Hyperglycemic Clamp Assay for Insulin Secretion

One-step hyperglycemic clamps were performed on conscious animals as described [41] (link). A 20% dextrose solution was infused through the jugular vein to clamp plasma glucose at 18 mM for 100 min and was adjusted based on glucose measurements (Roche Accu-Check; Roche, Indianapolis, IN). At 90 min, an arginine bolus injection was performed (1 mmol/kg; Sandoz Canada) to assess the maximal insulin response. Plasma samples were collected from the tail at several time points during the clamp for insulin measurements using a mouse insulin enzyme-linked immunosorbent assay (ELISA) kit (Alpco Diagnostics, Salem, NH). Plasma samples for C-peptide measurements were collected at 75 min and analyzed using a mouse C-peptide ELISA kit (Alpco Diagnostics). Two-hour hyperinsulinemic-euglycemic clamps were performed in ASK1 wild-type and knockout mice as previously described [41] (link). Briefly, following a 1-min bolus insulin infusion (85 mU/kg; Humulin R), insulin was infused at 5 mU/kg/min. Twenty percent dextrose was infused starting 5 min after the insulin infusion to clamp glycemia at ∼6.5 mM. The insulin sensitivity index (M/I) was calculated as the glucose infusion rate (M) divided by the average insulinemia during the last 60 min of the clamp (I).