Brevinin-1 and multiple insulin-releasing peptides in the skin of the frog Rana palustris L Marenah, P R Flatt, D F Orr, S M c Clean, C Shaw and Y H A Abdel-Wahab School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, N Ireland BT52 1SA, UK (Requests for offprints should be addressed to Dr L Marenah; Email: l.marenah@ulster.ac.uk) Abstract Few studies have comprehensively examined amphibian granular gland secretions for novel insulinotropic peptides. This study involved isolation and characterisation of bio- logically active peptides from the skin secretions of Rana palustris frogs. Crude secretions obtained by mild electrical stimulation from the dorsal skin surface were purified by reversed-phase HPLC on a semipreparative Vydac C18 column, yielding 80 fractions. These fractions were assayed for insulin-releasing activity using glucose- responsive BRIN-BD11 cells. Acute 20 min incubations were performed in Krebs Ringer bicarbonate buffer sup- plemented with 5·6 mmol/l glucose in the absence (con- trol) and presence of various fractions. Fractions 29–54 and fractions 68–75 showed significant 2·0–6·5-fold increases in insulin-releasing activity (P ,0·001). The fractions showing most prominent insulinotropic activity were further purified to single homogeneous peaks, which, on testing, evoked 1·5–2·8-fold increases in insulin release (P ,0·001). The structures of the purified peptides were determined by mass spectrometry and N-terminal amino acid sequencing. Electrospray ionisation ion-trap mass spectrometry analysis revealed molecular masses of 2873·5–8560·4 Da. Sufficient material was isolated to determine the primary amino acid sequence of the 2873·5 Da peptide, revealing a 27 amino acid sequence, ALSILRGLEKLAKMGIALTNCKATKKC, repressing palustrin-1c. The database search for this peptide showed a 48% homology with brevinin-1, an antimicrobial peptide isolated from various Rana species, which itself stimulated insulin release from BRIN-BD11 cells in a concentration- dependent manner. In conclusion, the skin secretions of R. palustris frogs contain a novel class of peptides with insulin-releasing activity that merit further investigation. Journal of Endocrinology (2004) 181, 347–354 Introduction Amphibian skin secretions are a rich source of biologically active peptides which participate in mechanisms of frogs and toads to defend against microbial infection and being eaten by predators (Bevins & Zasloff 1990). These peptides generally belong to families of bioactive peptides, which through evolution have given rise to counter- parts in mammals, such as bradykinins (Chen et al. 2002), caerulein/cholecystokinin (Anastasi et al. 1968), bombesin/gastrin-releasing peptide (Anastasi et al. 1972), exendin-4/glucagon like peptide-1 (Chen & Drucker 1997), tachykinins (Falconieri Erspamer et al. 1984), calcitonin gene-related peptide (Seon et al. 2000), and opioids and neuropeptides (Broccarda et al. 1981). Frogs of the genus Rana represent an extremely diverse and widely distributed group with 250 estimated species worldwide, including 36 species that have been identified in North America (Duellman & Trueb 1994). Previous work on the skin secretions of different Rana species has led to the characterisation of various antimicrobial peptides (Clark et al. 1994, Goraya et al. 1998, 2000). Most of these peptides are cationic in nature and have the potential to form amphipathic -helices either in aqueous solution or upon interaction with the lipid bilayers of bacterial cell membranes (Hancock et al. 1995, Hancock & Lehrer 1998). As well as antimicrobial peptides, the skin secretions of Rana species have yielded peptides that are either identical or structurally related to peptides syn- thesised in neuroendocrine tissues of mammals (Erspamer et al. 1986, Roseghini et al. 1988, 1989, Basir et al. 2000). The pickerel frog Rana palustris is widely distributed from eastern Texas to South Carolina and to south-eastern Canada (Hardy & Raymond 1991, Moler 1992). These frogs commonly inhabit cool, wooded streams and springs, although they are also found in many other habitats (Conant & Collins 1998). The body of R. palustris varies in colour from yellow green to light bronze. These frogs produce toxic skin secretions that are irritating to humans and can be fatal to some predators such as snakes and birds (Duellman & Trueb 1994). This study describes the bioassay-led isolation, purification and structural 347 Journal of Endocrinology (2004) 181, 347–354 0022–0795/04/0181–347  2004 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology.org