Inactivation of MAPK in epididymal fat and amelioration of triglyceride secretion by injection of GRK2 siRNA in ob/ob mice.

Abnormal G protein-coupled receptor kinase 2 (GRK2) accumulation has a crucial role in the development of insulin resistance and diabetes. Although GRK2 siRNA transfection in the liver improves insulin resistance-related vascular complications, the effects of GRK2 siRNA in lipid metabolism and obesity remain unknown. To investigate how GRK2 siRNA affects obesity, ob/ob mice were transfected with GRK2 siRNA, mainly in the liver, by using a hydrodynamic-based procedure. Epididymal fat, glucose, triglyceride, non-esterified fatty acid (NEFA), and alanine transaminase activity were higher in the control siRNA-transfected ob/ob mice than in the control siRNA-transfected Lean mice, but these parameters were reduced by GRK2 siRNA transfection into the ob/ob mice. GRK2 expression in epididymal fat was not altered among the 3 groups, although hepatic GRK2 expression was higher in the control siRNA-transfected ob/ob mice than in the control siRNA-transfected Lean mice. Additionally, we found that Akt interacted with GRK2 in the liver. Furthermore, phosphorylation levels of ERK1/2 and JNK were higher in the epididymal fats from the control siRNA-transfected ob/ob mice than in those from the control siRNA-transfected Lean mice, but they were lowered by transfection with GRK2 siRNA. The study results showed that GRK2 siRNA improved blood triglyceride levels and abnormal or excessive activity of mitogen-activated protein kinases in epididymal fat. This effect may be promoted by inhibition of the NEFA production pathway in the liver. Therefore, the interaction of organs (hepatic GRK2-epididymal fat) may help improve insulin resistance and diabetes-associated pathophysiology.

Suppression of GRK2 expression reduces endothelial dysfunction by restoring glucose homeostasis.

Despite the associations between diabetic complications and vascular endothelial dysfunction, a direct therapeutic method targeting endothelial dysfunction remains poorly characterized. We have previously shown that chemical inhibition of G-protein-coupled receptor kinase 2 (GRK2) slightly enhances insulin sensitivity and reduces endothelial dysfunction in type 2 diabetic mice. In this study, we identified GRK2 as a novel therapeutic target of diabetic endothelial dysfunction and investigated the effect on diabetic endothelial dysfunction through the systemic administration of GRK2 siRNA using a hydrodynamic-based procedure, resulting in suppression of increased GRK2 protein levels in the liver. Suppressed GRK2 levels in the liver markedly improved glucose homeostasis, as well as improved the impaired endothelial Akt/eNOS-dependent signal activation (insulin-stimulated phosphorylation of Akt and eNOS) and vascular responses (clonidine-induced and insulin-induced endothelial-dependent relaxation response and phenylephrine-induced contractile response) in type 2 diabetic aortas. Interestingly, insulin-stimulated Akt/eNOS signaling was increased only by normalizing the glucose concentration in human umbilical vein endothelial cells (HUVECs) with GRK2 overexpression, suggesting of an important role of hepatic GRK2. Our results clarified the relationship among hepatic GRK2, glucose homeostasis, and vascular endothelial function. Liver-targeting GRK2 siRNA delivery represents a novel therapeutic tool to restore glucose homeostasis and reduce endothelial dysfunction.

Glucose and angiotensin II-derived endothelial extracellular vesicles regulate endothelial dysfunction via ERK1/2 activation.

In various diseases, including diabetes, extracellular vesicles (EVs) have been detected in circulation and tissues. EVs are small membrane vesicles released from various cell types under varying conditions. Recently, endothelial cell-derived EVs (EEVs) were identified as a marker of endothelial dysfunction in diabetes, but the ensuing mechanisms remain poorly understood. In this study, we dissected the ensuing pathways with respect to nitric oxide (NO) production under the condition of type 2 diabetes. Human umbilical vein endothelial cells (HUVECs) were stimulated with glucose alone and with glucose in combination with angiotensin II (Ang II) for 48 h. In supernatants from glucose + Ang II-stimulated HUVECs, release of EEVs was assessed using Western blotting with an anti-CD144 antibody. EEV release was significantly increased after stimulation of HUVECs, and high glucose + Ang II-derived EEVs impaired ACh-induced vascular relaxation responses and NO production in mice aortic rings. Furthermore, high glucose + Ang II-derived EEVs induced ERK1/2 signalling and decreased endothelial NO synthase (eNOS) protein expression in mice aortas. Furthermore, in the presence of the MEK/ERK1/2 inhibitor PD98059, high glucose plus Ang II treatment stimulated EEVs in HUVECs and those EEVs prevented the impairments of ACh-induced relaxation and NO production in mice aortas. These data strongly indicate that high glucose and Ang II directly affect endothelial cells and the production of EEVs; the resultant EEVs aggravate endothelial dysfunction by regulating eNOS protein levels and ERK1/2 signalling in mice aortas.

Dietary polyphenol morin rescues endothelial dysfunction in a diabetic mouse model by activating the Akt/eNOS pathway.

SCOPE: Endothelial dysfunction is a critical factor during the initiation of diabetic cardiovascular complications. Polyphenols may represent beneficial dietary components eliciting cardiovascular protection. Although we previously reported that the polyphenol morin (MO) ameliorated diabetes-induced endothelial dysfunction, the underlying mechanism remains unclear. Here, we investigated protective effects and mechanisms of MO in streptozotocin STZ induced diabetic aorta endothelial dysfunction. METHODS AND RESULTS: Diabetes was induced by tail vein injection of STZ (200 mg/kg). At 12 wk after injection, the thoracic aorta was isolated and endothelial function was assessed by acetylcholine (ACh) induced, endothelial-dependent vasorelaxation in aortas. Nitric oxide (NO) levels and endothelial NO synthase (eNOS), phosphorylated-eNOS (p-eNOS), Akt, and phosphorylated-Akt (p-Akt) levels were also evaluated in aortas. Diabetic aortas showed attenuated endothelial function, which was improved by MO treatment. MO treatment alone increased NO levels and endothelial-dependent relaxation responses via Akt signaling, although ACh did not activate this pathway. Moreover, MO upregulated p-Akt (at Ser473 and Thr308) and p-eNOS (at Ser1177) expression in diabetic aortas, but ACh stimulation had no effect on p-Akt and p-eNOS levels. CONCLUSION: These results indicate a novel role for MO in protection against endothelial dysfunction in diabetes. The protective effects of MO are dependent on Akt-dependent activation of eNOS signaling.

Resveratrol Ameliorates Clonidine-Induced Endothelium-Dependent Relaxation Involving Akt and Endothelial Nitric Oxide Synthase Regulation in Type 2 Diabetic Mice.

Diabetic vascular complication is one of the manifestations of endothelial dysfunction. Resveratrol (RV) is considered to be beneficial in protecting endothelial function. However, the exact protective effect and mechanisms involved have not been fully clarified. In this study, we investigated the relationship between Akt/endothelial nitric oxide synthase (eNOS) activation and RV in diabetes-induced endothelial dysfunction. Aortas were dissected and placed in organ chambers, and nitric oxide (NO) production in response to acetylcholine (ACh) and RV was measured. ACh-induced endothelium-dependent relaxation was markedly increased in controls by RV pretreatment. Furthermore, RV caused NO-dependent relaxation via the Akt signaling pathway, which was weaker in the aortas of diabetic mice than age-matched controls. To further examine the underlying mechanisms, we measured the phosphorylation of Akt and eNOS by Western blotting. RV caused the phosphorylation of Akt and eNOS in aortas, which was decreased in diabetic mice. However, RV augmented the impaired clonidine-induced relaxation in diabetic mice. Interestingly, the phosphorylation of Akt and eNOS was increased under stimulation with RV and clonidine only in diabetic mice. Thus, either RV or clonidine causes Akt-dependent NO-mediated relaxation, which is weaker in diabetic mice than controls. However, additional exposure to RV and clonidine has an augmenting effect on the Akt/eNOS signaling pathway under diabetic conditions. RV-induced Akt/eNOS activity may be a common link involved in the clonidine-induced Akt/eNOS activity, so RV and clonidine may have a synergistic effect.

Effect of short-term polyphenol treatment on endothelial dysfunction and thromboxane A2 levels in streptozotocin-induced diabetic mice.

Diabetes is characterized by the development of endothelial dysfunction, which affects both nitric oxide (NO)-mediated relaxation and endothelium-derived contracting factors, associated with vascular oxidative stress. There is a growing body of evidence suggesting that polyphenols have several beneficial effects, such as antioxidant and anti-inflammatory activities. This study investigated whether short-term treatment with polyphenols (chlorogenic acid (CA), morin (MO), resveratrol (RV)) can improve endothelial dysfunction related to diabetes. Aorta reactivity was determined in organ chambers, and we measured NO production and thromboxane B2 (TXB2; a metabolite of TXA2) from aortas in response to acetylcholine (ACh). Streptozotocin (STZ)-induced diabetic mice (16 weeks) were injected with solvent (ethanol, 10% v/v; intraperitoneally (i.p.)), CA (0.03 mmol/kg/d), MO (0.03 mmol/kg/d), and RV (0.03 mmol/kg/d) for 5 d. The ACh-induced endothelium-dependent relaxation was markedly reduced in rings of STZ-induced diabetic mice compared to controls. The treatment with polyphenols (significantly: MO, tendency: CA and RV) for only 5 d improved the NO components of relaxation, but did not normalize ACh-stimulated NO production. However, polyphenol treatment suppressed the ACh-stimulated level of TXB2 in aortas from STZ-induced diabetic mice. Thus, treatment with polyphenols caused basal NO production and a prompt improvement of the endothelial function in diabetic mice, and this may involve the normalization of TXA2 levels, not NO production, under ACh stimulation.