Unacylated Ghrelin Induces Oxidative Stress Resistance in a Glucose Intolerance and Peripheral Artery Disease Mouse Model by Restoring Endothelial Cell miR-126 Expression

Abstract

Reactive oxygen species (ROS) are crucial in longterm diabetes complications, including peripheral artery disease (PAD). In this study, we have investigated the potential clinical impact of unacylated ghrelin (UnAG) in a glucose intolerance and PAD mouse model. We demonstrate that UnAG is able to protect skeletal muscle and endothelial cells (ECs) from ROS imbalance in hind limb ischemiasubjected ob/ob mice. This effect translates into reductions in hind limb functional impairment. We show that UnAG rescues sirtuin 1 (SIRT1) activity and superoxide dismutase-2 (SOD-2) expression in ECs. This leads to SIRT1-mediated p53 and histone 3 lysate 56 deacetylation and results in reduced EC senescence in vivo. We demonstrate, using small interfering RNA technology, that SIRT1 is also crucial for SOD-2 expression. UnAG also renews micro-RNA (miR)-126 expression, resulting in the posttranscriptional regulation of vascular cell adhesion molecule 1 expression and a reduced number of infiltrating inflammatory cells in vivo. Loss-of-function experiments that target miR-126 demonstrate that miR-126 also controls SIRT1 and SOD-2 expression, thus confirming its role in driving UnAG-mediated EC protection against ROS imbalance. These results indicate that UnAG protects vessels from ROS imbalance in ob/ob mice by rescuing miR-126 expression, thus emphasizing its potential clinical impact in avoiding limb loss in PAD.

Unacylated Ghrelin Reduces Skeletal Muscle Reactive Oxygen Species Generation and Inflammation and Prevents High-Fat Diet–Induced Hyperglycemia and Whole-Body Insulin Resistance in Rodents

“…The results show that UnAG negatively regulates skeletal muscle ROS production and inflammation, and these effects are indirectly supported by previous in vitro observations in nonmuscle cells (14,16,19). In another study, UnAG reduced endothelial oxidative stress in models of peripheral artery disease by restoring SOD expression (15,16). Skeletal muscle SOD expression and antioxidant enzyme activities were, however, unchanged by UnAG in the current model, indicating lower mitochondrial ROS generation rather than enhanced antioxidant defenses as a key mediator of UnAG-induced muscle antioxidant activity.…”

“…Finally, UnAG effects in vitro are abolished by autophagy inhibition, thereby indicating mechanistic involvement of autophagy in UnAG activities. The results show that UnAG negatively regulates skeletal muscle ROS production and inflammation, and these effects are indirectly supported by previous in vitro observations in nonmuscle cells (14,16,19). In another study, UnAG reduced endothelial oxidative stress in models of peripheral artery disease by restoring SOD expression (15,16).…”

“…Although no specific UnAG receptor has yet been identified, UnAG counteracts glucogenic effects of AG as well as AG-induced hyperglycemia (10), and negative associations have been reported between circulating UnAG and markers of whole-body insulin resistance in humans (12,13). Of interest, emerging antioxidant effects have been reported for UnAG in various cell types (14)(15)(16)(17), and we have demonstrated that UnAG stimulates autophagy in rodent muscle, thereby also potentially lowering muscle oxidative stress through disposal of damaged mitochondria (18). No information is available, however, on 1) the impact of UnAG on skeletal muscle ROS generation, inflammation, and insulin action and 2) whether UnAG prevents altered oxidative stress, inflammation, and insulin action in obesity and diabetes.…”

Epigenetic Programming of Cardiovascular Disease by Perinatal Hypoxia and Fetal Growth Restriction

“…In ob/ob mice, antioxidant treatment induces a miR-126-dependent anti-inflammatory and antioxidant vascular response [98], an effect also observed in HUVEC [99]. Both miRNAs, miR-21 and miR-126, are upregulated by unidirectional shear stress, protecting EC from apoptosis and increasing the activation of eNOS [100].…”

Sirtuins, aging, and cardiovascular risks

“…The activity of SIRT1 has been shown to be rescued by several compounds, such as resveratrol (D'Onofrio et al 2015;Sanchez-Fidalgo 2012), unacylated ghrelin (D'Onofrio et al 2015;Togliatto et al 2014), and ginsenoside Rb1 (D'Onofrio et al 2015;Song et al 2014), a steroid glycoside found in ginseng and paeonol (D'Onofrio et al 2015;Jamal et al 2014). Resveratrol, a polyphenolic antioxidant found in red wine, activating SIRT1 (Villalba and Alcain 2012;Wallerath et al 2002) prevents oxidative stress and coronary VSMC proliferation inhibiting extracellular signal regulated kinase (ERK) activation (Chong et al 2012;El-Mowafy et al 2008).…”