Tissue-Specific Regulation of Iron Release in Response to Body Iron Status Under Erythropoietic Stimulation

Introduction:

In a previous study, we demonstrated that dietary iron uptake and mobilization of stored iron are both up-regulated through suppression of serum hepcidin levels during erythropoietic stimulation by administration of Epoetin beta pegol (C.E.R.A.), a long-acting erythropoiesis-stimulating agent. It was also demonstrated that up-regulation of ferroportin (FPN) in reticuloendothelial macrophages and up-regulation of divalent metal transporter 1 (DMT1) and FPN in enterocytes are followed by hepcidin suppression; however, the quantitative contribution of dietary iron for erythropoiesis were undetermined.

In this study, we investigated how utilization of dietary iron for erythropoiesis is regulated under erythropoietic stimulation by C.E.R.A. in mice with different body iron status. To quantitatively estimate utilization of dietary iron for hemoglobin synthesis, we used a dietary iron tracing method using the stable iron isotope ⁵⁷Fe.

Methods:

To assess dietary iron-derived hemoglobin synthesis, a diet containing 200 ppm of ⁵⁷Fe instead of natural iron (⁵⁷Fe-diet) was used. A diet containing 200 ppm of natural iron (native Fe diet) was used as a control. C57BL/6NCrl mice were fed the native Fe diet and were intravenously administered 0.5 or 1.0 mg/mouse of iron dextran (iron-loaded condition) or dextran (control). Five days after iron loading, the diet was switched to the ⁵⁷Fe-diet immediately after intravenous injection of 10 µg/kg of C.E.R.A. or vehicle. On Day 5 and 8 after C.E.R.A. treatment, mice were euthanized by exsanguination under anesthesia with isoflurane, and hemoglobin levels were measured. Expression levels of DMT1 and FPN in control and iron-loaded mice (1.0 mg/mouse) on Day 5 were estimated by immunohistochemistry. Serum hepcidin levels on Day 5 were also measured by liquid column chromatography-tandem mass spectrometry (LC-MS/MS). To quantify dietary iron-derived hemoglobin synthesis, the content of hemoglobin containing ⁵⁷Fe (⁵⁷Fe-hemoglobin) was measured on Day 8 by inductively coupled plasma mass spectrometry (ICP-MS).

Results:

Hemoglobin levels on Day 8 were significantly higher in the C.E.R.A.-treated groups than in the vehicle-treated groups for each iron conditions. In the C.E.R.A.-treated groups, although iron loading did not affect hemoglobin levels, ⁵⁷Fe-hemoglobin levels were significantly decreased with iron loading. The serum hepcidin levels were significantly suppressed in each of the C.E.R.A.-treated groups. However, iron loading increased serum hepcidin levels on Day 5 in both the vehicle- and C.E.R.A.-treated groups. The expression levels of hepatic and splenic iron exporter FPN were not significantly changed by iron loading in the C.E.R.A.-treated group. In contrast, the expression levels of intestinal iron transporters DMT1 and FPN were significantly reduced by iron loading in the C.E.R.A.-treated group.

Conclusion:

Iron loading reduced utilization of dietary iron for hemoglobin synthesis under erythropoietic stimulation by C.E.R.A. treatment. However, iron loading did not affect total hemoglobin levels, indicating that the contribution of dietary iron and stored iron for erythropoiesis is properly controlled in response to body iron status. This was attributed to the tissue-specific regulatory mechanisms of iron transporters in iron absorptive tissue (intestine) and iron storage tissue (liver and spleen) in response to iron loading even FPN on both tissues is known to be commonly down-regulated by hepcidin-binding. Sensitive inactivation of iron importers and exporters in the duodenum under conditions of iron loading may effectively contribute to iron not being excessively incorporated under erythropoietic stimulation.