Analyses of insulin-potentiating fragments of human growth hormone by computative simulation; essential unit for insulin-Involved biological responses

Abstract

We analyzed the structural features of insulin-potentiating fragments of human growth hormone by computative simulations. The peptides were designated from the N-terminus sequences of the hormone positions at 1–15 (hGH1–15; H2N-Phe1-Pro2-Thr3-Ile4-Pro5-Leu6-Ser7-Arg8-Leu9-Phe10-Asp11-Asn12-Ala13-Met14-Leu15-COOH), 6–13 (hGH6–13), 7–13 (hGH7–13) and 8–13 (hGH8–13), which enhanced insulin-producing hypoglycemia. In these peptide molecules, ionic bonds were predicted to form between 8th-arginyl residue and 11th-aspartic residue, and this intramolecular interaction caused the formation of a macrocyclic structure containing a tetrapeptide Arg8-Leu9-Phe10-Asp11. The peptide positions at 6–10 (hGH6–10), 9–13 (hGH9–13) and 10–13 (hGH10–13) did not lead to a macrocyclic formation in the molecules, and had no effect on the insulin action. Although β-Ala13hGH1–15, in which the 13th-alanine was replaced by a β-alanyl residue, had no effect on insulin-producing hypoglycemia, the macrocyclic region (Arg8-Leu9-Phe10-Asp11) was observed by the computative simulation. An isothermal vibration analysis of both of β-Ala13hGH1–15 and hGH1–15 peptide suggested that β-Ala13hGH1–15 molecule was more flexible than hGH1–15; C-terminal carboxyl group of Leu15 easily accessed to Arg8 and inhibited the ionic bond formation between Arg8 and Asp11 in β-Ala13hGH1–15. The peptide of hGH8–13 dose-dependently enhanced the insulin-involved fatty acid synthesis in rat white adipocytes, and stabilized the C6-NBD-PC (1-acyl-2-[6-[(7-nitro-2,1,3benzoxadiazol-4-yl)amino]-caproyl]-sn-glycero-3-phosphatidylcholine) model membranes. In contrast, hGH9–13 had no effect both on the fatty acid synthesis and the membrane stability. In the same culture conditions as the fatty acid synthesis assay, hGH8–13 had no effect on the transcript levels of glucose transporter isoforms (GLUT 1, 4) and hexokinase isozymes (HK I, II) in rat white adipocytes. Judging from these results we considered that the macrocyclic structure in human growth hormonal peptides is regarded with the modification of insulin action, and hGH8–13 is an essential sequence for the modification of insulin action. This hGH8–13 peptide modifies the insulin action via stabilizing the cell membrane, and does not directly act on the insulin-involved glucose metabolism.