Fig 1.
Etomoxir (EX) inhibits most of fatty acid oxidation (FAO) at a 10 μM concentration in BT549 cells but does not affect cellular proliferation until much higher concentrations are used.
(A) The pool sizes of acylcarnitines (ACs) decrease by over 80% at 10 μM etomoxir. Additional small decreases are observed at 200 μM etomoxir (n = 3). (B) Isotopologue distribution pattern of citrate after BT549 cells were labeled with 100 μM U-13C palmitate for 24 hours. The M+2 isotopologue reflects FAO activity (n = 3). (C) Growth curve of BT549 cells when treated with vehicle control, 10 μM etomoxir, or 200 μM etomoxir (n = 4) (doubling time [DT]). All data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2.
Mitochondrial respiration and nutrient utilization do not show a dose response to etomoxir because 200 μM etomoxir (EX) has an off-target effect on respiratory complex I.
(A) Nutrient utilization after BT549 cells were treated with vehicle control, 10 μM etomoxir, or 200 μM etomoxir for 48 hours (n = 3). (B) Mitochondrial stress test of whole cells (BT549) after treatment with vehicle control, 10 μM etomoxir, or 200 μM etomoxir for 1 hour (n = 4). (C) Measured and calculated parameters of mitochondrial respiration (generated from data in Fig 2B). (D) 200 μM etomoxir leads to changes in state I respiration but does not affect state II respiration, indicating that 200 μM directly inhibits complex I of the electron transport chain (n = 3). (E) Isotopologue distribution pattern of citrate after BT549 cells were labeled with U-13C glucose for 12 hours (n = 3). (F) Isotopologue distribution pattern of citrate after BT549 cells were labeled with U-13C glutamine for 6 hours (n = 3). All data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. The oxygen consumption rate (OCR) was corrected for nonmitochondrial respiration. AA, antimycin A; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhdrazone; oligo, oligomycin; rot, rotenone; suc, succinate.
Fig 3.
Knockdown of CPT1A inactivates most of fatty acid oxidation (FAO) and decreases cellular proliferation.
(A) Isotopologue distribution pattern of citrate in BT549 cells with scrambled small interfering RNA (siRNA) (scrambled, black) or after CPT1A knockdown (KD, red). Cells were labeled with 100 μM U-13C palmitate for 24 hours, starting at 48 hours after siRNA knockdown. The M+2 peak reflects FAO activity (n = 3). (B) Growth curve of control and CPT1AKD BT549 cells (n = 4) (DT, doubling time). (C) The decrease in cellular proliferation cannot be rescued by various concentrations of acetate (n = 5). Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001.
Fig 4.
Knockdown of CPT1A causes mitochondrial uncoupling.
(A) CPT1AKD cells (KD, red) uptake more glucose, glutamine, glutamate, and fatty acids relative to scrambled small interfering RNA (siRNA) controls (scrambled, black). CPT1AKD cells also excrete more lactate (n = 4). (B) Mitochondrial stress test for scrambled siRNA controls and CPT1AKD cells (n = 3). (C) Measured and calculated mitochondrial respiration parameters (generated from data in Fig 4B). Data are presented as mean ± SEM and normalized to the final number of cells after respiration measurements to account for differences in proliferation. We note that coupling efficiencies are calculated as the ratio of the oxygen consumption rate (OCR) required for ATP production to basal OCR in the same sample and therefore are independent of the sample normalization method. *p < 0.05, **p < 0.01, ***p < 0.001. The OCR was corrected for nonmitochondrial respiration. AA, antimycin A; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhdrazone; oligo, oligomycin; rot, rotenone;.
Fig 5.
Imaging mitochondrial dysfunction in CPT1AKD cells.
(A, B) Mitochondria were stained by Mitotracker red, and nuclei were stained by DAPI. Images from scrambled small interfering RNA (siRNA) controls (A) show less fluorescence intensity of Mitotracker red compared to CPT1AKD cells (B). (C, D) Representative electron microscopy (EM) images of normal mitochondria in wild-type BT549 cells (C) and the abnormal vesicular morphology of mitochondria in CPT1AKD cells (D).
Fig 6.
The levels of complex lipids are altered in the mitochondria of CPT1AKD cells.
(A) Scatter plot comparing the integrated intensities of 77 lipid species altered between scrambled small interfering RNA (siRNA) controls and CPT1AKD cells. All lipids profiled that showed a fold difference ≥ 1.5, a p-value ≤ 0.01, and a signal intensity ≥ 10,000 are displayed. The diagonal line represents the equation y = x, so that points below the line represent the 66 lipids that decrease in abundance in CPT1AKD cells. (B) The identities and absolute concentrations of dysregulated lipids were determined and the relative differences plotted. Signaling lipids are displayed on top, and structural lipids on bottom. CL, cardiolipin; Cer, ceramide; DG, diacylglycerol; Gal/GlcCer, galactosyl/glucosylceramide; KD, knockdown; LacCer, lactosylceramide; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine; SM, sphingomyelin. Data are presented as mean ± SEM (n = 3). *p < 0.05, **p < 0.01.
Fig 7.
Model for the anabolic role of carnitine palmitoyltransferase I (CPT1) in mitochondrial metabolism.
Acyl-CoA species have anabolic fates in the cytosol (1), in addition to catabolic (2) and anabolic (3) fates in the mitochondrial matrix (e.g., phospholipid sidechain remodeling and protein acylation). ACSL, acyl-CoA synthetase; CAT, carnitine-acylcarnitine translocase; CPT1, carnitine palmitoyltransferase I; CPT2, carnitine palmitoyltransferase II; FA, fatty acid.