Figure 4

2-AB increases intracellular GSH levels by altering metabolism and AMPK activation as well as exerting cardioprotective effects against oxidative stress.

(a,b) Intracellular 2-AB (a) and GSH (b) levels in H9c2 cells after incubation with 5 mM 2-AB for 30 min. (c) Intracellular metabolite levels in the partial metabolism of GSH. (d) Western blot of H9c2 cells incubated with 5 mM 2-AB. Phosphorylated ACC and ACC protein levels were quantified using Image Studio^TM software. Bars indicate the mean ± s.d. (n = 3). *P < 0.05. P-values were determined by unpaired two-tailed Student’s t-tests. (e) Effect of AMPK inhibitor on 2-AB induced GSH production. Cells were pretreated with 5 μM AMPK inhibitor for 4 h and incubated with 5 mM 2-AB for 30 min. Bars indicate the mean ± s.d. (n = 3). ***P < 0.0001. P-values were determined by ANOVA with Tukey’s multiple comparisons post-test. (f) Intracellular levels of AMP in 2-AB stimulated cells. The cells were incubated with 5 mM 2-AB for 30 min. The abundance of AMP was determined using a quantification kit. Bars indicate the mean ± s.d. (n = 7). **P < 0.01. P-values were determined by unpaired two-tailed Student’s t-tests. (g) Effect of 2-AB treatment on H9c2 cell viability. 3PG, glycerate-3-phosphate; SEP, o-phosphoserine; Ser, serine; Gly, glycine; GSH, glutathione; Cys, cysteine; Glu, glutamic acid; Gln, glutamine; ND, not detected; pACC, phosphorylated acetyl-CoA carboxylase; ACC, acetyl-CoA carboxylase. Bars indicate the mean ± s.d. (n = 3). *P < 0.05, **P < 0.01, ***P < 0.0001. n.s., not significant. P-values were determined by unpaired two-tailed Student’s t-tests.