Activation of 12-lipoxygenase in proinflammatory cytokine-mediated beta cell toxicity.
OBJECTIVE
Beta cell inflammation and cytokine-induced toxicity are central to autoimmune diabetes development. Lipid mediators generated upon lipoxygenase (LO) activation can participate in inflammatory pathways. 12LO-deficient mice are resistant to streptozotocin-induced diabetes. This study sought to characterise the cellular processes involving 12LO-activation lipid inflammatory mediator production in cytokine-treated pancreatic beta cells.


METHODS
Islets and beta cell lines were treated with a combination of IL-1beta, IFN-gamma and TNF-alpha, or the 12LO product 12(S)-hydroxyeicosatetraenoic acid (HETE). Insulin secretion was measured using an enzyme immunoassay, and cell viability was evaluated using an in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay. 12LO activity was evaluated and 12LO protein levels were determined using immunoblotting with a selective leucocyte type 12LO antibody. Cellular localisation of 12LO was evaluated using immunocytochemistry.


RESULTS
Basal expression of leucocyte type 12LO protein was found in human and mouse islets and in several rodent beta cell lines. In mouse beta-TC3 cells, and in human islets, cytokines induced release of 12-HETE within 30 min. Cytokine addition also induced a rapid translocation of 12LO protein from the cytosol to the nucleus of beta-TC3 cells as shown by subcellular fractionation and immunostaining. Cytokine-induced cell death and inhibition of insulin secretion were partially reversed by baicalein, a 12LO inhibitor. 12(S)-HETE inhibited beta-TC3 cell insulin release in a time- and concentration-dependent manner. Incubating beta-TC3 cells with 100 nmol/l of 12(S)-HETE resulted in a 57% reduction in basal insulin release (6 h), and a 17% increase in cell death (18 h) as compared with untreated cells. 12(S)-HETE activated the stress-activated protein kinase c-Jun N-terminal kinase and p38 within 15 min, as judged by increased kinase protein phosphorylation.


CONCLUSIONS
The data suggest that inflammatory cytokines rapidly activate 12LO and show for the first time that cytokines induce 12LO translocation. The effects of 12-HETE on insulin secretion, cytotoxicity and kinase activation were similar to the effects seen with cytokines. The results provide mechanistic information of cytokine-induced toxic effects on pancreatic beta cells and support the hypothesis that blocking 12LO activation could provide a new therapeutic way to protect pancreatic beta cells from autoimmune injury.
Chen
M
M
Department of Medicine, Division of Endocrinology and Metabolism, School of Medicine, University of Virginia, PO Box 801405, Charlottesville, VA, 22908-1405, USA.
Yang
Z D
ZD
Smith
K M
KM
Carter
J D
JD
Nadler
J L
JL
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000494:pharmacology; D001092:Arachidonate 12-Lipoxygenase / Q000378:metabolism*; D002460:Cell Line; D016207:Cytokines / Q000633:toxicity*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D021381:Protein Transport / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016207:Cytokines; D007375:Interleukin-1; D014409:Tumor Necrosis Factor-alpha; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D007371:Interferon-gamma; D001092:Arachidonate 12-Lipoxygenase
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