Vitamin B4 (Adenine) Alleviates Iron Overload By CAMP/PKA Mediated Hepcidin Upregulation In Mice

BackgroundIron is an essential trace element that participates in forming oxygen-transporting storage molecules,such as hemoglobin and myoglobin,and plays roles in many physiological processes.Iron deficiency causes anemia,while iron overload leads to hemochromatosis,the later characterized by excess iron induced tissues damage and fibrosis.Hepcidin,a small peptide hormone synthesized and excreted by the liver,has recently been identified as a master regulator of iron homeostasis.In the human body,hepcidin binds to ferroportin(FPN),the mammalian iron exporter,leads to its internalization and degradation,and subsequently inhibits intestinal iron absorption,liver iron mobilization and macrophage iron recycling.Hepcidin is up-regulated in response to inflammation or iron overload,and is down-regulated in response to increased erythropoiesis,iron deficiency,or hypoxia.Consequently,insufficient production of hepcidin is associated with many iron overload disorders,such as hereditary hemochromatosis and thalassemia.Therefore,hepcidin has been proposed as an attractive target to treat iron overload disorders.For example,a hepcidin derivative,mini-hepcidin was found to ameliorate iron over load phenotype in Hbbth3/+ mice(a classic ?-thalassemia model),Hepcidin is mainly regulated by BMP/SMAD pathways.HFE,HJV(Hemojuvelin),TFR2(Transferrin receptor 2),HAMP(hepcidin),and FPN gene mutations in BMP/SMAD and HAMP/FPN pathways resulted in human hereditary hemochromatosis.In addition,hepcidin expression is also regulated by cytokine IL-6 activated phosphorylation of JAK2/STAT3.Notably,recent studies have linked vitamins A,D,E,and C to iron metabolism that is potentially controlled by hepatic hepcidin,a critical regulator of iron homeostasis.Rats on a vitamin A deficient diet treated exhibited decreased expression of hepatic hepcidin as well as serum iron and increased spleen iron content.Treatment of cultured hepatocytes vitamin D decreased expression of hepcidin mRNA by 0.5-fold.In a pilot study,this response was associated with a 34%decrease in circulating levels of hepcidin within 24 hours of vitamin D supplementation.Vitamin E abolished both the alcohol-mediated down regulation of hepcidin expression in the liver and the up-regulation of duodenal divalent metal transporter 1 in mice.Vitamin C was reported to inhibit hepcidin expression in HepG2 cells.These studies suggested a possible interplay between vitamins and iron metabolism,yet the molecular mechanism is still unclear.Objective1.To discover novel hepcidin regulators by systemic screening vitamins.2.To explore the molecular mechanisms of vitamin B4(adenine)increasing hepcidin.MATERIALS AND METHODSChemicalsAll vitamins and 8-Br-cAMP were purchased from Sigma Chemical Company.Recombinant human BMP6(No.507-BP-020)was purchased from R&D Systems.LDN193189,U0126 and H89 were purchased from Selleck Chemicals Company.Animals8-week-old.male C57BL/6 mice(SLRC Laboratory Animal Co.,Ltd.,Shanghai,China)were housed under SPF conditions with free access to AIN-76A standard mouse diet.The mice in the experimental groups were treated AIN-76A diet added.0.2%vitamin B4(adenine)after which the mice were euthanized under anesthesia(5%chloral hydrate,10 ml/kg body weight by i.p.injection).Whole blood was collected,and the liver and spleen were removed for further analysis.In another experiment,Hfe-/-mice were fed a diet containing 0.1%(w/w)vitamin B4(adenine)for up to 30 days and 60 days,then sacrificed and analyzed.For iron preloading,5-week-old wild type mice were fed a high iron diet for 10 days,and in the next 10 days,the control group was fed with high iron diet only,while the vitamin B4(adenine)-treated group was fed with high iron diet containing 0.2%(w/w)vitamin B4(adenine).Each experimental group contained 10 mice.All animal experiments were approved by the Institutional Animal Care and Use Committee of Zhejiang University.Western blot analysisLysates from cells and tissues dissolved in RIPA buffer containing a protease inhibitor cocktail(Roche Applied Science,Indianapolis,IS,USA)were quantified using the BCA protein assay kit(Pierce Biotechnology,Rockford MA,USA)as previously described.Blots were incubated with antibodies:rabbit anti-pSMAD1/5/8(1:1000;Cell Signaling Technology);rabbit anti-SMAD1(1:1000;Cell Signaling Technology);rabbit anti-pSTAT3(1:1000;Cell Signaling Technology);rabbit anti-STAT3(1:1000;Cell Signaling Technology);rabbit anti-pERKl/2(1:1000;Cell Signaling Technology);rabbit anti-ERKl/2(1:1000;Cell Signaling Technology);mouse anti-gapdh(1:2000;Sigma-Aldrich).Quantitative Real-time PCRQuantitative measurement of Hamp,Tfrl,BMP6 and Epo expression was carried out with 480 Real-time PCR(Roche)equipped with SDS software.HPRT was used as internal control.Analysis of iron parameters Serum iron,serum transferrin saturation and tissue non-heme iron content were measured.Statistical analysis Results were expressed as the mean ± standard error.Comparison between groups analyzed using the Student's t-test or one-way ANOVA with the Tukey post hoc test.Differences with P<0.05 were considered to be statistically significant.ResultsOur unbiased vitamin screen for hepcidin modulators identifies vitamin B4(adenine)as a potent hepcidin agonist.Further studies in both human cell lines and in mice validated the effect of vitamin B4(adenine)on hepcidin upregulation.Consistently,vitamin B4(adenine)dietary supplement(0.1%w/w)in mice led to an increase of hepatic hepcidin expression compared with normal diet-fed mice via BMP/SMAD and ERK1/2 pathways.Notably,vitamin B4(adenine)-rich diet(0.1%w/w)significantly ameliorated iron overload accompanied by the enhanced hepcidin expression in both high iron-fed mice and in Hfe-/-mice,the murine model of hereditary hemochromatosis.Moreover,we uncovered an essential role of cAMP/PKA-dependent axis in triggering vitamin B4(adenine)-induced hepcidin expression in primary hepatocytes.These findings suggest a potential therapeutic role of vitamin B4(adenine)for iron overload diseases.Conclusions1.Unbiased vitamin screen for hepcidin modulators identifies vitamin B4(adenine)as a potent hepcidin agonist.2.Vitamin B4(adenine)increased HAMP expression through cAMP/PKA-dependent BMP/SMAD1/5/8 and MEK/ERK1/2 pathways.3.Vitamin B4 attenuated hemochromatosis of Hfe-/-mice.