P0500

A palmitic acid stock solution was prepared using a previously described method [74 (link)]. Briefly, a 100 mM solution of PA (P0500; Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M NaOH solution (194-02191; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was heated at 70 °C in a shaking water bath. In an adjacent water bath, at 55 °C, a 10% (w/v) PA-free BSA solution (CultureSure, 034-25462; FUJIFILM Wako Pure Chemical Corporation) was prepared in ddH₂O. A 5 mM PA/10% (w/v) BSA stock solution was prepared by adding 250 μL of the 100 mM palmitate solution dropwise to 4.75 mL of the 10% (w/v) BSA solution at 55 °C, followed by vortex mixing for 10 s and 10 min incubation at 55 °C. The PA/BSA complex solution was cooled to room temperature and sterile filtered (0.45 μm pore size membrane filter). At the same time, the PA-free BSA stock solution was prepared as a control. The complex solution was stored at −20 °C, where it was stable for 3–4 weeks. The stored 5 mM PA/10% BSA stock solutions were heated for 15 min at 55 °C and then cooled to room temperature before use.

Palmitate or sodium oleate (P-0500 and Fluka-75165, respectively; Sigma-Aldrich, Saint-Quentin Fallavier, France) were pre-complexed with 10% (wt/vol.) fatty acid-free BSA (Roche Diagnostic, Grenoble, France) in DMEM at 50°C for 2 h to prepare stock solutions of 8 mmol/l. The stock solutions were filtered and diluted in serum-free culture medium to a final concentration of 0.5 mmol/l, as lower concentrations of palmitate or oleate did not affect insulin-induced Akt phosphorylation (see electronic supplementary material [ESM] Fig. 1). The concentration of glucose in the medium (4.5 g/l vs 1 g/l) did not modify alteration of insulin-induced Akt phosphorylation induced by palmitate. For the analysis of insulin signalling, treated myotubes were incubated with or without 10⁻⁷ mol/l of insulin for 20 min.

HEK293T cells in 24-well plates were incubated with 100 μm of either palmitic acid (P0500, Sigma) or C16:0-azide (16 (link)) in 350 μl of serum-free Dulbecco's modified Eagle's medium supplemented with 1 mg/ml defatted BSA (A7030, Sigma) for 4 h at 37 °C.

Palmitic acid (PA, P0500, Sigma-Aldrich) was dissolved in 50% ethanol to a concentration of 20 mM stock solution. 30% BSA (A8020, Solarbio, China) is stock solution. Then PA solution and BSA solution were mixed together in DMEM to obtain 0.4 mM working solution and 1% BSA working solution. PA (0.4 mM) was added to cultured cells for 24 hours. The cells were then stained with Oil red O (G1262, Solarbio, China) to examine the amount of lipid accumulation. Intracellular TG and TC levels were measured using a commercially available TG Assay Kit (A110-1-1, Nanjing jiancheng Bioengineering Institute, China) and TC Assay Kit (A111-1-1, Nanjing jiancheng Bioengineering Institute, China) according to the manufacturer’s protocol.

Oleate (OA, O1008, Sigma-Aldrich, Darmstadt, Germany) and palmitate (P0500, Sigma-Aldrich, Darmstadt, Germany) were solubilized in a phosphate-buffered saline (PBS, 20012050, Gibco, New York, NY, USA) solution by heating it to 55 °C and 65 °C, respectively. Solubilized fatty acids were conjugated with 10% fatty acid free-bovine serum albumin (BSA, B2064, Sigma-Aldrich, Darmstadt, Germany) in culture medium to generate a fatty acid stock solution. TGF-β1 (100-21-10, Peprotech, Suzhou, Jiangsu, China) was dissolved according to the manufacturer’s instructions. CCl₄ was added to the full medium and dissolved completely after being positioned in a CO₂ incubator for 48 h to form a saturated solution. For the model of the hepatocytes treated with causative agents, L02, Huh7, and HepG2 cells were seeded in 6-well plates, respectively. After adherence, the cells were incubated with palmitate, OA, CCl₄, lipopolysaccharide, or TGF-β1 at different concentrations for another 24 h, respectively. For the proinflammatory factors assay, L02 cells were seeded in 6-well plates and transfected with indicated plasmids in the figure legends. After 6 h of transfection, the cells were incubated with fresh medium for 24 h, and then they were collected for further analysis.