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In vitro and in vivo insulinotropic properties of the multifunctional frog skin peptide hymenochirin-1B: a structure–activity study

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Abstract

Hymenochirin-1b (Hym-1B; IKLSPETKDNLKKVLKGAIKGAIAVAKMV.NH2) is a cationic, α-helical amphibian host-defense peptide with antimicrobial, anticancer, and immunomodulatory properties. This study investigates the abilities of the peptide and nine analogues containing substitutions of Pro5, Glu6, and Asp9 by either l-lysine or d-lysine to stimulate insulin release in vitro using BRIN-BD11 clonal β cells or isolated mouse islets and in vivo using mice fed a high-fat diet to produce obesity and insulin resistance. Hym-1B produced a significant and concentration-dependent increase in the rate of insulin release from BRIN-BD11 cells without cytotoxicity at concentrations up to 1 µM with a threshold concentration of 1 nM. The threshold concentrations for the analogues were: [P5K], [E6K], [D9K], [P5K, E6K] and [E6K, D9k] 0.003 nM, [E6K, D9K] and [D9k] 0.01 nM, [P5K, D9K] 0.1 nM and [E6k] 0.3 nM. All peptides displayed cytotoxicity at concentrations ≥1 µM except the [P5K] and [D9k] analogues which were non-toxic at 3 µM. The potency and maximum rate of insulin release from mouse islets produced by the [P5K] peptide were significantly greater than produced by Hym-1B. Neither Hym-1B nor the [P5K] analogue at 1 µM concentration had an effect on membrane depolarization or intracellular Ca2+. The [P5K] analogue (1 µM) produced a significant increase in cAMP concentration in BRIN-BD11 cells and stimulated GLP-1 secretion from GLUTag cells. Down-regulation of the protein kinase A pathway by overnight incubation with forskolin completely abolished the insulin-releasing effects of [P5K]hym-1B. Intraperitoneal administration of the [P5K] and [D9k] analogues (75 nmol/kg body weight) to high-fat-fed mice with insulin resistance significantly enhanced glucose tolerance with a concomitant increase in insulin secretion. We conclude that [P5K]hym-1B and [D9k]hym-1B show potential for development into anti-diabetic agents.

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Abbreviations

LDH:

Lactate dehydrogenase

Hym-1B:

Hymenochirin-1b

[Ca2+]i:

Intracellular calcium concentration

IBMX:

3-Isobutyl-1-methylxanthine

PKC:

Protein kinase C

PKA:

Protein Kinase A

GLP-1:

Glucagon-like peptide 1

TNF-α:

Tumor necrosis factor α

IL-17:

Interleukin 17

CPF-6:

Caerulein precursor fragment 6

PMA:

Para-methoxyamphetamine

MALDI-TOF:

Matrix-assisted laser desorption/ionization time of flight

EGTA:

Ethylene glycol tetraacetic acid

CCK8:

Cholecystokinin 8

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

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Acknowledgments

This study was supported by the University of Ulster Research Strategy Funding and an award of a University Vice Chancellor Research Studentship to DKS. We thank Professor D. Drucker for access to GLUTag cells.

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Authors and Affiliations

  1. SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA, Northern Ireland, UK

    Bosede O. Owolabi, Opeolu O. Ojo, Dinesh K. Srinivasan, J. Michael Conlon, Peter R. Flatt & Yasser H. A. Abdel-Wahab

  2. School of Health, Sport and Bioscience, University of East London, Stratford, E15 4LZ, UK

    Opeolu O. Ojo

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  1. Bosede O. Owolabi
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  2. Opeolu O. Ojo
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  4. J. Michael Conlon
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  5. Peter R. Flatt
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  6. Yasser H. A. Abdel-Wahab
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Correspondence to Yasser H. A. Abdel-Wahab.

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Owolabi, B.O., Ojo, O.O., Srinivasan, D.K. et al. In vitro and in vivo insulinotropic properties of the multifunctional frog skin peptide hymenochirin-1B: a structure–activity study. Amino Acids 48, 535–547 (2016). https://doi.org/10.1007/s00726-015-2107-x

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  • DOI: https://doi.org/10.1007/s00726-015-2107-x

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