## 4 Discussion
The findings in the current study support the notion of the cAMP pathway not playing a significant role in BCL2 mRNA gene expression induced by CGS21680, because Forskolin left BCL2 mRNA expression unchanged. Therefore, it is possible that CGS21680's effect occurs through \(\mathbb{G}\beta\gamma\) protein coupling or through transport. Of note, the concentration of forskolin (100 nM) used in the present study is low. However, it is clear that the potency of forskolin (100 nM) induces cAMP accumulation and increases the expression of downstream signalings (such as NR4A) in skeletal muscle cell systems, as was previously demonstrated [14,19]. Hence, the concentration of forskolin (100 nM) used in the present study is the same. It was expected that treatment with insulin would contribute to cell death prevention by increasing the expression, in skeletal muscle tissue, of antiapoptotic genes, including BCL2. However, in this study, the skeletal muscle cell culture model was not given nutrients for one week (without FBS). During this week, insulin resistance was perceived in terms of inflammatory and metabolic effects. Previous studies found insulin to have a possible antiapoptotic effect [26,27]. For instance, streptozotocin-induced diabetic rats treated with insulin showed increased expression of BCL2 in the hippocampus. It was proposed that this was the antiapoptotic effect of insulin targeting a possible mechanism for hippocampal neuronal cell death induced by hyperglycemia [26,27]. This study did not support this assertion, as it found that BCL2 expression was not modulated by insulin in skeletal muscle cells in one-week-starved rats. Starvation-induced insulin resistance in skeletal muscle cells and differences in tissue type might explain this. Consistent with the current study, the adenosine A2A receptors represent a key mediator of adenosine function as an antiapoptotic agent. For example, one researcher reported that adenosine A2A receptor activation had a protective effect in small-for-size liver transplantation [16]. Another researcher also reported that A2A adenosine receptor activation was associated with attenuation of reperfusion lung injury and apoptosis through modulation of BCL2 expression and activation of extracellular signal-regulated kinases [13]. Other works highlighted the role of the adenosine A2B (pro-apoptotic) or the adenosine A3 (anti-apoptotic) receptors. For example, extracellular adenosine induced apoptosis of human arterial smooth muscle cells via adenosine A2B receptors [28]. Moreover, cytoprotective roles of adenosine A3 receptors were also reported in skeletal muscle ischemia and reperfusion injury [29]. The expression levels of A1, A2A, A2B and A3 receptors in this rat skeletal muscle cell line were previously reported [32; 33]. In fact, it was interesting to examine other adenosine receptor ligands, in particular, for adenosine A1 and A3 receptors. However, the aim in this current study was to evaluate the adenosine A2 ligands only. This did not exclude the scientific importance of other adenosine receptors ligands. Of note, the rationale for the study of adenosine A2 receptors ligands (but not adenosine A1 and A3) was that previous research studies performed in this field showed positive responses to adenosine A2 receptor activation/inhibition, in particular, with CGS21680 and PSB 603, [18; 20; 21; 22]. Actually, the aim of the present study was to investigate the potential role of highly selective adenosine A2 receptor ligands (CGS21680 and PSB 603) but not the potential role of adenosine A2 receptors themselves in skeletal muscle cells. Therefore, for the purpose of future work, it would be worthwhile to define the role of adenosine A2A receptor, using selective antagonists such as SCH58261 or SCH442416, and to investigate the modulation of BCL2 expression by those ligands.