inhibitor cocktail 16 (Roche, Laval, QC, Canada), Na3VO4 1 mM and NaF 1 mM) using a polytron homogenizer. Samples were centrifuged in a microfuge at 4uC, at full speed for 10 min and supernatants were collected and frozen at 280uC. A hundred fifty mg of proteins of each sample were loaded on a NuPAGE Bis-Tris 4– 12% gradient precast polyacylamide gel (Invitrogen, Burlington, ON, Canada), electrophoresed, blotted on a nitrocellulose mem- brane and probed overnight at 4uC with an antibody to phospho- AMPKa (Thr172) (cat. #: 2531, Cell Signalling Technology, Danvers, MA, USA). Membrane was stripped in stripping buffer (SDS 2%, Tris 62.5mM pH 6.8, b-mercaptoethanol 100 mM) at 55uC for 30 min, washed, blocked and reprobed overnight at 4uC with an antibody to AMPKa (23A3) (cat. #: 2603, Cell Signalling Technology, Danvers, MA, USA). The day after, the membrane was reprobed for an hour at room temperature with an antibody to a- tubulin (cat. #: 2125, Cell Signalling Technology, Danvers, MA, USA), and AMPK and a-tubulin signals were revealed together. Signals were quantitated by densitometry using the ImageJ software (NIH, Bethesda, MD, USA). Blood glucose and insulin measurements Two to three months old mice were fasted for 24 hours and blood was collected between 9am and 11am from the saphenous vein. For the refeeding experiments, mice were left at least one week to recover and were fasted again for 24 hours, refed between 9am and 11am and then, blood was collected 3 hours later. Blood glucose concentration was measured immediately when blood was collected using a One Touch II glucometer (LifeScan Canada Ltd., Burnaby, BC, Canada) and serum was frozen at 280uC. Serum insulin was measured using an ELISA kit (cat. #: EZRMI- 13K, Linco Research Inc., MO, USA), according to the manufacturer’s instructions. 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