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School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, Northern Ireland BT52 1SA, UK

(Requests for offprints should be addressed to L Marenah; Email: l.marenah{at}ulster.ac.uk)

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Skin secretions of Rana saharica were evaluated for the isolation and characterisation of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reverse phase HPLC yielding 80 fractions. In acute 20-min incubations with glucose responsive BRIN-BD11 cells, fractions 36–43, 46–54 and 57–63 significantly stimulated insulin release by 2- to 8-fold compared with 5.6 mM glucose alone. Pooled fractions in the latter two bands were rechromatographed to reveal 9 homogenous peaks, which elicited significant 1.3- to 3.5-fold increases in insulin release (P < 0.05). Structural analysis of the most potent non-toxic peptides was performed by mass spectrometry and automated Edman degradation. This revealed four major insulin-releasing peaks with molecular masses of 2676.9 Da, 3519.3 Da, 4920.4 Da and 4801.2 Da respectively. These peptides were found to be identical to brevinin-1E, brevinin-2EC, esculentin-1 and esculentin-1B, which belong to the group of antimicrobial peptides isolated from skin secretions of various Rana frog species. Preliminary studies on the mechanism underlying the insulinotropic actions of esculentins-1 and -1B suggested possible involvement of both cyclic AMP–protein kinase A and –C-dependent G-protein sensitive pathways. These data indicate that the skin secretions of Rana saharica frogs contain bioactive molecules with significant insulin-releasing activity. Relatives of the brevinin/esculentin peptide family merit further investigation as novel insulin secretagogues.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The granular glands of amphibians are known to release a broad spectrum of peptides with diverse biological activities. Skin secretions are released upon stress or injury as a result of contraction of mycocytes surrounding the glands (Simmaco et al. 1998). Previous studies have led to the isolation of gaegurins from the Korean frog, Rana rugosa (Park et al. 1994), brevinins from the Japanese frog, Rana brevipoda porsa (Morikawa et al. 1992), and esculentins and brevinins (Simmaco et al. 1994) from the European frog, Rana esculenta. These peptides are thought to play various roles, either in the regulation of physiological functions of the skin, or in the defence against predators or microorganisms (Barthalmus 1994). Indeed, many of the regulatory peptides generally belong to families of bioactive peptides, which have mammalian counterparts, such as caerulein/cholecystokinin (Anastasi et al. 1968), bombesin/gastrin-releasing peptide (Anastasi et al. 1972), and exendin-4/glucagon-like peptide-1 (Chen & Drucker 1997). The observation that many of these peptides have counterparts in the mammalian gastrointestinal tract or brain (Erspamer & Melchiorri 1980) has provided further stimulus to the study of frog-skin peptides. Recently, we have isolated various insulinotropic peptides including analogues of bombesin from skin secretions of Bombina variegata (Marenah et al. 2004a), Agalychnis litodryas (Marenah et al. 2004b) and Phyllomedusa trinitatis (Marenah et al. 2004c).

Frogs of the genus Rana are diverse, widely distributed worldwide (Duellman & Trueb 1994) and various antimicrobial peptides have been isolated from their skin secretions (Clark et al. 1994, Goraya et al. 1998, 2000, Marenah et al. 2004d). Most of these peptides share common properties, such as an overall cationic character and the tendency to adopt a helical conformation often resulting in an amphipathic behaviour. These properties are believed to play important roles in the interaction of the peptides with the membrane of target cells and/or in the mechanism that eventually causes cell lysis (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 synthesised in neuroendocrine tissues of mammals (Erspamer et al. 1986, Roseghini et al. 1988, 1989, Basir et al. 2000).

Rana saharica is a large sized frog being about 10–12 cm in body length. The species is widely distributed in the bigger oases in the Sahara from Algeria across to Egypt (Frost 1985). This study describes the purification, structural and biological characterisation of multiple peptides with insulin releasing activity from electrically stimulated skin secretions of Rana saharica frogs. Such peptides may be of therapeutic interest, as illustrated by the enthusiasm for the clinical treatment of type 2 diabetes with exendin-4 and related peptides isolated from the venom of the lizard, Heloderma suspectum (Kolterman et al. 2003, Green et al. 2004).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

RPMI-1640 tissue culture medium, foetal bovine serum, penicillin and streptomycin were all purchased from Gibco (Paisley, Strathclyde, UK). Phorbol-12-myrisate-13-acetate (PMA), forskolin, pertussis toxin and verapamil were obtained from the Sigma Chemical Company Ltd (Poole, Dorset, UK). High-performance liquid chromatography (HPLC) grade acetonitrile was obtained from Rathburn (Walkerburn, Scotland). Sequencing grade trifluoroacetic acid was obtained from Aldrich (Poole, Dorset, UK). All chemicals employed in the operation of the 491 Procise gas phase sequencer were supplied by Perkin Elmer Applied Biosystems (Warrington, Cheshire, UK). All other chemicals used were of the highest purity available.

Collection of skin secretions

Four young captive bred Rana saharica were maintained in terraria at 24 °C under a 12 h light/12 h darkness cycle and were fed on crickets. The skin secretions were obtained from the frogs by gentle electrical stimulation (4-ms pulse width, 50 Hz, 5 V) using platinum electrodes rubbed over the moistened dorsal skin surface for 10 s. Secretions were washed off into a glass beaker, using deionised water. The resultant secretions were freeze-dried in a Hetosicc 2.5 freeze dryer (Heto, UK). Approximately 50 mg, dry weight, of skin secretion was obtained. This procedure was carried out in accordance with the UK Animals (Scientific Procedures) Act 1986. It is a non-invasive technique causing no distress to the frog.

Purification of peptides

The lyophilised crude venom (20 mg) was dissolved in 0.12% trifluoroacetic acid (TFA)/water (2 ml) and 1 ml of this was chromatographed on a Vydac 218TP510 semi-preparative C-18 column (25 x 1 cm) Grace Vydac (Hesperia, CA, USA). The column was equilibrated with 0.12% (v/v) TFA/water at a flow rate of 2 ml/min. Using 0.1% (v/v) TFA in 70% acetonitrile/water, the concentration of acetonitrile in the eluting solvent was raised to 80% (v/v) over 80 min using linear gradients. Absorbance was monitored at 214 nm with collection of 2 ml fractions. Fractions that showed major insulin releasing activity were pooled and rechromatographed using a Vydac 208TP54 analytical C-18 column (25 x 0.46 cm). The column was equilibrated with 0.12% (v/v) TFA/water at a flow rate of 1 ml/min. Using 0.1% (v/v) TFA in 70% acetonitrile/ water, the concentration of acetonitrile in the eluting solvent was raised to 30% (v/v) over 10 min and to 60% (v/v) over 40 min using linear gradients. Absorbance was monitored at 214 nm and peaks were hand collected and prepared for acute insulin release studies. The peaks showing insulin-releasing activity were pooled and further purified to a single homogenous peak using a Vydac 208TP54 analytical C-18 column (25 x 0.46 cm). The concentration of acetonitrile in the eluting solvent was raised to 15% (v/v) over 5 min and to 80% (v/v) over 70 min using linear gradients. Absorbance was monitored at 214 nm.

Culture of insulin secreting cells

BRIN-BD11 cells were cultured in RPMI-1640 tissue culture medium containing 10% (v/v) foetal calf serum, 1% (v/v) antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin) and 11.1 mM glucose. The production and characterisation of BRIN-BD11 cells have been described elsewhere (McClenaghan et al. 1996). This robust, glucose-responsive cell line has been shown to respond to an array of established insulinotropic peptides (McClenaghan et al. 1996, O’Harte et al. 1998a,b, Abdel-Wahab et al. 1999). Cells were maintained in sterile tissue culture flasks (Corning Glass Works, Sunderland, UK) at 37 °C in an atmosphere of 5% CO₂ and 95% air using a LEEC incubator (Laboratory Technical Engineering, Nottingham, UK). In one experimental series, cells were cultured overnight with 25 µM forskolin, 10 nM PMA or 0.1 µg/ml pertussis toxin prior to acute tests.

Acute insulin release studies

Insulin release from BRIN-BD11 cells was determined using cell monolayers (McClenaghan et al. 1996). The cells were harvested with the aid of trypsin/EDTA (Gibco), seeded into 24-multiwell plates (Nunc, Rosklide, Denmark) at a density of 1.5 x 10⁶ cells per well, and allowed to attach overnight. Prior to the acute test, cells were preincubated for 40 min at 37 °C in a 1.0 ml Krebs Ringer bicarbonate buffer (115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 10 mM NaHCO₃, 5 g/l bovine serum albumin, pH 7.4) supplemented with 1.1 mM glucose. Test incubations were performed for three independent observations for each group. They were incubated for 20 min at 37 °C using the same buffer supplemented with 5.6 mM glucose in the absence (control) and presence of various venom fractions, peaks (equivalent to 25 µl dried HPLC fraction) or test agents as indicated in the figures. Cell viability after 20-min test incubations was assessed by modified neutral red assay (Hunt et al. 1987). After incubation, aliquots of buffer were removed and stored at –20 °C for insulin radioimmunoassay as detailed elsewhere (Flatt & Bailey 1981). Briefly, insulin was measured by a modified dextran-coated charcoal radioimmunoassay using crystalline rat insulin standard and guinea-pig antiporcine insulin antiserum. Intra- and interassay coefficients of variation were 4% and 7% respectively.

Molecular mass determination

The molecular masses of peptides in the purified insulin releasing peaks were determined using Electrospray ionisation quadripole ion-trap mass spectrometry (ESI-MS). Samples were infused at a flow rate of 5 µl/min. Mass spectra were recorded on a Thermo Finnigan LCQ benchtop quadripole ion-trap mass spectrometer (Thermo Finningan, Hemel Hempstead, Herts, UK). Spectra were collected using full ion scan mode over the mass-to-charge (m/z) range 150 to 2000. The heated capillary temperature was 220 °C and the spray voltage was set to 5 Kv. Nitrogen gas for the LCQ was delivered from a Whatman nitrogen generator (Whatman Inc, Haverhill, MA, USA) while helium damping gas, present in the ion-trap, was obtained from BOC Medical Gases (Guildford, Surrey, UK). Ions were detected and analysed in the positive mode as a function of their m/z ratio. The molecular masses of the peaks/peptides were determined from ESI-MS profiles using prominent multiple charged ions and the following equation: M_r=iM_i – iM_h where M_r=molecular mass, M_i=m/z ratio, i=number of charges and M_h=mass of a proton.

Structural analysis by automated Edman degradation

The primary structure of the purified peptide was determined by automated Edman degradation, using an Applied Biosystems Procise 491 microsequencer. Standard operating procedures were used (Applied Biosystems Model 491 Protein Sequencers User Manual). The limit for detection of phenylthiohydantoin amino acids was 0.2 pmol. Primary structures were compared with those deposited in the SWISSPROT database (www.hgmp.mrc.ac.uk/Registered/Option/gcg.html).

Statistical analysis

Results are expressed as means ± S.E.M. Values were compared using one-way ANOVA followed by Dunnett’s post hoc test. Groups of data were considered to be significantly different if P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation of insulin releasing peptides

Purification of crude venom by HPLC yielded 80 fractions (Fig. 1A). As shown in Fig. 1B, fractions 36–43, 46–54 (band 1) and 57–63 (band 2) exhibited highly significant insulin-releasing activity (P < 0.01, n=3) compared with the 5.6 mM glucose control. Fractions 36–43 were not assessed further in this study, but the individual fractions in each of the other more potent bands of activity were pooled and rechromatographed, yielding peaks 1.1–1.21 from band 1 and peaks 2.1–2.22 from band 2 (Fig. 2A). These peaks were rescreened for insulin-releasing activity, revealing 1.5- to 18-fold increases (P < 0.01) in insulin release with peaks 1.3, 1.4, 1.6–1.21, 2.2–2.7 and 2.13–2.22 (Fig. 2B).

View larger version (25K): [in this window] [in a new window]   Figure 1 (A) Reversed-phase HPLC of the crude venom of Rana saharica. The crude venom (20 mg) was dissolved in 0.12% trifluoroacetic acid/water (2 ml) and 1 ml was applied to a semi-preparative Vydac C18 column as described in Materials and Methods. The dashed line shows the concentration of acetonitrile in the eluting solvent. Bands 1 and 2 correspond to fractions eluting at 46–54 min and 57–63 min respectively. (B) Effects of various semi-preparative C18 HPLC fractions of Rana saharica crude venom on insulin secretion from BRIN-BD11 cells. Incubations were performed at 5.6 mM glucose using fractions shown in (A). Values are the means ± S.E.M. for 3 separate observations. *P < 0.01 compared with 5.6 mM glucose alone. Bands 1 and 2 correspond to fractions eluting at 46–54 min and 57–63 min respectively.

View larger version (20K): [in this window] [in a new window]   Figure 2 (A) Reversed-phase HPLC purification of the pooled fractions of Rana saharica from bands 1 and 2 in Fig. 1A. Fractions were applied to an analytical Vydac C18 column as described in Materials and Methods. The dashed lines show the concentration of acetonitrile in the eluting solvent. Bands 3, 4, 5 and 6 correspond to peaks 1.8–1.12, 1.13–1.21, 2.2–2.7 and 2.12–2.22 respectively. (B) Effects of various semi-preparative C18 HPLC fractions of Rana saharica crude venom on insulin secretion from BRIN-BD11 cells. Incubations were performed at 5.6 mM glucose using fractions shown in (A). Values are the means ± S.E.M. for 3 separate observations. *P < 0.01 compared with 5.6 mM glucose alone. Bands 1 and 2 correspond to fractions eluting at 46–54 min and 57–63 min respectively.

View larger version (23K): [in this window] [in a new window]   Figure 3 (A) Final reversed-phase HPLC purification of the pooled fractions of Rana saharica from bands 3, 4, 5 and 6 in Fig. 2A. Fractions were applied to an analytical Vydac C18 column as described in Materials and Methods. Individual peaks were hand collected. The dashed lines show the concentration of acetonitrile in the eluting solvent. (B) Effects of peptides isolated from Rana saharica venom on insulin secretion from BRIN-BD11 cells. Incubations were performed at 5.6 mM glucose using peptide peaks shown in (A). Values are the means ± S.E.M. for 3 separate observations. *P < 0.05 and **P < 0.01 compared with 5.6 mM glucose alone.

Each of the purified non-toxic peaks with significant insulin-releasing activity (Fig. 3B) was subjected to mass spectral analysis. Mass spectrometry of peaks 3.1, 3.4, 5.4, and 6.3 revealed molecular masses of 4920.4 Da, 4801.2 Da, 3519.3 Da and 2676.9 Da respectively (Fig. 4). Peptides with mass spectral data were subjected to N-terminal amino acid sequence analysis on an Applied Biosystems 491 Procise Protein Sequencer. The primary amino acid sequence for peaks 3.1 and 3.4 were successfully determined as 46-amino acid peptides (Table 1). A search in the Swiss-Prot FASTA database using the GCG sequence analysis programme for peaks 3.1 and 3.4 revealed them to be identical to esculentins-1 and -1B respectively, antimicrobial peptides originally isolated from the skin secretions of Rana esculenta (Simmaco et al. 1994). Similarly, the sequences for peaks 5.4 and 6.3 were identical to another class of antimicrobial peptides, brevinins-2EC and -1E respectively (Table 1).

View larger version (13K): [in this window] [in a new window]   Figure 4 Electrospray ion-trap mass spectrometry analysis of purified insulin-releasing peptides of Rana saharica eluted from HPLC as shown in Fig. 3A. Samples were applied and molecular masses determined as described in Materials and Methods.

Dilutions of each of the two non-toxic purified peaks 5.4 (brevinin-2EC) and 6.3 (brevinin-1E) by more than 1:10 resulted in loss of insulin releasing activity. In contrast, the activities of the 46 amino acid peptides 4920.4 Da (esculentin-1, peak 3.1) and 4801.2 Da (esculentin-1B, peak 3.4) were preserved at 1:500 but not at 1:1000 dilution (data not shown). Additional tests carried out at 1:10 dilution showed that the stimulatory effects of these peptides were abolished in cells cultured overnight with forskolin, PMA or pertussis toxin (Fig. 5). Thus, the overall impression is that esculentin-1 and -1B (peaks 3.1 and 3.4 respectively) operate through mixed pathways involving both protein kinase (PK) A and PKC. Interestingly, the insulin-releasing actions of esculentin-1 (peak 3.1) and esculentin-1B (peak 3.4) were not affected by 50 µM verapamil and were clearly evident in cells depolarised with 30 mM KCl (Table 2).

View larger version (26K): [in this window] [in a new window]   Figure 5 Acute effects of peaks 3.1 (esculentin-1) and 3.4 (esculentin-1B) from Rana saharica, forskolin and PMA on insulin secretion from BRIN-BD11 cells cultured overnight in the absence (control) and the presence of 25 µM forskolin, 10 nM PMA or 0.1 µg/ml pertussis toxin. Acute incubations were performed at 5.6 mM glucose. Values are the means ± S.E.M. for 8 separate observations. **P < 0.05 and ***P < 0.01 compared with 5.6 mM glucose alone under the same culture conditions. P < 0.05 and P < 0.01 compared with respective test reagent following control culture.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The potency of the amphibian peptides relative to established insulinotropic agents is hard to judge because the concentrations of the former are not known. However, their effects are broadly comparable to those induced by brain-gut peptides in the same clonal BRIN-BD11 cell line (O’Harte et al. 1998a,b, Abdel-Wahab et al. 1999). The insulinotropic actions of two peptides (peaks 3.1 and 3.4) were retained at a further 1:500 dilution, but potency of the other peptides isolated was lost at more than 1:10 dilution of the original sample.

Structural analysis of the most potent insulinotropic peptides isolated was carried out by electrospray ionisation mass spectrometry and automated Edman degradation. The mass spectrometry analysis of 4 of the insulinotropic peptides (peaks 3.1, 3.4, 5.4 and 6.3) were successfully determined, revealing molecular masses of between 2676.9 and 4920.4 Da. These experimental masses corresponded closely to calculated theoretical values, indicating the absence of any post-translational modifications of constituent amino acids such as phosphorylation, sulphation or glycation.

The primary amino acid sequences for peaks 3.1 and 3.4 (Table 1) were found to be identical to esculentin-1 and esculentin-1B respectively. Esculentins represent a family of related peptides consisting of 46 amino acid residues and a cysteine-bridge cyclic heptapeptide region at the C-terminal isolated from the skin secretions of various Rana species (Simmaco et al. 1993, 1994, Ponti et al. 1999). The mechanisms through which the family of esculentin causes bacterial death are not fully understood. However, the presence of a C-terminal cationic loop linked by a disulphide bridge containing 7 amino acids is presumed to play an important role in the antimicrobial activities (Clark et al. 1994, Simmaco et al. 1994).

Analysis of insulinotropic peaks 5.4 and 6.3 revealed primary structures that were found to be an exact match for brevinin-2EC and brevinin-1E respectively (Table 1). Brevinins are a family of related peptides containing a cysteine-bridge cyclic heptapeptide region at the C-terminal, which have been isolated from the skin secretions of various Rana species (Morikawa et al. 1992, Park et al. 1994, Conlon et al. 1999, Goraya et al. 2000). These cationic peptides exert antimicrobial properties against a wide variety of microorganisms including Gram-positive and Gram-negative bacteria (Hancock & Lehrer 1998, Kwon et al. 1998).

Determination of toxic effects of the isolated peptides on BRIN-BD11 cell viability, as assessed by vital neutral red staining, indicates that the observed secretory actions cannot be simply attributed to cell lysis or toxicity. It therefore follows that these peptides stimulate insulin release through regulated pathways. Blockade of voltage-dependent Ca²⁺ channels with verapamil did not affect secretory effectiveness of esculentin-1 and esculentin-1B (peaks 3.1 and 3.4). Similarly, a powerful insulin response was observed using cells depolarised with 30 mM KCl, indicating a degree of independence from changes in ion permeability. However, down-regulation of cyclic AMP–PKA- and –PKC-dependent pathways by overnight culture of BRIN-BD11 cells with forskolin (Altman et al. 1987, Gromada et al. 1998) and PMA (Hii et al. 1987, Yamatani et al. 1988, Persaud et al. 1989, Wolf et al. 1989) respectively, blocked the acute stimulatory effects of both peptides. Additionally, the stimulatory actions of esculentin-1 and -1B were inhibited by overnight culture with pertussis toxin (Seaquist et al. 1992), indicating the involvement of pertussis toxin-sensitive G-protein in their stimulatory action. Additional studies are required to assess the actions of esculentin-1 and -1B on normal pancreatic beta cells and to determine the exact mechanism through which these peptides trigger secretion. This will necessitate further peptide isolation from R. saharica skin secretions or solid phase peptide synthesis. The latter approach is complicated by the presence of a disulphide bridge at the C-terminal and the high aggregation and charge spread across the peptides. Thus, the preferred approach probably involves application of recombinant technology for the generation of large quantities for both in vitro and in vivo biological testing.

In conclusion, this study has shown that the skin secretions of the frog, Rana saharica, contain various insulin-releasing peptides including two classes of antimicrobial peptides, esculentins and brevinins, which appear to trigger secretion through physiological pathways. Further studies are required to assess relatives of the brevinin/esculentin peptide family as possible novel insulin secretagogues.

	Acknowledgements

 
These studies were supported, in part, by University of Ulster Research Strategy Funding and the Research and Development Office of the Northern Ireland Department of Health and Personal Social Services. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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Received 14 September 2005
Accepted 24 October 2005
Made available online as an Accepted Preprint 16 November 2005

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