Ca test

Blood concentrations of lactate, catecholamines (including adrenaline, noradrenaline, and dopamine), cortisol, GH, IGF-1, and BDNF in the collected blood samples were analyzed. Biochemical analyses for all the indices except lactate were blindly conducted by SRL Inc. (Tokyo, Japan). Specifically, concentrations of catecholamines were measured using high-performance liquid chromatography (CA Test TOSOH; Tosoh Corporation, Tokyo, Japan), those of cortisol, GH, and IGF-1 were measured using electrochemiluminescence immunoassay (cortisol: Elecsys^® Cortisol; Roche Diagnostic Systems, Basel, Switzerland, GH: Elecsys^® hGH; Roche Diagnostic Systems, Basel, Switzerland, IGF-1: IGF-I IRMA DAIICHI; Fujirebio Inc., Tokyo, Japan), and the concentration of BDNF was measured using an enzyme-linked immunosorbent assay (Quantikine^TM ELISA Human Free BDNF Immunoassay; R&D Systems Inc., Minneapolis, MN, USA). As mentioned above, LA was measured at each blood collection using an enzyme electrode method (Lactate Pro™2 LT-1730; ARKRAY Inc., Kyoto, Japan).

Human regular insulin (Eli Lilly, Indianapolis, IN) was injected intravenously at a dose of 0.05–0.10 U/kg body weight. Blood samples for glucose and epinephrine were collected before the injection and at 15, 30, 45, 60, 90 and 120 min after the injection; for T-TAS blood samples were collected before the injection and at 15, 30, 60, 90 and 120 min; and for WBC blood samples were collected before the injection and at 30 and 60 min after the injection. For measurement of epinephrine, the CA Test 「TOSOH」 (Tosoh Corporation, Tokyo, Japan) and HPLC system (SHIMADZU CORPORATION, Kyoto, Japan) were used.