Fig. 8

MscL-G22S activation increases the energy metabolism of cultured SCs through the PI3K/AKT/mTOR pathway. A-C PI3K/AKT/mTOR pathway contributes to the regulation of cellular metabolism. The western blot analysis showed significantly higher phosphorylated levels of the PI3K, AKT, mTOR, and their downstream molecule, p70S6K, in MscL-G22S-activated SCs than the controls. Notably, higher levels of phosphorylated Akt but not the PI3K, mTOR and p70S6K were observed in stretched SCs than those without stimulus, indicating relatively mild effects of mechanical stretching only on cultured SCs. D-E Mechanical stretching induced significant upregulation of HIF-1α and c-Myc expression in cultured SCs, especially for the SCs with MscL-G22S expression. F-O LY294002, a potent PI3K inhibitor, was used to further validate the roles of PI3K signaling pathway in MscL-G22S activation-induced upregulation of energy metabolism in cultured SCs. As indicated, inhibition of PI3K significantly reduced the MscL-G22S activation-induced upregulation of cell viability but did not affect the Ca²⁺ influx in the SCs with mechanical stretching for 10 min (F-G). Similarly, after a culture of 24 h, expression of HIF-1α and c-Myc, the levels of glucose uptake, glucose-6-phosphate, pyruvate and lactate (H-M), NAD⁺ and NAD⁺/NADH ratio (N-O) in MscL-G22S-activated SCs were significantly reduced by inhibition of PI3K. The data shown were representative of at least three independent experiments. NC, negative control; MscL, MscL-G22S-expressing SCs; NC-stretch, negative control with mechanical stretching; MscL-stretch, MscL-G22S-expressing SCs with mechanical stretching. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001