Hyperinsulinemic-Euglycemic Clamp Studies

Participants returned to the CRC within 1 month of the screening visit on the evening prior to their clamp study. At 10:00 p.m., all participants began an overnight fast. Upon beginning the overnight fast, participants with type 1 diabetes received an intravenous infusion of regular human insulin according to the protocol of Goldberg et al. (28), modified for use in healthy patients with type 1 diabetes (see Supplementary Data). The protocol targeted a plasma glucose concentration of 90–120 mg/dL by the next morning. Patients taking long-acting basal insulin used either insulin glargine or detemir, which was last given 24 h prior to beginning the intravenous insulin infusion. Patients taking continuous subcutaneous insulin infusions suspended and disconnected their pumps 15–30 min before starting the intravenous insulin infusion. Participants with GCK-MODY required no overnight insulin, but CRC staff monitored glucose every 2 h overnight. Female participants were studied on day 2–10 of their menstrual cycle.

Each clamp study commenced at 7:30 a.m. Experiments consisted of a 90-min equilibration period for [6,6-²H₂]glucose tracer infusion, a 60-min basal sampling period, and then two consecutive, 150-min experimental periods (Fig. 1B).

Insulin was infused intravenously at 12 mU/m²/min in the first experimental period (period 1) and at 40 mU/m²/min in the second (period 2). These rates were chosen to partially suppress lipolysis and hepatic glucose production at the end of period 1 and to completely inhibit lipolysis and hepatic glucose production while near-maximally stimulating muscle glucose uptake in period 2. In both experimental periods, somatostatin and glucagon were infused intravenously at 60 ng/kg/min and 0.65 ng/kg/min, rates selected to ensure glucagon remained at basal levels and equal between participants. A glucose tracer solution was prepared by dissolving 2.19 g (12.0 mmol) of [6,6-²H₂]glucose (Cambridge Isotope Laboratories, Tewksbury, MA) in 60 mL of isotonic saline. Participants received a [6,6-²H₂]glucose priming dose of 22 µmol/kg over the first 10 min of the equilibration period, followed by infusion at 0.22 µmol/kg/min through the end of the basal sampling period. This rate was lowered to 0.11 µmol/kg/min during period 1 and discontinued during period 2. A 0.5-mL aliquot of arterialized blood was drawn and centrifuged every 10 min during period 1 and every 5 min during period 2 to sample plasma glucose and adjust a 20% dextrose solution infusion to maintain plasma glucose between 95 and 100 mg/dL. The 20% dextrose solution was spiked with glucose tracer by adding 6.9 g (37.8 mmol) of [6,6-²H₂]glucose to 1,500 mL of stock 20% dextrose solution.

Research staff drew blood to assess metabolic and hormonal parameters three times during each of three 30-min steady-state sampling periods: during the basal sampling period and during the last 30 min of both 150-min experimental periods.