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We have previously shown that the insulinotropic imidazoline compound RX871024 induces death of insulinoma MIN6 cells, an effect involving stimulation of c-Jun N-terminal kinase (JNK) and caspase 3. It has also been reported that AMP-activated protein kinase (AMPK) activates JNK and induces β-cell death. Here we show that RX871024, but not another insulinotropic imidazoline compound (BL11282), suppressed AMPK activity in MIN6 cells. The inhibitory effect of RX871024 on AMPK was supported by the observation that the imidazoline induced lipid droplet formation in the cytoplasm of MIN6 cells. This reflects stimulation of anabolic pathways and inhibition of catabolic pathways in the cell that happen under conditions when AMPK is inhibited. Activation of AMPK by 5-aminoimidazole-4-carboxamide riboside (AICAR) elevated basal and cytokine-induced death in primary β-cells and in insulinoma MIN6 cells. RX871024 aggravated AICAR-induced insulinoma MIN6 cell death regardless of the presence of pro-inflammatory cytokines. The specific cytotoxic effect of imidazoline compound RX871024 on insulinoma cell death but not primary β-cell death is independent of its action on AMPK and may suggest the possibility of using this type of compound in the treatment of insulinomas.

An insufficient number of insulin-producing beta-cells is a major cause of defective control of blood glucose in both type 1 and type 2 diabetes. The aim of this study was to clarify whether the insulinotropic imidazolines can affect the survival of highly proliferating insulin-secreting cells, here exemplified by the MIN6 cell line. Our data demonstrate that RX871024, but not efaroxan, triggered MIN6 cell death and potentiated death induced by a combination of the pro-inflammatory cytokines interleukin-1beta, interferon- gamma and tumor necrosis factor-alpha. These effects did not involve changes in nitric oxide production but correlated with stimulation of c-jun N-terminal kinase (JNK) activity and activation of caspases-1, -3, -8 and -9. Our results suggest that the imidazoline RX871024 causes death of highly proliferating insulin-secreting cells, putatively via augmentation of JNK activity, a finding that may impact on the possibility of using compounds of similar activity in the treatment of diabetes.

Apoptosis was monitored in intact insulin-producing cells both with microfluorometry and with two-photon laser scanning microscopy (TPLSM), using a fluorescent protein based on fluorescence resonance energy transfer (FRET). TPLSM offers three-dimensional spatial information that can be obtained relatively deep in tissues. This provides a potential for future in vivo studies of apoptosis. The cells expressed a fluorescent protein (C-DEVD-Y) consisting of two fluorophores, enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), linked by the amino acid sequence DEVD selectively cleaved by caspase-3–like proteases. FRET between ECFP and EYFP in C-DEVD-Y could therefore be monitored on-line as a sensor of caspase-3 activation. The relevance of using caspase-3 activation to indicate β-cell apoptosis was demonstrated by inhibiting caspase-3–like proteases with Z-DEVD-fmk and thereby showing that caspase-3 activation was needed for high-glucose–and cytokine-induced apoptosis in the β-cell and for staurosporine-induced apoptosis in RINm5F cells. In intact RINm5F cells expressing C-DEVD-Y and in MIN6 cells expressing the variant C-DEVD-Y2, FRET was lost at 155 ± 23 min (n = 9) and 257 ± 59 min (n = 4; mean ± SE) after activation of apoptosis with staurosporine (6 μmol/l), showing that this method worked in insulin-producing cells.

Pancreatic beta cell damage caused by proinflammatory cytokines interleukin-1beta (IL-1beta), interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha) is a key event in the pathogenesis of type 1 diabetes. The suppressor of cytokine signaling-1 (SOCS-1) blocks IFNgamma-induced signaling and prevents diabetes in the non-obese diabetic mouse. Here, we investigated if SOCS-1 overexpression in primary beta cells provides protection from cytokine-induced islet cell dysfunction and death. We demonstrate that SOCS-1 does not prevent increase in NO production and decrease in glucose-stimulated insulin secretion in the presence of IL-1beta, IFNgamma, TNFalpha. However, it decreases the activation of caspase-3, -8 and -9, and thereby, promotes a robust protection from cytokine-induced beta cell death. Our data suggest that SOCS-1 overexpression may not be sufficient in preventing all the biological activities of IFNgamma in beta cells. In summary, we show that interference with IFNgamma signal transduction pathways by SOCS-1 inhibits cytokine-stimulated pancreatic beta cell death.

The imidazoline compound RX871024 reduces IL-1beta-induced NO production thereby protecting against IL-1beta-induced beta-cell apoptosis. The aim of this study was to evaluate whether imidazolines RX871024 and efaroxan protect beta-cells against death in the presence of a combination of the cytokines IL-1beta, IFNgamma, and TNFalpha. To address this issue, experiments involving different methods for detection of cell death, different concentrations of the cytokines, and a variety of conditions of preparation and culturing of ob/ob mouse islets and beta-cells have been carried out. Thoroughly performed experiments have not been able to demonstrate a protective effect of RX871024 and efaroxan on beta-cell death induced by the combination of cytokines. However, the inhibitory effect of RX871024 on NO production in ob/ob mouse islets and beta-cells was still observed in the presence of all three cytokines and correlated with the decrease in p38 MAPK phosphorylation. Conversely, efaroxan did not affect cytokine-induced NO production. Our data indicate that a combination of pro-inflammatory cytokines IL-1beta, IFNgamma, and TNFalpha, conditions modelling those that take place in type 1 diabetes, induces pancreatic beta-cell death that does not directly correlate with NO production and cannot be counteracted with imidazoline compounds.