Clamps were performed in conscious unrestrained animals as previously described (Berglund et al., 2009; Berglund et al., 2012; Holland et al., 2011). Clamps in DIO mice were initiated by primed continuous infusion of insulin (4mU/kg/min) and glucose was maintained constant at ~150 mg/dL during the clamped state via variable infusion of dextrose (50%). Clamps in Lepob/ob mice were initiated by primed continuous infusion of insulin (10mU/kg/min) and glucose was maintained constant at ~250 mg/dL during the clamped state. FGF21 or PBS was initiated with the onset of 3H-glucose tracer infusion (5 μCi bolus + 0.05 μCi/min) to allow for a 90 minute-pretreatment. Continuous infusion of FGF21 was maintained after initiating insulin. [3-3H]glucose was increased to 0.1 μCi/min at the onset of insulin delivery to account for changes in specific activity. Glucose turnover was determined and hepatic glucose output was calculated as whole body glucose disposal less glucose infusion rate as previously described (Hill et al., 2010). To generate samples for the analysis of insulin signaling intermediates FGF21 or PBS was infused identically, and insulin stimulation was allowed to proceed for 15 minutes prior to sacrifice and rapid freezing of tissues.
