Unacylated Ghrelin Promotes Skeletal Muscle Regeneration Following Hindlimb Ischemia via SOD‐2–Mediated miR‐221/222 Expression

Abstract

BackgroundSurgical treatment of peripheral artery disease, even if successful, does not prevent reoccurrence. Under these conditions, increased oxidative stress is a crucial determinant of tissue damage. Given its reported antioxidant effects, we investigated the potential of unacylated‐ghrelin (UnAG) to reduce ischemia‐induced tissue damage in a mouse model of peripheral artery disease.Methods and ResultsWe show that UnAG but not acylated ghrelin (AG) induces skeletal muscle regeneration in response to ischemia via canonical p38/mitogen‐actived protein kinase signaling UnAG protected against reactive oxygen species–induced cell injuries by inducing the expression of superoxide dismutase‐2 (SOD‐2) in satellite cells. This led to a reduced number of infiltrating CD68+ cells and was followed by induction of the myogenic process and a reduction in functional impairment. Moreover, we found that miR‐221/222, previously linked to muscle regeneration processes, was up‐regulated and negatively correlated with p57Kip2 expression in UnAG‐treated mice. UnAG, unlike AG, promoted cell‐cycle entry in satellite cells of mice lacking the genes for ghrelin and its receptor (GHSR1a). UnAG‐induced p38/mitogen‐actived protein kinase phosphorylation, leading to activation of the myogenic process, was prevented in SOD‐2–depleted SCs. By siRNA technology, we also demonstrated that SOD‐2 is the antioxidant enzyme involved in the control of miR‐221/222–driven posttranscriptional p57Kip2 regulation. Loss‐of‐function experiments targeting miR‐221/222 and local pre–miR‐221/222 injection in vivo confirmed a role for miR‐221/222 in driving skeletal muscle regeneration after ischemia.ConclusionsThese results indicate that UnAG‐induced skeletal muscle regeneration after ischemia depends on SOD‐2–induced miR‐221/222 expression and highlight its clinical potential for the treatment of reactive oxygen species–mediated skeletal muscle damage.

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.…”

“…In the current studies with prolonged hormone incubation, highest AG doses selectively induced a moderate increase of GSK-3b S9 phosphorylation but failed to reduce ROS generation and to enhance downstream insulin signaling. Also consistent with these findings, AG is a weaker autophagy inducer than UnAG and fails to stimulate both mitophagy (35) and ischemia-induced skeletal muscle regeneration (15). On the basis of the available knowledge, differential muscle effects of ghrelin forms may depend on still uninvestigated acylation-selective and time-dependent AG activities.…”

“…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.…”

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

“…We have previously shown that the more abundant circulating form of ghrelin, unacylated grelin (UnAG), exerts antioxidant effects on endothelial progenitor cells (EPCs) (17,18). A proof of concept is provided by UnAG administration, which induces SOD-2 expression and improves mitochondrial dysfunction in muscles subjected to ischemia (19). UnAG is one of the ghrelin forms that is mainly produced in the stomach (20).…”

“…UnAG is one of the ghrelin forms that is mainly produced in the stomach (20). UnAG differs from acylated ghrelin (AG) in term of its biological activity (17)(18)(19)21). Moreover, while both AG and UnAG are released into circulation (22), their ratio (AG/UnAG) varies and ranges from 1:2 to 1:9 (23), while a relative excess of AG has interestingly been found in individuals suffering from insulin resistanceconnoted metabolic disorders (24).…”