Insulin-releasing properties of the frog skin peptide pseudin-2 and its [Lys¹⁸]-substituted analogue

Pseudin-2 is a cationic α-helical peptide that was first isolated from the skin of the paradoxical frog Pseudis paradoxa on the basis of its antimicrobial activity. We have investigated the insulin-releasing properties and cytotoxicity of the peptide, together with selected analogues with increased cationicity and hydrophobicity. At concentrations in the range 10 ^-9-10^-6 m, pseudin-2, and its [Lys¹⁸], [Phe⁸], and [d-Lys³,d-Lys¹⁰,d-Lys¹⁴] derivatives, stimulated insulin release from the BRIN-BD11 clonal β-cell line without increasing release of lactate dehydrogenase. The [Lys¹⁸] analogue was the most potent (46% increase in insulin release at 10 ^-9 m) and the most effective (215% increase in insulin release at 10^-6 m). The more cationic [Lys³,Lys¹⁰,Lys ¹⁴] and [Lys³,Lys¹⁰,Lys¹⁴,Lys ²¹] analogues lacked insulinotropic action and the more hydrophobic [Phe¹⁶] analogue was cytotoxic at concentrations ≥10^-7 m. Pseudin-2 and [Lys¹⁸]-pseudin-2 had no effect on intracellular calcium concentrations and stimulated insulin release in the absence of external calcium. [Lys¹⁸]-pseudin-2 (10^-8 m) stimulated insulin release in the presence of diazoxide and verapamil. Our results demonstrate that pseudin-2 stimulates insulin secretion from BRIN-BD11 cells by a mechanism involving Ca²⁺-independent pathways and identify [Lys ¹⁸]-pseudin-2 as a peptide that may have potential for development as a therapeutically valuable insulinotropic agent for the treatment of type 2 diabetes.