Plasma concentrations of free glycerol and triglycerides (Sigma), β-hydroxybutyrate (Stanbio Laboratory), and nonesterified fatty acid (Wako Diagnostics) were measured using commercial assay kits. Liver triglyceride was extracted and measured as previously described (38 (link)). Plasma insulin was measured using an ELISA kit (CrystalChem). Glucose and insulin tolerance tests were performed as previously described (39 (link)). For insulin signaling studies, mice were fed HFD for 8 weeks before receiving a single dose of intravenous injection of saline or insulin (1.5 units/kg). Tissues were rapidly dissected 10 min after injection for immunoblotting analyses.
For gene expression analysis, total RNA from WAT was extracted using a commercial kit from Invitrogen. Total RNA from other tissues and cultured cells was extracted using the TRIzol method. For qPCR analysis, equal amount of RNA was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase followed by qPCR reactions using SYBR Green (Life Technologies). Relative abundance of mRNA was normalized to ribosomal protein 36B4. Adipose tissue and liver gene expression was analyzed using specific primers (Supplementary Table 1 ). Statistical significance was determined by Student t test.
For gene expression analysis, total RNA from WAT was extracted using a commercial kit from Invitrogen. Total RNA from other tissues and cultured cells was extracted using the TRIzol method. For qPCR analysis, equal amount of RNA was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase followed by qPCR reactions using SYBR Green (Life Technologies). Relative abundance of mRNA was normalized to ribosomal protein 36B4. Adipose tissue and liver gene expression was analyzed using specific primers (
