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Ferroportin (Fpn)-expressed at the plasma membrane of macrophages, enterocytes, and hepatocytes-mediates the transfer of cellular iron into the blood plasma. Under the control of the iron-regulatory hormone hepcidin, Fpn serves a critical role in systemic iron homeostasis. Although we have previously characterized human Fpn, a great deal of research in iron homeostasis and disorders uses mouse models. By way of example, the flatiron mouse, a model of classical ferroportin disease, bears the mutation H32R in Fpn and is characterized by systemic iron deficiency and macrophage iron retention. The flatiron mouse also appears to exhibit a manganese phenotype, raising the possibility that mouse Fpn serves a role in manganese metabolism. At odds with this observation, we have found that human Fpn does not transport manganese, so we considered the possibility that a species difference could explain this discrepancy. We tested the hypothesis that mouse but not human Fpn can transport manganese and performed a comparative analysis of mouse and human Fpn. We examined the functional properties of human Fpn, mouse Fpn, and mutant mouse Fpn by using radiotracer assays in RNA-injected Xenopus oocytes. We found that neither mouse nor human Fpn transports manganese. Mouse and human Fpn share identical properties with respect to substrate profile, calcium dependence, optimal pH, and hepcidin sensitivity. We have also demonstrated that Fpn is not an ATPase pump. Our findings validate the use of mouse models of ferroportin function in iron homeostasis and disease.

Current markers of iron deficiency (ID), such as ferritin and hemoglobin, have shortcomings, and hepcidin and erythroferrone (ERFE) could be of clinical relevance in relation to early assessment of ID. Here, we evaluate whether exposure to altitude-induced hypoxia (2,320 m) alone, or in combination with recombinant human erythropoietin (rHuEPO) treatment, affects hepcidin and ERFE levels before alterations in routine ID biomarkers and stress erythropoiesis manifest. Two interventions were completed, each comprising a 4-wk baseline, a 4-wk intervention at either sea level or altitude, and a 4-wk follow-up. Participants ( n = 39) were randomly assigned to 20 IU·kg body wt -1 rHuEPO or placebo injections every second day for 3 wk during the two intervention periods. Venous blood was collected weekly. Altitude increased ERFE ( P ≤ 0.001) with no changes in hepcidin or routine iron biomarkers, making ERFE of clinical relevance as an early marker of moderate hypoxia. rHuEPO treatment at sea level induced a similar pattern of changes in ERFE ( P < 0.05) and hepcidin levels ( P < 0.05), demonstrating the impact of accelerated erythropoiesis and not of other hypoxia-induced mechanisms. Compared with altitude alone, concurrent rHuEPO treatment and altitude exposure induced additive changes in hepcidin ( P < 0.05) and ERFE ( P ≤ 0.001) parallel with increases in hematocrit ( P < 0.001), demonstrating a relevant range of both hepcidin and ERFE. A poor but significant correlation between hepcidin and ERFE was found ( R 2 = 0.13, P < 0.001). The findings demonstrate that hepcidin and ERFE are more rapid biomarkers of changes in iron demands than routine iron markers. Finally, ERFE and hepcidin may be sensitive markers in an antidoping context.

Androgen deprivation therapy (ADT) is a mainstay of treatment for prostate cancer (PCa). As androgens stimulate erythropoiesis, ADT is associated with a reduction in hematocrit, which in turn contributes to fatigue and related morbidity. However, the mechanisms involved in ADT-induced reduction in erythropoiesis remain unclear. We conducted a 6-mo prospective cohort study and enrolled men with PCa about to undergo ADT (ADT-Group) and a control group of men who had previously undergone prostatectomy for localized PCa and were in remission (Non-ADT Group). All participants had normal testosterone levels at baseline. Fasting blood samples were collected at baseline, 12 wk, and 24 wk after initiation of ADT; samples were obtained at the same intervals from enrollment in the Non-ADT group. Blood count, iron studies, erythropoietin, erythroferrone, and hepcidin levels were measured. Seventy participants formed the analytical sample (31 ADT, 39 Non-ADT). ADT was associated with a significant reduction in erythrocyte count (estimated mean difference = -0.2×10 6 cells/µl, 95%CI = -0.3 to -0.1×10 6 cells/µl, P < 0.001), hematocrit (-1.9%, 95%CI = -2.7 to -1.1%, P < 0.001), and hemoglobin (-0.6 g/dl, 95%CI = -0.8 to -0.3 g/dl, P < 0.001). Serum hepcidin concentration increased in the ADT-group (18 ng/ml, P < 0.001); however, iron concentrations did not change (-1.1 µg/dl, P = 0.837). Ferritin levels increased in men on ADT (60 ng/ml, P < 0.001). Iron binding capacity, transferrin saturation, erythroferrone, and erythropoietin did not change. Nine men undergoing ADT developed new-onset anemia. In conclusion, reduced proliferation of marrow erythroid progenitors leads to ADT-induced reduction in erythropoiesis. Future studies should evaluate the role of selective androgen receptor modulators in the treatment of ADT-induced anemia.

Iron homeostasis is tightly regulated, and the peptide hormone hepcidin is considered to be a principal regulator of iron metabolism. Previous studies in a limited number of mouse strains found equivocal sex- and strain-dependent differences in mRNA and serum levels of hepcidin and reported conflicting data on the relationship between hepcidin ( Hamp1 ) mRNA levels and iron status. Our aim was to clarify the relationships between strain, sex, and hepcidin expression by examining multiple tissues and the effects of different dietary conditions in multiple inbred strains. Two studies were done: first, Hamp1 mRNA, liver iron, and plasma diferric transferrin levels were measured in 14 inbred strains on a control diet; and second, Hamp1 mRNA and plasma hepcidin levels in both sexes and iron levels in the heart, kidneys, liver, pancreas, and spleen in males were measured in nine inbred/recombinant inbred strains raised on an iron-sufficient or high-iron diet. Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). However, liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice fed iron-sufficient or high-iron diets, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males. We also measured plasma erythroferrone, performed RNA-sequencing analysis of liver samples from six inbred strains fed the iron-sufficient, low-iron, or high-iron diets, and explored differences in gene expression between the strains with the highest and lowest hepcidin levels. NEW & NOTEWORTHY Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). Liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males., (Copyright © 2017 the American Physiological Society.)

In the setting of normal kidney function, iron deficiency is associated with increased FGF23 production and cleavage, altering circulating FGF23 levels. Our objective was to determine how chronic kidney disease (CKD) and dietary iron intake affect FGF23 production and metabolism in wild-type (WT) and hepcidin knockout (HKO) mice. For 8 wk, the mice were fed diets that contained adenine (to induce CKD) or no adenine (control group), with either low-iron (4 ppm) or standard-iron (335 ppm) concentrations. The low-iron diet induced iron deficiency anemia in both the WT and HKO mice. Among the WT mice, in both the control and CKD groups, a low-iron compared with a standard-iron diet increased bone Fgf23 mRNA expression, C-terminal FGF23 (cFGF23) levels, and FGF23 cleavage as manifested by a lower percentage intact FGF23 (iFGF23). Independent of iron status, CKD was associated with inhibition of FGF23 cleavage. Similar results were observed in the HKO control and CKD groups. Dietary iron content was more influential on FGF23 parameters than the presence or absence of hepcidin. In the CKD mice (WT and HKO, total n = 42), independent of the effects of serum phosphate, iron deficiency was associated with increased FGF23 production but also greater cleavage, whereas worse kidney function was associated with increased FGF23 production but decreased cleavage. Therefore, in both the WT and HKO mouse models, dietary iron content and CKD affected FGF23 production and metabolism., (Copyright © 2016 the American Physiological Society.)

Iron homeostasis is achieved by regulating the intestinal absorption of the metal and its recycling by macrophages. Iron export from enterocytes or macrophages to blood plasma is thought to be mediated by ferroportin under the control of hepcidin. Although ferroportin was identified over a decade ago, little is understood about how it works. We expressed in Xenopus oocytes a human ferroportin-enhanced green fluorescent protein fusion protein and observed using confocal microscopy its exclusive plasma-membrane localization. As a first step in its characterization, we established an assay to detect functional expression of ferroportin by microinjecting oocytes with (55)Fe and measuring efflux. Ferroportin expression increased the first-order rate constants describing (55)Fe efflux up to 300-fold over control. Ferroportin-mediated (55)Fe efflux was saturable, temperature-dependent (activation energy, Ea ≈ 17 kcal/mol), maximal at extracellular pH ≈ 7.5, and inactivated at extracellular pH < 6.0. We estimated that ferroportin reacts with iron at its intracellular aspect with apparent affinity constant < 10(-7) M. Ferroportin expression also stimulated efflux of (65)Zn and (57)Co but not of (64)Cu, (109)Cd, or (54)Mn. Hepcidin treatment of oocytes inhibited efflux of (55)Fe, (65)Zn, and (57)Co. Whereas hepcidin administration in mice resulted in a marked hypoferremia within 4 h, we observed no effect on serum zinc levels in those same animals. We conclude that ferroportin is an iron-preferring cellular metal-efflux transporter with a narrow substrate profile that includes cobalt and zinc. Whereas hepcidin strongly regulated serum iron levels in the mouse, we found no evidence that ferroportin plays an important role in zinc homeostasis.

Hepcidin, a small peptide synthesized in the liver, controls extracellular iron by regulating its intestinal absorption, placental transport, recycling by macrophages, and release from stores. Hepcidin inhibits the cellular efflux of iron by binding to and inducing the degradation of ferroportin, the sole iron exporter in iron-transporting cells. In turn, hepcidin synthesis is increased by iron loading and decreased by anemia and hypoxia. Hepcidin is markedly induced during inflammation, trapping iron in macrophages, decreasing plasma iron concentrations, and contributing to the anemia of inflammation. Hepcidin deficiency due to the dysregulation of its synthesis causes most known forms of hemochromatosis.

Serum calcium levels are regulated by the action of parathyroid hormone (PTH). Major drivers of PTH hypersecretion and parathyroid cell proliferation are the hypocalcemia and hyperphosphatemia that develop in chronic kidney disease patients with secondary hyperparathyroidism (SHPT) as a result of low calcitriol levels and decreased kidney function. Increased PTH production in response to systemic hypocalcemia is mediated by the calcium-sensing receptor (CaR). Furthermore, as SHPT progresses, reduced expression of CaRs and vitamin D receptors (VDRs) in hyperplastic parathyroid glands may limit the ability of calcium and calcitriol to regulate PTH secretion. Current treatment for SHPT includes the administration of vitamin D sterols and phosphate binders. Treatment with vitamin D is initially effective, but efficacy often wanes with further disease progression. The actions of vitamin D sterols are undermined by reduced expression of VDRs in the parathyroid gland. Furthermore, the calcemic and phosphatemic actions of vitamin D mean that it has the potential to exacerbate abnormal mineral metabolism, resulting in the formation of vascular calcifications. Effective new treatments for SHPT that have a positive impact on mineral metabolism are clearly needed. Recent research shows that drugs that selectively target the CaR, calcimimetics, have the potential to meet these requirements.