Brevinin-1 and multiple insulin-releasing peptides in the skin of the frog Rana palustris

Abstract

Few studies have comprehensively examined amphibian granular gland secretions for novel insulinotropic peptides. This study involved isolation and characterisation of biologically 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 glucoseresponsive BRIN-BD11 cells. Acute 20 min incubations were performed in Krebs Ringer bicarbonate buffer supplemented with 5·6 mmol/l glucose in the absence (control) 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 concentrationdependent manner. In conclusion, the skin secretions of R. palustris frogs contain a novel class of peptides with insulin-releasing activity that merit further investigation.

Tigerinin-1R: a potent, non-toxic insulin-releasing peptide isolated from the skin of the Asian frog, Hoplobatrachus rugulosus

“…Recent studies have shown that several frog skin peptides that were first identified on the basis of their ability to inhibit the growth of bacteria also stimulate insulin release in vitro from BRIN‐BD11 rat clonal β ‐cells at low concentrations that are not cytotoxic to the cells. Such insulin‐releasing peptides have been identified in skin secretions and/or skin extracts from the species Hylarana güntheri [8], Lithobates catesbeianus [9], Lithobates palustris [10], Lithobates pipiens [11], Lithobates septentrionalis [12] and Pelophylax saharicus [13] belonging to the family Ranidae and from Hylomantis lemur [14] and Pseudis paradoxa [15] belonging to the family Hylidae. In addition, brevinin‐2GUb from H. güntheri [8], phylloseptin‐L2 from H. lemur [14] and brevinin‐2–related peptide (B2‐RP) from L. septentrionalis [12] have been shown to be active in vivo, increasing plasma insulin concentrations and improving glucose tolerance in mice.…”

An Overview of Brevinin Superfamily: Structure, Function and Clinical Perspectives

“…The main advantage of using amphibian skin as the object of investigation is the use of gentle methods (e.g. skin stimulation by norepinephrine (Mechkarska et al 2011) or gentle electrical stimulation (Marenah et al 2004) for sample preparation. Hence, there is no need to harm or kill animals for this work.…”

“…This new function may give an additional protection for frogs by stimulating insulin secretion and causing hypoglycaemia in attacking predators (Marenah et al 2004). …”

Insulin Releasing Properties of the Temporin Family of Antimicrobial Peptides

“…In particular, skin secretions may contain peptides with cytolytic properties that serve to protect the organism against invasion by bacteria, fungi and protozoa and to deter ingestion by predators [4,5]. Such insulin-releasing peptides have been identified in secretions from the frogs Phyllomedusa trinitatis [6], Agalychnis litodryas [7], Bombina variegata [8], Rana saharica [9], R. palustris [10], and R. pipiens [11]. Such insulin-releasing peptides have been identified in secretions from the frogs Phyllomedusa trinitatis [6], Agalychnis litodryas [7], Bombina variegata [8], Rana saharica [9], R. palustris [10], and R. pipiens [11].…”