Abstract
Several amphibian peptides that were first identified on the basis of their antimicrobial or cytotoxic properties have subsequently shown potential for development into agents for the treatment of patients with Type 2 diabetes. A strategy is presented for the isolation and characterization of such peptides that are present in norepinephrine-stimulated skin secretions from a range of frog species. The methodology involves (1) fractionation of the secretions by reversed-phase HPLC, (2) identification of fractions containing components that stimulate the rate of release of insulin from BRIN-BD11 clonal β-cells without simultaneously stimulating the release of lactate dehydrogenase, (3) identification of active peptides in the fractions in the mass range 1–6 kDa by MALDI-ToF mass spectrometry, (4) purification of the peptides to near homogeneity by further reversed-phase HPLC on various column matrices, and (5) structural characterization by automated Edman degradation. The effect of synthetic replicates of the active peptides on glucose homeostasis in vivo may be evaluated in appropriate animal models of Type 2 diabetes such as db/db mice and mice fed a high fat diet to produce obesity, glucose intolerance, and insulin resistance.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bailey CJ, Flatt PR, Conlon JM (2023) An update on peptide-based therapies for type 2 diabetes and obesity. Peptides 161:170939. https://doi.org/10.1016/j.peptides.2023.170939
Xu X, Lai R (2015) The chemistry and biological activities of peptides from amphibian skin secretions. Chem Rev 115:1760–1846. https://doi.org/10.1021/cr4006704
Conlon JM, Mechkarska M, Abdel-Wahab YH, Flatt PR (2018) Peptides from frog skin with potential for development into agents for type 2 diabetes therapy. Peptides 100:275–281. https://doi.org/10.1016/j.peptides.2017.09.001
Conlon JM, Mechkarska M, Leprince J (2019) Peptidomic analysis in the discovery of therapeutically valuable peptides in amphibian skin secretions. Expert Rev Proteomics 16:897–908. https://doi.org/10.1080/14789450.2019.1693894
McClenaghan NH, Barnett CR, Ah-Sing E, Abdel-Wahab YHA, O’Harte FP, Yoon TW, Swanston-Flatt SK, Flatt PR (1996) Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BD11, produced by electrofusion. Diabetes 45:1132–1140. https://doi.org/10.2337/diab.45.8.1132
Rowlands J, Walz N, Rowles JE, Keane KN, Carlessi R, Newsholme P (2019) Method protocols for metabolic and functional analysis of the BRIN-BD11 β-cell line: a preclinical model for type 2 diabetes. Methods Mol Biol 1916:329–340. https://doi.org/10.1007/978-1-4939-8994-2_32
Owolabi BO, Musale V, Ojo OO, Moffett RC, McGahon MK, Curtis TM, Conlon JM, Flatt PR, Abdel-Wahab YHA (2017) Actions of PGLa-AM1 and its [A14K] and [A20K] analogues and their therapeutic potential as anti-diabetic agents. Biochimie 138:1–12. https://doi.org/10.1016/j.biochi.2017.04.004
McLaughlin CM, Lampis S, Mechkarska M, Coquet L, Jouenne T, King JD, Mangoni ML, Lukic ML, Scorciapino MA, Conlon JM (2016) Purification, conformational analysis, and properties of a family of tigerinin peptides from skin secretions of the crowned bullfrog Hoplobatrachus occipitalis. J Nat Prod 79:2350–2356. https://doi.org/10.1021/acs.jnatprod.6b00494
Musale V, Guilhaudis L, Abdel-Wahab YHA, Flatt PR, Conlon JM (2019) Insulinotropic activity of the host-defense peptide frenatin 2D: conformational, structure-function and mechanistic studies. Biochimie 156:12–21. https://doi.org/10.1016/j.biochi.2018.09.008
Owolabi BO, Ojo OO, Srinivasan DK, Conlon JM, Flatt PR, Abdel-Wahab YH (2016) In vitro and in vivo insulinotropic properties of the multifunctional frog skin peptide hymenochirin-1B: a structure-activity study. Amino Acids 48:535–547. https://doi.org/10.1007/s00726-015-2107-x
Vasu S, Ojo OO, Moffett RC, Conlon JM, Flatt PR, Abdel-Wahab YHA (2017) Anti-diabetic actions of esculentin-2CHa(1-30) and its stable analogues in a diet-induced model of obesity-diabetes. Amino Acids 49:1705–1717. https://doi.org/10.1007/s00726-017-2469-3
Ojo OO, Srinivasan DK, Owolabi BO, McGahon MK, Moffett RC, Curtis TM, Conlon JM, Flatt PR, Abdel-Wahab YH (2016) Molecular mechanisms mediating the beneficial metabolic effects of [Arg4]tigerinin-1R in mice with diet-induced obesity and insulin resistance. Biol Chem 397:753–764. https://doi.org/10.1515/hsz-2016-0120
Musale V, Moffett RC, Owolabi B, Conlon JM, Flatt PR, Abdel-Wahab YHA (2021) Mechanisms of action of the antidiabetic peptide [S4K]CPF-AM1 in db/db mice. J Mol Endocrinol 66:115–128. https://doi.org/10.1530/JME-20-0152
Heydemann A (2016) An overview of murine high fat diet as a model for type 2 diabetes mellitus. J Diabetes Res 2016:2902351. https://doi.org/10.1155/2016/2902351
Do OH, Low JT, Gaisano HY, Thorn P (2014) The secretory deficit in islets from db/db mice is mainly due to a loss of responding beta cells. Diabetologia 57:1400–1409. https://doi.org/10.1007/s00125-014-3226-8
Musale V, Moffett RC, Conlon JM, Flatt PR, Abdel-Wahab YH (2021) Beneficial actions of the [A14K] analog of the frog skin peptide PGLa-AM1 in mice with obesity and degenerative diabetes: a mechanistic study. Peptides 136:170472. https://doi.org/10.1016/j.peptides.2020.170472
Conlon JM, Sonnevend A (2010) Antimicrobial peptides in frog skin secretions. Methods Mol Biol 618:3–14. https://doi.org/10.1007/978-1-60761-594-1_1
Conlon JM, Leprince J (2010) Identification and analysis of bioactive peptides in amphibian skin secretions. Methods Mol Biol 615:145–157. https://doi.org/10.1007/978-1-60761-535-4_12
Conlon JM, Musale V, Attoub S, Mangoni ML, Leprince J, Coquet L, Jouenne T, Abdel-Wahab YHA, Flatt PR, Rinaldi AC (2017) Cytotoxic peptides with insulin-releasing activities from skin secretions of the Italian stream frog Rana italica (Ranidae). J Pept Sci 23:769–776. https://doi.org/10.1002/psc.3025
Frost DR (2023) Amphibian species of the world: an online reference. Version 6.1 electronic database accessible at http://research.amnh.org/herpetology/amphibia/index.html. American Museum of Natural History, New York https://doi.org/10.5531/db.vz.0001. Accessed January 2023
Green AD, Vasu S, Flatt PR (2018) Cellular models for beta-cell function and diabetes gene therapy. Acta Physiol (Oxf) 222(3). https://doi.org/10.1111/apha.13012
Flatt PR, Bailey CJ (1981) Abnormal plasma glucose and insulin responses in heterozygous lean (ob/+) mice. Diabetologia 20:573–577. https://doi.org/10.1007/bf00252768
Conlon JM (2007) Purification of naturally occurring peptides by reversed-phase HPLC. Nat Protoc 2:191–197. https://doi.org/10.1038/nprot.2006.437
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Conlon, J.M., Moffett, R.C., Flatt, P.R., Leprince, J. (2024). Strategy for the Identification of Host-Defense Peptides in Frog Skin Secretions with Therapeutic Potential as Antidiabetic Agents. In: Schrader, M., Fricker, L.D. (eds) Peptidomics. Methods in Molecular Biology, vol 2758. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3646-6_15
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3646-6_15
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3645-9
Online ISBN: 978-1-0716-3646-6
eBook Packages: Springer Protocols