Sea044ra

Rats (n = 10 per group, half male and half female) that had fasted for 8 h received acute or chronic intraperitoneal injections of different doses of RFRP-3 as described above. Blood glucose was immediately measured using a blood glucose meter (FreeStyle Optium Neo, Abbott, CN) from tail vein blood samples at specific time points (15 min prior to and 0–120 min after an injection of RFRP-3). Fifteen minutes after injection, serum insulin, glucagon, epinephrine, leptin, and growth hormone levels were assessed by enzyme-linked immunosorbent assay (catalog No. CEA448Ra, CEB266Ra, CEA858Ge, SEA084Ra, and SEA044Ra; Cloud-Clone Corp, CN). The enzyme-linked immunosorbent assay sensitivity of insulin, glucagon, epinephrine, leptin and growth hormone was 50.2, 7.44, 8.92 pg/ml, 0.129 and 0.055 ng/ml, separately.

Growth hormone was assessed from the primary culture supernatant using electro-chemiluminiscence immunoassay (COBAS, e601, Roche-Hitachi, USA) with lowest detection limit of 0.03 ng/ml and the inter and intra-assay coefficient of variation was < 2.5%. The human growth hormone differs structurally from the rat growth hormone. GH from GH3 cell line supernatant was measured using ELISA (Cloud-Clone Corp, SEA044Ra, Hubei, China).