Overexpression of sphingosine-1-phosphate lyase protects insulin-secreting cells against cytokine toxicity.

Increasing evidence suggests a crucial role of inflammation in cytokine-mediated ß-cell dysfunction and death in type 1 diabetes mellitus, although the mechanisms are incompletely understood. Sphingosine 1-phosphate (S1P) is a multifunctional bioactive sphingolipid involved in the development of many autoimmune and inflammatory diseases. Here, we investigated the role of intracellular S1P in insulin-secreting INS1E cells by genetically manipulating the S1P-metabolizing enzyme S1P lyase (SPL). The expression of spl was down-regulated by cytokines in INS1E cells and rat islets. Overexpression of SPL protected against cytokine toxicity. Interestingly, the SPL overexpression did not suppress the cytokine-induced NFκB-iNOS-NO pathway but attenuated calcium leakage from endoplasmic reticulum (ER) stores as manifested by lower cytosolic calcium levels, higher expression of the ER protein Sec61a, decreased dephosphorylation of Bcl-2-associated death promoter (Bad) protein, and weaker caspase-3 activation in cytokine-treated (IL-1ß, TNFα, and IFNγ) cells. This coincided with reduced cytokine-mediated ER stress, indicated by measurements of CCAAT/enhancer-binding protein homologous protein (chop) and immunoglobulin heavy chain binding protein (bip) levels. Moreover, cytokine-treated SPL-overexpressing cells exhibited increased expression of prohibitin 2 (Phb2), involved in the regulation of mitochondrial assembly and respiration. SPL-overexpressing cells were partially protected against cytokine-mediated ATP reduction and inhibition of glucose-induced insulin secretion. siRNA-mediated spl suppression resulted in effects opposite to those observed for SPL overexpression. Knockdown of phb2 partially reversed beneficial effects of SPL overexpression. In conclusion, the relatively low endogenous Spl expression level in insulin-secreting cells contributes to their extraordinary vulnerability to proinflammatory cytokine toxicity and may therefore represent a promising target for ß-cell protection in type 1 diabetes mellitus.

Improved antioxidative defence protects insulin-producing cells against homocysteine toxicity.

Homocysteine (HC) is considered to play an important role in the development of metabolic syndrome complications. Insulin-producing cells are prone to HC toxicity and this has been linked to oxidative stress. However, the exact mechanisms remain unknown. Therefore it was the aim of this study to determine the nature of reactive oxygen species responsible for HC toxicity. Chronic exposure of RINm5F and INS1E insulin-producing cells to HC decreased cell viability and glucose-induced insulin secretion in a concentration-dependent manner and led to a significant induction of hydrogen peroxide generation in the cytosolic, but not the mitochondrial compartment of the cell. Cytosolic overexpression of catalase, a hydrogen peroxide detoxifying enzyme, provided a significant protection against viability loss and hydrogen peroxide generation, while mitochondrial overexpression of catalase did not protect against HC toxicity. Overexpression of CuZnSOD, a cytosolic superoxide dismutating enzyme, also protected against HC toxicity. However, the best protection was achieved in the case of a combined overexpression of CuZnSOD and catalase. Incubation of cells in combination with alloxan resulted in a significant increase of HC toxicity and an increase of hydrogen peroxide generation. Overexpression of CuZnSOD or catalase protected against the toxicity of HC plus alloxan, with a superior protection achieved again by combined overexpression. The results indicate that HC induces oxidative stress in insulin-producing cells by stimulation of superoxide radical and hydrogen peroxide generation in the cytoplasm. The low antioxidative defence status makes the insulin-producing cells very vulnerable to HC toxicity.