Nph insulin

NPH insulin (Novo Nordisk A/S) was used for daily treatment in diabetic rats. For in vivo experiments, human insulin (Novo Nordisk A/S), and a long‐acting glucagon‐analogue (Novo Nordisk A/S) were used. Both were formulated in 5 mmol/L phosphate, 140 mmol/L NaCl, 70 ppm polysorbate‐20, pH 7.4. For in vitro experiments human insulin (Novo Nordisk internal reference solution) and native glucagon (Novo Nordisk A/S) dissolved in 20% H₂O and 80% DMSO were used.

Diabetes was induced in NOD.scid mice by single intraperitoneal (IP) injection of STZ at 180 mg/kg body weight. One week after STZ treatment, nonfasting blood glucose levels were measured daily at 8.30–11.00 AM in tail vein blood using a Bayer Contour™ Glucometer (Bayer HealthCare, Tarrytown, NY, USA). Diabetes was diagnosed when blood glucose was >400 mg/dL (22.2 mmol/L) for two consecutive days. These mice were diabetic for at least two weeks before transplantation and were treated with 0.5 U Novolin R and 0.5 U NPH insulin (Novo Nordisk, Copenhagen, Denmark) daily. Normoglycaemia after transplantation was defined as the nonfasting blood glucose level in recipient mice <200 mg/dL for two consecutive days and thereafter.

Adult male Munich-Wistar rats obtained from a local facility, with initial body weights (BW) ranging from 230 to 250 g, were used in this study. Rats received a single i.v. injection of STZ (Sigma Chemical, St. Louis, MO, United States), 65 mg/Kg. DM was confirmed 2 days later by reflectometric measurement of blood glucose concentration (BG) in tail blood samples. All diabetic rats received evening injections of NPH insulin (Novo Nordisk, Kalundborg, Denmark), individually adjusted to maintain BG between 350 and 450 mg/dL, in order to model the usual clinical scenario, in which control of blood glucose levels is imperfect, rather than non-existent. Daily insulin doses ranged from 1 to 4 units/rat. Non-diabetic rats matched for initial age and BW, and given no pharmacological treatment, were used as controls (C). All rats had free access to tap water and standard rodent chow containing 0.5% Na and 22% protein (Nuvital Labs, Curitiba, Brazil), and were kept at 22 ± 1°C and 60 ± 5% relative air humidity under an artificial 12:12-h light–dark cycle. All experimental procedures were specifically approved by the local Research Ethics Committee (CAPPesq, process no. 034/15) and followed strictly international standards for manipulation and care of laboratory animals.