Aprotinin

Necropsy was performed on day 41 of treatment. The rats were anesthetized with diethyl ether, and blood samples were collected from the portal vein, transferred into tubes containing a DPP4 inhibitor (Millipore, Billerica, MA, USA) and aprotinin (Wako Pure Chemicals, Osaka, Japan) on ice, and centrifuged at 4°C, 10,000 ×g, for 5 min to obtain plasma. The concentration of GLP-1 in plasma was measured using a GLP-1 ELISA kit (Linco Research Inc.).

Immunoprecipitation and small RNA isolation were performed as described previously^{48 (link)}. In brief, OSCs or BmN4 cells ware lysed in buffer (30 mm HEPES-KOH (pH 7.4), 500 mm NaCl, 150 mm KOAc, 5 mm Mg(OAc)₂, 5 mm DTT, 0.1% NP-40, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 0.5% aprotinin (Wako)), and then centrifuged. The supernatants were incubated with Dynabeads Protein G (Thermo Fisher Scientific) bound to an anti-DDDDK-tag mAb (MBL, FLA-1). The beads were washed twice with the buffer and twice with the buffer without 500 mm NaCl, and then treated with Proteinase K and phenol-chloroform. The liberated RNAs were precipitated with ethanol, dephosphorylated with Antarctic Phosphatase (NEB), and then radiolabeled with ³²P-γ-ATP (PerkinElmer) and T4 PNK (NEB).

Rats were given an i.v. injection of either GLP-1 (3 nmol/100 µl saline) or ghrelin (1.5 nmol/100 µl saline) into the right femoral vein, then 30 min later they were given the other peptide into the left femoral vein. Blood was withdrawn from the right jugular vein at:  1, 2.5, 5, 10, 20, 30, 32.5, 35, 40, 50, and 60 min after the first injection. Blood was collected into tubes containing 1.25 mg EDTA and aprotinin (500 kallikrein-inhibiting units/ml blood) (Wako Pure Chemical Industries), and all samples were centrifuged within 2 h of collection. For GLP-1 assay, DPP4 inhibitor (10 µl/ml) (Millipore) was added to blood immediately after collection. For ghrelin assay, 1 M HCl (100 µl/ml) was added to the plasma. All samples were stored at − 70 °C until analysis. GLP-1 was measured with a Glucagon-Like Peptide-1 (Active) ELISA kit (Millipore), and ghrelin in an automated enzyme immunoassay system (AIA-600 II Immunoassay Analyzer, TOSOH Bioscience).

After administration of BCE followed by IP glucose solution as described in Section 2.3, blood samples were collected from the portal vein under anesthesia (isoflurane) 15 and 30 min after glucose loading, or 45 and 60 min after vehicle or BCE administration. Blood was drawn into a syringe containing EDTA disodium salt (final concentration, 1 mg/ml; Dojindo, Kumamoto, Japan), aprotinin (final concentration, 500 KIU/ml; Wako), and dipeptidyl peptidase‐IV inhibitor (final concentration, 100 μmol/L; Diprotin A; Peptide Institute, Inc., Osaka, Japan) (Kato, Nakanishi et al., 2017; Kato, Nishikawa, et al., 2017; Nagamine et al., 2014). Samples were centrifuged at 1,600g for 15 min at 4°C, and plasma total GLP‐1 concentrations were measured using an ELISA (GLP‐1 Total ELISA kit, Millipore, St. Charles, MS) according to the manufacturer's instructions. At the same time (15 and 30 min after glucose loading), ilea (ileum is defined as a 25 cm section of the cecum) were removed, and intestinal contents were obtained by washing with 5 ml ice‐cold saline containing 1% trifluoroacetic acid (TFA). The intestinal contents were homogeneously mixed and immediately stored at −80°C until use.

After overnight fasting, we measured blood glucose before- and 15, 30, 60 and 120 minutes after D-glucose injection (2 mg/g body weight, intraperitoneally) by a Glucocard DIA meter (GT1641) (Arkray). To evaluate insulin release 15 min after glucose challenge, blood was centrifuged in the presence of aprotinin (100 kIE/ml blood; Wako Chemicals), and serum insulin was measured with a mouse insulin ELISA kit (U-type) (Shibayagi).