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Tissue factor/factor VIIa signalling promotes cytokine-induced beta cell death and impairs glucose-stimulated insulin secretion from human pancreatic islets.

Edén D, Siegbahn A, Mokhtari D - Diabetologia (2015)

Bottom Line: The effect of TF/FVIIa on cytokine-induced beta cell death was found to be dependent on the stress kinase JNK, since FVIIa addition potentiated cytokine-induced JNK activation and JNK inhibition abolished the effect of TF/FVIIa on cytokine-induced beta cell death.Moreover, TF/FVIIa signalling resulted in inhibition of GSIS from human pancreatic islets.These results indicate that TF/FVIIa signalling has a negative effect on beta cell function and promotes beta cell death in response to cytokines.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Sciences, Clinical Chemistry, Science for Life Laboratory, University Hospital, Uppsala University, Entr. 61 3rd floor, S-751 85, Uppsala, Sweden.

ABSTRACT

Aims/hypothesis: Patients diagnosed with type 1 or type 2 diabetes have elevated levels of coagulation factor VIIa (FVIIa) and its receptor tissue factor (TF) in their bloodstream. This may affect the fate of the beta cells. We aimed to study the effects of TF/FVIIa signalling on cytokine-induced beta cell death and islet function in vitro.

Methods: Human pancreatic islets and MIN-6 beta cells were used to study TF mRNA and protein expression using real-time PCR, immunoblotting and flow cytometry. The effects of TF/FVIIa on cytokine-induced beta cell death were studied in MIN-6 cells and human pancreatic islets using cell-death ELISA and propidium iodide and cleaved caspase-3 staining. Effects of TF/FVIIa on the phosphorylation of p38, extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK) were investigated by immunoblotting. Glucose-stimulated insulin secretion (GSIS) from human islets was measured with an insulin ELISA.

Results: A combination of the cytokines IL-1β, TNF-α and IFN-γ induced TF expression in human pancreatic islets and in beta cells. TF/FVIIa did not affect basal beta cell death but, independently of downstream coagulation activity, augmented beta cell death in response to cytokines. The effect of TF/FVIIa on cytokine-induced beta cell death was found to be dependent on the stress kinase JNK, since FVIIa addition potentiated cytokine-induced JNK activation and JNK inhibition abolished the effect of TF/FVIIa on cytokine-induced beta cell death. Moreover, TF/FVIIa signalling resulted in inhibition of GSIS from human pancreatic islets.

Conclusions/interpretation: These results indicate that TF/FVIIa signalling has a negative effect on beta cell function and promotes beta cell death in response to cytokines.

No MeSH data available.


Related in: MedlinePlus

Cytokines induce TF expression in human islets and MIN-6 cells. Human islets and MIN-6 cells were left untreated (Control) or treated with a cytokine mixture of IL-1β + TNF-α + IFN-γ (Cyt.) for the indicated time periods. TF mRNA levels related to RPLP0 in (a) human islets and (b) MIN-6 cells. Immunoblot and graphs showing TF/GAPDH ratio using TF and GAPDH antibodies on (c, d) human islets and (e, f) MIN-6 cells. (g) MIN-6 cells were left untreated (Control) or were treated with cytokine mixture (Cyt.) for 6 h. Cells were incubated with mouse TF antibody (Ab) and TF cell surface expression was analysed using flow cytometry detecting forward scatter height (FSC-H) and fluorescence in fluorescence channel 1 height (FL1-H). Normal goat IgG was used as isotype control. Results are means ± SEM from four (a–f) or three (g) independent experiments. *p < 0.05 vs control, using paired Student’s t test
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Fig1: Cytokines induce TF expression in human islets and MIN-6 cells. Human islets and MIN-6 cells were left untreated (Control) or treated with a cytokine mixture of IL-1β + TNF-α + IFN-γ (Cyt.) for the indicated time periods. TF mRNA levels related to RPLP0 in (a) human islets and (b) MIN-6 cells. Immunoblot and graphs showing TF/GAPDH ratio using TF and GAPDH antibodies on (c, d) human islets and (e, f) MIN-6 cells. (g) MIN-6 cells were left untreated (Control) or were treated with cytokine mixture (Cyt.) for 6 h. Cells were incubated with mouse TF antibody (Ab) and TF cell surface expression was analysed using flow cytometry detecting forward scatter height (FSC-H) and fluorescence in fluorescence channel 1 height (FL1-H). Normal goat IgG was used as isotype control. Results are means ± SEM from four (a–f) or three (g) independent experiments. *p < 0.05 vs control, using paired Student’s t test

Mentions: We first studied the influence of cytokines on TF expression in islets and beta cells. In human islets and MIN-6 cells, a significant increase in TF mRNA levels (≈ twofold) was observed following 3 h of exposure to a cytokine mixture (IL-1β + TNF-α + IFN-γ) when compared with control (Fig. 1a, b). A further increase (≈ fourfold) was seen after exposure to cytokines for 6 and 24 h (Fig. 1a, b). This was paralleled by an induction in TF protein levels in human islets and MIN-6 cells following 6 and 24 h of cytokine exposure (Fig. 1c–f). A large pool (61.6 ± 4.9%) of untreated MIN-6 cells stained positive for TF cell surface expression (Fig. 1g). Cytokine treatment for 6 h increased the number of TF-positive cells (82.4 ± 6.8%) when compared with untreated cells (Fig. 1g), indicating that TF total and cell surface expression in beta cells are upregulated in the presence of cytokines.Fig. 1


Tissue factor/factor VIIa signalling promotes cytokine-induced beta cell death and impairs glucose-stimulated insulin secretion from human pancreatic islets.

Edén D, Siegbahn A, Mokhtari D - Diabetologia (2015)

Cytokines induce TF expression in human islets and MIN-6 cells. Human islets and MIN-6 cells were left untreated (Control) or treated with a cytokine mixture of IL-1β + TNF-α + IFN-γ (Cyt.) for the indicated time periods. TF mRNA levels related to RPLP0 in (a) human islets and (b) MIN-6 cells. Immunoblot and graphs showing TF/GAPDH ratio using TF and GAPDH antibodies on (c, d) human islets and (e, f) MIN-6 cells. (g) MIN-6 cells were left untreated (Control) or were treated with cytokine mixture (Cyt.) for 6 h. Cells were incubated with mouse TF antibody (Ab) and TF cell surface expression was analysed using flow cytometry detecting forward scatter height (FSC-H) and fluorescence in fluorescence channel 1 height (FL1-H). Normal goat IgG was used as isotype control. Results are means ± SEM from four (a–f) or three (g) independent experiments. *p < 0.05 vs control, using paired Student’s t test
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4589554&req=5

Fig1: Cytokines induce TF expression in human islets and MIN-6 cells. Human islets and MIN-6 cells were left untreated (Control) or treated with a cytokine mixture of IL-1β + TNF-α + IFN-γ (Cyt.) for the indicated time periods. TF mRNA levels related to RPLP0 in (a) human islets and (b) MIN-6 cells. Immunoblot and graphs showing TF/GAPDH ratio using TF and GAPDH antibodies on (c, d) human islets and (e, f) MIN-6 cells. (g) MIN-6 cells were left untreated (Control) or were treated with cytokine mixture (Cyt.) for 6 h. Cells were incubated with mouse TF antibody (Ab) and TF cell surface expression was analysed using flow cytometry detecting forward scatter height (FSC-H) and fluorescence in fluorescence channel 1 height (FL1-H). Normal goat IgG was used as isotype control. Results are means ± SEM from four (a–f) or three (g) independent experiments. *p < 0.05 vs control, using paired Student’s t test
Mentions: We first studied the influence of cytokines on TF expression in islets and beta cells. In human islets and MIN-6 cells, a significant increase in TF mRNA levels (≈ twofold) was observed following 3 h of exposure to a cytokine mixture (IL-1β + TNF-α + IFN-γ) when compared with control (Fig. 1a, b). A further increase (≈ fourfold) was seen after exposure to cytokines for 6 and 24 h (Fig. 1a, b). This was paralleled by an induction in TF protein levels in human islets and MIN-6 cells following 6 and 24 h of cytokine exposure (Fig. 1c–f). A large pool (61.6 ± 4.9%) of untreated MIN-6 cells stained positive for TF cell surface expression (Fig. 1g). Cytokine treatment for 6 h increased the number of TF-positive cells (82.4 ± 6.8%) when compared with untreated cells (Fig. 1g), indicating that TF total and cell surface expression in beta cells are upregulated in the presence of cytokines.Fig. 1

Bottom Line: The effect of TF/FVIIa on cytokine-induced beta cell death was found to be dependent on the stress kinase JNK, since FVIIa addition potentiated cytokine-induced JNK activation and JNK inhibition abolished the effect of TF/FVIIa on cytokine-induced beta cell death.Moreover, TF/FVIIa signalling resulted in inhibition of GSIS from human pancreatic islets.These results indicate that TF/FVIIa signalling has a negative effect on beta cell function and promotes beta cell death in response to cytokines.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Sciences, Clinical Chemistry, Science for Life Laboratory, University Hospital, Uppsala University, Entr. 61 3rd floor, S-751 85, Uppsala, Sweden.

ABSTRACT

Aims/hypothesis: Patients diagnosed with type 1 or type 2 diabetes have elevated levels of coagulation factor VIIa (FVIIa) and its receptor tissue factor (TF) in their bloodstream. This may affect the fate of the beta cells. We aimed to study the effects of TF/FVIIa signalling on cytokine-induced beta cell death and islet function in vitro.

Methods: Human pancreatic islets and MIN-6 beta cells were used to study TF mRNA and protein expression using real-time PCR, immunoblotting and flow cytometry. The effects of TF/FVIIa on cytokine-induced beta cell death were studied in MIN-6 cells and human pancreatic islets using cell-death ELISA and propidium iodide and cleaved caspase-3 staining. Effects of TF/FVIIa on the phosphorylation of p38, extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK) were investigated by immunoblotting. Glucose-stimulated insulin secretion (GSIS) from human islets was measured with an insulin ELISA.

Results: A combination of the cytokines IL-1β, TNF-α and IFN-γ induced TF expression in human pancreatic islets and in beta cells. TF/FVIIa did not affect basal beta cell death but, independently of downstream coagulation activity, augmented beta cell death in response to cytokines. The effect of TF/FVIIa on cytokine-induced beta cell death was found to be dependent on the stress kinase JNK, since FVIIa addition potentiated cytokine-induced JNK activation and JNK inhibition abolished the effect of TF/FVIIa on cytokine-induced beta cell death. Moreover, TF/FVIIa signalling resulted in inhibition of GSIS from human pancreatic islets.

Conclusions/interpretation: These results indicate that TF/FVIIa signalling has a negative effect on beta cell function and promotes beta cell death in response to cytokines.

No MeSH data available.


Related in: MedlinePlus