Skin secretions of Rana saharica frogs reveal antimicrobial peptides esculentins-1 and -1B and brevinins-1E and -2EC with novel insulin releasing activity

Abstract

Skin secretions of Rana saharica were evaluated for the isolation and characterisation of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reverse phase HPLC yielding 80 fractions. In acute 20-min incubations with glucose responsive BRIN-BD11 cells, fractions 36–43, 46–54 and 57–63 significantly stimulated insulin release by 2- to 8-fold compared with 5.6 mM glucose alone. Pooled fractions in the latter two bands were rechromatographed to reveal 9 homogenous peaks, which elicited significant 1.3- to 3.5-fold increases in insulin release (P < 0.05). Structural analysis of the most potent non-toxic peptides was performed by mass spectrometry and automated Edman degradation. This revealed four major insulin-releasing peaks with molecular masses of 2676.9 Da, 3519.3 Da, 4920.4 Da and 4801.2 Da respectively. These peptides were found to be identical to brevinin-1E, brevinin-2EC, esculentin-1 and esculentin-1B, which belong to the group of antimicrobial peptides isolated from skin secretions of various Rana frog species. Preliminary studies on the mechanism underlying the insulinotropic actions of esculentins-1 and -1B suggested possible involvement of both cyclic AMP–protein kinase A and –C-dependent G-protein sensitive pathways. These data indicate that the skin secretions of Rana saharica frogs contain bioactive molecules with significant insulin-releasing activity. Relatives of the brevinin/esculentin peptide family merit further investigation as novel insulin secretagogues.