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

Egefjord L, Jensen JL, Bang-Berthelsen CH, Petersen AB, Smidt K, Schmitz O, Karlsen AE, Pociot F, Chimienti F, Rungby J, Magnusson NE - BMC Endocr Disord (2009)

Bottom Line: The effect was even more pronounced when mixing the cytokines.TNF-alpha had little effect on zinc transporter expression.Particularly ZnT8, which has been associated with the development of diabetes, seems to be cytokine sensitive.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Aarhus, Aarhus, Denmark. le@studmed.au.dk

ABSTRACT

Background: Beta-cells are extremely rich in zinc and zinc homeostasis is regulated by zinc transporter proteins. beta-cells are sensitive to cytokines, interleukin-1beta (IL-1beta) has been associated with beta-cell dysfunction and -death in both type 1 and type 2 diabetes. This study explores the regulation of zinc transporters following cytokine exposure.

Methods: The effects of cytokines IL-1beta, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) 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.

Results: Our results showed a dynamic response of genes responsible for beta-cell zinc homeostasis to cytokines: IL-1beta 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-alpha had little effect on zinc transporter expression. IFN-gamma down regulated a number of zinc transporters. Insulin expression was down regulated by all cytokines. ZnT8 over expressing cells were more sensitive to IL-1beta induced apoptosis whereas no differences were observed with IFN-gamma, TNF-alpha, or a mixture of cytokines.

Conclusion: The zinc transporting system in beta-cells is influenced by the exposure to cytokines. Particularly ZnT8, which has been associated with the development of diabetes, seems to be cytokine sensitive.

No MeSH data available.


Related in: MedlinePlus

Transfection of INS-1E cells with human ZnT8: Left panel shows fluorescence (green) marked ZnT8 RNA-probe positivity in transfected cells, contrasting the void control cells, middle panel. Right panel shows the light microscopic appearance of the same cells.
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Figure 1: Transfection of INS-1E cells with human ZnT8: Left panel shows fluorescence (green) marked ZnT8 RNA-probe positivity in transfected cells, contrasting the void control cells, middle panel. Right panel shows the light microscopic appearance of the same cells.

Mentions: INS-1 and INS-1E cells were cultured in a 5% CO2 atmosphere in complete RPMI 1640 supplemented with 11 mM glucose, 10% heat-inactivated fetal bovine serum, 50 μM beta-mercaptoethanol, 2 mM L-Glutamine, 100 U/mL penicillin, and 100 g/mL streptomycin. For stimulation assays cells were plated (106 cells/well) into 6-well plates (NUNC, Roskilde, Denmark). The INS-1 cells were treated with 60–180 U recombinant mouse IL-1β/mL, 200 U TNF-α/mL, 200 U recombinant Rat IFN-γ/mL or a mixture, consisting of 180 U IL-1β/mL, 200 U TNF-α/mL and 200 U Rat IFN-γ/mL (PharMingen International, San Diego, CA, USA). For INS-1 cells treatments were given for 1 and 24 hours, in replicas of 6. For INS-1E and INS-1E-ZnT8-EGFP cells treatments were given for 6 and 24 hours in replicas of 3. Controls were incubated with RPMI 1640 medium and 11 mM glucose. The stably transfected cell line INS-1E-ZnT8-EGFP was maintained as described above with the addition of 75 μM G418 to maintain a pure culture of cells expressing the ZnT8-EGFP construct. Expression of the fusion protein was controlled by fluorescence microscopy and by Q-PCR using ZnT8-EGFP specific primers (figure 1) [16].


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

Egefjord L, Jensen JL, Bang-Berthelsen CH, Petersen AB, Smidt K, Schmitz O, Karlsen AE, Pociot F, Chimienti F, Rungby J, Magnusson NE - BMC Endocr Disord (2009)

Transfection of INS-1E cells with human ZnT8: Left panel shows fluorescence (green) marked ZnT8 RNA-probe positivity in transfected cells, contrasting the void control cells, middle panel. Right panel shows the light microscopic appearance of the same cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651882&req=5

Figure 1: Transfection of INS-1E cells with human ZnT8: Left panel shows fluorescence (green) marked ZnT8 RNA-probe positivity in transfected cells, contrasting the void control cells, middle panel. Right panel shows the light microscopic appearance of the same cells.
Mentions: INS-1 and INS-1E cells were cultured in a 5% CO2 atmosphere in complete RPMI 1640 supplemented with 11 mM glucose, 10% heat-inactivated fetal bovine serum, 50 μM beta-mercaptoethanol, 2 mM L-Glutamine, 100 U/mL penicillin, and 100 g/mL streptomycin. For stimulation assays cells were plated (106 cells/well) into 6-well plates (NUNC, Roskilde, Denmark). The INS-1 cells were treated with 60–180 U recombinant mouse IL-1β/mL, 200 U TNF-α/mL, 200 U recombinant Rat IFN-γ/mL or a mixture, consisting of 180 U IL-1β/mL, 200 U TNF-α/mL and 200 U Rat IFN-γ/mL (PharMingen International, San Diego, CA, USA). For INS-1 cells treatments were given for 1 and 24 hours, in replicas of 6. For INS-1E and INS-1E-ZnT8-EGFP cells treatments were given for 6 and 24 hours in replicas of 3. Controls were incubated with RPMI 1640 medium and 11 mM glucose. The stably transfected cell line INS-1E-ZnT8-EGFP was maintained as described above with the addition of 75 μM G418 to maintain a pure culture of cells expressing the ZnT8-EGFP construct. Expression of the fusion protein was controlled by fluorescence microscopy and by Q-PCR using ZnT8-EGFP specific primers (figure 1) [16].

Bottom Line: The effect was even more pronounced when mixing the cytokines.TNF-alpha had little effect on zinc transporter expression.Particularly ZnT8, which has been associated with the development of diabetes, seems to be cytokine sensitive.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Aarhus, Aarhus, Denmark. le@studmed.au.dk

ABSTRACT

Background: Beta-cells are extremely rich in zinc and zinc homeostasis is regulated by zinc transporter proteins. beta-cells are sensitive to cytokines, interleukin-1beta (IL-1beta) has been associated with beta-cell dysfunction and -death in both type 1 and type 2 diabetes. This study explores the regulation of zinc transporters following cytokine exposure.

Methods: The effects of cytokines IL-1beta, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) 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.

Results: Our results showed a dynamic response of genes responsible for beta-cell zinc homeostasis to cytokines: IL-1beta 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-alpha had little effect on zinc transporter expression. IFN-gamma down regulated a number of zinc transporters. Insulin expression was down regulated by all cytokines. ZnT8 over expressing cells were more sensitive to IL-1beta induced apoptosis whereas no differences were observed with IFN-gamma, TNF-alpha, or a mixture of cytokines.

Conclusion: The zinc transporting system in beta-cells is influenced by the exposure to cytokines. Particularly ZnT8, which has been associated with the development of diabetes, seems to be cytokine sensitive.

No MeSH data available.


Related in: MedlinePlus