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. 2017 Nov;66(11):2875-2887.
doi: 10.2337/db17-0215. Epub 2017 Aug 25.

Inhibition of 12/15-Lipoxygenase Protects Against β-Cell Oxidative Stress and Glycemic Deterioration in Mouse Models of Type 1 Diabetes

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Free PMC article

Inhibition of 12/15-Lipoxygenase Protects Against β-Cell Oxidative Stress and Glycemic Deterioration in Mouse Models of Type 1 Diabetes

Marimar Hernandez-Perez et al. Diabetes. .
Free PMC article

Abstract

Islet β-cell dysfunction and aggressive macrophage activity are early features in the pathogenesis of type 1 diabetes (T1D). 12/15-Lipoxygenase (12/15-LOX) is induced in β-cells and macrophages during T1D and produces proinflammatory lipids and lipid peroxides that exacerbate β-cell dysfunction and macrophage activity. Inhibition of 12/15-LOX provides a potential therapeutic approach to prevent glycemic deterioration in T1D. Two inhibitors recently identified by our groups through screening efforts, ML127 and ML351, have been shown to selectively target 12/15-LOX with high potency. Only ML351 exhibited no apparent toxicity across a range of concentrations in mouse islets, and molecular modeling has suggested reduced promiscuity of ML351 compared with ML127. In mouse islets, incubation with ML351 improved glucose-stimulated insulin secretion in the presence of proinflammatory cytokines and triggered gene expression pathways responsive to oxidative stress and cell death. Consistent with a role for 12/15-LOX in promoting oxidative stress, its chemical inhibition reduced production of reactive oxygen species in both mouse and human islets in vitro. In a streptozotocin-induced model of T1D in mice, ML351 prevented the development of diabetes, with coincident enhancement of nuclear Nrf2 in islet cells, reduced β-cell oxidative stress, and preservation of β-cell mass. In the nonobese diabetic mouse model of T1D, administration of ML351 during the prediabetic phase prevented dysglycemia, reduced β-cell oxidative stress, and increased the proportion of anti-inflammatory macrophages in insulitis. The data provide the first evidence to date that small molecules that target 12/15-LOX can prevent progression of β-cell dysfunction and glycemic deterioration in models of T1D.

Figures

Figure 1
Figure 1
Toxicity of 12/15-LOX inhibitors in mouse islets. A: Chemical structure of ML351 and caspase activity measurements performed with mouse islets in the presence of varying doses of ML351 for 24 h. B: Chemical structure of ML127 and caspase activity measurements performed with mouse islets in the presence of varying doses of ML127 for 24 h. C: Distribution of CANDOCK scores with the number of human proteins predicted to bind to ML127 and ML351. Scores less than a cutoff of −39.13 are predicted strong binders for both compounds. D: Number of predicted strong binders for ML127 and ML351 distribution in eight druggable protein classes. E: 12-HETE levels in media from mouse islets incubated with the indicated concentrations of ML351 and proinflammatory cytokines for 4 h. Data are mean ± SEM (n = 3 independent experiments). *P < 0.05 for the comparisons indicated. GPCR, G-protein–coupled receptor; RFU, relative fluorescence unit.
Figure 2
Figure 2
12/15-LOX inhibition protects against cytokine-induced islet dysfunction and oxidative stress. A: Mouse islets were incubated for 24 h with cytokines, ML351, and 12-HETE before exposure to low (2.5 mmol/L) and high (25 mmol/L) glucose, after which insulin levels in the medium were measured (n = 3–4 independent experiments). *P < 0.05 compared with control conditions (no cytokines, no ML351, no 12-HETE). B: Total islet insulin content for the insulin release experiments shown in A. C: Volcano plot of RNA sequencing analysis of mouse islets treated with cytokines + 10 μmol/L ML351 vs. cytokines alone for 24 h (n = 3 independent experiments; red, genes significantly altered [P < 0.05]; yellow, genes altered more than twofold; green, genes significantly altered more than twofold). D: Gene ontology pathway analysis of data shown in C. E: βTC3 β-cells were transfected with Nrf2-Luc reporter and incubated with ML351 at the concentrations indicated and then treated for 6 h with cytokine before luciferase activity measurement (n = 10 independent experiments). Data are luciferase activity normalized to control conditions (no cytokines, no ML351). *P < 0.05 compared with untreated control (no cytokines, no ML351). #P < 0.05 compared with cytokine treatment alone (no ML351). F: Mouse islets stained for ROS (CellROX reagent) (red) and DAPI (blue) upon treatment with or without cytokines and 10 μmol/L ML351. G: Human islets stained for ROS (CellROX reagent) (red) and DAPI (blue) upon treatment with or without cytokines and 10 μmol/L ML355. Original magnification ×200; scale bars = 100 μm. Bar graphs show quantitation of CellROX intensity in 10–16 islets from three independent mouse islet preparations (or human donors). *P < 0.05 compared with untreated islets (no cytokines, no ML compound). Data are mean ± SEM. PIC, proinflammatory cytokines.
Figure 3
Figure 3
12/15-LOX inhibition protects against glycemic deterioration after multiple low-dose STZ injections. Nine-week-old male C57BL/6J mice were treated with vehicle or ML351. Multiple low-dose STZ injections (55 mg/kg) were given for 5 days (n = 5–15). A: Study design. B: Serum drug levels. C: Change in body weight from the start of the study. D: Random fed blood glucose measurements performed throughout the study. E: Results of GTTs performed at day 8. F: AUC from GTTs presented in E. G: GSIS in vivo. H: Results of ITTs performed at day 18. I: Area over the curve (AOC) from ITTs presented in H. Data are mean ± SEM. *P < 0.05 compared with no-STZ + M0. #P < 0.05 compared with STZ + M0.
Figure 4
Figure 4
Effect of 12/15-LOX inhibition on β-cell mass and serum insulin in the multiple low-dose STZ diabetes mouse model. Nine-week-old male C57BL/6J mice were treated with vehicle or ML351. Multiple low-dose STZ injections (55 mg/kg) were given for 5 days (n = 5–15). A: Representative images of pancreata immunostained for insulin (brown) and counterstained for hematoxylin (blue). Original magnification ×100; scale bars = 200 μm. B: β-Cell mass of pancreata harvested at day 21. C: Random fed serum insulin levels. Data are mean ± SEM. *P < 0.05 compared with no-STZ + M0.
Figure 5
Figure 5
Inhibition of 12/15-LOX protects against islet oxidative stress in the multiple low-dose STZ diabetes mouse model. Nine-week-old male C57BL/6J mice were treated with vehicle or ML351. Multiple low-dose STZ injections (55 mg/kg) were given for 5 days (n = 5–15). Fixed pancreatic sections were subjected to immunofluorescence staining. Shown are representative images and the pixel density or nuclear/cytoplasmic ratio from analysis of at least three animals per group. A: Oxidative stress marker 4-HNE (magenta), insulin (green), and DAPI (blue). B: Nrf2 (magenta), insulin (green), and DAPI (blue). C: Antioxidant enzyme GPX1 (magenta), insulin (green), and DAPI (blue). Original magnification ×200; scale bars = 50 μm. *P < 0.05 compared with no-STZ + M0.
Figure 6
Figure 6
12/15-LOX inhibition mitigates glycemic deterioration in prediabetic NOD mice. Six-week-old NOD and CD1 female mice were injected daily for 2 weeks with vehicle or 24 mg/kg ML351 (n = 5). A: GTTs performed at 8 weeks of age. B: AUC of GTTs presented in A. C: Random fed blood glucose at 8 weeks of age. D: Representative images of pancreata immunostained for insulin (brown) and counterstained with hematoxylin (blue) from prediabetic 8-week-old mice. Original magnification ×100; scale bars = 100 μm. E: β-Cell mass of pancreata harvested at week 8. F: Average insulitis score. G: Representative images of pancreata immunostained for glucagon (magenta), insulin (green), and DAPI (gray). Original magnification ×200; scale bars = 100 μm. *P < 0.05 compared with CD1 + M0. #P < 0.05 compared with NOD + M0. INS, insulin; NS, not significant.
Figure 7
Figure 7
12/15-LOX inhibition reduces islet oxidative stress in prediabetic NOD mice. Fixed pancreatic sections from CD1 and NOD mice treated with vehicle or 24 mg/kg ML351 were subjected to immunofluorescence staining. Shown are representative images and the pixel density or nuclear/cytoplasmic ratio from analysis of at least three animals per group. A: Oxidative stress marker 4-HNE (magenta), insulin (green), and DAPI (blue). B: Nrf2 (magenta), insulin (green), and DAPI (blue). C: Antioxidant enzyme GPX1 (magenta), insulin (green), and DAPI (blue). Original magnification ×200; scale bars = 100 μm. *P < 0.05 compared with CD1 + M0.

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