Fig. 4

Succinyl-metabolites do not accumulate in fibroblasts and urines from LND patients, and AMPK is not activated in LND fibroblasts. A Fibroblasts were incubated for 7 days in Plasmax-PV medium with 15% FBS containing 2200 nM or 25 nM FA. Cell extracts were obtained with 0.4 N PCA and succinyl-AICAR/succinyl-AMP levels determined by HPLC. Graphs represent the mean ± SEM of at least 3 control individuals and 3 patients with LND, expressing the results as nmol/mg protein. Two-way ANOVA, Fisher’s multiple comparison test shows no significant differences. B PCA metabolites extraction was performed in urines from control individuals (N = 6), patients with LND (N = 14), HND (N = 8), HRH (N = 2), or ADSL deficiency (N = 1). Succinyl-AICAr and succinyl-adenosine levels were determined by HPLC. Results are expressed as nmol/mg creatinine. Graphs represent the mean ± SEM. One-way ANOVA, Fisher’s multiple comparison test shows no statistical differences. ADSL deficiency urine sample has not been included in the statistical analysis. C AMPK activity determined by Western blot, as the ratio pAMPK/AMPK, in total cell extracts from control and LND fibroblasts cultivated with Plasmax-PV containing different levels of FA. 80 µg of protein were loaded per well. The results are the mean ± SEM of 3 controls and 4 LND patients. *P < 0.05. Two-way ANOVA, Fisher’s multiple comparison test. D Fibroblasts were incubated for 7 days in Plasmax-PV medium with 15% FBS containing 2200 nM or 25 nM FA. Cell extracts were obtained with 0.4 N PCA and AMP and ATP levels determined by HPLC. Results of AMP and ATP expressed as nmol/mg protein, and the AMP/ATP ratio is the mean ± SEM of 3 control individuals and 4 patients with LND. A two-way ANOVA shows no significant differences