Investigation of insulinotropic and cytoprotective effects of selected amphibian skin peptides

Type 2 diabetes mellitus (T2DM) accounts for over 90% of all cases of diabetes. It is characterized by persistently high blood sugar levels, insulin resistance, and ongoing pancreatic β-cell failure, primarily due to oxidative stress and lipotoxicity. Current treatments face several issues, such as severe side effects, high costs, limited access in regions with higher disease rates, and the inability of existing drugs to control blood sugar effectively. The search for new diabetes treatments from natural sources has gained momentum, following the development of exendin-4 in the early 2000 derived from the saliva of the Gila monster (Heloderma suspectum), into a clinically available anti-diabetic drug. Previous studies have demonstrated the anti-diabetic effects of amphibian peptides such as tigerinin-1R, magainin-AM2, and alyteserin-2a. However, the mechanisms underlying their actions are not yet fully understood. Moreover, the protective effect of this study aims at the confirmation of the in vitro and in vivo actions of tigerinin-1R, magainin-AM2, and alyteserin-2a, as well as the investigation of the protective effects of these peptides on high glucose and lipid-induced oxidative stress in clonal pancreatic cells, BRIN-BD11, and microglial cells, BV2. The purity of commercially synthesised selected peptides was confirmed using reversed-phase HPLC. Insulin-releasing effects of graded concentrations of selected peptide (0 - 3μM) were studied in a 20 min acute test using BRIN-BD11 cells. For the investigation of mechanisms associated with peptides’ insulinotropic effects, BRIN-BD11 cells were incubated with and without known modulators of insulin secretion, in buffers containing increasing glucose concentrations (1.1 – 16.7mM) or in the absence of extracellular calcium. Insulin levels in cell supernatants were measured by ELISA. The release of lactate dehydrogenase in the supernatant of cells incubated with peptides was used as a marker of cytotoxicity, while cell viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Effects of peptides on membrane potential, intracellular calcium and on levels of markers of antioxidative status (catalase, total thiol and glutathione levels) in BRIN-BD11 and BV2 cells were also investigated. Furthermore, acute of all peptides and long-term effects of alyteserin-2a were analysed in mice with diet-induced obesity-diabetes, using liraglutide as the positive control. The results obtained revealed that all peptides are of acceptable purity levels. All peptides tested stimulated non-toxic insulin release at concentrations ≥0.01 μM in a dose-dependent manner. Alyeserin-2a produced the highest significant insulin secretion (3.5-fold, P<0.001, at 3 μM). Insulinotropic effects of all peptides tested increased with increasing glucose concentration and were reduced in the presence of diazoxide, verapamil, and in a calcium-free buffer. All peptides enhanced the membrane potential and increased intracellular calcium concentration. These results suggest the involvement of the KATP-dependent channel in the insulin-releasing effects of the tested peptides. Following the assessment of acute in vivo effects, all peptides improved glucose tolerance (28.0 %, P<0.001 for tigerinin-1R; 27 % P<0.001 for magainin-AM2; and 12.8%, P<0.01 for alyteserin-2a) and insulin secretion (1.5-fold, P<0.01 for tigerinin-1R; 1.4-fold, P<0.01 for magainin-AM2; and 1.2-fold, P<0.05 for alyteserin-2a. The assessment of the long-term effects of alyteserin-2a revealed improved glucose tolerance (36.7%, P<0.001) and insulin secretion (1.5-fold, P<0.05) in high-fat-fed mice after 15 days of treatment. Results obtained for the cytoprotective effects of the selected peptide on glucose-induced and lipid-induced toxicity showed that all the peptides inhibited the decrease of catalase activities, reduced levels of total thiols, and glutathione caused by high glucose and fatty acid in both BRIN-BD11 and BV2 cells. However, the exact mechanisms by which the peptides produce these effects are still not fully understood, highlighting the need for future research to clarify them. Altogether, these results indicate the potential of selected peptides as candidate agents that can be further developed for future clinical applications.

Citation

Edeani, J.N. (2026) Investigation of insulinotropic and cytoprotective effects of selected amphibian skin peptides. https://wlv.openrepository.com/handle/2436/626423

Publisher

University of Wolverhampton

URI

https://wlv.openrepository.com/handle/2436/626423

Type

Thesis or dissertation

Language

en

Description

A thesis presented for the degree of Doctor of Philosophy in the School of Sciences, Faculty of Science and Engineering, University of Wolverhampton.

Collections

Theses and Dissertations