Structure of ACSLs and the ACSL1 mutant. (A) Sequence comparison of mouse ACSL family members and the N-terminal-deleted ACSL1 mutant (ACSL1mt). (B) ACSL1 expression was detected by western blot analysis after transfection of pc-HA-ACSL1 wild type (wt) and pc-HA-ACSL1 mutant (mt) into COS7 cells. (C) Immunofluorescence micrographs after transfection of pc-HA-ACSL1wt or pc-HA-ACSL1mt in COS7 cells. Scale bars = 10 μm. (D) The ratio of mitochondrial co-localized HA-ACSL1 to total HA-ACSL1 was measured. n = 4, *P < 0.05 vs HA-ACSL1wt, by t-test.|@|~(^,^)~|@|Intracellular distribution of wild type ACSL1 (ACSL1wt) and mutant ACSL1 (ACSL1mt) in C2C12 myotubes. (A) Immunofluorescence micrographic analysis was performed after C2C12 myotubes were infected with Ad-HA-ACSL1wt or Ad-HA-ACSL1mt. Scale bars = 10 μm. (B) Subcellular fractionation was performed after infection with Ad-HA-ACSL1wt or Ad-HA-ACSL1mt into C2C12 myotubes. One half of the mitochondrial fraction was treated with protease K. Mitochondrial fractionation was confirmed by western blotting with antibodies against enolase (cytosol) and prohibitin (mitochondria). (C) Immunofluorescence micrographic analysis was performed after HepG2 cells were infected with Ad-HA-ACSL1wt or Ad-HA-ACSL1mt. Scale bars = 10 μm.|@|~(^,^)~|@|ACSL1 interacts with a mitochondrial protein CPT1b in myotubes. (A) Immunofluorescence micrographs were conducted after C2C12 myotubes were treated with siNS (nonspecific) or siCPT1b for 48h. Scale bars = 10 μm. (B) Relative mRNA levels of CPT1b after treatment with siCPT1b in myotubes. n = 3, *P < 0.05 vs control, by t-test. (C) COS7 cells were co-transfected with pc-HA-ACSL1wt or pc-HA-ACSL1mt and pCMV-CPT1b-Myc. Immunoprecipitation (IP) was conducted with an HA antibody, and then western blotting was performed with HA or Myc antibodies.|@|~(^,^)~|@|ACSL1 mainly contributes to palmitate-induced FAO. (A) C2C12 myotubes were treated with 500 μmol/L palmitate for 24 h and relative mRNA levels of ACSL isoforms were determined using qPCR. The ACSL1 mRNA level of untreated cells (Veh) was set to 1, and others were expressed as its relative values. Data are expressed as the mean ± SEM, n = 3, *P < 0.05 vs Veh, by t-test. (B and C) C2C12 myotubes were transfected with siRNAs against ACSL1 and ACSL3 or siNS for 24 h and then treated with palmitate for 24 h. The mRNA levels of ACSLs (B) and FAO (C) were then determined. Data are expressed as mean ± SEM, n = 3, *P < 0.05 vs siNS/Veh, _# P < 0.05 vs siNS/Palmitate, by ANOVA.|@|~(^,^)~|@|ACSL1 overexpression ameliorates palmitate-induced insulin resistance. (A-D) Acyl CoA Synthase activity and FAO were measured after myotubes were infected with Ad-HA-ACSL1wt, Ad-HA-ACSL1mt or Ad-GFP (control) (100 moi) for 24 h. Viral infection was compared by detecting green fluorescent protein (GFP) expression, Scale bars = 100 μm (A), and expression of HA-ACSL1wt and HA-ACSL1mt was confirmed by western blotting (B). (C) Acyl-CoA synthase activity was measured, and H2O2 production rates were normalized by the amount of proteins in the cell lysates. Data are expressed as mean ± SEM, n = 3, *P < 0.05 vs Ad-GFP, by ANOVA. (D) FAO rates were measured. Data are expressed as mean ± SEM, n = 4, *P < 0.05 vs Ad-GFP, _# P < 0.05 vs Ad-ACSL1wt, by ANOVA. (E and F) C2C12 myotubes were infected with Ad-HA-ACSL1wt, Ad-HA-ACSL1mt, or Ad-GFP (100 moi) for 24 h followed by palmitate (250 μmol/L) treatment for another 24 h, then stimulated with or without insulin (100 nmol/L) for 30 min. (E) Western blot analyses were performed with specific antibodies against pAKT (Ser473), AKT, HA, or γ-tubulin. (F) The ratio of pAKT to total AKT in the western blots was measured. The value of insulin stimulated pAKT/AKT of Ad-GFP-infected without palmitate-treated cells was set to 100, and the others were expressed as its relative values. Data are expressed as mean ± SEM, n = 3, *P < 0.05 vs Ad-GFP with insulin/Veh, _# P < 0.05 vs Ad-GFP with insulin/palmitate, by ANOVA. (G and H) C2C12 myotubes were infected with Ad-HA-ACSL1wt, Ad-HA-ACSL1mt, or Ad-GFP (100 moi) for 24 h, and then intracellular TG (G) and DAG (H) levels were measured. Data are expressed as mean ± SEM, n = 5 (TG), n = 3 (DAG), *P < 0.05 vs Veh, _# P < 0.05 vs Ad-GFP/Palmitate, by ANOVA.

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