title: Tonic inhibition by G protein-coupled receptor kinase 2 of Akt/endothelial nitric-oxide synthase signaling in human vascular endothelial cells under conditions of hyperglycemia with high insulin levels.
abstract: G protein-coupled receptor kinase 2 (GRK2) participates together with β-arrestins in the regulation of G protein-coupled receptor signaling, but emerging evidence suggests that GRK2 can interact with a growing number of proteins involved in signaling mediated by other membrane receptor families under various pathologic conditions. We tested the hypothesis that GRK2 may be an important contributor to vascular endothelial dysfunction in diabetes. Human umbilical venous endothelial cells (HUVECs) were exposed to high glucose and high insulin (HG/HI) to mimic insulin-resistant diabetic conditions. GRK2 expression and membrane translocation were up-regulated under HG/HI conditions. HG/HI did not modify activation of Akt or endothelial nitric-oxide synthase (eNOS), but GRK2 inhibitor or small interfering RNA (siRNA) resulted in an increase in Akt and eNOS activation in HUVECs exposed to HG/HI. Extracellular signal-regulated kinase 1/2 (ERK1/2) activation was increased after exposure to HG/HI, which was prevented by GRK2 inhibitor or siRNA. ERK1/2-mediated GRK2 phosphorylation at Ser-670 confirmed that ERK1/2 participated in a negative feedback regulatory loop. In human embryonic kidney 293T cells that overexpressed GRK2, Akt activity was unchanged, whereas ERK1/2 activity was raised. The effect of GRK inhibitor treatment on Akt/eNOS signaling was associated with membrane translocation of β-arrestin 2. The experiments with β-arrestin 2 siRNA showed that β-arrestin 2 may act as a positive modulator of Akt/eNOS signaling. Our studies reveal that GRK2, which is up-regulated by HG/HI, leads to a tonic inhibition of the insulin Akt/eNOS pathway in endothelial cells. We provide new insights into the pathogenesis of diabetes-associated vascular endothelial dysfunction.
authors.affiliation: Departments of Molecular and Medical Pharmacology (K.T., K.S., W.O., T.I., Y.H.) and Diagnostic Pathology (J.I.), Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
authors.forename: Kumiko
authors.initials: K
authors.lastname: Taguchi
authors.forename: Kimimasa
authors.initials: K
authors.lastname: Sakata
authors.forename: Wakana
authors.initials: W
authors.lastname: Ohashi
authors.forename: Takahiro
authors.initials: T
authors.lastname: Imaizumi
authors.forename: Joji
authors.initials: J
authors.lastname: Imura
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Hattori
mesh_terms: D019390:Arrestins / Q000378:metabolism; D003920:Diabetes Mellitus / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007333:Insulin Resistance; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D009581:Nitrofurans / Q000494:pharmacology; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D014753:Vinyl Compounds / Q000494:pharmacology; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605254:ARRB2 protein, human; D019390:Arrestins; D007328:Insulin; D009581:Nitrofurans; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D014753:Vinyl Compounds; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; C000589464:methyl 5-(2-(5-nitro-2-furyl)vinyl)-2-furoate; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt; D054769:G-Protein-Coupled Receptor Kinase 2; D005947:Glucose