Hepcidin Gene 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.

The potency of Thymus vulgaris seed extract in alleviating obesity complications and iron overload in obese rats

Natural products have long been recognized as valuable sources of bioactive compounds in therapeutic agents. Thymus vulgaris, commonly known as thyme and belonging to the Lamiaceae family, is widely distributed and extensively used in Egyptian folk medicine for its antibroncholitic, antispasmodic, carminative, and diuretic properties. However, there is a lack of research on the effects of T. vulgaris seed extract in mitigating obesity-induced iron overload in rats. Given its potent bioactive constituents, this study aimed to investigate the impact of T. vulgaris seed extract on iron overload associated with obesity by evaluating lipid profiles, iron status parameters—including plasma ferritin, total iron-binding capacity (TIBC), plasma transferrin, transferrin saturation percentage (TS%), and hepcidin gene expression in adipose tissue. About 30 flavonoids were identified in T. vulgaris seed extract using LC-MS. Among these, methoxyflavonoids were the most abundant metabolites. The administration of T. vulgaris seed extract to obese rats resulted in significant reductions in anthropometric features, hepcidin gene expression, cholesterol, triglycerides, low-density lipoprotein (LDL), TIBC, plasma ferritin, leptin, and iron and copper content in adipose tissue. Moreover, treatment with T. vulgaris extract led to a notable decrease in interleukin-6 (IL-6) levels (76.33 ± 4.31 ng/L and 40.83 ± 1.82 ng/L for low and high doses, respectively) compared to the obese control group (110.83 ± 5.82 ng/L). Conversely, plasma adiponectin levels were significantly increased with T. vulgaris supplementation (123.6 ± 7.9 µg/L and 217.4 ± 6.1 µg/L for low and high doses, respectively) compared to the obese group (54.6 ± 4.9 µg/L). Additionally, adipose tissue zinc levels were significantly elevated in response to T. vulgaris supplementation (2.54 ± 0.20 µg/g and 3.75 ± 0.14 µg/g for low and high doses, respectively) compared to the obese group (1.45 ± 0.23 µg/g). Our findings suggest that T. vulgaris could be a promising novel therapeutic agent for managing obesity-induced iron overload.

Direct and macrophage stimulation mediated effects of active, inactive, and cell-free supernatant forms of Akkermansia muciniphila and Faecalibacterium duncaniae on hepcidin gene expression in HepG2 cells.

Hepcidin is a crucial regulator of iron homeostasis with protective effects on liver fibrosis. Additionally, gut microbiota can also affect liver fibrosis and iron metabolism. Although the hepatoprotective potential of Akkermansia muciniphila and Faecalibacterium duncaniae, formerly known as F. prausnitzii, has been reported, however, their effects on hepcidin expression remain unknown. We investigated the direct and macrophage stimulation-mediated effects of active, heat-inactivated, and cell-free supernatant (CFS) forms of A. muciniphila and F. duncaniae on hepcidin expression in HepG2 cells by RT-qPCR analysis. Following stimulation of phorbol-12-myristate-13-acetate (PMA) -differentiated THP-1 cells with A. muciniphila and F. duncaniae, IL-6 concentration was assessed via ELISA. Additionally, the resulting supernatant was treated with HepG2 cells to evaluate the effect of macrophage stimulation on hepcidin gene expression. The expression of genes mediating iron absorption and export was also examined in HepG2 and Caco-2 cells via RT-qPCR. All forms of F. duncaniae increased hepcidin expression while active and heat-inactivated/CFS forms of A. muciniphila upregulated and downregulated its expression, respectively. Active, heat-inactivated, and CFS forms of A. muciniphila and F. duncaniae upregulated hepcidin expression, consistent with the elevation of IL-6 released from THP-1-stimulated cells as a macrophage stimulation effect in HepG2 cells. A. muciniphila and F. duncaniae in active, inactive, and CFS forms altered the expression of hepatocyte and intestinal iron-mediated absorption /exporter genes, namely dcytb and dmt1, and fpn in HepG2 and Caco-2 cells, respectively. In conclusion, A. muciniphila and F. duncaniae affect not only directly but also through macrophage stimulation the expression of hepcidin gene in HepG2 cells. These findings underscore the potential of A. muciniphila and F. duncaniae as a potential therapeutic target for liver fibrosis by modulating hepcidin and intestinal and hepatocyte iron metabolism mediated gene expression.

Iron Fortification and Inulin Supplementation in Early Infancy: Evaluating the Impact on Iron Metabolism and Trace Mineral Status in a Piglet Model

BackgroundInfant formula in the United States contains abundant iron, raising health concerns about excess iron intake in early infancy. ObjectivesUsing a piglet model, we explored the impact of high iron fortification and prebiotic or synbiotic supplementation on iron homeostasis and trace mineral bioavailability. MethodsTwenty-four piglets were stratified and randomly assigned to treatments on postnatal day 2. Piglets were individually housed and received an iron-adequate milk diet (AI), a high-iron milk diet (HI), HI supplemented with 5% inulin (HI with a prebiotic [HIP]), or HIP with an oral gavage of Ligilactobacillus agilis YZ050, an inulin-fermenting strain, every third day (HI with synbiotic [HIS]). Milk was provided in 14 meals daily, mimicking formula feeding in infants. Fecal consistency score and body weight were recorded daily or every other day. Blood and feces were sampled weekly, and tissues collected on postnatal day 29. Data were analyzed using mixed model analysis of variance with repeated measures whenever necessary. ResultsDiet did not affect growth. HI increased hemoglobin, hematocrit, and serum iron compared to AI. Despite marginal adequacy, AI upregulated iron transporter genes and maintained satisfactory iron status in most pigs. HI upregulated hepcidin gene expression in liver, caused pronounced tissue iron deposition, and markedly increased colonic and fecal iron. Inulin supplementation, regardless of L. agilis YZ050, not only attenuated hepatic iron overload but also decreased colonic and fecal iron without altering pH or the expression of iron regulatory genes. HI lowered zinc (Zn) and copper (Cu) in the duodenum and liver compared to AI, whereas HIP and HIS further decreased Zn and Cu in the liver and diminished colonic and fecal trace minerals. ConclusionsEarly-infancy excessive iron fortification causes iron overload and compromises Zn and Cu absorption. Inulin decreases trace mineral absorption likely by enhancing gut peristalsis and stool frequency.

Participation of Hepcidins in the Inflammatory Response Triggered by λ-Carrageenin in Gilthead Seabream (Sparus aurata)

The role of hepcidins, antimicrobial peptides involved in iron metabolism, immunity, and inflammation, is studied. First, gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs) were incubated with λ-carrageenin to study the expression of hepcidin and iron metabolism-related genes. While the expression of most of the genes studied was upregulated, the expression of ferroportin gene (slc40a) was downregulated. In the second part of the study, seabream specimens were injected intramuscularly with λ-carrageenin or buffer (control). The expression of the same genes was evaluated in the head kidney, liver, and skin at different time points after injection. The expression of Hamp1m, ferritin b, and ferroportin genes (hamp1, fthb, and slc40a) was upregulated in the head kidney of fish from the λ-carrageenin-injected group, while the expression of Hamp2C and Hamp2E genes (hamp2.3 and hamp2.7) was downregulated. In the liver, the expression of hamp1, ferritin a (ftha), slc40a, Hamp2J, and Hamp2D (hamp2.5/6) genes was downregulated in the λ-carrageenin-injected group. In the skin, the expression of hamp1 and (Hamp2A Hamp2C) hamp2.1/3/4 genes was upregulated in the λ-carrageenin-injected group. A bioinformatic analysis was performed to predict the presence of transcription factor binding sites in the promoter region of hepcidins. The primary sequence of hepcidin was conserved among the different mature peptides, although changes in specific amino acid residues were identified. These changes affected the charge, hydrophobicity, and probability of hepcidins being antimicrobial peptides. This study sheds light on the poorly understood roles of hepcidins in fish. The results provide insight into the regulatory mechanisms of inflammation in fish and could contribute to the development of new strategies for treat inflammation in farm animals.Graphical

The association of TMPRSS6 gene polymorphism with iron status in Egyptian children (a pilot study)

Several studies have shown association of single nucleotide polymorphisms (SNPs) of hepcidin regulatory pathways genes with impaired iron status. The most common is in the TMPRSS6 gene. In Africa, very few studies have been reported. We aimed to investigate the correlation between the common SNPs in the transmembrane protease, serine 6 (TMPRSS6) gene and iron indicators in a sample of Egyptian children for identifying the suitable candidate for iron supplementation.Patients and methods One hundred and sixty children aged 5–13 years were included & classified into iron deficient, iron deficient anemia and normal healthy controls. All were subjected to assessment of serum iron, serum ferritin, total iron binding capacity, complete blood count, reticulocyte count, serum soluble transferrin receptor and serum hepcidin. Molecular study of TMPRSS6 genotyping polymorphisms (rs4820268, rs855791 and rs11704654) were also evaluated.Results There was an association of iron deficiency with AG of rs855791 SNP, (P = 0.01). The minor allele frequency for included children were 0.43, 0.45 & 0.17 for rs4820268, rs855791 & rs11704654 respectively. Genotype GG of rs4820268 expressed the highest hepcidin gene expression fold, the lowest serum ferroportin & iron store compared to AA and AG genotypes (p = 0.05, p = 0.05, p = 0.03 respectively). GG of rs855791 had lower serum ferritin than AA (p = 0.04), lowest iron store & highest serum hepcidin compared to AA and AG genotypes (p = 0.04, p = 0.01 respectively). Children having CC of rs11704654 had lower level of hemoglobin, serum ferritin and serum hepcidin compared with CT genotype (p = 0.01, p = 0.01, p = 0.02) respectively.Conclusion Possible contribution of SNPs (rs855791, rs4820268 and rs11704654) to low iron status.

Molecular characterization of HAMP rs10421768 gene and phenotypic expression of hepcidin; a case-control study among sickle cell anaemia patients in Ghana.

The sporadic nature of blood transfusion therapy coupled with the alteration of HAMP genes may exacerbate the risk of iron burden in sickle cell anaemia (SCA) patients. The study determined the polymorphic distribution of the HAMP promoter gene rs10421768 and hepcidin levels in SCA patients. Sixty participants aged ≥12years [45 SCA patients and 15 controls (HbA)] were recruited from 15th March, 2023 to 20th July, 2023 for a case-control study at Methodist Hospital Wenchi, Ghana. Complete blood count and hepcidin levels assessment were done using haematology analyzer and ELISA, respectively. Genomic DNA was extracted using the Qiagen Kit, and HAMP gene rs10421768 (c.-582 A>G) was sequenced using the MassARRAY method. Data were analysed using SPSS version 26.0. The frequencies of the HAMP promoter rs10421768 genotypes AA, AG, and GG were 64.4%, 33.3%, and 2.2% in SCA patients, and 86.7%, 13.3%, and 0% in the controls, respectively. Serum hepcidin levels were significantly higher among controls than cases [204.0 (154.1-219.3) vs 150.2 (108.1-195.6)μg/L, p<0.010]. Participants with HAMP rs10421768 homozygous A genotype had higher serum levels of hepcidin compared with those in the wild genotypes (AG/GG) group [(188.7 (130.9-226.9) vs 136.8 (109.7-157.8)μg/L, p<0.016]. Disease severity and blood cell parameters were not associated with the HAMP variants (p>0.05). The HAMP promoter rs10421768 AA genotype has the highest frequency of distribution and the GG genotype with the least distribution. Participants with HAMP rs10421768 G allele (c.-582A>G) had reduced levels of hepcidin. HAMP rs10421768 genotypes had no association with blood cell parameters and disease severity. The HAMP rs10421768 genotypes may influence serum levels of hepcidin. Further study is required to elucidate the potential effect of the G allele on hepcidin transcription.

Hypoxia-inducible factor-prolyl hydroxylase inhibitor Roxadustat (FG-4592) reduces renal fibrosis in Dahl salt-sensitive rats.

Although hypoxia-inducible factor-prolyl hydroxylase (HIF-PH) inhibitors have been developed for the treatment of renal anemia, their effects on cardiac and renal dysfunction remain unknown. We previously reported on Dahl salt-sensitive rats, in a rat model of salt-sensitive hypertension, that exhibited anemia and impaired expression of duodenal iron transporters after the development of hypertensive cardiac and renal dysfunction. Therefore, we investigated the effects of Roxadustat (FG-4592), an HIF-PH inhibitor, on anemia, iron regulation, and cardiac and renal dysfunction in Dahl salt-sensitive rats. Six-week-old male Dahl salt-sensitive rats were fed a normal or high-salt diet for 8 weeks. A further subset of Dahl salt-sensitive rats, that were fed a high-salt diet, was administered Roxadustat for 8 weeks. Dahl salt-sensitive rats fed a high-salt diet developed hypertension, cardiac and renal dysfunction, and anemia after 8 weeks of feeding. Roxadustat increased hemoglobin and serum erythropoietin levels in Dahl salt-sensitive rats fed a high-salt diet. With regard to the iron-regulating system, Roxadustat lowered hepatic hepcidin gene expression and increased the gene expression of duodenal iron transporters, such as cytochrome b and divalent metal transporter 1 , in Dahl salt-sensitive rats fed a high-salt diet. Roxadustat did not affect the development of hypertension and cardiac hypertrophy in Dahl salt-sensitive rats with a high-salt diet; however, Roxadustat treatment attenuated renal fibrosis in these rats. Roxadustat ameliorated anemia with affecting the gene expression of the iron-regulating system, and did not affect cardiac hypertrophy but attenuated renal fibrosis in Dahl salt-sensitive rats fed a high-salt diet.

Hepcidin from Onychostoma macrolepis: Response to Aeromonas hydrophila infection by down-regulating expression of inflammatory factor genes and regulating iron homeostasis potentially

Hepcidin, as an antimicrobial peptide, is associated with innate immunity and is considered a potential antibiotic substitute. In the present study, the hepcidin gene from the cavefish - Onychostoma macrolepis was identified and analyzed. The recombinant hepcidin protein (rOmhepc) was obtained by prokaryotic expression, evaluating the inhibitory effect of 5 pathogenic bacteria in vitro. Sixty O. macrolepis injected with 100 μL A. hydrophila (1.5 × 108 CFU/mL) were randomly divided into the therapeutic group and infection group, and therapeutic group was injected with 100 μL rOmhepc (100 μg/mL) at 6 and 18 h. The survival rates of O. macrolepis and bacterial load in liver were measured at 24 h. The liver tissues were collected at 0, 6, 12, and 24 h after A. hydrophila injection for investigating expression levels of immune-related, inflammatory factor genes and FPN1 gene. The results demonstrated that the hepcidin CDS contained 279 bp and encoded 93 aa. Hepcidin protein has a hydrophobic surface formed by multiple hydrophobic residues (CCGCCYC), and the theoretical pI was 7.53. Omhepc gene was expressed at varying levels in tested tissues, with the liver showing the highest expression, followed by the spleen. The expression of hepcidin gene following A. hydrophila infection was up-regulated and then down-regulated in liver, and the highest expression level was found at 12 h with a 10.93-fold. The rOmhepc remarkably inhibited the growth of A. hydrophila, Staphylococcus aureus, and Streptococcus agalactiae, with inhibition rates reaching 69.67 %, 42.97 %, and 65.74 % at 100 μg/mL. The mortality rates of O. macrolepis and bacterial load in liver were significantly decreased in the therapeutic group than that of infection group (p < 0.05). After the rOmhepc therapeutic, interleukin-1β (IL-1β) and interleukin-6 (IL-6) were significantly down-regulated with 14.4-fold and 106.07-fold at 24 h. Furthermore, the expression of immune-related genes (C3, TNF-α, IFN-γ) and Ferroportin gene (FPN1) significantly decreased (p < 0.05).The integrated analyses indicated that the rOmhepc could significantly inhibit the growth of A. hydrophila both in vitro and in vivo, attenuating the over-expression of inflammatory factor, FPN1 and immune-related genes.

Stimulatory effects of seaweed Laminaria digitata polysaccharides additives on growth, immune-antioxidant potency and related genes induction in Rohu carp (Labeo rohita) during Flavobacterium columnare infection

Stimulatory effects of dietary supplementation of large brown alga, Laminaria digitata polysaccharides (LDPs) at doses of 0, 100, 200, and 300 mg/kg diets were assessed on growth, hemato-biochemical, and antioxidant-immune variables as well as related genes expression in Indian rohu carp (Labeo rohita) during Flavobacterium columnare infection. Fish growth indices were significantly (P < 0.05) influenced by dietary LDPs and bacterial infection but not their interaction. However, growth indices of rohu carp in normal unchallenged (NuC) increased pointedly at treatments of 200 or 300 mg/kg diets with no significant difference between them. In normal challenged bacterial (NbC) groups, highest fish growth was observed at the treatment of 300 mg LDPs/kg diets. Feed intake and FCR values were significantly (P < 0.05) affected by dietary LDPs, bacterial infection, and their interaction. The highest feed intake accompanied with lowest FCR value was observed in the treatment of 200 mg LDPs/kg diets. No mortality was seen in NuC groups but lowest fish survival was observed in NbC groups fed with 0.0 and 100 mg LDPs/kg diets. Highest values of white blood cell, total protein, albumin, and globulin found in NuC fish groups administrated with 200 or 300 mg/kg diets with no differences between them. Feeding NbC fish groups on 300 mg LDPs/kg diet recovered the adverse effects of bacterial infection. It is noted that levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and reduced glutathione (GSH) were significantly (P < 0.05) affected by dietary LDPs, bacterial infection, and their interaction. The values of antioxidant indices were significantly (P < 0.05) higher in NuC fish groups received 200 or 300 mg LDPs/kg diets with no significant (P > 0.05) differences between them. In NbC fish groups, values of antioxidant indices were significantly (P < 0.05) lower that those in NuC fish groups. Malondialdehyde (MDA) was significantly influenced by dietary LDPs alone and its lowest values were observed in both NuC and NbC fish groups fed with 200 or 300 mg/kg diets with no differences between them. Phagocytic activity, alternate complement pathway, lysozyme, respiratory burst, peroxidase, complement C3, and immunoglobulin M values improved significantly (P < 0.05) in both NuC and NbC fish groups fed with 200 or 300 mg/kg diets with no differences between them. The mRNA manifestation of hepatic SOD, CAT, and GPx genes were significantly (P < 0.05) upregulated in both NuC fish groups fed on 200 and 300 mg LDPs/kg diets but feeding NbC fish on 300 mg LDPs/kg diets enhanced their mRNA expression. The HSP70, HSP90, TNF-α, IL-1β, and IL-8 mRNA expression in the NuC fish groups showed no significant (P > 0.05) differences due to dietary supplementation of LDPs to ruho carp, but their expressions were significantly (P < 0.05) higher in NbC fish groups; particularly at the treatment of 100 mg LDPs/kg diets. The mRNA expression of IL-10, IgM, hepcidin, and Mx1 genes were significantly (P < 0.05) upregulated in NuC fish group fed with 200 or 300 mg LDPs/kg diets. The bacterial infection downregulated their expressions but the dietary LDPs recovered the adverse effects of bacterial infection especially at 200 or 300 mg LDPs/kg diets. These results shown that the use of LDPs (200 or 300 mg/kg diets) has a valuable role in enhancing the growth, antioxidant-immune status, and inflammatory responses in rohu carp during F. columnare infection.

CREB-H is a stress-regulator of hepcidin gene expression during early postnatal development.

Hepcidin, the hepatic iron hormone, is the central regulator of iron homeostasis. Cyclic AMP-Responsive Element-Binding protein 3-like 3 (CREB3L3/CREB-H) is a liver homeostatic regulator of essential nutrients (i.e. glucose and lipids) and has been previously involved in hepcidin response to pathologic stress signals. Here, we asked whether CREB-H has also a physiologic role in iron homeostasis through hepcidin. To this end, we analyzed hepcidin gene expression and regulation in the liver of wild type and Creb3l3 knockout mice during early postnatal development, as a model of "physiologic" stressful condition. The effect of iron challenge in vivo and BMP6 stimulation in vitro have been also addressed. In addition, we investigated the BMP signaling pathway and hepcidin promoter activity following CREB3L3 silencing and hepcidin promoter mutation in HepG2 cells. Creb3l3 knockout suckling and young-adult mice showed a prominent serum and hepatic iron accumulation, respectively, due to impaired hepcidin mRNA expression which progressively returned to normal level in adult mice. Interestingly, upon iron challenge, while the upstream BMP/SMAD signaling pathway controlling hepcidin was equally responsive in both strains, hepcidin gene expression was impaired in knockout mice and more iron accumulated in the liver. Accordingly, hepcidin gene response to BMP6 was blunted in primary CREB-H knockout hepatocytes and in HepG2 cells transfected with CREB-H siRNA or carrying a hepcidin promoter mutated in the CREB-H binding site. In conclusion, CREB-H has a role in maintaining the homeostatic balance of iron traffic through hepcidin during the critical postnatal period and in response to iron challenge. KEY MESSAGES: CREB-H KO mice develop liver iron overload shortly after weaning that normalizes in adulthood. CHEB-H is involved in hepcidin gene response to oral iron in vivo. CREB-H loss hampers hepcidin promoter response to BMP6. CREB-H is a key stress-sensor controlling hepcidin gene transcription in physiologic and pathophysiologic states.

Structure-based discovery of pyrazolamides as novel ERRγ inverse agonists

Estrogen-related receptor-gamma (ERRγ) is an orphan nuclear receptor with high structural similarity to estrogen receptors (ERα and β). The endogenous ligand of the receptor has yet to be identified. Only two classes of molecules—stilbene (diethylstilbestrol, 4-hydroxytamoxifen, and GSK5182) and flavonol (kaempferol) have been known to modulate the transcriptional activity of the receptor to date. Further, these agents lack selectivity to ERRγ suggesting the need for a new inverse agonist. Thus, virtual screening was used to identify pyrazolamide 7 as a novel ERRγ inverse agonist. Structure-based diversification and optimization of the compound further led to the identification of derivative 19 as a potent inverse agonist of ERRγ with selectivity over other nuclear receptors including those of ERR family. Pyrazolamide 19 exhibits strong affinity towards ERRγ and inhibits the expression of hepcidin, fibrinogen and gluconeogenic genes, which suggests that these compounds may have antimicrobial, anti-coagulant and antidiabetic activities.

REDD1 deletion and treadmill running increase liver hepcidin and gluconeogenic enzymes in male mice.

The iron-regulatory hormone hepcidin is transcriptionally up-regulated by gluconeogenic signals. Recent evidence suggeststhat increases in circulating hepcidin may decrease dietary iron absorption following prolonged exercise, however evidence is limited on whether gluconeogenic signals contribute to post-exercise increases in hepcidin. Mice with genetic knockout of regulated in development and DNA response-1 (REDD1) display greater glycogen depletion following exercise, possibly indicating greater gluconeogenesis. The objective of the present study was to determine liver hepcidin, markers of gluconeogenesis and iron metabolism in REDD1 knockout and wild-type mice following prolonged exercise. Twelve-week-old male REDD1 knockout and wild-type mice were randomised to rest or 60 min treadmill running with 1, 3 or 6 h recovery (n = 5-8/genotype/group). Liver gene expression of hepcidin (Hamp) and gluconeogenic enzymes (Ppargc1a, Creb3l3, Pck1, Pygl) were determined by qRT-PCR. Effects of genotype, exercise and their interaction were assessed by two-way ANOVAs with Tukey's post-hoc tests, and Pearson correlations were used to assess the relationships between Hamp and study outcomes. Liver Hamp increased 1- and 4-fold at 3 and 6 h post-exercise, compared to rest (P-adjusted < 0⋅009 for all), and was 50% greater in REDD1 knockout compared to wild-type mice (P = 0⋅0015). Liver Ppargc1a, Creb3l3 and Pck1 increased with treadmill running (P < 0⋅0001 for all), and liver Ppargc1a, Pck1 and Pygl were greater with REDD1 deletion (P < 0⋅02 for all). Liver Hamp was positively correlated with liver Creb3l3 (R = 0⋅62, P < 0⋅0001) and Pck1 (R = 0⋅44, P = 0⋅0014). In conclusion, REDD1 deletion and prolonged treadmill running increased liver Hamp and gluconeogenic regulators of Hamp, suggesting gluconeogenic signalling of hepcidin with prolonged exercise.

PECTIN MODULATES HEMATO-BIOCHEMICAL PARAMETER, HISTOPATHOLOGY, OXIDATIVE STRESS BIOMARKERS, CYTOKINES AND EXPRESSION OF HEPCIDN GENE IN LEAD INTOXICATED RATS

Publications concerning the protective effect of pectin against lead induced toxicity in rats are not available. In order to study such effect, 40 male rats were divided into 3 groups. The first group was contained 10 rats that kept as control group. The second group was contained 10 rats that received pectin at dose of 100 mg/kg BW during experimental period (8 weeks). The third group was contained 20 rats that received 400mg/kg BW of lead acetate daily for 4 weeks then divided into two subgroups (3A and 3B). Subgroup 3A contained 10 rats that still receive lead acetate in the same dosage whereas, subgroup B co-treated with lead acetate and pectin daily for another 4 weeks. Blood samples were collected after 2, 4 and 8 weeks from the start of the experiment. Liver, kidney and bone marrows were collected only at the end of the experiment. Lead acetate induced anemia only after 4 weeks of administration as reflected on decreased values of Hb, PCV, MCV, MCH and MCHC. These indices remained at lower levels in lead acetate treated groups until the end of the experiment. Concentrations of serum ferritin, iron, total antioxidant capacity (TAC) and reduced glutathione (GSH) and the expression of hepatic hepcidin gene were decreased significantly in lead acetate intoxicated rats compared to control. Activities of ALT and AST and concentrations of urea, creatinine, Nitric oxide (NO), TNF-α, IL-6, total iron binding capacity (TIBC) and lead were increased significantly in lead acetate intoxicated group compared to control. Hepatic degeneration and hemorrhage, renal lytic necrosis and apoptosis of myeloid cells were most prominent changes in lead intoxicated rats. Lead acetated related changes were improved by co-treatment with pectin however; normal control values have not been achieved. Conclusively, pectin is recommended to protect against lead acetate toxicity in rats. Key words: lead acetate; toxicity; pectin; hepcidin; oxidative stress biomarkers; histopathology

Immunometabolic involvement of hepcidin genes in iron homeostasis, storage, and regulation in gilthead seabream (Sparus aurata)

Iron is essential for all living things, especially marine organisms, due to its low availability in the marine environment. Iron regulation is key in all vertebrates and is controlled by hepcidin–ferroportin. To improve the knowledge of iron homeostasis in fish, an iron overload was induced in gilthead seabream (Sparus aurata), which was chosen as a study species because of its high interest in Mediterranean aquaculture. The amount of iron in different tissues was measured to determine its biodistribution and/or bioaccumulation. Since the liver is directly involved in iron metabolism, the morphological changes induced in this organ as a consequence of the iron increase were studied. The bactericidal activity of fish skin mucus was also determined, observing that it decreased in fish with high iron levels compared to control fish. In addition, to better understand iron regulation, the gene expression of hepcidin, ferroportin, transferrin, and ferritin was evaluated in the head kidney (the main hematopoietic organ in this species) and in the liver. Special interest was taken in the study of the multiple copies of the hamp2 gene present in the gilthead seabream genome. Bioinformatic analysis of the protein sequences derived from these hepcidin genes allowed us to determine the presence of one type I hepcidin and 12 type II hepcidins, all of them with antimicrobial potential. This number of mature hepcidin sequences found in gilthead seabream is the highest within Eupercaria described to date. All the results obtained indicate that the modulation of iron in seabream seems to be much more complicated than in other vertebrates, probably due to the possible involvement of the different hepcidins as mediators between iron metabolism and host immune response.

DISC-0974, an Anti-Hemojuvelin (HJV) Monoclonal Antibody, Reduced Hepcidin and Improved Anemia in a Rat Model of Chronic Kidney Disease

Introduction DISC-0974 is a humanized monoclonal antibody against hemojuvelin (HJV) and is currently in Phase I clinical studies. HJV is a pathway-specific coreceptor for bone morphogenetic protein (BMP) signaling that regulates hepcidin expression and iron metabolism. Loss of function mutations of the HFE2 gene encoding HJV in humans cause profound reductions in hepcidin synthesis and severe iron overload. Consequently, it is hypothesized that pharmacological reduction of HJV activity will lead to reduction of hepcidin synthesis and increased iron availability. This hypothesis has been confirmed in studies of DISC-0974 in rodents and non-human primates and in healthy human volunteers. We have conducted studies to evaluate the effect of DISC-0974 in an animal model of CKD anemia. Anemia is a common complication in patients with CKD and has been associated with multiple adverse outcomes in this population. In CKD hepcidin is increased because of both reduced renal clearance and increased synthesis. This increase is believed to be a central contributor to the development of anemia by reducing the availability of iron from systemic iron stores and by reducing dietary iron absorption. Supporting this hypothesis, treatment with hepcidin lowering drugs has been demonstrated to increase serum iron and to increase hemoglobin (Sheetz et al, Br J Clin Pharmacol. 2019;85:935-948). We hypothesize that by downregulating hepcidin, DISC-0974 will have beneficial effects in treating anemia in the CKD patients because it will make iron available for improved erythropoiesis and increased hemoglobin synthesis as well as reducing erythropoietin resistance. Material and Methods The effect of DISC-0974 on improving anemia was evaluated in an adenine-induced rat model of CKD. In this study, renal impairment was induced by giving Sprague Dawley rats a diet containing 0.75% adenine for 3 weeks to induce kidney injury and then switched to a control diet for the remainder of the study (i.e., 3 more weeks). DISC-0974 (20 mg/kg) or vehicle (n-5/group) was administered intravenously once per week starting on Day 7 and continuing to Day 35. An additional group of rats (n=5/group) was fed with the control diet for the entire study and dosed intravenously with vehicle. Rats were euthanized on Day 42. Blood was collected on Days 0, 7, 21, 35, and 42 for evaluation of hematology parameters, as well as serum iron and hepcidin. Livers were also collected at study end for evaluation of hepcidin gene (HAMP) mRNA expression level. Results Consistent with a previous report (Sun et al, Nephrol Dial Transplant. 2013;28:1733-1743), the 0.75% adenine diet induced kidney dysfunction, as evidenced by marked increase in both serum creatinine and blood urea nitrogen. Rats on adenine diet developed reduced serum iron, reduced red cell production, and lowered hemoglobin with a hypochromic microcytic profile reflecting reduced iron availability. CKD rats appeared to tolerate DISC-0974 treatment well, as DISC-0974 @ 20mg/kg did not affect body weight. Consistent with the mechanism of action of blocking the formation of the BMP/BMPR/HJV complex, DISC-0974 reduced HAMP gene expression in the liver as measured at the end the study, which led to reduced serum hepcidin and increased serum iron concentration. Cellular hemoglobinization measured as reticulocyte Hemoglobin was increased. The maximum improvement in hemoglobin by DISC-0974 compared to the vehicle group was 17g/L in the rat CKD model. Conclusions We conclude that treatment with DISC-0974 at a dose that reduces hepcidin gene expression is able to increase iron availability and substantially prevents the reduction in hemoglobin that is seen in animals that develop adenine-induced renal impairment. This study supports the development of DISC-0974 for the treatment of patients with CKD anemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genetic regulation of iron homeostasis in sideropenic patients with mild COVID-19 disease under a new oral iron formulation: Lessons from a different perspective

BackgroundSevere Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) needs iron to replicate itself. Coronaviruses are able to upregulate Chop/Gadd153 and Arg1 genes, consequently leading to CD8 lymphocytes decrease, degradation of asparagine and decreased nitric oxide (NO), thus impairing immune response and antithrombotic functions. Little is known about regulation of genes involved in iron metabolism in paucisymptomatic patients with COVID-19 disease or in patients with iron deficiency treated with sucrosomial iron. MethodsWhole blood was taken from the COVID-19 patients and from patients with sideropenic anemia, treated or not (control group) with iron supplementations. Enrolled patients were: affected by COVID19 under sucrosomal iron support (group A), affected by COVID-19 not under oral iron support (group B), iron deficiency not under treatment, not affected by COVID19 (control group). After RNA extraction and complementary DNA (cDNA) synthesis of Arg1, Hepcidin and Chop/Gadd153, gene expression from the 3 groups was measured by qRT-PCR. M2 macrophages were detected by cytofluorimetry using CD163 and CD14 markers. ResultsForty patients with COVID-19 (group A), 20 patients with iron deficiency treated with sucrosomial iron (group B) and 20 patients with iron deficiency not under treatment (control group) were enrolled. In all the patients supported with oral sucrosomial iron, the gene expression of Chop, Arg1 and Hepcidin genes was lower than in sideropenic patients not supported with iron, M1 macrophages polarization and functional iron deficiency was also lower in group A and B, than observed in the control group. ConclusionsNew oral iron formulations, as sucrosomial iron, are able to influence the expression of genes like Chop and Arg1 and to influence M2 macrophage polarization mainly in the early phase of COVID-19 disease.

Evaluate Iron Parameters Change and Gene Expression of Hepcidin and Ferritin in Pre- and Post- Menopausal Iraqi Women with Type 2 Diabetes Mellitus

Evaluate Iron Parameters Change and Gene Expression of Hepcidin and Ferritin in Pre- and Post- Menopausal Iraqi Women with Type 2 Diabetes Mellitus

Massive gene expansion of hepcidin, a host defense peptide, in gilthead seabream (Sparus aurata)

Host defense peptides (HDP) are among the most ancient immune molecules in animals and clearly reflect an ancestral evolutionary history involving pathogen-host interactions. Hepcidins are a very widespread family of HDPs among vertebrates and are especially diverse in teleosts. We have investigated the identification of new hepcidins in gilthead seabream (Sparus aurata), a fish farmed in the Mediterranean. Targeted gene predictions supported with expressed sequence tags (ESTs) derived from Hidden Markov Models were used to find the hamp genes in the seabream genome. The results revealed a massively clustered hamp duplication on chromosome 17. In fact, the seabream genome contains the largest number of hepcidin copies described in any vertebrate. The evolutionary history of hepcidins in seabream, and vertebrates generally, clearly indicates high adaptation in teleosts and novel subgroups within hepcidin type II. Furthermore, basal hepcidin gene expression analysis indicates specific-tissue expression profiles, while the presence and distribution of transcription factor binding sites (TFBS) in hamp promoters as well as their transcription profile upon bacterial challenge indicates different immune roles depending on the type of hepcidin and tissue. This massive duplication of HDP genes in a bony fish could point to a far more specific and adaptive innate immune system than assumed in the classic concept of immunity in mammals. Hence, a new world of knowledge regarding hepcidins in fish and vertebrates is being initiated.

Melatonin Regulates Iron Homeostasis by Inducing Hepcidin Expression in Hepatocytes.

The pineal hormone, melatonin, plays important roles in circadian rhythms and energy metabolism. The hepatic peptide hormone, hepcidin, regulates iron homeostasis by triggering the degradation of ferroportin (FPN), the protein that transfers cellular iron to the blood. However, the role of melatonin in the transcriptional regulation of hepcidin is largely unknown. Here, we showed that melatonin upregulates hepcidin gene expression by enhancing the melatonin receptor 1 (MT1)-mediated c-Jun N-terminal kinase (JNK) activation in hepatocytes. Interestingly, hepcidin gene expression was increased during the dark cycle in the liver of mice, whereas serum iron levels decreased following hepcidin expression. In addition, melatonin significantly induced hepcidin gene expression and secretion, as well as the subsequent FPN degradation in hepatocytes, which resulted in cellular iron accumulation. Melatonin-induced hepcidin expression was significantly decreased by the melatonin receptor antagonist, luzindole, and by the knockdown of MT1. Moreover, melatonin activated JNK signaling and upregulated hepcidin expression, both of which were significantly decreased by SP600125, a specific JNK inhibitor. Chromatin immunoprecipitation analysis showed that luzindole significantly blocked melatonin-induced c-Jun binding to the hepcidin promoter. Finally, melatonin induced hepcidin expression and secretion by activating the JNK-c-Jun pathway in mice, which were reversed by the luzindole treatment. These findings reveal a previously unrecognized role of melatonin in the circadian regulation of hepcidin expression and iron homeostasis.