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Despite its abundance in the environment, iron is poorly bioavailable and subject to strict conservation and internal recycling by most organisms. In vertebrates, the stability of iron concentration in plasma and extracellular fluid, and the total body iron content are maintained by the interaction of the iron-regulatory peptide hormone hepcidin with its receptor and cellular iron exporter ferroportin (SLC40a1). Ferroportin exports iron from duodenal enterocytes that absorb dietary iron, from iron-recycling macrophages in the spleen and the liver, and from iron-storing hepatocytes. Hepcidin blocks iron export through ferroportin, causing hypoferremia. During iron deficiency or after hemorrhage, hepcidin decreases to allow iron delivery to plasma through ferroportin, thus promoting compensatory erythropoiesis. As a host defense mediator, hepcidin increases in response to infection and inflammation, blocking iron delivery through ferroportin to blood plasma, thus limiting iron availability to invading microbes. Genetic diseases that decrease hepcidin synthesis or disrupt hepcidin binding to ferroportin cause the iron overload disorder hereditary hemochromatosis. The opposite phenotype, iron restriction or iron deficiency, can result from genetic or inflammatory overproduction of hepcidin.
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International Journal of
Molecular Sciences
Review
Hepcidin-Ferroportin Interaction Controls Systemic
Iron Homeostasis
Elizabeta Nemeth 1and Tomas Ganz 2, *


Citation: Nemeth, E.; Ganz, T.
Hepcidin-Ferroportin Interaction
Controls Systemic Iron Homeostasis.
Int. J. Mol. Sci. 2021,22, 6493.
https://doi.org/10.3390/ijms22126493
Academic Editor: Wojciech Bal
Received: 2 June 2021
Accepted: 14 June 2021
Published: 17 June 2021
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Copyright: © 2021 by the authors.
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Attribution (CC BY) license (https://
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4.0/).
1Department of Medicine, University of California, Los Angeles, CA 90095, USA; enemeth@mednet.ucla.edu
2Departments of Medicine and Pathology, University of California, Los Angeles, CA 90095, USA
*Correspondence: tganz@mednet.ucla.edu
Abstract:
Despite its abundance in the environment, iron is poorly bioavailable and subject to
strict conservation and internal recycling by most organisms. In vertebrates, the stability of iron
concentration in plasma and extracellular fluid, and the total body iron content are maintained by
the interaction of the iron-regulatory peptide hormone hepcidin with its receptor and cellular iron
exporter ferroportin (SLC40a1). Ferroportin exports iron from duodenal enterocytes that absorb
dietary iron, from iron-recycling macrophages in the spleen and the liver, and from iron-storing
hepatocytes. Hepcidin blocks iron export through ferroportin, causing hypoferremia. During
iron deficiency or after hemorrhage, hepcidin decreases to allow iron delivery to plasma through
ferroportin, thus promoting compensatory erythropoiesis. As a host defense mediator, hepcidin
increases in response to infection and inflammation, blocking iron delivery through ferroportin to
blood plasma, thus limiting iron availability to invading microbes. Genetic diseases that decrease
hepcidin synthesis or disrupt hepcidin binding to ferroportin cause the iron overload disorder
hereditary hemochromatosis. The opposite phenotype, iron restriction or iron deficiency, can result
from genetic or inflammatory overproduction of hepcidin.
Keywords: iron deficiency; iron overload; hemochromatosis; anemia; metal transport
1. The Roles of Iron
Iron is an essential trace element for nearly all living organisms. Even though iron
is one of the most abundant elements in the Earth’s crust, ferric iron, an oxidized form
common in the oxygen-rich environment on the surface of the Earth, is poorly soluble
and difficult to access by most life forms. Accordingly, biological organisms have evolved
mechanisms that conserve iron and recycle it internally. In adult humans, total body iron
content is approximately 3–4 g, whereas normal daily losses are only 1–2 mg. To remain in
iron balance, healthy humans must absorb a similar amount of iron from their diets.
The ability of iron to donate or accept an electron in cellular and extracellular envi-
ronments is what makes it a versatile catalytic component of many enzymes involved in
energy-producing bioreactions and critical biosynthetic pathways, as well as in enzymes
that generate reactive oxygen species for host defense. Iron also coordinates oxygen in
hemoglobin and myoglobin, molecules involved in oxygen transport and its cellular storage.
In biological systems, iron carries out its function in association with three common types
of moieties: iron coordinated by protein side chains, iron complexed within the porphyrin
ring of heme, and iron within iron-sulfur clusters. Outside of these controlled chemical
environments, iron displays promiscuous reactivity that can damage cells and tissues.
2. Iron Compartments and Flows
Biological organisms closely regulate intracellular and extracellular iron concentra-
tions, navigating between the twin threats of inadequate iron supply that would limit
critical functions, and uncontrolled iron excess that could be toxic to the organism. Sys-
temic iron homeostasis, best understood in humans and laboratory rodents, is maintained
Int. J. Mol. Sci. 2021,22, 6493. https://doi.org/10.3390/ijms22126493 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2021,22, 6493 2 of 13
by regulating intestinal iron absorption, the concentration of iron in blood plasma and
extracellular fluid, the distribution of iron among organs and tissues, and the amount
of iron kept in stores. Iron dyshomeostasis can manifest as total body iron deficit (iron
deficiency) or excess (iron overload), as well as iron maldistribution among tissues in
which individual tissues or organs may become iron-deficient or iron-overloaded. Such
iron disorders may be caused by genetic lesions that directly impair iron regulation, or
conditions that impact iron regulation indirectly.
Although all cells in a multicellular organism contain iron, systemic iron homeostasis
is primarily affected by the following compartments: the erythron (red blood cells and
their precursors in erythropoietic organs), two types of stores (hepatocytes of the liver
and macrophages of the spleen and the liver), blood plasma which moves iron between
tissues and organs, and absorptive enterocytes in the duodenum through which iron
enters the body, ordinarily to replace small losses from the body caused by shedding of
iron-containing cells (Figure 1).
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 13
2. Iron Compartments and Flows
Biological organisms closely regulate intracellular and extracellular iron concentra-
tions, navigating between the twin threats of inadequate iron supply that would limit crit-
ical functions, and uncontrolled iron excess that could be toxic to the organism. Systemic
iron homeostasis, best understood in humans and laboratory rodents, is maintained by
regulating intestinal iron absorption, the concentration of iron in blood plasma and extra-
cellular fluid, the distribution of iron among organs and tissues, and the amount of iron
kept in stores. Iron dyshomeostasis can manifest as total body iron deficit (iron deficiency)
or excess (iron overload), as well as iron maldistribution among tissues in which individ-
ual tissues or organs may become iron-deficient or iron-overloaded. Such iron disorders
may be caused by genetic lesions that directly impair iron regulation, or conditions that
impact iron regulation indirectly.
Although all cells in a multicellular organism contain iron, systemic iron homeostasis
is primarily affected by the following compartments: the erythron (red blood cells and
their precursors in erythropoietic organs), two types of stores (hepatocytes of the liver and
macrophages of the spleen and the liver), blood plasma which moves iron between tissues
and organs, and absorptive enterocytes in the duodenum through which iron enters the
body, ordinarily to replace small losses from the body caused by shedding of iron-con-
taining cells (Figure 1).
Figure 1. The key iron flows and compartments.
The largest of these iron compartments is the erythron (erythrocytes and their pre-
cursors), which contains approximately 2–3 g of iron, representing about 2/3–3/4 of the
total body iron in adult humans. Erythroid iron is almost entirely contained within hemo-
globin, at the concentration of 1 g of iron per liter of packed erythrocytes. The second
largest compartment are the hepatocyte stores, where up to 1 g of iron is contained in
cytoplasmic ferritin of hepatocytes. The stores are highly variable and can be nearly de-
pleted in many women of reproductive age because of menstrual blood loss combined
with low dietary intake. The recycling macrophages in the spleen, liver and marrow func-
tion as a rapid turnover compartment [1]. The spleen, which represents a substantial por-
tion of the macrophage storage compartment, normally contains only about 0.05 g of iron
but has the capacity for much larger amounts [2]. Only about 0.3 g of iron are contained
in the other tissues, in myoglobin of muscles and in iron-containing enzymes.
Figure 1. The key iron flows and compartments.
The largest of these iron compartments is the erythron (erythrocytes and their precur-
sors), which contains approximately 2–3 g of iron, representing about 2/3–3/4 of the total
body iron in adult humans. Erythroid iron is almost entirely contained within hemoglobin,
at the concentration of 1 g of iron per liter of packed erythrocytes. The second largest
compartment are the hepatocyte stores, where up to 1 g of iron is contained in cytoplas-
mic ferritin of hepatocytes. The stores are highly variable and can be nearly depleted in
many women of reproductive age because of menstrual blood loss combined with low
dietary intake. The recycling macrophages in the spleen, liver and marrow function as a
rapid turnover compartment [
1
]. The spleen, which represents a substantial portion of the
macrophage storage compartment, normally contains only about 0.05 g of iron but has the
capacity for much larger amounts [
2
]. Only about 0.3 g of iron are contained in the other
tissues, in myoglobin of muscles and in iron-containing enzymes.
Iron is transported around the body on blood plasma carrier protein transferrin, whose
two iron-binding sites are normally 20–45% occupied. Each cell in the body has transferrin
receptors (TfR1) that are endocytosed into acidified endosomes where iron dissociates from
the TfR1-transferrin complex and is transported across the endosomal membrane into the
cytoplasm. The transferrin compartment in blood plasma holds only 2–3 mg of iron but
delivers to target tissues 20–25 mg/day so its iron content turns over every few hours.
Despite changes in dietary iron content and tissue demand for iron, the concentration of
iron in the blood plasma in healthy humans is remarkably stable, as is the total body iron
content. Moreover, duodenal iron absorption increases several-fold after blood loss or
Int. J. Mol. Sci. 2021,22, 6493 3 of 13
exposure to hypoxia, or in response to iron deficiency. These observations provided early
evidence that the absorption and tissue distribution of iron must be subject to endocrine
regulation [3].
3. Molecular Basis of Systemic Iron Homeostasis
The absorption and tissue distribution of iron is principally controlled by the in-
teraction of the hepatic hormone hepcidin with ferroportin. Ferroportin is expressed in
iron-storing and iron-transporting tissues [
4
] and functions both as the hepcidin recep-
tor and the sole known cellular exporter of elemental iron in multicellular organisms
(
Figure 2
). The 25 amino-acid hepcidin peptide (MW 2.8 kD) is synthesized by hepatocytes
and secreted into blood plasma, with concentrations in healthy humans ranging from
approximately 2–20 nM [
5
,
6
], around hundred-fold higher than the concentration of the
similarly-sized peptide hormones insulin, glucagon or parathyroid hormone.
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 13
Iron is transported around the body on blood plasma carrier protein transferrin,
whose two iron-binding sites are normally 20–45% occupied. Each cell in the body has
transferrin receptors (TfR1) that are endocytosed into acidified endosomes where iron dis-
sociates from the TfR1-transferrin complex and is transported across the endosomal mem-
brane into the cytoplasm. The transferrin compartment in blood plasma holds only 2–3
mg of iron but delivers to target tissues 20–25 mg/day so its iron content turns over every
few hours. Despite changes in dietary iron content and tissue demand for iron, the con-
centration of iron in the blood plasma in healthy humans is remarkably stable, as is the
total body iron content. Moreover, duodenal iron absorption increases several-fold after
blood loss or exposure to hypoxia, or in response to iron deficiency. These observations
provided early evidence that the absorption and tissue distribution of iron must be subject
to endocrine regulation [3].
3. Molecular Basis of Systemic Iron Homeostasis
The absorption and tissue distribution of iron is principally controlled by the inter-
action of the hepatic hormone hepcidin with ferroportin. Ferroportin is expressed in iron-
storing and iron-transporting tissues [4] and functions both as the hepcidin receptor and
the sole known cellular exporter of elemental iron in multicellular organisms (Figure 2).
The 25 amino-acid hepcidin peptide (MW 2.8 kD) is synthesized by hepatocytes and se-
creted into blood plasma, with concentrations in healthy humans ranging from approxi-
mately 2–20 nM [5,6], around hundred-fold higher than the concentration of the similarly-
sized peptide hormones insulin, glucagon or parathyroid hormone.
Figure 2. The interaction of hepcidin with ferroportin controls iron flows into plasma.
4. Ferroportin Structure
Ferroportin, a member of the solute carrier family, is systematically named SLC40A1.
The human protein has 571 amino acids for a molecular weight of around 65–70 kD [7–9].
The variation in molecular mass is likely caused by tissue-specific glycosylation but the
functional consequences of the glycosylation differences between the forms purified from
the duodenal enterocytes, hepatocytes and macrophages are not yet understood [10].
Structurally, ferroportin consists of two 6-transmembrane-helix bundles (N-lobe and C-
lobe) joined by a cytoplasmic loop, with both C- and N-termini located in the cytoplasm
(Figure 3). The two helix bundles enclose a cavity through which iron is thought to exit
the cell. The shape and similarity to more completely characterized family members sug-
gest that ferroportin exports cellular iron by an alternating access mechanism, wherein
Figure 2. The interaction of hepcidin with ferroportin controls iron flows into plasma.
4. Ferroportin Structure
Ferroportin, a member of the solute carrier family, is systematically named SLC40A1.
The human protein has 571 amino acids for a molecular weight of around 65–70 kD
[79]
.
The variation in molecular mass is likely caused by tissue-specific glycosylation but the
functional consequences of the glycosylation differences between the forms purified from
the duodenal enterocytes, hepatocytes and macrophages are not yet understood [
10
].
Structurally, ferroportin consists of two 6-transmembrane-helix bundles (N-lobe and C-
lobe) joined by a cytoplasmic loop, with both C- and N-termini located in the cytoplasm
(
Figure 3
). The two helix bundles enclose a cavity through which iron is thought to exit
the cell. The shape and similarity to more completely characterized family members sug-
gest that ferroportin exports cellular iron by an alternating access mechanism, wherein
ferroportin alternates between an open-inward conformation which binds intracellular
iron, and an open-outward conformation which releases the iron to the extracellular space
(
Figure 3
). The remarkable feat of structural characterization of a ferroportin from the bac-
terium Bdellovibrio bacteriovorus in both open-outward and open-inward conformations [
11
]
represents strong support for such a model. The exported species is almost certainly ferrous
iron [
12
] but the energetics of iron export is not yet well characterized. Because iron is
exported against the force exerted by the transmembrane electric field, and there is no
evidence of direct coupling of the transport to ATP hydrolysis, the export of iron is likely
facilitated by the coupled “downhill” transport of another ion or small molecule [13].
Int. J. Mol. Sci. 2021,22, 6493 4 of 13
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 13
ferroportin alternates between an open-inward conformation which binds intracellular
iron, and an open-outward conformation which releases the iron to the extracellular space
(Figure 3). The remarkable feat of structural characterization of a ferroportin from the bac-
terium Bdellovibrio bacteriovorus in both open-outward and open-inward conformations
[11] represents strong support for such a model. The exported species is almost certainly
ferrous iron [12] but the energetics of iron export is not yet well characterized. Because
iron is exported against the force exerted by the transmembrane electric field, and there is
no evidence of direct coupling of the transport to ATP hydrolysis, the export of iron is
likely facilitated by the coupled “downhill” transport of another ion or small molecule
[13].
Structural analyses of ferroportin indicate that there are two divalent metal-binding
sites, one in each lobe, facing the internal cavity of ferroportin (Figure 3) [11,13,14]. How
these sites mediate the export of iron is not yet clear. The site in the N-lobe can bind cal-
cium which may have a modulatory role on iron transport [13]. Although calcium itself is
not transported, it is required for human ferroportin transport activity, and is thought to
directly bind to ferroportin and facilitate a conformational change critical to the transport
cycle. Another area of interest is how cytoplasmic iron destined for export reaches ferro-
portin. Current evidence indicates that cytoplasmic iron is predominantly present as a
complex of ferrous iron with reduced glutathione (summarized in [15]), and that the com-
plex of ferrous iron and reduced glutathione may be delivered to ferroportin via the cyto-
plasmic iron chaperone PCBP2 [16].
Figure 3. A current model of hepcidin (orange) interaction with ferroportin (blue) in which bind-
ing is dependent on iron (green). The framework of helices that make up ferroportin are depicted
as cylinders connected by extracellular and intracellular disordered loops. The side chains that
make up the binding sites for iron are shown. Modified from [14].
5. Ferroportin Interactions with Hepcidin
The flow of iron out of the cells is controlled by hepcidin through two known mech-
anisms: occlusion of the open-outward conformation of ferroportin by hepcidin [17], and
hepcidin-induced endocytosis and degradation of ferroportin [4]. The occlusion mecha-
nism would be effective at hepcidin concentrations where most ferroportin molecules re-
main occluded by hepcidin most of the time, and would be rapidly reversible when hep-
cidin concentrations are decreased. The endocytosis mechanism could be initiated by even
transient binding of hepcidin to ferroportin, and would cause the permanent removal of
ferroportin from the cell surface that would require resynthesis of ferroportin for the re-
covery of iron transport. The second mechanism would therefore be expected to occur at
lower concentrations of hepcidin, and to have a prolonged effect, even if hepcidin concen-
trations subsequently decrease. Although hepcidin peptide injected into mice is cleared
from blood circulation within minutes, its plasma iron lowering effect lasts for 24–48 h,
supporting the importance of the endocytic mechanism for ferroportin physiology [18].
Hepcidin regulation of ferroportin by the endocytic mechanism resembles the ge-
neric ligand-induced receptor endocytosis. It appears to require hepcidin-induced confor-
mational change in ferroportin that triggers the ubiquitination of the lysine-rich cytoplas-
Figure 3.
A current model of hepcidin (orange) interaction with ferroportin (blue) in which binding
is dependent on iron (green). The framework of helices that make up ferroportin are depicted as
cylinders connected by extracellular and intracellular disordered loops. The side chains that make up
the binding sites for iron are shown. Modified from [14].
Structural analyses of ferroportin indicate that there are two divalent metal-binding
sites, one in each lobe, facing the internal cavity of ferroportin (Figure 3) [
11
,
13
,
14
]. How
these sites mediate the export of iron is not yet clear. The site in the N-lobe can bind
calcium which may have a modulatory role on iron transport [
13
]. Although calcium
itself is not transported, it is required for human ferroportin transport activity, and is
thought to directly bind to ferroportin and facilitate a conformational change critical to
the transport cycle. Another area of interest is how cytoplasmic iron destined for export
reaches ferroportin. Current evidence indicates that cytoplasmic iron is predominantly
present as a complex of ferrous iron with reduced glutathione (summarized in [
15
]), and
that the complex of ferrous iron and reduced glutathione may be delivered to ferroportin
via the cytoplasmic iron chaperone PCBP2 [16].
5. Ferroportin Interactions with Hepcidin
The flow of iron out of the cells is controlled by hepcidin through two known mecha-
nisms: occlusion of the open-outward conformation of ferroportin by hepcidin [
17
], and
hepcidin-induced endocytosis and degradation of ferroportin [
4
]. The occlusion mech-
anism would be effective at hepcidin concentrations where most ferroportin molecules
remain occluded by hepcidin most of the time, and would be rapidly reversible when
hepcidin concentrations are decreased. The endocytosis mechanism could be initiated by
even transient binding of hepcidin to ferroportin, and would cause the permanent removal
of ferroportin from the cell surface that would require resynthesis of ferroportin for the
recovery of iron transport. The second mechanism would therefore be expected to occur at
lower concentrations of hepcidin, and to have a prolonged effect, even if hepcidin concen-
trations subsequently decrease. Although hepcidin peptide injected into mice is cleared
from blood circulation within minutes, its plasma iron lowering effect lasts for 24–48 h,
supporting the importance of the endocytic mechanism for ferroportin physiology [18].
Hepcidin regulation of ferroportin by the endocytic mechanism resembles the generic
ligand-induced receptor endocytosis. It appears to require hepcidin-induced conforma-
tional change in ferroportin that triggers the ubiquitination of the lysine-rich cytoplasmic
segment connecting the two 6-helix domains of ferroportin [
19
,
20
]. Ubiquitinated ferro-
portin is then targeted to lysosomes and proteasomes for degradation. A recent study [
21
]
provide convincing evidence that Rnf217 is an important E3 ubiquitin ligase that triggers
the degradation of ferroportin in response to hepcidin binding.
Recent advances in structural understanding of the hepcidin-ferroportin interaction
confirmed the simple models generated by targeted mutagenesis of hepcidin and ferro-
portin but also provided surprising new details [
14
]. In a nanodisc membrane model,
hepcidin is seen to bind to the C-lobe of ferroportin (Figure 3), with the highly variable
hepcidin loop largely extracellular, and the relatively conserved C- and N-termini of hep-
cidin deeply buried in the central cavity. The model also revealed an important unexpected
feature, the indication that the suspected hepcidin-binding site in the C-lobe, centered on
the critically important C325 thiol cysteine, utilizes an iron atom to coordinate hepcidin
Int. J. Mol. Sci. 2021,22, 6493 5 of 13
binding [
14
]. It is not yet clear how this contributes to hepcidin physiology but it can be
anticipated that the mechanism could provide selectivity for hepcidin to bind to ferroportin
molecules actively transporting iron as opposed to those that may be in a resting state. One
indication that this mechanism may have a physiological effect is the recently reported
inhibition of hepcidin-induced ferroportin degradation by the intracellular iron chelator
deferiprone but not by the extracellularly acting chelator deferoxamine [
22
]. How the
new structural model accommodates other hepcidin agonists ranging from minihepcidin
peptides [23] to small molecules [24] remains to be determined.
6. Hepcidin Synthesis and Elimination
Hepcidin production is principally transcriptionally regulated. The main systemic
regulators of hepcidin include plasma iron concentrations (mainly through the interaction
of diferric transferrin with transferrin receptors TFR1 and TFR2 in the liver), hepatic
iron stores, systemic inflammation mainly communicated to hepatocytes by IL-6, and
erythroid activity conveyed by the concentrations of the erythroid hormone erythroferrone
(
Figure 4
). The mechanisms were reviewed in recent publications [
25
,
26
] and will not be
discussed here.
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 13
mic segment connecting the two 6-helix domains of ferroportin [19,20]. Ubiquitinated fer-
roportin is then targeted to lysosomes and proteasomes for degradation. A recent study
[21] provide convincing evidence that Rnf217 is an important E3 ubiquitin ligase that trig-
gers the degradation of ferroportin in response to hepcidin binding.
Recent advances in structural understanding of the hepcidin-ferroportin interaction
confirmed the simple models generated by targeted mutagenesis of hepcidin and ferro-
portin but also provided surprising new details [14]. In a nanodisc membrane model, hep-
cidin is seen to bind to the C-lobe of ferroportin (Figure 3), with the highly variable hep-
cidin loop largely extracellular, and the relatively conserved C- and N-termini of hepcidin
deeply buried in the central cavity. The model also revealed an important unexpected
feature, the indication that the suspected hepcidin-binding site in the C-lobe, centered on
the critically important C325 thiol cysteine, utilizes an iron atom to coordinate hepcidin
binding [14]. It is not yet clear how this contributes to hepcidin physiology but it can be
anticipated that the mechanism could provide selectivity for hepcidin to bind to ferro-
portin molecules actively transporting iron as opposed to those that may be in a resting
state. One indication that this mechanism may have a physiological effect is the recently
reported inhibition of hepcidin-induced ferroportin degradation by the intracellular iron
chelator deferiprone but not by the extracellularly acting chelator deferoxamine [22]. How
the new structural model accommodates other hepcidin agonists ranging from minihep-
cidin peptides [23] to small molecules [24] remains to be determined.
6. Hepcidin Synthesis and Elimination
Hepcidin production is principally transcriptionally regulated. The main systemic
regulators of hepcidin include plasma iron concentrations (mainly through the interaction
of diferric transferrin with transferrin receptors TFR1 and TFR2 in the liver), hepatic iron
stores, systemic inflammation mainly communicated to hepatocytes by IL-6, and
erythroid activity conveyed by the concentrations of the erythroid hormone erythrofer-
rone (Figure 4). The mechanisms were reviewed in recent publications [25,26] and will not
be discussed here.
Figure 4. Iron, erythropoiesis and inflammation regulate hepcidin transcription through their ef-
fects on hepatocytes and by modulating the paracrine signaling between hepatic sinusoidal endo-
thelium and hepatocytes.
The size of hepcidin and its mechanism of action suggest two catabolic mechanisms.
One is efficient filtration of hepcidin as a small peptide through the renal glomerular
Figure 4.
Iron, erythropoiesis and inflammation regulate hepcidin transcription through their effects
on hepatocytes and by modulating the paracrine signaling between hepatic sinusoidal endothelium
and hepatocytes.
The size of hepcidin and its mechanism of action suggest two catabolic mechanisms.
One is efficient filtration of hepcidin as a small peptide through the renal glomerular
membrane [
27
], followed by reuptake and degradation of filtered hepcidin in the proximal
tubule, utilizing a generic mechanism for recycling the amino acid content of filtered
proteins. Efficient filtration of hepcidin in the glomerulus is facilitated by the relatively
low fraction of hepcidin that is bound by plasma protein, estimated at about 40% [
28
]. A
small percentage of filtered hepcidin survives the uptake and degradation in the proximal
tubule and can be detected in urine, similarly to other peptide hormones. In support of the
important role of the kidneys in hepcidin removal, serum hepcidin concentrations are very
high in patients with chronic renal failure, but are effectively lowered by hemodialysis [
29
].
This effect of hemodialysis is brief, as hepatic hepcidin synthesis is sufficiently rapid
to restore hepcidin concentrations within hours. The second mechanism of hepcidin
clearance depends on the endocytosis of the hepcidin-ferroportin complexes by the cells
that express high concentrations of ferroportin [
30
,
31
]. The quantitative contribution of the
two mechanisms as well as any possible alternative mechanisms of hepcidin catabolism
are not known.
Int. J. Mol. Sci. 2021,22, 6493 6 of 13
7. Evidence for Exclusivity of the Hepcidin-Ferroportin Ligand-Receptor Dyad
Ferroportin is evolutionarily ancient and can be found in plants and multicellular
animals as simple as hydra. By contrast, hepcidin is a vertebrate hormone, with no known
antecedent before fish. Remarkably, the essential conserved amino acid at the ferroportin
binding site for hepcidin, C326 (human numbering), first appears in cartilaginous fish
(Figure 5) some of which have a second ferroportin gene that has another amino acid in
this position. C326 is strictly conserved in all vertebrates that have hepcidin but another
amino acid is in this position in invertebrates, indicating that hepcidin and its binding site
on ferroportin co-evolved. A second hepcidin gene is found in some vertebrates, mostly
fish, but is not involved in iron regulation, and is hypothesized to have another function,
perhaps as an antimicrobial peptide [32].
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 13
membrane [27], followed by reuptake and degradation of filtered hepcidin in the proximal
tubule, utilizing a generic mechanism for recycling the amino acid content of filtered pro-
teins. Efficient filtration of hepcidin in the glomerulus is facilitated by the relatively low
fraction of hepcidin that is bound by plasma protein, estimated at about 40% [28]. A small
percentage of filtered hepcidin survives the uptake and degradation in the proximal tu-
bule and can be detected in urine, similarly to other peptide hormones. In support of the
important role of the kidneys in hepcidin removal, serum hepcidin concentrations are
very high in patients with chronic renal failure, but are effectively lowered by hemodial-
ysis [29]. This effect of hemodialysis is brief, as hepatic hepcidin synthesis is sufficiently
rapid to restore hepcidin concentrations within hours. The second mechanism of hepcidin
clearance depends on the endocytosis of the hepcidin-ferroportin complexes by the cells
that express high concentrations of ferroportin [30,31]. The quantitative contribution of
the two mechanisms as well as any possible alternative mechanisms of hepcidin catabo-
lism are not known.
7. Evidence for Exclusivity of the Hepcidin-Ferroportin Ligand-Receptor Dyad
Ferroportin is evolutionarily ancient and can be found in plants and multicellular
animals as simple as hydra. By contrast, hepcidin is a vertebrate hormone, with no known
antecedent before fish. Remarkably, the essential conserved amino acid at the ferroportin
binding site for hepcidin, C326 (human numbering), first appears in cartilaginous fish
(Figure 5) some of which have a second ferroportin gene that has another amino acid in
this position. C326 is strictly conserved in all vertebrates that have hepcidin but another
amino acid is in this position in invertebrates, indicating that hepcidin and its binding site
on ferroportin co-evolved. A second hepcidin gene is found in some vertebrates, mostly
fish, but is not involved in iron regulation, and is hypothesized to have another function,
perhaps as an antimicrobial peptide [32].
Figure 5. Although ferroportin is evolutionarily ancient, the thiol cysteine required for hepcidin
binding by ferroportin appears first in early vertebrate ferroportins. Cartilaginous fish ferroportin
that contains the C326-equivalent is denoted with species name in red. Second ferroportin in carti-
laginous fish that has another amino acid in this position is denoted with species name in blue.
Invertebrate ferroportin lacks C326 and is denoted with species name in black.
Consistent with the dyadic relationship between hepcidin and ferroportin, the iso-
steric C326S mutation in mice and humans, which causes complete loss of binding of hep-
cidin to ferroportin, phenocopies severe hepcidin deficiency [33]. It is therefore likely that
Figure 5.
Although ferroportin is evolutionarily ancient, the thiol cysteine required for hepcidin
binding by ferroportin appears first in early vertebrate ferroportins. Cartilaginous fish ferroportin that
contains the C326-equivalent is denoted with species name in red. Second ferroportin in cartilaginous
fish that has another amino acid in this position is denoted with species name in blue. Invertebrate
ferroportin lacks C326 and is denoted with species name in black.
Consistent with the dyadic relationship between hepcidin and ferroportin, the isosteric
C326S mutation in mice and humans, which causes complete loss of binding of hepcidin to
ferroportin, phenocopies severe hepcidin deficiency [
33
]. It is therefore likely that hepcidin
has no other nonredundant function than to bind to ferroportin and regulate its ability to
transport iron.
8. Regulation of Ferroportin by Intracellular Signals
In addition to systemic control of ferroportin by circulating hepcidin, ferroportin
is also regulated by intracellular conditions. These local regulatory mechanisms may
decrease toxicity from excessive cellular iron, preserve cellular iron under conditions of
systemic iron deficiency, or amplify the effect of systemic hepcidin changes. In addition,
inflammatory stimuli in the form of Toll-like receptor ligands can directly suppress cellular
ferroportin mRNA levels and decrease cellular iron export to blood plasma [
34
]. Table 1
summarizes the local regulators of ferroportin and the setting in which their activities have
been characterized.
Int. J. Mol. Sci. 2021,22, 6493 7 of 13
Table 1. Hepcidin-independent mechanisms that regulate ferroportin.
Mechanism Mode Cell Type Cellular Effect
on FPN Note
IRE-IRP [35] translational macrophage cellular iron =
FPN
This mechanism is
bypassed by
ferroportin transcripts
lacking the 50IRE, in
erythroid cells and
duodenal enterocytes
Iron via Nrf2 [36] transcriptional macrophage cellular iron =
FPN
Requires high iron
concentrations
Heme via BACH
and Nrf2 [36]transcriptional macrophage
cellular heme =
FPN
Induces ferroportin in
macrophages that
recycle iron from
hemoglobin and heme
miR-485–3p [37] translational multiple cell types cellular iron =
FPN
Physiological role is
uncertain
TLR [34] transcriptional macrophage Ligands FPN
Unclear how much this
mechanism contributes
to responses to
infections
HIF2α[38] transcriptional enterocyte cellular iron =
FPN
May induce
ferroportin mRNA in
enterocytes during low
hepcidin states
miR-20a and
miR-20b [39,40]translational enterocyte, lung miRNA = FPN Physiological role is
uncertain
The IRE-IRP system functions in most cells to coordinate ferroportin translation with
intracellular iron levels. Ferroportin mRNA contains 5
0
IRE. Under conditions of iron
deficiency, binding of IRP1/2 to 5
0
IRE inhibits ferroportin translation, thus limiting iron
export and conserving cellular iron. In duodenal enterocytes, however, the existence
of ferroportin transcripts that lack the 5
0
IRE would make these short-lived cells less
responsive to their own cellular iron levels, and by implication more responsive to systemic
iron requirements communicated to enterocytes by the concentration of hepcidin in blood
plasma [
35
]. Low hepcidin levels during iron deficiency and anemia allow continuous iron
export from enterocytes, resulting in activation of the enterocytes HIF system (specifically
HIF2
α
), which further increases ferroportin transcription [
38
]. HIF2
α
also mediates increase
in other mRNAs encoding proteins involved in the apical absorption of dietary iron (DCYTB
and DMT1) thus leading to a coordinated increase in apical and basolateral iron transport
and overall increase in duodenal iron absorption.
The 5
0
IRE-lacking form of ferroportin mRNA is also found in erythroblasts, and the
erythroid cells supply essential iron to other organs during iron deficiency [
35
]. Here
ferroportin may also function to mitigate iron toxicity by releasing excess iron not used for
hemoglobin synthesis.
Iron-recycling macrophages, found in the spleen (but also in the liver and the marrow),
are intermittently confronted with large boluses of hemoglobin that must be digested to
release heme, which is then degraded via heme oxygenase to release iron. This process
is dependent on the derepression of multiple genes involved in this process, including
ferroportin that is required to export the recycled iron. The primary stimulus for the dere-
pression of these genes is heme, sensed by the transcriptional repressor Bach1 (Btb And Cnc
Homology 1) that binds heme and dissociates from its partner the small Maf protein bound
to the promoter element MARE/ARE (Maf-recognition element/antioxidant response
element). Bach1 is replaced by the transcriptional inducer Nrf2 which is stabilized by heme,
translocated into the nucleus, and stimulates the transcription of the ferroportin gene and
other genes involved in the recycling of heme to iron [
36
,
41
]. At high concentrations of
Int. J. Mol. Sci. 2021,22, 6493 8 of 13
iron, ferroportin mRNA and protein are increased, presumably because of oxidative stress
causing Nrf2-mediated induction of ferroportin [42,43].
There is evidence that microRNAs such as miR-485-3p, miR-20a and miR-20b, can
posttranscriptionally downregulate ferroportin but the physiological role of this mechanism
is uncertain [37,39,40].
9. Local Functions of Ferroportin and Its Autocrine/Paracrine Regulation by Hepcidin
The “professional” iron exporting cell types, i.e., duodenal enterocytes, iron-recycling
macrophages and iron-storing hepatocytes, all deliver iron to blood plasma for use in
hemoglobin synthesis by erythroblasts as well as for other uses in iron-consuming tis-
sues. However, ferroportin is also found in other tissues where it appears to serve a
cell-autonomous function of releasing unneeded and potentially toxic iron.
Ferroportin is expressed at a remarkably high level in erythroblasts and mature
erythrocytes. In erythroblasts, it may release unobligated iron and here it is subject to
hepcidin regulation [
44
]. In mature erythrocytes, which lack an endocytic mechanism and
may therefore be less sensitive to inhibition of iron export by systemic hepcidin, ferroportin
may serve as a safety valve to release labile iron leaking from hemoglobin molecules
damaged by repeated cycling between high and low oxygen states. The importance of these
mechanism is illustrated by the adverse consequences of erythroid-specific disruption of
the ferroportin gene in mice, which suffer from mild hemolytic anemia caused by increased
sensitivity of erythrocytes to hemolytic stress [45,46].
Ferroportin is required for normal functioning of cardiac myocytes [
47
], presumably
to remove excess iron. Cardiac myocytes also produce hepcidin but this does not contribute
significantly to systemic hepcidin levels. Rather, hepcidin acts as an autocrine or paracrine
regulator of ferroportin that may protect tissues from loss of iron through ferroportin and
extreme iron deficiency when systemic hepcidin concentrations are very low [47].
Macrophage ferroportin also plays an important role in iron recycling after muscle
injury [
48
]. Selective loss of macrophage ferroportin in mice with muscle injury lim-
ited local iron recycling, impaired muscle healing and resulted in smaller myofibers and
fat accumulation.
In a mouse model of colitis, the hepcidin-ferroportin interaction in the intestine was
shown to be important for mucosal healing [
49
]. Absence of local hepcidin production
or insensitivity of macrophage ferroportin to hepcidin is thought to result in higher local
extracellular iron concentrations, affecting the growth of luminal and tissue-infiltrating
bacteria and worsening DSS-induced colitis.
During pregnancy, ferroportin is very highly expressed on the basal surface of syncy-
tiotrophoblast in the placenta [
7
,
50
], where it plays an indispensable role in iron transfer to
the fetus. Placental ferroportin appears to be strongly regulated by the trophoblast intracel-
lular iron concentrations, via the IRE-IRP system [
50
]. Interestingly, in normal pregnancy,
fetal hepcidin concentrations are very low in mice [50] and do not appear to contribute to
baseline iron homeostasis or affect iron transport through placental ferroportin. However,
fetal hepatic production of hepcidin may be sufficient to act on fetal hepatic ferroportin in
an autocrine/paracrine manner to retain iron in the fetal liver [
51
] where it is used for fetal
erythropoiesis until erythropoiesis transitions to the marrow, close to birth.
10. Functions of the Hepcidin-Ferroportin Axis in Host Defense
Infection and inflammation induce hepcidin, predominantly through the effects of the
cytokine interleukin-6 (IL-6) [
52
], and increased concentrations of hepcidin then inhibit
ferroportin activity, leading to depletion of iron in plasma and extracellular fluid (hypofer-
remia). Independently of hepcidin, microbial molecules that stimulate toll-like receptors
suppress cellular ferroportin mRNA transcription [
34
] sufficiently to cause hypoferremia
but it is not clear how much this effect contributes to hypoferremia generated during
infections
in vivo
. As ferroportin is much older on the evolutionary timeline than hepcidin,
Int. J. Mol. Sci. 2021,22, 6493 9 of 13
studies of the hypoferremic effects of infection and inflammation in invertebrates, which
lack hepcidin, should be illuminating.
Hypoferremia has long been suspected of having a host defense function but direct
evidence for this is quite recent [
53
]. Since transferrin-bound iron is poorly accessible to
most microbes, the most important iron species that supports microbial growth is non-
transferrin bound iron (NTBI) [
54
]. During infections, increased cytokine concentrations
inhibit erythropoiesis and activate the production of myeloid cells important for host
defense [
55
]. The inhibition of erythropoiesis decreases the consumption of plasma iron, just
as increased recycling of damaged erythrocytes and other cells during infections increases
iron delivery to plasma. The imbalance would raise the saturation of transferrin by iron
and enhance the production of NTBI, thereby increasing the risk of microbial outgrowth.
However, inflammatory increase of hepcidin and the effects of inflammation on ferroportin
transcription prevent the generation of NTBI by sequestering iron, predominantly inside
macrophages. This form of “nutritional immunity” is particularly important in infections
with gram negative bacteria and some fungi ([
54
,
56
58
]. Hepcidin can also kill microbes on
contact
in vitro
[
59
,
60
] but the concentrations required for this direct microbicidal activity
exceeded its concentrations in extracellular fluid, even in inflamed models.
11. Disorders of the Hepcidin-Ferroportin System
The hepcidin-ferroportin system, in analogy to other endocrine systems, can be dis-
rupted by mutations that affect the quantity or functionality of the ligand hepcidin or
its receptor ferroportin. Loss-of-function mutations in positive regulators of hepcidin or
the hepcidin gene itself (Table 2) cause hereditary hemochromatosis [
61
], a set of diseases
characterized by increased absorption of dietary iron, often causing massively increased
body iron content, and high plasma iron concentrations that saturate the iron-binding
capacity of transferrin, leading to the appearance of non-transferrin-bound iron (NTBI).
In affected patients who are not treated, NTBI is taken up avidly through alternative iron
transporters [
62
] in the liver, pancreas, heart and endocrine glands, causing progressive
tissue injury, organ failure, and even premature death. Two forms of hemochromatosis
define its clinical spectrum. At one extreme is the severe juvenile form that can cause severe
heart disease, endocrinopathy and death in young adults and at the other extreme are much
less severe adult forms which can even be clinically silent and not cause organ damage.
Loss-of-function ferroportin mutations cause iron overload that is usually localized to
macrophages, cells that use ferroportin to export iron recycled from senescent erythrocytes,
and causes increased serum ferritin but not clinically significant disease.
Table 2. Forms of hereditary hemochromatosis.
Mutated Gene Function Hepcidin Tissue Iron Clinical Severity
HFE Loss Low Liver, heart, endocrine Moderate, adult form
TFR2 Loss Low Liver, heart, endocrine Moderate, adult form
Hemojuvelin Loss Very low Heart, endocrine, liver Severe, juvenile form
Hepcidin Loss Very low to absent Heart, endocrine, liver Severe, juvenile form
Ferroportin Gain High Liver, heart, endocrine Variable, some severe
Ferroportin Loss Normal or low Spleen macrophages Mild
Loss-of-function mutations in the negative regulator of hepcidin transcription, the
transmembrane serine protease TMPRSS6 (also called matriptase 2), in mice or humans
cause a disease “iron-refractory iron deficiency anemia” (IRIDA) where hepcidin is ex-
cessive despite severe iron deficiency [
63
,
64
]. Elevated hepcidin inhibits intestinal iron
absorption and the release of stored and recycled iron, leading to low plasma iron concen-
trations and a microcytic anemia.
Hepcidin concentrations can also become dysregulated as a consequence of diseases
that affect iron absorption from the diet or cause a loss of blood from the body (each 1 mL of
packed erythrocytes represents about 1 mg of iron), produce systemic inflammation, make
Int. J. Mol. Sci. 2021,22, 6493 10 of 13
erythropoiesis ineffective (lots of erythrocyte precursors but few mature) or cause anemia
requiring erythrocyte transfusions without matching blood loss. These disease pathways
are summarized in Table 3. Iron deficiency, iron deficiency anemia [
65
] and anemia of
inflammation [66] are very common disorders affecting a large portion of the population.
Table 3. Disorders with dysregulated hepcidin production.
Disease State Serum Hepcidin Plasma Iron Body Iron Common Phenotypes
Iron deficiency Low Low Low Fatigue, anemia
Systemic inflammation High Low Normal Fatigue, anemia
Ineffective erythropoiesis Low High Increased Like hemochromatosis
Erythrocyte transfusions High High Increased Like hemochromatosis
12. Summary
The hepcidin-ferroportin system evolved to maintain stable body iron content and
plasma iron concentrations. The system also plays an important role in innate immunity,
particularly in host defense against gram-negative bacteria and some fungi. Dysregulation
of hepcidin or ferroportin production or their interaction underlies the pathogenesis of
a spectrum of iron disorders, from iron restrictive anemias to iron overload conditions.
Thus, plasma hepcidin measurements may be useful for diagnosing iron disorders, and
therapeutic targeting of the hepcidin-ferroportin system is a promising new direction to
develop improved treatments for iron disorders.
Author Contributions:
Conceptualization, T.G. and E.N. Writing, T.G. and E.N. All authors have
read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest:
E.N. and T.G. are shareholders of and scientific advisors for Intrinsic Life-
Sciences and Silarus Therapeutics and are consultants for Ionis Pharmaceuticals, Protagonist Thera-
peutics, Akebia, Vifor Pharma, and Disc Medicine.
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... Hepcidin deficiency is the cause of iron overload in hereditary hemochromatosis, iron-loading anemia, and its excess is associated with anemia of inflammation, chronic disease and iron deficiency anemia (IDA) (Pagani et al., 2019). Liver is the main source of Hepc (Nemeth and Ganz, 2021). In addition to the liver, other organs also produce Hepc peptides to a very less extent, including the heart, brain, lung, prostate, adrenal gland, thyroid, urinary bladder, intestines and kidney (Sukhbaatar and Weichhart, 2018). ...
... The hepcidin-ferroportin axis plays a lucid role in the development of cancers, particularly in the growth of tumors and metastases . Ferroportin is an iron exporter present on the surface of absorptive enterocytes, macrophages, hepatocytes, and placental cells (Nemeth and Ganz, 2021). This binding led to internalize and degrade of ferroportin, leading to decreased export of cellular iron; so, when hepc levels increase the iron absorption will decrease (Gammella et al., 2021). ...
... Hepcidin importance in iron metabolism stems from its ability to control iron export from cells (Nemeth and Ganz, 2021). This action of hepc is realized through the actions of ferroportin (FPN) (Vela and Vela-Gaxha, 2018). ...
Thesis
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Abstract Endometrial carcinoma (EC) is a common malignancy in female reproductive system, and its incidence is increasing. Surgery and postoperative radiotherapy are routinely used methods for treating this condition, but there is still a lack of effective treatment for recurrent or progressive EC. Thus, there is an urgent need to identify additional biological markers for the prognostic prediction of EC. Fifty seven patients with EC and 55 healthy women (with age up to 35 year) were involved in this study during their attendance at the Medicine City Hospital of Baghdad and Al-Yarmok hospital. The study was conducted from October 2020 to October 2021, written informal consent was obtained from all patients and the study was approved by ethical committees: Ref.: CSEC/0122/0061, January 20, 2022 of department of Biology, College of Science, University of Baghdad. Blood samples were collected from each participants via vein puncture for assessment the follwing interleukins: interleukin-27 (IL-27), interleukin-31 (IL31), interleukin -33 (IL-33), interleukin- 35 (IL-35), interleukin-36 (IL-36) and interleukin-38 (IL38). Some proteins also assesment in this study, include: Transforming growth factor-beta (TGF�β), Hepcidin (Hepc), Phosphatase and Tensin Homolog (PTEN), C-reactive protein (CRP) and Tumor marker Human epididymis protein 4 (HE4). As well as, the fresh blood was collected for deoxyribonucleic acid (DNA) extraction. PCR products: was performed for sequencing by the genetic analyzer, for forward and reverse primers, in macrogen company; (South Korea) to detect the polymorphism in of rs2735343 SNP of PTEN gene in EC patients. The results in present study showed a highly significant (P≤0.01) increase in IL-27, IL-31, IL-33, IL-35, IL-36 and IL-38 concentration in women with EC compared with healthy controls. Also, statistical analyses of this study showed that there was a highly significant (P≤0.01) increase II of TGF- β, HE4 hepc hormone level and CRP in women with EC compared with healthy controls. While, PTEN protein concentration was show highly significant (P≤0.01) decrease in EC patients compared with healthy control. DNA isolated from: patients and controls was amplification of PTEN by using new set of primers to amplify 450 bp from rs2735343 SNP for being use in sequencing. The genotypes frequency of C allele is in higher in EC rs2735343 (0.54) than healthy (0.17), while G allele was lowest percentage, in patients (0.46) than in healthy women (0.83). The SNP rs2735343 genotypes frequency in patients describe as follow: GG (18.18%), GC (56.36%) and CC (25.45%). While, in healthy group, the genotypes frequency was: GG (70.18%), GC (26.32%) and CC (3.50%). The SNP rs2735343 genotypes frequency showed a highly significant different (P≤0.01) in Wild type (GG) in control grop when compered with patients of EC. And highly, significant (P≤0.01) increase in mutant genotype (CC) in EC patient when compered with healthy control. The result show significant (P≤0.05) increase in heterozygous (GC) genotype in EC patient compered with healthy women. It can be concluded that the increase the level of some interleukins like: IL-27, 31, 33, 35, 36 and 38 could be used in the early diagnosis for EC and in accurate prognostic diagnosis. Also, TGF- β and HE-4 proteins could be used in the early diagnosis for EC. Addition of hepcidin and CRP estimation to TGF- β and HE-4 proteins results in accurate prognostic diagnosis. Polymorphesm of rs2735343 SNP in PTEN gene also can help in predignosis of EC.
... High-dose vitamin D supplementation decreases serum levels of hepcidin in healthy adults [40]. Hepcidin production is enhanced due to extra plasma iron, ultimately preventing dietary iron uptake, thereby avoiding further iron absorption [41]. ...
Article
We investigated the correlations of serum and dietary intake of iron with low-grade inflammation as well as with circulating hepcidin in adult Arabs with or without type 2 diabetes mellitus (T2DM). Three hundred and twelve (N=312) Saudi adult males and females with a mean age of 56.3 ± 6.5 years were included and divided into two groups, the control group (n=151, 43 males, 108 females), and T2DM group (n=161, 58 males, 103 females). Data included demographic characteristics, medical history, and dietary intake using food frequency and a 24-hour dietary recall for 1 day. Anthropometric measurements were noted and fasting blood samples extracted for the analysis of glucose, lipids, iron indices, hepcidin, 25(OH)D and endotoxin using commercially available assays. Hepcidin levels among T2DM participants were significantly higher than the control group (P<0.001). In all participants, serum hepcidin was positively associated with WHR, HbA1c, TG and TSAT while inversely associated with LDL-C and ferritin. Using hepcidin as dependent variable and age, anthropometrics, blood pressure, glucose, lipids, 25(OH)D, serum iron, transferrin and ferritin as independent variables showed that only glucose and WHR significantly predicted hepcidin by as much as 33.5% of the variances perceived (P<0.001). Sub-analysis in female participants revealed that endotoxin, iron and 25(OH)D were significant predictors of hepcidin, predicting 26.8% of the variances perceived (P<0.001). To conclude, the present study suggests that hepcidin is significantly linked with major cardiometabolic parameters, while its influence in iron indices, including low grade inflammation, appears to be stronger in females.
... An excess of iron stimulates the production of hepcidin that in turn inhibits the absorption of iron from the intestine. On the contrary, hepcidin is suppressed in iron deficiency, allowing dietary iron absorption and replenishment of iron storage [7,8]. In inflammation and infection, an increase in the hepcidin level can be observed, likely linked to the host defense mechanism to subtract iron from the infecting microorganisms [9]. ...
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Coronavirus disease 2019 (COVID-19) presents a clinical spectrum that ranges from a mild condition to critical illness. Patients with critical illness present respiratory failure, septic shock and/or multi-organ failure induced by the so called “cytokine storm”. Inflammatory cytokines affect iron metabolism, mainly inducing the synthesis of hepcidin, a hormone peptide not routinely measured. High levels of hepcidin have been associated with the severity of COVID-19. The aim of this study was to analyze, retrospectively, the levels of hepcidin in a group of COVID-19 patients admitted to the intensive care unit (ICU) of the Policlinico Tor Vergata of Rome, Italy. Thirty-eight patients from November 2020 to May 2021 were enrolled in the study. Based on the clinical outcome, the patients were assigned to two groups: survivors and non-survivors. Moreover, a series of routine laboratory parameters were monitored during the stay of the patients in the ICU and their levels correlated to the outcome. Statistical differences in the level of hepcidin, D-dimer, IL-6, LDH, NLR, neutrophils level, CRP, TNF-α and transferrin were observed between the groups. In particular, hepcidin values showed significantly different median concentrations (88 ng/mL vs. 146 ng/mL) between survivors and non-survivors. In addition, ROC curves analysis revealed sensitivity and specificity values of 74% and 76%, respectively, at a cut-off of 127 (ng/mL), indicating hepcidin as a good biomarker in predicting the severity and mortality of COVID-19 in ICU patients.
... ID can be characterized by (i) depleted iron stores linked with a decrease in the total body iron supply due to insufficient nutritional iron intake, impaired absorption or chronic blood loss, i.e. absolute ID; and/or (ii) reduced circulating iron, which can be linked to a persistent inflammatory state as in many CV diseases, i.e. functional ID. 2 Inflammation leads to an increased release of hepcidin, a liverexpressed type II acute phase protein and a major player in iron homeostasis. 3 Hepcidin regulates the degradation of ferroportin, a transmembrane protein which transports iron absorbed after dietary intake from the inside of the mucosal cells in the small intestine to the bloodstream, and also mediates the release of recycled iron from macrophages in the spleen and the liver 4 ( Figure 1). Therefore, ID observed with CV diseases may be due to the increased levels of hepcidin linked with a chronic inflammatory status, which leads to decreased iron absorption and mobilization from the reticuloendothelial system, i.e. functional ID. ...
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Iron deficiency (ID) is common in patients with cardiovascular disease. Up to 60% of patients with coronary artery disease, and an even higher proportion of those with heart failure (HF) or pulmonary hypertension have ID; the evidence for cerebrovascular disease, aortic stenosis and atrial fibrillation is less robust. The prevalence of ID increases with the severity of cardiac and renal dysfunction and is probably more common amongst women. Insufficient dietary iron, reduced iron absorption due to increases in hepcidin secondary to the low-grade inflammation associated with atherosclerosis and congestion or reduced gastric acidity, and increased blood loss due to anti-thrombotic therapy or gastro-intestinal or renal disease may all cause ID. For older people in the general population and patients with HF with reduced ejection fraction (HFrEF), both anaemia and ID are associated with a poor prognosis; each may confer independent risk. There is growing evidence that ID is an important therapeutic target for patients with HFrEF, even if they do not have anaemia. Whether this is also true for other HF phenotypes or patients with cardiovascular disease in general is currently unknown. Randomized trials showed that intravenous ferric carboxymaltose improved symptoms, health-related quality of life and exercise capacity and reduced hospitalizations for worsening HF in patients with HFrEF and mildly reduced ejection fraction (<50%). Since ID is easy to treat and is effective for patients with HFrEF, such patients should be investigated for possible ID. This recommendation may extend to other populations in the light of evidence from future trials.
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Postcovid syndrome is a serious public health problem affecting millions of people worldwide. There is a growing body of evidence that people may exhibit symptoms after organ damage developed during the acute phase of COVID‑19, while others experience new symptoms after a mild infection, without any evidence of acquired organ or tissue damage. In this regard, understanding the determinants and regulators of COVID‑19 and postcovid syndrome pathology is an important clinical challenge that will enable better management of future variants and pandemic waves. There is presumably a close relationship between iron homeostasis, COVID‑19, and postcovid syndrome, the pathogenetic aspects of which have yet to be determined. Nevertheless, the available literature already indicates that iron deficiency and iron deficiency anemia (without inflammatory anemia) in patients with postcovid syndrome occur in 30% and 9% of cases, respectively. Given the importance and urgency of this problem and the fact that one in three patients with postcovid syndrome may have iron deficiency, this article presents a case from clinical practice in which a patient with postcovid syndrome was found to have iron deficiency and iron deficiency anemia, and treatment of this condition and disease resulted in improvement in general well-being and regression of symptoms. Thus, the effects of COVID‑19 on iron metabolism exist, and they can be corrected. The use of oral iron preparations, in particular iron sulfate, allows optimal therapeutic and clinical effects in this clinical situation along with a good tolerability and safety profile.
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“Eryptosis” of mature mammalian red blood cells (RBCs) is widely acknowledged. As compared to mammalians, the teleost RBCs possess a nucleus and other cellular organelles, but the mode of death of teleost RBCs remains unclear. We isolated the RBCs from circulating blood of grass carp (Ctenopharyngodon idella) and cultured in saline solution (to simulate the cells lacking of nutrients) and normal L15 medium (to simulate the cells in circulation), respectively, to investigate the changes in cell morphology during death, which showed the death of RBCs were accompanied the presence of autolysosome analogues. Flow cytometry analysis revealed that the prolonged exposure time in saline solution or L15 medium increased the accumulation of reactive oxygen species and nitric oxide in the cells, promoted cell death. To further explore which death model occurred in the RBCs, four death inhibitors were used, which showed the ferroptosis inhibitor, Fer-1, to play a key role in rescuing the viability of RBCs, indicated ferroptosis to be the main player in the death of RBCs. The results of quantitative real-time PCR demonstrated that the exposure time modulated the expression of iron metabolism-related genes in RBCs. In addition, prolonged exposure of RBCs was accompanied by an increase in lipid peroxidation and decreased GSH-PX activity. Taken together, the above data suggest that ferroptosis may be the main mode of death of RBCs in grass carp, shedding new light on the biological characteristics of teleost RBCs.
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With aging, iron will accumulate in the brain, catalyzing oxidative radicals that damage brain neurons and induceAlzheimer's disease. In this experiment, we observed that in the brains of senile mice, the iron-exporting protein ferriportin was decreased. Further investigation indicated that H3K27 of the slc40a1 gene was methylated and that the methyltransferase Ezh2 was activated. To discharge the excessive iron in the brains of senile mice, we developed the YP peptide, and with it, we demethylated H3K27me of Slc40a1 by phosphorylation, activating the demethylase Kdm6a and inhibiting methyltransferase Ezh2. Consequently, the transcription of Slc40a1 was enhanced,and ferriportin was resumed. Thesenile brain reacquired the function of iron excretion, and as a result, the radicals in the brain weredecreased,and neuron damage was reduced, which finally restored the intelligence of dementia senile mice. Thus, YP could be a potential drug in the prevention of Alzheimer's disease
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We report herein a novel mechanism, unraveled by proteomics and validated by in vitro and in vivo studies, of the aberrant aging-associated upregulation of ovarian transferrin and ferritin in rat ovaries. The ovarian mass and serum estradiol titer plummeted while the ovarian labile ferrous iron and total iron levels escalated with age in rats. Oxidative stress markers, such as nitrite/nitrate, 3-nitrotyrosine, and 4-hydroxy-2-nonenal, accumulated in the aging ovaries due to an aberrant upregulation of the ovarian transferrin, ferritin light/heavy chains, and iron regulatory protein 2(IRP2)-mediated transferrin receptor 1 (TfR1). Ferritin inhibited estradiol biosynthesis in ovarian granulosa cells in vitro via the upregulation of a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and p65/p50-induced oxidative and inflammatory factor inducible nitric oxide synthase (iNOS). An in vivo study demonstrated how the age-associated activation of NF-κB induced the upregulation of iNOS and the tumor necrosis factor α (TNFα). The downregulation of the keap1-mediated nuclear factor erythroid 2-related factor 2 (Nrf2), that induced a decrease in glutathione peroxidase 4 (GPX4), was observed. The aberrant transferrin and ferritin upregulation triggered an iron accumulation via the upregulation of an IRP2-induced TfR1. This culminates in NF-κB-iNOS-mediated ovarian oxi-inflamm-aging and serum estradiol decrement in naturally aging rats. The iron accumulation and the effect on ferroptosis-related proteins including the GPX4, TfR1, Nrf2, Keap1, and ferritin heavy chain, as in testicular ferroptosis, indicated the triggering of ferroptosis. In young rats, an intraovarian injection of an adenovirus, which expressed iron regulatory proteins, upregulated the ovarian NF-κB/iNOS and downregulated the GPX4. These novel findings have contributed to a prompt translational research on the ovarian aging-associated iron metabolism and aging-associated ovarian diseases.
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Background/aims Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide. Iron overload has been implicated in chronic non-communicable liver diseases, but its relationship with NAFLD remains unclear. This study aimed to investigate the underlying roles of iron overload in the development of NAFLD. Methods Male Sprague Dawley rats were fed with a high-fat diet (HFD) and/or iron for 8, 12, and 20 weeks. Some rats fed with HFD plus iron also received intraperitoneal injection of deferoxamine (DFO) for 8 weeks. Liver steatosis, lipid metabolism and injury were evaluated. Results A NAFLD model, including typical liver steatosis, was established by feeding rats with a HFD, while iron overload alone is not enough to induce severe NAFL. Compared with rats fed a HFD, excess iron further increased lipid accumulation, serum levels of lipids, enzymes of liver function, and expression levels of CD36 and FAS in rat liver. In addition, iron overload decreased the activities of antioxidative enzymes in liver compared with HFD rats. The levels of CPT1 and the ratios of p-ACC/ACC were also decreased by iron overload. DFO effectively reversed the abnormal lipid metabolism and liver damage induced by a high-fat, high-iron diet. Conclusion A HFD plus iron overload might synergistically aggravate lipid metabolism disorders, liver injury, and oxidative damage, compared with a HFD alone. DFO might help to alleviate lipid metabolism dysfunction and improve the pathogenesis of NAFLD.
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Ferroportin (FPN), the body's sole iron exporter, is essential for maintaining systemic iron homeostasis. In response to either increased iron or inflammation, hepatocyte-secreted hepcidin binds to FPN, inducing its internalization and subsequent degradation. However, the E3 ubiquitin ligase that underlies FPN degradation has not been identified. Here, we report the identification and characterization of a novel mechanism involving the RNF217-mediated degradation of FPN. A combination of two different E3 screens revealed that the Rnf217 gene is a target of Tet1, mediating the ubiquitination and subsequent degradation of FPN. Interestingly, loss of Tet1 expression causes an accumulation of FPN and an impaired response to iron overload, manifested by increased iron accumulation in the liver together with decreased iron in the spleen and duodenum. Moreover, we found that the degradation and ubiquitination of FPN could be attenuated by mutating RNF217. Finally, using two conditional knockout mouse lines, we found that knocking out Rnf217 in macrophages increases splenic iron export by stabilizing FPN, whereas knocking out Rnf217 in intestinal cells appears to increase iron absorption. These findings suggest that the Tet1-RNF217-FPN axis regulates iron homeostasis, revealing new therapeutic targets for FPN-related diseases.
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The serum iron level in humans is tightly controlled by the action of the hormone hepcidin on the iron efflux transporter ferroportin. Hepcidin regulates iron absorption and recycling by inducing ferroportin internalization and degradation1. Aberrant ferroportin activity can lead to diseases of iron overload, such as hemochromatosis, or iron limitation anemias2. Here, we determined cryogenic electron microscopy (cryo-EM) structures of ferroportin in lipid nanodiscs, both in the apo state and in complex with cobalt, an iron mimetic, and hepcidin. These structures and accompanying molecular dynamics simulations identify two metal binding sites within the N- and C-domains of ferroportin. Hepcidin binds ferroportin in an outward-open conformation and completely occludes the iron efflux pathway to inhibit transport. The carboxy-terminus of hepcidin directly contacts the divalent metal in the ferroportin C-domain. We further show that hepcidin binding to ferroportin is coupled to iron binding, with an 80-fold increase in hepcidin affinity in the presence of iron. These results suggest a model for hepcidin regulation of ferroportin, where only iron loaded ferroportin molecules are targeted for degradation. More broadly, our structural and functional insights are likely to enable more targeted manipulation of the hepcidin-ferroportin axis in disorders of iron homeostasis.
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β-thalassemia is a genetic anemia caused by partial or complete loss of β-globin synthesis leading to ineffective erythropoiesis and RBCs with short life-span. Currently, there is no efficacious oral medication modifying anemia for patients with beta-thalassemia. The inappropriately low levels of the iron regulatory hormone hepcidin enable excessive iron absorption by ferroportin, the unique cellular iron exporter in mammals, leading to organ iron overload and associated morbidities. Correction of unbalanced iron absorption and recycling by induction of hepcidin synthesis or treatment with hepcidin mimetics ameliorates β-thalassemia. However, hepcidin modulation or replacement strategies currently in clinical development all require parenteral drug administration. We identified oral ferroportin inhibitors by screening a library of small molecular weight compounds for modulators of ferroportin internalization. Restricting iron availability by VIT-2763, the first clinical stage oral ferroportin inhibitor, ameliorated anemia and the dysregulated iron homeostasis in the Hbbth3/+ mouse model of beta-thalassemia intermedia. VIT-2763 not only improved erythropoiesis but also corrected the proportions of myeloid precursors in spleens of Hbbth3/+ mice. VIT-2763 is currently developed as an oral drug targeting ferroportin for the treatment of β-thalassemia.
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Erythroferrone (ERFE) is the main erythroid regulator of hepcidin, the homeostatic hormone controlling plasma iron levels and total body iron. When the release of erythropoietin from the kidney stimulates the production of new red blood cells, it also increases the synthesis of ERFE in bone marrow erythroblasts. Increased ERFE then suppresses hepcidin synthesis, thereby mobilizing cellular iron stores for use in heme and hemoglobin synthesis. Recent mechanistic studies have shown that ERFE suppresses hepcidin transcription by inhibiting bone morphogenetic protein signaling in hepatocytes. In ineffective erythropoiesis, pathological overproduction of ERFE by an expanded population of erythroblasts suppresses hepcidin and causes iron overload, even in non‐transfused patients. ERFE may be a useful biomarker of ineffective erythropoiesis and an attractive target for treating its systemic effects.
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Background Iron export via the transport protein ferroportin (Fpn) plays a critical role in the regulation of dietary iron absorption and iron recycling in macrophages. Fpn plasma membrane expression is controlled by the hepatic iron-regulated hormone hepcidin in response to high iron availability and inflammation. Hepcidin binds to the central cavity of the Fpn transporter to block iron export either directly or by inducing Fpn internalization and lysosomal degradation. Here, we investigated whether iron deficiency affects Fpn protein turnover. Methods We ectopically expressed Fpn in HeLa cells and used cycloheximide chase experiments to study basal and hepcidin-induced Fpn degradation under extracellular and intracellular iron deficiency. Conclusions/General significance We show that iron deficiency does not affect basal Fpn turnover but causes a significant delay in hepcidin-induced degradation when cytosolic iron levels are low. These data have important mechanistic implications supporting the hypothesis that iron export is required for efficient targeting of Fpn by hepcidin. Additionally, we show that Fpn degradation is not involved in protecting cells from intracellular iron deficiency.
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Iron-containing proteins rely on the incorporation of a set of iron cofactors for activity. The cofactors must be synthesized or assembled from raw materials located within the cell. The chemical nature of this pool of raw material - referred to as the labile iron pool – has become clearer with the identification of micro- and macro-molecules that coordinate iron within the cell. These molecules function as a buffer system for the management of intracellular iron and are the focus of this review, with emphasis on the major iron chaperone protein coordinating the labile iron pool: poly C-binding protein 1.
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In the adult, the liver-derived hormone hepcidin (HAMP) controls systemic iron levels by blocking the iron-exporting protein ferroportin (FPN) in the gut and spleen, the sites of iron absorption and recycling respectively. Impaired HAMP expression or FPN responsiveness to HAMP result in iron overload. HAMP is also expressed in the fetal liver but its role in controlling fetal iron stores is not understood. To address this question in a manner that safeguards against the confounding effects of altered maternal iron homeostasis, we generated fetuses harbouring a paternally-inherited ubiquitous knock-in of the HAMP-resistant fpnC326Y. Additionally, to safeguard against any confounding effects of altered placental iron homeostasis, we generated fetuses with a liver-specific knock-in of fpnC326Y or knockout of the hamp gene. These fetuses had reduced liver iron stores and hemoglobin, and markedly increased FPN in the liver, but not in the placenta. Thus, fetal liver HAMP operates cell-autonomously to increase fetal liver iron stores. Our findings also suggest that FPN in the placenta is not actively regulated by fetal liver HAMP under normal physiological conditions.
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Ironing out the details of mucosal healing Anemia is a frequent complication of disorders such as inflammatory bowel disease, occurring in part as a result of increased bleeding into the intestine. Bessman et al. show that the peptide hormone hepcidin, which regulates systemic iron homeostasis, is required for intestinal repair in a mouse model of inflammatory bowel disease (see the Perspective by Rescigno). This effect was independent of hepatocyte-produced hepcidin and systemic iron levels. Instead, production of hepcidin by conventional dendritic cells was necessary and sufficient to promote local iron sequestration by macrophages, which in turn modulated the makeup of the gut microbiota to one with a more beneficial distribution of species. Science , this issue p. 186 ; see also p. 129
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Iron deficiency is common worldwide and is associated with adverse pregnancy outcomes. The increasing prevalence of indiscriminate iron supplementation during pregnancy also raises concerns about the potential adverse effects of iron excess. We examined how maternal iron status affects the delivery of iron to the placenta and fetus. Using mouse models, we documented maternal homeostatic mechanisms which protect the placenta and fetus from maternal iron excess. We determined that under physiological conditions or in iron deficiency, fetal and placental hepcidin does not regulate fetal iron endowment. With maternal iron deficiency, critical transporters mediating placental iron uptake (transferrin receptor 1, TFR1) and export (ferroportin, FPN) were strongly regulated. In mice, not only was TFR1 increased but FPN was surprisingly decreased to preserve placental iron, in the face of fetal iron deficiency. In human placentas from pregnancies with mild iron deficiency, TFR1 was increased but without a change in FPN. However, induction of more severe iron deficiency in human trophoblast in vitro resulted in the regulation of both TFR1 and FPN, similarly to the mouse model. This placental adaptation prioritizing placental iron is mediated by the iron-regulatory protein 1 and is important for the maintenance of mitochondrial respiration, thus ultimately protecting the fetus from the potentially dire consequences of generalized placental dysfunction.