Endocrine Abstracts

Aim: Previous reports indicate therapeutic potentials of tigerinin-1R, isolated from the skin secretions of Hoplobatrachus rugulosus, as an antidiabetic agent. Despite the report of its insulin-releasing effects, the peptide’s mechanism of actions is poorly understood. This study investigates the impact of conjugation of tigerinin-1R with (dAla2)-GIP on the cellular uptake and anti-diabetic actions of the peptide.

Method: Amphipathicity, net charge and theoretical isoelectric point of the hybrid peptide were evaluated. Insulin-releasing effects of the hybrid (0 –3µ M) were investigated using BRIN-BD11. Cellular uptake, cytotoxicity and cell viability were assessed Effects of the peptide on erythrocyte haemolysis, glucose stimulated insulin-secretion, intracellular calcium concentration, membrane depolarisation, and glucose tolerance in high-fat fed mice were assessed.

Result: The conjugation of tigerinin-1R with (dAla2) GIP reduced the net charge from +1 to -1.9 and the theoretical pI reduced from 8.3 to 4.4. No significant change in amphipathicity was observed. The hybrid peptide stimulated non-toxic concentration-dependent insulin secretion at concentrations ≥10nM (P<0.05 to P<0.001). At 3µ M, the stimulatory effect of G-TGN was higher than that of tigerinin-1R (1.5-fold, P<0.01) and GIP (1.3-fold, P<0.05). The hybridization of GIP and tigerinin-1R did not affect cell viability nor cause erythrocyte haemolysis. Effects of G-TGN increased with increasing glucose concentration (1.1mM to 5.6mM, 1.3-fold, P<0.05 and 5.6mM to 16.7mM, 1.7-fold, P<0.01) and in the presence of KCl (30mM, 3.6-fold, P<0.001) and tolbutamide (200µ M, 2.4-fold, P<0.01). Verapamil (50nM, 23%, P<0.05), diazoxide (300µ M, 27%, P<0.05) and removal of extracellular calcium (19%, P<0.05) inhibited but not abolish the effects of G-TGN. The peptide enhanced intracellular calcium (23%, P<0.01) and increased membrane depolarisation (19%, P<0.05). Improvement in glucose tolerance in mice receiving G-TGN was higher compared with tigerinin-1R (19%, P<0.05) and GIP (13%, P<0.05). Cellular uptake of G-TGN increased by 3.6-fold compared to tigerinin-1R.

Conclusion: The conjugation of (dAla2) GIP with tigerinin-1R significantly enhanced its potential and encourages its development as a novel anti-diabetic drug.