Members of other units:
Dott.ssa Elisa ARNOLETTI
Dott.ssa Alessandra BARAGLI
Prof. Roberto MAGGIO
Dott. Mario ROSSI
University of L’Aquila
Obestatin (Ob) is a peptide coded by the ghrelin gene, identified in 2005. It displays cyto-trophic, cyto-protective and regenerative effects in numerous cell types and metabolic tissues such as pancreas, adipose tissue, cardiac and skeletal muscles, whose structural and functional damage is associated with metabolic syndrome and degenerative diseases. Some evidence indicates that the activation of the orphan receptor GPR39 is responsible for these positive activities, while others point toward the glucagon-like peptide 1 receptor (GLP1-R), such that there is no clear and univocal opinion on this merit. Indeed, while in pre-adipocytes and pancreatic β-cells Ob could exert its biological effects by activating GLP1-R, at the muscle level GPR39 seems involved. For this purpose, we will use skeletal muscle satellite cells (SC), myogenic precursors quiescent in the healthy adult muscle, but activated to proliferate and differentiate into myotubes subsequent to an injury. These cells express Ob, GLP1-R and GPR39 and may play a key role in the cell therapy against metabolic-based or not metabolic-based skeletal muscle degenerative diseases. Among these, we will consider muscle dystrophies, characterized by progressive muscle weakness and subsequent tissue degeneration caused by genetic mutations, and drug-induced myopathies. Interestingly, it has been reported that Ob, GPR39 and GLP1-R expression is markedly increased during muscle regeneration, suggesting the presence of an active autocrine/paracrine loop in this event. Nevertheless, a specific and receptor-mediated activity of Ob on SC has not been yet investigated. Thus, the first step of the project will be to verify if Ob exerts a direct biological activity on primary SC from healthy mice skeletal muscles. The effect of Ob treatment will be evaluated by measuring its ability to modulate cell proliferation and differentiation into myotubes. If a direct, specific and positive effect of Ob in SC biology will be revealed, we will determine Ob receptors molecular identity and the signaling pathways involved in its action. Moreover, we will monitor Ob effects on the regenerative process of drug-injured muscles (we will use myolesive drugs such as statins, and the myotoxin cardiotoxin) or caused by genetic defects (SC isolated from a murine model of Duchenne Muscolar Distrophy, SC-mdx), a subject not yet investigated. In this settings we will study the potential cyto-protective, anti-apoptotic and regenerative properties of Ob. Particularly, in SC-mdx we will look for the modulation of myostatin expression, whose lack improves muscle regeneration in the mdx model, and of utrophin expression, a dystrophin-homolog, which may functionally replace it in dystrophic conditions. As regards to Ob-receptors characterization, we will use cross-linking procedures to purify and enrich the Ob-receptor complex. Biochemical techniques will be used to assess the presence of the GLP1-R/GPR39 heterodimer. In the case of a negative result, we plan to analyze the protein complex through MALDI-TOF in order to identify molecules with high-homology sequences. To validate the obtained results, the putative receptor cDNAs will be over-expressed in cell lines and their pharmacological, functional and molecular profile assessed. The involvement of these receptors in mediating Ob effects will be confirmed by mean of their silencing and by monitoring their internalization dynamics. In addition, we will study and compare signalosome formation following receptor activation in SC deriving from healthy, statin-treated and mdx mice. Once these studies will be completed, our attention will be focused on in vivo studies to evaluate the local Ob-induced functional muscle recovery in drug-treated and mdx mice, and on screening for compounds pharmacologically active on the identified receptors.
The main objectives of our study are:
1) To demonstrate a direct biological activity of obestatin (Ob) on skeletal muscle primary satellite cells (SC). Given Ob cyto-trophic and pro-differentiative properties shown in studies on pancreatic progenitors, β-cells, pre-adipocites and cardiomyoblasts/cytes, we expect that in vitro Ob treatment will initially burst the proliferation rate, then increase SC differentiation potential.
2) To verify Ob anti-apoptotic, cyto-throphic and pro-differentiative properties on SC treated with miolesive and miotoxic stimuli, or isolated from mdx mice (SC-mdx). Given Ob regenerative properties highlighted in a recent study on the gastrocnemious muscle (Gurriarán-Rodríguez U., JBC, 2012), we expect Ob to have beneficial effects stimulating injured SC survival and recovery of their function.
3) To assess the molecular identity of receptors mediating Ob effects on SC, clarifying their pharmacological properties, their signaling and internalization dynamic. Based on pre-existing evidence and our preliminary results we expect that they correspond to the GLP1-R/GPR39 hetero-dimer.
4) To screen for compounds pharmacologically active on the receptors and selective for them.
5) To evaluate in vivo the improvement of the regeneration process and recovery of muscle function promoted by Ob administration to mice treated with miolesive and miotoxic stimuli and in mdx mice.
Muscle dystrophies are among the most disabling skeletal muscle genetic diseases, being characterized by myofibers degeneration leading to progressive paralysis up to death for respiratory complications. This situation causes a marked reduction in the quality of life of affected people and discomfort in their families. Clinical signs become manifest during childhood, upon depletion of skeletal muscle satellite cells (SC) regenerative potential and number, such that the replacement of injured myofibers is not anylonger guaranteed. To date the available interventions are not curative, though they permit an improvement of life expectancy. Patients and their families, therefore, need stable assistance. The Duchenne muscular dystrophy, the most common dystrophy, accounts for 19-29 clinical cases for 100,000 newborns, with considerable costs for the national health service. At present, scientific research in this field has already highlighted the importance of cell therapy and SC represent promising candidates. Our project is intended to clarify the direct action of the ghrelin gene product obestatin (Ob) on SC, in physiological and pathological conditions, since it has been already reported that Ob shows cyto-protective and mytogenic effects on pancreatic β-cells in diabetic settings and in ischemia/reperfusion injured cardiomyocytes. The pharmacological identification and characterization of Ob receptors on SC may have important implications in the control of muscle regeneration, as well as may allow the identification of analogues with agonist, antagonist or allosteric properties, useful to promote this process.
In the best situation, if the GLP1-R/GPR39 heterodimer were the Ob receptor in SC, we might find specific molecules acting selectively on it, bringing a new therapeutic approach in this area.
Our studies could thus favor the development of pharmacological therapies in order to improve skeletal muscle regeneration in various neuro-myo-degenerative diseases, of genetic or toxic origin, with a remarkable impact on their management and progression.
G-protein coupled receptor pharmacology, Signal transduction, Skeletal muscle satellite cells, Regenerative medicine, Dystrophy