Endocrine Abstracts

Aim: Our previous studies indicated the potential antidiabetic actions of the amphibian host-defence peptide, tigerinin-1. Tigerinin-1R was conjugated with gastric inhibitory polypeptide (GIP) as part of strategies for further development of its therapeutic effects. This study characterises the insulin-releasing and cytotoxic properties of the resulting hybrid peptide.

Method: Pure G-TGN was commercially synthesized G-TGN. Insulin releasing effects were assessed using BRIN-BD11 cells incubated with graded concentrations (0 – 3 µM) of the peptide. Cytotoxicity and effect of G-TGN on cell viability were assessed by LDH and MTT assays respectively. Actions of G-TGN on erythrocyte haemolysis, in the presence of increasing glucose concentration, on intracellular calcium level and membrane depolarisation, and on glucose tolerance in diet-induced diabetic mice were investigated.

Results: G-TGN stimulated non-toxic insulin secretion at concentrations ≥10 nM (P<0.05 to P<0.001). At 3 µM, G-TGN was more potent than native tigerinin-1R (1.5-fold, P<0.01) and GIP (1.3-fold, P<0.05). G-TGN did not affect cell viability nor cause erythrocyte lysis. Insulin-releasing effect of G-TGN was glucose dependent (1.1 mM to 5.6 mM, 1.3-fold, P<0.05 and 5.6 mM to 16.7 mM, 1.7-fold, P<0.01) and increase in incubations containing KCl (30 mM, 3.6-fold, P<0.001) and tolbutamide (200 µM, 2.4-fold, P<0.01). Verapamil (50 nM, 23%, P<0.05), diazoxide (300 µM, 27%, P<0.05) and absence of calcium (19%, P<0.05) reduced the effects of G-TGN. G-TGN enhanced intracellular calcium (23%, P<0.01) and increased membrane depolarisation (19%, P<0.05), and improved glucose tolerance in vivo (versus tigerinin-1R, 19%, P<0.05; versus GIP, 13%, P<0.05).

Conclusion: Hybridization of tigerinin-1R with GIP enhanced its potential and encourages further development of its therapeutic potentials.