Ghrelin is an acyl-peptide gastric hormone acting on the pituitary and hypothalamus to stimulate growth hormone (GH) release, adiposity, and appetite. Ghrelin endocrine activities are entirely dependent on its acylation and are mediated by GH secretagogue (GHS) receptor (GHSR)-1a, a G protein–coupled receptor mostly expressed in the pituitary and hypothalamus, previously identified as the receptor for a group of synthetic molecules featuring GH secretagogue (GHS) activity. Des-acyl ghrelin, which is far more abundant than ghrelin, does not bind GHSR-1a, is devoid of any endocrine activity, and its function is currently unknown. Ghrelin, which is expressed in heart, albeit at a much lower level than in the stomach, also exerts a cardio protective effect through an unknown mechanism, independent of GH release. Here we show that both ghrelin and des-acyl ghrelin inhibit apoptosis of primary adult and H9c2 cardiomyocytes and endothelial cells in vitro through activation of extracellular signal–regulated kinase-1/2 and Akt serine kinases. In addition, ghrelin and des-acyl ghrelin recognize common high affinity binding sites on H9c2 cardiomyocytes, which do not express GHSR-1a. Finally, both MK-0677 and hexarelin, a nonpeptidyl and a peptidyl synthetic GHS, respectively, recognize the common ghrelin and des-acyl ghrelin binding sites, inhibit cell death, and activate MAPK and Akt.These findings provide the first evidence that, independent of its acylation, ghrelin gene product may act as a survival factor directly on the cardiovascular system through binding to a novel, yet to be identified receptor, which is distinct from GHSR-1a.

Cachexia is a wasting syndrome associated with cancer, AIDS, multiple sclerosis, and several other disease states. It is characterized by weight loss, fatigue, loss of appetite, and skeletal muscle atrophy and is associated with poor patient prognosis, making it an important treatment target. Ghrelin is a peptide hormone that stimulates growth hormone (GH) release and positive energy balance through binding to the receptor GHSR-1a. Only acylated ghrelin (AG), but not the unacylated form (UnAG), can bind GHSR-1a; however, UnAG and AG share several GHSR-1a-independent biological activities. Here we investigated whether UnAG and AG could protect against skeletal muscle atrophy in a GHSR-1a-independent manner. We found that both AG and UnAG inhibited dexamethasone-induced skeletal muscle atrophy and atrogene expression through PI3Kβ-, mTORC2-, and p38-mediated pathways in myotubes. Upregulation of circulating UnAG in mice impaired skeletal muscle atrophy induced by either fasting or denervation without stimulating muscle hypertrophy and GHSR-1a-mediated activation of the GH/IGF-1 axis. In Ghsr-deficient mice, both AG and UnAG induced phosphorylation of Akt in skeletal muscle and impaired fasting-induced atrophy. These results demonstrate that AG and UnAG act on a common, unidentified receptor to block skeletal muscle atrophy in a GH-independent manner.

Ghrelin is an acylated peptidyl gastric hormone acting on the pituitary and hypothalamus to stimulate appetite, adiposity, and growth hormone release, through activation of growth hormone secretagogue receptor (GHSR)-1a receptor. Moreover, ghrelin features several activities such as inhibition of apoptosis, regulation of differentiation, and stimulation or inhibition of proliferation of several cell types. Ghrelin acylation is absolutely required for both GHSR-1a binding and its central endocrine activities. However, the unacylated ghrelin form, des-acyl ghrelin, which does not bind GHSR-1a and is devoid of any endocrine activity, is far more abundant than ghrelin in plasma, and it shares with ghrelin some of its cellular activities. In here we show that both ghrelin and des-acyl ghrelin stimulate proliferating C2C12 skeletal myoblasts to differentiate and to fuse into multinucleated myotubes in vitro through activation of p38. Consistently, both ghrelin and des-acyl ghrelin inhibit C2C12 proliferation in growth medium. Moreover, the ectopic expression of ghrelin in C2C12 enhances differentiation and fusion of these myoblasts in differentiation medium. Finally, we show that C2C12 cells do not express GHSR-1a, but they do contain a common high-affinity binding site recognized by both acylated and des-acylated ghrelin, suggesting that the described activities on C2C12 are likely mediated by this novel, yet unidentified receptor for both ghrelin forms.

In platelets stimulated by cross-linking of Fc␥RIIA, inhibition of Rap1B activation by ADP scavengers could be overcome by the simultaneous recruitment of the G i -coupled ␣ 2A -adrenergic receptor by epinephrine. By contrast, serotonin, which binds to a G q -coupled receptor, could not restore activation of Rap1B. When tested alone, epinephrine was found to be able to induce GTP binding to Rap1B, whereas serotonin produced only a slight effect. Finally, activation of Rap1B induced by stimulation of the G qcoupled thromboxane A 2 receptor by U46619 was completely inhibited by ADP scavengers under conditions in which intracellular Ca 2؉ mobilization was unaffected. Inhibition of U46619-induced Rap1B activation was also observed upon blockade of the P2Y12 but not of the P2Y1 receptor for ADP. These results demonstrate that stimulation of a G i -dependent signaling pathway by either ADP of epinephrine is necessary and sufficient to activate the small GTPase Rap1B.

The small GTP-binding protein Rap1B is activated in human platelets upon stimulation of a G i -dependent signaling pathway. In this work, we found that inhibition of platelet adenylyl cyclase by dideoxyadenosine or SQ22536 did not cause activation of Rap1B and did not restore Rap1B activation in platelets stimulated by cross-linking of Fc␥ receptor IIA (Fc␥RIIA) in the presence of ADP scavengers. Moreover, elevation of the intracellular cAMP concentration did not impair the G idependent activation of Rap1B. Two unrelated inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, totally prevented Rap1B activation in platelets stimulated by cross-linking of Fc␥RIIA, by stimulation of the P2Y 12 receptor for ADP, or by epinephrine. However, in platelets from PI3K␥-deficient mice, both ADP and epinephrine were still able to normally stimulate Rap1B activation through a PI3K-dependent mechanism, suggesting the involvement of a different isoform of the enzyme. Moreover, the lack of PI3K␥ did not prevent the ability of epinephrine to potentiate platelet aggregation through a G i -dependent pathway. The inhibitory effect of wortmannin on Rap1B activation was overcome by addition of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ), but not PtdIns(3,4)P 2 , although both lipids were found to support phosphorylation of Akt. Moreover, PtdIns(3,4,5)P 3 was able to relieve the inhibitory effect of apyrase on Fc␥RIIA-mediated platelet aggregation. We conclude that stimulation of a G i -dependent signaling pathway causes activation of the small GTPase Rap1B through the action of the PI3K product PtdIns(3,4,5)P 3 , but not PtdIns(3,4)P 2 , and that this process may contribute to potentiation of platelet aggregation.Rap1B is a small GTP-binding protein highly expressed in human platelets (1). In resting cells, it is mainly located at the membrane, but it translocates to the cytosol upon phosphorylation by protein kinase A (2). In activated platelets, Rap1B rapidly interacts with the reorganized actin-based cytoskeleton (3). As other GTPases, Rap1B is activated by binding of GTP. Platelet stimulation by different agonists, such as thrombin, collagen, and ADP, induces the rapid binding of GTP to Rap1B (4, 5). An increase in the intracellular Ca 2ϩ concentration in stimulated platelets has been shown to be sufficient to promote Rap1B activation, and specific Ca 2ϩ /calmodulin-sensitive guanine nucleotide exchange factors for Rap1B have been identified (5, 6). We (7) and others (8) have recently described a new pathway for Rap1B activation that is initiated by stimulation of membrane G i -coupled receptors and that is independent of intracellular Ca 2ϩ increases. In fact, the sole binding of ADP to the P2Y 12 receptor, as well as the interaction of epinephrine with the ␣ 2A -adrenergic receptor, is sufficient to trigger Rap1B activation. Moreover, we have found that agonists that activate platelets through stimulation of G q -coupled receptors, such as the thromboxane A 2 analog U46619, or through stimu...