Amphibian skin secretions are considered to be one of the richest resources of bioactive molecules in the animal kingdom for pharmaceutical prospecting. This thesis has investigated amphibian skin peptides of eight different species of anurans (frogs and toads) for insulin-releasing and anti-diabetic properties. The skins of Pseudis paradoxa, Hylarana guntheri, Hylomantis lemur and Leptodactylus laticeps, were tested for the presence of peptides with stimulatory effects on insulin-release from the clonal BRIN-BDll cell line. Upon purification of the skin secretions/extract, multiple insulin-releasing peptides were isolated and fully characterised by mass spectrometry along with N-terminal amino acid sequencing by Edman degradation. A number of synthetic peptides demonstrated potent in vitro biological activities, possessing the ability to stimulate insulin release up to a concentration of 3 flM without any association of cellular toxicity. For example, brevinin-2GUb isolated from the skin extract of H. guntheri stimulated insulin release 3.7 fold compared to 5.6 mmol/l glucose control (P<O.OOI). Under similar experimental conditions, the insulin output of several synthetic peptides identified, were akin to the well-characterised incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-l (GLP-I). Structure/function studies presented suggest the insulin-releasing potency of a peptide was related to charge and hydrophobicity. Mechanistic studies on the following synthetic peptides; pseudin-2 (ps-2), [Lys-18]_ps_2, [Phe 8]_ps_2, brevinin-2GUb, phylloseptin-L2, and [Asp23]ocellatin-Ll suggest that their stimulatory effects on insulin secretion were mediated via calcium independent pathways. Additionally, when tested in vivo each of these peptides, displayed valuable anti-diabetic properties, lowering blood glucose and increasing insulin following an intraperitoneal glucose load (18 mmol/kg). The skin extract of Rana luteiventris has previously been established as a rich source of antimicrobial peptides. Fourteen purified fractions with varying degrees of antimicrobial activity were tested for their ability to stimulate insulin-release. Serial dilutions gave an insight to the potency of the peptides. Multiple insulin- releasing non-toxic peptides belonging to ranatuerin, brevinin, temporin and esculentin families were identified. Mechanistic studies indicated that, ranatuerin- 2Lb, esculentin-2L, brevinin-1Lb, and 3 unclassified peptides stimulated insulin release via Ca2+ independent pathways. In contrast, bradykinin, ternporin-I La and temporin-l Lb appeared to stimulate insulin secretion via a calcium dependent mechanism. The most effective insulinotropic peptide identified from the skin extract of R. luteiventris was esculentin-2L. Skin extracts of Bufo arabicus, Buergeria buergeri and Hoplobatrachus rugulosus were also investigated. Following reverse-phase HPLC purification, a number of non-toxic insulin-releasing peptides were identified. This included peptides isolated from the skin of B. arabicus and B. buergeri as well as two from H. rugulosus (Molecular mass; 745.1, 1758.3, 1305.8 and 1368.5 Da, respectively). In conclusion, this work has identified novel peptides from the skins of various amphibians with stimulatory effects on insulin release and, in some cases, promising glucose lowering effects in vivo. Such an approach into nature's untapped resources may ultimately offer novel agents with valuable anti-diabetic properties for the future treatment of type 2 diabetes.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:554281 |
| Date | January 2009 |
| Creators | Power, Gavin Jude |
| Publisher | University of Ulster |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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