Gago glucose oxidase kit

HGP was measured, essentially as previously described,25 using mouse primary hepatocytes. Viable cells were allowed to settle for 4‐6 hours before serum starvation in M199 media containing dexamethasone (100 nM). Following overnight serum free conditions, cells were washed once in PBS before incubation for 8 hours in glucose‐free DMEM (Invitrogen, Paisley, UK) containing lactate (10 mM), pyruvate (1 mM), dexamethasone (100 nM) plus stimuli, as indicated in the Figure legends. Media was harvested for measurement of glucose and hepatocytes were lysed. The amount of glucose present in the media was measured using a GAGO glucose oxidase kit (Sigma). Glucose assays were performed in a 96‐well plate format. 50 μL of cell culture media was incubated with 100 μL of glucose oxidase/peroxidase assay reagent for 30 minutes at 37°C before the reaction was stopped by addition of 100 μL 12 N H₂SO₄. Absorbance was measured at 405 nm and the amount of glucose present was determined using a glucose standard curve generated in the same assay. The final glucose concentration was normalized to total protein content per well and data are presented as mg HGP per μg protein.

AICAR and 4‐Nitrobenzylthioinosine (NBMPR) were purchased from Tocris (Abingdon, UK). Dibutyryl cAMP, uridine and metformin were purchased from Sigma (Dorset, UK). LC‐MS materials were obtained from Fisher Scientific (Loughborough, UK) unless otherwise stated. Glucagon was obtained from Novo Nordisk (Bagsvaerd, Denmark). 8CPT‐cAMP was purchased from Calbiochem (Nottingham, UK) and 8CPT‐2‐Methyl‐O‐cAMP (8CPT‐2MeO‐cAMP) was purchased from Biolog Life Sciences Institute (Bremen, Germany). Donkey anti‐goat 680RD, donkey anti‐mouse 680RD, donkey anti‐rabbit 800CW secondary antibodies were purchased from LI‐COR Biosciences, UK. The GAGO glucose oxidase kit was purchased from Sigma. [5,6‐³H] uridine was purchased from Perkin Elmer (Beaconsfield, UK). pAMPK (Thr172) and total ACC antibodies were purchased from Cell Signalling Technologies (Leiden, UK). Total AMPK was purchased from Abcam (Cambridge, UK) and actin was purchased from Proteintech (Manchester, UK). pACC was made in‐house by Division of Signal Transduction Therapy (University of Dundee).