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First published online 19 September 2006
doi: 10.1242/jcs.03164

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In vivo expression and functional characterization of the zinc transporter ZnT8 in glucose-induced insulin secretion

¹ DRFMC/SCIB/LAN, UMR-E3 CEA/UJF, CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble CEDEX 9, France
² Mellitech, DRFMC/SCIB, 17 rue des Martyrs, 38054 Grenoble CEDEX 9, France
³ Thérapie Cellulaire du Diabète, INSERM ERIT-M 0106 Faculté de Médecine, Place de Verdun, 59045 Lille, France
⁴ Gene Expression Unit, Division of Biochemistry, Katholieke Universiteit Leuven, 3000 Leuven, Belgium
⁵ Laboratoire Canaux Ioniques et Signalisation, DRDC/LCI, CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble CEDEX 9, France

View larger version (4K): [in a new window]   Fig. 1. Expression of ZNT8 mRNA in mouse tissues. ZNT8 mRNA expression was assessed by expression microarrays (Affymetrix) using RNA extracted ex vivo from mouse pancreatic islets, panreactic acini, pituitary, brain, adrenal gland, liver, skeletal muscle, epididymal adipose tissue, lung, kidney and spleen.

View larger version (92K): [in a new window]   Fig. 2. Expression of ZnT-8 in human islets at the protein level. (A) Western blot with anti-human ZnT-8 antibody showing the presence of the protein in total extracts of human pancreas. Lane 1, HeLa ZnT-8-EGFP (positive control); lane 2, HeLa-EGFP (negative control); lane 3, rat insulinoma INS-1E cells; lane 4, human pancreatic islet extracts. (B) The ZnT-8 protein is detected (red) by immunochemistry of pancreatic islet cells. Nuclei were counterstained with Carrazi's hematoxylin. Bar, 30 µm.

View larger version (10K): [in a new window]   Fig. 3. Colocalization of ZnT-8 and insulin in human islet cells. Analysis of islet cell cytospins using anti-insulin and anti-ZnT-8 antibodies by confocal fluorescence microscopy. Insulin (green, left) displayed a characteristic punctuate staining. ZnT-8 staining (red, middle) completely coincided with the staining of insulin. Superimposition of the two images demonstrates the colocalization of ZnT-8-GFP and insulin (yellow, right). Bars, 20 µm.

View larger version (61K): [in a new window]   Fig. 4. ZnT-8 overexpression is INS-1E cells. (A) Western blot with anti-human ZnT-8 antibody showing the presence of the fusion protein at the expected size (i.e. 67 kDa) in ZnT-8-expressing cells but not in control EGFP-expressing cells. (B) Confocal fluorescence microscopy of ZnT-8-EGFP-expressing INS-1E cells. A representative cell displaying punctuated staining consistent with insulin colocalization. The panel on the right is a magnified image of the selected area of the cell on the left, showing that ZnT-8-EGFP is mainly localized in granules in close proximity to the plasma membrane. (C) Fluorescence microscopy of ZnT-8-expressing cells. Arrows indicate localization of ZnT-8-EGFP at the plasma membrane. Bars, 10 µm (C, left panel in B); 5 µm (B, right panel).

View larger version (47K): [in a new window]   Fig. 5. Determination of ZnT-8 membrane topology. (A) Confocal microscopy images of INS-1E-ZnT-8-EGFP cells, incubated with anti-ZnT-8 antibodies 9A or 9B (see Materials and Methods) before or after membrane permeabilization. The EGFP signal is present in both permeabilized and non-permeabilized cells, whereas antibodies recognize the ZnT-8 protein only after permeabilization. Bars, 15 µm. (B) Proposed model for the membrane topology of ZnT-8. Gray boxes indicate location of antibody epitopes, 9A, 9B. HIS, histidine-rich region; Ct, C-terminus; Nt, N-terminus.

View larger version (21K): [in a new window]   Fig. 6. Effect of extracellular zinc concentration on zinc content and cell viability. (A) Zinc concentrations in cells were measured by electrothermal atomic absorption spectrophotometry in either normal or zinc-supplemented medium. Values are expressed as zinc concentration normalized for protein content. *P<0.01 vs control; **P<0.05 vs non-supplemented cells. White bars, control cells; gray bars, INS-1E-ZnT8-EGFP cells. (B) After incubation with different concentrations of zinc sulphate, the cell viability was determined by MTT assay. Values are the mean ± s.e.m. of at least ten independent experiments White bars, control cells; gray bars, INS-1E-ZnT8-EGFP cells. (C) After incubation with different concentrations of the zinc chelator TPEN, the cell viability was determined by MTT assay. Values are the mean ± s.e.m. of at least ten independent experiments. *P<0.05 vs control; **P<0.005 vs control. White bars, control cells; gray bars, INS-1E-ZnT8-EGFP cells.

View larger version (5K): [in a new window]   Fig. 7. Influence of ZnT-8 on glucose-induced insulin secretion. INS-1E-EGFP and INS-1E-ZnT-8-EGFP cells were used to assess insulin secretion in response to glucose. After incubation in glucose-free buffer (see Materials and Methods), cells were exposed to different concentrations of glucose for 30 minutes. Intracellular and secreted insulin were measured by ELISA. White bars, control cells; gray bars, INS-1E-ZnT8-EGFP cells. Values are the mean ± s.e.m. of at least five experiments, *P<0.01 vs control, **P<0.005 vs control.

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