The plasma membrane metal-ion transporter ZIP14 contributes to nontransferrin-bound iron uptake by human β-cells

Abstract

The relationship between iron and β-cell dysfunction has long been recognized as individuals with iron overload display an increased incidence of diabetes. This link is usually attributed to the accumulation of excess iron in β-cells leading to cellular damage and impaired function. Yet, the molecular mechanism(s) by which human β-cells take up iron has not been determined. In the present study, we assessed the contribution of the metal-ion transporters ZRT/IRT-like protein 14 and 8 (ZIP14 and ZIP8) and divalent metal-ion transporter-1 (DMT1) to iron uptake by human β-cells. Iron was provided to the cells as nontransferrin-bound iron (NTBI), which appears in the plasma during iron overload and is a major contributor to tissue iron loading. We found that overexpression of ZIP14 and ZIP8, but not DMT1, resulted in increased NTBI uptake by βlox5 cells, a human β-cell line. Conversely, siRNA-mediated knockdown of ZIP14, but not ZIP8, resulted in 50% lower NTBI uptake in βlox5 cells. In primary human islets, knockdown of ZIP14 also reduced NTBI uptake by 50%. Immunofluorescence analysis of islets from human pancreatic sections localized ZIP14 and DMT1 nearly exclusively to β-cells. Studies in primary human islets suggest that ZIP14 protein levels do not vary with iron status or treatment with IL-1β. Collectively, these observations identify ZIP14 as a major contributor to NTBI uptake by β-cells and suggest differential regulation of ZIP14 in primary human islets compared with other cell types such as hepatocytes.

iron is an essential trace mineral necessary for numerous biological functions, including dioxygen transport and oxidation-reduction reactions. Iron is utilized for these functions in part because of its ability to readily donate or accept electrons. Yet, the redox capacity of iron is not without consequences as iron can also catalyze the generation of highly reactive hydroxyl radicals (40), which can damage lipids, protein, and DNA (21, 41). Due to the duality of iron redox chemistry, iron transport and homeostasis are tightly regulated in vivo to prevent/mitigate iron-catalyzed generation of reactive oxygen species (11). However, in genetic disorders such as hemochromatosis, in which excessive amounts of dietary iron are absorbed, or β-thalassemia major, which requires lifelong blood transfusions, excess iron overwhelms the normal mechanisms of iron transport and homeostasis (4). One such consequence is the appearance of plasma nontransferrin-bound iron (NTBI), a form of iron that appears when the carrying capacity of transferrin, the circulating iron transport protein, becomes exceeded (3). The exact chemical nature of NTBI in the plasma is not known but is thought to consist mainly of ferric citrate and other low-molecular-weight iron species (9, 12). Although it is generally believed that NTBI is a pathologic species that appears only when transferrin saturation exceeds 75% (24), plasma NTBI has been reported to be commonly present in diabetics with transferrin saturations below 60% (25).

Studies in mice have shown that plasma NTBI is rapidly cleared mostly by the liver and, to a lesser extent, the pancreas, kidney, and heart (2, 6, 43). Accordingly, NTBI is a major contributor to iron loading of the liver and other tissues in iron-overload disorders. In the liver and pancreas, NTBI is taken up mainly by hepatocytes and acinar cells via ZRT/IRT-like protein 14 (ZIP14; SLC39A14) (19). How NTBI is taken up by the kidney, heart, and other organs/cell types remains to be established.

Studies of iron-loaded human pancreata have revealed that iron not only accumulates in acinar cells but also in β-cells of the islets (20, 28, 37). Iron loading of the β-cell has been proposed to contribute to the well-known β-cell dysfunction and diabetes in individuals with clinical iron overload (20, 22). Given the known role of NTBI uptake to iron loading of various organs and cells, we hypothesize that human β-cells are able to take up NTBI. The aim of the present study was to examine the potential roles of the transmembrane transporters divalent metal-ion transporter-1 (DMT1; SLC11A2), ZIP14, and ZIP8 (SLC39A8) in NTBI uptake by human β-cells. We focused on these three transporters because of their well-documented roles in NTBI uptake/iron metabolism (13, 14, 19, 27, 42) and because they have been reported to be expressed in pancreatic islets or β-cells (15, 16, 22, 35). We examined β-cell NTBI uptake by using βlox5 cells, a human β-cell line, and primary human islets. We further investigated the expression and distribution of ZIP14, DMT1, and ZIP8 in human islets by performing immunofluorescence analysis of human pancreas sections.

MATERIALS AND METHODS

Cell culture and treatments.

βlox5 cells were cultured in low glucose (1 g/l) Dulbecco’s modified Eagle’s medium (Cellgro) containing 100 U/ml penicillin, 100 μg/ml streptomycin, 10% fetal bovine serum (FBS; Atlanta Biologicals), 1% minimum Eagle’s medium non-essential amino acids (Corning), and 15 mM HEPES (Cellgro). Primary human islets from nondiabetic organ donors of at least 90% purity and viability were obtained from the Integrated Islet Distribution Program (IIDP) and were cultured in Ultra-Low attachment plates (Corning) with CMRL1066 medium containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Depletion of cellular iron or iron loading was performed by treating islets with 50 µM deferoxamine mesylate (Hospira) or 100 µM ferric ammonium citrate (MP Biomedicals) and 1 mM ascorbate (EMD Millipore), respectively, for 48 h. Where indicated, recombinant interleukin-1β (IL-1β; Peprotech) was added to the medium at 100 U/ml for 24 h. Before transfection, islets were dissociated by incubating in Accutase (Life Technologies) for 15 min at 37°C followed by pipetting to ensure a single cell suspension. All cells were maintained in 5% CO₂ at 37°C.

Determination of DMT1, ZIP8, and ZIP14 mRNA copy numbers.

Total RNA was isolated from primary human islets obtained from nondiabetic donors by using RNAzol (Molecular Research Center) following the manufacturer’s protocol. cDNA was synthesized from isolated RNA by using the High Capacity cDNA Archive Kit (Life Technologies). Quantitative RT-PCR was performed by using SYBR Select Master Mix (Life Technologies) and a CFX96 Real-Time PCR Detection System (Bio-Rad). Copy numbers of DMT1, ZIP8, and ZIP14 were calculated by comparing the C_t values from human islet cDNA samples to standard curves generated from known quantities of the plasmids pBluescriptR-hDMT1 (BC100014; Addgene), pCMV-Sport6-hZIP8 (BC012125; Open Biosystems), and pCMV-XL4-hZIP14 (BC015770; Open Biosystems). The following primers were used: DMT1 (forward, 5′-TGCATCTTGCTGAAGTATGTCACC-3′ and reverse, 5′-CTCCACCATCAGCCACAGGAT-3′); ZIP14 (forward, 5′-CAAGTCTGCAGTGGTGTTTG-3′ and reverse, 5′-GTGTCCATGATGATGCTCATTT-3′), and ZIP8 (forward, 5′-CAGTGTGGTATCTCTACAGGATGGA-3′ and reverse, 5′-CAGTTTGGGCCCCTTCAAA-3′). The primers, which target all known mRNA transcripts of DMT1, ZIP14, and ZIP8, were designed by using NCBI-Primer BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast).

siRNA knockdown of DMT1, ZIP8, and ZIP14.

SMARTpool siRNA targeting either human DMT1 or ZIP14 (Thermo Scientific) and Flexitube siRNA targeting ZIP8 (Qiagen) were used to suppress mRNA expression. Transfection was performed by using Lipofectamine RNAiMAX (Life Technologies) and Opti-MEM Medium (Life Technologies) for siRNA and reagent suspension following the manufacturer’s protocol to yield a final concentration of 12 nM siRNA after addition of the complex to plated cells. In brief, Opti-MEM medium was added to separate vials of either siRNA or Lipofectamine RNAiMAX, after which the contents of each vial were combined and incubated for 15 min. After incubation, 500 µl of the transfection mixture was added to each well of a six-well plate containing 2 ml of cell culture medium and cultured for 48 h before collection. Successful knockdown was confirmed by immunoblotting.

Overexpression of DMT1, ZIP8, and ZIP14.

Cultured βlox5 cells were transiently transfected with either pcDNA3.1hDMT1–1A/IRE+ (generously contributed by Dr. Natascha Wolff, University of Witten/Herdecke, Witten, Germany), pCMV-Sport6-hZip14 (BC015770), pCMV-Sport6-hZIP8 (BC012125), or pCMV-Sport6-empty vector by using Effectene Transfection Reagent (Qiagen) according to the manufacturer’s protocol. After 24 h, cells were harvested for confirmation of overexpression or used in iron uptake experiments. Isolation of cell-surface proteins was accomplished by using the Pierce Cell Surface Protein Isolation Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. In brief, cells were incubated with a cell-impermeable biotinylation reagent that was quenched before cell lysis, ensuring that only proteins located on the cell surface were biotinylated. Cell-surface proteins were then separated from intracellular proteins by incubating the cell lysates with NeutrAvidin Agarose Resin (Thermo Fisher Scientific) followed by column filtration, to remove unbound nonbiotinylated proteins, and elution of biotinylated cell-surface proteins.

Immunoblotting.

Cells were lysed and sonicated in RIPA buffer containing 150 mM sodium chloride, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris-base, and Complete, Mini Protease Inhibitor Cocktail (Roche). The RC DC Protein Assay Kit (Bio-Rad) was used to determine lysate protein concentrations. Lysate samples were mixed with Laemmli buffer and incubated at 37°C for 20 min before immunoblot analysis for ZIP14, ZIP8, and DMT1 or incubated at 95°C for 10 min for other proteins. The immunoblotting procedure and chemiluminescence detection were performed as previously described (5) with the exception of nitrocellulose replacing PVDF membranes. Primary antibodies used were rabbit anti-DMT1 (1:1,000, generously contributed by Dr. Francois Canonne-Hergaux, INSERM, Toulouse, France), rabbit anti-ZIP8 (1:5,000; Prestige Antibodies; Sigma-Aldrich), rabbit anti-ZIP14 (1:5,000; Prestige Antibodies, Sigma-Aldrich), rabbit anti-CCS (1:200; Santa Cruz Biotechnology), mouse anti-Na⁺-K⁺-ATPase (1:200; Santa Cruz Biotechnology), goat anti-ferritin light chain (1:4,000; Novus Biologicals), or mouse anti-α tubulin (1:5,000; Sigma-Aldrich).

Immunofluorescence.

Paraffin-embedded tail sections of human pancreata from nondiabetic organ donors were obtained through the Network for Pancreatic Organ Donation (nPOD; University of Florida). Paraffin was cleared with xylene and tissues were rehydrated in stages. After hydration, slides were subjected to heat-induced epitope retrieval in buffer containing 10 mM sodium citrate, 0.05% Tween 20, and adjusted to pH 6.0 with HCl. Slides were then briefly cooled in distilled H₂0 and washed with TBS to remove residual sodium citrate buffer. Washed slides were then incubated in blocking buffer containing 2% goat serum for 30 min to prevent nonspecific binding of secondary antibody. During the primary antibody incubation, human sections were triple stained for insulin, glucagon, and either DMT1, ZIP8, or ZIP14 by using guinea pig anti-insulin (1:200; Abcam), mouse anti-glucagon (1:1,000; Abcam), and either rabbit anti-DMT1 (1:1,000; Prestige Antibodies; Sigma-Aldrich), rabbit anti-Zip8 (1:250; Peprotech), or rabbit anti-ZIP14 (1:1000; Prestige Antibodies; Sigma-Aldrich) antibodies. During staining for ZIP8, slides were also permeabilized with 5% Triton X-100 for 15 min after antigen retrieval. To control for nonspecific primary antibody binding, serial sections were also triple stained with nonimmune rabbit IgG replacing the primary antibody for DMT1, ZIP8, or ZIP14 at the same concentration. Primary antibody incubations were performed at 4°C overnight in a humidified chamber. After primary incubation, slides were washed with TBS and incubated for 2 h at room temperature with the secondary antibodies, either goat anti-guinea pig Alexa Fluor 594 (1:250; Life Technologies) for insulin, goat anti-mouse Pacific Blue (1:250; Life Technologies) for glucagon, or goat anti-rabbit Alexa Fluor 488 (1:250; Life Technologies) for either DMT1, ZIP8, or ZIP14 to obtain a fluorescent signal. After this incubation period, slides were washed with TBS and coverslips were mounted. Confocal microscopy was performed and images obtained by using an Olympus IX2-DSU spinning disk confocal fluorescent microscope equipped with a Hamamatsu ORCA-AG camera and 3i SlideBook v4.2 software.

Cellular NTBI uptake.

Cultured cells were washed twice with serum-free medium (SFM) and incubated for 1 h in SFM containing 2% bovine serum albumin to bind residual transferrin and prevent iron uptake via transferrin-bound iron endocytosis. After incubation, cells were again washed with SFM before addition of medium containing 2 μM ferric ammonium citrate (MP Biomedicals) radiolabeled with ⁵⁹Fe and 1 mM ascorbate. Cells were incubated with radiolabeled media for 2 h during siRNA experiments and for 1 h during overexpression experiments. After the incubation period, medium was aspirated and cells were washed three times with an iron chelator solution containing 1 mM diethylenetriaminepentaaetic acid and 1 mM bathophenanthroline disulfonate to remove any residual radiolabeled iron. Cells were then lysed in SDS lysis buffer and assessed for radioactivity by using a WIZARD2 gamma counter (PerkinElmer). Counts for each sample were normalized to cellular protein concentrations.

Statistical analysis.

Data were analyzed for statistical significance using one-way ANOVA and Tukey’s multiple-comparison post hoc test or Student’s t-test (GraphPad Prism) where indicated. Unequal variance between groups was accounted for by log transformation, where applicable, to normalize variance before statistical analysis.

RESULTS

Overexpression of NTBI transporters in human β-cells.

To determine whether the expression of established NTBI transporters could promote iron uptake in β cells, ZIP14, ZIP8, and DMT1 were overexpressed in βlox5 cells, a human β-cell line (7), and NTBI uptake (from [⁵⁹Fe]ferric citrate) was measured at pH 7.4, the pH of blood plasma. We found that overexpression of ZIP14 or ZIP8, but not DMT1, increased the ability of βlox5 cells to take up NTBI when compared with cells transfected with empty vector control (Fig. 1). To explore the possibility that the lack of DMT1-mediated NTBI transport in βlox5 cells results from poor DMT1 expression at the cell surface, we isolated cell-surface proteins from cells overexpressing DMT1. Western blotting analysis of isolated cell-surface proteins and total-cell lysate revealed that DMT1 was detectable at the plasma membrane (Fig. 2A), despite not increasing NTBI uptake (Fig. 1B). Overexpressed ZIP14 and ZIP8 were detectable at the cell surface (Fig. 2, B and C), and this was associated with increased NTBI uptake (Fig. 1B). The proteins Na⁺-K⁺-ATPase and copper chaperone for superoxide dismutase (CCS) were measured to indicate cell-surface and intracellular protein fractions, respectively.

Fig. 1.

ZIP14 and ZIP8, but not DMT1, overexpression increases iron uptake by βlox5 cells. A: Western blot analysis of cell lysates from βlox5 cells transfected with pCMV-Sport6-empty vector (EV), DMT1, ZIP14, or ZIP8. Tubulin is shown to indicate lane loading. B: effect of ZIP14, ZIP8, or DMT1 overexpression on the uptake of iron by βlox5 cells. To measure iron uptake, cells were incubated for 1 h in serum-free medium containing 2 μM [⁵⁹Fe]ferric citrate and 1 mM ascorbate and the cellular uptake of ⁵⁹Fe was measured by gamma counting. Data represent means ± SE of 3 independent experiments performed in triplicate. Group means were compared by unpaired Student’s t-test. *P < 0.05, statistically significant differences relative to cells transfected with EV (ZIP14 P = 0.02, ZIP8 P = 0.005, and DMT1 P = 0.19).

Fig. 2.

When overexpressed in βlox5 cells, DMT1, ZIP14, and ZIP8 localize to the plasma membrane. Western blot analysis of DMT1, ZIP14, ZIP8, Na⁺-K⁺-ATPase, and copper chaperone for superoxide dismutase (CCS) in total-cell lysate (TCL) or cell-surface (CS) proteins isolated from βlox5 cells transfected with either empty vector (EV), DMT1 (A), ZIP14 (B), or ZIP8 (C). Plasma membrane proteins were labeled with sulfo-NHS-SS-biotin and affinity purified by using streptavidin-agarose columns before Western blotting. Na⁺-K⁺-ATPase and CCS serve as markers for plasma membrane and cytosolic proteins, respectively. Images shown are representative of Western blots from 3 independent experiments.

siRNA knockdown of NTBI transporters in human β-cells.

To define the contribution of endogenous ZIP14, ZIP8, and DMT1 to NTBI uptake by human β-cells, siRNA was used in combination with NTBI uptake measurements in βlox5 cells. siRNA-mediated suppression of ZIP14 expression decreased NTBI uptake by ~50% (Fig. 3). By contrast, siRNA knockdown of ZIP8 did not affect uptake (Fig. 3), suggesting that endogenous ZIP8-mediated NTBI uptake is negligible in βlox5 cells. We were unable to achieve successful knockdown of DMT1 in this cell line because the cells died shortly after transfection. Quantification of mRNA transcript abundance in primary human islets indicates that the copy number of mRNA transcripts encoding ZIP14 is approximately two and four times the number of ZIP8 and DMT1 transcripts, respectively (Fig. 4A). Similar to βlox5 cells (Fig. 3), knockdown of ZIP14 in primary human islets decreased NTBI uptake by ~50% (Fig. 4, B and C), further suggesting that ZIP14 is a major route of NTBI uptake in human β-cells. Although ZIP14 has a predicted molecular mass of ~54 kDa, Western blot analysis of ZIP14 in human β-cells detects the protein at ~130 kDa (Figs. 1–4), similar to ZIP14 from mouse and rat tissues, including pancreas. The reader is referred to our previous publication (32) for anti-ZIP14 antibody validation, a full-length Western blot, and a discussion of molecular weight.

Fig. 3.

NTBI uptake by βlox5 cells is decreased by siRNA knockdown of endogenous ZIP14 but not ZIP8. A: Western blot analysis of lysates from βlox5 cells transfected with negative control siRNA (siNC) or siRNA targeting either ZIP14 (siZIP14, left) or ZIP8 (siZIP8, right). Long exposures of the same blots are also shown to more clearly show the degree of siRNA knockdown. B: to measure NTBI uptake, cells were incubated for 2 h in serum-free medium containing 2 μM [⁵⁹Fe]ferric citrate and 1 mM ascorbate and the cellular uptake of ⁵⁹Fe was measured by gamma counting. Data represent means ± SE of 3 independent experiments performed in triplicate. Group means were compared by unpaired Student’s t-test. *P < 0.05, statistically significant differences relative to cells transfected with siNC (ZIP14 P = 0.03 and ZIP8 P = 0.58).

Fig. 4.

Human islets express ZIP14 and siRNA knockdown of ZIP14 decreases NTBI uptake by primary human islets. A: quantitative RT-PCR analysis of mRNA copy numbers of ZIP14, ZIP8, and DMT1 in isolated human islets. B: Western blot analysis of cell lysates from isolated human islets transfected with either negative control siRNA (siNC) or siRNA targeting ZIP14 (siZIP14). C: to measure iron uptake, cells were incubated for 2 h in serum-free medium containing 2 μM [⁵⁹Fe]ferric citrate and 1 mM ascorbate and the cellular uptake of ⁵⁹Fe was measured by gamma counting. Data represent means ± SE of 3 independent experiments performed in triplicate for iron uptake determination and means ± SE of 4 individual islet donors measured in duplicate for mRNA copy number measurement. Group means were compared by unpaired Student’s t-test. *P = 0.002, statistically significant differences relative to cells transfected with siNC.

Cellular localization of NTBI transporters in human islets.

To determine whether ZIP14, DMT1, and ZIP8 are expressed in β-cells and/or other islet cell types we performed immunofluorescence analysis of human pancreas sections (focusing on islets). In the case of ZIP14, we found that protein expression is largely restricted to β-cells with negligible expression in α-cells (Fig. 5A). ZIP14 staining in β-cells displayed a diffuse speckled pattern throughout the cytosol. Staining for DMT1 in human pancreatic islets indicated that its expression was restricted to β-cells with no signal detected from α-cells (Fig. 5B). DMT1 displayed a punctate, granular staining pattern suggesting an intracellular localization, consistent with the known role of DMT1 in endosomal iron transport, at least in some cell types (10). Staining for ZIP8 in the human pancreas revealed only low-level diffuse staining in pancreatic acinar cells but not β-cells (Fig. 5C). It should be noted that the weak staining for ZIP8 in the pancreas is not due a lack of sensitivity of the anti-ZIP8 antibody because ZIP8 was readily detectable in human placenta sections analyzed in parallel as a positive control (Fig. 5D).

Fig. 5.

Immunofluorescence analysis of ZIP14, DMT1, and ZIP8 in human pancreatic islets. Human pancreatic tail sections with islets were stained for either ZIP14 (AI, green), DMT1 (BI, green), or ZIP8 (CI, green) along with insulin (A-CII, red) or insulin and glucagon (A-CIII, red and blue, respectively). D: human placenta section stained for ZIP8 and DAPI nuclear stain (DI, green and blue, respectively). Serial sections were analyzed in parallel with nonimmune IgG replacing the primary antibody for ZIP14, DMT1, or ZIP8 (A-CIV: pancreatic sections; DII: placenta section). Images taken at either ×60 (pancreas ZIP14 and DMT1), ×20 (pancreas ZIP8), or ×40 (placenta ZIP8) original magnification were obtained by using a spinning disk confocal fluorescent microscope system. Images shown are from Network for Pancreatic Organ Donation (nPOD) cases 6001 (ZIP14) and 6104 (DMT1 and ZIP8) and are representative of 3–4 individual cases.

Modulation of ZIP14 expression by iron and IL-1β in human β-cells.

To determine whether ZIP14 levels are induced by iron loading in human β-cells, we treated primary human islets with ferric ammonium citrate and ascorbate (FAC) and measured ZIP14 protein expression. We found that cellular iron loading, confirmed by elevated ferritin and decreased transferrin receptor 1 (TFR1) protein levels, did not increase ZIP14 protein expression in primary islets (Fig. 6A). Additionally, depletion of cellular iron levels by deferoxamine mesylate (DFO), as indicated by elevated TFR1 protein levels, did not affect ZIP14 protein expression (Fig. 6A).

Fig. 6.

Cellular iron loading and treatment with IL-1β do not increase ZIP14 levels in primary human islets. A: Western blot analysis of ZIP14, TFR1, and ferritin in human-islet lysates 48 h after treatment with CON medium or medium containing 50 µM deferoxamine mesylate (DFO), or 100 µM ferric ammonium citrate + 1 mM ascorbate (FAC). Lysates from βlox5 cells transfected with either siNC or siZIP14 siRNA are shown to confirm the band size of ZIP14 protein. B: Western blot analysis for ZIP14 in human-islet lysates after incubation in CON medium or medium containing 100 U/ml recombinant human IL-1β for 24 h. Lysates from βlox5 cells transfected with either siNC or siZIP14 siRNA are shown to confirm the band size. Tubulin is shown to indicate lane loading. Images are representative of 2 independent experiments using islets from individual, nondiabetic donors.

ZIP14 mRNA levels have been observed to increase in response to IL-1β in isolated mouse hepatocytes (26). To determine if IL-1β induces ZIP14 expression in human β cells, primary human islets were treated with IL-1β for 24 h. Treatment of human islets with IL-1β resulted in no induction of ZIP14 at the protein level (Fig. 6B).

DISCUSSION

Disorders of iron overload in humans are associated with β-cell iron accumulation (20, 28, 37), which is thought to impair β-cell function (31). While β-cell iron loading has been documented during these disorders, little is known regarding the mechanism(s) by which β-cells take up iron (17). The present study is the first to examine the contribution of the established NTBI transport proteins DMT1, ZIP14, and ZIP8 to β-cell NTBI uptake. The observation that suppression of ZIP14 expression decreased NTBI uptake by ~50% in the human pancreatic β-cell line βlox5 suggests that ZIP14 is a major route of NTBI uptake by human β-cells. A similar reduction in NTBI uptake was observed after suppression of ZIP14 expression in isolated primary human islets, which express ZIP14 in β-cells. Iron loading in human islets is reported to be restricted to β-cells (28, 37), in line with the pattern of ZIP14 expression in human islets as determined by immunofluorescence analysis, suggesting that the lack of iron accumulation in α-cells may be due to a lack of ZIP14 expression. Additionally, the restriction of ZIP14 within human islets to β-cells suggests that the reduction in iron uptake measured after siRNA inhibition of ZIP14 is likely due to reduced iron uptake by β-cells rather than another islet cell type. Although ZIP14 in the human pancreas is detected in β-cells, more robust ZIP14 staining was observed in surrounding acinar cells (Fig. 5A), similar to our previous studies of ZIP14 expression in rat pancreas (32). Indeed, the prominent expression of ZIP14 in acinar cells likely explains why iron preferentially loads in the exocrine pancreas during iron overload (19). However, in contrast to the pattern of ZIP14 expression in human pancreas, ZIP14 in the rat pancreas was not detectable in β-cells (32). Based on these observations, we speculate that the lack of β-cell ZIP14 in rodents accounts for the fact that rodent β-cells are resistant to loading iron, even in the context of massive iron overload (1, 33, 36, 38, 39). We are aware of only two studies that have documented iron loading by Perls’ staining in rodent β-cells, but these studies utilized nonphysiologic models of iron loading, either portacaval shunting (18) or repeated intraperitoneal injections of ferric nitrilotriacetic acid (29).

Previous reports have indicated that ZIP14 protein levels are modulated by cellular iron status. For example, in human hepatoma HepG2 cells, ZIP14 protein levels are induced by iron loading with ferric ammonium citrate (FAC) (32, 44). ZIP14 protein levels are also elevated in iron-loaded rat liver and pancreas (32). The studies in HepG2 cells also showed that ZIP14 is downregulated by treatment with DFO (44), which causes iron depletion as indicated by increased levels of TFR1, a marker of cellular iron status. In the present study with primary human islets, neither iron depletion nor iron loading (confirmed by clear alterations in TFR1 levels) affected ZIP14 protein levels, thus suggesting differences in iron-dependent regulation of ZIP14 between cultured primary islets and other cell types.

Studies of primary mouse hepatocytes have indicated that ZIP14 is additionally regulated by the inflammatory cytokine IL-1β, which has been reported to be produced by β-cells from individuals with type-2 diabetes (28). Thus one could envision a scenario in which IL-1β increases β-cell ZIP14 levels, which in diabetics with plasma NTBI could increase β-cell NTBI uptake. However, in the present study, we found that treatment with IL-1β had no consistent effect on ZIP14 protein levels in primary human islets, again highlighting differential responses between islets and other cell populations. In line with this finding, others have reported that mRNA expression of ZIP14 in MIN6 cells, a mouse β-cell line, in unaffected by IL-1β treatment (8).

The observation that robust knockdown of ZIP14 did not completely abolish NTBI uptake suggests that β-cells possess ZIP14-independent pathways of NTBI uptake. A role for DMT1 in β-cell NTBI uptake seems unlikely given that overexpression of DMT1 in βlox5 cells did not stimulate NTBI uptake despite localizing to the cell surface. The lack of increased NTBI transport agrees with previous studies showing that DMT1 transports iron poorly at plasma pH of 7.4 (14, 30). A role for ZIP8 also seems unlikely as knockdown of endogenous ZIP8 did not affect NTBI uptake in βlox5 cells and because ZIP8 was not detectable in human islets by immunofluorescence analysis. Alternative candidates for NTBI uptake by β-cells include L- and T-type Ca²⁺ channels, which have been reported to participate in NTBI uptake by cardiomyocytes (23, 34).

In conclusion, our data identify ZIP14, but not DMT1 or ZIP8, as a significant contributor to NTBI uptake by human β cells, thus revealing a potential target for inhibitors that may mitigate/prevent β-cell iron accumulation, and possibly β-cell dysfunction, in disorders of iron overload.

GRANTS

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-080706 (to M. D. Knutson).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

R.C. and M.D.K. conceived and designed the research; R.C. performed experiments; R.C. and M.D.K. analyzed data; R.C. and M.D.K. interpreted results of experiments; R.C. and M.D.K. prepared figures; R.C. and M.D.K. drafted the manuscript; R.C. and M.D.K. edited and revised the manuscript; R.C. and M.D.K. approved final version of the manuscript.