Mechanisms of action of the antidiabetic peptide [S4K]CPF-AM1 in db/db mice.

The antidiabetic effects and mechanisms of action of an analogue of a frog skin host-defence peptide belonging to the caerulein-precursor fragment family, [S4K]CPF-AM1 were investigated in db/db mice with a genetically inherited form of degenerative diabetes-obesity. Twice-daily treatment with the peptide (75 nmol/kg body weight) for 28 days significantly decreased blood glucose (P < 0.01) and HbA1c (P < 0.05) and increased plasma insulin (P < 0.05) concentrations with no effect on body weight, energy intake, body composition or plasma lipid profile. Peptide administration improved insulin sensitivity and intraperitoneal glucose tolerance. Elevated biomarkers of liver and kidney function associated with the db/db phenotype were significantly lowered by [S4K]CPF-AM1 administration. Peptide treatment significantly (P < 0.05) increased pancreatic insulin content and improved the responses of isolated islets to established secretagogues. Elevated expression of genes associated with insulin signalling (Slc2a4, Insr, Irs1, Akt1, Pik3ca, Ppm1b) in the skeletal muscle of db/db mice were significantly downregulated by peptide treatment. Genes associated with insulin secretion (Abcc8, Kcnj11, Slc2a2, Cacn1c, Glp1r, Gipr) were significantly upregulated by treatment with [S4K]CPF-AM1. Studies with BRIN-BD1I clonal ß-cells demonstrated that the peptide evoked membrane depolarisation, increased intracellular Ca2+ and cAMP and activated the protein kinase C pathway. The data indicate that the antidiabetic properties of [S4K]CPF-AM1 mice are mediated by direct insulinotropic action and by regulation of transcription of genes involved in both the secretion and action of insulin.

Isolation and characterization of cytotoxic and insulin-releasing components from the venom of the black-necked spitting cobra Naja nigricollis (Elapidae).

Four peptides with cytotoxic activity against BRIN-BD11 rat clonal ß-cells were purified from the venom of the black-necked spitting cobra Naja nigricollis using reversed-phase HPLC. The peptides were identified as members of the three-finger superfamily of snake toxins by ESI-MS/MS sequencing of tryptic peptides. The most potent peptide (cytotoxin-1N) showed strong cytotoxic activity against three human tumor-derived cell lines (LC50 = 0.8 ± 0.2 µM for A549 non-small cell lung adenocarcinoma cells; LC50 = 7 ± 1 µM for MDA-MB-231 breast adenocarcinoma cells; and LC50 = 9 ± 1 µM for HT-29 colorectal adenocarcinoma cells). However, all the peptides were to varying degrees cytotoxic against HUVEC human umbilical vein endothelial cells (LC50 in the range 2-22 µM) and cytotoxin-2N was moderately hemolytic (LC50 = 45 ± 3 µM against mouse erythrocytes). The lack of differential activity against cells derived from non-neoplastic tissue limits their potential for development into anti-cancer agents. In addition, two proteins in the venom, identified as isoforms of phospholipase A2, effectively stimulated insulin release from BRIN-BD11 cells (an approximately 6-fold increase in rate compared with 5.6 mM glucose alone) at a concentration (1 µM) that was not cytotoxic to the cells suggesting possible application in therapy for Type 2 diabetes.

Conformational analysis and in vitro immunomodulatory and insulinotropic properties of the frog skin host-defense peptide rhinophrynin-27 and selected analogs.

The study investigates conformational analysis and the in vitro cytokine-mediated immunomodulatory and insulin-releasing activities of rhinophrynin-27 (ELRLPEIARPVPEVLPARLPLPALPRN; RP-27), a proline-arginine-rich peptide first isolated from skin secretions of the Mexican burrowing toad Rhinophrynus dorsalis (Rhinophrynidae). In both water and 50% trifluoroethanol-water, the peptide adopts a polyproline type II helical conformation with a high degree of deviation from the canonical collagen-like folding and a pronounced bend in the molecule at the Glu13 residue. Incubation of mouse peritoneal cells with RP-27 significantly (P < 0.05) inhibited production of the pro-inflammatory cytokines TNF-α and IL-1ß and stimulated production of the anti-inflammatory cytokine IL-10. The peptide significantly (P < 0.01) stimulated release of insulin from BRIN-BD11 rat clonal ß-cells at concentrations ≥ 1 nM while maintaining the integrity of the plasma membrane and also stimulated insulin release from isolated mouse islets at a concentration of 10-6 M. Increasing the cationicity of RP-27 by substituting glutamic acid residues in the peptide by arginine and increasing hydrophobicity by substituting alanine residues by tryptophan did not result in analogues with increased activity with respect to cytokine production and insulin release. The combination of immunosuppressive and insulinotropic activities together with very low cytotoxicity suggests that RP-27 may represent a template for the development of an agent for use in anti-inflammatory and Type 2 diabetes therapies.

Immunomodulatory, insulinotropic, and cytotoxic activities of phylloseptins and plasticin-TR from the Trinidanian leaf frog Phyllomedusa trinitatis.

The aim of the study was to determine the in vitro immunomodulatory, cytotoxic, and insulin-releasing activities of seven phylloseptin-TR peptides and plasticin-TR, first isolated from the frog Phyllomedusa trinitatis. The most cationic peptides, phylloseptin-1.1TR and phylloseptin-3.1TR, showed greatest cytotoxic potency against A549, MDA-MB231, and HT-29 human tumor-derived cells and against mouse erythrocytes. Phylloseptin-4TR was the most hydrophobic and the most effective peptide at inhibiting production of the proinflammatory cytokines TNF-α and IL-1ß by mouse peritoneal cells but was without effect on production of the antiinflammatory cytokine IL-10. Phylloseptin-2.1TR and phylloseptin-3.3TR were the most effective at stimulating the production of IL-10. The noncytotoxic peptide, plasticin-TR, inhibited production of TNF-α and IL-1ß but was without effect on IL-10 production. The results of CD spectroscopy suggest that the different properties of plasticin-TR compared with the immunostimulatory activities of the previously characterized plasticin-L1 from Leptodactylus laticeps may arise from greater ability of plasticin-TR to oligomerize and adopt a stable helical conformation in a membrane-mimetic environment. All peptides stimulated release of insulin from BRIN-BD11 rat clonal ß cells with phylloseptin-3.2TR being the most potent and effective and phylloseptin-2.1TR the least effective suggesting that insulinotropic potency correlates inversely with helicity. The study has provided insight into structure-activity relationships among the phylloseptins. The combination of immunomodulatory and insulinotropic activities together with low cytotoxicity suggests that phylloseptin-3.3TR and plasticin-TR may represent templates for the development of agents for use in antiinflammatory and type 2 diabetes therapies.

Peptidomic analysis of the host-defense peptides in skin secretions of Rana graeca provides insight into phylogenetic relationships among Eurasian Rana species.

Peptidomic analysis of norepinephrine-stimulated skin secretions from the Greek stream frog Rana graeca Boulenger, 1891 led to the identification and structural characterization of a range of host-defense peptides. These comprised brevinin-1GRa, brevinin-1GRb and an N-terminally extended form of brevinin-1GRb, ranatuerin-2GR together with its oxidized form and (11-28) fragment, temporin-GRa, temporin-GRb and its non-amidated form, and a melittin-related peptide, MRP-GR and its (1-18) fragment. The most abundant peptide, MRP-GR significantly (P < 0.001) stimulated insulin release from BRIN-BD11 clonal ß-cells at concentrations ≥0.1 nM. Rana graeca (formerly Rana graeca graeca) and the morphologically similar Italian stream frog Rana italica Dubois, 1987 (formerly Rana graeca italica) were originally regarded as sub-species. However, the primary structures of the host defense peptides from both frogs support the claim based upon comparisons of the nucleotide sequences of S1 satellite DNA that R. graeca and R. italica are separate species. Cladistic analyses based upon the primary structures of the brevinin-1 and ranatuerin-2 peptides from Eurasian frogs indicate a close phylogenetic relationship between R. graeca and Rana latastei whereas R. italica is most closely related to Rana dalmatina.

Insulinotropic activity of the host-defense peptide frenatin 2D: Conformational, structure-function and mechanistic studies.

Of four naturally occurring frenatin peptides tested, frenatin 2D (DLLGTLGNLPLPFI.NH2) from Discoglossus sardus was the most potent and effective in producing concentration-dependent stimulation of insulin release from BRIN-BD11 rat clonal ß-cells without displaying cytotoxicity. The peptide also stimulated insulin release from 1.1B4 human-derived clonal ß-cells and isolated mouse islets and improved glucose tolerance concomitant with increased circulating insulin concentrations in mice following intraperitoneal administration. The insulinotropic activity of frenatin 2D was not associated with membrane depolarization or an increase in intracellular [Ca2+] but incubation of the peptide (1 µM) with BRIN-BD11 cells produced a modest, but significant (P < 0.05), increase in cAMP production. Stimulation of insulin release was abolished in protein kinase A-downregulated cells but maintained in protein kinase C-downregulated cells. Circular dichroism studies showed that, in the presence of dodecylphosphocholine micelles, frenatin 2D exhibited a helical content of 35% and a turn content of 28%. Substitution of the Thr5, Asn8, Pro10, and Ile14 residues in frenatin-2D by Trp and interchange of Pro12 and Phe13 led to loss of insulinotropic activity but the [D1W] and [G7W] analogues were as potent and effective as the native peptide. Frenatin 2D (1 µM) also stimulated proliferation of BRIN-BD11 cells and provided significant protection of the cells against cytokine-induced apoptosis. It is concluded that the insulinotropic activity of frenatin 2D is mediated predominantly, if not exclusively, by the KATP channel-independent pathway.

Insulinotropic, glucose-lowering, and beta-cell anti-apoptotic actions of peptides related to esculentin-1a(1-21).NH2.

Long-standing Type 2 diabetes is associated with loss of both ß-cell function and ß-cell mass. Peptides derived from the frog-skin host-defense peptide esculentin-1 have been shown to exhibit potent, broad-spectrum antimicrobial activity. The aim of the present study is to determine whether such peptides also show insulinotropic and ß-cell protective activities. Esculentin-1a(1-21).NH2, esculentin-1b(1-18).NH2, and esculentin-1a(1-14).NH2 produced concentration-dependent stimulations of insulin release from BRIN-BD11 rat clonal ß-cells, 1.1B4 human-derived pancreatic ß-cells, and isolated mouse islets with no cytotoxicity at concentrations of up to 3 µM. The mechanism of insulinotropic action involved membrane depolarization and an increase in intracellular Ca2+ concentrations. The analogue [D-Lys14, D-Ser17]esculentin-1a(1-21).NH2 (Esc(1-21)-1c) was less potent in vitro than the all L-amino acid containing peptides and esculentin-1a(9-21) was inactive indicating that helicity is an important determinant of insulinotropic activity. However, intraperitoneal injection of Esc(1-21)-1c (75 nmol/kg body weight) together with a glucose load (18 mmol/kg body weight) in C57BL6 mice improved glucose tolerance with a concomitant increase in insulin secretion, whereas administration of esculentin-1a(1-21).NH2, esculentin-1b(1-18).NH2, and esculentin-1a(1-14) was without significant effect on plasma glucose levels. Esc(1-21)-1c (1 µM) protected BRIN-BD11 cells against cytokine-induced apoptosis (P < 0.01) and augmented proliferation of the cells (P < 0.01) to a similar extent as glucagon-like peptide-1. The data demonstrate that the multifunctional peptide Esc(1-21)-1c, as well as showing therapeutic potential as an anti-infective and wound-healing agent, may constitute a template for development of compounds for treatment of patients with Type 2 diabetes.

Cytotoxic peptides with insulin-releasing activities from skin secretions of the Italian stream frog Rana italica (Ranidae).

Peptidomic analysis of norepinephrine-stimulated skin secretions from Italian stream frog Rana italica led to the purification and characterization of two host-defense peptides differing by a single amino acid residue belonging to the brevinin-1 family (brevinin-1ITa and -1ITb), a peptide belonging to the temporin family (temporin-ITa) and a component identified as prokineticin Bv8. The secretions contained relatively high concentrations of the methionine-sulphoxide forms of brevinin-1ITa and -1ITb suggesting that these peptides may have a role as antioxidants in the skin of this montane frog. Brevinin-1ITa (IVPFLLGMVPKLVCLITKKC) displayed potent cytotoxicity against non-small cell lung adenocarcinoma A549 cells (LC50  = 18 µM), breast adenocarcinoma MDA-MB-231 cells (LC50  = 8 µM) and colorectal adenocarcinoma HT-29 cells (LC50  = 18 µM), but the peptide was also strongly hemolytic against mouse erythrocytes (LC50  = 7 µM). Temporin-ITa (VFLGAIAQALTSLLGKL.NH2 ) was between three and fivefold less potent against these cells. Brevinin-1ITa inhibited growth of both Gram-positive Staphylococcus epidermidis and Gram-negative Escherichia coli as well as a strain of the opportunist yeast pathogen Candida parapsilosis, whereas temporin-ITa was active only against S. epidermidis and C. parapsilosis. Both peptides stimulated the release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥1 nM, but brevinin-1ITa was cytotoxic to the cells at concentrations ≥3 µM. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Actions of PGLa-AM1 and its [A14K] and [A20K] analogues and their therapeutic potential as anti-diabetic agents.

PGLa-AM1 (GMASKAGSVL10GKVAKVALKA20AL.NH2) was first identified in skin secretions of the frog Xenopus amieti (Pipidae) on the basis of its antimicrobial properties. PGLa-AM1 and its [A14K] and [A20K] analogues produced a concentration-dependent stimulation of insulin release from BRIN-BD11 rat clonal ß-cells without cytotoxicity at concentrations up to 3 µM. In contrast, the [A3K] analogue was cytotoxic at concentrations ≥ 30 nM. The potency and maximum rate of insulin release produced by the [A14K] and [A20K] peptides were significantly greater than produced by PGLa-AM1. [A14K]PGLa-AM1 also stimulated insulin release from mouse islets at concentrations ≥ 1 nM and from the 1.1B4 human-derived pancreatic ß-cell line at concentrations > 30 pM. PGLa-AM1 (1 µM) produced membrane depolarization in BRIN-BD11 cells with a small, but significant (P < 0.05), increase in intracellular Ca2+ concentrations but the peptide had no direct effect on KATP channels. The [A14K] analogue (1 µM) produced a significant increase in cAMP concentration in BRIN-BD11 cells and down-regulation of the protein kinase A pathway by overnight incubation with forskolin completely abolished the insulin-releasing effects of the peptide. [A14K]PGLa-AM1 (1 µM) protected against cytokine-induced apoptosis (p < 0.001) in BRIN-BD11 cells and augmented (p < 0.001) proliferation of the cells to a similar extent as GLP-1. Intraperitoneal administration of the [A14K] and [A20K] analogues (75 nmol/kg body weight) to both lean mice and high fat-fed mice with insulin resistance improved glucose tolerance with a concomitant increase in insulin secretion. The data provide further support for the assertion that host defense peptides from frogs belonging to the Pipidae family show potential for development into agents for the treatment of patients with Type 2 diabetes.