9 research outputs found
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
Impaired anticipatory postural adjustments due to experimental infrapatellar fat pad pain
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
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) Zn<sup>2+</sup> 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 Zn<sup>2+</sup> from the INS-1E cells. (C) 40×. Bar = 50 µm. (D) 100×. Bar = 20 µm.</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
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
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<sub>0–120</sub> 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