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.

Keywords: ZIP14; diabetes; hemochromatosis; iron; β-cell.

Copyright © 2017 the American Physiological Society.

Figures

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.

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.

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.

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.