Sphingosine 1-phosphate affects cytokine-induced apoptosis in rat pancreatic islet beta-cells.

Cytokines mediate pancreatic islet beta-cell apoptosis and necrosis, leading to loss of insulin secretory capacity and type 1 diabetes mellitus. The cytokines, IL-1beta and interferon-gamma, induced terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining of rat islet cells within 48 h by about 25-30%, indicative of apoptosis and/or necrosis. Sphingosine 1-phosphate (S1P) at nanomolar concentrations significantly reduced islet cell cytokine-induced TUNEL staining. Similar effects were observed in INS-1 cells. The dihydro analog of S1P also reduced the percentage of TUNEL stained islet and INS-1 cells, whereas the S1P receptor antagonist BML-241 blocked the protective effects. Pertussis toxin did not affect the S1P protective response. In the presence of a phospholipase C antagonist, U73122, there was significant inhibition of the S1P protective effects against apoptosis/necrosis. S1P stimulated INS-1 cell protein kinase C activity. Carbamylcholine chloride acting through muscarinic receptors also inhibited cytokine-induced TUNEL staining in pancreatic islet cells. S1P and/or dihydro-S1P also antagonized cytokine-induced increases in cytochrome c release from mitochondria and caspase-3 activity in INS-1 cells, which are indicative of cell apoptosis vs. necrosis. S1P failed to affect nitric oxide synthase activity after 48 h. Thus, the evidence suggests that S1P acting on S1P receptors coupled to G(q) mediates protective effects on islet beta-cells against cytokine-induced apoptosis.

Sphingosine kinase activity and sphingosine-1 phosphate production in rat pancreatic islets and INS-1 cells: response to cytokines.

Sphingosine-1 phosphate (S1P) is a bioactive sphingolipid with the potential to mobilize Ca2+, to inhibit apoptosis, and to promote mitogenesis. Sphingosine kinase (SPHK) and S1P were characterized in INS-1 insulinoma cells and isolated rat islets of Langerhans. SPHK activity increased in INS-1 cell homogenates treated with interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha (TNF-alpha), and responses were additive. IL-1beta or TNF-alpha increased islet SPHK activity within 15 min to 1 h; activity remained elevated after 8 h. SPHK2 was the predominant active isoform in INS-1 cells; little or no SPHK1 activity was detected. Cytokines increased endogenous S1P biosynthesis in 32P(i)-prelabeled INS-1 cells, and cycloheximide inhibited the response after 8 h, suggesting that protein synthesis mediated the response. There was no [32P]S1P release from cells. Compared with basal values, IL-1beta and TNF-alpha induced increases in SPHK1a mRNA levels relative to 18S ribosomal RNA in INS-1 cells within 1 h; relative SPHK2 mRNA levels were unchanged after cytokine treatment. IL-1beta, but not TNF-alpha, induced relative SPHK1a mRNA expression levels within 1 h in islets, whereas SPHK2 mRNA levels were unchanged. Thus, IL-1beta and TNF-alpha induced an early and sustained increase in SPHK activity in INS-1 cells and isolated islets, suggesting that S1P plays a role in the pathological response of pancreatic beta-cells to cytokines.

Endothelial differentiation gene receptors in pancreatic islets and INS-1 cells.

The endothelial differentiation gene (EDG) receptors are a class of G protein-coupled receptors. EDG-1, -3, -5, -6, and -8 bind the bioactive lipid sphingosine-1-phosphate (SPP) as the primary signaling ligand. EDG-2, -4, and -7 bind the ligand lysophosphatidic acid. EDG-1, -2, -3, -5, -6, and -7, but not -8, mRNAs were expressed in isolated rat pancreatic islets, whereas INS-1 insulinoma cells expressed only EDG-1, -2, -3, and -5 mRNAs. EDG-4 mRNA was expressed in mouse islets. EDG-1 mRNA but not EDG-3 mRNA was rapidly induced relative to 18S rRNA after stimulation of isolated islets with phorbol 12-myristate 13-acetate (PMA) or cholecystokinin-8S for 2 h. The protein kinase C inhibitor GF 109203X blocked the EDG-1 induction by PMA. Similarly, in islets stimulated for 2 h with 17 mmol/l glucose, the relative EDG-1 mRNA levels increased almost twofold compared with levels in control islets at 5.5 mmol/l glucose. In contrast, after 11 mmol/l glucose stimulation for 7 days, the relative levels of rat islet EDG-1 mRNA were significantly reduced to 54% below that of islets cultured at 5.5 mmol/l glucose. There was no change in relative EDG-3 mRNA levels. Stimulation of EDG receptors in islets and INS-1 cells with SPP inhibited glucagon-like peptide 1 (GLP-1)-stimulated cAMP production and insulin secretion in a concentration-dependent manner. Pertussis toxin antagonized the SPP effects on insulin release. Thus, EDG receptors are expressed in pancreatic islet beta-cells and G(i) seems to mediate the inhibition by SPP of adenylyl cyclase and cAMP formation and inhibition of the stimulation of insulin secretion by GLP-1.