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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

Log2 expression levels relative to UBC7 for beta-Actin, Cyclophilin A, BAX, BCL2, and insulin in INS-1E and INS-1E-ZnT8-EGFP cells (n = 3). The last plot indicates the expression level of the normalizing gene UBC7. Black; INS-1E cells, red; INS-1E-ZnT8-EGFP cells. Circles; 6 hour treatments, triangles; 24 hour treatments.
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Figure 4: Log2 expression levels relative to UBC7 for beta-Actin, Cyclophilin A, BAX, BCL2, and insulin in INS-1E and INS-1E-ZnT8-EGFP cells (n = 3). The last plot indicates the expression level of the normalizing gene UBC7. Black; INS-1E cells, red; INS-1E-ZnT8-EGFP cells. Circles; 6 hour treatments, triangles; 24 hour treatments.

Mentions: The sensitivity to cytokines in INS-1E and INS-1E-ZnT8-EGFP cells was estimated by measuring mitochondrial activity (figure 3). No differences between the cell lines were found after exposure to TNF-α. Compared to INS-1E, IFN-γ decreased survival after 6 hours (but not after 12 or 24 hours). IL-1β decreased survival after 12 and 24 hours. Cytokine mixtures decreased survival after 6 hours (but not after 12 or 24 hours). These results were verified by an independent assay specifically measuring apoptosis and necrosis at 12, 24, and 36 hours with IL-1β and cytokine mixture. Apoptosis was increased by 4 and 2 fold in the ZnT8 transfected cell line following treatment with IL-1β for 24 and 36 hours, respectively (table 5). No differences were observed between the cell lines using the cytokine mixture. In addition, BCL2 and BAX mRNA expressions were compared between the cell lines at 6 and 24 hours (figure 4). Two of the housekeeping genes (beta-Actin and Cyclophilin A) showed a marked difference between the two cell lines and the two time points. Expression of the housekeeping gene UBC7 showed no difference between the two cell lines and a minor difference between the two time points. None of the housekeeping genes showed a difference between the treatments. UBC7 was therefore used as the normalizing gene. BAX and BCL2 showed no response to cytokine treatment (p = 0.07 and p = 0.13), but both genes showed a strong time effect (p < 0.0001 and p = 0.0001), and BAX exhibited a higher expression in the INS-1E-ZnT8-EGFP cells (p < 0.0001). Insulin gene expression showed a difference between the two cell lines and a difference between the two time points. On top of that IL-1β decreased the expression of insulin in INS-1E-ZnT8-EGFP cells (p = 0.0001) and a mixture of cytokines decreased the expression for both cell lines (p < 0.0001).


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)

Log2 expression levels relative to UBC7 for beta-Actin, Cyclophilin A, BAX, BCL2, and insulin in INS-1E and INS-1E-ZnT8-EGFP cells (n = 3). The last plot indicates the expression level of the normalizing gene UBC7. Black; INS-1E cells, red; INS-1E-ZnT8-EGFP cells. Circles; 6 hour treatments, triangles; 24 hour treatments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Log2 expression levels relative to UBC7 for beta-Actin, Cyclophilin A, BAX, BCL2, and insulin in INS-1E and INS-1E-ZnT8-EGFP cells (n = 3). The last plot indicates the expression level of the normalizing gene UBC7. Black; INS-1E cells, red; INS-1E-ZnT8-EGFP cells. Circles; 6 hour treatments, triangles; 24 hour treatments.
Mentions: The sensitivity to cytokines in INS-1E and INS-1E-ZnT8-EGFP cells was estimated by measuring mitochondrial activity (figure 3). No differences between the cell lines were found after exposure to TNF-α. Compared to INS-1E, IFN-γ decreased survival after 6 hours (but not after 12 or 24 hours). IL-1β decreased survival after 12 and 24 hours. Cytokine mixtures decreased survival after 6 hours (but not after 12 or 24 hours). These results were verified by an independent assay specifically measuring apoptosis and necrosis at 12, 24, and 36 hours with IL-1β and cytokine mixture. Apoptosis was increased by 4 and 2 fold in the ZnT8 transfected cell line following treatment with IL-1β for 24 and 36 hours, respectively (table 5). No differences were observed between the cell lines using the cytokine mixture. In addition, BCL2 and BAX mRNA expressions were compared between the cell lines at 6 and 24 hours (figure 4). Two of the housekeeping genes (beta-Actin and Cyclophilin A) showed a marked difference between the two cell lines and the two time points. Expression of the housekeeping gene UBC7 showed no difference between the two cell lines and a minor difference between the two time points. None of the housekeeping genes showed a difference between the treatments. UBC7 was therefore used as the normalizing gene. BAX and BCL2 showed no response to cytokine treatment (p = 0.07 and p = 0.13), but both genes showed a strong time effect (p < 0.0001 and p = 0.0001), and BAX exhibited a higher expression in the INS-1E-ZnT8-EGFP cells (p < 0.0001). Insulin gene expression showed a difference between the two cell lines and a difference between the two time points. On top of that IL-1β decreased the expression of insulin in INS-1E-ZnT8-EGFP cells (p = 0.0001) and a mixture of cytokines decreased the expression for both cell lines (p < 0.0001).

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