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. 2010 Apr;140(4):723-30.
doi: 10.3945/jn.109.117077. Epub 2010 Feb 17.

Anemic Copper-Deficient Rats, but Not Mice, Display Low Hepcidin Expression and High Ferroportin Levels

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Anemic Copper-Deficient Rats, but Not Mice, Display Low Hepcidin Expression and High Ferroportin Levels

Supak Jenkitkasemwong et al. J Nutr. .
Free PMC article

Abstract

The transmembrane protein ferroportin (Fpn) is essential for iron efflux from the liver, spleen, and duodenum. Fpn is regulated predominantly by the circulating iron regulatory hormone hepcidin, which binds to cell surface Fpn, initiating its degradation. Accordingly, when hepcidin concentrations decrease, Fpn levels increase. A previous study found that Fpn levels were not elevated in copper-deficient (CuD) mice that had anemia, a condition normally associated with dramatic reductions in hepcidin. Lack of change in Fpn levels may be because CuD mice do not display reduced concentrations of plasma iron (holotransferrin), a modulator of hepcidin expression. Here, we examined Fpn protein levels and hepcidin expression in CuD rats, which exhibit reduced plasma iron concentrations along with anemia. We also examined hepcidin expression in anemic CuD mice with normal plasma iron levels. We found that CuD rats had higher liver and spleen Fpn levels and markedly lower hepatic hepcidin mRNA expression than did copper-adequate (CuA) rats. In contrast, hepcidin levels did not differ between CuD and CuA mice. To examine potential mediators of the reduced hepcidin expression in CuD rats, we measured levels of hepatic transferrin receptor 2 (TfR2), a putative iron sensor that links holotransferrin to hepcidin production, and transcript abundance of bone morphogenic protein 6 (BMP6), a key endogenous positive regulator of hepcidin production. Diminished hepcidin expression in CuD rats was associated with lower levels of TfR2, but not BMP6. Our data suggest that holotransferrin and TfR2, rather than anemia or BMP6, are signals for hepcidin synthesis during copper deficiency.

Figures

FIGURE 1
FIGURE 1
Hemoglobin (A) and plasma iron (B) concentrations in CuA and CuD rats and mice on P49 in the postweaning rat model, P25 in the perinatal rat model, and P20 in the perinatal mouse model. Values represent means ± SEM, n = 4–6. *Different from respective CuA group, P < 0.05.
FIGURE 2
FIGURE 2
Hepatic Fpn protein (A,B) and hepcidin mRNA (C,D) levels in postweaning (A,C) and perinatal (B,D) CuA and CuD rats. To indicate protein loading in A and B, each blot was stripped and reprobed for SR-B1, an integral membrane protein. Values below Western blots indicate relative intensities of Fpn-immunoreactive bands. Relative hepcidin mRNA levels in postweaning (C) and perinatal (D) copper deficiency. Values represent means ± SEM, n = 4. *Different from respective CuA group, P < 0.05.
FIGURE 3
FIGURE 3
Splenic Fpn protein levels in postweaning CuA and CuD rats. Western blot analysis of splenic Fpn in postweaning (A) and perinatal (B) rats. To indicate protein loading, the blot was stripped and reprobed for SR-B1. Values below Western blots (A,B) indicate relative intensities of Fpn-immunoreactive bands. Values represent mean ± SEM, n = 4. *Different from respective CuA group, P < 0.05.
FIGURE 4
FIGURE 4
Fpn protein and hepcidin mRNA levels in CuA and CuD mice. Western blot analysis of hepatic Fpn (A) and splenic Fpn (B) in perinatal mice. To indicate protein loading, the blot was stripped and reprobed for SR-B1. (C) Hepatic hepcidin mRNA abundance. Values represent mean ± SEM, n = 4.
FIGURE 5
FIGURE 5
Hepatic TfR2 protein and BMP6 mRNA levels in CuA and CuD rats. Western blot analysis of liver TfR2 in postweaning (A) and perinatal (B) copper deficiency. To indicate protein loading, the blot was stripped and reprobed for SR-B1. Values below Western blots indicate relative intensities of TfR2-immunoreactive bands. Relative BMP6 mRNA levels in postweaning (C) and perinatal (D) copper deficiency. Values represent mean ± SEM, n = 4. *Different from respective CuA group, P < 0.05.
FIGURE 6
FIGURE 6
Effect of CuD on hepcidin regulation in rats and mice. In rats, CuD decreases Cp ferroxidase activity, which likely reduces plasma iron concentrations. Low plasma iron levels limit iron supply to the bone marrow, resulting in anemia, a suppressor of hepcidin expression. Diminished plasma iron concentrations are associated with lower levels of hepatic TfR2, a putative body iron sensor that modulates hepcidin expression according to plasma iron concentrations. Although CuD decreases Cp activity in mice, plasma iron levels are normal, possibly due to additional plasma ferroxidases. The anemia in CuD does not appear to result from limited iron supply to the bone marrow but rather to a defect in bone marrow iron utilization. Yet despite the anemia, CuD mice have normal levels of hepcidin, possibly related to normal plasma iron levels. The normal levels of hepcidin in anemic, CuD mice likely accounts for the normal Fpn levels in these animals. The question marks identify gaps in our understanding of these relationships.

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