Fig. 2
Basic amino acids cause mitochondrial depolarisation and decrease intracellular ATP levels in isolated mouse pancreatic acinar cells. (a–f) Isolated pancreatic acinar cells were incubated for 1, 2, 3 and 6 h without or with 40 mM L-Lys, L-Arg or L-Orn. We used the fluorescence probe tetramethylrhodamine methyl ester (TMRM) to measure the mitochondrial membrane potential (ΔΨm). The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) was added at the end of the experiments to induce maximum depolarisation as the positive control. Representative traces show the relative TMRM fluorescence of cells treated with L-Lys (a–b), L-Arg (d–e) and L-Orn (g–h), and raw fluorescent data is presented in supplementary table S6. The difference between TMRM fluorescence before and after the addition of the FCCP was normalised to the value after the addition of FCCP. Bar graphs show the quantification of the results (c, f, i) values are denoted as mean ± SEM from 6 to 8 cell preparations, for details of exact values and parallels per group please see the supplementary table S2. Two-way ANOVA was performed followed by the Šídák’s post-hoc test (c, f, i), and statistically significant differences were detected between the control and amino acid treatments and marked with ** P < 0.01, *** P < 0.001, or **** P < 0.0001. (j) For the measurement of intracellular ATP levels, cells were incubated for 6 h without and with 40 mM L-Lys, L-Orn or L-Arg. ATP level in the control cells immediately after isolation (0 h) was considered as 100% (not shown). Values are mean ± SEM from 6 to 8 cell preparations, for details of exact values and parallels per group please see the supplementary table S2. (j) One-way ANOVA was performed followed by Dunnett’s post-hoc test, and statistically significant differences were detected and marked with * for P < 0.05 or ** for P < 0.01. Abbreviations: Ctrl, control; L-Arg, L-Arginine; L-Lys, L-Lysine; L-Orn, L-Ornithine; norm., normalized.
