Antsense 3

BALB/c strain of Rag-2/Jak3 double-deficient (BRJ) mice⁸ were used. WT BRJ or Kuma (Ins2^+/Q104del) BRJ mice were housed and monitored in our animal research facility according to institutional guidelines in Kumamoto University or Tokyo Institute of Technology. All methods were performed following the institutional guidelines in Kumamoto University or Tokyo Institute of Technology. Kuma mouse is maintained either by crossing heterozygous females with wild type male BRJ mice (only the early generations) or by in vitro fertilization.
Mice were allowed free access to food and water except when being fasted. Genotyping was performed using the genomic polymerase chain reaction (PCR) analysis of the Insulin (Ins) 2 gene using the primer pair indicated in Fig. 1C. The amplified PCR fragments were subjected to Pvu II restriction enzyme cut. Mutant mice were identified to be resistant for Pvu II. Fasting blood glucose levels and body weights were measured from 2 weeks of age onwards in mice that had been fasted for 2 h using a portable glucose monitor (Life check sensor, Gunze, Tokyo, Japan) or blood glucose meter ANTSENSE III (Horiba, Kyoto Japan), by making an incision in the tail.

In this study, diabetes was induced as reported previously [22 (link)]. Briefly, rats were injected with STZ dissolved in 0.03 mol/L citrate buffer at pH 4.5, which was used at a dose of 50 mg/kg (i.v.). Age-matched non-diabetic rats were injected with vehicle alone. After 1 week, non-fasting serum glucose levels were measured using Antsense III (Horiba, Kyoto, Japan), and rats with values above 300 mg/dL were considered diabetic. Rats were assigned based on body weight (BW) and blood glucose level. The rats that did not meet the experimental criterion were euthanized using the method of inhalation of carbon dioxide (CO₂).

Blood samples were taken from the tail vein for blood glucose measurement. BG (mg/dl) was measured using an automatic analyzer (Antsense III; Horiba, Kyoto, Japan) at the beginning (20 weeks of age) and end of the experiment. Changes in BG levels were compared among and within the groups at 2 or 6 weeks.

Mice were fasted overnight (16 h) and orally administered glucose solution (2 g/kg body weight) for the analysis of glucose tolerance. Fed and fasting blood glucose levels were measured through the tail blood using Antsense III (HORIBA, Kyoto, Japan) between 9:00 and 11:00 a.m. The levels of HbA_1c in whole blood, glycated serum proteins in serum, and 3-hydroxybutyric acid in plasma were determined using Direct Enzymatic HbA_1c Assay (Diazyme Laboratories, Poway, CA, USA), Glycated Serum Protein Assay (Diazyme Laboratories), and Autokit 3-HB (FUJIFILM Wako, Osaka, Japan), respectively, according to the manufacturers' instructions. To determine insulin levels, insulin was extracted from the frozen pancreas using 70% (v/v) ethanol containing 0.18 N HCl. The neutralized samples were quantified using the insulin high-range HTRF kit (PerkinElmer—Cisbio, Waltham, MA, USA) according to the manufacturer's instructions.