ZIP14 and DMT1 in the liver, pancreas, and heart are differentially regulated by iron deficiency and overload: implications for tissue iron uptake in iron-related disorders

Abstract

ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n . Collectively, these data suggest that ZIP14 may not only function during iron overload to take up NTBI, but also under normal or iron-deficient conditions when cells take up iron via endocytosis of transferrin. The aim of the present study was to determine how iron deficiency and overload affect the expression of ZIP14 and DMT1 in the liver, pancreas, and heart. The localization of ZIP14 in liver and pancreas was also determined. Knowledge of where ZIP14 is expressed in these organs and how ZIP14 and DMT1 are regulated in vivo by iron will help us to better assess the contribution of these transporters to tissue iron uptake. Design and Methods Animals, diets, and non-heme iron determinationRats were made iron-deficient, iron-adequate, or iron-loaded as described previously. 10 Briefly, weanling (21-day-old) male Sprague-Dawley rats were fed modified AIN-93G purified diets containing iron at 10 ppm (iron deficient, FeD), 50 ppm (iron adequate, FeA), or 18,916 ppm (iron overload, FeO) for 3 weeks. Male Zip14 (Slc39a14) knockout and wild-type control mice maintained on the 129+Ter/SvJcl x C57BL/6 background 11 were analyzed at 6 weeks of age. Male and female hypotransferrinemic (hpx) mice and wild-type controls maintained on the BALB/cJ background were analyzed at 16 weeks of age. Homozygous hpx mice were given a weekly intraperitoneal injection of human apo-transferrin (0.6-1.8 mg) (EMD Chemicals). All mice consumed a commercial rodent diet containing 240 ppm iron (Teklad 7912, Harlan Laboratories). At the end of the studies, animals were anesthetized with vaporized isoflurane and sacrificed by exsanguination via the descending aorta. Tissues were harvested, immediately frozen in liquid nitrogen, and stored at -80°C until use. Animal protocols were approved by the University of Florida Institutional Animal Care and Use Committee. Tissue non-heme iron concentrations were determined colorimetrically after acid digestion of tissues. 12 Generation of ZIP14 antibodyRabbit anti-ZIP14 antiserum was generated against peptide ENEQTEEGKPSAIEVC corresponding to amino acids 138-153 of rat ZIP14. Antibodies specific to the ZIP14 peptide immunogen were affinity purified by using a peptide-agarose column of SulfoLink coupling gel (Pierce). Sample preparation and western blot analysisThe preparation of samples and western blot analysis are described in the Online Supplementary Design and Methods. Transfection, immunoprecipitation, and enzymatic deglycosylationEffectene reagent (Qiagen) was used to transiently transfect HEK 293T cells with either empty pCMV-Sport2 (control) or pCMV-Sport6 containing rat ZIP14 cDNA. To immunoprecipitate ZIP14, anti-ZIP14 antibody (400 mg) was covalently linked to AminoLink Plus Coupling Resin (Thermo Scientific) and added to a Pierce Spin Column according to the manufacturer's protocol. Immunoprecipitations were performed using the CoImmunoprecipitation Kit (Pierce) according to the manufacturer's instructions. To assess protein glycosylati...

Hepatic metal ion transporter ZIP8 regulates manganese homeostasis and manganese-dependent enzyme activity

“…Compared with control mice, we observed that ZIP8-iKO mice had a decreased abundance of fully glycosylated N-glycan species (26.6% in WT vs. 16.2% in ZIP8-iKO mice) and an increased abundance of truncated N-glycan species, especially under-galactosylated N-glycan species including monosialo-monogalacto-biantennary glycans (1.0% in WT vs. 3.7% in ZIP8-iKO mice), asialo-monogalacto-biantennary glycans (0 in WT vs 1.7% in ZIP8-iKO mice), and asialo-agalactobiantennary glycans (0 in WT vs 1.4% in ZIP8-iKO mice). Similarly, compared with control mice, ZIP8-LSKO mice had a decreased abundance of fully glycosylated N-glycan species (41.0% in WT vs. 35.5% in ZIP8-LSKO mice) and an increased abundance of truncated N-glycan species, especially under-galactosylated N-glycan species monosialo-monogalacto-biantennary glycans (0.7% vs. 2.6%). Mn deficiency has also been found to moderately impair N-acetylglucosaminylation (32) mediated by another Mn-dependent glycosyltransferase, N-acetyl-glucosaminyltransferase II (33).…”

“…The current model of Mn metabolism by the liver is that hepatocytes take up Mn from blood at the basolateral surface and excrete it into the bile at the apical surface. ZIP14, a close family member of ZIP8, may be responsible for the uptake of Mn from blood, as it has been reported to be localized on the basolateral membrane of hepatocytes (35) and has affinity for Mn in vitro (28,36). Furthermore, patients carrying SLC39A14 mutations showed excessive Mn accumulation in the whole blood and brain but a lack of Mn in the liver, possibly due to the bypassing of hepatic uptake by Mn and subsequent biliary excretion in the absence of SLC39A14 (37).…”

An iron-regulated and glycosylation-dependent proteasomal degradation pathway for the plasma membrane metal transporter ZIP14

“…In keeping with its functions, ZIP14 is localized at the plasma membrane and in transferrin-containing endosomal compartments (1,2,4). Recent studies demonstrate that the level of ZIP14 protein is increased in the liver of rats fed a high iron diet and in ironloaded human hepatoma cells, suggesting that ZIP14 contributes to tissue iron loading under high-iron conditions (6). A tissue expression array shows that ZIP14 mRNA is ubiquitously expressed with high levels in the liver, pancreas, and heart (2).…”

Iron transport proteins: Gateways of cellular and systemic iron homeostasis

“…NTBI refers to a heterogeneous mixture of low-molecular-weight forms of iron that become detectable in plasma when transferrin saturations exceed 75% (71). NTBI is taken up by the liver via ZRT/IRT-like protein-14 (ZIP14/SLC39A14), a transmembrane metal-ion transporter located on the sinusoidal membrane of hepatocytes (72). ZIP14 was originally identified as a zinc transporter, but subsequent studies showed that it could also transport iron (73,74) and that its protein levels in liver increase in response to iron loading (72).…”

“…NTBI is taken up by the liver via ZRT/IRT-like protein-14 (ZIP14/SLC39A14), a transmembrane metal-ion transporter located on the sinusoidal membrane of hepatocytes (72). ZIP14 was originally identified as a zinc transporter, but subsequent studies showed that it could also transport iron (73,74) and that its protein levels in liver increase in response to iron loading (72). Slc39a14-null mice display markedly impaired uptake of intravenously administered 59 Fe-NTBI and fail to load iron in hepatocytes when the mice are fed an iron-loaded diet or crossed with mouse models of hereditary hemochromatosis (75).…”