Fig. 6

Metabolic alterations in LND fibroblasts under physiological conditions. A ZMP levels in fibroblasts treated with AICAr. Fibroblasts were maintained with Plasmax-PV medium containing 25 nM FA for 7 days and treated with or without AICAr at different concentrations (0.1 mM, 0.25 mM, 0.5 mM, and 1 mM) for 24 h. Cell extracts were obtained with 0.4 N PCA and ZMP levels determined by HPLC, and results expressed as nmol/mg protein. B, C Mitochondrial potential is decreased in LND fibroblasts. Cells were incubated with DilC or DilC plus CCCP and signal intensity was quantified by cytometry. The results are the mean ± SEM of 3 controls and 4 LND patients. A two-way ANOVA shows significant differences with P < 0.05 between control and LND groups. *P < 0.05; P** < 0.01; P*** < 0.005 ****P < 0.001. Two-way ANOVA, Fisher’s multiple comparison test. D Mitochondrial respiration was evaluated in control and LND fibroblasts cultivated with Plasmax-PV containing different levels of FA by seahorse assay as described in Material and Methods. Basal mitochondrial respiration, maximal respiration and ATP production are expressed as the oxygen consumption rate (pmol/min) normalized by the number of cells in the field of view. Analysis or the extracellular acidification rate (ECAR) is expressed as the fold change after oligomycin treatment. The results are the mean ± SEM of 3 controls and 3 LND patients. *P < 0.05; **P < 0.01. Two-way ANOVA, Fisher’s multiple comparison test