View Article Online View Journal Metallomics IntegratedAccepted biometal Manuscript science This article can be cited before page numbers have been issued, to do this please use: L. Jin, D. M. Frazer, Y. Lu, S. J. Wilkins, S. Ayton, A. I. Bush and G. Anderson, Metallomics, 2019, DOI: 10.1039/C8MT00370J. Volume 8 Number 1 January 2016 Pages 1–136 This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Metallomics Accepted Manuscripts are published online shortly after www.rsc.org/metallomics acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the author guidelines. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s ISSN 1756-5901 standard Terms & Conditions and the ethical guidelines, outlined in our author and reviewer resource centre, still apply. In no PAPER David P. Giedroc et al. The S2 Cu(I) site in CupA from Streptococcus pneumoniae is required for cellular copper resistance Indexed in event shall the Royal Society of Chemistry be held responsible Medline! for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. rsc.li/metallomics Page 1 of 12 Metallomics 1 2 3 Significance to metallomics statement View Article Online 4 DOI: 10.1039/C8MT00370J 5 6 Manganese is an essential metal, but how its trafficking is regulated within the body remains 7 incompletely understood. Ferroportin is best known as an iron export protein, although it can also 8 9 transport a number of other divalent metal ions, including potentially manganese. However, the 10 11 literature is divided in this area, and in particular, there are few data on how ferroportin might 12 contribute to manganese homeostasis under physiological conditions. By taking advantage of two 13 14 knockout mouse models with altered ferroportin levels, we have been able to provide new insights 15 16 into the contribution this transporter makes to manganese distribution in vivo. 17 18 19 20 21 22 23 24 25 26 Manuscript 27 28 29 30 31 32 33 34 35 36 Accepted 37 38 39Published on 13 March 2019. Downloaded 3/13/2019 11:02:47 PM. 40 41 42 43 44 45 46 47 48 Metallomics 49 50 51 52 53 54 55 56 57 58 59 60 Please doMetallomics not adjust margins Page 2 of 12 1 2 3 View Article Online 4 Metallomics DOI: 10.1039/C8MT00370J 5 6 7 8 ARTICLE 9 10 11 12 Mice overexpressing hepcidin suggest ferroportin does not play a 13 major role in Mn homeostasis 14 \Received 00th January 20xx, a,b a a a c c 15 Accepted 00th January 20xx Lian Jin , David M Frazer , Yan Lu , Sarah J Wilkins , Scott Ayton , Ashley Bush , Gregory J 16 Andersona,b,d 17 DOI: 10.1039/x0xx00000x 18 www.rsc.org/ Manganese is an essential metal that is required for a wide range of biological functions. Ferroportin (FPN), the only 19 known cellular exporter of iron, has also been proposed to play a role in manganese export, but this relationship is 20 incompletely understood. To investigate this in more detail in vivo, we examined the relative distributions of manganese 21 and iron in TMPRSS6 deficient mice, which are characterized by constitutively high expression of the iron regulatory 22 hormone hepcidin and, consequently, very low FPN levels in their tissues. Tmprss6-/- mice showed frank iron deficiency 23 and reduced iron levels in most tissues, consistent with FPN playing an important role in the distribution of this metal, but 24 manganese levels were largely unaffected. Associated studies using intestine-specific FPN knockout mice showed that loss 25 of FPN significantly reduced the dietary absorption of iron, but had no effect on manganese intake. Taken together, our 26 data suggest that FPN does not play a major role in Mn transport in vivo. They do not exclude a minor role for FPN in Manuscript 27 manganese homeostasis, nor the possibility that the transporter may be relevant at high Mn levels, but at physiological 28 levels of this metal, other transport proteins appear to be more important. 29 30 metals. For both Fe and Mn, the liver plays a central regulatory 31 Introduction role.8, 9 In broad terms, the regulation of Fe traffic in the body is 32 Metals such as iron (Fe) and manganese (Mn) are essential for better studied than that of Mn. Dietary Fe is taken up by 33 a wide range of biological functions. Iron, for example, is intestinal enterocytes, then released into the circulation across 34 required for oxygen delivery, energy production and DNA the enterocyte basolateral membrane through ferroportin 35 synthesis,1 and manganese is required for the activity of many (FPN; SLC40A1), the only known mammalian iron export Accepted 36 enzymes, including those involved in glucose and lipid protein.10 In the circulation, iron binds to transferrin and is 37 metabolism and antioxidant defence.2 Both Fe and Mn levels then distributed to cells throughout the body. In addition to 38 in the body must be strictly controlled as a deficiency or excess Published on 13 March 2019. Downloaded 3/13/2019 11:02:47 PM. being important for intestinal Fe absorption, FPN is required 39 of either metal can be harmful. Iron deficiency can lead to for Fe export from most body cells. This iron export process is 40 anemia and its associated pathological consequences, regulated by the hepatic hormone hepcidin, which binds to 41 including compromised neurological development when FPN and leads to its internalization and degradation.11 When 42 present in infancy.3, 4 In contrast, since Fe can catalyze the body iron levels rise, hepcidin production is increased and FPN 43 formation of toxic oxygen radicals, excess iron can damage levels decline.12 This in turn reduces iron absorption and iron 44 vital organs including the liver and heart. Mn is also toxic when accumulates in intestinal enterocytes. Iron also accumulates in 45 present in excess, and its neurotoxicity is particularly notable.2 other cells in the body. Conversely, when body iron levels are 46 Significantly, high Mn levels have been associated with depleted, hepcidin expression is downregulated to allow for 47 neurological disorders such as hepatic encephalopathy and increased dietary Fe absorption and mobilization of body Fe Metallomics 48 parkinsonism.5-7 Because of this dual nature, being essential, stores, both of which require active FPN on the cell surface.13, 49 yet being toxic in excess, sophisticated mechanisms have 14 Pathological disturbances leading to either increased or 50 developed to maintain the cellular and body levels of Fe and decreased hepcidin expression lead to anemia and 51 Mn within the optimum physiological range. This is largely hemochromatosis respectively.15-17 52 achieved by regulating the cellular intake and efflux of the 53 FPN has been shown to transport several divalent metal 54 ions other than Fe2+, including Co2+ and Zn2+, but not others a. Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, such as Cu2+ or Cd2+.18 The literature on whether FPN is able to 55 Brisbane, Queensland, Australia. 56 b. Faculty of Medicine, University of Queensland, Brisbane, Australia transport Mn is confusing and apparently contradictory. Some 57 c. Melbourne Dementia Research Centre, Florey Institute of Neuroscience and in vitro studies have suggested that FPN transports minimal Mental Health, University of Melbourne, Melbourne, Victoria, Australia amounts of Mn,18, 19 but others have suggested otherwise.20, 21 58 d. School of Chemistry and Molecular Biosciences, University of Queensland, 59 Brisbane, Australia. In vivo studies have generally suggested that FPN is able to 60 This journal is © The Royal Society of Chemistry 2019 Metallomics, 2019, 00, 1-3 | 1 Please do not adjust margins Page 3 of 12 Please doMetallomics not adjust margins 1 ARTICLE Journal Name 2 3 transport Mn, but its influence is usually modest. For example, protease which acts upstream of hepcidin toView reduce Article Online its 4 it has been reported that the flatiron mouse (ffe/+), a model of expression.46 Tmprss6-/- mice lack DOI:the 10.1039/C8MT00370J protease and 5 genetic FPN deficiency, albeit with an unusual underlying consequently hepcidin is expressed at a constitutively high 6 mechanism, has a reduced capacity to absorb orally level.47 This in turn reduces FPN expression and leads to iron 7 administered Mn and has reduced Mn levels in the blood, liver deficiency in the mice. These animals represent an ideal model 8 and bile.22 In addition, there is evidence that intestinal in which to study the effects of increased hepcidin (and hence 9 absorption of Mn is increased in Hfe knockout mice, a mouse reduced FPN) on Mn metabolism. Using both these mice and 10 model of HFE-related hemochromatosis23 where FPN mice carrying an intestine-specific deletion of FPN, we showed 11 expression is increased secondary to diminished hepcidin that FPN likely plays a minimal role in body Mn homeostasis. 12 expression.24, 25 While these studies provide some evidence to Metal transporters other than FPN appear to be more 13 implicate FPN in Mn homeostasis, its relative contribution at important in regulating Mn flux in the body under normal 14 physiological levels of Mn remains unclear.