The essential role of endothelial nitric oxide synthase activation in insulin-mediated neuroprotection against ischemic stroke in diabetes Shiang-Suo.

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The essential role of endothelial nitric oxide synthase activation in insulin-mediated neuroprotection against ischemic stroke in diabetes Shiang-Suo Huang, PhD, Yi-Jhu Lu, MS, Jiung-Pang Huang, MS, Yang-Tzu Wu, BS, Yuan-Ji Day, MD, PhD, Li-Man Hung, PhD Journal of Vascular Surgery Volume 59, Issue 2, Pages 483-491 (February 2014) DOI: 10.1016/j.jvs.2013.03.023 Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 1 Body weight and plasma levels of glucose and insulin. Increased plasma glucose (A) and decreased plasma insulin level (B) and body weight (C) resulting from streptozotocin (STZ) injection were reversed by insulin treatment. Values are expressed as mean ± standard error of the mean. CONT, Age-matched nondiabetic controls (n = 28); DM, STZ-induced diabetic rats (n = 29); DMI, insulin-treated diabetic rats (n = 28); FC I/R, focal cerebral ischemia-reperfusion. Journal of Vascular Surgery 2014 59, 483-491DOI: (10.1016/j.jvs.2013.03.023) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 2 Focal cerebral ischemia-reperfusion (FC I/R)-induced brain infarction and neurologic deficits. FC I/R-induced brain infarction (A and B) and neurologic deficits (C and D) exacerbated by diabetes were rescued by insulin treatment. A, Shows representative coronal sections and (B) shows mean ± standard error of the mean of quantitative infarct volume. Neurologic functions were assessed by Bederson score (C) and grip test (D) in age-matched nondiabetic controls (CONT, n = 19), STZ-induced diabetic rats (DM, n = 21), and insulin-treated diabetic rats (DMI, n = 12). Values are expressed as mean ± standard error of the mean. Journal of Vascular Surgery 2014 59, 483-491DOI: (10.1016/j.jvs.2013.03.023) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 3 Cerebral glucose uptake and glucose transporter (GLUT) protein expression. Cerebral tissues from ischemia-reperfusion (I/R) side (Ipsilateral) and the non-I/R counterpart (Contralateral) were harvested from controls (CONT, n = 7), diabetic (DM, n = 9), and insulin-treated diabetic (DMI, n = 6) rats for biochemical analyses. Diabetes and focal cerebral (FC) I/R injury-impaired cerebral glucose uptake (A) and GLUT1 translocation to the plasma membrane (B and C), which were not affected by insulin treatment. Both GLUT3 and GLUT8 were not altered by experimental interventions (B). All experiments were performed in triplicates from at least four animals. Values are expressed as mean ± standard error of the mean. Journal of Vascular Surgery 2014 59, 483-491DOI: (10.1016/j.jvs.2013.03.023) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 4 Cerebral Akt and endothelial nitric oxide synthase (eNOS) phosphorylation. Cerebral tissues from ischemia-reperfusion (I/R) side (Ipsilateral) and the non-I/R counterpart (Contralateral) were harvested from controls (CONT), diabetic (DM), and insulin-treated diabetic (DMI) rats for protein analyses. The reduced cerebral eNOS (A and B) and Akt (C and D) phosphorylation in diabetes with or without focal cerebral ischemia-reperfusion (FC I/R) injury was corrected by insulin treatment. All experiments were performed in triplicates from at least four animals. Values are expressed as mean ± standard error of the mean. p-eNOS, Phosphorylated eNOS; iNOS, inducible NOS; nNOS, neuronal NOS. Journal of Vascular Surgery 2014 59, 483-491DOI: (10.1016/j.jvs.2013.03.023) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 5 Effect of endothelial nitric oxide synthase (eNOS) inhibition on infarct size and neurologic function. A, Shows representative coronal sections, and (B) shows quantitative infarct volume. The neurologic functions were assessed by Bederson score (C) and grip test (D) in insulin-treated diabetic rats (DMI, n = 10) and DMI cotreated with N-iminoethyl-L-ornithine (L-NIO) (DMI + L-NIO, n = 5) 24 hours prior to focal cerebral ischemia-reperfusion (FC I/R) injury. Insulin-mediated prevention of FC I/R-induced brain infarction and neurologic deficits in diabetes was antagonized by administration of the eNOS inhibitor L-NIO. Values are expressed as mean ± standard error of the mean. Journal of Vascular Surgery 2014 59, 483-491DOI: (10.1016/j.jvs.2013.03.023) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 6 Effect of N-iminoethyl-L-ornithine (L-NIO) on body weight and plasma levels of insulin and glucose. The plasma levels of glucose (A) and insulin (B) and the body weight (C) in diabetic rats were not affected by administration of the endothelial nitric oxide synthase (eNOS) inhibitor L-NIO. Values are expressed as mean ± standard error of the mean. DMI, Insulin-treated diabetic rats (n = 10); DMI + L-NIO, DMI cotreated with L-NIO 24 hours prior to focal cerebral ischemia-reperfusion (FC I/R) injury (n = 5). Journal of Vascular Surgery 2014 59, 483-491DOI: (10.1016/j.jvs.2013.03.023) Copyright © 2014 Society for Vascular Surgery Terms and Conditions