Activation of 12-lipoxygenase in proinflammatory cytokine-mediated beta cell toxicity

Abstract

Aims/hypothesis: 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-1β, IFN-γ and TNF-α, 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 β-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 β-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 β-TC3 cell insulin release in a time-and concentration-dependent manner. Incubating β-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/interpretation: 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.

Selective inhibition of 12-lipoxygenase protects islets and beta cells from inflammatory cytokine-mediated beta cell dysfunction

“…Review of 12-lipoxygenase and links to biological systems highlights the important role of 12-lipoxygenase in the development of type 1 diabetes [11,15,17,23,33,34]. Studies of mouse models with transgenic deletion of 12-lipoxygenase suggest that activity of the 12-lipoxygenase enzyme strongly associates with beta cell dysfunction [17,19,33]. Islet-specific deletion of 12-lipoxygenase confers protection of islets from high fat, streptozotocin and cytokines [35].…”

“…12-Lipoxygenase is expressed in rodent and human islets [11,[15][16][17][18] and is upregulated under conditions of metabolic and cytokine stress. Direct addition of 12-S-HETE can impair beta cell function or can lead to loss of human beta cell viability [13,15,16,[19][20][21][22]. Recent evidence indicates that increased expression of 12-lipoxygenase in islets is a common feature of both rodent and human models of type 1 and type 2 diabetes [11,13,15,17,22].…”

Systemic alterations in the metabolome of diabetic NOD mice delineate increased oxidative stress accompanied by reduced inflammation and hypertriglyceremia

“…134000 Ϯ 88000 106000 Ϯ 1e ϩ05 0.79 0.02 C18-diol (4) 124 Ϯ 80 85 Ϯ 89 0.69 0.00 C18-ketone (2) 9920 Ϯ 11000 5480 Ϯ 3600 0.55 0.001 C20-hydroxy (10) 9480 Ϯ 5600 4230 Ϯ 5200 0.45 Ͻ0.00001 C20-ketone (3) 3050 Ϯ 2400 1260 Ϯ 1600 0.41 Ͻ0.00001 LPC (12) 7970000 Ϯ 2600000 3270000 Ϯ 1800000 0.41 Ͻ0.00001 PC (50) 53500000 Ϯ 1.1e ϩ07 39300000 Ϯ 1.6e ϩ07 0.73 Ͻ0.00001 PE (10) 210000 Ϯ 78000 141000 Ϯ 79000 0.67 0.002 PI (6) 1050000 Ϯ 290000 478000 Ϯ 320000 0.46 Ͻ0.00001 Prostacyclin (4) 125 Ϯ 82 49.8 Ϯ 42 0.4 Ͻ0.00001 SM (20) 2380000 Ϯ 660000 1410000 Ϯ 600000 0.59 Ͻ0.00001 Sterol (9) 1030000 Ϯ 240000 656000 Ϯ 280000 0.64 Ͻ0.00001 Triol (2) 23 Ϯ 23 10.7 Ϯ 9.6 0.47 0.01 FC, fold change in means in diabetic vs. control animals. *Based on Mann-Whitney U-test.…”

“…1). Free fatty acids exhibit complex interactions with ␤-cells, acting as both secretagogues (14) and agents of apoptotic signaling (9,26,42,55). Moreover, the development of T1D is known to have a strong inflammatory component (3,34,43).…”

“…Moreover, the development of T1D is known to have a strong inflammatory component (3,34,43). Cyclooxygenase (COX)-2 and 12-lipoxygenase (LOX) derived products of AA, such as prostaglandin E2 (PGE2) and 12-hydroxyeicosatetraenoic acid (12-HETE), have been shown to play critical roles in cytokine-induced human ␤-cell destruction (9,26,42,55). Thus, one may anticipate that the reduction in circulating AA represents a shift in equilibrium toward eicosanoid production.…”