Pparα

Total RNA was extracted with an Axyprep multisource RNA miniprep kit (Axygen, America), and cDNA was synthesized using TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing). Quantitative real-time PCR was performed using a SYBR Green kit (TransGen Biotech, Beijing) with a StepOnePlus real-time PCR system (ABI), and the primer sets used for MTTP, ApoA1, ApoB, ApoC2, CD31, TGFβ, TSP1, VEGFR1, IL-1β, TNFα, IL-6, IL-10, CCL2, PPARα, SIRT1, and β-actin (Sangon Biotech, Shanghai) are listed in Table 1. Relative gene expression was measured with triplicates for each sample and normalized to β-actin.

The BCA protein content detection kit was bought from KeyGen Biotech (Nanjing, China). Hypersensitive ECL chemiluminescence kit, protease, and phosphatase inhibitor were bought from NCM Biotech (Suzhou, China). The protein extraction kit was obtained from Beyotime Institute of Biotechnology (Shanghai, China). Non-esterified fatty acids (NEFA) and blood lipid test kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Revert Aid First Strand cDNA Synthesis Kit was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Power SYBR Green PCR master mix was from Invitrogen (Carlsbad, CA, United States). The primers of AMPKα, SIRT1, PGC-1α, PPARα, GLUT4, UCP3, and β-actin were designed and synthesized by Sangon Biotech (Shanghai, China). Both primary antibodies to AMPKα, PGC-1α, PPARα, GLUT4, UCP3, GAPDH, Lamin B1, and Na, K-ATPase (Cat no: 66536-1-Ig, 66369-1-Ig, 15540-1-AP, 66846-1-Ig, 10750-1-AP, 60004-1-Ig, 66095-1-Ig, and 14418-1-AP) and secondary antibodies (Cat no: SA00001-1 and SA00001-2) were purchased from the Proteintech Group (Chicago, IL, United States). The antibody to Phospho-AMPKα (Thr172, Cat #: 50081S) was purchased from the Cell Signaling Technology (Boston, MA, United States). The antibody to SIRT1 (Cat #: ab189494) was purchased from Abcam (Cambridge, United Kingdom).

Adipose and liver tissues were lysed with RIPA lysis buffer (1 mM MgCl₂, 10 mM Tris-HCl at pH 7.4, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), and 1% Non-idet P40 cocktail). The proteins were separated by 5∼12% SDS polyacrylamide gel electrophoresis and transferred to cellulose membranes. The membranes were incubated overnight with the following primary antibodies: GAPDH (Proteintech, United States), ATGL (Proteintech, United States), PPARα (Sangon, Shanghai, China), FABP4 (Sangon, Shanghai, China), and CPT2 (Sangon, Shanghai, China). They were then immunoblotted with secondary antibody (Immunoway, United States). Immunoreactivity was visualized with enhanced chemiluminescence and analyzed with the Quantity One System (BioRad, United States) (Kim et al., 2020 (link); Liang et al., 2020 (link)).