Ferroportin Q248H Mutation Leads to a Moderate Iron Load in Mutant Slc40a1^Q248H/Q248H Mice

BACKGROUND: In Africans, ferroportin (Scl40a1 or FPN) Q248H mutation is associated with increased iron load. FPN Q248H has high allele frequency (2.2-13.4%) and is linked to reduction of inflammation and reduced anemia in Africa. We previously showed that FPN Q248H resists degradation by physiologic hepcidin concentrations and thus may facilitate higher iron uptake.

OBJECTIVES: to analyze the effect of FPN Q248H mutation in vivo in a knock-in Slc40a1^Q248H mouse model on normal and high iron diet.

METHODS: The study was approved by Howard University IACUC committee. The Slc40a1^Q248H/WT mice were generated by Flp- FRT recombination on the C57BL/6 background and homozygous Slc40a1^Q248H/Q248H mutant mice were produced in Howard University animal facility by breeding. Twenty-three weeks old homozygous and wild type C57BL/6 (WT) females were fed normal or 2% carbonyl iron diet. Total iron concentration and ferritin were measured in serum by ELISA. Expression of FPN, transferrin receptor (TFR), ferritin heavy chain (FHC), divalent metal transporter 1 (DMT1), duodenal cytochrome B (Dcytb), hepcidin, and IL-1β, IL-6, and IL-18 cytokines were measured by real-time RT-PCR. Iron deposition was determined by Perl's staining. Hepatic oxidative stress was measured by malondialdehyde ELISA.

RESULTS: On normal iron diet, Slc40a1^Q248H/Q248H mice grew normally and showed a tendency to increased serum ferritin, decreased serum iron and reduced hematocrit compared to WT mice. Levels of FPN,Dcytb and DMT1 mRNA were increased in duodenum of mutant mice. In the spleen of Slc40a1^Q248H/Q248H mice, FPN,FTH1, TFR and DMT1 mRNAs were also elevated. In contrast, in the liver of Slc40a1^Q248H/Q248H mice, levels of FPN and TFR mRNA were decreased, but hepcidin levels were increased compared to WT mice. There was a trend towards hepatic FTH1 level increase in the mutant mice. Hepatic expression of pro-inflammatory IL-1β, IL-6, and IL-18 cytokines mRNA was similar between mutant and WT mice. No iron accumulation was found in the liver, and iron accumulation in the spleen was comparable between WT and Slc40a1^Q248H/Q248H mice.

On high iron diet, Slc40a1^Q248H/Q248H mice showed significant increase in serum ferritin compared to WT mice on normal diet. Levels of FPN mRNA remained increased whereas Dcytb and DMT1 mRNA levels were decreased in duodenum of the mutant mice. In the liver, FPN mRNA levels were further decreased in the mutant mice, and FTH1 levels were equally increased in WT and Slc40a1^Q248H/Q248H mice. In the spleen, FPN mRNA levels were equally increased for WT and Slc40a1^Q248H/Q248H mice. Spleen FTH1 mRNA levels were further elevated in mutant mice compared to WT mice. Higher iron accumulation was found in the liver and spleen of Slc40a1^Q248H/Q248H mice compared to WT mice. Elevated levels of hepatic oxidative stress were observed in the liver of Slc40a1^Q248H/Q248H mice. Expression of pro-inflammatory IL-1β, IL-6, and IL-18 cytokines mRNA remained low in the liver of Slc40a1^Q248H/Q248H mice.

CONCLUSION: Upregulation of duodenal FPN, Dcytb and DMT1 and splenic FPN,FTH1, TFR and DMT1 expression indicated increased iron absorption and recycling in female Slc40a1^Q248H/Q248H mice. The mutantmice showed tendency to mild iron load in tissues as indicated by increased serum ferritin and higher ferritin levels in the spleen and liver. High iron diet led to significant iron accumulation in the liver and increased hepatic stress compared to WT mice. Surprisingly, hepatic levels of pro-inflammatory cytokines were reduced in the mutant mice. Taken together, our study of the first in vivo model of FPNQ248H mutation indicates that it alters iron metabolism and might reduce liver inflammation.

ACKNOWLEDGEMENTS: This work was supported by NIH grants 1R01HL125005, 5U54MD007597, 1P30AI117970-06 and 1SC1HL150685.

Alhakami:Jazan University, Saudi Arabia: Other: Scholarship awardee.