The chondrichthyan glucagon-like peptide 3 regulates hepatic ketone metabolism in the Pacific spiny dogfish Squalus suckleyi.

Chondrichthyans have a novel proglucagon-derived peptide, glucagon-like peptide (GLP)-3, in addition to GLP-1 and GLP-2 that occur in other vertebrates. Given that the GLPs are important regulators of metabolic homeostasis across vertebrates, we sought to investigate whether GLP-3 displays functional actions on metabolism within a representative chondrichthyan, the Pacific spiny dogfish Squalus suckleyi. There were no observed effects of GLP-3 perfusion (10 nM for 15 min) on the rate of glucose or oleic acid acquisition at the level of the spiral valve nor were there any measured effects on intermediary metabolism within this tissue. Despite no effects on apparent glucose transport or glycolysis in the liver, a significant alteration to ketone metabolism occurred. Firstly, ketone flux through the perfused liver switched from a net endogenous production to consumption following hormone application. Accompanying this change, significant increases in mRNA transcript abundance of putative ketone transporters and in the activity of ß-hydroxybutyrate dehydrogenase (a key enzyme regulating ketone flux in the liver) were observed. Overall, while these results show effects on hepatic metabolism, the physiological actions of GLP are distinct between this chondrichthyan and those of GLP-1 on teleost fishes. Whether this is the result of the particular metabolic dependency on ketone bodies in chondrichthyans or a differential function of a novel GLP remains to be fully elucidated.

Purification, conformational analysis and cytotoxic activities of host-defense peptides from the Tungara frog Engystomops pustulosus (Leptodactylidae; Leiuperinae).

The amphibian family Leptodactylidae is divided into three sub-families: Leiuperinae, Leptodactylinae, and Paratelmatobiinae. Host-defense peptides (HDPs) present in the skins of frogs belonging to the Leptodactylinae have been studied extensively, but information is limited  regarding peptides from Leiuperinae species. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Tungara frog Engystomops pustulosus (Leiuperinae) collected in Trinidad led to the isolation and structural characterization of previously undescribed pustulosin-1 (FWKADVKEIG KKLAAKLAEELAKKLGEQ), [Q28E] pustulosin-1 (pustulosin-2), and pustulosin-3 (DWKETAKELLKKIGAKVAQVISDKLNPAPQ). The primary structures of these peptides do not resemble those of previously described frog skin HDPs. In addition, the secretions contained tigerinin-1EP (GCKTYLIEPPVCT) with structural similarity to the tigerinins previously identified in skin secretions from frogs from the family Dicroglossidae. Pustulosin-1 and -3 adopted extended α-helical conformations in 25% trifluoroethanol-water and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). Pustulosin-1 and -3 displayed cytotoxic activity against a range of human tumor-derived cell lines (A549, MDA-MB-231, and HT29), but their therapeutic potential for development into anti-cancer agents is limited by their comparable cytotoxic activity against non-neoplastic human umbilical vein endothelial cells. The peptides also displayed weak antimicrobial activity against Escherichia coli (MIC = 125 µM) but were inactive against Staphylococcus aureus. Tigerinin-1EP was inactive against both the tumor-derived cells and bacteria.

Antimicrobial, cytotoxic, and insulin-releasing activities of the amphibian host-defense peptide ocellatin-3N and its L-lysine-substituted analogs.

The host-defense peptide ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH2 ), first isolated from the Caribbean frog Leptodactylus nesiotus, inhibited growth of clinically relevant Gram-positive and Gram-negative bacteria as well as a strain of the major emerging yeast pathogen Candida parapsilosis. Increasing cationicity while maintaining amphipathicity by the substitution Asp4 →Lys increased potency against the microorganisms by between 4- and 16-fold (MIC ≤3 µM) compared with the naturally occurring peptide. The substitution Ala18 →Lys and the double substitution Asp4 →Lys and Ala18 →Lys had less effects on potency. The [D4K] analog also showed 2.5- to 4-fold greater cytotoxic potency against non-small-cell lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells (LC50 values in the range of 12-20 µM) compared with ocellatin-3N but was less hemolytic to mouse erythrocytes. However, the peptide showed no selectivity for tumor-derived cells [LC50 = 20 µM for human umbilical vein endothelial cells (HUVECs)]. Ocellatin-3N and [D4K]ocellatin-3N stimulated the release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥1 nM, and [A18K]ocellatin-3N, at concentrations ≥0.1 nM. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 µM, indicating that plasma membrane integrity had been preserved. The three peptides produced an increase in intracellular [Ca2+ ] in BRIN-BD11 cells when incubated at a concentration of 1 µM. In view of its high insulinotropic potency and relatively low hemolytic activity, the [A18K] ocellatin analog may represent a template for the design of agents with therapeutic potential for the treatment of patients with type 2 diabetes.

Effects of long-acting analogues of lamprey GLP-1 and paddlefish glucagon on alpha- to beta-cell transdifferentiation in an insulin-deficient transgenic mouse model.

The abilities of the long-acting, dual-agonist anti-diabetic peptides [D-Ala2 ]palmitoyl-lamprey GLP-1 and [D-Ser2 ]palmitoyl-paddlefish glucagon to induce α-cell to ß-cell transdifferentiation were investigated in GluCreERT2 ;ROSA26-eYFP mice. These animals have been genetically engineered so that yellow fluorescent protein is specifically expressed in glucagon-producing α-cells, thereby allowing cell lineage tracing. Insulin deficiency was produced by treatment of the mice with multiple low doses of streptozotocin. Administration of the peptides (twice daily intraperitoneal injections of 25 nmol/kg body weight over 10 days) to streptozotocin-treated mice produced significant (P < 0.05) increases in pancreatic insulin content and plasma insulin concentrations compared with control mice. Immunohistochemical studies demonstrated a significant (P < 0.05) increase in the % of cells staining for both insulin and fluorescent protein in islets located in the head region of the pancreas (from 10.0 ± 1.3% of total cells in untreated mice to 20.0 ± 3.85% in mice treated with D-Ala2 ]palmitoyl-lamprey GLP-1 and to 17.3 ± 1.1% in mice treated with [D-Ser2 ]palmitoyl-paddlefish glucagon). Corresponding effects upon islets in the tail region were not significant. The data indicate an improvement in ß-cell mass and positive effects on transdifferentiation of glucagon-producing to insulin-producing cells. The study provides further evidence that proglucagon-derived peptides from phylogenetical ancient fish show therapeutic potential for treatment of diabetes.

Peptidomic analysis in the discovery of therapeutically valuable peptides in amphibian skin secretions.

Introduction: The emergence of multidrug-resistant microorganisms has necessitated a search for new antimicrobial agents. Skin secretions of many frog species contain peptides that possess potent, broad-spectrum antibacterial and antifungal activities and so show promise for development into anti-infective agents. Several such peptides also possess cytokine-mediated anti-inflammatory properties and a range of anti-diabetic activities.Areas covered: A peptidomic approach, involving reversed-phase HPLC and MALDI mass spectrometry, to the comprehensive identification of peptides of potential therapeutic importance in frog skin secretions is described and its advantages over analyses involving bioassays discussed. Peptidomic studies relating to the characterization of peptides with demonstrated antimicrobial, anti-inflammatory and anti-diabetic properties in skin secretions of frogs belonging to the extensive Pipidae and Ranidae families are reviewed.Expert commentary: The initial promise of frog skin peptides as agents to treat infections produced by drug-resistant microorganisms has not been fulfilled although topical applications to treat skin diseases and a role in promoting wound healing remains a possibility. Future directions are more likely to involve the application of such peptides in the treatment of patients with sepsis and related inflammatory conditions and as a component of a therapeutic regime for Type 2 diabetes.

Insights into conformation and membrane interactions of the acyclic and dicarba-bridged brevinin-1BYa antimicrobial peptides.

Brevinin-1BYa is a 24-amino acid residue host-defense peptide, first isolated from skin secretions of the foothill yellow-legged frog Rana boylii. The peptide is of interest, as it shows broad-spectrum antimicrobial activity, and is particularly effective against opportunistic yeast pathogens. Its potential for clinical use, however, is hindered by its latent haemolytic activity. The structures of two analogues, the less haemolytic [C18S,C24S]brevinin-1BYa and the more potent cis-dicarba-brevinin-1BYa, were investigated in various solution and membrane-mimicking environments by [Formula: see text] spectroscopy and molecular modelling techniques. Neither peptide possesses a secondary structure in aqueous solution. In both the membrane-mimicking sodium dodecyl sulphate micelles and 33% 2,2,2-trifluoroethanol ([Formula: see text] solvent mixture, the peptides' structures are characterised by two [Formula: see text]-helices connected by a flexible hinge located at the [Formula: see text] residues. With the aid of molecular dynamics simulations and paramagnetic probes, it was determined that the peptides' helical segments lie parallel to the micellar surface, with their hydrophobic residues facing towards the micelle core and the hydrophilic residues pointing outwards, suggesting that both peptides exert their biological activity by a non-pore-forming mechanism. Unlike that of the dicarba analogue, the C-terminus of the acyclic peptide is only weakly associated with the micellar surface and is in direct contact with the surrounding aqueous solvent.

Structural and positional studies of the antimicrobial peptide brevinin-1BYa in membrane-mimetic environments.

Brevinin-1BYa (FLPILASLAAKFGPKLFCLVTKKC), first isolated from skin secretions of the foothill yellow-legged frog Rana boylii, shows broad-spectrum activity, being particularly effective against opportunistic yeast pathogens. The structure of brevinin-1BYa was investigated in various solution and membrane-mimicking environments by proton nuclear magnetic resonance (1 H-NMR) spectroscopy and molecular modelling. The peptide does not possess a secondary structure in aqueous solution. In a 33% 2,2,2-trifluoroethanol (TFE-d3 )-H2 O solvent mixture, as well as in membrane-mimicking sodium dodecyl sulfate and dodecylphosphocholine micelles, the peptide's structure is characterised by a flexible helix-hinge-helix motif, with the hinge located at the Gly13 /Pro14 residues, and the two α-helices extending from Pro3 to Phe12 and from Pro14 to Thr21 . Positional studies involving the peptide in sodium dodecyl sulfate and dodecylphosphocholine micelles using 5-doxyl-labelled stearic acid and manganese chloride paramagnetic probes show that the peptide's helical segments lie parallel to the micellar surface, with the residues on the hydrophobic face of the amphipathic helices facing towards the micelle core and the hydrophilic residues pointing outwards, suggesting that the peptide exerts its biological activity by a non-pore-forming mechanism.

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.

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.

Assessment of the potential of temporin peptides from the frog Rana temporaria (Ranidae) as anti-diabetic agents.

Temporin A (FLPLIGRVLSGIL-NH2 ), temporin F (FLPLIGKVLSGIL-NH2 ), and temporin G (FFPVIGRILNGIL-NH2 ), first identified in skin secretions of the frog Rana temporaria, produced concentration-dependent stimulation of insulin release from BRIN-BD11 rat clonal ß-cells at concentrations ≥1 nM, without cytotoxicity at concentrations up to 3 µM. Temporin A was the most effective. The mechanism of insulinotropic action did not involve an increase in intracellular Ca2+ concentrations. Temporins B, C, E, H, and K were either inactive or only weakly active. Temporins A, F, and G also produced a concentration-dependent stimulation of insulin release from 1.1B4 human-derived pancreatic ß-cells, with temporin G being the most potent and effective, and from isolated mouse islets. The data indicate that cationicity, hydrophobicity, and the angle subtended by the charged residues in the temporin molecule are important determinants for in vitro insulinotropic activity. Temporin A and F (1 µM), but not temporin G, protected BRIN-BD11 cells against cytokine-induced apoptosis (P < 0.001) and augmented (P < 0.001) proliferation of the cells to a similar extent as glucagon-like peptide-1. Intraperitoneal injection of temporin G (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 temporin A and F administration was without significant effect on plasma glucose levels. The study suggests that combination therapy involving agents developed from the temporin A and G sequences may find application in Type 2 diabetes treatment.

Anti-diabetic actions of esculentin-2CHa(1-30) and its stable analogues in a diet-induced model of obesity-diabetes.

Actions of esculentin-2CHa(1-30) (GFSSIFRGVAKFASKGLGKDLAKLGVDLVA) and its analogues, ([D-Arg7, D-Lys15, D-Lys23]-esculentin-2CHa(1-30) and [Lys15-octanoate]-esculentin-2CHa(1-30), were evaluated in high-fat fed NIH Swiss mice with impaired glucose tolerance and insulin resistance. Twice-daily i.p. administration of the esculentin-2CHa(1-30) peptides (75 nmol/kg body weight) or exendin-4 (25 nmol/kg) for 28 days reduced body weight, without altering cumulative energy intake. All peptides reduced blood glucose levels by 6-12 mmol/l concomitant with lower plasma insulin levels, with significance evident from day 6. All peptides improved glucose tolerance, insulin sensitivity, blood glucose profile over 24 h and decreased HbA1c to a similar extent as exendin-4. The peptides also reduced high fat diet-induced increases in plasma GLP-1 and glucagon. None of the peptides altered bone mineral density/content or lean mass but decreased fat mass. Islets isolated from peptide-treated mice exhibited improved glucose-, alanine- and GLP-1-stimulated insulin secretion. Islet morphometric analyses revealed that exendin-4 and the esculentin-2CHa(1-30) peptides significantly reduced islet, beta and alpha cell areas compared to high-fat controls. Esculentin-2CHa(1-30) peptides markedly reduced high fat diet-induced increase in beta cell proliferation and apoptosis. Peptide treatments had beneficial effects on expression of islet genes (Ins1, Slc2a2, Pdx1) and skeletal muscle genes involved in insulin action (Slc2a4, Pdk1, Irs1, Akt1). High-fat diet significantly increased LDL cholesterol which was reduced by the acylated esculentin-2CHa(1-30) analogue. Peptide treatments did not alter circulating concentrations of amylase and marker enzymes of liver function, indicating a lack of toxicity. These data indicate that esculentin-2CHa(1-30) and its analogues may be useful for improvement of blood glucose control and weight loss in 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.

The Potential of Frog Skin-Derived Peptides for Development into Therapeutically-Valuable Immunomodulatory Agents.

The aim of this article is to review the immunoregulatory actions of frog skin-derived peptides in order to assess their potential as candidates for immunomodulatory or anti-inflammatory therapy. Frog skin peptides with demonstrable immunomodulatory properties have been isolated from skin secretions of a range of species belonging to the families Alytidae, Ascaphidae, Discoglossidae, Leptodactylidae, Pipidae and Ranidae. Their effects upon production of inflammatory and immunoregulatory cytokines by target cells have been evaluated ex vivo and effects upon cytokine expression and immune cell activity have been studied in vivo by flow cytometry after injection into mice. The naturally-occurring peptides and/or their synthetic analogues show complex and variable actions on the production of proinflammatory (TNF-α, IL-1ß, IL-12, IL-23, IL-8, IFN-γ and IL-17), pleiotropic (IL-4 and IL-6) and immunosuppressive (IL-10 and TGF-ß) cytokines by peripheral and spleen cells, peritoneal cells and/or isolated macrophages. The effects of frenatin 2.1S include enhancement of the activation state and homing capacity of Th1-type lymphocytes and NK cells in the mouse peritoneal cavity, as well as the promotion of their tumoricidal capacities. Overall, the diverse effects of frog skin-derived peptides on the immune system indicate their potential for development into therapeutic agents.

Molecular mechanisms mediating the beneficial metabolic effects of [Arg4]tigerinin-1R in mice with diet-induced obesity and insulin resistance.

The frog skin host-defense peptide tigerinin-1R stimulates insulin release in vitro and improves glucose tolerance and insulin sensitivity in animal models of type 2 diabetes. This study extends these observations by investigating the molecular mechanisms of action underlying the beneficial metabolic effects of the analogue [Arg4]tigerinin-1R in mice with diet-induced obesity, glucose intolerance and insulin resistance. The study also investigates the electrophysiological effects of the peptide on KATP and L-type Ca2+ channels in BRIN-BD11 clonal ß cells. Non-fasting plasma glucose and glucagon concentrations were significantly (p<0.05) decreased and plasma insulin increased by twice daily treatment with [Arg4]tigerinin-1R (75 nmol/kg body weight) for 28 days. Oral and intraperitoneal glucose tolerance were significantly (p<0.05) improved accompanied by enhanced secretion and action of insulin. The peptide blocked KATP channels and, consistent with this, improved beta cell responses of isolated islets to a range of secretagogues. Peptide administration resulted in up-regulation of key functional genes in islets involved insulin secretion (Abcc8, Kcnj11, Cacna1c and Slc2a2) and in skeletal muscle involved with insulin action (Insr, Irs1, Pdk1, Pik3ca, and Slc2a4). These observations encourage further development of tigerinin-1R analogues for the treatment of patients with type 2 diabetes.

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

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 ß 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.

Purification, Conformational Analysis, and Properties of a Family of Tigerinin Peptides from Skin Secretions of the Crowned Bullfrog Hoplobatrachus occipitalis.

Four host-defense peptides belonging to the tigerinin family (tigerinin-1O: RICTPIPFPMCY; tigerinin-2O: RTCIPIPLVMC; tigerinin-3O: RICTAIPLPMCL; and tigerinin-4O: RTCIPIPPVCF) were isolated from skin secretions of the African crowned bullfrog Hoplobatrachus occipitalis. In aqueous solution at pH 4.8, the cyclic domain of tigerinin-2O adopts a rigid amphipathic conformation that incorporates a flexible N-terminal tail. The tigerinins lacked antimicrobial (MIC > 100 µM) and hemolytic (LC50 > 500 µM) activities but, at a concentration of 20 µg/mL, significantly (P < 0.05) inhibited production of interferon-γ (IFN-γ) by peritoneal cells from C57BL/6 mice without affecting production of IL-10 and IL-17. Tigerinin-2O and -4O inhibited IFN-γ production at concentrations as low as 1 µg/mL. The tigerinins significantly (P ≤ 0.05) stimulated the rate of insulin release from BRIN-BD11 clonal ß-cells without compromising the integrity of the plasma membrane. Tigerinin-1O was the most potent (threshold concentration 1 nM) and the most effective (395% increase over basal rate at a concentration of 1 µM). Tigerinin-4O was the most potent and effective peptide in stimulating the rate of glucagon-like peptide-1 release from GLUTag enteroendocrine cells (threshold concentration 10 nM; 289% increase over basal rate at 1 µM). Tigerinin peptides have potential for development into agents for the treatment of patients with type 2 diabetes.

Conformational Analysis of the Host-Defense Peptides Pseudhymenochirin-1Pb and -2Pa and Design of Analogues with Insulin-Releasing Activities and Reduced Toxicities.

Pseudhymenochirin-1Pb (Ps-1Pb; IKIPSFFRNILKKVGKEAVSLIAGALKQS) and pseudhymenochirin-2Pa (Ps-2Pa; GIFPIFAKLLGKVIKVASSLISKGRTE) are amphibian peptides with broad spectrum antimicrobial activities and cytotoxicity against mammalian cells. In the membrane-mimetic solvent 50% (v/v) trifluoroethanol-H2O, both peptides adopt a well-defined α-helical conformation that extends over almost all the sequence and incorporates a flexible bend. Both peptides significantly (p < 0.05) stimulate the rate of release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥ 0.1 nM but produce loss of integrity of the plasma membrane at concentrations ≥ 1 µM. Increasing cationicity by the substitution Glu(17) → l-Lys in Ps-1Pb and Glu(27) → l-Lys in Ps-2Pa generates analogues with increased cytotoxicity and reduced insulin-releasing potency. In contrast, the analogues [R8r]Ps-1Pb and [K8k,K19k]Ps-2Pa, incorporating d-amino acid residues to destabilize the α-helical domains, retain potent insulin-releasing activity but are nontoxic to BRIN-BD11 cells at concentrations of 3 µM. [R8r]Ps-1Pb produces a significant increase in insulin release rate at 0.3 nM and [K8k,K19k]Ps-2Pa at 0.01 nM. Both analogues show low hemolytic activity (IC50 > 100 µM) but retain broad-spectrum antimicrobial activity and remain cytotoxic to a range of human tumor cell lines, albeit with lower potency than the naturally occurring peptides. These analogues show potential for development into agents for type 2 diabetes therapy.

Recent advances in peptide-based therapies for obesity and type 2 diabetes.

Options for the treatment of type 2 diabetes mellitus (T2DM) and obesity have recently been expanded by the results of several large clinical trials with incretin-based peptide therapies. Most of these studies have been conducted with the glucagon-like peptide-1 (GLP-1) receptor agonist semaglutide, which is available as a once weekly subcutaneous injection and once daily tablet, and the once weekly injected dual agonist tirzepatide, which interacts with receptors for GLP-1 and glucose-dependent insulinotropic polypeptide (GIP). In individuals with T2DM these therapies have achieved reductions of glycated haemoglobin (HbA1c) by > 2% and lowered body weight by > 10%. In some studies, these agents tested in non-diabetic, obese individuals at much higher doses have lowered body weight by > 15%. Emerging evidence suggests these agents can also offer cardio-protective and potentially reno-protective effects. Other incretin-based peptide therapies in early clinical development, notably a triple GLP-1/GIP/glucagon receptor agonist (retatrutide) and a combination of semaglutide with the amylin analogue cagrilintide (CagriSema), have shown strong efficacy. Although incretin therapies can incur adverse gastrointestinal effects these are for most patients mild-to-moderate and transient but result in cessation of treatment in some cases. Thus, the efficacy of new incretin-based peptide therapies is enhancing the opportunity to control body weight and blood glucose and improve the treatment of T2DM and obesity.

Strategy for the Identification of Host-Defense Peptides in Frog Skin Secretions with Therapeutic Potential as Antidiabetic Agents.

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 ß-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.

Amphibian host-defense peptides with potential for Type 2 diabetes therapy - an updated review.

Investigations conducted since 2018 have identified several host-defense peptides present in frog skin secretions whose properties suggest the possibility of their development into a new class of agent for Type 2 diabetes (T2D) therapy. Studies in vitro have described peptides that (a) stimulate insulin release from BRIN-BD11 clonal ß-cells and isolated mouse islets, (b) display ß-cell proliferative activity and protect against cytokine-mediated apoptosis and (c) stimulate production of the anti-inflammatory cytokine IL-10 and inhibit production of the pro-inflammatory cytokines TNF-α and IL-1ß. Rhinophrynin-27, phylloseptin-3.2TR and temporin F are peptides with therapeutic potential. Studies in vivo carried out in db/db and high fat-fed mice have shown that twice-daily administration of [S4K]CPF-AM1 and [A14K]PGLa-AM1, analogs of peptides first isolated from the octoploid frog Xenopus amieti, over 28 days lowers circulating glucose and HbA1c concentrations, increases insulin sensitivity and improves glucose tolerance and lipid profile. Peptide treatment produced potentially beneficial changes in the expression of skeletal muscle genes involved in insulin signaling and islet genes involved in insulin secretion in these murine models of T2D. Lead compounds uncovered by the study of frog HDPs may provide a basis for the design of new types of agents that can be used, alone or in combination with existing therapies, for the treatment of T2D.