Two studies....dealing with muscle....Long read if your bored
Ghrelin and Des-Acyl Ghrelin Promote Differentiation and Fusion of C2C12 Skeletal Muscle Cells, Nicoletta Filigheddu, Molecular Biology of the Cell Vol. 18, 986–994, March 200
DISCUSSION
[This is a very good description of the process of muscle creation]
Skeletal muscle satellite cells are quiescent mononucleated myoblasts, located between the sarcolemma and the basal membrane of terminally differentiated adult muscle fibers. On muscle diseases or direct injury, quiescent satellite cells are activated to undergo proliferation and eventually differentiate to allow muscle regeneration.
Skeletal muscle regeneration involves, sequentially, satellite cell proliferation, commitment to terminal differentiation, cell fusion into multinucleated syncitia, and muscle fiber formation.
Such mechanisms leading to muscle regeneration are poorly understood; they seem to recapitulate the embryonic program of differentiation, although the extracellular factors regulating such processes may be different.
Satellite cell differentiation into skeletal muscle can be subdivided into temporally separable events, coordinated by the expression of proteins of the muscle regulatory factors family, such as myogenin, and of cyclin-dependent kinase inhibitor of the p21 family (Andres and Walsh, 1996), resulting in cell cycle exit and commitment to terminal differentiation. Later on, expression of muscle contractile proteins, such as MHCs and myosin light chains (MLCs), are hallmarks of phenotypic differentiation. Finally, fusion of myocytes into multinucleated myotubes is the terminal step of muscle differentiation.
The growing interest in skeletal muscle regeneration is associated to the opening of new therapeutic strategies for several muscular degenerative pathologies such as dystrophies, muscular atrophy, and cachexia associated to aging, cancer, chronic heart failure, and acquired immunodeficiency syndrome as well as the treatments of skeletal muscle injury after trauma.
Although Ghrelin (GHR) is a circulating hormone mainly secreted by the stomach, it is also synthesized in a number of tissues, suggesting both endocrine and paracrine effects (Gnanapavan et al., 2002).
The evidence that 1) Ghrelin (GHR) up-regulation is specifically associated to either congestive heart failure (CHF)- or cancer- induced cachexia (Nagaya et al., 2001, Shimizu et al., 2003) and that its administration strongly prevents CHF associated cachexia (Nagaya et al., 2004); 2) GHR, (Des-Acyl Ghrelin) D-GHR, and Growth Hormone Secretagogues (GHSs) inhibit apoptosis of cardiac myocytes (Filigheddu et al., 2001; Baldanzi et al., 2002); and 3) skeletal muscle features high binding sites for synthetic GHSs (Papotti et al., 2000), lead us to speculate that GHR and D-GHR may act directly also on skeletal muscle. Indeed, we observed that both GHR and D-GHR stimulate tyrosine phosphorylation of several proteins and activate ERK-1/2 and Akt (data not shown), indicating that both factors could exert a biological activity on these cells.
Here, we show that nanomolar concentrations of both GHR and D-GHR induce the differentiation of proliferating skeletal myoblasts in a concentration-dependent manner and promote their fusion into multinucleated syncitia in vitro. The cellular and molecular mechanisms by which GHR and D-GHR elicit these responses are not known. Cell cycle withdrawal is a prerequisite for myogenic terminal differentiation (Walsh and Perlman, 1997). Indeed, the ability of GHR and D-GHR to reduce DNA synthesis of proliferating C2C12 myoblasts is highly consistent with their prodifferentiative activity. However, inhibition of cell proliferation is not sufficient to elicit muscle differentiation. For example, myostatin inhibits both proliferation and differentiation of C2C12 myoblasts, through down-regulation of MyoD and myogenin expression (Joulia et al., 2003). Conversely, GHR and D-GHR, beyond inhibiting cell proliferation, induce the expression of myogenin, which is required for the complete program of differentiation of skeletal myoblasts to proceed (Zhang et al., 1999). To our knowledge this is the first evidence for an extracellular factor able to induce muscle differentiation of proliferating skeletal myoblasts in GM.
In proliferating C2C12 myoblasts, activation of p38 pathway obtained by overexpression of constitutively active MKK6 is sufficient to induce myogenin expression, cell cycle exit, and skeletal muscle terminal differentiation (Wu et al., 2000). Thus, we investigated whether GHR and D-GHR prodifferentiative activity is mediated by p38. Consistently, inhibition of p38 by cell treatment with SB203580 resulted in the partial albeit significant inhibition of GHR and D-GHR induced differentiative activity. In addition, we also showed that both GHR and D-GHR activate p38. Altogether, these data demonstrate that GHR and D-GHR act as antiproliferative and prodifferentiative factors by stimulating the p38 pathway.
The lack of expression of Growth Hormone Secretagogue Receptor One-A (GHSR-1a) in either C2C12 myoblasts and skeletal muscle tissue (Gnanapavan et al., 2002) as well as the activity exerted by D-GHR suggest that GHR and D-GHR–differentiating activities are mediated by a yet unidentified receptor, common to both acylated and unacylated peptide and distinct from GHSR-1a. Indeed, here we showed that C2C12 cells feature high-affinity common binding sites for both GHR and D-GHR. Such binding sites are specific, because they do not recognize either N-terminal truncated ghrelin or motilin, which are unable to induce differentiation. These studies also demonstrate that the N-terminal portion of the GHR peptide is required for binding and induction of C2C12 muscular differentiation. Together, these data provide further evidence for novel GHR receptor subtypes, which do not discriminate between the acylated and unacylated peptide. Although evidence for common GHR and D-GHR receptors have been reported in several cells, including a cardiomyocyte-derived cell line (Baldanzi et al., 2002), this is the first evidence for their expression in skeletal muscle.
We also verified whether the ghrelin gene is up-regulated in C2C12 myoblasts induced to differentiate in DM. However, no difference of ghrelin expression was detected by real-time RT-PCR between proliferating and differentiating cells (data not shown), suggesting that GHR gene product is not involved in DM-induced skeletal muscle differentiation in vitro.
By showing that GHR and D-GHR stimulate terminal differentiation of skeletal myoblasts in vitro, we may raise the hypothesis that the function of GHR gene may be involved in skeletal muscle differentiation in vivo. However, the lack of a consistent phenotype in GHR knockout mice, suggests that GHR function is not required for myogenesis during development. Consistently, we have not detected any GHR expression in somites or related structures during embryonic development by in situ hybridization (data not shown). However, although not essential for embryo development, GHR might be involved in the complex process of myogenesis in the adulthood, i.e., in regenerative processes of skeletal muscle. This hypothesis is consistent with the data showing that FGF6 is not required for muscle development, but is required in the adult for damage-induced muscle regeneration (Floss et al., 1997).
Upon muscular injury, skeletal myoblasts are activated to terminally differentiate through an autocrine/paracrine loop. We may speculate that GHR would contribute to skeletal muscle plasticity, promoting the differentiation and fusion of myoblasts in the damaged muscles. If this hypothesis would be proved, the activation of the receptor mediating GHR and D-GHR differentiative activity as well as the overexpression of the hormone may provide novel therapeutic strategies for the reduction or retardation of several skeletalmuscle pathologies, including dystrophies, atrophies, and cachexia.
GHRP-2, a GHS-R agonist, directly acts on myocytes to attenuate the dexamethasone-induced expressions of muscle-specific ubiquitin ligases, Atrogin-1 and MuRF1, Daisuke Yamamoto, et al., Life Sciences 82 (2008) 460–466
Introduction
A variety of diseases and conditions, including sepsis, cancer, renal failure, excess of glucocorticoid, denervation and disuse of muscle, can cause muscle atrophy. In these diverse conditions, the atrophying muscles show increased protein degradation through activation of the ubiquitin (Ub)-proteasome pathway (Baracos et al., 1995; Kayali et al., 1987; Price et al., 1996; Tiao et al., 1997; Tischler et al., 1990). It is recently reported that the expressions of Atrogin-1 and MuRF1, both of which are musclespecific Ub-ligases, are involved in protein degradation in muscle and increased in these diverse conditions causing muscle atrophy (Bodine et al., 2001; Gomes et al., 2001; Lecker et al., 2004). Atrogin-1 is a muscle-specific F-box type E3 ligase and reported to be induced 8 to 40 fold in muscle atrophy during fasting, diabetes, cancer and renal failure (Bodine et al., 2001), up to 3 fold in hind limb suspension, immobilization and denervation, and up to 10 fold in cachetic or dexamethasone administration model (Gomes et al., 2001). MuRF1 is a Ring Finger type muscle-specific E3 ligase that is initially found in association with the myofibril (Kandarian and Jackman, 2006) and suggested to play an important role in the myofibrillar proteins breakdown. Both muscle-specific E3 ligases are considered to play a pivotal role in muscle atrophy because knockout mice lacking these E3 ligases are prevented from muscle atrophy (56% sparing for atrogin-1-/- and 36% for MuRF1-/-) (Bodine et al., 2001).
On the other hand, several protective factors for muscle atrophy have been reported. One of the potent protective factors is IGF-I. IGF-I prevents muscle atrophy induced by glucocorticoid (Kanda et al., 1999; Schakman et al., 2005), disuse (Alzghoul et al., 2004) and denervation (Day et al., 2002). IGF-I has a potency to inhibit Atrogin-1 and MuRF1 expressions in atrophying muscle (Sacheck et al., 2004; Stitt et al., 2004). The protective effect of IGF-I for muscle atrophy, at least partly, is exerted by this mechanism (Bodine et al., 2001; Lecker et al., 2004).
Ghrelin stimulates GH release from the pituitary through the GH secretagogue receptor (GHS-R) (Kojima et al., 1999). Also, Growth Hormone Releasing Peptide-2 (GHRP-2), a synthetic ligand for GHS-R, stimulates GH release from the pituitary (Wu et al., 1996). GHRP-2 administration increases plasma GH levels in rats (Sawada et al., 1994) and humans (Pihoker et al., 1995). As a result, plasma IGF-I levels are reported to increase in some studies (Bowers et al., 2004). Thus, GHRP-2 is expected to have a protective action against muscle atrophy via IGF-I. Indeed, a recent report suggested that GHRP-2 was able to prevent arthritis-induced increase in Atrogin-1 and MuRF1 expressions in rat muscle (Granado et al., 2005).
On the other hand, there are reports suggesting the presence of GHS-R in muscle (Papotti et al., 2000; Pierno et al., 2003) and the signal transduction mechanism of ghrelin is partly similar to those of IGF-I and insulin (Murata et al., 2002). Hence GHS-R ligands may play a role in the process of muscle atrophy.
In the present study, we have examined the effect of GHRP-2 on Atrogin-1 and MuRF1 mRNA levels in dexamethasoneinduced muscle atrophy in the rats, as a model of muscle atrophy that is often observed during steroid hormone-treatment in human. We have further tested whether the effect is a direct action on myocytes through GHS-R and found for the first time that GHRP-2 directly acted on myocytes
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Discussion
In the present experiment, we found that GHRP-2 attenuated Atrogin-1mRNA level induced by dexamethasone in ratmuscles. Although the mechanism by which dexamethasone causes muscle atrophy is unknown, one possibility is via enhancement of glutamine synthetase activity (Falduto et al., 1992a,b) and the other is via induction of Atrogin-1 expression (Bodine et al., 2001; Lecker et al., 2004).
GHRP-2 has an action to stimulate GH secretion from pituitary, which in turn could increase plasma IGF-I levels. Since IGF-I has been reported to be a growth factor causing muscle hypertrophy (Kanda et al., 1999; Schakman et al., 2005), the elevation of plasma IGF-I levels may affect dexamethasone induced muscle atrophy. Interestingly IGF-I has been already reported to attenuate Atrogin-1 expression in vivo (Sacheck et al., 2004; Stitt et al., 2004). In the present study, however, plasma IGF-I levels were not changed by the treatment with GHRP-2. This finding was consistent with previous reports that GHRP-2 did not increase plasma IGF-I levels in mice (Tschop et al., 2002) and humans (Nijland et al., 1998), suggesting that GHRP-2 does not always increase plasma IGF-I levels. Our data rather suggested that the reduced mRNA levels of Atrogin-1 and MuRF1 in muscle by GHRP-2 was not due to the rise of circulating IGF-I levels. In addition, IGF-I expression in soleus muscles was not affected by GHRP-2 in the present study. Recently, Granado et al. (2005) reported that subcutaneous daily administration of GHRP-2 (100 ug/kg) decreased expression of Atrogin-1 and MuRF1 in atrophic muscle of adjuvant-induced arthritis rats. In their report, plasma IGF-I level wasmuch lower in arthritis rats than in normal control and GHRP-2 did not increase muscle IGF-I mRNA level. Their findings, consistent with our findings, suggested that GHRP-2 decreased Atrogin-1 and MuRF1 mRNA levels through a pathway other than circulating IGF-I and local IGF-I production.
Binding assay using GHS-R ligands has shown specific binding sites in muscle (Papotti et al., 2000) and in vitro application of ghrelin or ghrelin agonists modulated chloride and potassium conductance in rat muscle (Pierno et al., 2003). These findings suggest the presence of GHS-R in skeletal muscle. In this experiment, we found the expression of GHSR1a in differentiated C2C12 cells. We have already reported that intracellular signal pathways of ghrelin were partly similar to those of insulin and IGF-I (Murata et al., 2002). From the above reasons, we speculated GHRP-2 might work in myocytes to suppress Atrogin-1 and MuRF1 mRNA levels like IGF-I and examined whether GHRP-2 has a direct action on myocytes to inhibit Atrogin-1 and MuRF1 mRNA expressions. GHRP-2 dose-dependently suppressed dexamethasone-induced Atrogin- 1 and MuRF1 expressions in C2C12 cells. These findings indicate that GHRP-2 directly acts on myocytes and attenuates the level of Atrogin-1 and MuRF1 mRNA.
To further clarify a direct suppressive effect of GHRP-2 on Atrogin-1 and MuRF1 mRNA levels, [D-Lys3]-GHRP-6, a GHS-R1a antagonist was used in C2C12 cells. There are two types of ghrelin receptors, GHS-R1a and GHS-R1b (Howard et al., 1996; Mckee et al., 1997). GHS-R1a is an active receptor mediating ghrelin action. GHS-R1b, a splicing variant of GHSR1a, does not mediate ghrelin signal. We examined the specificity of GHRP-2 action using [D-Lys3]-GHRP-6. We found that [D-Lys3]-GHRP-6 partly and completely reversed the suppressive effects of GHRP-2 on Atrogin-1 and MuRF1 mRNA levels, respectively. These results suggest that GHRP-2 directly inhibits Atrogin-1 and MuRF1 mRNA level through GHS-R1a.
Since C2C12 cells produce IGF-I (Frost et al., 2003), paracrine or autocrine action of IGF-I may be involved in the suppressive effect of GHRP-2 on Atrogin-1 and MuRF1 mRNA level. To elucidate this possibility, we measured IGF-I mRNA level in C2C12 cells. However, we were not able to find the increase in IGF-I mRNA in C2C12 cells in response to GHRP-2, suggesting that locally produced-IGF-I in C2C12 cells is not involved in the suppressive effect of GHRP-2 on Atrogin-1 and MuRF1 mRNA levels. Dexamethasone also did not influence IGF-I mRNA level in C2C12 cells, although it decreased IGF-I mRNA level in vivo soleus muscle. These results suggest that dexamethasone has an indirect action to reduce IGF-I mRNA level in muscles in in vivo animals. Glucocorticoid is reported to inhibit pulsatile GH secretion (Giustina and Veldhuis, 1998) and reduce GH receptor expression (King and Carter-Su, 1995). As a result, IGF-I mRNA level was thought to decrease in vivo experiment in the present study. Dexamethasone has been reported to reduce the expression in in vivo animals (Gilson et al., 2007), being consistent with our in vivo result.
In summary, GHRP-2 suppressed dexamethasone-induced Atrogin-1 mRNA expressions in in vivo rats without elevating plasma IGF-I and IGF-I mRNA in muscle. Furthermore GHRP-2 decreased dexamethasone-induced Atrogin-1 and MuRF1 expressions in C2C12 myocytes. This effect was blocked by the addition of [D-Lys3]-GHRP-6, a GHS-R1a antagonist. These findings suggest that a direct action of GHRP-2 through GHSR1a suppresses Atrogin-1 and MuRF1 mRNA levels in C2C12 cells. GHRP-2 might lead to the protection of muscle atrophy induced by dexamethasone.
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