Eriza S. Secondes

Normal systemic iron homeostasis in mice with macrophage-specific deletion of transferrin receptor 2

Iron is an essential element, since it is a component of many macromolecules involved in diverse physiological and cellular functions, including oxygen transport, cellular growth, and metabolism. Systemic iron homeostasis is predominantly regulated by the liver through the iron regulatory hormone hepcidin. Hepcidin expression is itself regulated by a number of proteins, including transferrin receptor 2 (TFR2). TFR2 has been shown to be expressed in the liver, bone marrow, macrophages, and peripheral blood mononuclear cells. Studies from our laboratory have shown that mice with a hepatocyte-specific deletion of Tfr2 recapitulate the hemochromatosis phenotype of the global Tfr2 knockout mice, suggesting that the hepatic expression of TFR2 is important in systemic iron homeostasis. It is unclear how TFR2 in macrophages contributes to the regulation of iron metabolism. We examined the role of TFR2 in macrophages by analysis of transgenic mice lacking Tfr2 in macrophages by crossing Tfr2(f/f) mice with LysM-Cre mice. Mice were fed an iron-rich diet or injected with lipopolysaccharide to examine the role of macrophage Tfr2 in iron- or inflammation-mediated regulation of hepcidin. Body iron homeostasis was unaffected in the knockout mice, suggesting that macrophage TFR2 is not required for the regulation of systemic iron metabolism. However, peritoneal macrophages of knockout mice had significantly lower levels of ferroportin mRNA and protein, suggesting that TFR2 may be involved in regulating ferroportin levels in macrophages. These studies further elucidate the role of TFR2 in the regulation of iron homeostasis and its role in regulation of ferroportin and thus macrophage iron homeostasis.

Ferroportin expression in adipocytes does not contribute to iron homeostasis or metabolic responses to a high calorie diet

Background & Aims Iron has an increasingly recognized role in the regulation of adipose tissue function, including the expression of adipokines involved in the pathogenesis of nonalcoholic fatty liver disease. The cellular iron exporter, ferroportin, has been proposed as being a key determinant of adipocyte iron homeostasis. Methods We studied an adipocyte-specific ferroportin (Fpn1) knockout mouse model, using an Adipoq-Cre recombinase driven Fpn1 deletion and fed mice according to the fast food diet model of nonalcoholic steatohepatitis. Results We showed successful selective deletion of Fpn1 in adipocytes, but found that this did not lead to increased adipocyte iron stores as measured by atomic absorption spectroscopy or histologically quantified iron granules after staining with 3,3’-diaminobenzidine–enhanced Perls’ stain. Mice with adipocyte-specific Fpn1 deletion did not show dysregulation of adiponectin, leptin, resistin, or retinol-binding protein-4 expression. Similarly, adipocyte-specific Fpn1 deletion did not affect insulin sensitivity during hyperinsulinemic–euglycemic clamp studies or lead to histologic evidence of increased liver injury. We have shown, however, that the fast food diet model of nonalcoholic steatohepatitis generates an increase in adipose tissue macrophage infiltration with crown-like structures, as seen in human beings, further validating the utility of this model. Conclusions Ferroportin may not be a key determinant of adipocyte iron homeostasis in this knockout model. Further studies are needed to determine the mechanisms of iron metabolism in adipocytes and adipose tissue.

Evaluation of a bone morphogenetic protein 6 variant as a cause of iron loading

BackgroundAtypical iron overload without variation in the five clinically associated hereditary hemochromatosis genes is now recognized; however, their etiology remains unknown. Since the identification of iron overload in the bone morphogenetic protein 6 (Bmp6) knockout mouse, the search has been on for clinically pathogenic variants in the BMP6 gene. A recent report proposes that variants in the pro-peptide region of BMP6 are the underlying cause of several cases of iron overload. We performed targeted next-generation sequencing on three cases of atypical iron overload with Asian ethnicity and identified a p.Q118dup (aka p.E112indelEQ, p.Q115dup, p.Q118_L119insQ) variant in BMP6. The purpose of this study was to characterize the molecular function of the identified BMP6 variant. Molecular characterization by immunofluorescence microscopy and Western blotting of transfected cells, bioinformatics, and population analyses was performed.ResultsIn contrast to reports for other BMP6 pro-peptide variants in this region, our data indicates that this variant does not affect the function of the mature BMP6 protein.ConclusionsOur data suggest that assignment of disease causation in clinical cases of iron overload to pro-peptide variants in BMP6 should thus be treated with caution and requires biological characterization.