Mechanism of protection by soluble epoxide hydrolase inhibition in type 2 diabetic stroke.

Inhibition of soluble epoxide hydrolase (sEH) is a potential target of therapy for ischemic injury. sEH metabolizes neuroprotective epoxyeicosatrienoic acids (EETs). We recently demonstrated that sEH inhibition reduces infarct size after middle cerebral artery occlusion (MCAO) in type 1 diabetic mice. We hypothesized that inhibition of sEH would protect against ischemic injury in type 2 diabetic mice. Type 2 diabetes was produced by combined high-fat diet, nicotinamide and streptozotocin in male mice. Diabetic and control mice were treated with vehicle or the sEH inhibitor t-AUCB then subjected to 60-min MCAO. Compared to chow-fed mice, high fat diet-fed mice exhibited an upregulation of sEH mRNA and protein in brain, but no differences in brain EETs levels were observed between groups. Type 2 diabetic mice had increased blood glucose levels at baseline and throughout ischemia, decreased laser-Doppler perfusion of the MCA territory after reperfusion, and sustained larger cortical infarcts compared to control mice. t-AUCB decreased fasting glucose levels at baseline and throughout ischemia, improved cortical perfusion after MCAO and significantly reduced infarct size in diabetic mice. We conclude that sEH inhibition, as a preventative treatment, improves glycemic status, post-ischemic reperfusion in the ischemic territory, and stroke outcome in type 2 diabetic mice

High fat diet increases sEH expression in brain.

<p>Mice were treated with a high fat (60% fat) or chow diet (13% fat) for 15 weeks. A) EPHX2 mRNA expression was measured via qPCR in cortex. EPHX2 expression was normalized to 18S expression. *p<0.05 vs. Chow, n = 7 per group. B) sEH protein expression was measured via Western blot in brain. sEH protein expression was normalized to beta actin protein expression. *p<0.01 vs. Chow, n = 4 per group.</p

sEH inhibition decreased glucose levels in type 2 diabetic mice before, during, and after ischemia.

<p>Control and HFD+STZ/NA mice were treated with the sEH inhibitor t-AUCB (1 mg/kg i.p. daily, 7 days) or vehicle and subjected to middle cerebral artery occlusion (60 min). Blood glucose levels were measured at baseline, 30 min into the occlusion, and 30 min post-reperfusion. *p<0.001 vs. Control Vehicle, #p<0.01 vs. HFD+STZ/NA Vehicle. ∧p<0.05 vs. baseline of same treatment group. N = 4–5 per group.</p

2 mg/kg t-AUCB prevented reduced infarct size in control mice following MCAO.

<p>Control mice were treated with the sEH inhibitor t-AUCB (1 mg/kg or 2 mg/kg i.p. daily, 7 days) or vehicle and subjected to middle cerebral artery occlusion (60 min). Brains were harvested at 24 h post-MCAO, and infarct size measured by TTC staining and corrected for edema as described in methods. Infarct size is shown for separately for cortex, striatum, and total hemisphere. *p<0.05 vs. Control Vehicle of same brain region via 2-way ANOVA post-hoc test, #p<0.01 vs. Control Vehicle Hemisphere via t-test only, N = 7 per group.</p

Pre-diabetic mice have increased body weight, increased insulin levels and decreased glucose tolerance.

<p>A) Body weight B) serum insulin levels, and C) blood glucose during i.p. glucose tolerance test (GTT) were measured in mice fed a low fat (Chow; n = 5) or high fat (n = 4) diet for 15 weeks. Mice were fasted overnight prior to the GTT. Glucose (2 g/kg body weight) was injected i.p. at time 0. D) Area under the curve from GTT test results was calculated for each treatment group. *p<0.001 vs. Chow.</p

Baseline Characteristics of Type 2 Diabetic Mice and Controls.

<p>Age, weight, and body temperature, monitored via rectal temperature probe, were measured in control and HFD+STZ/NA mice treated with vehicle or t-AUCB treatment (n = 11–16 mice per group).</p

sEH inhibition decreased fasting glucose levels in type 2 diabetic mice.

<p>Fasting blood glucose levels were obtained from control and HFD+STZ/NA mice before (A) and after (B) treatment with the sEH inhibitor t-AUCB (1 mg/kg i.p. daily, 7 days) or vehicle. C) Serum insulin levels were also measured in t-AUCB or vehicle treated mice 24 h after ischemia. *p<0.001 vs. Control Vehicle, #p<0.05 vs. HFD+STZ/NA Vehicle. A) n = 20–22 per group. B) n = 5–8 per group. C) n = 2–6 per group.</p