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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

FVIIa promotes cytokine-induced caspase-3 cleavage in primary human beta cells. (a) Untreated or FVIIa pre-treated (10 nmol/l, 6 h) human islets were stimulated with a cytokine mixture of IL-1β, TNF-α and IFN-γ (Cyt.) for 72 h, then mildly dispersed using trypsin to generate single islet cells. Cells were stained with DAPI (blue), insulin antibody (red) and cleaved caspase-3 antibody (green) and visualised by fluorescence microscopy. Islets from five different donors were used and images show results from one representative donor. Scale bar, 50 μm. (b) Cells (~2,000/group) were counted and the number of cells double-positive for insulin and cleaved caspase-3 are depicted in the graph. Results are means ± SEM from five experiments. *p < 0.05 for indicated comparison, using paired Student’s t test
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Fig3: FVIIa promotes cytokine-induced caspase-3 cleavage in primary human beta cells. (a) Untreated or FVIIa pre-treated (10 nmol/l, 6 h) human islets were stimulated with a cytokine mixture of IL-1β, TNF-α and IFN-γ (Cyt.) for 72 h, then mildly dispersed using trypsin to generate single islet cells. Cells were stained with DAPI (blue), insulin antibody (red) and cleaved caspase-3 antibody (green) and visualised by fluorescence microscopy. Islets from five different donors were used and images show results from one representative donor. Scale bar, 50 μm. (b) Cells (~2,000/group) were counted and the number of cells double-positive for insulin and cleaved caspase-3 are depicted in the graph. Results are means ± SEM from five experiments. *p < 0.05 for indicated comparison, using paired Student’s t test

Mentions: Finally, we investigated the effects of FVIIa in primary human islet cells. Untreated islets or islets treated with cytokine mixture for 72 h were dispersed to generate single islet cells and then co-stained using insulin and cleaved caspase-3 antibodies. The number of insulin-positive cells varied between 30% and 80% using five different donor preparations (Fig. 3). The cytokine mixture induced caspase-3 cleavage in beta cells when compared with control cells and caspase-3 cleavage was further increased by FVIIa pre-treatment (Fig. 3b).Fig. 3


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)

FVIIa promotes cytokine-induced caspase-3 cleavage in primary human beta cells. (a) Untreated or FVIIa pre-treated (10 nmol/l, 6 h) human islets were stimulated with a cytokine mixture of IL-1β, TNF-α and IFN-γ (Cyt.) for 72 h, then mildly dispersed using trypsin to generate single islet cells. Cells were stained with DAPI (blue), insulin antibody (red) and cleaved caspase-3 antibody (green) and visualised by fluorescence microscopy. Islets from five different donors were used and images show results from one representative donor. Scale bar, 50 μm. (b) Cells (~2,000/group) were counted and the number of cells double-positive for insulin and cleaved caspase-3 are depicted in the graph. Results are means ± SEM from five experiments. *p < 0.05 for indicated comparison, using paired Student’s t test
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig3: FVIIa promotes cytokine-induced caspase-3 cleavage in primary human beta cells. (a) Untreated or FVIIa pre-treated (10 nmol/l, 6 h) human islets were stimulated with a cytokine mixture of IL-1β, TNF-α and IFN-γ (Cyt.) for 72 h, then mildly dispersed using trypsin to generate single islet cells. Cells were stained with DAPI (blue), insulin antibody (red) and cleaved caspase-3 antibody (green) and visualised by fluorescence microscopy. Islets from five different donors were used and images show results from one representative donor. Scale bar, 50 μm. (b) Cells (~2,000/group) were counted and the number of cells double-positive for insulin and cleaved caspase-3 are depicted in the graph. Results are means ± SEM from five experiments. *p < 0.05 for indicated comparison, using paired Student’s t test
Mentions: Finally, we investigated the effects of FVIIa in primary human islet cells. Untreated islets or islets treated with cytokine mixture for 72 h were dispersed to generate single islet cells and then co-stained using insulin and cleaved caspase-3 antibodies. The number of insulin-positive cells varied between 30% and 80% using five different donor preparations (Fig. 3). The cytokine mixture induced caspase-3 cleavage in beta cells when compared with control cells and caspase-3 cleavage was further increased by FVIIa pre-treatment (Fig. 3b).Fig. 3

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