Oil red o

The murine mesenchymal stem cell line, C3H10T1/2, was kindly donated by Prof. Eduardo Domínguez Medina (University of Santiago de Compostela, Spain) and cultured at 37 °C under 5% CO₂ in DMEM (4.5 g/L glucose, Corning, USA) containing 10% fetal bovine serum (FBS; Sigma-Aldrich, MO, USA) and 1% penicillin and streptomycin (P/S, Biowest) until differentiation into adipocytes, as previously described [19 (link)]. In brief, differentiation was performed after cell confluence with induction medium [maintenance medium supplemented with 1 µM dexamethasone, 0.5 mM isobutylmethylxanthine, 1 µM rosiglitazone (Sigma-Aldrich, MO, USA), and 5 µg/mL insulin (Actrapid, NovoNordisk)]. Two, four, and 6 days after induction, the medium was replaced with maintenance medium containing 5 µg/mL insulin (Actrapid, NovoNordisk). The accumulation of cytoplasmic triglycerides in these cells was detected by staining with Oil Red O (Sigma-Aldrich, MO, USA).
A high-glucose and high-insulin (HG/HI) insulin resistance, and lipid hypertrophy/insulin resistance cell models were established as described previously [11 (link), 20 (link)]. Briefly, cells were differentiated until day 6, washed three times with PBS, and incubated for 2 h in low-glucose (1 g/L) and serum-free cell culture medium. Cells were then cultured in high glucose (4.5 g/L) and high insulin (100 nM), or with palmitate (500 µM in 2% fatty acid-free BSA [Capricorn Scientific, USA]) or oleic acid [1 mM, conjugated in 0.5 mM fatty acid-free BSA [Capricorn Scientific, USA]) in serum-free medium for 24 h in oleic and HG/HI, and 18 h in palmitate. For vesicle isolation, the treatment was prolonged to 48 h. For pAkt/Akt pathway analysis, cells were washed three times in PBS and stimulated with insulin (100 nM) for 10 min.
The murine macrophages Raw 264.7 cells were cultured in DMEM (4.5 g/L glucose, Lonza) containing 10% fetal bovine serum (Sigma-Aldrich, MO, USA) and 1% P/S (Biowest) at 37 °C under 5% CO₂.
Human macrophage THP-1 cells were cultured in RPMI (without L-Glutamine, Lonza) containing 10% FBS (Sigma-Aldrich, MO, USA), 1% P/S (Biowest) and 0.05 mM β-mercaptoethanol at 37 °C under 5% CO₂. THP-1 monocytes were differentiated into macrophages by adding 5 µg/mL Phorbol Myristate Acetate (PMA) for 24 h. For RNA extraction, the treatment was performed for 24 h, whereas for the isolation of vesicles in the cell secretome, the treatment was prolonged to 48 h.
The human hepatocytes HepaRG cells were cultured in William’s E medium supplemented with GlutaMAX (Gibco) containing 10% fetal bovine serum (FBS; Sigma-Aldrich, MO, USA) and 1% P/S (Biowest) at 37 °C under 5% CO₂. However, before differentiation, HepaRG cells were cultured in proliferation medium [maintenance medium with 5 µg/mL insulin (Actrapid, NovoNordisk) and 0.5 µM hydrocortisone hemisuccinate sodium salt (Sigma-Aldrich, MO, USA) for 6 days, renewing the medium every two days. HepaRG cells were differentiated using differentiation medium [maintenance medium with 5 µg/mL insulin (Actrapid, NovoNordisk), 50 µM hydrocortisone hemisuccinate sodium salt, and 2% dimethyl sulfoxide (DMSO, Thermo Fisher Scientific, Massachusetts, USA)] for 15 days, renewing the medium for the first three days and then every three days. Once differentiated, HepaRG cells were washed twice with PBS and serodeprived for 2 h. The cells were then cultured with high glucose (4.5 g/L) and high insulin (100 nM), with palmitate [500 µM in 2% fatty acid-free BSA (Capricorn Scientific, USA)], oleic acid [1 mM, conjugated in 0.5 mM fatty acid-free BSA (Capricorn Scientific, USA)], or with combination treatment [glucose (4.5 g/L) and insulin (100 nM), 250 µM palmitate (Sigma-Aldrich, MO, USA) in 2% fatty acid-free BSA (Capricorn Scientific, USA) and 0.5 mM oleic acid (Sigma-Aldrich, MO, USA), conjugated in 0.5 mM fatty acid-free BSA (Capricorn Scientific)]. For RNA or protein extraction, the treatment was performed for 24 h, whereas for the isolation of vesicles from the cell secretome, the treatment was prolonged to 48 h.
Murine hepatocyte primary cell culture was obtained by hepatic perfusion of 14-week-old male C57BL/6 mice weighing 20 g following previously described protocols [21 (link), 22 (link)]. Hepatocytes from the perfusion were resuspended in 100 mL of complete medium [DMEM (Gibco) with 10% FBS (Lonza) and 1% P/S (Gibco)], divided into 2 × 50 mL tubes, and centrifuged at 400 rpm (SorvallTM ST16, Fisher Scientific) for 4 min. The pellet was resuspended in 30 mL complete medium and 15 mL 90% Percoll (GE Healthcare) and centrifuged at 500 rpm for 10 min. Subsequently, the cell pellet from both tubes was transferred to a single tube, resuspended in complete medium, and centrifuged three times at 500 rpm for 5 min 3 times. Finally, the pellet of hepatocytes was resuspended in 50 mL of complete medium to analyze cell viability by Trypan Blue staining on slides (Cell Counting Chamber Slides, Invitrogen) for cell counting using the Countess® equipment (Invitrogen). Once the viability of the hepatocytes was obtained, 250.000 cells were seeded in 9.2 cm² plates (Corning, Thermo Fisher Scientific, Massachusetts, USA) previously treated with collagen (Sigma-Aldrich, MO, USA).