G Protein–Coupled Receptor Kinase 2, With β-Arrestin 2, Impairs Insulin-Induced Akt/Endothelial Nitric Oxide Synthase Signaling in ob/ob Mouse Aorta

Abstract

In type 2 diabetes, impaired insulin-induced Akt/endothelial nitric oxide synthase (eNOS) signaling may decrease the vascular relaxation response. Previously, we reported that this response was negatively regulated by G protein–coupled receptor kinase 2 (GRK2). In this study, we investigated whether/how in aortas from ob/ob mice (a model of type 2 diabetes) GRK2 and β-arrestin 2 might regulate insulin-induced signaling. Endothelium-dependent relaxation was measured in aortic strips. GRK2, β-arrestin 2, and Akt/eNOS signaling pathway proteins and activities were mainly assayed by Western blotting. In ob/ob (vs. control [Lean]) aortas: 1) insulin-induced relaxation was reduced, and this deficit was prevented by GRK2 inhibitor, anti-GRK2 antibody, and an siRNA specifically targeting GRK2. The Lean aorta relaxation response was reduced to the ob/ob level by pretreatment with an siRNA targeting β-arrestin 2. 2) Insulin-stimulated Akt and eNOS phosphorylations were decreased. 3) GRK2 expression in membranes was elevated, and, upon insulin stimulation, this expression was further increased, but β-arrestin 2 was decreased. In ob/ob aortic membranes under insulin stimulation, the phosphorylations of Akt and eNOS were augmented by GRK2 inhibitor. In mouse aorta, GRK2 may be, upon translocation, a key negative regulator of insulin responsiveness and an important regulator of the β-arrestin 2/Akt/eNOS signaling, which is implicated in diabetic endothelial dysfunction.

Improvement of vascular insulin sensitivity by downregulation of GRK2 mediates exercise-induced alleviation of hypertension in spontaneously hypertensive rats

“…For gene silencing assay, small interfering RNA (siRNA) specifically targeting GRK2 mRNA was purchased from Santa Cruz Biotechnology. Arteriolar tissues were transfected with siRNA specific to GRK2 or scrambled control by Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer's instructions (29,33). At 48 h after transfection, arteriolar segments were treated with 10 Ϫ7 M insulin for 30 min.…”

Effect of Short-term Polyphenol Treatment on Endothelial Dysfunction and Thromboxane A₂ Levels in Streptozotocin-Induced Diabetic Mice

“…15,16,27) Briefly, the thoracic aortas were carefully isolated from mice, dissected from surrounding fat and connective tissue, cut into circular segments (2 mm long), and immediately placed in Krebs-Henseleit Solution (KHS), concentration in mmol/L: NaCl, 118.0; KCl, 4.7; NaHCO 3 , 25.0; CaCl 2 , 1.8; NaH 2 PO 4 , 1.2; MgSO 4 , 1.2; glucose, 11.0. Aortic rings were suspended in 10 mL jacketed organ baths filled with 10 mL of KHS continuously aerated with a mixture of 95% O 2 and 5% CO 2 at 37°C.…”

“…[8][9][10][11][12][13][14][15][16]27,28) Plasma samples were stored at −20°C until analysis. Briefly, plasma glucose, cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, and serum non-esterified fatty acid (NEFA) levels were each determined with a commercially available enzyme kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan).…”

“…The endothelium plays a central role in physiological maintenance of the vascular function by regulating vascular tone, leukocyte adhesion, and platelet activation, as a result of the release of vasoactive substances such as nitric oxide (NO), prostacyclin, and thromboxane A 2 (TXA 2 ). 1,[6][7][8][9][10][11][12][13][14][15][16] Several natural polyphenols have been evaluated for their ability to protect against endothelial dysfunction and, thus, for their effectiveness in preventing cardiovascular disease. 17) Indeed, we have also shown that long-term chlorogenic acid (CA) treatment (2 months) can normalize the impaired endothelium-dependent relaxation seen in diabetic rats.…”

Vascular Endothelial σ₁-Receptor Stimulation With SA4503 Rescues Aortic Relaxation via Akt/eNOS Signaling in Ovariectomized Rats With Aortic Banding

“…23 Translocation of G protein-coupled receptor kinase 2 to the plasma membrane interferes with β-arrestin 2-mediated activation of the Akt/eNOS pathway in the ob/ob mouse aorta. 36 Moreover, Fernández-Hernando et al reported that genetic ablation of Akt1 on an apolipoprotein E knockout background (ApoE -/-Akt1 -/-) increases aortic lesion expansion and promotes coronary atherosclerosis relative to ApoE -/-single nulls. 37 Akt1 loss increases the levels of inflammatory mediators and reduces eNOS phosphorylation.…”