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

Abstract

Pseudin-2 is a cationic alpha-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 beta-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,Lys14] and [Lys3,Lys10,Lys14,Lys21] analogues lacked insulinotropic action and the more hydrophobic [Phe16] analogue was cytotoxic at concentrations > or =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 [Lys18]-pseudin-2 as a peptide that may have potential for development as a therapeutically valuable insulinotropic agent for the treatment of type 2 diabetes.

Structure-Activity Relationships of Peptide Antibiotics with Improved Bacterial Cell Selectivity of Pseudin

“…6 The molecular mass of peptide was determined by matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (Shimadzu, Kyoto, Japan). .…”

“…Minimum inhibitory concentrations (MICs) of Ps-4K-P peptide against these bacteria were determined using a broth microdilution assay and compared with those of pseudin analogues as described in our previous report. 6 Hemolytic activity-Hemolytic activity of the peptides was tested against human red blood cells (hRBCs) as described previously. 7 Controls for no hemolysis (blank) and 100% hemolysis consisted of human red blood cells suspended in PBS and 0.1% Triton-X 100, respectively.…”

“…We designed Ps-P analogues with substitution of Gly 11 with Pro as well as Ps-K14-K18-P with further substitution of E14 and L18 with Lys to increase bacterial cell selectivity. 6 These Ps analogues exhibited reduced cytotoxicity but antimicrobial activity was also decreased. In this study, we designed new analogue, Ps-4K-P with four Lys substitution for Glu, Gln, and Leu, retaining high antibacterial activity with bacterial cell selectivity.…”

“…5 We have determined the tertiary structure of Pseudin-2 (GLNALKKVFQGIHEAIKLINNHVQ) and it revealed that pseudin has a linear α-helix from Leu 2 to Glu24. 6 Since pseudin has high cytotoxicity against mammalian cells, we tried to design more potent peptides. We designed Ps-P analogues with substitution of Gly 11 with Pro as well as Ps-K14-K18-P with further substitution of E14 and L18 with Lys to increase bacterial cell selectivity.…”

Antimicrobial peptides from amphibians

“…Ocellatin L2 and plasticin-L1 are devoid of antimicrobial activity but both have the ability to release insulin from BRIN-BD11 cells (101). Pseudin-2 isolated from the skin of the paradoxical frog Pseudis paradoxa is a cationic and a-helical peptide that can stimulate insulin release from the BRIN-BD11 clonal b cell line without hemolytic activity (102). Exendin-4 isolated from venom of a reptile has been used as therapeutic agent for type 2 diabetes (103,104).…”