Figure 1.

Effect of Adiponectin on S1P metabolism and signalling. (a) Quantitative mRNA analysis was performed by real-time PCR in total RNA extracted from C2C12 myotubes stimulated or not with 1 µg/mL Adn for 15 h. mRNA quantitation of S1P metabolism enzymes (SK1, SK2, SPL, SPP1 and SPP2), S1PR (S1P₁, S1P₂, S1P₃, S1P₄ and S1P₅) and S1P specific transporter Spns2 was based on the 2^−ΔΔCt method, using individual enzyme/receptor/S1P transporter of the unchallenged specimen as calibrator. Data are the mean ± SEM of three independent experiments, each performed in triplicate. (b) C2C12 myotubes were incubated for 24 h in the absence or in the presence of 1 µg/mL Adn. Aliquots of total cell lysates were used to perform Western analysis, using specific anti-SK1, anti-SK2, anti-SPL and anti-Spns2 antibodies. A representative blot is shown. The histogram represents the densitometric analysis of at least three independent experiments, each performed in triplicate. Data are the mean ± SEM and are reported as protein expression normalized to GAPDH, fold change over control (set as 1). The effect of Adn on S1P₄ mRNA and SK2 protein levels was statistically significant by Student’s t-test (* p < 0.05).