the Effect of Fatty Acids in Hepatic Insulin resistance-part2


Monounsaturated fatty acid (MUFA) oleic acid (OA) has been reported to reverse saturated fatty acid palmitic acid (PA)-induced hepatic insulin resistance (IR). However, the detailed molecular mechanism remains elusive. In addition, previous research also showed that -3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA), exerted opposite effects to PA in muscle IR, but whether it plays the same role in hepatic IR and the possible mechanism involved needs to be further explored. Here, we confirmed that EPA reversed PA-induced IR in HepG2 cells and compared the proteome change after different free fatty acids (FFAs) treatments. For MUFA, 234 proteins were identified as differentially expressed proteins, and their functions were mainly related to response to stress and endogenous stimuli, lipid metabolic process and protein binding. For PUFA, the PA-induced expression change of 1326 proteins could be reversed by EPA and 415 of them were mitochondrial proteins, which covered most of the functional proteins in oxidative phosphorylation (OXPHOS) and tricarboxylic acid cycle (TCA). Mechanism study revealed that c-Jun and ROS played a role in OA- and EPA-reversed PA-induced IR, respectively. EPA or OA alleviated PA-induced abnormal ATP production, ROS generation, and calcium content. Importantly, H2O2-activated production of ROS increased the expression of c-Jun, further resulting in IR of HepG2 cells. Besides, we provided more feasible candidates as potential c-Jun-targeted “responders” for FFAs treatments. Taken together, we demonstrated that ROS/c-Jun is a common pathway to different FFAs-regulated IR in HepG2 cells.

Sample Processing Protocol

SILAC labeling was performed as we previously described [19]. To generate triple SILAC labeling states, DMEM medium without arginine and lysine (Invitrogen) was supplemented with L-[12C6,14N2]-lysine (Lys0) and L-[12C6,14N4]-arginine (Arg0) (Sigma) as “light” labeling, L-[2H4]-lysine (Lys4) and L-[13C6]-arginine (Arg6) as “medium” labeling, and L-[13C6,15N2]-lysine (Lys8) and L-[13C6,15N4]-arginine (Arg10) as “heavy” labeling. HepG2 cells were cultured for at least six cell population doublings in SILAC DMEM medium with 10% dialyzed fetal bovine serum (PAN Biotech UK Ltd), 100 U/mL penicillin and 100 mg/mL streptomycin to allow complete isotope incorporation (>95% labeling efficiency) before treatments with different FFAs. The SILAC strategy for investigating the effect of OA was conducted as we previously described [19]. Briefly, for forward labeling experiments, cells with light amino acids labeling were treated with 0.5 mM PA, ones with medium amino acids labeling were treated with the vehicle control, and ones with heavy amino acids labeling were treated with 0.5 mM PA plus 0.2 mM OA. For reverse labeling experiments, cells with light amino acids labeling were treated with 0.5 mM PA plus 0.2 mM OA, ones with medium amino acids labeling were treated with the vehicle control, and ones with heavy amino acids labeling were treated with 0.5 mM PA (Figure 2A). Following treatment with different FFAs for 12 hours, these different amino acids-labeled cells were incubated with 100 nM insulin for 20 minutes, washed twice with phosphate-buffered saline (PBS) and then scraped into lysis buffer. After sonication and centrifugation, the supernatant was collected for measuring protein concentration. The SILAC strategy for investigating the effect of EPA on PA-induced IR was similar to the strategy of OA as described above. The only difference was that the concentration of EPA used was 0.15 mM.

Data Processing Protocol

The raw mass spectrometric data were analyzed with Proteome Discoverer (version 1.4). Sequest HT was used for protein identification and Percolator was used for evaluation of false discovery rate (FDR) of protein identification. Uniprot human protein database (updated on 02-2016 including 92382 entries) supplemented with 247 known contaminants was used for database search. The database search parameters were as follows: 1) trypsin was chosen as enzyme, and no more than two missed cleavages were allowed; 2) 10 ppm and 0.02 Da were used for the mass tolerance of precursor ions and product ions, respectively; 3) cysteine carbamidomethylation was chosen as a fixed modification; 4) stable isotope-labeled arginine (Arg8, Arg10) and lysine (Lys4, Lys6) for SILAC and methionine oxidation were set as variable modifications. The peptides confidence was set as high. FDR <1% was set for protein identification.


Yaping Sun, the institute of biophysics chinese academy of science
Yaping Sun, Key Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China ( lab head )

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Shotgun proteomics


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