Fig 1.
CPT II activities and thermal instability in CPT II-deficient fibroblasts.
Fibroblast lysates were preincubated at 37°C or 41°C. Enzymatic reactions commenced by the addition of substrates at 37°C. Data are the means of five separate experiments. The average of three independent experiments is shown ± SEM (*P<0.05).
Fig 2.
CPT II expression and the dominant—negative effect of CPT II variants on substrate-dependent kinetics.
(A) CPT II expression was analyzed by Western Blotting with an anti-CPT II antibody. (B) Dominant—negative effect of CPT II variants was analyzed by substrate-dependent kinetics. (C) Relative protein expression of control and variant CPT II. (D) Real-time PCR analysis of control and variant CPT II expression. Lane 1, control fibroblasts; lane 2, V368I (heterozygous); lane 3, V368I (homozygous); lane 4, F352C (heterozygous) + V368I (homozygous). CPT II protein was expressed relative to β-actin. Vmax and Km values were obtained from kinetic analysis (1/V versus 1/[S] plots) by varying the concentrations of L-[methyl-3H] carnitine between 0–300 μM at a fixed 50 μM palmitoyl-CoA concentration. (●) control, (▲) V368I (Homo), (◆) F352C (Hetero) + V368I (Homo). Data are means of three separate experiments. The average of three independent experiments is shown ± SEM (*P<0.05)
Fig 3.
Pulse-chase (left) and half-lives (right) of control and variant CPT II in fibroblasts.
Cultured fibroblasts were pulse-labeled with L-[35S] methionine for 2 h and chased for 0, 6, 12, and 18 h. CPT II from fibroblast lysates was immunoprecipitated with anti-CPT II antibodies, then subjected to SDS-PAGE followed by autoradiography. (●) control, (■) V368I (homozygous), (▲) F352C (heterozygous) + V368I (homozygous). The average of three independent experiments is shown ± SEM.
Fig 4.
Reduction of mitochondrial membrane potential of control and variant CPT IIs in cultured fibroblasts.
Control (A, D), V368I (homozygous) (B, E), and F352C (heterozygous) + V368I (homozygous) (C, F) fibroblasts were cultured at 37°C and 41°C. Mitochondrial depolarization was monitored by 15 min treatment with 10 μM of JC-1 in the dark and visualized under a fluorescence microscope. Scale bars, 100 μm.
Fig 5.
Cell apoptosis analysis of CPT II variants in fibroblasts.
(A) Apoptotic fibroblasts were measured by flow cytometry. Fibroblasts incubated with DMSO were used as a control. (B) LDH assay of control and CPT II variants in fibroblasts. (C) Apoptotic factor release was analyzed by Western Blotting with antibodies against caspase-3, caspase-8, cytochrome c, and Bid for control and variant CPT IIs in control and CPT II-deficient fibroblasts. CPT II proteins are expressed relative to β-actin. Data are means of three separate experiments. The average of three independent experiments is shown ± SEM (*P<0.05).
Fig 6.
Correlation between CPT II activity and β-oxidation/ATP production at 41°C and 37°C in CPT II-deficient fibroblasts.
(●) control, (■) V368I (homozygous), (△) F352C (heterozygous) + V368I (homozygous). CPT II activity, β-oxidation, and ATP production are expressed as % of control values of control fibroblasts at 37°C. Data areand means of five separate experiments: 0.53 nmol/min/mg protein of CPT II activity; 14.5 nmol of released CO2/h/106 cells; 8.12 nmol of ATP/106 cells. The average of three independent experiments is shown ± SEM.
Fig 7.
A proposed energy crisis pathway induced by CPT II deficiency.