Skin Secretions Of Frogs Research Articles

Potential skin protective effects after UVB irradiation afforded by an antioxidant peptide from Odorrana andersonii

With increasing demand, the development of new natural antioxidants has become a primary direction of scientific research. We previously identified a short gene-encoded peptide (OA-VI12) from Odorrana andersonii frog skin secretions that exerted direct scavenging capacity against free radicals, suggesting a possible function in protecting skin against photodamage caused by their high-altitude habitat. In the current research, we examined the effects of OA-VI12 on both UVB-irradiation and hydrogen peroxide-induced oxidative stress models established with human immortalized keratinocytes. In addition, we identified the differentially expressed genes (DEGs) in the oxidative stress and OA-VI12 groups and further performed transcriptome as well as functional and pathway enrichment analyses. Results showed that OA-VI12 protected cell viability, promoted the release of catalase, and decreased the levels of lactate dehydrogenase and reactive oxygen species. Moreover, the peptide promoted the production of superoxide dismutase and glutathione, alleviated epidermis and dermis thickness, and decreased the production of light spots and collagen fibers in skin from the photo-injured mouse model. Kyoto Encyclopedia of Genes and Genomes analysis showed mitogen-activated protein kinase (MAPK) to be the most abundant signaling pathway. Gene Ontology (GO) analysis indicated that the top 55 significantly enriched GO terms mainly involved cellular processes, parts, and binding. These results revealed the beneficial role of the small molecule gene-encoding antioxidant peptide (OA-VI12) and its potential application as a protective agent against photodamage.

Peptide and non-peptide mimetics as potential therapeutics targeting oral bacteria and oral biofilms.

The development of the oral biofilm requires a complex series of interactions between host tissues and the colonizing bacteria as well as numerous interspecies interactions between the organisms themselves. Disruption of normal host–microbe homoeostasis in the oral cavity can lead to a dysbiotic microbial community that contributes to caries or periodontal disease. A variety of approaches have been pursued to develop novel potential therapeutics that are active against the oral biofilm and/or target specific oral bacteria. The structure and function of naturally occurring antimicrobial peptides from oral tissues and secretions as well as external sources such as frog skin secretions have been exploited to develop numerous peptide mimetics and small molecule peptidomimetics that show improved antimicrobial activity, increased stability and other desirable characteristics relative to the parent peptides. In addition, a rational and minimalist approach has been developed to design small artificial peptides with amphipathic α‐helical properties that exhibit potent antibacterial activity. Furthermore, with an increased understanding of the molecular mechanisms of beneficial and/or antagonistic interspecies interactions that contribute to the formation of the oral biofilm, new potential targets for therapeutic intervention have been identified and both peptide‐based and small molecule mimetics have been developed that target these key components. Many of these mimetics have shown promising results in in vitro and pre‐clinical testing and the initial clinical evaluation of several novel compounds has demonstrated their utility in humans.

Bioevaluation of Ranatuerin-2Pb from the Frog Skin Secretion of Rana pipiens and its Truncated Analogues.

Antimicrobial peptides (AMPs) are considered as a promising agent to overcome the drug-resistance of bacteria. Large numbers of AMPs have been identified from the skin secretion of Rana pipiens, including brevinins, ranatuerins, temporins and esculentins. In this study, the cDNA precursor of a broad-spectrum antimicrobial peptide, ranatuerin-2Pb, was cloned and identified. Additionally, two truncated analogues, RPa and RPb, were synthesised to investigate the structure-activity relationship of ranatuerin-2Pb. RPa lost antimicrobial activity against Candida albicans, MRSA, Enterococcus faecalis and Pseudomonas aeruginosa, while RPb retained its broad-spectrum antimicrobial activity. Additionally, ranatuerin-2Pb, RPa and RPb demonstrated inhibition and eradication effects against Staphylococcus aureus biofilm. RPb showed a rapid bacterial killing manner via membrane permeabilization without damaging the cell membrane of erythrocytes. Moreover, RPb decreased the mortality of S. aureus infected Galleria mellonella larvae. Collectively, our results suggested that RPb may pave a novel way for natural antimicrobial drug design.

Enhancing disease resistance in poplar through modification of its natural defense pathway.

Modification of the poplar defense pathway through pathogen-induced expression of an amphibian host defense peptide modulates plant innate immunity and confers robust and reliable resistance against a major poplar pathogen, Septoria musiva. Host defense peptides (HDPs), also known as cationic antimicrobial peptides, represent a diverse group of small membrane-active molecules that are part of the innate defense system of their hosts against pathogen invasion. Here we describe a strategy for development of poplar plants with enhanced HDP production and resistance to the commercially significant fungal pathogen Septoria musiva. The naturally occurring linear amphipathic α-helical HDP dermaseptin B1, which has 31 residues and originated from the skin secretion of arboreal frogs, was N-terminally modified (MsrA2) and evaluated in vitro for antifungal activity and phytotoxicity. The MsrA2 peptide inhibited germination of S. musiva conidia at physiologically relevant low micromolar concentrations that were non-toxic to poplar protoplasts. The nucleotide sequence of MsrA2, optimized for expression in plants, was introduced into the commercial hybrid poplar Populus nigra L.×P. maximowiczii A. Henry (NM6) via Agrobacterium-mediated transformation. Transgene expression was regulated by the pathogen-inducible poplar promoter win3.12T, a part of the poplar innate defense system. Most importantly, the induced accumulation of MsrA2 peptide in poplar leaves was sufficient to confer resistance against S. musiva. The antifungal resistance of plants with high MsrA2 expression and MsrA2 accumulation was strong and reproducible, and without deleterious effects on plant growth and development. These results provide an insight into development of new technologies for engineering durable disease resistance against major pathogens of poplar and other plants.

A novel membrane-disruptive antimicrobial peptide from frog skin secretion against cystic fibrosis isolates and evaluation of anti-MRSA effect using Galleria mellonella model

Antimicrobial peptides from amphibian skin secretion are a promising source for the development of alternative antibiotics against the urgent antibiotic resistance. Methicillin-resistant S. aureus (MRSA) has been found to persist in both early and late disease course of cystic fibrosis (CF). Japonicin-2LF was isolated from the skin secretion of Fujian Large-headed Frog (Limnonectes fujianensis) via the combination of cDNA cloning and MS/MS sequencing. The antimicrobial and anti-biofilm activities of Japonicin-2LF were evaluated using both reference and clinic isolated strains. The permeability of the cell membrane treated by the peptide was revealed by fluorescent staining. The cytotoxicity was examined by haemolysis, MTT and LDH assays. Wax moth larvae (Galleria mellonella) infection model was applied to assess the efficacy of Japonicin-2LF against the reference and clinic MRSA isolates in vivo. Japonicin-2LF exhibited potent antimicrobial activity, particularly against Gram-positive bacteria Staphylococcus aureus and MRSA, killing the bacteria via membrane permeabilisation. Additionally, Japonicin-2LF demonstrated the inhibition and eradication of biofilms, particularly against the biofilm of MRSA by eradicating the biofilm matrix as well as killing all the sessile bacteria. In the in vivo assay, Japonicin-2LF significantly decreased the mortality of MRSA acute infected larvae. In conclusion, it is a novel antimicrobial peptide discovered from the skin secretion of Limnonectes fujianensis, and particularly effective against both planktonic and sessile MRSA. The further in vivo study suggests that Japonicin-2LF could be a potential drug candidate to control the MRSA infection in cystic fibrosis patients.

Biochemical and Biological Profile of Parotoid Secretion of the Amazonian Rhinella marina (Anura: Bufonidae).

Skin secretions of frogs have a high chemical complexity. They have diverse types of biomolecules, such as proteins, peptides, biogenic amines, and alkaloids. These compounds protect amphibians' skin against growth of bacteria, fungi, and protozoa and participate in defense system against attack from predators. Therewith, this work performed biochemical and biological profile of macroglands parotoid secretion from cane toad. For poison analysis, we performed molecular exclusion and reverse phase chromatography, electrophoresis, and mass spectrometry. Antimicrobial, antiplasmodial, leishmanicidal, cytotoxicity, genotoxicity, and inflammatory activity of crude and/or fractions of R. marina secretion were also evaluated. Fractionation prior to filtration from poison showed separation of low mass content (steroids and alkaloids) and high molecular mass (protein). Material below 10 kDa two steroids, marinobufagin and desacetylcinobufagin, was detected. Crude extract and fractions were active against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Plasmodium falciparum, Leishmania guyanensis, and Leishmania braziliensis. Crude extract was also active against cancer cells although it was not cytotoxic for normal cells. This extract did not show significant DNA damage but it showed an important inflammatory effect in vivo. The information obtained in this work contributes to the understanding of the constituents of R. marina secretion as well as the bioactive potential of these molecules.

Activation of the Bile Acid Pathway and No Observed Antimicrobial Peptide Sequences in the Skin of a Poison Frog.

The skin secretions of many frogs have genetically-encoded, endogenous antimicrobial peptides (AMPs). Other species, especially aposematic poison frogs, secrete exogenously derived alkaloids that serve as potent defense molecules. The origins of these defense systems are not clear, but a novel bile-acid derived metabolite, tauromantellic acid, was recently discovered and shown to be endogenous in poison frogs (Mantella, Dendrobates, and Epipedobates). These observations raise questions about the evolutionary history of AMP genetic elements, the mechanism and function of tauromatellic acid production, and links between these systems. To understand the diversity and expression of AMPs among frogs, we assembled skin transcriptomes of 13 species across the anuran phylogeny. Our analyses revealed a diversity of AMPs and AMP expression levels across the phylogenetic history of frogs, but no observations of AMPs in Mantella. We examined genes expressed in the bile-acid metabolic pathway and found that CYP7A1 (Cytochrome P450), BAAT (bile acid-CoA: amino acid N-acyltransferase), and AMACR (alpha-methylacyl-CoA racemase) were highly expressed in the skin of M. betsileo and either lowly expressed or absent in other frog species. In particular, CYP7A1 catalyzes the first reaction in the cholesterol catabolic pathway and is the rate-limiting step in regulation of bile acid synthesis, suggesting unique activation of the bile acid pathway in Mantella skin. The activation of the bile acid pathway in the skin of Mantella and the lack of observed AMPs fuel new questions about the evolution of defense compounds and the ectopic expression of the bile-acid pathway.

Isolation of a trefoil factor BmTFF3 from frog skin secretions that can form a functional complex with aerolysin-like pore-forming protein in host defensive system

Isolation of a trefoil factor BmTFF3 from frog skin secretions that can form a functional complex with aerolysin-like pore-forming protein in host defensive system

Antibacterial activity of the skin secretion of Phyllomedusa azurea (Anura: Hylidae) from the Central Brazil Cerrado

Phyllomedusa azurea is a frog species well distributed geographically in South America, including Brazilian biomes as Pantanal and Cerrado. Compared with other anurans from the Phyllomedusinae family, there are few reports on the bioactive potential of skin-derived molecules from this species. In this perspective, the aim of the present study was to evaluate the in vitro antibacterial activity of skin secretion of P. azurea by detection of Minimum Inhibitory Concentration (MIC) of the growth of bacterial indicator strains and to determine if occurs a changing in the bacterial cell envelope permeability. The MIC determination was carried out by the microdilution plate method. The absorbance was measured and analyzed statistically using the t-test to compare two groups (0.05 % of significance). The impact of the crude extract on cell envelope permeability of Staphylococcus aureus ATCC 25923 was conducted by the crystal violet assay, and the absorbance was measured spectrophotometry followed by the calculation of the crystal violet uptake percentage. The specific MIC for S. aureus ATCC 25923 and Escherichia coli ATCC 25922 was 31.25 µg/mL, while for Bacillus subtilis ATCC 6633 was 125 µg/mL and Pseudomonas aeruginosa ATCC 27853 was 250 µg/mL. The treatment with crescent concentrations of frog skin secretion increased the crystal violet uptake by S. aureus ATCC 25923 cells, suggesting an action on the cell plasma membrane. The results demonstrated that the skin secretion of P. azurea presents antibacterial activity and merit further investigations to characterize the bioactive molecules.

Structure-activity relationship of an antimicrobial peptide, Phylloseptin-PHa: balance of hydrophobicity and charge determines the selectivity of bioactivities.

BackgroundAntimicrobial peptides (AMPs) from the skin secretions of amphibians are now considered as a potential alternative to conventional antibiotics. Phylloseptins are a family of AMPs identified in the skin secretions of Phyllomedusinae tree frogs which exhibit highly conserved structural characteristics. This study examines the structure–activity relationship of the newly discovered phylloseptin, Phylloseptin-PHa (PSPHa) from Pithecopus hypochondrialis.Materials and methodsPSPHa and modified analogs were produced by solid phase synthesis and purified by reverse-phase HPLC. Rationally designed modified analogs incorporating changes in significant physicochemical parameters such as hydrophobicity, hydrophobic moment and net charge were investigated to determine their influence on secondary structure, antimicrobial activity, membrane permeabilization and cytotoxicity.ResultsOverall, we found that when rationally designing AMPs by altering their primary structure it is important to keep a balance between hydrophobicity and charge.ConclusionThis study provides new insights which will help in the future development of AMPs as alternatives to conventional antibiotics for the treatment of Staphylococcus aureus and methicillin-resistant S. aureus infections.

Preliminary investigation of antimicrobial activity of the skin secretions of frogs from Bangladesh

Abstract Amphibians, like some animals and plants, defend themselves against various pathogenic organisms by producing and secreting various peptides and small molecules from granular skin glands. In this study, we evaluated for the first time, the antibiotic activity of the skin secretions of 8 different frog species (Euphlyctis cyanophlyctis, E. hexadactylus, Fejervarya teraiensis, F. asmati, F. syhadrensis, Hoplobatrachus tigerinus, Microhyla ornata and Polypedates leucomystax) from Bangladesh. These secretions were collected by a nonlethal approach through chemical stimulation and the antibacterial activity was evaluated by broth macrodilution method against some Gram-positive and Gram-negative bacteria. Our study revealed that all the skin secretions (8 out of 8) from the selected frogs have antimicrobial activity against Gram-positive bacteria and 7 out of 8 skin secretions possess antibacterial activity against Gram-negative bacteria tested. Further analysis of data showed that these secretions are significantly more effective against Gram-positive bacteria than Gram-negative bacteria.

Batrachotoxin acts as a stent to hold open homotetrameric prokaryotic voltage-gated sodium channels.

Batrachotoxin (BTX), an alkaloid from skin secretions of dendrobatid frogs, causes paralysis and death by facilitating activation and inhibiting deactivation of eukaryotic voltage-gated sodium (Nav) channels, which underlie action potentials in nerve, muscle, and heart. A full understanding of the mechanism by which BTX modifies eukaryotic Nav gating awaits determination of high-resolution structures of functional toxin-channel complexes. Here, we investigate the action of BTX on the homotetrameric prokaryotic Nav channels NaChBac and NavSp1. By combining mutational analysis and whole-cell patch clamp with molecular and kinetic modeling, we show that BTX hinders deactivation and facilitates activation in a use-dependent fashion. Our molecular model shows the horseshoe-shaped BTX molecule bound within the open pore, forming hydrophobic H-bonds and cation-π contacts with the pore-lining helices, leaving space for partially dehydrated sodium ions to permeate through the hydrophilic inner surface of the horseshoe. We infer that bulky BTX, bound at the level of the gating-hinge residues, prevents the S6 rearrangements that are necessary for closure of the activation gate. Our results reveal general similarities to, and differences from, BTX actions on eukaryotic Nav channels, whose major subunit is a single polypeptide formed by four concatenated, homologous, nonidentical domains that form a pseudosymmetric pore. Our determination of the mechanism by which BTX activates homotetrameric voltage-gated channels reveals further similarities between eukaryotic and prokaryotic Nav channels and emphasizes the tractability of bacterial Nav channels as models of voltage-dependent ion channel gating. The results contribute toward a deeper, atomic-level understanding of use-dependent natural and synthetic Nav channel agonists and antagonists, despite their overlapping binding motifs on the channel proteins.

Alanine Scanning Studies of the Antimicrobial Peptide Aurein 1.2

Antimicrobial peptides (AMPs) are compounds widely distributed in nature that display activity against a broad spectrum of pathogens. Amphibian skin, as an organ rich in pharmacologically active peptides, appears to be an interesting source of novel AMPs. Aurein 1.2 (GLFDIIKKIAESF-NH2) is a short 13-residue antimicrobial peptide primarily isolated from the skin secretions of Australian bell frogs. In this study, the alanine scan of aurein 1.2 was performed to investigate the effect of each amino acid residue on its biological and physico-chemical properties. The biological studies included determination of minimum inhibitory concentration, activity against biofilm, and inhibitory effect on its formation. Moreover, the hemolytic activity as well as serum stability was determined. The hydrophobicity of peptides and their self-assembly were investigated using reversed-phase chromatography. In addition, their helicity was calculated from circular dichroism spectra. The results not only provided information on structure-activity relationship of aurein 1.2 but also gave insights into design of novel analogs of AMPs in the future.

Skin secretion transcriptome remains in chromatographic fractions suitable for molecular cloning

Traditional sources of mRNA for molecular cloning on amphibian skin secretion studies have been the frog's skin and skin secretions. Here, we demonstrate that mRNA isolated from chromatographic fractions of skin secretions is amenable for molecular cloning assays. We identified precursor sequences of the Arg0, Trp5, Leu8-bradykinin and six antimicrobial peptides of Pelophylax esculentus (Ranidae). These results show that both transcriptomic and peptidomic analyses can be performed with a single sample reducing in half the amount of starting skin secretion required. This is a significant advantage when working with endangered or very rare amphibian species, where minimal samples are available.

YANTIMICROBIAL EFFECT OF CRUDE FROG (RANA RIDIBUNDA) SKIN SECRETIONS EXTRACT ON WOUNDS HEALING IN MICE

Amphibians cutaneous glands spread over the skin which contain different bioactive substances. So, the aim of this study is to study the effects of frog skin secretions on parasites and pathogenic bacterial isolates. Five adult frogs (Rana ridibunda) from AL-Jadria streams in Baghdad used in this study. The frogs were stimulated by single intraperitoneal injection of norepinephrine-HCl (40ng/gm) body weight. Frogs skin glands secreted bioactive substances then washed .The washing solution was centrifuged, lyophilized and re-suspended and sterilized. The result of the study revealed that the concentration (500mg/ml) inhibited bacterial growth so as Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Fungi so as Candida albicans, while the concentration (250mg/ml) inhibited the growth of Salmonella typhi. All concentrations of crude extract were cytotoxic for both of Leishmania tropica and Leishmania donovani cell culture by the evidence of significant regression and correlation (P <0.071).The treatment with crud extract (500mg/ml) was active to stimulate tissue cells proliferation and eradicate pathogens inside the wound, thereby may lead to enhance healing of cutaneous wounds.

Unveiling the Multifaceted Mechanisms of Antibacterial Activity of Buforin II and Frenatin 2.3S Peptides from Skin Micro-Organs of the Orinoco Lime Treefrog (Sphaenorhynchus lacteus).

Amphibian skin is a rich source of natural compounds with diverse antimicrobial and immune defense properties. Our previous studies showed that the frog skin secretions obtained by skin micro-organs from various species of Colombian anurans have antimicrobial activities against bacteria and viruses. We purified for the first time two antimicrobial peptides from the skin micro-organs of the Orinoco lime treefrog (Sphaenorhynchus lacteus) that correspond to Buforin II (BF2) and Frenatin 2.3S (F2.3S). Here, we have synthesized the two peptides and tested them against Gram-negative and Gram-positive bacteria, observing an effective bactericidal activity at micromolar concentrations. Evaluation of BF2 and F2.3S membrane destabilization activity on bacterial cell cultures and synthetic lipid bilayers reveals a distinct membrane interaction mechanism. BF2 agglutinates E. coli cells and synthetic vesicles, whereas F2.3S shows a high depolarization and membrane destabilization activities. Interestingly, we found that F2.3S is able to internalize within bacterial cells and can bind nucleic acids, as previously reported for BF2. Moreover, bacterial exposure to both peptides alters the expression profile of genes related to stress and resistance response. Overall, these results show the multifaceted mechanism of action of both antimicrobial peptides that can provide alternative tools in the fight against bacterial resistance.

Discovery of Novel Caeridins from the Skin Secretion of the Australian White's Tree Frog, Litoria caerulea.

Abundant biologically active peptides have been discovered from frog skin secretions, a rich natural source of bioactive compounds with great potential in drug discovery. In this study, three Caeridin peptides, namely, Caeridin-1, S5-Caeridin-1, and Caeridin-a1, were discovered from the skin secretion of the Australian White's tree frog, Litoria caerulea, for the first time, by means of combining transcriptomic and peptidomic analyses. It also represents the first report on bioactive Caeridins since this family of peptides was initially studied 20 years ago. Chemically synthetic versions of each natural Caeridin demonstrated promising bioactivities either on rat smooth muscles or against microbial growth. Specifically, Caeridin-1 produced contraction of rat bladder smooth muscle, while S5-Caeridin-1 induced relaxation of rat ileum smooth muscle, both at nanomolar concentrations. Moreover, Caeridin-a1 was shown to potently inhibit the growth of the planktonic Gram-positive bacteria Staphylococcus aureus (S. aureus), methicillin-resistant S. aureus (MRSA), and Enterococcus faecalis (E. faecalis), the Gram-negative bacterium, Escherichia coli (E. coli), and the yeast, Candida albicans (C. albicans). The discovery of these Caeridins may induce further intensive and systematic studies of frog skin peptides to promote the discovery of natural templates as lead compounds for drug discovery and therapeutic application.

Biological Properties, Current Applications and Potential Therapeautic Applications of Brevinin Peptide Superfamily.

The Brevinin peptides are antimicrobial agents obtained from frog skin secretions. Brevinin-2R has attracted many attentions due to its very low hemolytic activity, cationic property, and high affinity to cancer cells. Moreover, it has shown little toxicity against normal mammalian cells, while having killed several tumor cell lines by activation of lysosome-mitochondrial death pathway. In this review, we introduced the Brevinin superfamily with a focus on its therapeutic applications. Next, some unique properties of Brevinins were briefly discussed, including their ability to stimulate insulin secretion, dendritic cell maturation, and wound healing. In this context, we also provide information about the decoration of nanoparticles, such as cerium nano-oxide, by Brevinins. Finally, we addressed their potential for anti-tumor and drug design applications.

Role of Disulfide Bonds in Activity and Stability of Tigerinin-1R.

Tigerinin-1R (Arg–Val–Cys–Ser–Ala–Ile–Pro–Leu–Pro–Ile–Cys–His–NH2), a cationic 12-mer peptide containing a disulfide bond extracted from frog skin secretions, lacks antibacterial activity, but has the ability to stimulate insulin release both in vitro and in vivo. To study the structure–function relationships of tigerinin-1R, we designed and synthesized five analogs, including tigerinin-cyclic, tigerinin-1R-L4, tigerinin-linear, [C3K]tigerinin-1R, and [C11K]tigerinin-1R. Tigerinin-1R promoted insulin secretion in a concentration-dependent manner in INS-1 cells without obvious cytotoxicity. At a concentration of 10−5 M, [C11K]tigerinin-1R exhibited the highest stimulation ability, suggesting that the positive charge at the C-terminus may contribute to the in vitro insulin-releasing activity of tigerinin-1R. Tigerinin-1R peptides stimulated insulin release in INS-1 cells through a universal mechanism that involves mobilization of intracellular calcium without disrupting the cell membrane. In vivo experiments showed that both tigerinin-1R and [C11K]tigerinin-1R improved glucose tolerance in overnight-fasted mice. Due to its structural stability, tigerinin-1R showed superior hypoglycemic activity to [C11K]tigerinin-1R, which suggested a critical role of the disulfide bonds. In addition, we also identified a protective effect of tigerinin-1R peptides in apoptosis induced by oxidative stress. These results further confirm the potential for the development of tigerinin-1R as an anti-diabetic therapeutic agent in clinical practice.

Identification of Components in Frog Skin Secretions with Therapeutic Potential as Antidiabetic Agents.

Several peptides that were first identified on the basis of their antimicrobial or immunomodulatory 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 in norepinephrine-stimulated skin secretions from a range of frog species. The methodology involves fractionation of the secretions by reversed-phase HPLC, 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, identification of active peptides in the mass range 1-6kDa by MALDI-TOF mass spectrometry, purification of the peptides to near homogeneity by further HPLC, and structural characterization by automated Edman degradation. The effect of synthetic replicates of the active peptides on glucose homeostasis in vivo may be evaluated in mice fed a high fat diet to produce obesity, glucose intolerance, and insulin resistance.