NRF2-Dependent Glutamate-L-Cysteine Ligase Catalytic Subunit Expression Mediates Insulin Protection Against Hyperglycemia-Induced Brain Endothelial Cell Apoptosis

Abstract

Increased oxidative stress and susceptibility of brain endothelium are contributing factors in the development of central nervous system complications in neuro-degenerative disorders in diabetes, Alzheimer's and Parkinson's disease. The molecular mechanisms underpinning the vulnerability of brain endothelial cells to chronic oxidative challenge have not been elucidated. Here, we investigated the oxidative susceptibility of human brain endothelial cells (IHEC) to chronic hyperglycemic stress and insulin signaling and cytoprotection. Chronic hyperglycemia exacerbated IHEC apoptosis in accordance with exaggerated cytosolic and mitochondrial glutathione and protein-thiol redox imbalance, and actin/Keap-1 S-glutathionylation. Insulin attenuated hyperglycemia-induced apoptosis via restored cytosolic and mitochondrial redox. Insulin stimulated glutamate-L-cysteine ligase (GCL) activity by activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling, increased serine phosphorylation and nuclear translocation of nuclear NF-E2-related factor 2 (Nrf2), and upregulation of Nrf2-dependent GCL-catalytic (GCLc) subunit expression. Expression of the GCL-modulatory subunit (GCLm) was unchanged. Inhibitors of insulin receptor tyrosine kinase, PI3K, Akt and mTOR abrogated insulin-induced Nrf2-mediated GCLc expression, redox balance, and IHEC survival. Collectively, these results demonstrate that human brain endothelial cells exhibit vulnerability to hyperglycemic stress which is associated with marked cytosolic and mitochondrial redox shifts. Activation of insulin signaling through PI3K/Akt/mTOR/Nrf2/ GCLc pathway affords significant cell protection by maintaining cellular redox balance.

Dietary leucine improves flesh quality and alters mRNA expressions of Nrf2-mediated antioxidant enzymes in the muscle of grass carp ( Ctenopharyngodon idella )

“…This author found that TOR was involved in the synthesis of Nrf2 protein in HepG2 cells. An in vitro study in human brain endothelial cells showed that elevated mTOR and S6K1 expression could up-regulate Nrf2 expression (Okouchi et al, 2006). The result of present study showed that 13.3 g kg −1 leucine supplementation significantly up-regulated TOR and S6K1 gene expression in the muscle.…”

S-glutathionylation: relevance in diabetes and potential role as a biomarker

“…Nrf1, Nrf2, AP-1, NFκB and c-Myc seem to be involved in the regulation of the promoter activity of the GCLm gene (Wild et al, 1999;Yang et al, 2005;Benassi et al, 2006). Interestingly, it has been shown that insulin was unable to change the levels of GCLm mRNA (Okouchi et al, 2006), while GCLc levels were affected by this stimulus in the same report.…”

Hormonal Signaling Systems of the Brain in Diabetes Mellitus

“…Hence, being the most insulin-responsive brain area, the hypothalamus contributes to whole-body glucose homeostasis via IRS-PI 3-kinase signaling. The prime function of the other mechanism of neuroprotective action of insulin and IGF-1 realized via PI 3-kinase is to control the oxidative stress and susceptibility of the brain endothelium, the important contributing factors in the development of CNS disorders in DM (Okouchi et al, 2006). It was found that chronic hyperglycemia exacerbated apoptosis of human brain endothelial cells in accordance with exaggerated cytosolic and mitochondrial glutathione and protein-thiol redox imbalance.…”

“…Insulin activates the PI 3-kinase/AKT kinase/mTOR kinase cascade, increases serine phosphorylation and nuclear translocation of nuclear NF-E2-related factor 2 (Nrf2), and enhances the expression of catalytic subunit of Nrf2-dependent glutamate-L-cysteine ligase, a heterodimeric enzyme participating in glutathione metabolism, and, hence, attenuates hyperglycemia-induced apoptosis via the restored cytosolic and mitochondrial redox balance. Inhibitors of IR tyrosine kinase, PI 3-kinase, AKT kinase and mTOR kinase abrogate insulin-induced Nrf2-mediated glutamate-L-cysteine ligase expression, redox balance, and the survival of human brain endothelial cells (Okouchi et al, 2006). Insulin-regulated PI 3-kinase-dependent pathways are involved in the prevention of endoplasmic reticulum stress that contributes to DM and neurodegenerative disorders (Hosoi et al, 2007).…”