Quantitative Analysis of Metabolic Regulators

Real-time quantitative PCR was performed as described previously [26 (link)]. Forward and reverse primer pairs were: 5’-CACCTTGACACTACACCCTT-3′ and 5’-GTGGCT GTGAACACCTCT-3′ for glucose- 6- phosphatase (G6Pase); 5’-AGTCA CCATCAC TTCCTGGAAGA-3′ and 5’-GGTGCAGAATCGCGAGTT-3′ for phosphoenolpyruvate carboxykinase (PEPCK); 5′-AAGATGCCTCCTGTGACT-3′ and 5′-GATGACCGAAGTGCTTGT-3′ for proliferator-activated receptor γ co- activator 1α (PGC-1α); 5’-CCCTGAACCCTAAGGCCAACCGTGAAAA-3′ and 5’-TCTCCGGAGTCCATCACAATGCCTGTG- 3′ for β-actin. Protein analysis was performed with Western Blots as described previously [24 (link)]. Primary antibodies included anti-insulin receptor (InsR) (#3025)/ phospho-InsR (Tyr1150/1151, #3024), anti- AMP-dependent protein kinase (AMPK) (#2532)/ phosphor- AMPK (Thr172, #2531), and anti-AKT kinase (AKT) (#9272)/phospho-AKT (Ser473, #4060) (Cell Signaling Technology, Beverly, MA, USA); anti- PEPCK; and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA, sc-47778).