species. Two mouse lines were genetically selected for high (AIRmax) or low (AIRmin) acute inflammatory responses (AIR). AIRmax is susceptible, and AIRmin is resistant to DSS-induced colitis and colon carcinogenesis. Furthermore, AIRmin mice present polymorphism of the Slc11a1 gene. Here we investigated the possible modulating effect of the Slc11a1 R and S variants in DSS-induced colitis by using AIRmin mice homozygous for Slc11a1R (AIRminRR) or S (AIRminSS) alleles. We evaluated UC by the disease activity index (DAI), considering weight loss, diarrhea, blood in the anus or feces, cytokines, histopathology, and cell populations in the distal colon epithelium. AIRminSS mice have become susceptible to DSS effects, with higher DAI, IL6, G-CSF, and MCP-1 production and morphological and colon histopathological alterations than AIRminRR mice. The results point to a role of the Slc11a1 S allele in DSS colitis induction in the genetic background of AIRmin mice.

Effects of Iron and Vitamin C on Growth Performance, Iron Utilization, Antioxidant Capacity, Nonspecific Immunity, and Disease Resistance to Aeromonas hydrophila in Chinese Mitten Crab (Eriocheir sinensis)

Iron is an essential trace element with an abroad physiological function, and iron deficiency can impair animal health. Vitamin C (VC) has the potential to release iron from diets and increase iron uptake. This study evaluates the effects of dietary iron and vitamin C on the growth performance, iron utilization, antioxidant capacity, and nonspecific immunity of the Chinese mitten crab (Eriocheir sinensis). Juvenile E. sinensis ( 1.14 ± 0.01 g ) were fed six diets supplemented with three levels of iron (41.40, 92.25, and 143.00 mg/kg), and each iron level supplemented either 4.33 mg/kg or 700.90 mg/kg of vitamin C for 8 weeks. Crabs fed 41.40 mg/kg of iron diet had the lowest weight gain (WG) and specific growth rate (SGR) and had the highest feed conversion rate (FCR) regardless of dietary vitamin C levels. Dietary 700.90 mg/kg of vitamin C improved WG and SGR but decreased FCR when the diets were supplemented with 41.40 and 143.00 mg/kg of iron ( P &lt; 0.05 ). Dietary 700.90 mg/kg of vitamin C significantly improved the expression levels of divalent metal ion transporter 1, ferroportin, and iron regulation protein in the intestine and hepatopancreas when supplemented with 92.25 and 143.00 mg/kg of iron ( P &lt; 0.05 ) and enhanced the transferrin content in hemolymph and iron deposition in the hepatopancreas when supplemented with 92.25 and 143.00 mg/kg of iron ( P &lt; 0.05 ). Crabs fed the diet supplemented with 41.40 mg/kg of iron had the highest malondialdehyde content and the lowest superoxide dismutase activity in the hepatopancreas. Dietary 700.90 mg/kg of vitamin C supplementation also increased the acid phosphatase and bacteriolytic activity when diets were supplemented with 41.40 and 143.00 mg/kg of iron ( P &lt; 0.05 ). In addition, crabs fed 143.00 mg/kg of iron and 700.90 mg/kg of vitamin C diet had the highest survival rate after the Aeromonas hydrophila challenge. This study demonstrates that dietary 700.90 mg/kg of vitamin C could promote iron absorption and utilization, thereby increasing the growth, immunity, and disease resistance of E. sinensis.

Silver Nanoparticles Induce a Size-dependent Neurotoxicity to SH-SY5Y Neuroblastoma Cells via Ferritinophagy-mediated Oxidative Stress.

Silver nanoparticles (AgNPs) are widely used in a variety of consumer products because of their antibacterial and antifungal characteristics, but little is known about their toxicity to the brain. In this study, we investigated AgNP-induced neurotoxicity using the human neuroblastoma cancer (SH-SY5Y) cell line. After a 24 h treatment of AgNPs with two primary sizes (5 and 50nm labeled as Ag-5 and Ag-50, respectively), a series of toxicological endpoints including cell viability, expression of proteins and genes in amyloid precursor protein (APP) amyloid hydrolysis process and ferritinophagy signaling pathways, oxidative stress, intracellular iron levels, and molecular regulators of iron metabolism were evaluated. Our results showed that both Ag-5 and Ag-50 induced notable neurotoxic effects on SH-SY5Y cells indicated by cell proliferation inhibition, increased BACE1 protein expression, and decreased APP and ADAM10 gene expression. Activation of nuclear receptor coactivator 4-mediated ferritinophagy and blockade of autophagic flux were induced by AgNPs, accompanied by intracellular iron accumulation and overexpression of divalent metal-ion transporter-1 and ferroportin1 in SH-SY5Y cells. In addition, AgNPs significantly decreased glutathione peroxidase 4 protein expression but increased malondialdehyde concentration, suggesting that AgNP-induced iron accumulation may trigger oxidative stress by disruption of the intracellular oxidant and antioxidant systems. In addition, compared with Ag-50, Ag-5 with higher cellular uptake efficiency caused more detrimental effects on SH-SY5Y cells. In conclusion, our findings demonstrated a size-dependent neurotoxicity in SH-SY5Y cells by AgNPs via ferritinophagy-mediated oxidative stress.

Zinc induces hephaestin expression via a PI3K-CDX2 dependent mechanism to regulate iron transport in intestinal Caco-2 cells

Zinc stimulates intestinal iron absorption via induction of divalent metal ion transporter (DMT1) and hephaestin (HEPH). While the increase in DMT1 is mediated via a PI3K/IPR2 axis, the mechanisms of Zn-induced HEPH expression downstream of PI3K remain elusive. In the current study we probed the role of Caudal-related homeobox transcription factor-2 (CDX2) on Zn-induced HEPH expression. Zn treatment of Caco-2 cells increased CDX2 phosphorylation and HEPH protein and mRNA expression. siRNA-silencing of CDX2 inhibited Zn-induced HEPH expression. LY294002, an antagonist of PI3K inhibited Zn-induced phosphorylation of CDX2, and downstream HEPH expression. These results suggest that increased expression of HEPH in intestinal cells following Zn treatment is mediated via a PI3K-CDX2 pathway.

Cadmium Accumulation in the Goat Liver and Kidney Is Partially Promoted by the Upregulation of Metal Transporter Genes.

Simple SummaryThe pattern of cadmium (Cd) accumulation and the role of metal transport in tissues’ cadmium deposition using Cd from an organic source are not well-clarified in ruminants. The present study results clearly showed that dietary Cd exhibited different deposition rates between goat liver, kidney, and muscle. Cd concentration in the liver, kidney, and muscle of goats fed 1.07 mg Cd/kg of dry matter (DM) for 60 days remained within the scope of (1.0 mg/kg) the food safety standards of China (GB 2762-2017) and the European Commission Regulation 1881/2006 (amended by No. 629/2008). Dietary Cd promoted the expression of metal transporter genes in the liver and kidneys of goats. DMT1, ZIP8, and ZIP14 might play an imperative role in the uptake of Cd in the goat liver and kidney but not in the muscle.Metal transporters, including divalent metal-ion transporter-1 (DMT1), Zrt-/Irt-like protein 8 and 14 (ZIP8 and ZIP14), and ferroportin-1 (FPN1), reportedly participate in cellular cadmium (Cd) uptake, but those in farm animals remain unclarified. This study aimed to examine the growth, plasma biochemical indices, Cd accumulation, and expression of metal transporter genes in the liver, kidney, and muscle of goats exposed to rice paddies contaminated with different levels of Cd. Twenty-four goats were randomly assigned across three dietary treatments: 0.23, 0.63, and 1.07 mg of Cd/kg of dry matter (DM) for 60 days. The results showed that dietary Cd exposure increased (p < 0.05) both Cd accumulation and the mRNA expressions of metal transporter genes (DMT1, ZIP, and FPN1) in the liver and kidney but not in the muscle, suggesting dietary Cd exhibited different deposition rates between goat liver, kidney, and muscle. These outcomes suggest that high levels of dietary Cd stimulated the expression of metal transporter genes and thereby enhanced the uptake and accumulation of Cd in the goat liver and kidney. As such, higher Cd concentrations in the liver and kidney observed with Cd diets could be partly explained by upregulation of metal transport genes expression.

Intestinal Dmt1 is Required for Iron Loading in Mice Modeling β‐Thalassemia Intermedia

β‐thalassemia intermedia (βTI) is characterized by chronic anemia, ineffective erythropoiesis, and excessive intestinal iron absorption leading to systemic iron overload. Divalent metal‐ion transporter 1 (DMT1), the main intestinal iron transporter, may mediate excessive iron absorption in Th3/+ mice, which model βTI.ObjectiveTo determine if intestinal DMT1 is required for iron‐loading in beta‐thalassemia, and if so, to utilize a ginger nanoparticle‐derived lipid vector (GDLVs) siRNA delivery system to normalize iron absorption in Th3/+ mice.HypothesisDMT1 mediates excessive iron absorption in βTI mice and decreasing DMT1 expression by GDLVs‐siRNA will prevent iron accumulation in these mice.MethodsTh3/+ mice were crossed with mice lacking intestinal DMT1 generating double KOs. Also, weanling mice were gavaged daily for 16 days with GDLVs carrying functional DMT1 siRNA, and iron absorption and iron loading were assessed.ResultsLiver, spleen, kidney, heart, and duodenum non‐heme iron levels were decreased in Th3/+ mice lacking intestinal DMT1. Serum non‐heme iron content, serum ferritin levels, and transferrin saturation also decreased in Th3/+ mice lacking intestinal DMT1. Splenomegaly was alleviated in the double KO mice. Oral gavage of GDLVs carrying DMT1 siRNA blunted intestinal DMT1 expression and mitigated hepatic and splenic iron loading. Serum non‐heme iron levels, serum ferritin, and transferrin saturation also decreased in Th3/+ mice receiving GDLVs with DMT1 siRNA.ConclusionIntestinal DMT1 is required for iron‐loading in Th3/+ mice, and in vivo DMT1 knock down is an effective approach to blunt iron loading in mice modeling βTI.

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

Several common hereditary disorders are associated with toxic iron overload which leads to liver cirrhosis, cardiomyopathy, and endocrine disorders. Hereditary hemochromatosis arises from mutations in the genes coding for any of several proteins involved in iron sensing (e.g. Hfe), hepcidin production (e.g. hemojuvelin), or hepcidin action, resulting in elevated iron absorption relative to the iron need. Divalent metal‐ion transporter‐1 (DMT1) is a H+‐coupled Fe2+ transporter and the principal or only mechanism by which nonheme iron is taken up at the intestinal brush border [Shawki A et al (2015) Am J Physiol Gastrointest Liver Physiol 309, G635–G647]. DMT1 is a validated therapeutic target in treating iron overload. We have shown that Na+/H+ exchanger‐3 (NHE3) is required for adequate iron absorption, via its physiological role in generating at the intestinal brush border an acidic microclimate that energizes DMT1‐mediated H+‐coupled Fe2+ transport [Shawki A et al (2016) Am J Physiol Gastrointest Liver Physiol 311, G423–G430], so we have explored here the contribution of NHE3 to pathological iron loading. We tested the hypothesis that NHE3 activity is necessary for pathological iron loading. We examined the effect of ablating the SLC9A3 gene coding for NHE3 in the Hfe mouse model of hereditary hemochromatosis. We measured tissue iron levels, hematological and blood‐iron variables, and the hepatic expression of Hamp1 (coding for hepcidin) by using qPCR in male and female FVB/N mice, age ≍ 120 d, fed a normal diet (NIH‐07). We examined four genotypes in both sexes: wildtype mice, NHE3 knockout (NHE3−/−), Hfe knockout (hemochromatosis disease model, Hfe−/−), and double knockout (NHE3−/− | Hfe−/−) (n = 12–26 mice per group). We chose α = 0.05, and analyzed our data (mean, SD) by using multifactorial ANOVA. We found that ablation of NHE3 prevented the liver iron loading characteristic of the Hfe−/− mouse model, an effect that was independent of sex. We observed a similar effect on iron loading in the spleen. Ablation of NHE3 produced no sign of overt anemia. Hepatic expression of hepcidin was depressed in NHE3−/− and in Hfe−/− mice relative to wildtype mice, and remained low in the NHE3−/− | Hfe−/− mice, providing a plausible explanation for the observation that, despite correction of the tissue iron levels in NHE3−/− | Hfe−/− mice, iron levels remained high in the readily accessible serum iron pool (typically 0.1% of total body iron). We conclude that ablation of NHE3 prevents pathological tissue iron loading in the Hfe mouse model of hemochromatosis. Pharmacological blockade of NHE3 may offer a means of inhibiting iron absorption in hereditary hemochromatosis.

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.