A glycine-leucine-rich peptide structurally related to the plasticins from skin secretions of the frog Leptodactylus laticeps (Leptodactylidae)

International audienceA glycine-leucine-rich peptide was isolated from norepinephrine-stimulated skin secretions of the Sante Fe frog Leptodactylus laticeps (Leptodactylidae) whose primary structure (Gly-Leu-Val-Asn-Gly-Leu-Leu-Ser-Ser-Val-Leu-Gly-Gly-Gly-Gln-Gly-Gly-Gly-Gly-Leu-Leu-Gly-Gly-Ile-Leu) contains the (GXXXG)(3) motif found in the plasticins, previously identified only in phyllomedusid frogs (Hylidae). Circular dichroism studies showed that the secondary structure of the peptide, termed plasticin-L1, was markedly solvent-dependent displaying a random coil conformation in water, a beta-sheet structure in methanol, and an alpha-helical conformation in 50% trifluoroethanol-water. A synthetic replicate of the peptide did not inhibit the growth of Escherichia coli or Staphylococcus aureus or lyse human erythrocytes at concentrations up to 500 microM. At relatively high concentrations (>or=1 microM), the peptide produced a significant (PAsp. Ocellatin-L2 was devoid of antimicrobial and hemolytic activity but also showed significant activity in stimulating insulin release from BRIN-BD11 cells (181% of basal rate at 3 microM)

Muscarinic receptor subtypes mediate stimulatory and paradoxical inhibitory effects on an insulin-secreting β cell line

Acetylcholine (ACh), a major neurotransmitter from the autonomic nervous system, regulates the cholinergic stimulation of insulin secretion, through interactions with muscarinic receptors. The present study has characterised the individual involvement of muscarinic receptor subtypes in ACh-induced insulin secretion, using clonal β cells and selective muscarinic receptor antagonists. BRIN BD11 cells clearly expressed mRNA encoding m1–m4 whereas m5 was not detected by RT-PCR. Insulin release was measured from BRIN BD11 cells treated with ACh in the presence of muscarinic receptor antagonists at concentrations ranging from 3 nM to 1 μM. 300 nM of muscarinic toxin-3 (M4 antagonist) and 1 μM of methoctramine (M2 antagonist) increased ACh (100 μM) stimulated insulin secretion by 168% and 50% respectively (ANOVA, P<0.05). The antagonists alone had no effect on insulin secretion. In contrast, 300 nM of pirenzepine (M1 antagonist) and 30 nM of hexahydro-sila-difenidol p-fluorohydrochloride (M3 antagonist) inhibited ACh stimulation by 91% and 84% respectively (ANOVA, P<0.01). It is concluded that ACh acts on different receptor subtypes producing both a stimulatory and an inhibitory action on insulin release

Time-correlation between membrane depolarization and intracellular calcium in insulin secreting BRIN-BD11 cells: studies using FLIPR

Cytoplasmic Ca2+ ([Ca2+]i) and membrane potential changes were measured in clonal pancreatic beta cells using a fluorimetric imaging plate reader (FLIPR). KCl (30 mM) produced a fast membrane depolarization immediately followed by increase of [Ca2+]i in BRIN-BD11 cells. l-Alanine (10 mM) but not l-arginine (10 mM) mimicked the KCl profile and also produced a fast membrane depolarization and elevation of [Ca2+]i. Conversely, a rise in glucose from 5.6 mM to 11.1 or 16.7 mM induced rapid membrane depolarization, followed by a slower and delayed increase of [Ca2+]i. GLP-1 (20 nM) did not affect membrane potential or [Ca2+]i. In contrast, acetylcholine (ACh, 100 μM) induced fast membrane depolarization immediately followed by a modest [Ca2+]i increase. When extracellular Ca2+ was buffered with EGTA, ACh mobilized intracellular calcium stores and the [Ca2+]i increase was reduced by 2-aminoethoxydiphenyl borate but not by dantrolene, indicating the involvement of inositol triphosphate receptors (InsP3R). It is concluded that membrane depolarization of beta cells by glucose stimulation is not immediately followed by elevation of [Ca2+]i and other metabolic events are involved in glucose induced stimulus-secretion coupling. It is also suggested that ACh mobilizes intracellular Ca2+ through store operated InsP3R

Insulin-releasing and cytotoxic properties of the frog skin peptide, tigerinin-1R: a structure–activity study

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A potent, non-toxic insulin-releasing peptide isolated from an extract of the skin of the Asian frog, Hylarana guntheri (Anura:Ranidae)

International audiencePeptides in extract of the skin of the Asian frog Hylarana guntheri Boulenger,1882 were purified by reversed-phase HPLC and individual components analysed for their ability to release insulin from the rat BRIN-BD11 clonal beta cell line. The most potent peptide identified in the extract belonged to the brevinin-2 family (brevinin-2GUb; GVIIDTLKGAAKTVAAELLRKAHCKLTNSC). Other peptides with weaker insulin-releasing activity belonged to the brevinin-1 (2 peptides), brevinin-2 (2 peptides) and temporin (3 peptides) families. Only the brevinin-1 peptides showed cytolytic activity against the BRIN-BD11 cells, as demonstrated by an increased rate of release of the cytosolic enzyme, lactate dehydrogenase. A synthetic replicate of brevinin-2GUb produced a significant stimulation of insulin release (139% of basal rate; P<0.05) at a concentration of 100 nM with a maximum response of 373% of basal rate at a concentration of 3 microM) by a mechanism that did not involve mobilization of intracellular calcium. Brevinin-2GUb also inhibited the growth of microorganisms (MIC against Escherichia coli=32 microM, Staphylococcus aureus=64 microM, and Candida albicans=64 microM) but had only weak hemolytic activity against human erythrocytes (LC(50)=700 microM). Administration of brevinin-2GUb (75 nmol/kg body weight) into mice significantly (P<0.05) improved glucose tolerance following a intraperitoneal injection of glucose, thereby demonstrating that the peptide shows potential for development into a therapeutically valuable agent for the treatment of Type 2 diabetes

Cooperative enhancement of insulinotropic action of GLP-1 by acetylcholine uncovers paradoxical inhibitory effect of beta cell muscarinic receptor activation on adenylate cyclase activity

The cooperative effect of glucagon-like peptide 1 (GLP-1) and acetylcholine (ACh) was evaluated in a beta cell line model (BRIN BD11). GLP-1 (20 nM) and ACh (100 μM) increased insulin secretion by 24–47%, whereas in combination there was a further 89% enhancement of insulin release. Overnight culture with 100 ng/mL pertussis toxin (PTX) or 10 nM PMA significantly reduced the combined insulinotropic action (P&lt;0.05 and P&lt;0.001, respectively) and the sole stimulatory effects of GLP-1 (PTX treatment; P&lt;0.01) or ACh (PMA treatment; P&lt;0.05). Under control conditions, ACh (50 nM–1 mM) concentration-dependently inhibited by up to 40% (P&lt;0.001) the 10-fold (P&lt;0.001) elevation of cyclic 3′,5′-adenosine monophosphate (cAMP) induced by 20 nM GLP-1. The paradoxical inhibitory action of ACh was abolished by PTX pre-treatment, suggesting involvement of Gi and/or Go G protein alpha subunit. Effects of selective muscarinic receptor antagonists on the concentration-dependent insulinotropic actions of ACh (50 nM–1 mM) on 20 nM GLP-1 induced insulin secretion revealed inhibition by ρ-FHHSiD (M3 antagonist, P&lt;0.05), stimulation with pirenzepine (M1 antagonist, P&lt;0.001) and no significant effects of either methoctramine (M2 antagonist) or MT-3 (M4 antagonist). Antagonism of M2, M3 and M4 muscarinic receptor effects with methoctramine (3–100 nM), ρ-FHHSiD (3–30 nM) or MT-3 (10–300 nM) did not significantly affect the inhibitory action of ACh on GLP-1 stimulated cAMP production. In contrast, M1 receptor antagonism with pirenzepine (3–300 nM) resulted in a concentration-dependent decrease in the inhibitory action of ACh on GLP-1 stimulated cAMP production (P&lt;0.001). These data indicate an important functional cooperation between the cholinergic neurotransmitter ACh and the incretin hormone GLP-1 on insulin secretion mediated through the M3 muscarinic receptor subtype. However, the insulinotropic action of ACh was associated with a paradoxical inhibitory effect on GLP-1 stimulated cAMP production, achieved through a novel PTX- and pirenzepine-sensitive M1 muscarinic receptor activated pathway. An imbalance between these pathways may contribute to dysfunctional insulin secretion

Peptidomic analysis of skin secretions from the bullfrog Lithobates catesbeianus (Ranidae) identifies multiple peptides with potent insulin-releasing activity

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