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 Nitric Oxide–Mediated Induction of Ferritin Synthesis in J774 Macrophages by Inflammatory Cytokines: Role of Selective Iron Regulatory Protein-2 Downregulation

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 1059-1066 ◽  
Author(s):  
Stefania Recalcati ◽  
Donatella Taramelli ◽  
Dario Conte ◽  
Gaetano Cairo
Keyword(s):  
Nitric Oxide ◽  
Posttranscriptional Regulation ◽  
Iron Homeostasis ◽  
Regulatory Protein ◽  
Intracellular Iron ◽  
Iron Regulatory Proteins ◽  
Ferritin Synthesis ◽  
Direct Demonstration ◽  
J774 Cells

Cytokine-treated macrophages represent a useful model to unravel the molecular basis of reticuloendothelial (RE) iron retention in inflammatory conditions. In the present study, we showed that stimulation of murine macrophage J774 cells with interferon (IFN)-γ/lipopolysaccharide (LPS) resulted in a nitric oxide-dependent modulation of the activity of iron regulatory proteins (IRP)-1 and 2, cytoplasmic proteins which, binding to RNA motifs called iron responsive elements (IRE), control ferritin translation. Stimulation with cytokines caused a small increase of IRP-1 activity and a strong reduction of IRP-2 activity accompanied by increased ferritin synthesis and accumulation. Cytokines induced only a minor increase of H chain ferritin mRNA, thus indicating that IRP-2–mediated posttranscriptional regulation plays a major role in the control of ferritin expression. This was confirmed by direct demonstration that the translational repression function of IRP was impaired in stimulated cells. In fact, translation in cell-free extracts of a reporter transcript under the control of an IRE sequence was repressed less efficiently by IRP-containing lysates from cytokine-treated cells than by lysates from control cells. Our findings throw light on the role of IRP-2 showing that: (1) this protein responds to a stimulus in opposite fashion to IRP-1; (2) when abundantly expressed, as in J774 cells, IRP-2 is sufficient to regulate intracellular iron metabolism in living cells; and (3) by allowing increased ferritin synthesis, IRP-2 may play a role in the regulation of iron homeostasis in RE cells during inflammation.

HFE Downregulates Iron Uptake From Transferrin and Induces Iron-Regulatory Protein Activity in Stably Transfected Cells

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3915-3921 ◽  
Author(s):  
H.D. Riedel ◽  
M.U. Muckenthaler ◽  
S.G. Gehrke ◽  
I. Mohr ◽  
K. Brennan ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Iron Uptake ◽  
Iron Homeostasis ◽  
Hereditary Hemochromatosis ◽  
Regulatory Protein ◽  
Intracellular Iron ◽  
Iron Storage ◽  
Iron Regulatory Proteins ◽  
Cellular Iron

Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder of iron metabolism. More than 80% of HH patients are homozygous for a point mutation in a major histocompatibility complex (MHC) class I type protein (HFE), which results in a lack of HFE expression on the cell surface. A previously identified interaction of HFE and the transferrin receptor suggests a possible regulatory role of HFE in cellular iron absorption. Using an HeLa cell line stably transfected with HFE under the control of a tetracycline-sensitive promoter, we investigated the effect of HFE expression on cellular iron uptake. We demonstrate that the overproduction of HFE results in decreased iron uptake from diferric transferrin. Moreover, HFE expression activates the key regulators of intracellular iron homeostasis, the iron-regulatory proteins (IRPs), implying that HFE can affect the intracellular “labile iron pool.” The increase in IRP activity is accompanied by the downregulation of the iron-storage protein, ferritin, and an upregulation of transferrin receptor levels. These findings are discussed in the context of the pathophysiology of HH and a possible role of iron-responsive element (IRE)-containing mRNAs.

scholarly journals Crucial function of vertebrate glutaredoxin 3 (PICOT) in iron homeostasis and hemoglobin maturation

2013 ◽  
Vol 24 (12) ◽  
pp. 1895-1903 ◽  
Author(s):  
Petra Haunhorst ◽  
Eva-Maria Hanschmann ◽  
Lars Bräutigam ◽  
Oliver Stehling ◽  
Bastian Hoffmann ◽  
...  
Keyword(s):  
Iron Metabolism ◽  
Iron Homeostasis ◽  
Regulatory Protein ◽  
Iron Regulation ◽  
Iron Starvation ◽  
Intracellular Iron ◽  
Cellular Iron ◽  
Cellular Iron Uptake ◽  
S Proteins

The mechanisms by which eukaryotic cells handle and distribute the essential micronutrient iron within the cytosol and other cellular compartments are only beginning to emerge. The yeast monothiol multidomain glutaredoxins (Grx) 3 and 4 are essential for both transcriptional iron regulation and intracellular iron distribution. Despite the fact that the mechanisms of iron metabolism differ drastically in fungi and higher eukaryotes, the glutaredoxins are conserved, yet their precise function in vertebrates has remained elusive. Here we demonstrate a crucial role of the vertebrate-specific monothiol multidomain Grx3 (PICOT) in cellular iron homeostasis. During zebrafish embryonic development, depletion of Grx3 severely impairs the maturation of hemoglobin, the major iron-consuming process. Silencing of human Grx3 expression in HeLa cells decreases the activities of several cytosolic Fe/S proteins, for example, iron-regulatory protein 1, a major component of posttranscriptional iron regulation. As a consequence, Grx3-depleted cells show decreased levels of ferritin and increased levels of transferrin receptor, features characteristic of cellular iron starvation. Apparently, Grx3-deficient cells are unable to efficiently use iron, despite unimpaired cellular iron uptake. These data suggest an evolutionarily conserved role of cytosolic monothiol multidomain glutaredoxins in cellular iron metabolism pathways, including the biogenesis of Fe/S proteins and hemoglobin maturation.

scholarly journals Nitric Oxide–Mediated Induction of Ferritin Synthesis in J774 Macrophages by Inflammatory Cytokines: Role of Selective Iron Regulatory Protein-2 Downregulation

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 1059-1066 ◽  
Author(s):  
Stefania Recalcati ◽  
Donatella Taramelli ◽  
Dario Conte ◽  
Gaetano Cairo
Keyword(s):  
Nitric Oxide ◽  
Posttranscriptional Regulation ◽  
Iron Homeostasis ◽  
Regulatory Protein ◽  
Intracellular Iron ◽  
Iron Regulatory Proteins ◽  
Ferritin Synthesis ◽  
Direct Demonstration ◽  
J774 Cells

Abstract Cytokine-treated macrophages represent a useful model to unravel the molecular basis of reticuloendothelial (RE) iron retention in inflammatory conditions. In the present study, we showed that stimulation of murine macrophage J774 cells with interferon (IFN)-γ/lipopolysaccharide (LPS) resulted in a nitric oxide-dependent modulation of the activity of iron regulatory proteins (IRP)-1 and 2, cytoplasmic proteins which, binding to RNA motifs called iron responsive elements (IRE), control ferritin translation. Stimulation with cytokines caused a small increase of IRP-1 activity and a strong reduction of IRP-2 activity accompanied by increased ferritin synthesis and accumulation. Cytokines induced only a minor increase of H chain ferritin mRNA, thus indicating that IRP-2–mediated posttranscriptional regulation plays a major role in the control of ferritin expression. This was confirmed by direct demonstration that the translational repression function of IRP was impaired in stimulated cells. In fact, translation in cell-free extracts of a reporter transcript under the control of an IRE sequence was repressed less efficiently by IRP-containing lysates from cytokine-treated cells than by lysates from control cells. Our findings throw light on the role of IRP-2 showing that: (1) this protein responds to a stimulus in opposite fashion to IRP-1; (2) when abundantly expressed, as in J774 cells, IRP-2 is sufficient to regulate intracellular iron metabolism in living cells; and (3) by allowing increased ferritin synthesis, IRP-2 may play a role in the regulation of iron homeostasis in RE cells during inflammation.

HFE Downregulates Iron Uptake From Transferrin and Induces Iron-Regulatory Protein Activity in Stably Transfected Cells

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3915-3921 ◽  
Author(s):  
H.D. Riedel ◽  
M.U. Muckenthaler ◽  
S.G. Gehrke ◽  
I. Mohr ◽  
K. Brennan ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Iron Uptake ◽  
Iron Homeostasis ◽  
Hereditary Hemochromatosis ◽  
Regulatory Protein ◽  
Intracellular Iron ◽  
Iron Storage ◽  
Iron Regulatory Proteins ◽  
Cellular Iron

Abstract Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder of iron metabolism. More than 80% of HH patients are homozygous for a point mutation in a major histocompatibility complex (MHC) class I type protein (HFE), which results in a lack of HFE expression on the cell surface. A previously identified interaction of HFE and the transferrin receptor suggests a possible regulatory role of HFE in cellular iron absorption. Using an HeLa cell line stably transfected with HFE under the control of a tetracycline-sensitive promoter, we investigated the effect of HFE expression on cellular iron uptake. We demonstrate that the overproduction of HFE results in decreased iron uptake from diferric transferrin. Moreover, HFE expression activates the key regulators of intracellular iron homeostasis, the iron-regulatory proteins (IRPs), implying that HFE can affect the intracellular “labile iron pool.” The increase in IRP activity is accompanied by the downregulation of the iron-storage protein, ferritin, and an upregulation of transferrin receptor levels. These findings are discussed in the context of the pathophysiology of HH and a possible role of iron-responsive element (IRE)-containing mRNAs.

scholarly journals Effect of nitric oxide on expression of transferrin receptor and ferritin and on cellular iron metabolism in K562 human erythroleukemia cells

Blood ◽  
1995 ◽  
Vol 85 (10) ◽  
pp. 2962-2966 ◽  
Author(s):  
R Oria ◽  
L Sanchez ◽  
T Houston ◽  
MW Hentze ◽  
FY Liew ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Iron Metabolism ◽  
Transferrin Receptor ◽  
Regulatory Protein ◽  
Inhibitory Effect ◽  
Receptor Expression ◽  
Mrna Levels ◽  
Intracellular Iron ◽  
Cellular Iron ◽  
Erythroleukemia Cells

Nitric oxide (NO) is known to increase the affinity of the intracellular iron-regulatory protein (IRP) for iron-response elements (IREs) in transferrin receptor and ferritin mRNAs, suggesting that it may act as a regulator of cellular iron metabolism. In this study, exogenous NO produced by adding the NO-generator S-nitroso-N-acetyl penicillamine gave a dose-dependent upregulation of transferrin receptor expression by K562 erythroleukemia cells and increased levels of transferrin receptor mRNA. NO did not affect the affinity of transferrin binding by the transferrin receptor. NO alone did not alter intracellular ferritin levels, but it did abrogate the inhibitory effect of the iron chelator desferrioxamine and potentiated the stimulatory effect of additional iron. NO also caused some increase in ferritin mRNA levels, which might mask any IRP-/IRE-mediated inhibitory effect of NO on ferritin translation. Although NO did not affect net iron uptake, it increased release of iron from K562 cells pulsed previously with 59Fe, and subcellular fractionation showed that it also increased the proportion of intracellular iron bound to ferritin. These findings provide direct evidence that NO can affect cellular iron metabolism and suggest that NO produced in vivo by activated bone marrow macrophages might affect erythropoiesis.

Overexpression of mitochondrial ferritin causes cytosolic iron depletion and changes cellular iron homeostasis

Blood ◽  
2005 ◽  
Vol 105 (5) ◽  
pp. 2161-2167 ◽  
Author(s):  
Guangjun Nie ◽  
Alex D. Sheftel ◽  
Sangwon F. Kim ◽  
Prem Ponka
Keyword(s):  
Iron Homeostasis ◽  
Rna Binding ◽  
Binding Activity ◽  
Intracellular Iron ◽  
Free Radical Damage ◽  
Iron Regulatory Proteins ◽  
Cellular Iron ◽  
Cellular Iron Uptake ◽  
A Cell ◽  
Cellular Iron Homeostasis

AbstractCytosolic ferritin sequesters and stores iron and, consequently, protects cells against iron-mediated free radical damage. However, the function of the newly discovered mitochondrial ferritin (MtFt) is unknown. To examine the role of MtFt in cellular iron metabolism, we established a cell line that stably overexpresses mouse MtFt under the control of a tetracycline-responsive promoter. The overexpression of MtFt caused a dose-dependent iron deficiency in the cytosol that was revealed by increased RNA-binding activity of iron regulatory proteins (IRPs) along with an increase in transferrin receptor levels and decrease in cytosolic ferritin. Consequently, the induction of MtFt resulted in a dramatic increase in cellular iron uptake from transferrin, most of which was incorporated into MtFt. The induction of MtFt caused a shift of iron from cytosolic ferritin to MtFt. In addition, iron inserted into MtFt was less available for chelation than that in cytosolic ferritin and the expression of MtFt was associated with decreased mitochondrial and cytosolic aconitase activities, the latter being consistent with the increase in IRP-binding activity. In conclusion, our results indicate that overexpression of MtFt causes a dramatic change in intracellular iron homeostasis and that shunting iron to MtFt likely limits its availability for active iron proteins.

scholarly journals Linking Cancer Metabolic Dysfunction and Genetic Instability through the Lens of Iron Metabolism

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1077 ◽  
Author(s):  
Michael S. Petronek ◽  
Douglas R. Spitz ◽  
Garry R. Buettner ◽  
Bryan G. Allen
Keyword(s):  
Dna Damage ◽  
Steady State ◽  
Cancer Cells ◽  
Iron Metabolism ◽  
Genetic Instability ◽  
Cellular Proliferation ◽  
Intracellular Iron ◽  
Iron Regulatory Proteins ◽  
Wide Range ◽  
Labile Iron

Iron (Fe) is an essential element that plays a fundamental role in a wide range of cellular functions, including cellular proliferation, DNA synthesis, as well as DNA damage and repair. Because of these connections, iron has been strongly implicated in cancer development. Cancer cells frequently have changes in the expression of iron regulatory proteins. For example, cancer cells frequently upregulate transferrin (increasing uptake of iron) and down regulate ferroportin (decreasing efflux of intracellular iron). These changes increase the steady-state level of intracellular redox active iron, known as the labile iron pool (LIP). The LIP typically contains approximately 2% intracellular iron, which primarily exists as ferrous iron (Fe2+). The LIP can readily contribute to oxidative distress within the cell through Fe2+-dioxygen and Fenton chemistries, generating the highly reactive hydroxyl radical (HO•). Due to the reactive nature of the LIP, it can contribute to increased DNA damage. Mitochondrial dysfunction in cancer cells results in increased steady-state levels of hydrogen peroxide and superoxide along with other downstream reactive oxygen species. The increased presence of H2O2 and O2•− can increase the LIP, contributing to increased mitochondrial uptake of iron as well as genetic instability. Thus, iron metabolism and labile iron pools may play a central role connecting the genetic mutational theories of cancer to the metabolic theories of cancer.

scholarly journals Transcriptional regulation of Mycobacterium tuberculosis hupB gene expression

Microbiology ◽  
2014 ◽  
Vol 160 (8) ◽  
pp. 1637-1647 ◽  
Author(s):  
Satya Deo Pandey ◽  
Mitali Choudhury ◽  
Manjula Sritharan
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Iron Homeostasis ◽  
Iron Concentration ◽  
Dna Interaction ◽  
Intracellular Iron ◽  
Mobility Shift ◽  
Dna Footprinting ◽  
Sequence Of Events ◽  
Electrophoretic Mobility Shift Assays

The influence of iron levels on the transcription of the hupB gene in Mycobacterium tuberculosis is the focus of this study. Studies in our laboratory showed HupB to be co-expressed with the two siderophores in low-iron organisms. Mycobactin biosynthesis is repressed by the IdeR–Fe2+ complex that binds the IdeR box in the mbtB promoter. Recently, we demonstrated the positive regulatory effect of HupB on mycobactin biosynthesis by demonstrating its binding to a 10 bp HupB box in the mbtB promoter. Earlier, we observed that HupB, expressed maximally in low-iron media (0.02 µg Fe ml−1; 0.36 µM Fe) was still detectable at 8 µg Fe ml−1 (144 µM Fe) when the siderophores were absent and complete repression was seen only at 12 µg Fe ml−1 (216 µM Fe). In this study, we observed elevated levels of hupB transcripts in iron-limited organisms. IdeR, and not FurA, functioned as the iron regulator, by binding to two IdeR boxes in the hupB promoter. Interestingly, the 10 bp HupB box, first reported in the mbtB promoter, was identified in the hupB promoter. Using DNA footprinting and electrophoretic mobility shift assays, we demonstrated the functionality of the HupB box and the two IdeR boxes. The high hupB transcript levels expressed by the organism and the in vitro protein–DNA interaction studies led us to hypothesize the sequence of events occurring in response to changes in the intracellular iron concentration, emphasizing the roles played by IdeR and HupB in iron homeostasis.

scholarly journals Paradoxical Increase of Insulin Sensitivity Despite Blunted Adipose Tissue Browning in Genetic Ablation of IRP1 (OR09-08-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Mikyoung You ◽  
Jin-Seon Yook ◽  
Soonkyu Chung
Keyword(s):  
Adipose Tissue ◽  
Insulin Sensitivity ◽  
Iron Metabolism ◽  
Core Body Temperature ◽  
Serum Levels ◽  
Genetic Ablation ◽  
Ucp1 Expression ◽  
Core Body ◽  
Tissue Browning

Abstract Objectives Iron regulatory protein 1 (IRP1) plays a key regulator of cellular iron metabolism, systemic oxygen sensing, and erythropoiesis. Deletion of IRP1 leads to profound HIF2a-dependent abnormalities in erythropoiesis and iron metabolism. Previously, we demonstrated that modulation of adipose tissue iron metabolism is necessary for adipose tissue browning. However, the role of IRP1 in adipose tissue browning and its metabolic consequences are uncertain. This study aimed to investigate the role of IRP1 in regulating adipose tissue browning in a mouse model of genetic ablation of IPR1 (IRP1−/−). Methods The IRP1−/− mice and wildtype (WT) controls were kept either at room (25°C) or cold (6°C) temperature for 7 days. Adipose tissue browning was evaluated by UCP1 expression and prevalence of beige-like structure in inguinal fat. Thermogenic heat release captured by infrared camera and core body temperature was measured by a rectal thermometer. The modulation of iron metabolism was assessed by serum levels of ferritin, hematocrit, and erythropoietin levels by ELISA. To investigate the role of IRP1 on energy metabolism, IRP1−/− and WT controls were fed a high-fat diet (45%) for 14 weeks. Insulin sensitivity was determined by glucose and insulin tolerance test and HOMA-IR score. [3H]-2-deoxyglucose (DOG) was injected to determine the distribution of 3H-radioactivity was quantified. Results IRP1−/− mice dramatically increased serum levels of erythropoietin but decreased hepcidin. IRP1−/− developed polycythemia and reticulocytosis, which was not affected by cold exposure. IRP1−/− were completely blunted in cold-induced browning in the inguinal fat showing no changes in UCP1 and adipocyte morphology. Unexpectedly, IRP1−/− showed higher core body temperature and heat release than control independent of UCP1 expression. Chronic intake of HF diet paradoxically increased the insulin sensitivity regardless of obesity. 2-DOG distribution was significantly increased in red blood cells, suggesting that red blood cell-dependent energy expenditure significantly contributed to rapid glucose disposal. Conclusions Disruption of IRP1 blunted adipose tissue browning. The paradoxical rise in insulin sensitivity in IRP1−/− is likely due to red blood cells-mediated energy expenditure. Funding Sources None.

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