Hyperinsulinemic–euglycemic clamp in mice

Male FGF21KO and WT mice after rmFGF21 treatment or fat transplantation were studied by hyperinsulinemic–euglycemic clamp to assess GIR, endogenous glucose production and insulin-stimulated glucose uptake. Mice were catheterized in the right internal jugular vein 3–4 days prior to clamp experiment. After fasting for 6 h, [3-³H]glucose (PerkinElmer) was infused at 0.05 μCi min⁻¹ for 2 h of the pretreatment period and blood samples were collected at the end. Then, the infusion of [3-³H]glucose was continued for 2 h of clamp period at 0.1 μCi min⁻¹ together with a bonus infusion of 300 mU kg⁻¹ human insulin (Novolin R, Novo Nordisk) and then a continuous infusion of insulin at a rate of 6 mU kg⁻¹ min⁻¹ to raise plasma insulin within a physiological range. Plasma glucose concentration was measured at 10 min intervals, and 20% glucose was infused at variable rates to maintain plasma glucose at basal concentrations. Plasma glucose values were maintained stable during clamp experiments (Supplementary Fig. ^9b). At 120 min of clamp period, a bonus of 10 μCi 2-deoxy-D-[1-¹⁴C] glucose (2-[¹⁴C]DG) (PerkinElmer) was administered. After 35 min, the mice were killed and tissues (SAT, VAT, muscle and BAT) were taken for glucose uptake analysis. Basal and insulin-stimulated endogenous glucose production was estimated with glucose concentration and [3-³H]glucose level during the pretreatment and the clamp period. Individual glucose uptake for each tissue was estimated with 2-[¹⁴C]DG concentration in plasma and tissue sample. This protocol has been adapted from established protocol⁶¹ and our previous studies^62,63.