Lispro

Chemicals such as acetonitrile, glacial acetic acid, dimethylsulfoxide, formic acid, methanol, ammonium hydroxide, porcine insulin, bovine insulin, and [[13C6] Leu26, 30]-C-peptide (human) were obtained from Sigma (Schnelldorf, Germany). All aqueous buffers and solutions were prepared in purified water (MilliQ quality, Frankfurt, Germany). Recombinant human insulin was obtained from Aventis (Frankfurt, Germany). The labeled insulin (internal) standard [[2H10] LeuB6, B11, B15, B17]-insulin (human) was purchased from PeptaNova (Sandhausen, Germany). The used solid-phase extraction cartridges OASIS MCX (30 mg, 3 mL) were from Waters (Eschborn, Germany), and the synthetic insulin analogs lispro (Humalog), aspart (Novorapid), glulisine (Apidra), and insulin degludec (Tresiba) were supplied by Eli Lilly (Indianapolis, IN, USA), Novo Nordisk (Princeton, NJ, USA), and Aventis (Kansas City, MO, USA), respectively. The glargine metabolite (DesB31-32 glargine) was obtained from IBA (Warsaw, Poland).

Procedures were established to assess the hormonal responses to the sudden transition from hyperglycemia to hypoglycemia. At 120 minutes during the hyperglycemic clamp, a portion of the ferrets were continued on hyperglycemic infusion, whereas another portion of the ferrets underwent a hypoglycemic phase. In this group of ferrets, the glucose infusion was discontinued at 120 minutes and a continuous infusion of 20 mU/kg/min insulin lispro (Eli Lilly) initiated with an additional 50 mU/kg infused during the initial 5 minutes. insulin lispro does not cross-react with several assays that detect ferret insulin, as detailed in Results. As hypoglycemia ensued, the variable dextrose infusion was resumed with the goal of clamping the blood glucose at 45 mg/dL. The glycemic goal was based on extrapolation from human studies [29] (link) as a glucose level likely to influence islet secretion but avoid hypoglycemia severe enough to risk ferret morbidity. Blood samples (100 µl) were obtained at 120, 125, 130, 150 and 180 min for determination of plasma hormonal concentrations including ferret insulin.