Intracellular Iron Deficiency Research Articles

#1546 Mitochondrial oxidative phosphorylation in human proximal tubular epithelial cells is impaired by iron deficiency

Abstract Background and Aims We recently reported that iron deficiency may precede decline in renal function in patients with diabetes who had normal renal function at baseline but had a subsequent annual rate of eGFR decline of 3.3% or more (J Diabetes Investig. 2023;14:874-882.). Mitochondria contain many mitochondrial respiratory chain (MRC) enzymes that require iron as an active center, and consequently iron deficiency is known to impair mitochondrial function. Since the proximal tubular epithelial cells (PTECs) of the kidney are the cells with the greatest number of mitochondria in the body, we investigated whether iron deficiency impairs the mitochondrial capacity of the PTECs. Method Human cultured PTECs were analyzed in ‘control group’, ‘iron-deficient group ‘(co-cultured with deferiprone (DFP), an iron chelator), and ‘rescue group’ (co-cultured with DFP and FeCl3 which saturates the chelating ability of DFP), each with n ≥ 4 (MRC activities n = 4-5, others n ≥ 5). All analyses were performed after 24 hours of incubation. Values are shown as averages ± SEM. Results A Seahorse Extracellular Flux Analyzer showed that the basal oxygen consumption rates (OCRs) of control, iron-deficiency and rescue groups were 30.5 ± 1.2 pmol/min, 11.5 ± 0.7 pmol/min, and 31.1 ± 1.2 pmol/min, respectively, which revealed that basal OCRs of PTECs significantly decreased in the iron-deficient group. In addition, the analyzer also revealed that the activities of MRC for complexes I, II, and III, which have iron-sulfur clusters as their active centers, were significantly decreased in citrate synthase ratio to 77.6 ± 6.2%, 75.1 ± 6.4%, and 72.8 ± 9.3% of the control group, respectively, while complex IV was not significantly different at 84.9% of the control group. These reductions in MRC activities were not observed in the rescue group. Western blotting results also revealed that complex I, II, and III subunit proteins, NDUF9, SDHA, and UQCR2, were significantly reduced in the iron-deficient group (reduced to 38 ± 5.8%, 87 ± 8.8%, and 55 ± 5.7% of the control group, respectively). On the other hand, lactate concentrations in the culture medium of control group, iron-deficient group and rescue group were 1.73 ± 0.13 mmol, 3.01 ± 0.15 mmol and 1.61 ± 0.12 mmol, which revealed that glycolytic system was increased in the iron-deficient group, presumably to compensate for decreased oxidative phosphorylation. In response to intracellular iron deficiency, the iron regulatory protein 2 (IRP2) was shown in Western blotting to be unchanged in the rescue group (123 ± 4.0% of the control group), but increased in the iron-deficient group (170 ± 20% of the control group). The qRT-PCR analyses showed that mRNA expression of iron-sulfur cluster assembly enzyme (ISCU) was significantly decreased in the iron-deficiency group compared with the control group (41.8%), but not in the rescue group (95.1%), which is presumed that IRP2 suppressed ISCU expression, as has been proved previously. These results should indicate that iron deficiency suppressed iron utilization in the mitochondria of PTECs. The number of viable PTECs in the iron-deficient group after 24 hours was 1.99 ± 0.09 × 10^5 cells/well, which was significantly lower than that of the control (3.15 ± 0.15 × 10^5 cells/well) and rescue (2.68 ± 0.18 × 10^5 cells/well) groups. On the other hand, the number of dead PTECs was 0.042 ± 0.019 × 10^5 cells in the iron-deficient group, which was not significantly different from the control (0.023 ± 0.005 × 10^5 cells) and the rescue (0.020 ± 0.011 × 10^5 cells) groups. These data suggest that cell growth should be suppressed by iron deficiency. Conclusion Mitochondrial oxidative phosphorylation through the MRC was impaired by iron deficiency in PTECs. This impairment of mitochondrial capacity in PTECs may lead to PTECs dysfunction, potentially inducing renal damage such as cell death and interstitial injury. Further analysis of the effects of this mitochondrial dysfunction on ATP production capacity and reactive oxygen species production is needed.

Abstract 1892: KK2260: A fully human tetravalent bispecific antibody targeting TfR1 and EGFR for the treatment of EGFR-expressing tumors

Abstract Background: TfR1 is a transmembrane glycoprotein involved in iron uptake and regulation of cell growth. High TfR1 expression has been reported in different types of cancers, therefore recognized as a target molecule for cancer therapy. Despite the promise of potent efficacy of TfR1-targeted agents, there is a risk of side effects (i.e., severe anemia), since TfR1 is also highly expressed in certain normal cells (e.g., reticulocytes). KK2260 is a fully human tetravalent bispecific antibody (bsAb) based on REGULGENTTM technology targeting TfR1 and EGFR. This bsAb was designed for EGFR-expressing cell selective anti-cancer drug which could not be achieved by conventional monospecific Ab. In this study, we evaluated the mechanism of action (MOA), anti-cancer effect, and toxicity of KK2260. Methods: Binding activity of KK2260 to TfR1 and EGFR was evaluated by surface plasmon resonance method. Ligand blocking activity was evaluated by binding competition on esophageal squamous cell carcinoma (ESCC) OE21 cells. Growth inhibitory activity of KK2260 on cancer cells or hematopoietic progenitor cells was evaluated by proliferation inhibition assay. In some studies, excessive iron or anti-EGFR parent Ab was added. Effect on the cell surface target expression levels was evaluated by flow cytometry. The intracellular iron level was evaluated by cell imaging. Effect on EGF induced EGFR phosphorylation was evaluated by Western blotting. The in vivo efficacy of KK2260 was evaluated in cell line-derived and patient-derived xenograft (PDX) models of ESCC selected for high EGFR expression level. The toxicity of KK2260 was evaluated in cynomolgus monkeys. Results: KK2260 bound to TfR1 and EGFR with no ligand binding inhibition. KK2260 inhibited cell growth of moderate to high EGFR-expressing cancer cells, but not against EGFR-negative cancer cells or hematopoietic progenitor cells. KK2260 downregulated TfR1 expression on OE21 cells and the intracellular iron level decreased, but no effect was observed on EGFR phosphorylation. The growth inhibitory activity of KK2260 was abolished by adding excessive amounts of iron or reduced by inhibiting the binding to EGFR. Potent anti-tumor activity was observed in the ESCC PDX models with tumor regression. In the intermittent-dose toxicity study of cynomolgus monkeys, KK2260 was well-tolerated. Conclusions: KK2260 demonstrated potent anti-cancer effect both in vitro and in vivo. Intriguingly, KK2260 did not inhibit EGFR phosphorylation while EGFR-binding dependent TfR1 downregulation resulted in intracellular iron deficiency, which was considered as the MOA of KK2260. KK2260 was well tolerated in non-human primates. These preclinical data suggest the unique property of KK2260 is expected to provide significant benefit to patients with advanced ESCC and other EGFR-expressing tumors and Phase 1 study is currently on going in Japan (jRCT2031230372). Citation Format: Keisuke Mitamura, Hironori Otsuki, Keiko Nakahashi, Ayaka Kitagawa, Masayo Kamigaki, Yohei Kurio, Miho Inoue, Yasunori Uemura. KK2260: A fully human tetravalent bispecific antibody targeting TfR1 and EGFR for the treatment of EGFR-expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1892.

Iron Availability Influences Protein Carbonylation in Arabidopsis thaliana Plants.

Protein carbonylation is an irreversible form of post-translational modification triggered by reactive oxygen species in animal and plant cells. It occurs either through the metal-catalyzed oxidation of Lys, Arg, Pro, and Thr side chains or the addition of α, β-unsaturated aldehydes and ketones to the side chains of Cys, Lys, and His. Recent genetic studies concerning plants pointed to an implication of protein carbonylation in gene regulation through phytohormones. However, for protein carbonylation to stand out as a signal transduction mechanism, such as phosphorylation and ubiquitination, it must be controlled in time and space by a still unknown trigger. In this study, we tested the hypothesis that the profile and extent of protein carbonylation are influenced by iron homeostasis in vivo. For this, we compared the profile and the contents of the carbonylated proteins in the Arabidopsis thaliana wild-type and mutant-deficient in three ferritin genes under normal and stress conditions. Additionally, we examined the proteins specifically carbonylated in wild-type seedlings exposed to iron-deficient conditions. Our results indicated that proteins were differentially carbonylated between the wild type and the triple ferritin mutant Fer1-3-4 in the leaves, stems, and flowers under normal growth conditions. The profile of the carbonylated proteins was also different between the wild type and the ferritin triple mutant exposed to heat stress, thus pointing to the influence of iron on the carbonylation of proteins. Consistent with this, the exposure of the seedlings to iron deficiency and iron excess greatly influenced the carbonylation of certain proteins involved in intracellular signal transduction, translation, and iron deficiency response. Overall, the study underlined the importance of iron homeostasis in the occurrence of protein carbonylation in vivo.

Iron therapy mitigates chronic kidney disease progression by regulating intracellular iron status of kidney macrophages.

Systemic iron metabolism is disrupted in chronic kidney disease (CKD). However, little is known about local kidney iron homeostasis and its role in kidney fibrosis. Kidney-specific effects of iron therapy in CKD also remain elusive. Here, we elucidate the role of macrophage iron status in kidney fibrosis and demonstrate that it is a potential therapeutic target. In CKD, kidney macrophages exhibited depletion of labile iron pool (LIP) and induction of transferrin receptor 1, indicating intracellular iron deficiency. Low LIP in kidney macrophages was associated with their defective antioxidant response and proinflammatory polarization. Repletion of LIP in kidney macrophages through knockout of ferritin heavy chain (Fth1) reduced oxidative stress and mitigated fibrosis. Similar to Fth1 knockout, iron dextran therapy, through replenishing macrophage LIP, reduced oxidative stress, decreased the production of proinflammatory cytokines, and alleviated kidney fibrosis. Interestingly, iron markedly decreased TGF-β expression and suppressed TGF-β-driven fibrotic response of macrophages. Iron dextran therapy and FtH suppression had an additive protective effect against fibrosis. Adoptive transfer of iron-loaded macrophages alleviated kidney fibrosis, validating the protective effect of iron-replete macrophages in CKD. Thus, targeting intracellular iron deficiency of kidney macrophages in CKD can serve as a therapeutic opportunity to mitigate disease progression.

Differential Recovery of Macrophage Iron Deficiency By Oral Iron Formulations Determines Cell Inflammatory Response in Conditions of Iron Deficiency Anemia

Introduction: Sucrosomial iron (SI) is an innovative oral iron formulation which is absorbed intact through para and trans-cellular intestinal routes as well as via M-cells. Once in the circulation SI is internalized intact by reticulo-endothelial macrophages, which likely play a major role in iron recycling from the sucrosome shell and release into the bloodstream where iron joins the transferrin (Tf)-bound iron pool. By contrast, the common oral iron salt iron sulfate (FeSO4) is absorbed via the canonical inorganic iron pathway involving DMT1 and FPN and directly joins the Tf-bound iron pool. FeSO4 administration generates higher levels of free non-transferrin-bound iron (NTBI) to which macrophages can be exposed, leading to cell iron accumulation. Thus iron provided both as SI or FeSO4 can target macrophages. Recently, macrophage iron overload has been implicated in sterile inflammation through ROS-dependent cell pro-inflammatory activation. Here we investigated and compared how SI and FeSO4 shape the polarization status of macrophages and affect inflammation in conditions of iron deficiency anemia. Methods: To this aim, iron-deficient wild-type mice administered a low iron diet (<10ppm) were repeatedly treated with SI or FeSO4 every day for 2 weeks to correct their anemia, and hepatic and splenic macrophages were analyzed in term of cell iron status and inflammatory response. Results: Iron-deficient mice treated with SI showed an efficient but slightly slower recovery of anemia as monitored by blood parameters (e.g. Hb, RBC, HCT) compared to FeSO4-treated animals. The gradual anemia recovery by SI likely reflects the additional macrophage-dependent processing needed to recycle iron from the intact SI shell, which is not required following FeSO4 absorption. Indeed, Tf saturation and NTBI were more elevated in FeSO4-treated than SI-treated mice. Consistent with these observations, FeSO4 corrected intracellular iron deficiency in macrophages more rapidly than SI, as indicated by a more pronounced TfR1 suppression and bigger labile iron pool. This triggered more elevated ROS levels and increased apoptosis in macrophages from FeSO4-treated compared to SI-treated mice. Importantly, the faster cell iron deficiency recovery was associated with TNFα, IL1β and IL-6 release in macrophages from FeSO4-treated mice, which remained almost negligible in cells from SI-treated animals. In vivo findings are fully recapitulated in vitro in iron-deficient bone marrow-derived macrophages (BMDM) exposed to SI or FeSO4. FeSO4 corrected macrophage iron status faster than SI, as suggested by a quick rise in labile iron pool and suppression of TfR1 after 2h of treatment. By contrast, SI showed a slower and progressive ability to improve cell iron deficiency, modulating LIP and TfR1 after 5h of treatment, in agreement with a longer recycling process of iron from the SI shell. While FeSO4 exposure caused a massive increase in ROS levels and a significant elevation of inflammatory cytokines, SI minimally affected ROS and inflammatory cytokine production in BMDMs. Conclusions: Our data indicate that the gradual cell iron deficiency correction by SI exerts a protective effect in macrophages against iron-mediated inflammatory activation by limiting ROS formation, suggesting that the kinetic of cell iron accumulation determines macrophage inflammatory response. Overall, these studies show that SI is a superior oral iron formulation than iron salts in terms of reduced pro-oxidant and inflammatory action, with relevance for iron deficiency anemia treatment in individuals with pre-existing inflammatory conditions, including chronic kidney disease, anemia of cancer, inflammatory bowel disease, ulcerative colitis, post-operative anemia, celiac disease and obesity.

Iron redistribution induces oxidative burst and resistance in maize against Curvularia lunata.

ΔClnps6 induced iron redistribution in maize B73 leaf cells and resulted in reactive oxygen species (ROS) burst to enhance plant resistance against Curvularia lunata. Iron is an indispensable co-factor of various crucial enzymes that are involved in cellular metabolic processes and energy metabolism in eukaryotes. For this reason, plants and pathogens compete for iron to maintain their iron homeostasis, respectively. In our previous study, ΔClnps6, the extracellular siderophore biosynthesis deletion mutant of Curvularia lunata, was sensitive to exogenous hydrogen peroxide and virulence reduction. However, the mechanism was not studied. Here, we report that maize B73 displayed highly resistance to ΔClnps6. The plants recruited more iron at cell wall appositions (CWAs) to cause ROS bursts. Intracellular iron deficiency induced by iron redistribution originated form up-regulated expression of genes involved in intracellular iron consumption in leaves and absorption in roots. The RNA-sequencing data also showed that the expression of respiratory burst oxidase homologue (ZmRBOH4) and NADP-dependent malic enzyme 4 (ZmNADP-ME4) involved in ROS production was up-regulated in maize B73 after ΔClnps6 infection. Simultaneously, jasmonic acid (JA) biosynthesis genes lipoxygenase (ZmLOX), allene oxide synthase (ZmAOS), GA degradation gene gibberellin 2-beta-dioxygenase (ZmGA2OX6) and ABA degradation genes abscisic acid hydroxylase (ZmABH1, ZmABH2) involved in iron homeostasis were up-regulated expression. Ferritin1 (ZmFER1) positive regulated maize resistance against C. lunata via ROS burst under Fe-limiting conditions. Overall, our results showed that iron played vital roles in activating maize resistance in B73-C. lunata interaction.

Elucidation of the coping strategy in an OMP homozygous knockout mutant of Synechocystis 6803 defective in iron uptake.

The TonB-dependent outer membrane (OM) transporters import iron (Fe) under Fe-deficient growth conditions in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (S. 6803 or WT). The present study characterises the mechanisms employed by a homozygous Δslr1908 (Slr1908, an OM protein) mutant of S. 6803 to adapt to intracellular iron deficiency imposed due to lack of Slr1908 when grown under Fe sufficient conditions for short (1week) and long term (3weeks). Although the homozygous Δslr1908 cells showed symptoms of stress such as slow growth, high ROS and altered ultrastructure, they were transcriptionally as well as translationally modulating their physiology to abrogate the Fe limitation. The mutant displayed only 20% of the Fe content in comparison to the WT after 1week of growth, which increased to 50% by the end of the third week. The increase in intracellular Fe is ascribed to higher transcripts as well as corresponding protein of slr0042 which is the alternate OM route for Fe uptake. The mutant was also allocating its available Fe pools judiciously among Fe requiring proteins such as catalase, superoxide dismutase and cytochromes. The results elucidate the mechanism employed to tide over the Fe deficiency by Δslr1908 mutant and will be important in explaining the fine-tuning of Fe homeostasis in this model cyanobacterium.

Neurohormonal activation induces intracellular iron deficiency and mitochondrial dysfunction in cardiac cells

BackgroundIron deficiency (ID) is common in patients with heart failure (HF) and is associated with poor outcomes, yet its role in the pathophysiology of HF is not well-defined. We sought to determine the consequences of HF neurohormonal activation in iron homeostasis and mitochondrial function in cardiac cells.MethodsHF was induced in C57BL/6 mice by using isoproterenol osmotic pumps and embryonic rat heart-derived H9c2 cells were subsequently challenged with Angiotensin II and/or Norepinephrine. The expression of several genes and proteins related to intracellular iron metabolism were assessed by Real time-PCR and immunoblotting, respectively. The intracellular iron levels were also determined. Mitochondrial function was analyzed by studying the mitochondrial membrane potential, the accumulation of radical oxygen species (ROS) and the adenosine triphosphate (ATP) production.ResultsHearts from isoproterenol-stimulated mice showed a decreased in both mRNA and protein levels of iron regulatory proteins, transferrin receptor 1, ferroportin 1 and hepcidin compared to control mice. Furthermore, mitoferrin 2 and mitochondrial ferritin were also downregulated in the hearts from HF mice. Similar data regarding these key iron regulatory molecules were found in the H9c2 cells challenged with neurohormonal stimuli. Accordingly, a depletion of intracellular iron levels was found in the stimulated cells compared to non-stimulated cells, as well as in the hearts from the isoproterenol-induced HF mice. Finally, neurohormonal activation impaired mitochondrial function as indicated by the accumulation of ROS, the impaired mitochondrial membrane potential and the decrease in the ATP levels in the cardiac cells.ConclusionsHF characteristic neurohormonal activation induced changes in the regulation of key molecules involved in iron homeostasis, reduced intracellular iron levels and impaired mitochondrial function. The current results suggest that iron could be involved in the pathophysiology of HF.

The role of cellular iron deficiency in controlling iron export

BackgroundIron export via the transport protein ferroportin (Fpn) plays a critical role in the regulation of dietary iron absorption and iron recycling in macrophages. Fpn plasma membrane expression is controlled by the hepatic iron-regulated hormone hepcidin in response to high iron availability and inflammation. Hepcidin binds to the central cavity of the Fpn transporter to block iron export either directly or by inducing Fpn internalization and lysosomal degradation. Here, we investigated whether iron deficiency affects Fpn protein turnover. MethodsWe ectopically expressed Fpn in HeLa cells and used cycloheximide chase experiments to study basal and hepcidin-induced Fpn degradation under extracellular and intracellular iron deficiency. Conclusions/General significanceWe show that iron deficiency does not affect basal Fpn turnover but causes a significant delay in hepcidin-induced degradation when cytosolic iron levels are low. These data have important mechanistic implications supporting the hypothesis that iron export is required for efficient targeting of Fpn by hepcidin. Additionally, we show that Fpn degradation is not involved in protecting cells from intracellular iron deficiency.

Sensing of Liver Iron Content Requires Cell-Cell Communication between Hepatocytes and Liver Sinusoidal Endothelial Cells

The liver stores iron and senses systemic and tissue iron availability. Hepatocytes control iron homeostasis by producing the peptide hormone hepcidin that controls dietary iron absorption and iron release from intracellular stores. Recent data challenged the exclusive role of hepatocytes in controlling iron levels. Indeed, liver sinusoidal endothelial cells (LSECs) increase BMP2 and BMP6 levels in response to iron, which control hepcidin expression in a paracrine manner. However the molecular mechanism(s) of how BMPs respond to iron levels remain unknown. We established primary murine LSEC cultures and exposed these to iron sources. Unexpectedly, BMP2 mRNA expression is strongly reduced by iron treatment, while BMP6 levels are only mildly increased. This finding suggests that intracellular iron content cannot directly activate BMP2 transcription and only slightly contribute to BMP6 upregulation in LSEC cultures. However, if LSECs are co-cultured with iron-loaded primary hepatocytes the expression of BMP2 and BMP6 is increased and the fold induction of BMP6 is greater compared to LSECs cultured alone, suggesting that the iron status of hepatocytes instructs the LSEC BMP response. These data are supported by findings in a genetic mouse model of iron overload (Slc40a1C326S/C326S). Hepatocytes isolated from Slc40a1C326S/C326S mice display an iron-loaded molecular signature and the expected low mRNA expression of Transferrin Receptor 1 (Tfr1). By contrast, LSECs show high expression of Tfr1, indicating intracellular iron deficiency. Despite this, hepatic BMP levels are increased, suggesting that BMP2 and BMP6 expression are directly related to the intracellular iron content of hepatocytes but not LSECs. RNA-sequencing of isolated hepatic cell populations is ongoing to identify putative hepatocyte regulators involved in the iron-mediated BMP2 and BMP6 regulation. Disclosures Muckenthaler: Silence Therapeutics: Consultancy; Novartis: Research Funding.

Iron Deficiency as a Therapeutic Target in Cardiovascular Disease.

Iron deficiency is the most common nutritional disorder in the world. It is prevalent amongst patients with cardiovascular disease, in whom it is associated with worse clinical outcomes. The benefits of iron supplementation have been established in chronic heart failure, but data on their effectiveness in other cardiovascular diseases are lacking or conflicting. Realising the potential of iron therapies in cardiovascular disease requires understanding of the mechanisms through which iron deficiency affects cardiovascular function, and the cell types in which such mechanisms operate. That understanding has been enhanced by recent insights into the roles of hepcidin and iron regulatory proteins (IRPs) in cellular iron homeostasis within cardiovascular cells. These studies identify intracellular iron deficiency within the cardiovascular tissue as an important contributor to the disease process, and present novel therapeutic strategies based on targeting the machinery of cellular iron homeostasis rather than direct iron supplementation. This review discusses these new insights and their wider implications for the treatment of cardiovascular diseases, focusing on two disease conditions: chronic heart failure and pulmonary arterial hypertension.

Intracellular iron deficiency in pulmonary arterial smooth muscle cells induces pulmonary arterial hypertension in mice

Iron deficiency augments hypoxic pulmonary arterial pressure in healthy individuals and exacerbates pulmonary arterial hypertension (PAH) in patients, even without anemia. Conversely, iron supplementation has been shown to be beneficial in both settings. The mechanisms underlying the effects of iron availability are not known, due to lack of understanding of how cells of the pulmonary vasculature respond to changes in iron levels. The iron export protein ferroportin (FPN) and its antagonist peptide hepcidin control systemic iron levels by regulating release from the gut and spleen, the sites of absorption and recycling, respectively. We found FPN to be present in pulmonary arterial smooth muscle cells (PASMCs) and regulated by hepcidin cell autonomously. To interrogate the importance of this regulation, we generated mice with smooth muscle-specific knock in of the hepcidin-resistant isoform fpn C326Y. While retaining normal systemic iron levels, this model developed PAH and right heart failure as a consequence of intracellular iron deficiency and increased expression of the vasoconstrictor endothelin-1 (ET-1) within PASMCs. PAH was prevented and reversed by i.v. iron and by the ET receptor antagonist BQ-123. The regulation of ET-1 by iron was also demonstrated in healthy humans exposed to hypoxia and in PASMCs from PAH patients with mutations in bone morphogenetic protein receptor type II. Such mutations were further associated with dysregulation of the HAMP/FPN axis in PASMCs. This study presents evidence that intracellular iron deficiency specifically within PASMCs alters pulmonary vascular function. It offers a mechanistic underpinning for the known effects of iron availability in humans.

Elevated lactate in acute heart failure patients with intracellular iron deficiency as identifier of poor outcome.

We believe that there is a physiological link between intracellular iron status (assessed by soluble transferrin receptor [sTfR]) and efficiency of energy production/consumption (assessed by lactate, a product of anaerobic cell metabolism), which may further impact the outcome of patients with acute heart failure (AHF). To examine if elevated levels of lactate (> 2 mmol/L) accompanied by unmet cellular iron requirements (defined as sTfR > 1.59 mg/L) identify AHF patients with an unfavourable outcome. The study is a single-centre, retrospective analysis of AHF patients in whom lactate and iron status were assessed on admission. The endpoint of the study was one-year mortality. The study population consisted of 89 patients at a mean age of 65 ± 13 years. Mean systolic blood pressure and creatinine level were 135 ± 36 mmHg and 1.3 ± 0.6 mg/dL, respectively, and median [25th-75th quartiles] lactate level on admission was 2.0 [1.6-2.6] mmol/L. In 17 (19%) patients, both lactate and sTfR were below the cut-off values (group 1). In 38 (43%) individuals one of the markers was elevated (group 2) and in the remaining 34 (38%) patients both markers were above the predefined cut-off values (group 3). There was no difference in clinical and laboratory characteristics between the groups. During one-year follow-up 23 (26%) patients died. Mortality risk in group 3 was higher compared to the rest of the population (hazard ratio 5.6, 95% confidence interval 2.2-14, p = 0.0003), even after adjustments for well-defined prognostic factors. Patients with unmet iron cell requirements and hyperlactataemia on admission have significantly higher mortality risk compared to individuals without those pathologies.

Perturbation of Iron Metabolism by Cisplatin through Inhibition of Iron Regulatory Protein 2

Cisplatin is classically known to exhibit anticancer activity through DNA damage in the nucleus. Here we found a mechanism by which cisplatin affects iron metabolism, leading to toxicity and cell death. Cisplatin causes intracellular iron deficiency through direct inhibition of the master regulator of iron metabolism, iron regulatory protein 2 (IRP2) with marginal effects on IRP1. Cisplatin, but not carboplatin or transplatin, binds human IRP2 at Cys512 and Cys516 and impairs IRP2 binding to iron-responsive elements of ferritin and transferrin receptor-1 (TfR1) mRNAs. IRP2 inhibition by cisplatin caused ferritin upregulation and TfR1 downregulation leading to sustained intracellular iron deficiency. Cys512/516Ala mutant IRP2 made cells more resistant to cisplatin. Furthermore, combination of cisplatin and the iron chelator desferrioxamine enhanced cytotoxicity through augmented iron depletion in culture and xenograft mouse model. Collectively, cisplatin is an inhibitor of IRP2 that induces intracellular iron deficiency.

Dietary iron loading negatively affects liver mitochondrial function.

Iron is an essential co-factor for several metabolic processes, including mitochondrial respiration, and mitochondria are the major sites of iron-utilization. Cellular iron homeostasis must be tightly regulated, as intracellular iron deficiency can lead to insufficient energy production, whereas iron overload triggers ROS (reactive oxygen species) formation via the Fenton reaction. So far little is known on how iron imbalances affect mitochondrial function in vivo and the impact of the genotype on that, we studied the effects of dietary iron loading on mitochondrial respiratory capacity in liver by comparing two genetically divergent mouse strains, namely C57BL/6N and FVB mice. Both mouse strains differed in their basal iron levels and their metabolic responses to iron loading as determined by expression of iron trafficking proteins (ferritin was increased in livers of animals receiving high iron diet) as well as tissue iron content (2-fold increase, FVB p = 0.0013; C57BL/6N p = 0.0022). Dietary iron exposure caused a significant impairment of mitochondrial oxidative phosphorylation, especially regarding OXPHOS capacity (FVB p = 0.0006; C57BL/6N p = 0.0087) and S-ETS capacity (FVB p = 0.0281; C57BL/6N p = 0.0159). These effects were more pronounced in C57BL/6N than in FVB mice and were paralleled by an iron mediated induction of oxidative stress in mitochondria. The increased susceptibility of C57BL6/N mice to iron loading may be due to reduced expression of anti-oxidant defense mechanisms and altered iron trafficking upon dietary challenge pointing to a role of genetic modifiers for cellular and mitochondrial iron trafficking. Finally, iron-mediated induction of mitochondrial oxidative stress and reduction of oxidative phosphorylation may underlie fatigue in subjects with iron loading diseases.

Ferroportin mRNA is down-regulated in granulosa and cervical cells from infertile women

To explore the relationship between iron and infertility by investigating iron-related gene expression in granulosa and uterine cervical cells. Case-control study. Two tertiary hospitals. Two independent cohorts of fertile (n = 18 and n = 17) and infertile (n = 31 and n = 35) women. Invitro fertilization. Gene expression levels of ferritin light chain (FTL), ferritin heavy chain (FTH), transferrin receptor (TFRC), and ferroportin (SLC40A1) mRNA were analyzed in granulosa and cervical cells. In the first cohort, fertile and infertile women were similar in body mass index. Ferroportin mRNA levels were decreased in granulosa cells from infertile women in parallel with increased serum hepcidin levels. A positive association between ferroportin and TFRC mRNA, a gene associated with intracellular iron deficiency, was observed only in granulosa cells from fertile women. The major findings were replicated in a second independent cohort. Ferroportin mRNAs and circulating hepcidin identify a subset of infertile women and may constitute a target for therapy.

Endogenous siderophore 2,5-dihydroxybenzoic acid deficiency promotes anemia and splenic iron overload in mice.

Eukaryotes produce a siderophore-like molecule via a remarkably conserved biosynthetic pathway. 3-OH butyrate dehydrogenase (BDH2), a member of the short-chain dehydrogenase (SDR) family of reductases, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore 2,5-dihydroxybenzoic acid (2,5-DHBA). Depletion of the mammalian siderophore by inhibiting expression of bdh2 results in abnormal accumulation of intracellular iron and mitochondrial iron deficiency in cultured mammalian cells, as well as in yeast cells and zebrafish embryos We disrupted murine bdh2 by homologous recombination to analyze the effect of bdh2 deletion on erythropoiesis and iron metabolism. bdh2 null mice developed microcytic anemia and tissue iron overload, especially in the spleen. Exogenous supplementation with 2,5-DHBA alleviates splenic iron overload in bdh2 null mice. Additionally, bdh2 null mice exhibit reduced serum iron. Although BDH2 has been proposed to oxidize ketone bodies, we found that BDH2 deficiency did not alter ketone body metabolism in vivo. In sum, our findings demonstrate a key role for BDH2 in erythropoiesis.

Fine-tuned iron availability is essential to achieve optimal adipocyte differentiation and mitochondrial biogenesis.

Adipose tissue from obese and insulin-resistant individuals showed altered expression of several iron-related genes in a recent study, suggesting that iron might have an important role in adipogenesis. To investigate this possible role, we aimed to characterise the effects of iron on adipocyte differentiation. Intracellular iron deficiency was achieved using two independent approaches: deferoxamine administration (20 and 100 μmol/l) and transferrin knockdown (TF KD). The effects of added FeSO4, holo-transferrin and palmitate were studied during human and 3T3-L1 adipocyte differentiation. Finally, the relationship between iron-related and mitochondrial-related genes was investigated in human adipose tissue. Most adipose tissue iron-related genes were predominantly expressed in adipocytes compared with stromal vascular cells. Of note, transferrin gene and protein expression increased significantly during adipocyte differentiation. Both deferoxamine and TF KD severely blunted adipocyte differentiation in parallel with increased inflammatory mRNAs. These effects were reversed in a dose-dependent manner after iron supplementation. Palmitate administration also led to a state of functional intracellular iron deficiency, with decreased Tf gene expression and iron uptake during adipocyte differentiation, which was reversed with transferrin co-treatment. On the other hand, iron in excess impaired differentiation, but this antiadipogenic effect was less pronounced than under iron chelation. Of interest, expression of several genes involved in mitochondrial biogenesis occurred in parallel with expression of iron-related genes both during adipogenesis and in human adipose tissue. Precise and fine-tuned iron availability is essential to achieve optimal adipocyte differentiation, possibly modulating adipocyte mitochondrial biogenesis.

Desferrioxamine is both an iron chelator and a lysosomotropic amine

Iron regulatory protein 2 (IRP2) coordinates cellular regulation of iron metabolism by binding to iron responsive elements in mRNA. The protein is synthesized constitutively, but is rapidly degraded when iron stores are replete. Desferrioxamine has been widely used to block iron-dependent degradation of IRP2, presumably by inducing an intracellular iron deficiency. However, desferrioxamine is a weak basic alkylamine with pKa values of 8.32, 9.16, 9.94 and 11.44. Thus, we investigated whether desferrioxamine acts both as an iron chelator and as a lysosomotropic amine, such as chloroquine. Such amines accumulate in lysosomes and increase lysosomal pH, causing swelling of lysosomes and inhibition of lysosomal function. We exposed HEK293 cells to desferrioxamine or chloroquine and found that 100 μM desferrioxamine did induce swelling of lysosomes, although less than that caused by 150 μM chloroquine. Further, quantitative immunoblotting demonstrated that both chloroquine and desferrioxamine substantially increased intracellular IRP2 levels. Lysomotropic amines inhibit lysosomal proteases and also inhibit unloading of iron from the transferrin-transferrin receptor complex. Experiments in progress with specific protease inhibitors will allow dissection of these two potential mechanisms of action of chloroquine and desferrioxamine.

Pluripotent haemopoietic progenitor cells (CFU-Sd8) in peripheral blood of hereditarily anaemic Belgrade (b/b) rats.

The unique anaemic syndrome of the Belgrade laboratory (b/b) rat is due to an intracellular iron deficiency which is induced by a not yet defined mutation, resulting in impairment of haemopoiesis. We investigated the CFU-Sd8 number and concentration in the peripheral blood of b/b rats to study the relationship between medullary and extramedullary haemopoiesis in this anaemic syndrome. The results show normal concentration of CFU-Sd8 in the peripheral blood of b/b rats. This finding was unexpected in the state of severe anaemia and disturbed growth factor production in b/b rats, where the mobilization of CFU-Sd8 from bone marrow to blood is expected. The results suggest that severe anaemia is not regularly accompanied by the mobilization of pluripotent progenitors from bone marrow to the blood.