title: In vitro and in vivo insulinotropic properties of the multifunctional frog skin peptide hymenochirin-1B: a structure-activity study.
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 Pro(5), Glu(6), and Asp(9) 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 Ca(2+). 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.
authors.affiliation: SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA, Northern Ireland, UK.
authors.forename: Bosede O
authors.initials: BO
authors.lastname: Owolabi
authors.affiliation: SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA, Northern Ireland, UK.|School of Health, Sport and Bioscience, University of East London, Stratford, E15 4LZ, UK.
authors.forename: Opeolu O
authors.initials: OO
authors.lastname: Ojo
authors.affiliation: SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA, Northern Ireland, UK.
authors.forename: Dinesh K
authors.initials: DK
authors.lastname: Srinivasan
authors.affiliation: SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA, Northern Ireland, UK.
authors.forename: J Michael
authors.initials: JM
authors.lastname: Conlon
authors.affiliation: SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA, Northern Ireland, UK.
authors.forename: Peter R
authors.initials: PR
authors.lastname: Flatt
authors.affiliation: SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA, Northern Ireland, UK. y.abdel-wahab@ulster.ac.uk.
authors.forename: Yasser H A
authors.initials: YH
authors.lastname: Abdel-Wahab
mesh_terms: D029845:Amphibian Proteins / Q000494:pharmacology*; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000138:chemical synthesis / Q000494:pharmacology*; D001001:Anura; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy*; D059305:Diet, High-Fat; D052216:Glucagon-Like Peptide 1 / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism*; D007333:Insulin Resistance; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D009924:Organ Culture Techniques; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D013329:Structure-Activity Relationship
chemical_list: D029845:Amphibian Proteins; D023181:Antimicrobial Cationic Peptides; D007004:Hypoglycemic Agents; D007328:Insulin; C000589575:hymenochirin-1B, Hymenochirus boettgeri; D052216:Glucagon-Like Peptide 1; D000242:Cyclic AMP; D007770:L-Lactate Dehydrogenase; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D005947:Glucose; D002118:Calcium
keywords: Amphibian skin peptide; Hymenochirin-1b; Insulin release; Type 2 diabetes