The leukocyte 12-lipoxygenase (12-LO) gene is expressed in pancreatic β cells and macrophages. Products of the 12-LO enzyme are proinflammatory and may be involved in immune-mediated conditions, but formal proof of this hypothesis is lacking. Leukocyte 12-LO is preferentially expressed over 5-and 15-lipoxygenase in pancreatic β cells and may be involved in cytokine-mediated β-cell damage through generation of the lipid hydroperoxide 12-hydroperoxyeicosatetraenoic acid (12-HPETE) (1-7). Reduction of 12-HPETE to 12-hydroxyeicosatetraenoic acid (12-HETE) is facilitated by the enzyme glutathione peroxidase, and recent work has demonstrated that 12-LO inhibitors can prevent glutamate-induced neuronal cell death when intracellular glutathione stores are depleted (8). Both neuronal cell death and peroxide generation required 12-LO activity in this model and were prevented by treatment with a lipoxygenase inhibitor, baicalein (10 µM). Furthermore, the proinflammatory cytokine IL-1β was shown to increase 12-LO product formation in pancreatic islets (9, 10), whereas nordihydroguaiaretic, a lipoxygenase inhibitor, protected rat islet cells from cytokine-induced destruction (11). Given that 12-HPETE and/or other 12-LO products may damage pancreatic β cells through lipid peroxidation, it is possible that inhibition of the 12-LO pathway may protect pancreatic β cells from cytotoxic conditions.To test this hypothesis, we administered low-dose streptozotocin (STZ) to 12-LO knockout (12-LO KO) mice and genetic controls to evaluate the development of diabetes. In addition, we isolated pancreatic islets and tested them for sensitivity to cytokine-induced dysfunction. We also evaluated the capacity of macrophages to generate nitric oxide (NO) and superoxide when stimulated. We chose the low dose STZ-induced diabetic model because previous studies have demonstrated that β-cell destruction is mediated in part by generation of free radicals and lipid hydroperoxides as well as by immune activation (12, 13). 12-LO KO mice were generated by homologous recombination in embryonic stem cells, as described previously (14).In the present study, we found that 12-LO KO mice were significantly more resistant to STZ-induced diabetes than were C57BL/6 controls and that islets purified from 12-LO KO mice were protected from cytokine-induced dysfunction. Furthermore, macrophages from 12-LO KO mice generated ∼50% of the nitrate/nitrite compared with macrophages from control C57BL/6 mice. Leukocyte 12-lipoxygenase (12-LO) gene expression in pancreatic β cells is upregulated by cytotoxic cytokines like IL-1β. Recent studies have demonstrated that 12-LO inhibitors can prevent glutamateinduced neuronal cell death when intracellular glutathione stores are depleted. Therefore, 12-LO pathway inhibition may prevent β-cell cytotoxicity. To evaluate the role of 12-LO gene expression in immune-mediated islet destruction, we used 12-LO knockout (12-LO KO) mice. Male homozygous 12-LO KO mice and control C57BL/6 mice received 5 consecutive daily injections of low-...

Cyclooxygenase-2 (COX-2) gene and 12-lipoxygenase (12-LO) gene are preferentially expressed over other types of cyclooxygenase and lipoxygenase in pancreatic beta-cells. Inhibition of either COX-2 or 12-LO can prevent cytokine-induced pancreatic beta-cell dysfunction as defined by inhibition of glucose-stimulated insulin secretion. As cellular stress induces both genes and their respective end products in pancreatic beta-cells, we evaluated the role of 12-hydroxyeicosatetraenoic acid (HETE) on COX-2 gene expression, protein expression, and prostaglandin E2 (PGE2) production. We demonstrate that 12-HETE significantly increases COX-2 gene expression and consequent product formation, whereas a closely related lipid, 15-HETE, does not. In addition, IL-1beta-stimulated prostaglandin E2 production is completely inhibited by a preferential lipoxygenase inhibitor cinnaminyl-3,4-dihydroxy-alpha-cyanocinnamate. We then evaluated IL-1beta-induced PGE2 production in islets purified from control C57BL/6 mice and 12-LO knockout mice lacking cytokine-inducible 12-HETE. IL-1beta stimulated an 8-fold increase in PGE2 production in C57BL/6 islets but failed to stimulate PGE2 in 12-LO knockout islets. Addition of 12-HETE to 12-LO knockout islet cells produced a statistically significant rise in PGE2 production. Furthermore, 12-HETE, but not 15-HETE, stimulated COX-2 promoter and activator protein-1 binding activity. These data demonstrate that 12-HETE mediates cytokine-induced COX-2 gene transcription and resultant PGE2 production in pancreatic beta-cells.

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The leukocyte type of 12-lipoxygenase (12-LO) may play a role in inflammatory reactions in many cell types through the conversion of arachidonic acid to proinflammatory eicosanoids that include 12-hydroperoxyeicosatetraenoic acid and 12-hydroeicosatetraenoic acid. Previous studies demonstrating the presence of a functional 12-LO pathway in rat and human pancreatic beta-cells plus the recent cloning of a rat leukocyte type of 12-LO allowed us to evaluate whether inflammatory cytokines such as interleukin-1 beta (IL-1 beta) can regulate the beta-cell 12-LO enzyme pathway, thus providing a potential link between the cytotoxic effects of cytokines on pancreatic beta-cells and the proinflammatory effects of 12-LO products. We demonstrate that IL-1 beta induces 12-LO protein and messenger RNA (mRNA) expression in RIN m5F cells and 12-LO mRNA expression in rat islets. RIN m5F cells treated for 16 h with IL-1 beta (25, 50, and 100 ng/liter) showed a maximal 2-fold increase in the expression of a leukocyte form of 12-LO demonstrated by Western blots. A concomitant increase in 12-LO mRNA expression was seen at this time point using a highly sensitive competitive polymerase chain reaction assay. The increase in mRNA and protein expression was preceded by increased 12-LO pathway activity measured by a RIA for 12-S-HETE. Separate experiments using purified Sprague-Dawley rat islets also showed increased expression of 12-LO mRNA and enzyme activity in response to IL-1 beta. These results demonstrate that IL-1 beta can up-regulate 12-LO expression and activity in rat beta-cells.

Several autoantibodies related to Type 1 diabetes mellitus and their corresponding autoantigens have been previously identified. While peptide antigens are more widely recognized, lipid antigens like sulfatides and gangliosides are also known epitopes for the diabetic humoral immune response. Islet cell antibodies (ICA) in Type 1 diabetes are heterogeneous immunoglobulins directed against selected antigens in the islets of Langerhans. Moreover, ICA may be the best predictive marker of disease in family members of patients with Type 1 diabetes. The aims of this study were: (1) to purify lipids from porcine pancreas that contain ICA epitopes; (2) to characterize these lipid antigens, and (3) to use the purified lipids in an assay to detect antibodies in patients with Type 1 diabetes. A unique family of 4 lysophospholipids, 1 fully characterized as lysophosphatidylmyoinositol, partially inhibited ICA staining, and therefore, were considered to be candidate antigens for an ICA immunoassay. Using a dot blot immunoassay, we detected antibodies directed against these phospholipids in 28 out of 46 (61%) diabetic sera, while detecting only 1 false positive out of 28 nondiabetic sera (3.6%; p < 0.0001 comparing diabetic vs. nondiabetic serum). Therefore, lysophospholipid immunoassay positivity is present in sera of Type 1 diabetic patients. Furthermore, we detected 15 out of 23 ICA-negative diabetic sera (65.2%), showing that our phospholipid immunoassay does not correlate with ICA positivity.