Patent Application
20080058269
Collection of skin secretions: Captive bred of Agalychnis calcarifer, Agalychnis litodryas, Bombina variegata, Phyllomedusa trinitatis, Rana palustris, Rana pipiens and Rana saharica were maintained in terraria at 24° C. with 12 h/12 h light/dark cycle and fed on crickets. The skin secretions were obtained from groups of four amphibians from each species 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 for each species.
Purification of peptide: Lyophilized crude venom (20 mg) from each species was dissolved in 0.12% trifluoroacetic acid/water (2 ml) and 1 ml of it was chromatographed on a Vydac 218TP510 semi-preparative C-18 column (25×1 cm, Hesperia, Calif., USA). The column was equilibrated with 0.12% (v/v) trifluoroacetic acid/water at a flow rate of 2 ml/min. Using 0.1% (v/v) TFA in 70% acetonitrile/water, 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 which showed major insulin releasing activity were pooled and rechromatographed using a Vydac 208TP54 analytical C-18 column (25×0.46 cm). The column was equilibrated with 0.12% (v/v) trifluoroacetic acid/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 15% (v/v) over 5 min and to 80% (v/v) over 70 min using linear gradients. Absorbance was monitored at 214 mn.
Culture of insulin-secreting cells: Clonal rat insulin-secreting BRIN-BD11 cells were cultured in RPMI-1640 tissue culture medium containing 10% (v/v) fetal 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 are described elsewhere [5]. Cells were maintained in sterile tissue culture flasks (Corning, Glass Works, UK) at 37° C. in an atmosphere of 5% CO2 and 95% air using LEEC incubator (Laboratory Technical Engineering, Nottingham, UK). In three experimental series using purified peptides from Agalychnis calcarifer, Bombina variegata and Rana saharica 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 N H, Barnett C R, Ah-Sing E, Abdel-Wahab Y H, O'Harte F P, Yoon T W, Swanston-Flatt S K and Flatt P R 1996 Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BD11, produced by electrofusion. Diabetes 45 1132-1140]. The cells were harvested with the aid of trypsin/EDTA (Gibco), seeded into 24-multiwell plates (Nunc, Rosklide, Denmark) at a density of 1.5×106 cells per well, and allowed to attach overnight. Prior to 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 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 approx. 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 S M, Chrzanowska C, Barnnett C R, Brand H N and Fawell J K 1987 A comparison of in vitro cytotoxicity assays and their application to water samples. Alternatives to Laboratory Animals 15 20-29]. After incubation, aliquots of buffer were removed and stored at −20° C. for insulin radiomnimmunoassay [Flatt P R. and Bailey C J 1981 Abnormal plasma glucose and insulin responses in heterozygous lean (ob/+) mice. Diabetologia 20 573-577].
Molecular mass determination: The molecular masses of the purified individual non-toxic peaks exhibiting insulin releasing activity were determined using Matrix Assisted Laser Desorption Ionisation-Time of Flight (MALDI-TOF) mass spectrometry. Electrospray Ionisation quadripole ion-trap Mass Spectrometry (ESI-MS) was used for Agalychnis calcarifer, Bombina variegata, Rana pipiens and Rana saharica. Masses were recorded and compared with theoretical values calculated by the peptide calculator, a computer software package.
Depyroglutamation: Where necessary (Bombina variegata), pyroglutamate at the N-terminal was removed by adding 25 μl of pyroglutamnate aminopeptidase preparation (50 mM Na2HPO4, 10 mM β-mercaptoethanol, 1 mM dithiothreitol, and 1 mM EDTA adjusted to pH 7.3 with H3HPO4) containing 0.4 mg/ml pyroglutamate aminopeptidase to 100 μl of the lypophilised peptide. The reaction mix was incubated for 2 hours at 37° C. and then stored at −20° C. for subsequent amino acid determination by Edman degradation.
Structural analysis by automated Edman degradation: The primary structures of the purified peptides were determined by automated Edman degradation, using an Applied Biosystems Procise 491 microsequencer. Standard operating procedures were used (Applied Biosystems Model 491 Protein Sequencers Users Manual). The limit for detection of phenylthiohydantoin amino acids was 0.2 pmol. The primary structures were compared with those deposited in the SWISSPROT™ database.
Statistical analysis: Results are expressed as mean ±S.E.M. Values were compared using Student's unpaired t-test. Groups of data were considered to be significantly different if P<0.05.
Isolation, mass spectrometry and sequence analysis of insulin-releasing peptides: Skin secretions from the various amphibian species were purified by HPLC, yielding in each case multiple fractions that were subsequently screened for in vitro biological activity using BRIN-BD11 cells. The insulin-releasing profiles of peaks emerging from the primary HPLC separation are illustrated for Agalychnis calcarifer, Agalychnis litodryas, Bombina variegata, Phyllomedusa trinitatis, Rana palustris, Rana pipieizs and Rana saharica in
The major peaks of insulin-releasing activity were subjected to further HPLC purification steps, giving rise ultimately to the isolation of pure peptides with proven insulinotropic activity (Tables 1-7). Where sufficient sample was available, molecular masses and either partial or complete sequences were determined for each peptide as summarised for the various amphibian species in Tables 1-7. In instances where a complete sequence was obtained, the theoretical (calculated) molecular masses of the peptides were shown to corresponded closely to the experimental masses. This indicates the absence of any post-translational modification of constituent amino acids, such as phosphorylation, sulphation or glycation.
Six of the insulinotropic peptides have proved to be established structures. Peak 4.1 from Rana pipiens was identical to pipinin-1, peak 23 from Bombina variegata to bombesin, peak 2.10 from Phyllomedusa trinitatis to dermaseptin BIV precursor and peaks 5.4, 6.5 and 8.3 from Rana saharica matched Esculentin-1B, Brevinin-2EC and Brevinin-1E respectively. With these few exceptions, all other insulin-releasing peptides were novel structures as established using the SWISSPROT™ database. Even the functional observations with pipinin-1, dermaseptin BIV precursor esculentin-1B and brevinin-2EC and 1E were novel as these were totally unsuspected insulin releasing peptides.
Modest similarity existed between some of the isolated insulin-releasing peptides and amphibian antimicrobial peptides that are unsuspected insulin secretagogues such as brevinin, dermaseptin, Rugosin A and tryptophyllin. However, unlike the latter agents, no evidence was obtained of cell lysis or a toxic action that might account for insulin secretion. Thus the peptides reported herein appear to act through physiological mammalian processes controlling exocytosis of insulin.
To support this view, further studies were carried out using purified peptides from Agalychins calcarifer, Bombina variegata and Rana saharica to examine cellular mechanisms underlying the stimulation of insulin secretion. The stimulatory effects of the 1653.2 Dapeptide (peak 1.10) from Agalychnis calcarifer (
Agalychnis calcarifer on insulin secretion from BRIN-BD11 cells
The stimulatory effects of the 4920.4 and 4801.2 Da peptides (peaks 5.1 and 5.4) from Rana saharica were abolished in cells cultured overnight with forskolin, PMA or pertussis toxin (
This research describes for the first time the isolation and characterisation of peptides with insulin-releasing activity from the skin secretions of Agalychnis calcarifer, Agalychnis litodryas, Bombina variegala, Phyllomedusa trinitatis, Rana palustris, Rana pipiens and Rana saharica. It is notable that this work has not only uncovered a diverse range of novel peptides structures but it has also revealed that the skin secretions from each amphibian species studied represents an unsuspected and rich source of peptides capable of stimulating physiological insulin secretion from mammalian pancreatic beta cells.
The insulin output induced by amphibian peptides is approximately equivalent to that induced by established mammalian gut peptides, GLP-1, GIP or CCK-8 [Gault V A, O'Harte F P M, Harriott P, Mooney M H, Green, B D and Flatt P R 2003 Effects of the novel (Pro3) GIP antagonist and extending (9-39) amide on GIP- and GLP-1-induced cyclic AMP generation, insulin secretion and postprandial insulin release in obese diabetic (ob/ob) mice: evidence that GIP is the major physiological incretin. Diabetologia 46 222-230; O'Harte F P M, Abdel-Wahab Y H, Conlon J M and Flatt P R 1998 Glycation of glucagon-like peptide-1(7-36)amide: characterization and impaired action on rat insulin secreting cells. Diabetologia 41 1187-1193; Abdel-Wahab Y H, O'Harte F P M, Mooney M H, Conlon J M and Platt P R 1999 N-terminal glycation of cholecystokinin-8 abolishes its insulinotropic action on clonal pancreatic B-cells. Biochimica et Biophysica Acta 1452 60-67]. This indicates that the amphibian peptides isolated are at least as capable as physiological mammalian hormones in stimulating insulin secretion. It is also clear that these peptides may also trigger insulin secretion and have other beneficial actions on beta cells which involve novel secretory pathways as suggested by studies using peak 1.10 from Agalychnis calcarifer and peaks 21, 22, 23 and 24 from Bombina variegata. In these cases the secretagogues appeared to be mediated through both protein kinase A and G-protein independent pathways. In the case of peptides isolated from Rana saharica (peaks 5.1 and 5.4), the stimulatory effects were also independent of pathways triggered by protein kinase C.
It is apparent from the insulin stimulatory effects that specific receptors must exist for these amphibian peptides on mammalian insulin-secreting beta cells. This gives rise to two major and highly novel non-exclusive possibilities. The first is that these insulin-releasing amphibian peptides have homologous or closely related mammalian counter-parts.
The second important possibility arising from this research is that the novel amphibian peptides described in Tables 1-7, or fragments thereof may offer a therapeutically useful means of treating insulin secretory dysfunction and other beta cell disturbances typical of diabetes in humans. Diabetes is predicted to reach epidemic proportions throughout the world in the next 20 years and current treatments do not restore normal glucose homeostasis, therein resulting in debilitating diabetic complications and premature death. Amphibian peptides may therefore be a useful addition to the therapeutic arsenal for use either alone or in combination with other agents to improve diabetes control and decrease the risk of associated complications.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 0326720.0 | Nov 2003 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP04/13693 | 11/17/2004 | WO | 00 | 9/5/2006 |
