Immepip

αTC1.6 cells were seeded into a 24-well plate at a density of 2.0 × 10⁵ cells per well and then preincubated in 500 μL of Krebs–Ringer bicarbonate buffer (KRB) containing 20 mM glucose for 30 min at 37 °C in 5% CO₂. Subsequently, αTC1.6 cells were incubated in 500 μL of KRB containing 20 or 2.8 mM glucose ± 1 μM histamine (Sigma), 1 and 100 μM of the selective H₃R agonist immepip (Sigma) [24,25] , or the selective H₃R inverse agonist JNJ-5207852 (kindly gifted from Dr. Nicholas Carruthers, Johnson & Johnson Pharmaceutical Research and Development, USA) [26,27] , or KRB containing 20 mM KCl ± 1 μM immepip, for 1 h at 37 °C in 5% CO₂. immepip and JNJ-5207852 were dissolved in distilled water. We used a glucagon ELISA kit (Wako) to measure glucagon concentration in KRB.

Animals were intraperitoneally injected with the H3R antagonist thioperamide (2, 5, or 10 mg/kg/day, Sigma-Aldrich, USA) within 1 h of and then once per day after TBI. The H1R antagonist pyrilamine (10 mg/kg/day, Sigma-Aldrich, USA) and H2R antagonist cimetidine (10 mg/kg/day, Sigma-Aldrich, USA) were intraperitoneally injected 15 min before treatment with thioperamide. α-FMH (5 μg/day in saline, intracerebroventricularly [i.c.v.]) (Provensi et al., 2014 (link)) or the H3R agonist immepip (1 μg/day in saline, Sigma-Aldrich, USA, i.c.v.) was given at 15 min before treatment with thioperamide.