Hyperthermia-induced Hsp90·eNOS Preserves Mitochondrial Respiration in Hyperglycemic Endothelial Cells by Down-regulating Glut-1 and Up-regulating G6PD Activity

Abstract

Uncoupling of NO production from NADPH oxidation by endothelial nitric-oxide synthase (eNOS) is enhanced in hyperglycemic endothelium, potentially due to dissociation of heat shock proteins 90 (Hsp90), and cellular glucose homeostasis is enhanced by a ROS-induced positive feed back mechanism. In this study we investigated how such an uncoupling impacts oxygen metabolism and how the oxidative phosphorylation can be preserved by heat shock (42°C for 2 h, hyperthermia) in bovine aortic endothelial cells. Normal and heat-shocked bovine aortic endothelial cells were exposed to normoglycemia (NG, 5.0 mM) or hyperglycemia (30 mM). With hyperglycemia treatment, O 2 consumption rate was reduced (from V O 2 max ‫؍‬ 7.51 ؎ 0.54 to 2.35 ؎ 0.27 mm Hg/min/10 6 cells), whereas in heat-shocked cells, O 2 consumption rate remained unaltered (8.19 ؎ 1.01 mm Hg/min/10 ؋ 10 6 cells). Heat shock was found to enhance Hsp90/endothelial NOS interactions and produce higher NO. Moreover, ROS generation in the hyperglycemic condition was also reduced in heat-shocked cells. Interestingly, glucose uptake was reduced in heat-shocked cells as a result of decrease in Glut-1 protein level. Glucose phosphate dehydrogenase activity that gives rise to NADPH generation was increased by hyperthermia, and mitochondrial oxidative metabolism was preserved. In conclusion, the present study provides a novel mechanism wherein the reduced oxidative stress in heat-shocked hyperglycemic cells down-regulates Glut-1 and glucose uptake, and fine-tuning of this pathway may be a potential approach to use for therapeutic benefit of diabetes mellitus. Heat shock proteins (Hsps)2 play critical roles in endothelial function in the hyperglycemic state, although their exact function is not clearly established. Along with hyperglycemia, the production of superoxide, insulin resistance, and a decline in vascular bioavailability of nitric oxide (NO) contribute to extreme rates of morbidity and mortality (1-4). Various factors regulate endothelial nitric-oxide synthase (eNOS) signaling in endothelial cells by modulating its phosphorylation/de-phosphorylation dynamics and the coupling/uncoupling of its redox reactions. Protein kinase B (Akt) phosphorylates eNOS on serine 1177 and increases the production of NO (5), and it is found to be very critical in overall glucose metabolism and cell survival (6). Hsp90 binding to eNOS was found to be a prerequisite for successive Akt-mediated stimulation of eNOS, and indeed, Hsp90 can be considered as a scaffold between eNOS and Akt (5,7,8). Vascular endothelial growth factor enhances the recruitment of Hsp90 and facilitates Akt-dependent phosphorylation of eNOS, which results in increased NO production. NO generation is reduced under diabetic conditions due to NOS uncoupling (i.e. incomplete redox transformation of NADPH, arginine, and O 2 to NO, citrulline, and H 2 O, leading instead to the generation of reactive oxygen species) resulting in the generation of more superoxide, and Hsp levels, especially Hsp90, are reduced in hypergly...

Repetitive progressive thermal preconditioning hinders thrombosis by reinforcing phosphatidylinositol 3-kinase/Akt-dependent heat-shock protein/endothelial nitric oxide synthase signaling

“…37 Heat stimulation of endothelial cells at 42°C can enhance Hsp90/eNOS interactions, produce higher NO, and depress oxidative stress. 38 Our data in TP or 3-PTP displays a similar Hsp70/eNOS expression, vascular protection against FeCl 3 injury, oxidative stress, CX3CL1, ICAM-1 and t-PA and PAI-1 activity. We suggest that all these implications could evidence that PTP, like TP via action of Hsp/eNOS, affects oxidative stress, inflammation, and adhesion molecule expression.…”

Involvement of insulin-induced reversible chromatin remodeling in altering the expression of oxidative stress-responsive genes under hyperglycemia in 3T3-L1 preadipocytes

“…This places epigenetic mechanisms and heat shock elements to use for therapeutic benefit against diabetes and associated disorders. Consistent with this a recent report has also shown the role of heat shock elements in reducing oxidative stress associated with diabetes mellitus in bovine aortic endothelial cells (BAECs) (Presley et al, 2010). However, much more is still to be learned about the role of these genome-wide epigenetic alterations and chromatin remodeling events in the pathogenesis of hyperglycemia/hyperinsulinemia associated metabolic disorders.…”

Changes in glucose transporter expression and nitric oxide production are associated with liver injury in diabetes

“…On the other hand, increase of GLUT1 protein expression could be regulated on translation level by increasing translation of pre‐existing mRNAs. It is supposed that up‐regulation of GLUT1 by ROS happens on the post‐transcriptional level . GLUT1 expression is regulated in part via mTOR pathway; mTOR protein exerts its regulatory functions mainly by affecting translation factors .…”

“…It is supposed that up-regulation of GLUT1 by ROS happens on the post-transcriptional level. 39 GLUT1 expression is regulated in part via mTOR pathway; 40 mTOR protein exerts its regulatory functions mainly by affecting translation factors. 41 Similarly, a considerable increase of GLUT1 mRNA expression without increase of protein GLUT1 expression in the liver of rats fed with the high-fat diet could be one more manifestation of mTOR-dependent regulation.…”