Resistance to type 1 diabetes induction in 12-lipoxygenase knockout mice - PubMed (original) (raw)

Resistance to type 1 diabetes induction in 12-lipoxygenase knockout mice

D Bleich et al. J Clin Invest. 1999.

Abstract

Leukocyte 12-lipoxygenase (12-LO) gene expression in pancreatic beta cells is upregulated by cytotoxic cytokines like IL-1beta. Recent studies have demonstrated that 12-LO inhibitors can prevent glutamate-induced neuronal cell death when intracellular glutathione stores are depleted. Therefore, 12-LO pathway inhibition may prevent beta-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-dose streptozotocin to induce immune-mediated diabetes. Fasting serum glucose and insulin levels were measured at 7-day intervals, and the mice were followed up for 28 days. 12-LO KO mice were highly resistant to diabetes development compared with control mice and had higher serum insulin levels on day 28. Isolated pancreatic islets were treated with IL-1beta, TNF-alpha, and IFN-gamma for 18 hours. Glucose-stimulated insulin secretion in cytokine-treated islets from C57/BL6 mice decreased 54% from that of untreated islets. In marked contrast, the same cytokine mix led to only a 26% decrease in islets from 12-LO KO mice. Furthermore, cytokine-induced 12-hydroxyeicosatetraenoic acid (12-HETE) production was absent in 12-LO KO islets but present in C57/BL6 islets. Isolated peritoneal macrophages were stimulated for 48 hours with IFN-gamma + LPS and compared for nitrate/nitrite generation. 12-LO KO macrophages generated 50% less nitrate/nitrite when compared with C57BL/6 macrophages. In summary, elimination of leukocyte 12-LO in mice ameliorates low dose streptozotocin-induced diabetes by increasing islet resistance to cytokines and decreasing macrophage production of nitric oxide.

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Figures

Figure 1

Figure 1

Serum glucose levels in low dose STZ–treated 12-LO KO and C57BL/6 mice. Eight 12-LO KO and 12 C57BL/6 mice received 40 mg/kg of STZ for 5 consecutive days as shown. The mean fasting serum glucose ± SEM for each group of mice is shown at each time point. 12-LO KO mice were significantly more resistant to STZ-induced diabetes than C57BL/6 control mice. *P = 0.01 vs. 12-LO KO. **P = 0.003 vs. 12-LO KO.

Figure 2

Figure 2

Serum insulin levels in low dose STZ–treated 12-LO KO and C57BL/6 mice. 12-LO KO mice demonstrated a 45% decrease in insulin levels from day 0 to day 28, whereas C57BL/6 mice had a 90% decrease in insulin levels after low-dose STZ treatment. A statistically significant difference in insulin levels between 12-LO KO and C57BL/6 mice is seen on days 21 and 28.

Figure 3

Figure 3

Differential effect of cytokines on 12-HETE production in C57BL/6 and 12-LO KO islets. C57BL/6 islets demonstrated a 2.5-fold increase in 12-HETE production in the presence of cytokines, whereas 12-HETE production in 12-LO KO islets was unchanged. The number in parenthesis represents the number of experiments performed for each condition. *P < 0.05 vs. C57BL/6 islets.

Figure 4

Figure 4

Basal and glucose-stimulated insulin secretion in 12-LO KO and C57BL/6 islets. Islet preparations were assessed for basal and glucose-stimulated insulin secretion as described in Methods. 12-LO KO islets secreted 102 ± 2 ng insulin/20 islets/h in the basal state and 620 ± 50 ng insulin/20 islets/h when stimulated with 17 mM glucose. In comparison, C57BL/6 islets secreted 102 ± 4 ng insulin/20 islets/h in the basal state and 595 ± 101 ng insulin/20 islets/h when stimulated with 17 mM glucose. The number in parenthesis represents the number of experiments performed for each condition.

Figure 5

Figure 5

Effect of cytokine treatment on glucose-stimulated insulin secretion in 12-LO KO and C57BL/6 islets. Isolated islets were subjected to high-dose cytokine treatment as shown. Filled bars represent 17 mM glucose stimulation; hatched bars represent cytokine treatment (0.5 ng/mL IL-1β + 30 ng/mL TNF-α + 30 ng/mL IFN-γ) plus 17 mM glucose stimulation. Cytokine treatment decreased glucose-stimulated insulin secretion by 26% and 54% in 12-LO KO and C57BL/6 islets, respectively. Shown is 1 representative experiment in triplicate out of 3 separate experiments. *P < 0.03 vs. C57BL/6 glucose-stimulated islets.

Figure 6

Figure 6

Nitrate/nitrite determination in 12-LO KO and C57BL/6 peritoneal macrophages. Peritoneal macrophages were purified as described in Methods and stimulated with LPS (2 μg/mL) + IFN-γ (100 ng/mL) for 48 hours. We used Griess reagent to measure total medium nitrate/nitrite as an estimate of NO production. As shown, 12-LO KO macrophages generated ∼50% of the NO of C57BL/6 macrophages under the conditions described. Six individual experiments were performed with macrophages isolated from both strains of mice. *P < 0.0001 vs. C57BL/6 macrophages.

Figure 7

Figure 7

Generation of superoxide by 12-LO KO and C57BL/6 macrophages. Purified macrophages were treated with PMA (50 ng/mL), and superoxide generation was measured by monitoring the reduction of ferricytochrome c to ferrocytochrome_c_ at 550 λ over time. As shown, no difference was observed in superoxide generation. Eight individual experiments were performed with macrophages isolated from both strains of mice.

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