scholarly journals EGCG Protects against 6-OHDA-Induced Neurotoxicity in a Cell Culture Model

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Dan Chen ◽  
Anumantha G. Kanthasamy ◽  
Manju B. Reddy
Keyword(s):  
Iron Metabolism ◽  
Iron Homeostasis ◽  
Regulatory Gene ◽  
Cell Uptake ◽  
Dopamine Depletion ◽  
Intracellular Iron ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Sytox Green ◽  
Gene And Protein Expression

Background. Parkinson’s disease (PD) is a progressive neurodegenerative disease that causes severe brain dopamine depletion. Disruption of iron metabolism may be involved in the PD progression.Objective. To test the protective effect of (−)-epigallocatechin-3-gallate (EGCG) against 6-hydroxydopamine- (6-OHDA-) induced neurotoxicity by regulating iron metabolism in N27 cells.Methods. Protection by EGCG in N27 cells was assessed by SYTOX green assay, MTT, and caspase-3 activity. Iron regulatory gene and protein expression were measured by RT-PCR and Western blotting. Intracellular iron uptake was measured using55Fe. The EGCG protection was further tested in primary mesencephalic dopaminergic neurons by immunocytochemistry.Results. EGCG protected against 6-OHDA-induced cell toxicity. 6-OHDA treatment significantly (p<0.05) increased divalent metal transporter-1 (DMT1) and hepcidin and decreased ferroportin 1 (Fpn1) level, whereas pretreatment with EGCG counteracted the effects. The increased55Fe (by 96%,p<0.01) cell uptake confirmed the iron burden by 6-OHDA and was reduced by EGCG by 27% (p<0.05), supporting the DMT1 results. Pretreatment with EGCG and 6-OHDA significantly increased (p<0.0001) TH+cell count (~3-fold) and neurite length (~12-fold) compared to 6-OHDA alone in primary mesencephalic neurons.Conclusions. Pretreatment with EGCG protected against 6-OHDA-induced neurotoxicity by regulating genes and proteins involved in brain iron homeostasis, especially modulating hepcidin levels.

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

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Shiqi Zhang ◽  
Emmanouil Ntasis ◽  
Sarah Kabtni ◽  
Jaap van den Born ◽  
Gerjan Navis ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Umbilical Vein ◽  
Receptor Protein ◽  
Dependent Manner ◽  
Tubular Epithelial Cells ◽  
Intracellular Iron ◽  
Alternative Source ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Metal Transporter

Iron has been suggested to affect the clinical course of type 2 diabetes (T2DM) as accompanying increased intracellular iron accumulation may provide an alternative source for reactive oxygen species (ROS). Although carnosine has proven its therapeutic efficacy in rodent models of T2DM, little is known about its efficacy to protect cells from iron toxicity. We sought to assess if high glucose (HG) exposure makes cultured human umbilical vein endothelial cells (HUVECs) and renal proximal tubular epithelial cells (PTECs) more susceptible to metal induced toxicity and if this is ameliorated by L-carnosine. HUVECs and PTECs, cultured under normal glucose (5 mM, NG) or HG (30 mM), were challenged for 24 h with FeCl3. Cell viability was not impaired under HG conditions nor did HG increase susceptibility to FeCl3. HG did not change the expression of divalent metal transporter 1 (DMT1), ferroportin (IREG), and transferrin receptor protein 1 (TFRC). Irrespective of glucose concentrations L-carnosine prevented toxicity in a dose-dependent manner, only if it was present during the FeCl3challenge. Hence our study indicates that iron induced cytotoxicity is not enhanced under HG conditions. L-Carnosine displayed a strong protective effect, most likely by chelation of iron mediated toxicity.

Differential expression of divalent metal transporter DMT1 (Slc11a2) in the spermatogenic epithelium of the developing and adult rat testis

2005 ◽  
Vol 288 (1) ◽  
pp. C176-C184 ◽  
Author(s):  
Kathleen P. Griffin ◽  
Donald T. Ward ◽  
Wei Liu ◽  
Gavin Stewart ◽  
Ian D. Morris ◽  
...  
Keyword(s):  
Germ Cells ◽  
Male Fertility ◽  
Plasma Membranes ◽  
Iron Homeostasis ◽  
Divalent Metal ◽  
Intracellular Iron ◽  
Divalent Metal Transporter ◽  
Rat Testis ◽  
Metal Transporter ◽  
Adult Rat

Iron is essential for male fertility, and disruptions in iron balance lead to impairment of testicular function. The divalent metal transporter DMT1 is a key modulator of transferrin- and non-transferrin-bound iron homeostasis. As a first step in determining the role of DMT1 in the testis, we have characterized the pattern of DMT1 expression in the developing and adult rat testis. Northern blot analysis and RT-PCR of testis polyadenylated RNA revealed the presence of iron-responsive element (IRE) and non-IRE transcripts. Semiquantitative immunoblotting of immature and adult rat testis uncovered the expression of two distinct DMT1 protein species. Immunohistochemistry showed that DMT1 was widespread throughout each seminiferous tubule and was expressed in the intracellular compartment. In the adult rat testis, DMT1 was immunolocalized to both the Sertoli and germ cells. In contrast to the immature testis, expression was dependent on the stage of the spermatogenic cycle. DMT1 was not detected on any plasma membranes in either the developing or the adult testis, suggesting that DMT1 is not primarily responsible for translocating iron across this epithelium. Our data suggest an important role for DMT1 in intracellular iron handling during spermatogenesis and imply that germ cells have a need for a precisely targeted and timed supply of iron. We suggest that DMT1 may, as it does in other tissues, play a role in transporting iron between intracellular compartments and thus may play an important role in male fertility.

Effects of iron regulatory protein regulation on iron homeostasis during hypoxia

Blood ◽  
2003 ◽  
Vol 102 (9) ◽  
pp. 3404-3411 ◽  
Author(s):  
Brian D. Schneider ◽  
Elizabeth A. Leibold
Keyword(s):  
Iron Uptake ◽  
Iron Homeostasis ◽  
Rna Binding ◽  
Late Phase ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Iron Regulatory Proteins ◽  
Ft Synthesis

AbstractIron regulatory proteins (IRP1 and IRP2) are RNA-binding proteins that affect the translation and stabilization of specific mRNAs by binding to stem-loop structures known as iron responsive elements (IREs). IREs are found in the 5′-untranslated region (UTR) of ferritin (Ft) and mitochondrial aconitase (m-Aco) mRNAs, and in the 3′-UTR of transferrin receptor (TfR) and divalent metal transporter-1 (DMT1) mRNAs. Our previous studies show that besides iron, IRPs are regulated by hypoxia. Here we describe the consequences of IRP regulation and show that iron homeostasis is regulated in 2 phases during hypoxia: an early phase where IRP1 RNA-binding activity decreases and iron uptake and Ft synthesis increase, and a late phase where IRP2 RNA-binding activity increases and iron uptake and Ft synthesis decrease. The increase in iron uptake is independent of DMT1 and TfR, suggesting an unknown transporter. Unlike Ft, m-Aco is not regulated during hypoxia. During the late phase of hypoxia, IRP2 RNA-binding activity increases, becoming the dominant regulator responsible for decreasing Ft synthesis. During reoxygenation (ReO2), Ft protein increases concomitant with a decrease in IRP2 RNA-binding activity. The data suggest that the differential regulation of IRPs during hypoxia may be important for cellular adaptation to low oxygen tension.

scholarly journals Hepcidin Expression in Adipose Tissue Increases during Cardiac Surgery

2010 ◽  
pp. 393-400 ◽  
Author(s):  
M Vokurka ◽  
Z Lacinová ◽  
J Křemen ◽  
P Kopecký ◽  
J Bláha ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Iron Metabolism ◽  
Epicardial Adipose Tissue ◽  
Low Grade ◽  
Phase Reaction ◽  
Anemia Of Chronic Disease ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Hepcidin Expression ◽  
Massive Obesity

Hepcidin, a key regulator of iron metabolism, plays a crucial role in the pathogenesis of anemia of chronic disease. Although it is produced mainly in the liver, its recently described expression in adipose tissue has been shown to be enhanced in massive obesity due to chronic low-grade inflammation. Our objective was to study the changes in hepcidin expression in adipose tissue during acute-phase reaction. We measured hepcidin mRNA expression from isolated subcutaneous and epicardial adipose tissue at the beginning and at the end of the surgery. The expression of mRNAs for hepcidin and other iron-related genes (transferrin receptor 1, divalent metal transporter 1, ferritin, ferroportin) were measured by real-time RT-PCR. Hepcidin expression significantly increased at the end of the surgery in subcutaneous but not in epicardial adipose tissue. Apart from the increased levels of cytokines, the parameters of iron metabolism showed typical inflammation-induced changes. We suggest that acute inflammatory changes could affect the regulation of hepcidin expression in subcutaneous adipose tissue and thus possibly contribute to inflammation-induced systemic changes of iron metabolism.

scholarly journals Melatonin Alleviates Acute Sleep Deprivation-Induced Memory Loss in Mice by Suppressing Hippocampal Ferroptosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Xintong Wang ◽  
Zixu Wang ◽  
Jing Cao ◽  
Yulan Dong ◽  
Yaoxing Chen
Keyword(s):  
Sleep Deprivation ◽  
Antioxidant Enzyme ◽  
Iron Homeostasis ◽  
Memory Loss ◽  
Blue Staining ◽  
Signal Pathways ◽  
Divalent Metal Transporter 1 ◽  
Study Objective ◽  
Divalent Metal Transporter

Objectives: Memory decline caused by insufficient sleep is a critical public health issues and currently lacks effective treatments. This study objective was to explore alleviative effect of melatonin on sleep deprivation (SD)-induced deficiencies in learning and memory.Materials and Methods: A continuous 72 h SD mouse model, with or without melatonin or Fer-1 supplementation were established. The changes of cognitive function, iron homeostasis, lipid peroxidation and intracellular signal pathways in mice were detected by Morris water maze, antioxidant assay, immunohistochemistry, western blot, RT-PCR and Prussian blue staining. In vitro, we treated HT-22 cells with ferroptosis inducer (Erastin) to further explore the specific mechanism of melatonin in ferroptosis.Results: Mice subjected to SD had significantly elevated latency and path length to reach hidden platform, as well as a decrease in number of entries and time spent in the target zone when the hidden platform was removed (p &lt; 0.05). Nevertheless, supplementation with ferroptosis inhibitor (Fer-1) mitigated the memory impairment associated with SD. Further evaluation revealed an up-regulation of intracellular iron accumulation, transferrin receptor 1 and divalent metal transporter 1 expression and ROS and MDA production, and a down-regulation of ferroportin and antioxidant enzyme (GPX4 and SOD) expression in SD mice. SD decreased expression of MT2 receptor rather than of MT1, and inhibited ERK/Nrf2 signaling activation in the hippocampus (p &lt; 0.05). In contrast, the aforementioned SD-inductions were reversed by supplementation using 20 and 40 mg/kg melatonin in SD mice. In vitro, melatonin pretreatment reversed Erastin-induced ferroptosis, abnormalities in iron transporter protein and antioxidant enzyme expression and suppression of ERK/Nrf2 signaling in HT-22 cells, however this protective effect of melatonin was blocked by MT2-, ERK- and Nrf2-specific antagonists (p &lt; 0.05).Conclusion: Our finding suggested SD may induce ferroptosis, in turn leading to cognitive deficits. Melatonin alleviated memory loss and hippocampal ferroptosis caused by acute SD through binding to the MT2 receptor to activate ERK/Nrf2 signaling.

scholarly journals Iron-Response Element Binding to Iron Regulatory Protein (IRP) 1 and 2 Are Indispensable for Adipose Tissue Browning

2020 ◽  
Vol 4 (Supplement_2) ◽  
pp. 1283-1283
Author(s):  
Mikyoung You ◽  
Soonkyu Chung
Keyword(s):  
Adipose Tissue ◽  
Iron Metabolism ◽  
Knockout Mice ◽  
Iron Homeostasis ◽  
Regulatory Protein ◽  
Mrna Translation ◽  
Intracellular Iron ◽  
Mobility Shift ◽  
Iron Regulatory Proteins ◽  
Tissue Browning

Abstract Objectives Intracellular iron homeostasis is tightly regulated in posttranscriptional levels via iron regulatory proteins (IRPs). IRPs bind to the iron-responsive elements (IREs), leading to either mRNA translation or stability. Our recent study demonstrated that iron metabolism is intimately linked with adipose tissue browning and thermogenic activation. However, the role of IRP/IRE interactions in the adipose tissue is poorly understood. We aim to characterize the IRP/IRE interactions in the adipose tissue in terms of depot-specificity and thermogenic potential. Methods To induce adipocyte browning, mice were administrated with beta-3 adrenoceptor agonist CL316243 (CL) for 5 days, and different depots of adipose tissue of epididymal (eWAT), inguinal (iWAT), brown (BAT), and liver were collected. Iron metabolism and thermogenesis were evaluated. To investigate the IRP/IRE binding, electrophoretic mobility shift assay (EMSA) was performed in the cytosolic using the fluorescence-labeled IRE (IR-IRE). To distinguish the IRE binding with IRP1 and 2, the cytosolic fraction from IRP1 and 2 knockout mice were used as positive controls. Results In a normal temperature, the constitutive IRP/IRE binding was found in the BAT, but not in the eWAT and iWAT. In response to CL treatment, iron content and transferrin receptor levels significantly increased in the WAT. Accordingly, the IRE/IRPs binding significantly increased in the CL-treated iWAT. Genetic deletion of IRP1 or 2 poses a marginal impact on constitutively active BAT development, suggesting IRP1 and 2 plays a compensatory role. Unlikely to BAT, the deletion of either IRP1 or 2 failed to induce WAT browning in the IRP1 and 2 knockout mice with CL stimulation. Consistently, both IRE binding to IRP1 and 2 were manifest in the CL treated iWAT, implicating that IRP1 and 2 plays a separate and synergistic function for WAT browning. Conclusions Our study defined the depot-specific iron regulatory metabolism in the adipose tissue using an innovative EMSA method. We demonstrated that, for the first time in our knowledge, IRE binding to both IRP1 and IRP2 is indispensable for the thermogenic activation of WAT, which is distinct from the iron regulatory mechanism found in the BAT. We propose that iron metabolism in the WAT is a novel determinant for WAT browning and thermogenic energy expenditure. Funding Sources None.

scholarly journals Hypoxia-inducible factor 2α but not hypoxia-inducible factor-1α increases iron traffic in astrocytes

2020 ◽  
Author(s):  
Manman Xu ◽  
Zhiqiang Chen ◽  
Xizhen Ma ◽  
Hangrui Xu ◽  
Changsheng Fan ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ferrous Iron ◽  
Iron Homeostasis ◽  
Hypoxia Inducible Factor ◽  
Western Blots ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Kinase C ◽  
Cultured Astrocytes

Abstract Background Disrupted iron homeostasis in the substantia nigra (SN) is an important mechanism in Parkinson’s disease (PD). In our previous studies using cultured neurons, we showed that 6-hydroxydopamine (6-OHDA) treatment increased the expression of divalent metal transporter-1 (DMT1) gating iron influx and decreased the expression of ferroportin 1 (FPN1) gating iron efflux, leading to increased iron deposition. In astrocytes, 6-OHDA increased both DMT1 and FPN1 expressions and iron traffic. However, the underlying mechanisms mediating these processes remain elusive. Hypoxia-inducible factors (HIFs) are important regulators of iron homeostasis. Methods Using western blots, we observed HIFs, DMT1 and FPN1 expressions in primary cultured astrocytes and ventral mesencephalic (VM) treated with 6-OHDA, inhibitors of HIF-1α and HIF-2α, protein kinase C (PKC) inhibitor and PKC activator, radical scavenger and inducible NO synthase (iNOS) inhibitor. The ferrous iron traffic of astrocytes was determined by measuring the quenching or reversing of calcein fluorescence. Results Using primary rat cell cultures, we observed that 6-OHDA treatment significantly increased the expressions of HIF-1α and HIF-2α in cultured astrocytes but not in VM neurons. Moreover, we observed that HIF-2α inhibitor, but not HIF-1α inhibitor reversed the 6-OHDA-induced upregulation of DMT1 and FPN1 and ferrous iron traffic. Blocking protein kinase C (PKC) pathway by bisindolylmaleimide I hydrochloride (Bisl) or N-acetyl-l-cysteine (NAC) or Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) inhibits Phorbol 12-myristate 13-acetate (PMA) or 6-OHDA induced activation of HIF-2α, whereas activating by PMA or 6-OHDA was sufficient to activate HIF-2α. Notably, blocking of PKC delta (PKC δ) phosphorylation by NAC, or L-NAME inhibits 6-OHDA induced activation of HIF-2α. Conclusion Our data indicate that astrocytic HIF-2α, but not HIF-1α, may be an important regulator of DMT1 and FPN1 expressions by activating the PKC -dependent pathway.

DMT1 Expression Is Regulated by DMT1 Associated Protein (DAP) in K562 Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2661-2661
Author(s):  
Yasumasa Okazaki ◽  
Hong Yin ◽  
Yuxiang Ma ◽  
Emiko Okazaki ◽  
Mary Yeh ◽  
...  
Keyword(s):  
Mitochondrial Protein ◽  
Protoporphyrin Ix ◽  
K562 Cells ◽  
Benzodiazepine Receptor ◽  
Mrna Levels ◽  
Intracellular Iron ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Excess Iron ◽  
C Terminus

Abstract While iron is essential for cell growth and survival excess iron through oxidative stress may produce hepatitic cirrhosis and hepatocellular carcinoma, diabetes mellitus, and cardiomyopathy. Iron is absorbed across the duodenum with transport across the brush border mediated by DMT1 and across the basolateral surface by ferroportin with mechanisms that are inversely regulated by body iron concentrations. We have identified in rat intestine DAP, a novel protein that binds to the C-terminus of DMT1 (IRE) but not to the C-terminus of the non-IRE isoform (Blood, Nov 2004; 104: 53). DAP is a 526 amino acid protein that has been previously described as binding to the peripheral benzodiazepine receptor, an intrinsic mitochondrial protein involved in steriodogenesis and possibly in protoporphyrin IX transport into the mitochrondria. To investigate if DAP may have a role in regulation of intracellular iron transport DAP expression was down regulated using a vector containing a siRNA for DAP transfected into K562 cells by electroporation. Expression levels of DAP, transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1) and ferritin were examined by western blot and quantitative quantitivative PCR assays from days 1 to 6 after transfection. Following transfection with the DAP siRNA DAP mRNA levels were decreased 50% by day 1 with DAP protein levels decreasing by 50% at day 3. The DAP siRNA also decreased DMT1 protein expression by about 50% for the DMT1 (IRE) protein but had no effect on the protein derived from the non-IRE isoform. The leels of DMT1 mRNA were not affected by DAP siRNA. The decrease of DAP expression was not associated with any change in TfR1 or ferritin expression, suggesting that altered levels of DAP did not affect intracellular iron pools. Transfection with the DAP siRNA resulted also in more protean effects decreasing cell proliferation, the transition from S-phase to G2 in cell cycle, and protein synthesis. These data are consistent with DAP regulating DMT1 expression in K562 cells by modulating turnover of DMT1 (IRE) protein and also having more global effects on cellular metabolism.

Deregulation of proteins involved in iron metabolism in hepcidin-deficient mice

Blood ◽  
2005 ◽  
Vol 105 (12) ◽  
pp. 4861-4864 ◽  
Author(s):  
Lydie Viatte ◽  
Jeanne-Claire Lesbordes-Brion ◽  
Dan-Qing Lou ◽  
Myriam Bennoun ◽  
Gaël Nicolas ◽  
...  
Keyword(s):  
Iron Overload ◽  
Iron Homeostasis ◽  
Genetic Disorder ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Metal Transporter ◽  
The Common ◽  
Related Proteins ◽  
Deficient Mice

Abstract Evidence is accumulating that hepcidin, a liver regulatory peptide, could be the common pathogenetic denominator of all forms of iron overload syndromes including HFE-related hemochromatosis, the most prevalent genetic disorder characterized by inappropriate iron absorption. To understand the mechanisms whereby hepcidin controls iron homeostasis in vivo, we have analyzed the level of iron-related proteins by Western blot and immunohistochemistry in hepcidin-deficient mice, a mouse model of severe hemochromatosis. These mice showed important increased levels of duodenal cytochrome b (Dcytb), divalent metal transporter 1 (DMT1), and ferroportin compared with control mice. Interestingly, the level of ferroportin was coordinately up-regulated in the duodenum, the spleen, and the liver (predominantly in the Kupffer cells). Finally, we also evidenced a decrease of ceruloplasmin in the liver of hepcidin-deficient mice. We hypothesized that the deregulation of these proteins might be central in the pathogenesis of iron overload, providing key therapeutic targets for iron disorders. (Blood. 2005;105:4861-4864)

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