SLC30A3 Responds to Glucose- and Zinc Variations in ß-Cells and Is Critical for Insulin Production and In Vivo Glucose-Metabolism During ß-Cell Stress

BACKGROUND:Ion transporters of the Slc30A- (ZnT-) family regulate zinc fluxes into sub-cellular compartments. beta-cells depend on zinc for both insulin crystallization and regulation of cell mass. METHODOLOGY/PRINCIPAL FINDINGS:This study examined: the effect of glucose and zinc chelation on ZnT gene and protein levels and apoptosis in beta-cells and pancreatic islets, the effects of ZnT-3 knock-down on insulin secretion in a beta-cell line and ZnT-3 knock-out on glucose metabolism in mice during streptozotocin-induced beta-cell stress. In INS-1E cells 2 mM glucose down-regulated ZnT-3 and up-regulated ZnT-5 expression relative to 5 mM. 16 mM glucose increased ZnT-3 and decreased ZnT-8 expression. Zinc chelation by DEDTC lowered INS-1E insulin content and insulin expression. Furthermore, zinc depletion increased ZnT-3- and decreased ZnT-8 gene expression whereas the amount of ZnT-3 protein in the cells was decreased. Zinc depletion and high glucose induced apoptosis and necrosis in INS-1E cells. The most responsive zinc transporter, ZnT-3, was investigated further; by immunohistochemistry and western blotting ZnT-3 was demonstrated in INS-1E cells. 44% knock-down of ZnT-3 by siRNA transfection in INS-1E cells decreased insulin expression and secretion. Streptozotocin-treated mice had higher glucose levels after ZnT-3 knock-out, particularly in overt diabetic animals. CONCLUSION/SIGNIFICANCE:Zinc transporting proteins in beta-cells respond to variations in glucose and zinc levels. ZnT-3, which is pivotal in the development of cellular changes as also seen in type 2 diabetes (e.g. amyloidosis in Alzheimer's disease) but not previously described in beta-cells, is present in this cell type, up-regulated by glucose in a concentration dependent manner and up-regulated by zinc depletion which by contrast decreased ZnT-3 protein levels. Knock-down of the ZnT-3 gene lowers insulin secretion in vitro and affects in vivo glucose metabolism after streptozotocin treatment

Zinc transporter gene expression is regulated by pro-inflammatory cytokines: a potential role for zinc transporters in beta-cell apoptosis?

<p>Abstract</p> <p>Background</p> <p>β-cells are extremely rich in zinc and zinc homeostasis is regulated by zinc transporter proteins. β-cells are sensitive to cytokines, interleukin-1β (IL-1β) has been associated with β-cell dysfunction and -death in both type 1 and type 2 diabetes. This study explores the regulation of zinc transporters following cytokine exposure.</p> <p>Methods</p> <p>The effects of cytokines IL-1β, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) on zinc transporter gene expression were measured in INS-1-cells and rat pancreatic islets. Being the more sensitive transporter, we further explored ZnT8 (Slc30A8): the effect of ZnT8 over expression on cytokine induced apoptosis was investigated as well as expression of the insulin gene and two apoptosis associated genes, BAX and BCL2.</p> <p>Results</p> <p>Our results showed a dynamic response of genes responsible for β-cell zinc homeostasis to cytokines: IL-1β down regulated a number of zinc-transporters, most strikingly ZnT8 in both islets and INS-1 cells. The effect was even more pronounced when mixing the cytokines. TNF-α had little effect on zinc transporter expression. IFN-γ down regulated a number of zinc transporters. Insulin expression was down regulated by all cytokines. ZnT8 over expressing cells were more sensitive to IL-1β induced apoptosis whereas no differences were observed with IFN-γ, TNF-α, or a mixture of cytokines.</p> <p>Conclusion</p> <p>The zinc transporting system in β-cells is influenced by the exposure to cytokines. Particularly ZnT8, which has been associated with the development of diabetes, seems to be cytokine sensitive.</p

Relative gene expression of ZnT-3 and ZnT-8 after 24 hours of 100 µM DEDTC treatment.

<p>INS-1E cells were treated with DEDTC at 5 mM glucose. A) ZnT-3 gene expression normalised to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.05). N = 6. B) ZnT-8 gene expression normalised to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.01). N = 6.</p

High-dose streptozotocin for three days in ZnT-3−/− knockout mice and control mice.

<p>Following a series of low-dose exposures (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005684#pone-0005684-g008" target="_blank">Fig. 8</a>) ZnT-3−/− knock-out mice and control mice were treated with 200 mg/kg/day. A) Non-fasting morning blood glucose levels. Salt indicates sham saline injections. Data are mean and SEM (*p<0.01). B) Intraperitoneal glucose tolerance test in ZnT-3−/− knockout mice and control mice after high-dose streptozotozin. Blood glucose concentrations were measured before and 15, 30, 60 and 120 minutes after the glucose injection. Significantly higher AUC_0–120 in knock-out vs WT mice (p<0.01). N = 5 for ZnT-3−/− and n = 4 for wild type.</p

Insulin expression, content and secretion after 24 hours of 100 µM DEDTC treatment.

<p>INS-1E cells were treated with DEDTC at 5 mM glucose. A) Insulin gene expression normalized to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.01). N = 6. B) Insulin secretion related to insulin content in INS-1E cells. Data are mean and SEM (*p<0.01). N = 3.</p

Detection of apoptosis in INS-1E cells after 24 hours of hyperglycamia or zinc depletion.

<p>A–C) Glucose stimulation with 5 mM and 16 mM. A) Bax/Bcl-2 ratio of gene expression. Both genes were normalised to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.01). N = 6. B) Detection of intracellular DNA fragments in INS-1E cells (apoptosis) after 16 mM glucose stimulation. Data are mean and SEM. N = 4. C) Detection of DNA fragments in medium from INS-1E cells (necrosis) treated 16 mM glucose. Data are mean and SEM (*p<0.05). N = 4. D–F) Zinc chelation with 100 µM DEDTC. D) Bax/Bcl-2 ratio of gene expression. Both genes were normalised to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.01). N = 6. E) Detection of DNA fragments in INS-1E (apoptosis) after 100 µM DEDTC treatment. Data are mean and SEM (*p<0.01) N = 4. F) Detection of DNA fragments in medium from INS-1E cells (necrosis) treated with 100 µM DEDTC. Data are mean and SEM (*p<0.01). N = 4.</p

Zinc content of INS-1E cells.

<p>(A+B) Zn²⁺ autometallography of untreated INS-1E cells. (A) 40×. Bar = 50 µm. (B) 100×. Bar = 20 µm. (C+D) DEDTC treatment for 24 hours removed most autometallographically-detectable Zn²⁺ from the INS-1E cells. (C) 40×. Bar = 50 µm. (D) 100×. Bar = 20 µm.</p

Fasting glucose levels after low-dose streptozotozin in ZnT-3−/− knock-out mice.

<p>ZnT-3−/− knock-out mice and control mice were treated with 50 mg/kg/day for five days. Data are mean and SEM (*p<0.01). N = 15.</p

ZnT-3 protein in INS-1E cells and mouse islets.

<p>A) Western blot of ZnT-3 knockout tissue, and normal background strain tissue using the anti-ZnT-3 polyclonal antibody (20 µg per lane). B) Western blot with ZnT-3 antibody. Lane one shows the protein marker in kDa. Subsequent lanes: Control rat brain (10 µg protein) (lanes 2–4), mock transfected INS1-E cells (50 µg protein) (lanes 5–7), 100 µM DEDTC-treated INS1-E cells (50 µg protein) (lanes 8–10), ZnT-3 siRNA transfected INS1-E cells (50 µg protein) (lanes 11–13). Insert shows the quantification, brain tissue values are original multiplied by 5. C) Light micrograph of INS-1E cells exposed to ZnT-3 antibody. Silver enhanced colloidal gold (10 nm) particles attached to secondary antibodies against the ZnT-3 primary antibody are seen within the cells. There was no background stain and controls were negative (insert). Bar = 20 µm. D) Demonstration of ZnT3 antibody positivity in intact mouse islets (lane 1), compared with INS-1E cells before (lane 2) and after (lane 3) treatment with 100 µM DEDTC and brain tissue (lane 4). Each upload with 20 µg protein.</p