Iron Metabolism In Vertebrates Research Articles

Quantitative demonstration of the existence of two isoforms of canine hepcidin in the serum & urine of dogs

Hepcidin-25 is the key peptide hormone controlling vertebrate iron metabolism. However, in the last twenty years there was some disagreement in the literature over the structure of this compound. The aim of this research was to study whether more than one isoform of canine hepcidin-25 exists. For the purpose of comparison serum concentrations of hepcidin-25 were determined in the samples of 47 dogs sick with acute/chronic inflammation too. The study demonstrated that two isoforms of canine hepcidin-25 exist. A statistical correlation may indicate that both molecules are synthesised by dogs together. No statistically significant correlations were found between the measured concentrations of the two canine hepcidin-25 isoforms and the measured serum iron parameters in the sampled dogs, irrespective of the measurements were made in serum or urine. The mean urinary total hepcidin-25/creatinine ratio in healthy dogs was 1.08 ± 0.10. The mean serum total hepcidin-25 concentration was 79.8 ± 4.9 ng/ml, about 65% of which was the 25β version. The presence of inflammation results in a statistically significant increase in the serum concentration of both hepcidin varieties. The role and fate of the two molecules may need to be researched further to provide better understanding of their relation.

342-LB: Iron Regulatory Protein 2 Regulates Proinsulin Production in Pancreatic Beta Cells through Iron-Dependent Cdkal1-Catalyzed tRNA Modification

Iron regulatory protein 2 (Irp2) is a key regulator of cellular iron metabolism in vertebrates. Irp2 controls the post-transcriptional expression of the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-storage protein ferritin. Here we show that Irp2-/-mice develop diabetes characterized by glucose intolerance and impaired glucose-stimulated insulin secretion from pancreatic β-cells. Impaired glucose-stimulated insulin secretion in Irp2-/-β-cells is not due to insulin resistance but rather to a reduction in insulin stores caused by impaired proinsulin processing. Loss of Irp2 reduces TfR1 expression and iron content, and increases ferritin expression and iron sequestration causing cellular iron deficiency that impairs mitochondrial Fe-S cluster biosynthesis. This in turn reduces the function of Cdk5-regulated subunit-associated protein 1 like (Cdkal1), a 4Fe-4S cluster enzyme that catalyzes the 2-methythio (ms) modification of t6A37 of tRNALys(UUU)to ms2t6A37, which is critical for efficient translation of lysine codons in proinsulin. Irp2-deficient β cells show reduced ms2t6A37 levels and lysine incorporation into proinsulin. Increasing cellular iron content in Irp2-deficient β cells normalizes mitochondrial function, ms2t6A37 levels, proinsulin and insulin content, and insulin secretion. These study reveals a previously unidentified link between insulin processing and cellular iron homeostasis, and show that Irp2 is required for normal β cell function and whole body glucose homeostasis. Disclosure M.C. Ferreira dos Santos: None. M. Rajan: None. E. Leibold: None. Funding American Diabetes Association (1-18-PMF-020 to M.C.F.D.S.); National Institute of Diabetes and Digestive and Kidney Diseases

Iron-regulatory proteins: molecular biology and pathophysiological implications.

Iron is required for key cellular functions, and there is a strong link between iron metabolism and important metabolic processes, such as cell growth, apoptosis and inflammation. Diseases that are directly or indirectly related to iron metabolism represent major health problems. Iron-regulatory proteins (IRPs) 1 and 2 are key controllers of vertebrate iron metabolism and post-transcriptionally regulate expression of the major iron homeostasis genes. Here we discuss how dysregulation of the IRP system can result from both iron-related and unrelated effectors and explain how this can have important pathological consequences in several human disorders.

The zebrafish mutant gene chardonnay (cdy) encodes divalent metal transporter 1 (DMT1)

Iron is an essential nutrient required for the function of all cells, most notably for the production of hemoglobin in red blood cells. Defects in the mechanisms of iron absorption, storage, or utilization can lead to disorders of iron-limited erythropoiesis or iron overload. In an effort to further understand these processes, we have used the zebrafish as a genetic system to study vertebrate iron metabolism. Here we characterized the phenotype of chardonnay (cdy), a zebrafish mutant with hypochromic, microcytic anemia, and positioned the mutant gene on linkage group 11. The cdy gene was isolated by a functional genomics approach in which we used a combination of expression studies, sequence analyses, and radiation hybrid panel mapping. We identified erythroid-specific genes using a whole embryo mRNA in situ hybridization screen and placed these genes on the zebrafish genomic map. One of these genes encoded the iron transporter divalent metal transporter 1 (DMT1) and colocalized with the cdy gene. We identified a nonsense mutation in the cdy allele and demonstrated that, whereas wild-type zebrafish DMT1 protein can transport iron, the truncated protein expressed in cdy mutants is not functional. Our studies further demonstrate the conservation of iron metabolism in vertebrates and suggest the existence of an alternative pathway of intestinal and red blood cell iron uptake.

Ceruloplasmin Ferroxidase Activity Stimulates Cellular Iron Uptake by a Trivalent Cation-specific Transport Mechanism

The balance required to maintain appropriate cellular and tissue iron levels has led to the evolution of multiple mechanisms to precisely regulate iron uptake from transferrin and low molecular weight iron chelates. A role for ceruloplasmin (Cp) in vertebrate iron metabolism is suggested by its potent ferroxidase activity catalyzing conversion of Fe2+ to Fe3+, by identification of yeast copper oxidases homologous to Cp that facilitate high affinity iron uptake, and by studies of "aceruloplasminemic" patients who have extensive iron deposits in multiple tissues. We have recently shown that Cp increases iron uptake by cultured HepG2 cells. In this report, we investigated the mechanism by which Cp stimulates cellular iron uptake. Cp stimulated the rate of non-transferrin 55Fe uptake by iron-deficient K562 cells by 2-3-fold, using a transferrin receptor-independent pathway. Induction of Cp-stimulated iron uptake by iron deficiency was blocked by actinomycin D and cycloheximide, consistent with a transcriptionally induced or regulated transporter. Cp-stimulated iron uptake was completely blocked by unlabeled Fe3+ and by other trivalent cations including Al3+, Ga3+, and Cr3+, but not by divalent cations. These results indicate that Cp utilizes a trivalent cation-specific transporter. Cp ferroxidase activity was required for iron uptake as shown by the ineffectiveness of two ferroxidase-deficient Cp preparations, copper-deficient Cp and thiomolybdate-treated Cp. We propose a model in which iron reduction and subsequent re-oxidation by Cp are essential for an iron uptake pathway with high ion specificity.

Iron Regulatory Proteins, Iron Responsive Elements and Iron Homeostasis

The discovery of iron regulatory proteins (IRPs) has provided a molecular framework from which to more fully understand the coordinate regulation of vertebrate iron metabolism. IRPs bind to iron responsive elements (IREs) in specific mRNAs and regulate their utilization. The targets of IRP action now appear to extend beyond proteins that function in the storage (ferritin) or cellular uptake (transferrin receptor) of iron to include those involved in other aspects of iron metabolism as well as in the tricarboxylic acid cycle. To date, it appears that IRPs modulate the utilization of six mammalian mRNAs. Current studies are aimed at defining the mechanisms responsible for the hierarchical regulation of these mRNAs by IRPs. In addition, much interest continues to focus on the signaling pathways through which IRP function is regulated. Multiple factors modulate the RNA binding activity of IRP1 and/or IRP2 including iron, nitric oxide, phosphorylation by protein kinase C, oxidative stress and hypoxia/reoxygenation. Because IRPs are key modulators of the uptake and metabolic fate of iron in cells, they are focal points for the modulation of cellular iron homeostasis in response to a variety of agents and circumstances.

Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress.

As an essential nutrient and a potential toxin, iron poses an exquisite regulatory problem in biology and medicine. At the cellular level, the basic molecular framework for the regulation of iron uptake, storage, and utilization has been defined. Two cytoplasmic RNA-binding proteins, iron-regulatory protein-1 (IRP-1) and IRP-2, respond to changes in cellular iron availability and coordinate the expression of mRNAs that harbor IRP-binding sites, iron-responsive elements (IREs). Nitric oxide (NO) and oxidative stress in the form of H2O2 also signal to IRPs and thereby influence cellular iron metabolism. The recent discovery of two IRE-regulated mRNAs encoding enzymes of the mitochondrial citric acid cycle may represent the beginnings of elucidating regulatory coupling between iron and energy metabolism. In addition to providing insights into the regulation of iron metabolism and its connections with other cellular pathways, the IRE/IRP system has emerged as a prime example for the understanding of translational regulation and mRNA stability control. Finally, IRP-1 has highlighted an unexpected role for iron sulfur clusters as post-translational regulatory switches.

Translational regulation of mammalian and Drosophila citric acid cycle enzymes via iron-responsive elements.

The posttranscriptional control of iron uptake, storage, and utilization by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) provides a molecular framework for the regulation of iron homeostasis in many animals. We have identified and characterized IREs in the mRNAs for two different mitochondrial citric acid cycle enzymes. Drosophila melanogaster IRP binds to an IRE in the 5' untranslated region of the mRNA encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH). This interaction is developmentally regulated during Drosophila embryogenesis. In a cell-free translation system, recombinant IRP-1 imposes highly specific translational repression on a reporter mRNA bearing the SDH IRE, and the translation of SDH-Ip mRNA is iron regulated in D. melanogaster Schneider cells. In mammals, an IRE was identified in the 5' untranslated regions of mitochondrial aconitase mRNAs from two species. Recombinant IRP-1 represses aconitase synthesis with similar efficiency as ferritin IRE-controlled translation. The interaction between mammalian IRPs and the aconitase IRE is regulated by iron, nitric oxide, and oxidative stress (H2O2), indicating that these three signals can control the expression of mitochondrial aconitase mRNA. Our results identify a regulatory link between energy and iron metabolism in vertebrates and invertebrates, and suggest biological functions for the IRE/IRP regulatory system in addition to the maintenance of iron homeostasis.

Distribution of radioiron in larval lampreys, Petromyzon marinus L., following administration of 55ferrous citrate

Light microscopic autoradiography and quantitative tissue analyses were used to examine the distribution of radioiron in larvae (ammocoetes) of the lamprey Petromyzon marinus L. over time following the injection of 55ferrous citrate (55Fe). It was found that a single intraperitoneal injection of 55Fe is an efficient method of introducing iron into ammocoetes, and radioiron retention within the body remained high throughout the 28-d experiment. Immediately after radioiron administration the isotope was transported in the serum fraction of the blood to sites of iron storage and elimination. The liver is the most important target organ for iron in ammocoetes, and radioiron acquired from the alimentary canal, skin, carcass, and blood contributed to the initial increase in this organ. Data indicated only temporary storage in the liver; between 3 and 16 d, radioiron was redistributed by the blood to the skin, kidney, alimentary canal, and carcass. Biliary transport of radioiron from the liver to the lumen of the anterior intestine may also explain the lowering of iron concentrations in the liver and the rise in levels in the alimentary canal during this interval. The redeposition of 55Fe in the liver at 16 d may signify longer-term storage of the metal in this organ. There is movement of radioiron in and out of the carcass tissues over time, and most of the radioactivity is localized in the adipose tissue. The epithelial cells of the proximal tubules of the kidney absorb and concentrate the radioiron presumably present in the glomerular filtrate. This concentration probably reflects both the absorption of iron bound to ferritin and a mechanism necessary for maintaining the high serum iron levels of ammocoetes. The release of mucus from iron-laden mucous cells of the skin and exfoliation of radioiron-laden epithelial cells in specific regions of the posterior intestine are possible routes for iron elimination. However, percent incorporation of the injected radioactivity was still high at the end of the experiment, indicating that iron excretion in ammocoetes is relatively low or that it is a slow process. These data support the notion that lampreys provide a useful animal model for the study of iron metabolism in vertebrates.

Occurrence and Structure of Iron Inclusions in Adipocytes of Larval Lampreys

AbstractThe occurrence and structure of adipocytes in the larvae of two lamprey species, Geotria australis and Petromyzon marinus, were examined by electron microscopy. Adipocytes from both species possessed large electron‐dense inclusions which histochemical and energy dispersal X‐ray analyses show as containing iron. The greatest concentration of inclusions in adipocytes was found in the nephric fold of G. australis. While some iron is present in the cytoplasmic matrix as ferritin, the majority is seen in large ammocoetes in membrane‐bound dense aggregations of haemosiderin. The wide variety of inclusion types seen in smaller larvae may reflect on the method of formation of these inclusions within the cell. Because of the high level of iron loading in the larval lamprey nephric fold, this readily accessible tissue may provide a valuable model for studies of iron metabolism in vertebrates.

Distribution of55Fe in juvenile adult lampreys,Petromyzon marinus L., following oral intubation of the isotope

The capacity of a sanguivorous lamprey,Petromyzon marinus L., to deal with ingested iron was studied over time using autoradiography and scintillation counting of solubilized tissue samples after intubation of the oesophagus with a single dose of55ferrous citrate. A highly efficient mechanism for absorption in the anterior intestine was recognized with 17% of the intubated radioactivity absorbed into the body after only 5 min, 66% by 3 h, and almost 80% by 21 h. Iron concentration in the epithelial cells of the anterior intestine may be a factor in restricting iron absorption during spontaneous feeding. A decline in total body radioactivity over the 15 days following iron intubation probably results from transport of the metal in the blood and release of radioiron from the mucous cells of the posterior intestine. The kidneys appear to play a smaller but still significant role in iron loss. Gradual increases in radioiron concentration (cpm g−1 wet weight) and percent of total body radioactivity occur in the liver (2 to 26%), carcass (14 to 37%), and integument (4 to 12%) during the course of the experiment, indicating that these are the chief sites of iron storage during times of metal excess. However, eventually integument may also be a site of iron excretion. Significant fluctuations in radioiron concentration (cpm ml−1) in whole blood during the 15 day period can be correlated with transport of the metal to sites of storage and excretion, and maybe with incorporation into haemoglobin and with erythropoietic activity. Feeding adult lampreys represent a valuable system, with both general and unique characters, for studying iron metabolism in vertebrates.