Hepatic Hepcidin Expression Research Articles

Normalization of Fetal Cerebral and Hepatic Iron by Parental Iron Therapy to Pregnant Rats with Systemic Iron Deficiency without Anemia.

Iron (Fe) is a co-factor for enzymes of the developing brain necessitating sufficient supply. We investigated the effects of administering ferric derisomaltose/Fe isomaltoside (FDI) subcutaneously to Fe-deficient (ID) pregnant rats on cerebral and hepatic concentrations of essential metals and the expression of iron-relevant genes. Pregnant rats subjected to ID were injected with FDI on the day of mating (E0), 14 days into pregnancy (E14), or the day of birth (postnatal (P0)). The efficacy was evaluated by determination of cerebral and hepatic Fe, copper (Cu), and zinc (Zn) and gene expression of ferroportin, hepcidin, and ferritin H + L in pups on P0 and as adults on P70. Females fed an ID diet (5.2 mg/kg Fe) had offspring with significantly lower cerebral and hepatic Fe compared to female controls fed a standard diet (158 mg/kg Fe). Cerebral Cu increased irrespective of supplying a standard diet or administering FDI combined with the standard diet. Hepatic hepcidin mRNA was significantly lower following ID. Cerebral hepcidin mRNA was hardly detectable irrespective of iron status. In conclusion, administering FDI subcutaneously to ID pregnant rats on E0 normalizes fetal cerebral and hepatic Fe. When applied at later gestational ages, supplementation with additional Fe to the offspring is needed to normalize cerebral and hepatic Fe.

Foxo1 is an iron-responsive transcriptional factor regulating systemic iron homeostasis

AbstractThe liver plays a crucial role in maintaining systemic iron homeostasis by secreting hepcidin, which is essential for coordinating iron levels in the body. Imbalances in iron homeostasis are associated with various clinical disorders related to iron deficiency or iron overload. Despite the clinical significance, the mechanisms underlying how hepatocytes sense extracellular iron levels to regulate hepcidin synthesis and iron storage are not fully understood. In this study, we identified Foxo1, a well-known regulator of macronutrient metabolism, which translocates to the nucleus of hepatocytes in response to high-iron feeding, holo-transferrin, and bone morphogenetic protein 6 (BMP6) treatment. Furthermore, Foxo1 plays a crucial role in mediating hepcidin induction in response to both iron and BMP signals by directly interacting with evolutionally conserved Foxo binding sites within the hepcidin promoter region. These binding sites were found to colocalize with Smad-binding sites. To investigate the physiological relevance of Foxo1 in iron metabolism, we generated mice with hepatocyte-specific deletion of Foxo1. These mice exhibited reduced hepatic hepcidin expression and serum hepcidin levels, accompanied by elevated serum iron and liver nonheme iron concentrations. Moreover, high-iron diet further exacerbated these abnormalities in iron metabolism in mice lacking hepatic Foxo1. Conversely, hepatocyte-specific Foxo1 overexpression increased hepatic hepcidin expression and serum hepcidin levels, thereby ameliorating iron overload in a murine model of hereditary hemochromatosis (Hfe−/− mice). In summary, our study identifies Foxo1 as a critical regulator of hepcidin and systemic iron homeostasis. Targeting Foxo1 may offer therapeutic opportunities for managing conditions associated with aberrant iron metabolism.

The drug-specific properties of hypoxia-inducible factor-prolyl hydroxylase inhibitors in mice reveal a significant contribution of the kidney compared to the liver to erythropoietin induction

AimsKidney disease often leads to anemia due to a defect in the renal production of the erythroid growth factor erythropoietin (EPO), which is produced under the positive regulation of hypoxia-inducible transcription factors (HIFs). Chemical compounds that inhibit HIF-prolyl hydroxylases (HIF-PHs), which suppress HIFs, have been developed to reactivate renal EPO production in renal anemia patients. Currently, multiple HIF-PH inhibitors, in addition to conventional recombinant EPO reagents, are used for renal anemia treatment. This study aimed to elucidate the therapeutic mechanisms and drug-specific properties of HIF-PH inhibitors. Methods and key findingsGene expression analyses and mass spectrometry revealed that HIF-PH inhibitors (daprodustat, enarodustat, molidustat, and vadadustat) alter Epo gene expression levels in the kidney and liver in a drug-specific manner, with different pharmacokinetics in the plasma and urine after oral administration to mice. The drug specificity revealed the dominant contribution of EPO induction in the kidneys rather than in the liver to plasma EPO levels after HIF-PH inhibitor administration. We also found that several HIF-PH inhibitors directly induce duodenal gene expression related to iron intake, while these drugs indirectly suppress hepatic hepcidin expression to mobilize stored iron for hemoglobin synthesis through induction of the EPO-erythroferrone axis. SignificanceRenal EPO induction is the major target of HIF-PH inhibitors for their therapeutic effects on erythropoiesis. Additionally, the drug-specific properties of HIF-PH inhibitors in EPO induction and iron metabolism have been shown in mice, providing useful information for selecting the proper HIF-PH inhibitor for each renal anemia patient.

Matriptase-2 regulates iron homeostasis primarily by setting the basal levels of hepatic hepcidin expression through a nonproteolytic mechanism

Matriptase-2 (MT2), encoded by TMPRSS6, is a membrane-anchored serine protease. It plays a key role in iron homeostasis by suppressing the iron-regulatory hormone, hepcidin. Lack of functional MT2 results in an inappropriately high hepcidin and iron-refractory iron-deficiency anemia. Mt2 cleaves multiple components of the hepcidin-induction pathway in vitro. It is inhibited by the membrane-anchored serine protease inhibitor, Hai-2. Earlier in vivo studies show that Mt2 can suppress hepcidin expression independently of its proteolytic activity. In this study, our data indicate that hepatic Mt2 was a limiting factor in suppressing hepcidin. Studies in Tmprss6-/- mice revealed that increases in the dietary iron to ∼0.5% were sufficient to overcome the high hepcidin barrier and to correct the iron-deficiency anemia. Interestingly, the increased iron in Tmprss6-/- mice was able to further upregulate hepcidin expression to a similar magnitude as in wild-type mice. These results suggest that lack of Mt2 does not impact the iron induction of hepcidin. Additional studies of wild-type Mt2 and the proteolytic-dead form, fMt2S762A, indicated that the function of Mt2 is to lower the basal levels of hepcidin expression in a manner that primarily relies on its nonproteolytic role. This idea is supported by the studies in mice with the hepatocyte-specific ablation of Hai-2, which showed a marginal impact on iron homeostasis and no significant effects on iron regulation of hepcidin. Together, these observations suggest that the function of Mt2 is to set the basal levels of hepcidin expression and that this process is primarily accomplished through a nonproteolytic mechanism.

Association between iron metabolism and non-alcoholic fatty liver disease: results from the National Health and Nutrition Examination Survey (NHANES 2017–2018) and a controlled animal study

BackgroundIron metabolism may be involved in the pathogenesis of the non-alcoholic fatty liver disease (NAFLD). The relationship between iron metabolism and NAFLD has not been clearly established. This study aimed to clarify the relationship between biomarkers of iron metabolism and NAFLD.MethodsBased on the National Health and Nutrition Examination Survey (NHANES), restricted cubic spline models and multivariable logistic regression were used to examine the association between iron metabolism [serum iron (SI), serum ferritin (SF), transferrin saturation (TSAT), and soluble transferrin receptor (sTfR)] and the risk for NAFLD. In addition, stratified subgroup analysis was performed for the association between TSAT and NAFLD. Moreover, serum TSAT levels were determined in male mice with NAFLD. The expression of hepcidin and ferroportin, vital regulators of iron metabolism, were analyzed in the livers of mice by quantitative real-time PCR (qRT-PCR) and patients with NAFLD by microarray collected from the GEO data repository.ResultsPatients with NAFLD showed decreased SI, SF, and TSAT levels and increased STfR levels based on the NHANES. After adjusting for confounding factors, TSAT was significantly negatively correlated with NAFLD. Of note, the relationship between TSAT and NAFLD differed in the four subgroups of age, sex, race, and BMI (P for interaction < 0.05). Consistently, mice with NAFLD exhibited decreased serum TSAT levels. Decreased hepcidin and increased ferroportin gene expression were observed in the livers of patients and mice with NAFLD.ConclusionSerum TSAT levels and hepatic hepcidin expression were decreased in both patients and mice with NAFLD. Among multiple biomarkers of iron metabolism, lower TSAT levels were significantly associated with a higher risk of NAFLD in the U.S. general population. These findings might provide new ideas for the prediction, diagnosis, and mechanistic exploration of NAFLD.

Intracellular labile iron is a key regulator of hepcidin expression and iron metabolism.

Liver iron loading can induce hepatic expression of hepcidin and regulate iron metabolism. However, the mechanism by which hepatocyte senses iron loading and further regulates iron metabolism remains unclear. Intracellular labile iron is nonferritin-bound and redox active; it is transitory, and it serves as a crossroads of cellular iron metabolism, the effect of intracellular labile iron in iron metabolism regulation is particularly poorly understood. An intracellular labile iron overload cell model was established using ferric ammonium citrate (FAC) and the lipophilic iron chelator 8-hydroxyquinoline (8HQ/FAC). RNA-Seq was performed to screen the genes that were highly expressed exclusively in 8HQ/FAC-treated HepG2 cells. High-iron-diet mice model and Hfe knockout hemochromatosis mice were used to investigate the importance of tumor necrosis factor α (TNFα) in iron metabolism. Intracellular labile iron in hepatocytes had a dual function in iron metabolism: It induced hepatocytes to express hepcidin via endoplasmic reticulum stress-induced transcription factors, and it stimulated expression of bone morphogenic protein 6 (BMP6, regulator of iron metabolism) in liver sinusoidal endothelial cells (LSECs) via promoting the secretion of TNFα by the hepatocytes. Blockade of TNFα dysregulated iron metabolism during iron overload. Furthermore, administration of TNFα could reduce iron burden in Hfe knockout hemochromatosis mice. Our findings reveal the importance of intracellular labile iron in iron metabolism, and propose that TNFα might be a novel therapeutic target for HFE-associated hemochromatosis.

Influence of prazosin on systemic iron levels and the associated iron metabolic alterations in spontaneously hypertensive rats

The relationship between cardiovascular diseases and iron disorders has gained increasing attention; however, the effects of hypotensive drugs on iron metabolic alterations in hypertension are not well understood. The purpose of this study was to investigate iron metabolic changes after prazosin treatment of spontaneously hypertensive rats (SHRs) and Wistar–Kyoto (WKY) rats. Our second objective was to examine the effects of hypertension and anti‐hypertensive drugs on bone formation and resorption. SHRs and WKY rats were randomized into either prazosin‐treated groups (WKY + PZ and SHR + PZ) or untreated groups (WKY and SHR). After 7 days of intragastric prazosin administration, the rats were sacrificed for analysis; blood samples and organs (the duodenum, liver, kidneys, spleen, and femur) were collected. Both WKY + PZ and SHR groups exhibited iron deficiency in the serum and liver. Prazosin increased the iron levels in the bone tissue of SHRs. Prazosin stimulated the expression of hepcidin mRNA in the liver of SHRs and inhibited the expression of this iron‐regulatory hormone in WKY rats. FPN1 expression in the duodenum was increased significantly in SHRs, however markedly decreased after prazosin treatment. The expression of TLR4 and Ctsk was enhanced in the bone tissue of SHRs, whereas CLC‐7 expression was inhibited. Both hypotension and hypertension can lead to iron deficiency. Treatment with prazosin restored iron homeostasis in SHRs. The inverse impacts of prazosin on hepatic hepcidin expression in SHRs versus WKY rats indicates differing iron regulatory mechanisms between hypertensive and normal animals. The osteoclast activity was found to be enhanced in SHRs. Further study is needed to address whether the changes in osteoblast and osteoclast activity in SHRs correlates with the effects on iron metabolism.

Caffeine Decreases Hepcidin Expression to Alleviate Aberrant Iron Metabolism under Inflammation by Regulating the IL-6/STAT3 Pathway.

Caffeine is well-known as a psychostimulant, and it can also be beneficial in numerous diseases such as diabetes and different types of cancer. Previous studies have shown that caffeine can have a protective role in bacterial infection-induced inflammation and hyperoxia-mediated pulmonary inflammation. Hepcidin, which is regulated by the IL-6/STAT3 inflammation pathway, is a peptide hormone that maintains systemic iron homeostasis. We hypothesized that caffeine’s effects on inflammation may also influence hepcidin production and therefore systemic iron metabolism. To this end, we treated 2-month-old mice with caffeine by daily intragastric administration for 7 days, administering intraperitoneal LPS after the final caffeine treatment. Twelve hours after LPS treatment the mice were euthanized, and tissues were collected. We found that caffeine decreased hepatic hepcidin expression and attenuated LPS-induced hepatic hepcidin overexpression. IL-6 expression and STAT3 phosphorylation were also reduced upon caffeine administration. Additionally, hepatic and splenic FPN1 levels increased after caffeine treatment, leading to lower iron levels in liver and spleen tissues and higher iron levels in serum. Caffeine also prevented the increase in spleen weight and decrease in body weight after LPS treatment. Together, our findings suggest that caffeine decreases hepcidin expression via inhibiting inflammation and the activation of the IL-6/STAT3 pathway, thus presenting an attractive, potential therapeutic for the treatment of anemia of inflammation.

Ablation of Na+/H+ exchanger‐3 prevents tissue iron loading in the Hfe mouse model of hereditary hemochromatosis

Several common hereditary disorders are associated with toxic iron overload which leads to liver cirrhosis, cardiomyopathy, and endocrine disorders. Hereditary hemochromatosis arises from mutations in the genes coding for any of several proteins involved in iron sensing (e.g. Hfe), hepcidin production (e.g. hemojuvelin), or hepcidin action, resulting in elevated iron absorption relative to the iron need. Divalent metal‐ion transporter‐1 (DMT1) is a H+‐coupled Fe2+ transporter and the principal or only mechanism by which nonheme iron is taken up at the intestinal brush border [Shawki A et al (2015) Am J Physiol Gastrointest Liver Physiol 309, G635–G647]. DMT1 is a validated therapeutic target in treating iron overload. We have shown that Na+/H+ exchanger‐3 (NHE3) is required for adequate iron absorption, via its physiological role in generating at the intestinal brush border an acidic microclimate that energizes DMT1‐mediated H+‐coupled Fe2+ transport [Shawki A et al (2016) Am J Physiol Gastrointest Liver Physiol 311, G423–G430], so we have explored here the contribution of NHE3 to pathological iron loading. We tested the hypothesis that NHE3 activity is necessary for pathological iron loading. We examined the effect of ablating the SLC9A3 gene coding for NHE3 in the Hfe mouse model of hereditary hemochromatosis. We measured tissue iron levels, hematological and blood‐iron variables, and the hepatic expression of Hamp1 (coding for hepcidin) by using qPCR in male and female FVB/N mice, age ≍ 120 d, fed a normal diet (NIH‐07). We examined four genotypes in both sexes: wildtype mice, NHE3 knockout (NHE3−/−), Hfe knockout (hemochromatosis disease model, Hfe−/−), and double knockout (NHE3−/− | Hfe−/−) (n = 12–26 mice per group). We chose α = 0.05, and analyzed our data (mean, SD) by using multifactorial ANOVA. We found that ablation of NHE3 prevented the liver iron loading characteristic of the Hfe−/− mouse model, an effect that was independent of sex. We observed a similar effect on iron loading in the spleen. Ablation of NHE3 produced no sign of overt anemia. Hepatic expression of hepcidin was depressed in NHE3−/− and in Hfe−/− mice relative to wildtype mice, and remained low in the NHE3−/− | Hfe−/− mice, providing a plausible explanation for the observation that, despite correction of the tissue iron levels in NHE3−/− | Hfe−/− mice, iron levels remained high in the readily accessible serum iron pool (typically 0.1% of total body iron). We conclude that ablation of NHE3 prevents pathological tissue iron loading in the Hfe mouse model of hemochromatosis. Pharmacological blockade of NHE3 may offer a means of inhibiting iron absorption in hereditary hemochromatosis.

Targeted autophagy disruption reveals the central role of macrophage iron metabolism in systemic iron homeostasis

AbstractIron homeostasis depends on both intracellular control through iron-responsive proteins and the systemic level of iron through hepcidin-ferroportin axis. Indeed, the hormone hepcidin downregulates the ferroportin iron exporter to control iron recycling from macrophages and iron uptake from enterocytes. Here, we focused on the role of autophagy in macrophage iron metabolism and systemic iron homeostasis. Mice deficient for autophagy in macrophages (LysM-Atg5−/−) mimicked a primary iron overload phenotype, resulting in high ferroportin expression in both macrophages and enterocytes that correlated with marked parenchymal iron overload. Furthermore, LysM-Atg5−/− mice exhibited increased hematopoietic activity with no sign of anemia but correlating with rather high plasma iron level. Compared with wild-type cells, bone marrow–derived macrophages from LysM-Atg5−/− mice had significantly increased ferroportin expression and decreased iron content, confirming high iron export. In erythrophagocytic macrophages, autophagy regulates hemosiderin storage mechanisms as well as degradation of ferroportin and subsequently its plasma membrane localization and iron export; furthermore, ferroportin colocalization with hepcidin indicates hepcidin autocrine activity. Relatively high hepatic hepcidin expression and decreased hepcidin level in the spleen of LysM-Atg5−/− mice, correlating with low hemosiderin iron storage, as well as in erythrophagocytic Atg5−/− macrophages were evidenced. Therefore, our results highlight the critical role of autophagy in macrophages for iron trafficking and systemic iron homeostasis. We propose that in macrophages, autophagy restricts ferroportin level and iron export, resulting in hepcidin expression with an autocrine-paracrine effect that plays a role in the regulation of ferroportin expression in duodenal enterocytes.

SiRNA Dynamic PolyConjugates for the Targeting of Hepatocyte HAMP Genes as Potential Treatment for Anemia of Inflammation: A Research Protocol

Introduction: Anemia of inflammation (AI) is a condition caused by iron sequestration from invading pathogens, which is primarily caused by hepcidin upregulation. This results in low serum iron levels. The objective of this research protocol is to evaluate the potential of small interfering RNA (siRNA) Dynamic PolyConjugates (DPCs) in decreasing hepatic hepcidin expression for AI treatment. Methods: DPCs carrying Hepcidin Antimicrobial Peptide (HAMP) gene siRNA will be synthesized and injected into the tail veins of AI-induced mice on a standardized low-iron diet. Various experiments will then be conducted to verify that siRNA DPCs specifically target hepatocytes without causing significant toxicity. To evaluate the treatment’s efficacy, HAMP mRNA and serum iron levels will be measured using Reverse Transcription Quantitative Real- time Polymerase Chain Reaction (RT-qPCR) and a common calorimeter method, respectively. These measurements will determine the potential of siRNA to silence hepatic hepcidin expression and its resulting ability to increase serum iron levels. Results: It is anticipated that successful targeting of siRNA DPCs to hepatocytes will be confirmed through immunofluorescence and that toxicity levels induced by the treatment will be statistically insignificant. Moreover, we expect lower HAMP mRNA levels and thus higher serum iron concentrations in the experimental group compared to the control. Discussion: Hepatocyte-specific delivery of the siRNA DPC with minimal toxicity and effective silencing of the HAMP gene would deem this delivery vehicle to be a notable candidate in treating AI compared to other current conventional treatments. Certain limitations include confounding variables and potential toxicity, which should be further considered. Conclusion: Future implications of this study include human testing of siRNA DPC administration in AI patients as well as using DPCs conjugated to other siRNAs in the potential treatment of other gene-related pathologies associated with abnormal upregulation of specific proteins.

Effect of Recombinant Human Erythroferrone Protein on Hepcidin Gene (Hamp1) Expression in HepG2 and HuH7 Cells.

Iron is essential for all living organisms. It is strictly controlled by iron transporters, transferrin receptors, ferroportin and hepcidin. Erythroferrone (ERFE) is an iron-regulatory hormone which is highly expressed in erythroblasts by erythropoietin (EPO) stimulation and osteoblasts independently of EPO by sequestering bone morphogenetic proteins and inhibiting hepatic hepcidin expression. Although the hepcidin suppressive function of ERFE is known, its receptors still require investigation. Here, we aim to identify ERFE receptors on the HepG2 and Huh7 cells responsible for ERFE. Recombinant ERFE (rERFE) was first produced in HEK293 cells transfected with pcDNA3.1 + ERFE, then purified and detected by Western blot. The liver cells were treated with an rERFE-rich medium of transfected HEK293 cells and a purified rERFE-supplemented medium at various time points, and hepcidin gene (Hamp1) expression was determined using qRT-PCR. The results show that 37-kD rERFE was expressed in HEK293 cells. Hamp1 was suppressed at 3 h and 6 h in Huh7 cells after rERFE treatments (p < 0.05), then restored to the original levels. Hamp1 was activated after treatment with purified rERFE for 24 h and 48 h. Together, these results reveal that ERFE suppressed Hamp1 expression in liver cells, possibly acting on membrane ERFE receptor, which in Huh7 cells was more sensitive to the ERFE concentrate.

A Comprehensive Analysis of the Erythropoietin-erythroferrone-hepcidin Pathway in Hereditary Hemolytic Anemias.

A Comprehensive Analysis of the Erythropoietin-erythroferrone-hepcidin Pathway in Hereditary Hemolytic Anemias.

Calcitonin increases hepatic hepcidin expression through the BMP6 of kidney in mice

BackgroundOsteoporosis is frequently accompanied by iron disorders. Calcitonin (CT) was approved as a clinical drug to treat osteoporosis. Hepcidin is a peptide hormone that is secreted by the liver and controls body iron homeostasis. Hepcidin deficiency leads to iron overload diseases. This study was aimed at investigating the effect of CT on hepatic hepcidin and the mechanism by which CT modulates hepatic hepcidin pathways and iron metabolism. MethodRT-PCR, Western blot, ELISA and siRNA were used to detect the effect of CT on iron metabolism in vivo and in vitro. In addition, the regulatory signal molecules of hepcidin were measured to explore the molecular mechanism of its regulation. ResultsThe results showed that CT strongly increased hepcidin expression and altered iron homeostasis, after mice were intraperitoneal injection of CT. In response to CT administration, BMP6 level in kidney and the serum BMP6 was increased significantly. The phosphorylation of Smad1/5/8 proteins in liver was increased at 3 h and 6 h. Moreover, the Bmp inhibitor LDN-193,189 pretreatment significantly attenuated the CT-mediated increases in phosphorylated Smad1/5/8 and Hamp1 mRNA levels. Calcitonin receptor (CTR) siRNA transfection significant suppressed the role of CT on BMP6 expression in Caki-1 cells. ConclusionOur results suggest that CT strongly induces hepcidin expression and affected iron metabolism. It will provide a new strategy for the treatment of calcium iron related diseases.

Hepatocyte neogenin is required for hemojuvelin-mediated hepcidin expression and iron homeostasis in mice

AbstractNeogenin (NEO1) is a ubiquitously expressed multifunctional transmembrane protein. It interacts with hemojuvelin (HJV), a BMP coreceptor that plays a pivotal role in hepatic hepcidin expression. Earlier studies suggest that the function of HJV relies on its interaction with NEO1. However, the role of NEO1 in iron homeostasis remains controversial because of the lack of an appropriate animal model. Here, we generated a hepatocyte-specific Neo1 knockout (Neo1fl/fl;Alb-Cre+) mouse model that circumvented the developmental and lethality issues of the global Neo1 mutant. Results show that ablation of hepatocyte Neo1 decreased hepcidin expression and caused iron overload. This iron overload did not result from altered iron utilization by erythropoiesis. Replacement studies revealed that expression of the Neo1L1046E mutant that does not interact with Hjv, was unable to correct the decreased hepcidin expression and high serum iron in Neo1fl/fl;Alb-Cre+ mice. In Hjv−/− mice, expression of HjvA183R mutant that has reduced interaction with Neo1, also displayed a blunted induction of hepcidin expression. These observations indicate that Neo1-Hjv interaction is essential for hepcidin expression. Further analyses suggest that the Hjv binding triggered the cleavage of the Neo1 cytoplasmic domain by a protease, which resulted in accumulation of truncated Neo1 on the plasma membrane. Additional studies did not support that Neo1 functions by inhibiting Hjv shedding as previously proposed. Together, our data favor a model in which Neo1 interaction with Hjv leads to accumulation of cleaved Neo1 on the plasma membrane, where Neo1 acts as a scaffold to induce the Bmp signaling and hepcidin expression.

Central Nervous System Inflammation Induced by Lipopolysaccharide Up-Regulates Hepatic Hepcidin Expression by Activating the IL-6/JAK2/STAT3 Pathway in Mice.

It is known that lipopolysaccharide (LPS) triggers inflammatory response after intracerebroventricular (ICV) injection and elevates the expression of hepcidin through the interleukin 6/janus kinase 2/transducer and activator of the transcription 3 (IL-6/JAK2/STAT3) signaling pathway in the brain. This study was conducted to determine whether LPS ICV injection can regulate peripheral hepatic hepcidin expression and iron metabolism. Here, we studied the hepcidin expression in the liver, as well as serum iron and transferrin saturation, after LPS ICV injection. We also demonstrated the role of the IL-6/JAK2/STAT3 pathway in hepcidin expression in the livers of IL-6 knockout (IL-6–/– mice) and IL-6+/+ mice. AG490 was used to verify the effect of the IL-6/JAK2/STAT3 pathway on hepatic hepcidin expression. Our present study demonstrated that LPS ICV injection up-regulated hepatic hepcidin expression. This finding provides further evidence for highlighting the importance of the central inflammation on hepatic hepcidin expression and peripheral iron metabolism.

Effects of long-term ethanol ingestion on hepatic iron metabolism in two mouse strains.

The mechanisms responsible for dysregulation of iron metabolism in response to ethanol ingestion are poorly understood. Relatively brief ethanol exposures in rodents are associated with reduced hepatic hepcidin expression without increases in hepatic iron content. This study evaluated the effects of long-term ethanol treatment on hepatic iron metabolism in two mouse strains. Ethanol was administered in the drinking water to C57BL/6 and BALB/c mice for up to 11months. Hepatic histology and iron concentrations (HIC) were assessed, along with expression of relevant genes and proteins by real-time RT-PCR and western blot, respectively. The livers of ethanol-consuming mice of both strains showed mild steatosis without inflammation or fibrosis. Stainable hepatocyte iron was modestly increased in both strains ingesting ethanol, although hepatic iron concentrations were significantly higher only in C57BL/6 mice. Long-term ethanol did not affect hepcidin mRNA (Hamp1 or Hamp2) in either strain, nor was the expression of several oxidative stress-responsive genes (glutamate cysteine ligase, gamma-glutamyl transpeptidase, heme oxygenase-1 and growth differentiation factor 15) altered in response to ethanol, suggesting that oxidative stress and suppression of hepcidin expression in short-term ethanol feeding models may be transient phenomena that resolve as mice adapt to ethanol exposure. This murine model of chronic ethanol ingestion demonstrates modest increases in hepatic iron without changes in hepcidin expression, markers of oxidative stress or significant histologic liver injury. Further investigations are needed to characterize the mechanisms of dysregulated iron metabolism resulting from chronic ethanol ingestion.

The ectodomain of matriptase-2 plays an important nonproteolytic role in suppressing hepcidin expression in mice

AbstractMatriptase-2 (MT2), encoded by TMPRSS6, is a membrane-anchored serine protease that plays a key role in suppressing hepatic hepcidin expression. MT2 is synthesized as a zymogen and undergoes autocleavage for activation. Previous studies suggest that MT2 suppresses hepcidin by cleaving hemojuvelin and other components of the bone morphogenetic protein–signaling pathway. However, the underlying mechanism is still debatable. Here we dissected the contributions of the nonproteolytic and proteolytic activities of Mt2 by taking advantage of Mt2 mutants and Tmprss6−/− mice. Studies of the protease-dead full-length Mt2 (Mt2S762A) and the truncated Mt2 that lacks the catalytic domain (Mt2mask) indicate that the catalytic domain, but not its proteolytic activity, was required for Mt2 to suppress hepcidin expression. This process was likely accomplished by the binding of Mt2 ectodomain to Hjv and Hfe. We found that Mt2 specifically cleaved the key components of the hepcidin-induction pathway, including Hjv, Alk3, ActRIIA, and Hfe, when overexpressed in hepatoma cells. Nevertheless, studies of a murine iron-refractory iron-deficiency anemia–causing mutant (Mt2I286F) in the complement protein subcomponents C1r/C1s, urchin embryonic growth factor, and bone morphogenetic protein 1 domain indicate that Mt2I286F can be activated, but it exhibited a largely compromised ability to suppress hepcidin expression. Coimmunoprecipitation analysis revealed that Mt2I286F, but not Mt2S762A, had reduced interactions with Hjv, ActRIIA, and Hfe. In addition, increased expression of a serine protease inhibitor, the hepatocyte growth factor activator inhibitor-2, in the liver failed to alter hepcidin. Together, these observations support the idea that the substrate interaction with Mt2 plays a determinant role and suggest that the proteolytic activity is not an appropriate target to modulate the function of MT2 for clinical applications.

Nonclinical Characterization of the Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor Roxadustat, a Novel Treatment of Anemia of Chronic Kidney Disease.

Anemia of chronic kidney disease (CKD) is a multifactorial disorder caused by impaired erythropoietin (EPO) production and altered iron homeostasis associated with inflammation. Hypoxia-inducible factor (HIF) is a transcription factor that stimulates erythropoiesis via a coordinated response involving increased EPO production and enhanced iron availability for Hb synthesis. HIF degradation is regulated by HIF-prolyl hydroxylase (HIF-PH) enzymes. We hypothesized that roxadustat, an orally available small-molecule inhibitor of HIF-PH, would increase EPO production and promote erythropoiesis in animal models of anemia. In cells, roxadustat increased both HIF-1α and HIF-2α proteins, leading to an increase in EPO production, even in the presence of EPO-suppressing inflammatory cytokines. Roxadustat administered intermittently to healthy rats and cynomolgus monkeys increased circulating EPO levels, reticulocytes, blood Hb, and hematocrit in a dose-dependent manner. Roxadustat corrected anemia in a rat model of CKD after five-sixth nephrectomy and in a rat model of anemia of inflammation with impaired iron metabolism induced by peptidoglycan-polysaccharide (PG-PS). In the PG-PS model, roxadustat significantly decreased hepatic expression of hepcidin, a hormone responsible for iron sequestration and functional iron deficiency, and increased expression of two genes involved in duodenal iron absorption: divalent metal transporter 1 and duodenal cytochrome b. In conclusion, by activating the HIF pathway, roxadustat increased EPO production, elevated Hb, corrected anemia, and improved iron homeostasis. The coordinated erythropoietic response stimulated by roxadustat, involving both EPO production and mobilization of iron stores, makes this compound a promising treatment of anemia of CKD and anemia associated with functional iron deficiency. SIGNIFICANCE STATEMENT: Roxadustat is a novel orally available small-molecule inhibitor of HIF prolyl hydroxylase enzymes that reversibly stabilizes HIF-α, thus activating transcription of HIF-dependent genes, including EPO and regulators of iron homeostasis. Activation of the HIF pathway by roxadustat induces erythropoiesis in healthy rats and monkeys and corrects experimentally induced anemia in rats. The coordinated erythropoietic response that increases EPO production and mobilizes iron stores makes roxadustat a promising treatment for anemia of chronic kidney disease and anemia associated with functional iron deficiency.

Antibacterial and antioxidant activity of clove oil against Streptococcus iniae infection in Nile tilapia (Oreochromis niloticus) and its effect on hepatic hepcidin expression

This study was designed to evaluate the modulating effect dietary clove essential oil (CL) has on the antioxidant and immunological status of Nile tilapia following Streptococcus iniae (Si) infection. Fish were placed on either control or (1.5 and 3%) CL-supplemented diets for 4 weeks. After sampling, the remaining fish in the control group were divided into 2 groups: an unchallenged (negative control) and an Si-challenged positive control. On the other hand, the remaining fish in CL-supplemented groups were challenged with Si, and mortality was checked for two weeks before the final sampling. Serum immunological parameters, tissue antioxidants, and oxidative stress markers were determined. Moreover, hepatic hepcidin expression was also measured in different groups. The obtained results showed improvements in blood phagocytic, bactericidal, lysozyme, and respiratory burst activities in CL-supplemented fish before and after the Si challenge. Si-challenge caused a remarkable increase in tissue malondialdehyde (MDA) levels that was inhibited by CL supplementation. The activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) in tissues were significantly elevated in a dose-dependent manner in CL-supplemented groups in both pre- and post-challenge experiments; renal SOD did not show any differences. Hepatic nitric oxide (NO) level was significantly decreased in CL-supplemented fish in a dose-dependent manner. In the post-challenge experiment, nitrosative stress was apparent in the liver and kidney; however, CL supplementation was sufficient to reverse it. Interestingly, a remarkable induction of the hepatic hepcidin expression was observed in all CL-supplemented groups in the pre-challenge experiment and Si-challenged fish, underscoring the role of CL as an antibacterial through inducing hepatic hepcidin expression to combat S. iniae infection. CL-supplementation was associated with lower mortality rates after Si-challenge, which was more pronounced in CL-3% supplemented fish.In conclusion, our results demonstrate that CL has a potent antioxidant role via increasing antioxidant enzymes’ activities and antagonizing lipid peroxidation. Moreover, CL has an immune-stimulant effect by inducing the hepatic hepcidin expression and immunological markers in response to S. iniae infection.