The iron regulatory hormone hepcidin inhibits expression of iron release as well as iron uptake proteins in J774 cells

The mechanism by which hepcidin controls cellular iron release protein ferroportin 1 (Fpn1) in macrophages has been well established. However, little is known about the effects of hepcidin on cellular iron uptake proteins. Here, we demonstrated for the first time that hepcidin can significantly inhibit the expression of transferrin receptor 1 (TfR1) and divalent metal transporter 1 in addition to Fpn1, and therefore reduce transferrin-bound iron and non-transferrin-bound iron uptake and also iron release in J774 macrophages. Analysis of mechanisms using the iron-depleted cells showed that hepcidin has a direct inhibitory effect on all iron transport proteins we examined. Further studies demonstrated that the down-regulation of TfR1 induced by hepcidin is associated with cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA), probably being mediated by the cAMP–PKA pathway in J774 macrophages.     

Suppressed hepcidin expression correlates with hypotransferrinemia in copper-deficient rat pups but not dams

Copper deficiency leads to anemia but the mechanism is unknown. Copper deficiency also leads to hypoferremia, which may limit erythropoiesis. The hypoferremia may be due to limited function of multicopper oxidases (MCO) hephaestin in enterocytes or GPI-ceruloplasmin in macrophages of liver and spleen whose function as a ferroxidase is thought essential for iron transfer out of cells. Iron release may also be limited by ferroportin (Fpn), the iron efflux transporter. Fpn may be lower following copper deficiency because of impaired ferroxidase activity of MCO. Fpn is also dependent on the liver hormone hepcidin as Fpn is degraded when hepcidin binds to Fpn. Anemia and hypoferremia both down regulate hepcidin by separate mechanisms. Current studies confirmed and extended earlier studies with copper-deficient (CuD) rats that suggested low hepicidin resulted in augmented Fpn. However, current studies in CuD dams failed to confirm a correlation that hepcidin expression was associated with low transferrin receptor 2 (TfR2) levels and also challenged the dogma that holotransferrin can explain the correlation with hepcidin. CuD dams exhibited hypoferremia, low liver TfR2, anemia in some rats, yet no depression in Hamp expression, the hepcidin gene. Normal levels of GDF-15, the putative erythroid cytokine that suppresses hepcidin, were detected in plasma of CuD and iron-deficient (FeD) dams. Importantly, FeD dams did display greatly lower Hamp expression. Normal hepcidin in these CuD dams is puzzling since these rats may need extra iron to meet needs of lactation and the impaired iron transfer noted previously.     

Endogenous ghrelin‐O‐acyltransferase (GOAT) acylates local ghrelin in the hippocampus

Ghrelin is an appetite‐stimulating peptide. Serine 3 on ghrelin must be acylated by octanoate via the enzyme ghrelin‐O‐acyltransferase (GOAT) for the peptide to bind and activate the cognate receptor, growth hormone secretagogue receptor type 1a (GHSR1a). Interest in GHSR1a increased dramatically when GHSR1a mRNA was demonstrated to be widespread in the brain, including the cortex and hippocampus, indicating that it has multifaceted functions beyond the regulation of metabolism. However, the source of octanoylated ghrelin for GHSR1a in the brain, outside of the hypothalamus, is not well understood. Here, we report the presence of GOAT and its ability to acylate non‐octanoylated ghrelin in the hippocampus. GOAT immunoreactivity is aggregated at the base of the dentate granule cell layer in the rat and wild‐type mouse. This immunoreactivity was not affected by the pharmacological inhibition of GHSR1a or the metabolic state‐dependent fluctuation of systemic ghrelin levels. However, it was absent in the GHSR1a knockout mouse hippocampus, pointing the possibility that the expression of GHSR1a may be a prerequisite for the production of GOAT. Application of fluorescein isothiocyanate (FITC)‐conjugated non‐octanoylated ghrelin in live hippocampal slice culture (but not in fixed culture or in the presence of GOAT inhibitors) mimicked the binding profile of FITC‐conjugated octanoylated ghrelin, suggesting that extracellularly applied non‐octanoylated ghrelin was acylated by endogenous GOAT in the live hippocampus while GOAT being mobilized out of neurons. Our results will advance the understanding for the role of endogenous GOAT in the hippocampus and facilitate the search for the source of ghrelin that is intrinsic to the brain.

     

GOAT induced ghrelin acylation regulates hedonic feeding

Ghrelin is an orexigenic hormone that regulates homeostatic and reward-related feeding behavior. Recent evidence indicates that acylation of ghrelin by the gut enzyme ghrelin O-acyl transferase (GOAT) is necessary to render ghrelin maximally active within its target tissues. Here we tested the hypothesis that GOAT activity modulates food motivation and food hedonics using behavioral pharmacology and mutant mice deficient for GOAT and the ghrelin receptor (GHSR). We evaluated operant responding following pharmacological administration of acyl-ghrelin and assessed the necessity of endogenous GOAT activity for operant responding in GOAT and GHSR-null mice. Hedonic-based feeding behavior also was examined in GOAT-KO and GHSR-null mice using a “Dessert Effect” protocol in which the intake of a palatable high fat diet “dessert” was assessed in calorically-sated mice. Pharmacological administration of acyl-ghrelin augmented operant responding; notably, this effect was dependent on intact GHSR signaling. GOAT-KO mice displayed attenuated operant responding and decreased hedonic feeding relative to controls. These behavioral results correlated with decreased expression of the orexin-1 receptor in reward-related brain regions in GOAT-KO mice. In summary, the ability of ghrelin to stimulate food motivation is dependent on intact GHSR signaling and modified by endogenous GOAT activity. Furthermore, GOAT activity is required for hedonic feeding behavior, an effect potentially mediated by forebrain orexin signaling. These data highlight the significance of the GOAT–ghrelin system for the mediation of food motivation and hedonic feeding.     

The iron regulatory hormone hepcidin reduces ferroportin 1 content and iron release in H9C2 cardiomyocytes

Iron plays a key pathophysiological role in a number of cardiac diseases. Studies on the mechanisms of heart iron homeostasis are therefore crucial for understanding the causes of excessive heart iron. In addition to iron uptake, cellular iron balance in the heart also depends on iron export. We provided evidence for the existence of iron exporter ferroportin 1 (Fpn1) in the heart in a recent study. The presence of hepcidin, a recently discovered iron regulatory hormone, was also confirmed in the heart recently. Based on these findings and the inhibiting role of hepcidin on Fpn1 in other tissues, we speculated that hepcidin might be able to bind with, internalize and degrade Fpn1 and then decrease iron export in heart cells, leading to an abnormal increase in heart iron and iron mediated cell injury. We therefore investigated the effects of hepcidin on the contents of Fpn1 and iron release in H9C2 cardiomyocyte cell line. We demonstrated that hepcidin has the ability to reduce Fpn1 content as well as iron release in this cell. The similar regulation patterns of hepcidin on the Fpn1 and iron release suggested that the decreased iron release resulted from the decreased content of Fpn1 induced by hepcidin. We also found that hepcidin has no significant effects on ceruloplasmin (CP) and hephaestin (Heph) — two proteins required for iron release from mammalian cells. The data imply that Fpn1, rather than Heph and CP, is the limited factor in the regulation of iron release from heart cells under physiological conditions.     

Copper deficiency has minimal impact on ferroportin expression or function

Interactions between copper and iron homeostasis have been known since the nineteenth century when anemia in humans was first described due to copper limitation. However, the mechanism remains unknown. Intestinal and liver iron concentrations are usually higher following copper deficiency (CuD). This may be due to impaired function of the multicopper oxidases hephaestin or ceruloplasmin (Cp), respectively. However, iron retention could be due to altered ferroportin (Fpn), the essential iron efflux transporter in enterocytes and macrophages. Fpn mRNA is controlled partially by intracellular iron and IRE dependence. CuD should augment Fpn based on iron level. Some argue that Fpn stability is controlled partially by membrane ferroxidase (GPI-Cp). CuD should result in lower Fpn since GPI-Cp expression and function is reduced. Fpn turnover is controlled by hepcidin. CuD results in variable Hamp (hepcidin) expression. Fpn mRNA and protein level were evaluated following dietary CuD in rats and mice. To correlate with Fpn expression, measurements of tissue iron were conducted in several rodent models. Following CuD there was little change in Fpn mRNA. Previous work indicated that under certain circumstances Fpn protein was augmented in liver and spleen following CuD. Fpn levels in CuD did not correlate with either total iron or non-heme iron (NHI), as iron levels in CuD liver were higher and in spleen lower than copper adequate controls. Fpn steady state levels appear to be regulated by a complex set of factors. Changes in Fpn do not explain the anemia of CuD.     

利用FRET技术研究Hepcidin 和Fpn 相互作用

铁是生命必需的微量元素,ferroportin(Fpn)是小肠吸收细胞铁释放的重要蛋白。新近发现肝脏分泌的抗菌多肽 hepcidin 具有调节肠铁吸收的重要作用,但目前尚缺少Fpn和hepcidin发生作用的实验依据。应用荧光共振能量转移技术(fluorescence resonance energy transfer ,FRET)对hepcidin和Fpn之间的作用关系进行了深入研究。首先进行了hepcidin-CF P融合蛋白表达载体的构建及表达鉴定;然后对含YFP,Fpn-YFP基因动物细胞表达载体的构建、表达和FRET检测。实验结果证实hepcidin和Fpn之间存在直接的相互作用,并发现两种蛋白发生相互作用后hepcidin也在细胞质中有分布。为临床治疗铁代谢紊乱性疾病提供了新的治疗策略和重要理论依据。     

Leishmania donovani inhibits ferroportin translation by modulating FBXL5‐IRP2 axis for its growth within host macrophages

Hepcidin mediated ferroportin (Fpn) degradation in macrophages is a well adopted strategy to limit iron availability towards invading pathogens. Leishmania donovani (LD), a protozoan parasite, resides within macrophage and competes with host for availing iron. Using in vitro and in vivo model of infection, we reveal that LD decreases Fpn abundance in host macrophages by hepcidin independent mechanism. Unaffected level of Fpn‐FLAG in LD infected J774 macrophage confirms that Fpn down‐regulation is not due its degradation. While increased Fpn mRNA but decreased protein expression in macrophages suggests blocking of Fpn translation by LD infection that is confirmed by ³⁵S‐methionine labelling assay. We further reveal that LD blocks Fpn translation by induced binding of iron regulatory proteins (IRPs) to the iron responsive element present in its 5′UTR. Supershift analysis provides evidence of involvement of IRP2 particularly during in vivo infection. Accordingly, a significant increase in IRP2 protein expression with simultaneous decrease in its stability regulator F‐box and leucine‐rich repeat Protein 5 (FBXL5) is detected in splenocytes of LD‐infected mice. Increased intracellular growth due to compromised expressions of Fpn and FBXL5 by specific siRNAs reveals that LD uses a novel strategy of manipulating IRP2‐FBXL5 axis to inhibit host Fpn expression.     

Effects of hypoxic preconditioning on the expression of iron influx and efflux proteins in primary neuron culture

The mechanisms of neuroprotection induced by hypoxic preconditioning (HP) and the effects of HP on iron metabolism proteins in the brain have not been fully elucidated. Based on the accumulated information, we hypothesized that HP would be able to affect the expression of iron metabolism proteins in the brain and that the changes in the expression of these proteins induced by HP might be partly associated with the HP-induced neuroprotection. Here, we demonstrated for the first time that HP could induce a significant increase in the expression of HIF-1alpha as well as iron uptake (TfR1 and DMT1) and release (Fpn1) proteins and thus increase transferrin-bound iron (Tf-Fe) and non-transferrin-bound iron (NTBI) uptake and iron release, and also a progressive increase in cellular iron content in the cultured neurons. We concluded that HP has the ability to speed iron transport rate and proposed that the increase in iron transport rate and cellular iron in neurons might be one of the mechanisms involved in neuroprotection in the HP neurons. We also demonstrated that Fpn1 expression was significantly affected by HIF-1alpha, implying that the gene encoding this iron efflux protein is hypoxia-inducible.     

Different Characteristics of Hepcidin Expression in IL-6+/+ and IL-6−/− Neurons and Astrocytes Treated with Lipopolysaccharides

A region-specific regulation of inflammation on the expression hepcidin in the brain has been demonstrated, however, it remains unknown whether there is also a cell-specific regulation of inflammation on hepcidin in the brain. Here, we investigated the effects of lipopolysaccharides (LPS) on the expression of hepcidin mRNA and also the expression of IL-6 mRNA, the phosphorylation of STAT3 and the expression of ferroportin 1 (Fpn1) and ferritin light chain (Ft-L) proteins in neurons and astrocytes obtained from wild type (IL-6+/+) and IL-6 knockout (IL-6?/?) mice. We demonstrated that the responses of the expression of hepcidin and IL-6 mRNAs, the phosphorylation of STAT3, and the expression of Fpn1 protein to LPS in IL-6+/+ astrocytes and also the responses of the expression of hepcidin mRNA, the phosphorylation of STAT3 and the expression of Fpn1 protein to IL-6 in IL-6?/? astrocytes were much stronger than those in IL-6+/+ and IL-6?/? neurons. A significant increase in Ft-L was found in LPS-treated IL-6+/+ and IL-6-treated IL-6?/? astrocytes, but not in LPS-treated IL-6+/+ and IL-6-treated IL-6?/? neurons. Our findings provide in vitro evidence for the existence of a cell-specific regulation of LPS on the expression of hepcidin and also Ft-L in the brain.     

Duodenal Ferroportin Is Up-Regulated in Patients with Chronic Hepatitis C

Hepatitis C virus (HCV) infection is a leading cause of liver-related mortality. Chronic hepatitis C (CHC) is frequently associated with disturbances in iron homeostasis, with serum iron and hepatic iron stores being elevated. Accumulating evidence indicates that chronic HCV infection suppresses expression of hepatic hepcidin, a key mediator of iron homeostasis, leading to iron overload conditions. Since hepcidin mediates degradation of ferroportin, a basolateral transporter involved in the release of iron from cells, diminished hepcidin expression probably leads to up-regulation of ferroportin-1 (Fpn1) in patients with CHC. In this study, we determined the protein levels of duodenal Fpn1, and found that its expression was significantly up-regulated in patients with CHC. The expression of duodenal Fpn1 is negatively correlated with mRNA levels of hepcidin, and positively correlated with serum iron parameters. Although iron is a critical factor for growth of a variety of pathogenic bacteria, our results suggest that iron overload in blood does not increase the infection rate of bacteria in patients with CHC.     

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.     

Knockdown of ghrelin‐O‐acyltransferase attenuates colitis through the modulation of inflammatory factors and tight junction proteins in the intestinal epithelium

Ghrelin‐O‐acyltransferase (GOAT) is a membrane‐bound enzyme that attaches eight‐carbon octanoate to a serine residue in ghrelin and thereby acylates inactive ghrelin to produce active ghrelin. In this study, we investigated the function of GOAT in the intestinal mucosal barrier. The intestinal mucosal barrier prevents harmful substances such as bacteria and endotoxin from entering the other tissues, organs, and blood circulation through the intestinal mucosa. Here, we established 5% dextran sodium sulfate (DSS)‐induced colitis in mice and found that the body weight and colon weight were significantly decreased in these mice. Furthermore, increased inflammation and apoptosis were observed in the tissues of DSS‐induced colitis mice, with increased expression of tumor necrosis factor‐α, interleukin‐6, phosphorylation of nuclear factor kappa B‐p65 (p‐NF‐κB‐p65), and cleaved caspase‐3, and decreased expression of tight junction (TJ) proteins such as zonula occluden‐1 and occludin. The knockdown of GOAT significantly attenuated colitis‐induced inflammation responses and apoptosis, while GOAT overexpression significantly enhanced the induction of colitis. These results suggest that knockdown of GOAT may attenuate colitis‐induced inflammation, ulcers, and fecal occult blood by decreasing the intestinal mucosal permeability via the modulation of inflammatory factors and TJ proteins.     

Molecular Cloning and Expression Analysis of Porcine Ghrelin O-Acyltransferase

The peptide hormone ghrelin is secreted in the stomach, with unique N-octanoylation at serine 3, which is a requirement for its functionality. These functions include growth hormone release, appetite stimulation, gastrointestinal motility, glucose regulation, and cell proliferation. The enzyme responsible for ghrelin acylation was recently identified as ghrelin O-acyltransferase (GOAT). In this study, porcine GOAT was cloned and characterized. A full-length cDNA of GOAT of 2013 bp was obtained, which included a 70-bp 5' UTR, a 635-bp 3' UTR, and a 1308-bp open reading frame encoding a protein of 415 amino acids. The GOAT and ghrelin mRNAs are co-expressed in stomach, pancreas, and duodenum at high levels. GOAT was also detected in liver, lung, brain, testis, spleen, kidney, heart, muscle, lipid, and ovary. Our results provide an important basis for further research on GOAT function and the relationship between ghrelin and GOAT.     

The manganese superoxide dismutase Ala16Val dimorphism modulates iron accumulation in human hepatoma cells

The Ala/16Val dimorphism incorporates alanine (Ala) or valine (Val) in the mitochondrial targeting sequence of manganese superoxide dismutase (MnSOD), modifying MnSOD mitochondrial import and activity. In alcoholic cirrhotic patients, the Ala-MnSOD allele is associated with hepatic iron accumulation and an increased risk of hepatocellular carcinoma. The Ala-MnSOD variant could modulate the expression of proteins involved in iron storage (cytosolic ferritin), uptake (transferrin receptors, TfR-1 and-2), extrusion (hepcidin), and intracellular distribution (frataxin) to trigger hepatic iron accumulation. We therefore assessed the Ala/Val-MnSOD genotype and the hepatic iron score in 162 alcoholic cirrhotic patients. In our cohort, this hepatic iron score increased with the number of Ala-MnSOD alleles. We also transfected Huh7 cells with Ala-MnSOD-or Val-MnSOD-encoding plasmids and assessed cellular iron, MnSOD activity, and diverse mRNAs and proteins. In Huh7 cells, MnSOD activity was higher after Ala-MnSOD transfection than after Val-MnSOD transfection. Additionally, iron supplementation decreased transfected MnSOD proteins and activities. Ala-MnSOD transfection increased the mRNAs and proteins of ferritin, hepcidin, and TfR2, decreased the expression of frataxin, and caused cellular iron accumulation. In contrast, Val-MnSOD transfection had limited effects. In conclusion, the Ala-MnSOD variant favors hepatic iron accumulation by modulating the expression of proteins involved in iron homeostasis.     

A novel human ghrelin variant (In1-ghrelin) and ghrelin-O-acyltransferase are overexpressed in breast cancer: potential pathophysiological relevance

The human ghrelin gene, which encodes the ghrelin and obestatin peptides, contains 5 exons (Ex), with Ex1-Ex4 encoding a 117 amino-acid (aa) preproprotein that is known to be processed to yield a 28-aa (ghrelin) and/or a 23-aa (obestatin) mature peptides, which possess biological activities in multiple tissues. However, the ghrelin gene also encodes additional peptides through alternative splicing or post-translational modifications. Indeed, we previously identified a spliced mRNA ghrelin variant in mouse (In2-ghrelin-variant), which is regulated in a tissue-dependent manner by metabolic status and may thus be of biological relevance. Here, we have characterized a new human ghrelin variant that contains Ex0-1, intron (In) 1, and Ex2 and lacks Ex3-4. This human In1-ghrelin variant would encode a new prepropeptide that conserves the first 12aa of native-ghrelin (including the Ser3-potential octanoylation site) but has a different C-terminal tail. Expression of In1-variant was detected in 22 human tissues and its levels were positively correlated with those of ghrelin-O-acyltransferase (GOAT; p = 0.0001) but not with native-ghrelin expression, suggesting that In1-ghrelin could be a primary substrate for GOAT in human tissues. Interestingly, levels of In1-ghrelin variant expression in breast cancer samples were 8-times higher than those of normal mammary tissue, and showed a strong correlation in breast tumors with GOAT (p = 0.0001), ghrelin receptor-type 1b (GHSR1b; p = 0.049) and cyclin-D3 (a cell-cycle inducer/proliferation marker; p = 0.009), but not with native-ghrelin or GHSR1a expression. Interestingly, In1-ghrelin variant overexpression increased basal proliferation of MDA-MB-231 breast cancer cells. Taken together, our results provide evidence that In1-ghrelin is a novel element of the ghrelin family with a potential pathophysiological role in breast cancer.     

Differential distribution of ghrelin-O-acyltransferase (GOAT) immunoreactive cells in the mouse and rat gastric oxyntic mucosa

The enzyme that acylates ghrelin was recently identified in mice as the fourth member of the membrane-bound O-acyltransferases superfamily (MBOAT4) and named ghrelin-O-acyltransferase (GOAT). Only one report showed GOAT mRNA expression in ghrelin-expressing cells of the mouse stomach. We investigated the distribution of GOAT protein in peripheral tissues and co-expression with endocrine markers in the gastric mucosa using a custom-made anti-GOAT antibody. Tissues were collected from male Sprague-Dawley rats and C57BL/6 mice. Western blot revealed two immunoreactive bands in rat and mouse gastric corpus mucosal proteins, a 50 kDa band corresponding to the GOAT protein and a 100 kDa band likely corresponding to a dimer. Western blot also detected GOAT in the plasma and levels were strongly increased after 24-h fasting in mice and slightly in rats. GOAT-immunoreactive cells were located in the gastric corpus mucosa and the anterior pituitary gland, whereas other peripheral tissues of rats and mice examined were negative. In mice, GOAT-immunoreactive cells were mainly distributed throughout the middle portion of the oxyntic glands, whereas in rats they were localized mainly in the lower portion of the glands. Double labeling showed that 95 ± 1% of GOAT-immunoreactive cells in mice co-labeled with ghrelin, whereas in rats only 56 ± 4% of GOAT-positive cells showed co-expression of ghrelin. The remainder of the GOAT-immunopositive cells in rats co-expressed histidine decarboxylase (44 ± 3%). No co-localization was observed with somatostatin in rats or mice. These data suggest species differences between rats and mice in gastric GOAT expression perhaps resulting in a different role of the MBOAT4 enzyme in the rat stomach. Detection of GOAT in the plasma raises the possibility that ghrelin octanoylation may occur in the circulation and the fasting-induced increase in GOAT may contribute to the increase of acylated ghrelin after fasting.     

Smad6 and Smad7 are co-regulated with hepcidin in mouse models of iron overload

The inhibitory Smad7 acts as a critical suppressor of hepcidin, the major regulator of systemic iron homeostasis. In this study we define the mRNA expression of the two functionally related Smad proteins, Smad6 and Smad7, within pathways known to regulate hepcidin levels. Using mouse models for hereditary hemochromatosis (Hfe-, TfR2-, Hfe/TfR2-, Hjv- and hepcidin1-deficient mice) we show that hepcidin, Smad6 and Smad7 mRNA expression is coordinated in such a way that it correlates with the activity of the Bmp/Smad signaling pathway rather than with liver iron levels. This regulatory circuitry is disconnected by iron treatment of Hfe ?/? and Hfe/TfR2 mice that significantly increases hepatic iron levels as well as hepcidin, Smad6 and Smad7 mRNA expression but fails to augment pSmad1/5/8 levels. This suggests that additional pathways contribute to the regulation of hepcidin, Smad6 and Smad7 under these conditions which do not require Hfe.