Palustrin-1C
& Brevinin-1
two Insulin-releasing Peptides
Brevinin-1 and multiple insulin-releasing
peptides in the skin of the frog Rana palustris
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 glucose-responsive
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 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.
Marenah L, et al. J
Endocrinol. 2004 May;181(2):347-54
Effects of various
concentrations of synthetic brevinin-1 on insulin secretion
from BRIN-BD11 cells. Incubations were performed at 5·6
mM glucose in (A) normal KRB buffer and (B) Ca2+-free
buffer. Values are the meanS.E.M. for eight separate
observations. ***P,0·001 compared with 5·6 mM glucose
alone. OOP,0·01 and OOOP,0·001 compared with similar conditions
in (A).