Cell culture and adenovirus infection

For the in vitro study, porcine subcutaneous adipocytes were isolated from 3-day-old DLY pigs using previously published methods [23]. These cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose, KeyGEN BioTECH, Jiangsu, China) supplemented with 10% fetal bovine serum (FBS, GIBCO, New Zealand) and 100 U of penicillin and streptomycin at 37 °C in a humidified atmosphere of 5% CO₂. The adenoviruses encoding green fluorescent protein (GFP), pAdM-FH-GFP (the GFP control, abbreviated as CON) and pAdM-FH-GFP-CRTC3 (the CRTC3 overexpression construct, abbreviated as OE) with high transfection efficiency were purchased from Vigene Company (Vigene, Shandong, China). Before adenovirus infection, cells were seeded in 6-well or 12-well plates and cultured with complete high-glucose DMEM (DMEM/F12 + 10% fetal bovine serum + 100 U of penicillin + 100 U of streptomycin) at 37 °C in a humidified atmosphere of 95% air: 5% CO₂. After reaching confluence, cells were incubated with high glucose DMEM containing the CON and OE adenoviruses (0.25 μL/mL) for 6 h, after which the medium was replaced with complete medium. After 48 h, the cells were used for immunostaining, gene expression, metabolomics and RNA-seq analyses.