Betaine Alleviates High-Fat Diet-Induced Disruption of Hepatic Lipid and Iron Homeostasis in Mice

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive fat deposition in the liver, which is often associated with disrupted iron homeostasis. Betaine has been reported to be hepatoprotective, yet whether and how betaine ameliorates high-fat diet-induced disruption of hepatic lipid and iron homeostasis remains elusive. In this study, mice were fed either standard (CON) or high-fat diet (HFD) for 9 weeks to establish a NAFLD model. Mice raised on HF diet were then assigned randomly to HF and HFB groups, HFB group being supplemented with 1% (w/v) of betaine in the drinking water for 13 weeks. Betaine supplementation significantly alleviated excessive hepatic lipid deposition and restored hepatic iron content. Betaine partly yet significantly reversed HFD-induced dysregulation of lipogenic genes such as PRARγ and CD36, as well as the iron-metabolic genes including FPN and HAMP that encodes hepcidin. Similar mitigation effects of betaine were observed for BMP2 and BMP6, the up-stream regulators of hepcidin expression. Betaine significantly rectified disrupted expression of methyl transfer gene, including BHMT, GNMT and DNMT1. Moreover, HFD-modified CpG methylation on the promoter of PRARγ and HAMP genes was significantly reversed by betaine supplementation. These results indicate that betaine alleviates HFD-induced disruption of hepatic lipid and iron metabolism, which is associated with modification of CpG methylation on promoter of lipogenic and iron-metabolic genes.     

EnvIRONmental Aspects in Myelodysplastic Syndrome

Systemic iron overload is multifactorial in patients suffering from myelodysplastic syndrome (MDS). Disease-immanent ineffective erythropoiesis together with chronic red blood cell transfusion represent the main underlying reasons. However, like the genetic heterogeneity of MDS, iron homeostasis is also diverse in different MDS subtypes and can no longer be generalized. While a certain amount of iron and reactive oxygen species (ROS) are indispensable for proper hematological output, both are harmful if present in excess. Consequently, iron overload has been increasingly recognized as an important player in MDS, which is worth paying attention to. This review focuses on iron- and ROS-mediated effects in the bone marrow niche, their implications for hematopoiesis and their yet unclear involvement in clonal evolution. Moreover, we provide recent insights into hepcidin regulation in MDS and its interaction between erythropoiesis and inflammation. Based on Tet methylcytosine dioxygenase 2 (TET2), representing one of the most frequently mutated genes in MDS, leading to disturbances in both iron homeostasis and hematopoiesis, we highlight that different genetic alteration may have different implications and that a comprehensive workup is needed for a complete understanding and development of future therapies.     

Hemosiderin Accumulation in Liver Decreases Iron Availability in Tachycardia-Induced Porcine Congestive Heart Failure Model

Despite advances in the management of iron deficiency in heart failure (HF), the mechanisms underlying the effects of treatment remain to be established. Iron distribution and metabolism in HF pathogenesis need to be clarified. We used a porcine tachycardia-induced cardiomyopathy model to find out how HF development influences hepatic and myocardial iron storing, focusing on ferritin, the main iron storage protein. We found that cumulative liver congestion (due to the decrease of heart function) overwhelms its capacity to recycle iron from erythrocytes. As a consequence, iron is trapped in the liver as poorly mobilized hemosiderin. What is more, the ferritin-bound Fe³⁺ (reflecting bioavailable iron stores), and assembled ferritin (reflecting ability to store iron) are decreased in HF progression in the liver. We demonstrate that while HF pigs show iron deficiency indices, erythropoiesis is enhanced. Renin–angiotensin–aldosterone system activation and hepatic hepcidin suppression might indicate stress erythropoiesisinduced in HF. Furthermore, assembled ferritin increases but ferritin-bound Fe³⁺ is reduced in myocardium, indicating that a failing heart increases the iron storage reserve but iron deficiency leads to a drop in myocardial iron stores. Together, HF in pigs leads to down-regulated iron bioavailability and reduced hepatic iron storage making iron unavailable for systemic/cardiac needs.     

Matriptase-2 and Hemojuvelin in Hepcidin Regulation: In Vivo Immunoblot Studies in Mask Mice

Matriptase-2, a serine protease expressed in hepatocytes, is a negative regulator of hepcidin expression. The purpose of the study was to investigate the interaction of matriptase-2 with hemojuvelin protein in vivo. Mice lacking the matriptase-2 proteolytic activity (mask mice) display decreased content of hemojuvelin protein. Vice versa, the absence of hemojuvelin results in decreased liver content of matriptase-2, indicating that the two proteins interact. To further characterize the role of matriptase-2, we investigated iron metabolism in mask mice fed experimental diets. Administration of iron-enriched diet increased liver iron stores as well as hepcidin expression. Treatment of iron-overloaded mask mice with erythropoietin increased hemoglobin and hematocrit, indicating that the response to erythropoietin is intact in mask mice. Feeding of an iron-deficient diet to mask mice significantly increased spleen weight as well as the splenic content of erythroferrone and transferrin receptor proteins, indicating stress erythropoiesis. Liver hepcidin expression was decreased; expression of Id1 was not changed. Overall, the results suggest a complex interaction between matriptase-2 and hemojuvelin, and demonstrate that hepcidin can to some extent be regulated even in the absence of matriptase-2 proteolytic activity.     

Investigating the Molecular Mechanisms of Renal Hepcidin Induction and Protection upon Hemoglobin-Induced Acute Kidney Injury

Hemolysis is known to cause acute kidney injury (AKI). The iron regulatory hormone hepcidin, produced by renal distal tubules, is suggested to exert a renoprotective role during this pathology. We aimed to elucidate the molecular mechanisms of renal hepcidin synthesis and its protection against hemoglobin-induced AKI. In contrast to known hepatic hepcidin induction, incubation of mouse cortical collecting duct (mCCD_cl1) cells with IL-6 or LPS did not induce Hamp1 mRNA expression, whereas iron (FeS) and hemin significantly induced hepcidin synthesis (p < 0.05). Moreover, iron/heme-mediated hepcidin induction in mCCD_cl1 cells was caused by the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, as indicated by increased nuclear Nrf2 translocation and induced expression of Nrf2 downstream targets GCLM (p < 0.001), NQO1 (p < 0.001), and TXNRD1 (p < 0.005), which could be prevented by the known Nrf2 inhibitor trigonelline. Newly created inducible kidney-specific hepcidin KO mice demonstrated a significant reduction in renal Hamp1 mRNA expression. Phenylhydrazine (PHZ)-induced hemolysis caused renal iron loading and oxidative stress in both wildtype (Wt) and KO mice. PHZ treatment in Wt induced inflammatory markers (IL-6, TNFα) but not Hamp1. However, since PHZ treatment also significantly reduced systemic hepcidin levels in both Wt and KO mice (both p < 0.001), a dissection between the roles of systemic and renal hepcidin could not be made. Combined, the results of our study indicate that there are kidney-specific mechanisms in hepcidin regulation, as indicated by the dominant role of iron and not inflammation as an inducer of renal hepcidin, but also emphasize the complex interplay of various iron regulatory mechanisms during AKI on a local and systemic level.     

The Oral Ferroportin Inhibitor VIT-2763 Improves Erythropoiesis without Interfering with Iron Chelation Therapy in a Mouse Model of β-Thalassemia

In β-thalassemia, ineffective erythropoiesis leads to anemia and systemic iron overload. The management of iron overload by chelation therapy is a standard of care. However, iron chelation does not improve the ineffective erythropoiesis. We recently showed that the oral ferroportin inhibitor VIT-2763 ameliorates anemia and erythropoiesis in the Hbb^th3/+ mouse model of β-thalassemia. In this study, we investigated whether concurrent use of the iron chelator deferasirox (DFX) and the ferroportin inhibitor VIT-2763 causes any pharmacodynamic interactions in the Hbb^th3/+ mouse model of β-thalassemia. Mice were treated with VIT-2763 or DFX alone or with the combination of both drugs once daily for three weeks. VIT-2763 alone or in combination with DFX improved anemia and erythropoiesis. VIT-2763 alone decreased serum iron and transferrin saturation (TSAT) but was not able to reduce the liver iron concentration. While DFX alone had no effect on TSAT and erythropoiesis, it significantly reduced the liver iron concentration alone and in the presence of VIT-2763. Our results clearly show that VIT-2763 does not interfere with the iron chelation efficacy of DFX. Furthermore, VIT-2763 retains its beneficial effects on improving ineffective erythropoiesis when combined with DFX in the Hbb^th3/+ mouse model. In conclusion, co-administration of the oral ferroportin inhibitor VIT-2763 and the iron chelator DFX is feasible and might offer an opportunity to improve both ineffective erythropoiesis and iron overload in β-thalassemia.     

Iron Mining for Erythropoiesis

Iron is necessary for essential processes in every cell of the body, but the erythropoietic compartment is a privileged iron consumer. In fact, as a necessary component of hemoglobin and myoglobin, iron assures oxygen distribution; therefore, a considerable amount of iron is required daily for hemoglobin synthesis and erythroid cell proliferation. Therefore, a tight link exists between iron metabolism and erythropoiesis. The liver-derived hormone hepcidin, which controls iron homeostasis via its interaction with the iron exporter ferroportin, coordinates erythropoietic activity and iron homeostasis. When erythropoiesis is enhanced, iron availability to the erythron is mainly ensured by inhibiting hepcidin expression, thereby increasing ferroportin-mediated iron export from both duodenal absorptive cells and reticuloendothelial cells that process old and/or damaged red blood cells. Erythroferrone, a factor produced and secreted by erythroid precursors in response to erythropoietin, has been identified and characterized as a suppressor of hepcidin synthesis to allow iron mobilization and facilitate erythropoiesis.     

Effect of Erythropoietin on the Expression of Murine Transferrin Receptor 2

Erythropoietin (EPO) downregulates hepcidin expression to increase the availability of iron; the downregulation of hepcidin is mediated by erythroferrone (ERFE) secreted by erythroblasts. Erythroblasts also express transferrin receptor 2 (TFR2); however, the possible role of TFR2 in hepcidin downregulation is unclear. The purpose of the study was to correlate liver expression of hepcidin with the expression of ERFE and TFR2 in murine bone marrow and spleen at 4, 16, 24, 48, 72 and 96 h following administration of a single dose of EPO. Splenic Fam132b expression increased 4 h after EPO injection; liver hepcidin mRNA was decreased at 16 h. In the spleen, expression of TFR2 and transferrin receptor (TFR1) proteins increased by an order of magnitude at 48 and 72 h after EPO treatment. The EPO-induced increase in splenic TFR2 and TFR1 was associated with an increase in the number of Tfr2- and Tfr1-expressing erythroblasts. Plasma exosomes prepared from EPO-treated mice displayed increased amount of TFR1 protein; however, no exosomal TFR2 was detected. Overall, the results confirm the importance of ERFE in stress erythropoiesis, support the role of TFR2 in erythroid cell development, and highlight possible differences in the removal of TFR2 and TFR1 from erythroid cell membranes.     

Hepcidin-Induced Iron Deficiency Is Related to Transient Anemia and Hypoferremia in Kawasaki Disease Patients

Kawasaki disease (KD) is a type of systemic vasculitis that primarily affects children under the age of five years old. For sufferers of KD, intravenous immunoglobulin (IVIG) has been found to successfully diminish the occurrence of coronary artery lesions. Anemia is commonly found in KD patients, and we have shown that in appropriately elevated hepcidin levels are related to decreased hemoglobin levels in these patients. In this study, we investigated the time period of anemia and iron metabolism during different stages of KD. A total of 100 patients with KD and 20 control subjects were enrolled in this study for red blood cell and hemoglobin analysis. Furthermore, plasma, urine hepcidin, and plasma IL-6 levels were evaluated using enzyme-linked immunosorbent assay in 20 KD patients and controls. Changes in hemoglobin, plasma iron levels, and total iron binding capacity (TIBC) were also measured in patients with KD. Hemoglobin, iron levels, and TIBC were lower (p < 0.001, p = 0.009, and p < 0.001, respectively) while plasma IL-6 and hepcidin levels (both p < 0.001) were higher in patients with KD than in the controls prior to IVIG administration. Moreover, plasma hepcidin levels were positively and significantly correlated with urine hepcidin levels (p < 0.001) prior to IVIG administration. After IVIG treatment, plasma hepcidin and hemoglobin levels significantly decreased (both p < 0.001). Of particular note was a subsequent gradual increase in hemoglobin levels during the three weeks after IVIG treatment; nevertheless, the hemoglobin levels stayed lower in KD patients than in the controls (p = 0.045). These findings provide a longitudinal study of hemoglobin changes and among the first evidence that hepcidin induces transient anemia and hypoferremia during KD’s acute inflammatory phase.     

Transferrin Saturation/Hepcidin Ratio Discriminates TMPRSS6-Related Iron Refractory Iron Deficiency Anemia from Patients with Multi-Causal Iron Deficiency Anemia

Pathogenic TMPRSS6 variants impairing matriptase-2 function result in inappropriately high hepcidin levels relative to body iron status, leading to iron refractory iron deficiency anemia (IRIDA). As diagnosing IRIDA can be challenging due to its genotypical and phenotypical heterogeneity, we assessed the transferrin saturation (TSAT)/hepcidin ratio to distinguish IRIDA from multi-causal iron deficiency anemia (IDA). We included 20 IRIDA patients from a registry for rare inherited iron disorders and then enrolled 39 controls with IDA due to other causes. Plasma hepcidin-25 levels were measured by standardized isotope dilution mass spectrometry. IDA controls had not received iron therapy in the last 3 months and C-reactive protein levels were <10.0 mg/L. IRIDA patients had significantly lower TSAT/hepcidin ratios compared to IDA controls, median 0.6%/nM (interquartile range, IQR, 0.4–1.1%/nM) and 16.7%/nM (IQR, 12.0–24.0%/nM), respectively. The area under the curve for the TSAT/hepcidin ratio was 1.000 with 100% sensitivity and specificity (95% confidence intervals 84–100% and 91–100%, respectively) at an optimal cut-off point of 5.6%/nM. The TSAT/hepcidin ratio shows excellent performance in discriminating IRIDA from TMPRSS6-unrelated IDA early in the diagnostic work-up of IDA provided that recent iron therapy and moderate-to-severe inflammation are absent. These observations warrant further exploration in a broader IDA population.     

The Interplay between Drivers of Erythropoiesis and Iron Homeostasis in Rare Hereditary Anemias: Tipping the Balance

Rare hereditary anemias (RHA) represent a group of disorders characterized by either impaired production of erythrocytes or decreased survival (i.e., hemolysis). In RHA, the regulation of iron metabolism and erythropoiesis is often disturbed, leading to iron overload or worsening of chronic anemia due to unavailability of iron for erythropoiesis. Whereas iron overload generally is a well-recognized complication in patients requiring regular blood transfusions, it is also a significant problem in a large proportion of patients with RHA that are not transfusion dependent. This indicates that RHA share disease-specific defects in erythroid development that are linked to intrinsic defects in iron metabolism. In this review, we discuss the key regulators involved in the interplay between iron and erythropoiesis and their importance in the spectrum of RHA.     

Rare Gain-of-Function KCND3 Variant Associated with Cerebellar Ataxia,Parkinsonism, Cognitive Dysfunction,and Brain Iron Accumulation

Loss-of-function mutations in the K_V4.3 channel-encoding KCND3 gene are linked to neurodegenerative cerebellar ataxia. Patients suffering from neurodegeneration associated with iron deposition may also present with cerebellar ataxia. The mechanism underlying brain iron accumulation remains unclear. Here, we aim to ascertain the potential pathogenic role of KCND3 variant in iron accumulation-related cerebellar ataxia. We presented a patient with slowly progressive cerebellar ataxia, parkinsonism, cognitive impairment, and iron accumulation in the basal ganglia and the cerebellum. Whole exome sequencing analyses identified in the patient a heterozygous KCND3 c.1256G>A (p.R419H) variant predicted to be disease-causing by multiple bioinformatic analyses. In vitro biochemical and immunofluorescence examinations revealed that, compared to the human K_V4.3 wild-type channel, the p.R419H variant exhibited normal protein abundance and subcellular localization pattern. Electrophysiological investigation, however, demonstrated that the K_V4.3 p.R419H variant was associated with a dominant increase in potassium current amplitudes, as well as notable changes in voltage-dependent gating properties leading to enhanced potassium window current. These observations indicate that, in direct contrast with the loss-of-function KCND3 mutations previously reported in cerebellar ataxia patients, we identified a rare gain-of-function KCND3 variant that may expand the clinical and molecular spectra of neurodegenerative cerebellar disorders associated with brain iron accumulation.     

Breakthrough studies with mono- and binary-metal ion systems comprising of Fe(II) and As(III) using community prepared wooden charcoal packed columns

Groundwater of Assam (India) contains excessive amounts of As(III) and Fe(II). The rural and semi-urban population of Assam uses indigenous household iron filters fabricated using community prepared wooden charcoal (CPWC) to reduce Fe(II) concentration, however no efforts are made to reduce As(III) concentrations before use. The present work is directed toward assessing the potential of CPWC for metal removal from mono- and binary-metal ion systems comprising of Fe(II) and As(III) through continuous mode column studies. A decrease in breakthrough throughput volumes (V_B) from mono- and binary-metal ion systems is observed with increase in flow rates and decrease in bed depths. The order of breakthrough of metal ions observed as As(III) followed by Fe(II) from binary-metal ion system and hence the V_B for As(III) is termed as critical V_B as Fe(II) is yet to breakthrough through the beds. An early breakthrough for Fe(II) and As(III) from binary-metal ion system compared to respective mono-metal ion systems is observed for all the cases of flow rates and bed depths. It indicates impact on the uptake of a selected metal ion [either Fe(II) or As(III)] by the presence of the other metal ion [either As(III) or Fe(II)] present in the binary-metal ion system. The minimum and maximum errors involved between the predicted and experimental BDST curves for As(III) uptake till critical V_B from the binary-metal ion system varied between 1.0% and 24.8%.     

Expression Silencing of Glutathione Peroxidase 4 in Mouse Erythroleukemia Cells Delays In Vitro Erythropoiesis

Among the eight human glutathione peroxidase isoforms, glutathione peroxidase 4 (GPX4) is the only enzyme capable of reducing complex lipid peroxides to the corresponding alcohols. In mice, corruption of the Gpx4 gene leads to embryonic lethality and more detailed expression silencing studies have implicated the enzyme in several physiological processes (e.g., embryonal cerebrogenesis, neuronal function, male fertility). Experiments with conditional knockout mice, in which expression of the Gpx4 gene was silenced in erythroid precursors, indicated a role of Gpx4 in erythropoiesis. To test this hypothesis in a cellular in vitro model we transfected mouse erythroleukemia cells with a Gpx4 siRNA construct and followed the expression kinetics of erythropoietic gene products. Our data indicate that Gpx4 is expressed at high levels in mouse erythroleukemia cells and that expression silencing of the Gpx4 gene delays in vitro erythropoiesis. However, heterozygous expression of a catalytically inactive Gpx4 mutant (Gpx4^+/Sec46Ala) did not induce a defective erythropoietic phenotype in different in vivo and ex vivo models. These data suggest that Gpx4 plays a role in erythroid differentiation of mouse erythroleukemia cells but that heterozygous expression of a catalytically inactive Gpx4 is not sufficient to compromise in vivo and ex vivo erythropoiesis.     

New Cases of Hypochromic Microcytic Anemia Due to Mutations in the SLC11A2 Gene and Functional Characterization of the G75R Mutation

Divalent metal-iron transporter 1 (DMT1) is a mammalian iron transporter encoded by the SLC11A2 gene. DMT1 has a vital role in iron homeostasis by mediating iron uptake in the intestine and kidneys and by recovering iron from recycling endosomes after transferrin endocytosis. Mutations in SLC11A2 cause an ultra-rare hypochromic microcytic anemia with iron overload (AHMIO1), which has been described in eight patients so far. Here, we report two novel cases of this disease. The first proband is homozygous for a new SLC11A2 splicing variant (c.762 + 35A > G), becoming the first ever patient reported with a SLC11A2 splicing mutation in homozygosity. Splicing studies performed in this work confirm its pathogenicity. The second proband harbors the previously reported DMT1 G75R mutation in homozygosis. Functional studies with the G75R mutation in HuTu 80 cells demonstrate that this mutation results in improper DMT1 accumulation in lysosomes, which correlates with a significant decrease in DMT1 levels in patient-derived lymphoblast cell lines (LCLs). We also suggest that recombinant erythropoietin would be an adequate therapeutic approach for AHMIO1 patients as it improves their anemic state and may possibly contribute to mobilizing excessive hepatic iron.     

Modulatory Effects of Fractalkine on Inflammatory Response and Iron Metabolism of Lipopolysaccharide and Lipoteichoic Acid-Activated THP-1 Macrophages

Fractalkine (CX3CL1) acts as a chemokine as well as a regulator of iron metabolism. Fractalkine binds CX3CR1, the fractalkine receptor on the surface of monocytes/macrophages regulating different intracellular signalling pathways such as mitogen-activated protein kinase (MAPK), phospholipase C (PLC) and NFκB contributing to the production of pro-inflammatory cytokine synthesis, and the regulation of cell growth, differentiation, proliferation and metabolism. In this study, we focused on the modulatory effects of fractalkine on the immune response and on the iron metabolism of Escherichia coli and Pseudomonas aeruginosa lipopolysaccharides (LPS) and Staphylococcus aureus lipoteichoic acid (LTA) activated THP-1 cells to get a deeper insight into the role of soluble fractalkine in the regulation of the innate immune system. Pro-inflammatory cytokine secretions of the fractalkine-treated, LPS/LTA-treated, and co-treated THP-1 cells were determined using ELISArray and ELISA measurements. We analysed the protein expression levels of signalling molecules regulated by CX3CR1 as well as hepcidin, the major iron regulatory hormone, the iron transporters, the iron storage proteins and mitochondrial iron utilization. The results showed that fractalkine treatment alone did not affect the pro-inflammatory cytokine secretion, but it was proposed to act as a regulator of the iron metabolism of THP-1 cells. In the case of two different LPS and one type of LTA with fractalkine co-treatments, fractalkine was able to alter the levels of signalling proteins (NFκB, PSTAT3, Nrf2/Keap-1) regulating the expression of pro-inflammatory cytokines as well as hepcidin, and the iron storage and utilization of the THP-1 cells.