PMC

Rotenone induced GPAT expression. C2C12 cells were treated with 0 (control) or 5 nM rotenone for 24 h and harvested for determination of expression levels of glycerol-3-phosphate acyltransferase (GPAT) by real-time PCR (A) and Western blot (B) analysis, which were expressed as GPAT/β-actin ratios, respectively. Data represent means ± SEM from 4 separate experiments. *p < 0.05 compared to control.

Inhibition of complex I increased TAG deposition. Lipids from the following experiments were extracted and analyzed by shotgun lipidomics as described under the section of “Materials and Methods”. A. C2C12 cells were treated with 0 (control) or 1 nM (piericidin A) for 48 h. B and C. C2C12 cells were transfected with NDUFV1 siRNA for 48 h prior to being harvested for NDUFV1 protein and TAG analysis. Protein levels of NDUFV1 and TAG mass levels were analyzed by Western blot using antibody against NDUFV1 (B) and determined by shotgun lipidomics (C), respectively.

Rotenone induced triacylglycerol deposition in vitro. A. C2C12 cells were treated with different concentrations of rotenone for different intervals as indicated. TAG contents were determined by shotgun lipidomics and expressed as fold increase vs. control. Data represent means ± SEM from 3–5 separate experiments. *p < 0.05 and ^#p < 0.01 compared to control. B. Cells including C2C12, H9C2, Hepa1–6, HEK293, BE(2)-C, and 3T3-L1 were treated with rotenone (5, 10, 5, 5, 20, and 5 nM, respectively) for 48 h. The contents of TAG were determined by lipidomics and expressed as fold increases vs. controls which were treated with vehicle. The basal TAG levels are (nmol/mg protein): C2C12, 2.45 ± 0.22 (n = 14); H9C2, 3.90 ± 0.27 (n =3); Hep1–6, 11.75 ± 0.28 (n = 3); HEK293, 24.17 ± 1.80 (n = 3); BE(2)-C, 6.60 ± 0.20 (n = 3); and 3T3-L1, 17.53 ± 1.23 (n = 4). *p < 0.05 and ^#p < 0.01 compared to control.

Rotenone activated fatty acid synthesis. A. C2C12 cells were treated with different concentrations of rotenone as indicated for 24 h and processed for determination of activated ATP citrate lyase (ACL) with antibody against p-ACL, whereas β-actin was used as loading control. Data represent means ± SEM from 3 separate experiments. ^#p < 0.01 compared to control. B. C2C12 cells were treated with 0 (open bars) or 5 (solid bars) nM rotenone for 48 h. Cells were harvested for quantification of triacylglycerol (TAG) mass and fatty acyl composition by shotgun lipidomics as described under the section of “Materials and Methods”. The composition of palmitoleic (16:1), oleic (18:1), and linoleic (18:2) acids in total fatty acyl of TAG were expressed as percent of total fatty acids in TAG. Data represent means ± SEM from 5 separate experiments. ^#p < 0.01 compared with control.

Rotenone induced mitochondrial stresses. A. C2C12 cells (~10⁴) were plated in a Seahorse 96-well plate and treated with rotenone for 48 h in serum-free medium. Oxygen consumption rate (OCR) was determined with a Seahorse XF96 Analyzer in Seahorse assay medium. Data represent means ± SEM of three independent determinations and were expressed as percent of control which was the cells treated with vehicle. B. C2C12 cells were plated in 96-well tissue culture plate and treated with rotenone for 48 h. Mitochondrial membrane potential was assayed with a JC-10 kit from Abcam. Data represent means ± SD of three separate experiments. C. C2C12 cells were treated with rotenone for 6 (open bars) or 12 h (solid bars) at the concentrations as indicated. Cells were harvested in NADH lysis buffer and assayed for NADH and NAD⁺ contents as described under the section of “Materials and Methods”. Data represent means ± SEM from 8 separate experiments. ^*p < 0.05 and ^#p < 0.01 compared to control. C2C12 cells were treated with 0 or 5 nM rotenone for 48 h, and processed for measurement of acetyl carnitine (D) or organic acids (E) as described under the section of “Materials and Methods”. Data represent means ± SEM from 4 separate experiments for both (D) and (E). *p < 0.05 and ^#p < 0.01 compared to control. α-KG stands for α-ketoglutarate.

Rotenone induced triacylglycerol deposition in C2C12. Cells were treated with 5 nM rotenone for 48 h and harvested for TAG analysis. Lipids from the cells were extracted using a modified Bligh and Dyer procedure. Samples were diluted to a final concentration of ~500 fmol/µl by chloroform/methanol/isopropanol (1/2/4, v/v/v) with 4% LiOH to facilitate lithium adduct in neutral loss [268 (internal standard), 228, 252, 254, 256, 278, 280, 282, 284, 304, and 306] scans. The abundant ion peaks including 849.7, 809.7, 811.7, 813.7, 835.7, 837.7, 839.7, 841.7, 861.8, 863.8, 865.8, 867.8, 887.8, 889.8, 891.8, 893.8, 895.8, 913.8, 915.8, 917.8, 919.8, 921.8, 937.8, 939.8, and 941.8 were identified as lithiated TAG species using shotgun lipidomics as previously described (). The mass spectra (A and B) were displayed after normalization to the internal standard (IS) peak at m/z 849.7. Data represent 4 separate experiments. C2C12 cells were cultured treated with 0 or 5 nM of rotenone for 48 h. To visualize the accumulated intracellular lipid droplets, cells were fixed with 3.7% formaldehyde prior to staining with Oil-Red-O (C and D). The nucleus was stained with haematoxylin. Images represent 3 separate experiments.