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

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.

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.

Figure 2

Zinc transporter expression profiles for ZnT5, ZnT6, ZnT8, Zip5, and Zip6 following cytokine exposure in islets. The figure shows the time course for the log2 difference +/- SEM between the IL-1β stimulation and the IFN-γ stimulation, showing a systematic development over time for the indicated genes. (n = 4). Black; ZnT5, red; ZnT6, green; ZnT8, blue; Zip5, purple; Zip6.

Figure 3

Relative survival for INS-1E and INS-1E-ZnT8-EGFP cells following cytokine exposure after 6, 12, and 24 hours was estimatedby MTT. Values were calculated relative to control cells for each cell line +/- SEM (n = 6). (*) p < 0.01 indicates significance in two independent experiments.

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.