Essential role of systemic iron mobilization and redistribution for adaptive thermogenesis through HIF2-α/hepcidin axis

Iron is an essential biometal, but is toxic if it exists in excess. Therefore, iron content is tightly regulated at cellular and systemic levels to meet metabolic demands but to avoid toxicity. We have recently reported that adaptive thermogenesis, a critical metabolic pathway to maintain whole-body energy homeostasis, is an irondemanding process for rapid biogenesis of mitochondria. However, little information is available on iron mobilization from storage sites to thermogenic fat. This study aimed to determine the iron-regulatory network that underlies beige adipogenesis. We hypothesized that thermogenic stimulus initiates the signaling interplay between adipocyte iron demands and systemic iron liberation, resulting in iron redistribution into beige fat. To test this hypothesis, we induced reversible activation of beige adipogenesis in C57BL/6 mice by administering a β3-adrenoreceptor agonist CL 316,243 (CL). Our results revealed that CL stimulation induced the iron-regulatory protein–mediated iron import into adipocytes, suppressed hepcidin transcription, and mobilized iron from the spleen. Mechanistically, CL stimulation induced an acute activation of hypoxia-inducible factor 2-α (HIF2-α), erythropoietin production, and splenic erythroid maturation, leading to hepcidin suppression. Disruption of systemic iron homeostasis by pharmacological HIF2-α inhibitor PT2385 or exogenous administration of hepcidin-25 significantly impaired beige fat development. Our findings suggest that securing iron availability via coordinated interplay between renal hypoxia and hepcidin down-regulation is a fundamental mechanism to activate adaptive thermogenesis. It also provides an insight into the effects of adaptive thermogenesis on systemic iron mobilization and redistribution. Includes supplemental materials

Plasma iron in WT and Hepc−/− mice treated with PBS or LPS.

<p>Error bars represent SD for n = 3 mice in each group. Statistical significance is indicated by * symbols (***p< 0.0005).</p

Ferroportin expression in duodenum, and spleen of WT and Hepc−/− mice treated with PBS or LPS.

<p>Relative changes in ferroportin mRNA levels were quantified by real-time qRT-PCR. Graphed are mean values that were obtained from mRNA expression analyses regarding cyclophilin in the duodenum (duo), top panel, and the spleen, lower panel. Results are expressed relative to control PBS treated WT mice. Western Blot of ferroportin on membrane extracts were performed with 5 µg/lane for the duodenum and 20 µg/lane for the spleen. Expression was normalized to beta actin and quantified using Image J. Quantification of the blots are presented in % (100% set for Hepc+/+ and Hepc−/− mice, respectively). Error bars represent SD for n = 3 mice in each group. Statistical significance is indicated by * symbols (**p< 0.005, ***p< 0.0005).</p

Iron Homeostasis and Inflammatory Status in Mice Deficient for the Cystic Fibrosis Transmembrane Regulator.

Cystic Fibrosis (CF) is a frequent and lethal autosomal recessive disease caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Patients with CF suffer from chronic infections and severe inflammation, which lead to progressive pulmonary and gut diseases. Recently, an expanding body of evidence has suggested that iron homeostasis was abnormal in CF with, in particular, systemic iron deficiency and iron sequestration in the epithelium airway. The molecular mechanisms responsible for iron dysregulation and the relationship with inflammation in CF are unknown.We assessed the impact of CFTR deficiency on systemic and tissue iron homeostasis as well as inflammation in wildtype and CFTR knockout (KO) mice. First, in contrast to the systemic and intestinal inflammation we observed in the CFTR KO mice, we reported the absence of lung phenotype with regards to both inflammation and iron status. Second, we showed a significant decrease of plasma ferritin levels in the KO mice, as in CF patients, likely caused by a decrease in spleen ferritin levels. However, we measured unchanged plasma iron levels in the KO mice that may be explained by increased intestinal iron absorption.These results indicate that in CF, the lung do not predominantly contributes to the systemic ferritin deficiency and we propose the spleen as the major organ responsible for hypoferritinemia in the KO mouse. These results should provide a better understanding of iron dysregulation in CF patients where treating or not iron deficiency remains a challenging question

Relative mRNA levels and proteins of indicated genes in the liver and the duodenum of WT and Hepc−/− mice treated with PBS or LPS.

<p>Relative changes in mRNA levels were quantified by real-time qRT-PCR in the liver, top panel and the duodenum (duo), low panel. Western Blot of Dcytb and DMT1 on membrane extracts were performed with 25 µg/lane. Error bars represent SD for n = 3 mice in each group. Statistical significance is indicated by * symbols (*p< 0.05, **p< 0.005, ***p< 0.0005).</p

AMPK is not required for the effect of metformin on the inhibition of BMP6-induced hepcidin gene expression in hepatocytes

Abstract The biguanide metformin is used for its antidiabetic effect for many years but how metformin acts remains poorly understood and controversial. AMP-activated protein kinase (AMPK), a protein kinase that plays a key role in maintaining energy homeostasis, is assumed to be one of the prime targets of metformin. However, since our demonstration that AMPK is not required for the beneficial effects of metformin on the control of glycemia, the list of AMPK-independent actions of metformin is rapidly increasing. Given the conflicting results on the effects of metformin we sought, using our genetic mouse models deficient in the catalytic subunits of AMPK, to determine whether this kinase is involved in the effects of metformin on the expression of the iron-regulatory hormone hepcidin, as recently proposed. Here we demonstrate, using different approaches, either isolated hepatocytes that lack AMPK, or direct AMPK activators, that, AMPK activation is not necessary for metformin to inhibit BMP6-induced hepcidin gene expression. These results may shed new lights on the increasingly recognized AMPK-independent metformin’s molecular action, an important area of current research

Pulmonary Iron Homeostasis in Hepcidin Knockout Mice

Pulmonary iron excess is deleterious and contributes to a range of chronic and acute inflammatory diseases. Optimal lung iron concentration is maintained through dynamic regulation of iron transport and storage proteins. The iron-regulatory hormone hepcidin is also expressed in the lung. In order to better understand the interactions between iron-associated molecules and the hepcidin-ferroportin axis in lung iron balance, we examined lung physiology and inflammatory responses in two murine models of systemic iron-loading, either hepcidin knock-out (Hepc KO) or liver-specific hepcidin KO mice (Hepc KOliv), which do (Hepc KOliv) or do not (Hepc KO) express lung hepcidin. We have found that increased plasma iron in Hepc KO mice is associated with increased pulmonary iron levels, consistent with increased cellular iron uptake by pulmonary epithelial cells, together with an increase at the apical membrane of the cells of the iron exporter ferroportin, consistent with increased iron export in the alveoli. Subsequently, alveolar macrophages (AM) accumulate iron in a non-toxic form and this is associated with elevated production of ferritin. The accumulation of iron in the lung macrophages of hepcidin KO mice contrasts with splenic and hepatic macrophages which contain low iron levels as we have previously reported. Hepc KOliv mice with liver-specific hepcidin deficiency demonstrated same pulmonary iron overload profile as the Hepc KO mice, suggesting that pulmonary hepcidin is not critical in maintaining local iron homeostasis. In addition, the high iron load in the lung of Hepc KO mice does not appear to enhance acute lung inflammation or injury. Lastly, we have shown that intraperitoneal LPS injection is not associated with pulmonary hepcidin induction, despite high levels of inflammatory cytokines. However, intranasal LPS injection stimulates a hepcidin response, likely derived from AM, and alters pulmonary iron content in Hepc KO mice

Duodenal and spleen iron-related proteins in CFTR KO mice.

<p>Duodenum (A) and spleen (B, C) were collected from WT and CFTR KO mice. Duodenum DcytB, DMT1 and ferroportin were analyzed using proteins from membrane enriched-fractions and L-ferritin from cytosolic fractions. The loading control, ß-actin, is shown (A). Perls’ blue staining of spleen section from WT and KO mice (B). Spleen ferroportin was analyzed using proteins from membrane enriched-fractions and L-ferritin from cytosolic fractions. The loading control, ß-actin, is shown. The right panel represents the quantification of the blots (arbitrary units). Data are presented as mean ± SEM. * P<0.05.</p