CRFR1 activation protects against cytokine-induced β-cell death

Abstract

During the development of diabetes β-cells are exposed to elevated concentrations of proinflammatory cytokines, TNFα and IL1β, which in vitro induce β-cell death. The class B G-protein-coupled receptors (GPCRs): corticotropin-releasing factor receptor 1 (CRFR1) and CRFR2 are expressed in pancreatic islets. As downstream signaling by other class B GPCRs can protect against cytokine-induced β-cell apoptosis, we evaluated the protective potential of CRFR activation in β-cells in a pro-inflammatory setting. CRFR1/CRFR2 ligands activated AKT and CRFR1 signaling and reduced apoptosis in human islets. In rat and mouse insulin-secreting cell lines (INS-1 and MIN6), CRFR1 agonists upregulated insulin receptor substrate 2 (IRS2) expression, increased AKT activation, counteracted the cytokine-mediated decrease in BAD phosphorylation, and inhibited apoptosis. The anti-apoptotic signaling was dependent on prolonged exposure to corticotropin-releasing factor family peptides and followed PKA-mediated IRS2 upregulation. This indicates that CRFR signaling counteracts proinflammatory cytokine-mediated apoptotic pathways through upregulation of survival signaling in β-cells. Interestingly, CRFR signaling also counteracted basal apoptosis in both cultured INS-1 cells and intact human islets.

Keywords: CRFR; GPCR; apoptosis; cytokines; survival; urocortins; β cells.

Fig. 1. Effect of CRFR signalling on AKT activation

INS-1 cells (a) were treated with 50 nM of oCRF for various time points ranging from 1 h to 24 h. Lysates were subjected to immunoblotting using antibodies against phosphorylated (P-AKT) or total AKT (T-AKT). A representative blot is shown (n=3). INS-1 cells b) or MIN6 cells c) were treated with 50 nM of either oCRF, rUcn1 or mUcn3 for 16 h and lysates were treated as above (n = 4).

Fig. 2. Determination of receptor type and signalling components

a) INS-1 cells were treated with 5 μM of the CRFR1 antagonist, antalarmin 30 min prior to addition of 50 nM of oCRF, rUcn1 or mUcn3 and cultured for 16 h. Lysates were analysed by immunoblotting using antibodies against phosphorylated (P-AKT) or total AKT (T-AKT). A representative blot is shown (n=4). b) INS-1 cultured with 10 μM of the PI3K inhibitor LY294002 30 min prior to treatment with oCRF, rUcn1 or mUcn3 and handled as above. c) INS-1 cells were transiently transfected with a glucose-6-phosphatase promotor construct leading to luciferase expression and a constitutive active β-galactosidase construct. Cells were cultured with increasing concentration of oCRF. Data are presented as mean luciferase activity normalised to β-galactosidase ± SEM from three independent experiments each performed in triplicates.*p<0.05, ***p<0.001 vs. untreated cells (t test). d) INS-1 cells were treated with 50 nM of oCRF or rUcn1 for various time points ranging from 30 min to 24 hours. Lysates were subjected to immunoblotting using antibodies against IRS2 and the housekeeping protein β-actin. A representative blot from three independent experiments is shown (n=3). e) INS-1 cells were pre-treated with 10 μM of H89 for 30 min prior to addition of 50 nM of oCRF or rUcn1 and culturing for additional 16 h. Lysates were analysed for IRS2 expression, β-actin, phosphorylated AKT (P-AKT) and total AKT (T-AKT). The blot shown is a representative of three independent experiments (n=3).

Fig. 2. Determination of receptor type and signalling components

a) INS-1 cells were treated with 5 μM of the CRFR1 antagonist, antalarmin 30 min prior to addition of 50 nM of oCRF, rUcn1 or mUcn3 and cultured for 16 h. Lysates were analysed by immunoblotting using antibodies against phosphorylated (P-AKT) or total AKT (T-AKT). A representative blot is shown (n=4). b) INS-1 cultured with 10 μM of the PI3K inhibitor LY294002 30 min prior to treatment with oCRF, rUcn1 or mUcn3 and handled as above. c) INS-1 cells were transiently transfected with a glucose-6-phosphatase promotor construct leading to luciferase expression and a constitutive active β-galactosidase construct. Cells were cultured with increasing concentration of oCRF. Data are presented as mean luciferase activity normalised to β-galactosidase ± SEM from three independent experiments each performed in triplicates.*p<0.05, ***p<0.001 vs. untreated cells (t test). d) INS-1 cells were treated with 50 nM of oCRF or rUcn1 for various time points ranging from 30 min to 24 hours. Lysates were subjected to immunoblotting using antibodies against IRS2 and the housekeeping protein β-actin. A representative blot from three independent experiments is shown (n=3). e) INS-1 cells were pre-treated with 10 μM of H89 for 30 min prior to addition of 50 nM of oCRF or rUcn1 and culturing for additional 16 h. Lysates were analysed for IRS2 expression, β-actin, phosphorylated AKT (P-AKT) and total AKT (T-AKT). The blot shown is a representative of three independent experiments (n=3).

Fig. 3. Effect of CRFR signalling on survival pathways

a) INS-1 cells were pre-cultured with oCRF, rUcn1 or mUcn3 for 16 h. Subsequently, IL-1β (160 pg/ml) or TNFα (20ng/ml) was added to the cells and culture continued for another 24 hours. Cell extract was analysed for phosphorylated (P-AKT) or total (T-AKT) AKT by western blot analysis. A representative blot is shown (n=5). b) INS-1 cells were pre-treated as above and exposed to TNFα for 24 h. Levels of phosphorylated BAD and β-actin were analysed by western blot analysis. A representative blot of 3 independent experiments is shown (n=3).

Fig. 4. Effect of CRFR signalling on cytokine-induced MAPK and NFκB activation

a) INS-1 cells were pre-cultured with oCRF, rUcn1 or mUcn3 for 16 h followed by IL-1β (160 pg/ml) or TNFα (20 ng/ml) exposure for additional 20 min. Cell extracts were analysed for phosporylated (P) JNK, p38, ERK1/2. A representative blot from three independent experiments is shown (n=3). b) INS-1 cells were transiently co-transfected with NFκB-responsive reporter construct together with a constitutively active β-galactosidase construct, incubated with oCRF for 16 h and exposed to IL-1β or TNFα for another 6 hours. Data are presented as mean luciferase activity normalised to β-galactosidase ± SEM from four independent experiments each performed in triplicates (n=4).*p<0.05 vs. cytokine-treated cells (t test). White bars represent controls, black bars IL-1β-treated and grey bars TNFα-treated cells. c) INS-1 cells were treated as above and degradation of IκBα analysed by western blot analysis using whole cell lysates and antibodies against IκBα and the housekeeping protein β-actin. A representative blot from three independent experiments is shown (n=3).

Fig. 5. Effects of CRFR1 signalling on cytokine-induced apoptosis

a) MIN6 cells were pre-treated with 50 nM of oCRF, rUcn1 or mUcn3 for 16 h. Subsequently cells were exposed to IL-1β (160 pg/ml) in combination with TNFα for 24 h. Following lysis, western blotting was performed and apoptosis detected by antibodies recognising the cleaved form of caspase 3 as well as β-actin. A representative blot from three independent experiments is shown (n=3). b) INS-1 cells were pre-treated as for a) Subsequently TNFα (20 ng/ml) or was added and cells were cultured for another 24 h. c) MIN6 cells were treated as above and exposed to IL-1β (160 pg/ml) or TNFα (20 ng/ml) for 24 h. d) INS-1 cells were pretreated with vehicle (EtOH) or 5μM antalarmin for 30 min before stimulation with 50nM oCRF or rUCN1 for 60 hours. Apoptosis was detected by cell death detection ELISA and data are presented as mean fold induction of apoptosis ± SEM from three to four independent experiments (n=3–4).*p<0.05, ** p<0.01, *** p<0.001 vs. cytokine-stimulated cells (t test). White bars represent controls, black bars TNFα-treated cells and grey bars IL-1β-treated cells.

Fig. 6. Effect of CRFR signalling on human islets

a) Human islets were pre-treated with 50 nM of oCRF, rUcn1 or mUcn3 as indicated and analysed for levels of phosphorylated AKT (P-AKT) or total AKT (T-AKT) by western blot analysis. A representative of three independent experiments is shown (n=3). b) Human islets were pre-treated as described above and cultured for 6 days. Apoptosis was determined by the cell death detection ELISA. Experiment was performed independently on islets from three different donors and data presented as mean apoptosis ± SEM.*p<0.05, **p<0.01 vs. untreated islets (t test) (n=3).