Peptides (v.29, #6)

Dendritic cell acquisition of epitope cargo mediated by simple cationic peptide structures by Brendon Y. Chua; Emily M. Eriksson; Daniel P. Poole; Weiguang Zeng; David C. Jackson (881-890).
In this study we evaluate the uptake by murine dendritic cells (DCs) of different synthetic, branched cationic peptide structures with a view to facilitating peptide epitope delivery. The level of cell uptake by fluorescenated peptides was measured by flow cytometry following quenching of extracellular fluorescence with trypan blue. Branched peptides containing either N-terminal arginine or N-terminal lysine residues were able to mediate cell entry but the peptide containing four arginine residues in a branching configuration (R4) was found to be superior not only to other branched peptides in translocating to the cell interior and also to a peptide containing four arginine residues arranged linearly. Fluorescenated R4 was found to be localized within intracellular vesicle-like compartments as well as being distributed throughout the cell cytoplasm. Uptake of R4 utilized an energy-dependent process that appeared to involve phosphatidylinositol-3-kinase and could induce intermediate levels of DC maturation. R4 when conjugated to a T-helper cell and CTL epitope construct was able to induce antigen-specific CD8+ T-cell mediated immune responses in mice when administered in adjuvant as were DCs that were pulsed with this construct and then matured with LPS. Fluorescenated R4 was also found to translocate into the interior of other cell types indicating that it may be useful for the delivery of peptide cargo into other specialized cells.
Keywords: Dendritic cells; Synthetic vaccines; Cell entry; Peptide epitope;

The effect of pea albumin 1F on glucose metabolism in mice by Xin-Peng Dun; Fa-Fang Li; Jian-He Wang; Zheng-Wang Chen (891-897).
Pea albumin 1F (PA1F), a plant peptide isolated from pea seeds, can dramatically increase blood glucose concentration by subcutaneous injection with a dosage of 5 or 10 μg/g (body weight) in normal and type II diabetic mice (KK/upj-Ay). The voltage-dependent anion channel 1 (VDAC-1) has been identified as the PA1F binding protein from mice pancreatic cell membrane, which may be involved in the regulation of enhancing blood glucose in response to PA1F binding. The results clearly show that peptide-signaling molecules from plants can affect mammalian physiological functions, especially, in association with glucose metabolism.
Keywords: Pea albumin 1F; Isoforms; Glucose metabolism; Voltage-dependent anion channel 1 (VDAC-1);

Involvement of nitric oxide in insulin induced memory improvement by S. Choopani; M. Moosavi; N. Naghdi (898-903).
Although brain was considered as an insulin-insensitive organ, recently it has appeared that insulin has some interesting effects on some brain regions like hippocampus. It has been known that intra-hippocampally administered insulin can improve learning and memory. Knowing that insulin can stimulate nitric oxide (NO) synthesis via eNOS activation and also that NO synthase (NOS) inhibitors can affect learning and memory, the aim of this study was to assess if NO is involved in insulin induced memory improvement. Wistar male rats were intra-CA1 cannulated and the effect of post-training and pre-probe trial intra-hippocampal administration of N-nitro-l-arginine methyl ester (l-NAME) (5, 10, 30 μg), insulin +  l-NAME ±  l-arginine were assessed in a single-day testing version of Morris water maze (MWM) task. Our results show that, l-NAME can prevent insulin induced memory improvement. This drug had no effect on escape latency of a non-spatial visual discrimination task. Therefore, it seems that endogenous nitric oxide has a role in spatial learning and memory improvement caused by insulin.
Keywords: Insulin; Nitric oxide; Memory; Rat; Morris water maze;

Regulation of ghrelin structure and membrane binding by phosphorylation by Eva Dehlin; Jianhua Liu; Samuel H. Yun; Elizabeth Fox; Sandra Snyder; Cyrille Gineste; Leslie Willingham; Mario Geysen; Bruce D. Gaylinn; Julianne J. Sando (904-911).
The peptide hormone ghrelin requires Ser-3 acylation for receptor binding, orexigenic and anti-inflammatory effects. Functions of desacylghrelin are less well understood. In vitro kinase assays reveal that the evolutionarily conserved Ser-18 in the basic C-terminus is an excellent substrate for protein kinase C. Circular dichroism reveals that desacylghrelin is ∼12% helical in aqueous solution and ∼50% helical in trifluoroethanol. Ser-18-phosphorylation, Ser-18-Ala substitution, or Ser-3-acylation reduces the helical character in trifluoroethanol to ∼24%. Both ghrelin and desacylghrelin bind to phosphatidylcholine:phosphatidylserine sucrose-loaded vesicles in a phosphatidylserine-dependent manner. Phosphoghrelin and phosphodesacylghrelin show greatly diminished phosphatidylserine-dependent binding. These results are consistent with binding of ghrelin and desacylghrelin to acidic lipids via the basic face of an amphipathic helix with Ser-18 phosphorylation disrupting both helical character and membrane binding.
Keywords: Ghrelin structure; Ghrelin phosphorylation; Peptide-membrane binding; PKC substrate;

Effects of ghrelin on neuronal activity in the ventromedial nucleus of the hypothalamus in infantile rats: An in vitro study by Hiroki Yanagida; Takefumi Morita; Juhyon Kim; Keitaro Yoshida; Kazuki Nakajima; Yutaka Oomura; Matthew J. Wayner; Kazuo Sasaki (912-918).
Ghrelin is an endogenous ligand for the growth hormone (GH) secretagogue (GHS) receptor (GHS-R) and a potent stimulant for GH secretion even in infantile rats before puberty. Although the ventromedial nucleus of the hypothalamus (VMH) might be a site of action for ghrelin to induce GH release, the electrophysiological effect of ghrelin on VMH neurons in infantile rats remains to be elucidated. Thus, the purpose of the present study was to investigate the effect of ghrelin on VMH neurons using hypothalamic slices of infantile rats. Ghrelin excited a majority of VMH neurons in a concentration-dependent manner. VMH neurons that were excited by GH releasing peptide-6 (GHRP-6), a synthetic GHS, were also excited by ghrelin and vice versa. Repeated application of ghrelin to the same VMH neuron decreased progressively the excitatory responses depending on the number of times it was administered. The excitatory effect of ghrelin on VMH neurons in normal artificial cerebrospinal fluid (ACSF) persisted in low Ca2+-high Mg2+ ACSF. The present results indicate that (1) ghrelin excites a majority of VMH neurons dose-dependently and postsynaptically and (2) the excitatory effects of ghrelin are mimicked by GHRP-6 and desensitized by repeated applications of ghrelin.
Keywords: Ghrelin; VMH; GHS; GHRP-6; GHRH; Neuronal activity; Infantile rats;

Novel stable PACAP analogs with potent activity towards the PAC1 receptor by Steve Bourgault; David Vaudry; Béatrice Botia; Alain Couvineau; Marc Laburthe; Hubert Vaudry; Alain Fournier (919-932).
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38- or 27-amino acid neuropeptide with promising therapeutic applications for the treatment of several pathophysiological states related to neurodegenerative diseases. However, its use for therapeutic applications is actually limited by its restricted bioavailability and rapid degradation. Therefore, metabolically stable PACAP analogs represent promising tools to further investigate the physiological roles of PACAP and ascertain its usefulness in some clinical conditions. In this study, derivatives of PACAP27 and PACAP38 have been rationally designed to develop PAC1 receptor agonists resistant to peptidase action. Results showed that N-terminal modifications confer resistance to dipeptidyl peptidase IV, a major proteolytic process involved in PACAP degradation. Moreover, in vitro incubation of both PACAP isoforms in human plasma revealed that PACAP38 is rapidly metabolized, with a half-life of less than 5 min, while PACAP27 was stable in these experimental conditions. Hence, following the elucidation of its plasmatic metabolites, PACAP38 was modified at its putative endopeptidase and carboxypeptidase sites of cleavage. All peptide analogs were tested for their ability to bind the PAC1 receptor, as well as for their potency to induce calcium mobilization and inhibit PC12 cell proliferation through the PAC1 receptor. This approach revealed two leading compounds, i.e. acetyl-[Ala15, Ala20]PACAP38-propylamide and acetyl-PACAP27-propylamide, which exhibited improved metabolic stability and potent biological activity. This study describes innovative data related to PACAP metabolism in human plasma and depicts the development of a metabolically stable PACAP38 analog, acetyl-[Ala15, Ala20]PACAP38-propylamide, which behaves as a super-agonist towards the PAC1 receptor.
Keywords: PACAP; Stable analogs; PAC1 receptor agonists; Dipeptidyl peptidase IV; Plasma metabolites;

The endogenous brain opioid system is believed to play an important role in mediating reward mechanisms. Opioid innervation is high in many limbic regions and reinforcing actions of many drugs of abuse, including cocaine, are thought to be mediated via endogenous opioid system. The aim of the present study was to indicate whether the anti-opioid peptide, neuropeptide FF (NPFF; FLFQPQRF-NH2) was able to modify the rewarding effect of cocaine (5 mg/kg) measured in the expression of conditioned place preference (CPP) test in rats and the expression of sensitization to hyperlocomotor effect of cocaine (10 mg/kg) in mice. Our results indicate that NPFF (5, 10, and 20 nmol) given intracerebroventricularly (i.c.v.) inhibited the expression of cocaine-induced CPP at the dose of 10 nmol (P  < 0.01) and 20 nmol (P  < 0.001). Moreover, NPFF inhibited the expression of cocaine-induced sensitization to its hyperlocomotor effect at the dose of 20 nmol (P  < 0.05) and acute hyperlocomotor effect of cocaine at doses of 5 nmol (P  < 0.01), 10 nmol (P  < 0.01), and 20 nmol (P  < 0.05). Our study suggests that NPFF may participate in a rewarding effect of cocaine measured in the CPP paradigm. On the other hand, our experiments indicate that NPFF is involved in the mechanism of expression of sensitization to cocaine hyperlocomotion but this effect seems to be non-specific because NPFF also inhibited the acute hyperlocomotor effect of cocaine.
Keywords: Neuropeptide FF (NPFF); Cocaine; Conditioned place preference; Sensitization; Locomotion;

Hippocampal asymmetry in exploratory behavior to vasoactive intestinal polypeptide by Margarita Ivanova; Alexandar Ternianov; Stiliana Belcheva; Roman Tashev; Negrin Negrev; Iren Belcheva (940-947).
The effects of vasoactive intestinal polypeptide (VIP) microinjected uni- or bilaterally into the CA1 hippocampal area of male Wistar rats at a dose of 10, 50 and 100 ng on exploratory behavior were examined. VIP microinjected bilaterally at a high dose (100 ng) significantly decreased the horizontal movements, while at low doses (10 and 50 ng) had no effect on the exploratory activity. Microinjections of VIP into the left hippocampal CA1 area at doses 50 and 100 ng suppressed the exploratory activity, while right-side VIP administration at a dose 100 ng significantly increased horizontal movements compared to the respective controls. Vertical activity was stimulated only by VIP administered into the right hippocampal CA1 area at the three doses used. Neither bilateral nor left injections of VIP induced changes in the vertical movements. The main finding was the presence of hippocampal asymmetry in exploratory behavior to unilateral microinjections of VIP depending on the dose and the microinjected hemisphere.
Keywords: Vasoactive intestinal polypeptide; Hippocampus; Asymmetry; Exploratory behavior; Rat;

VIP balances innate and adaptive immune responses induced by specific stimulation of TLR2 and TLR4 by Alicia Arranz; Yasmina Juarranz; Javier Leceta; Rosa P. Gomariz; Carmen Martínez (948-956).
Crohn's disease (CD) is a chronic intestinal inflammatory pathology, which develops as a result of innate immune signals, such as the activation of Toll-like receptors (TLRs), and adaptive immune signals, including Th1 cytokine release. We have recently demonstrated in TNBS-induced colitis, a murine model of CD, that VIP plays a homeostatic role, by reducing TNBS-induced TLR2 and TLR4 expression to control levels. The purpose of this paper is to elucidate for the first time, the physiological relevance of VIP specific control of innate and adaptive immune responses through TLR2 and TLR4 ligands. In addition, we investigated the effect of VIP on regulatory activity of T regulatory (Treg) cells in the TNBS-colitis model. First, we found that VIP downregulated the inflammatory response elicited in mesenteric lymph node cell cultures by treatment with the TLR2 ligand Pam3Cys, or the TLR4 ligand lipopolysaccharide (LPS), reducing the production of the chemokine CXCL1. Also, treatment with VIP impaired the induction of Th1 responses by decreasing p70 interleukin (IL)-12 and interferon gamma (IFN-γ) levels after TLR2/TLR4 stimulation in culture. Besides, VIP treatment restored in vivo the numbers of TLR2 and TLR4 positive CD4+CD25+ T lymphocytes, augmented by TNBS administration, and increased the expression of molecules involved in regulatory T cell function, such as Foxp3 and TGF-β. In conclusion, the ability of VIP to down-regulate uncontrolled inflammation by targeting TLR-mediated responses and regulatory T cell activity could be used as a new alternative therapy for intestinal inflammatory/autoimmune disorders.
Keywords: Crohn's disease; Lymph node; LPS; Pam3Cys; TLRs;

A non-variable L1-peptide displays high sensitivity and specificity for detecting women having human papillomavirus-associated cervical lesions by Mauricio Urquiza; Tatiana Guevara; Ricardo Sanchez; Magnolia Vanegas; Manuel E. Patarroyo (957-962).
Anti-human papillomavirus (HPV) antibody detection is promising technique for detecting women at risk of suffering cervical cancer, since potentially oncogenic, persistent, long-term HPV-infections elicit an antibody response which is rarely detected in transitory HPV-infection patients. We have identified a non-variable C-terminus L1-peptide, belonging to an α-helix surface exposed on L1-protein, specifically recognized by antibodies from HPV-associated cervical lesion patients. This peptide tested against 313 sera presented higher reactivity with antibodies from cervical cancer (OD mean 0.43 ± 0.13) or cervical lesion patients (OD mean 0.41 ± 0.17) than antibodies from normal cytology patients (OD mean 0.17 ± 0.03). High-risk HPV-infected patients presented higher antibody reactivity (OD mean 0.36 ± 0.17) than high-risk HPV-non-infected patients (OD mean 0.22 ± 0.11). This peptide showed 88.36% sensitivity, 99.39% specificity and 94.21% correct classification of high risk-HPV cervical lesion or cervical cancer patients. This peptide should be taken into account for designing serological screening or diagnostic tests for use in a clinical scenario.
Keywords: Cervical cancer; HPV-infection; L1-peptides; Serological test;

Antitumor effects, cell selectivity and structure–activity relationship of a novel antimicrobial peptide polybia-MPI by Kai-rong Wang; Bang-zhi Zhang; Wei Zhang; Jie-xi Yan; Jia Li; Rui Wang (963-968).
A novel antimicrobial peptide, polybia-MPI, was purified from the venom of the social wasp Polybia paulista. It has potent antimicrobial activity against both Gram-positive and Gram-negative bacteria, but causing no hemolysis to rat erythrocytes. To date, there is no report about its antitumor effects on any tumor cell lines. In this study we synthesized polybia-MPI and studied its antitumor efficacy and cell selectivity. Our results revealed that polybia-MPI exerts cytotoxic and antiproliferative efficacy by pore formation. It can selectively inhibit the proliferation of prostate and bladder cancer cells, but has lower cytotoxicity to normal murine fibroblasts. In addition, to investigate the structure–activity relationship of polybia-MPI, three analogs in which Leu7, Ala8 or Asp9 replaced by l-Pro were designed and synthesized. l-Pro substitution of Leu7 or Asp9 significantly reduces the content of α-helix conformation, and l-Pro substitution of Ala8 can disrupt the α-helix conformation thoroughly. The l-Pro substitution induces a significant reduction of antitumor activity, indicating that the α-helix conformation of polybia-MPI is important for its antitumor activity. In summary, polybia-MPI may offer a novel therapeutic strategy in the treatment of prostate cancer and bladder cancer, considering its relatively lower cytoxicity to normal cells.
Keywords: Antimicrobial peptide; Polybia-MPI; Antitumor; Cell selectivity; Analog;

Bovine hemoglobin: An attractive source of antibacterial peptides by Naïma Nedjar-Arroume; Véronique Dubois-Delval; Estelle Yaba Adje; Jonathan Traisnel; François Krier; Patrice Mary; Mostafa Kouach; Gilbert Briand; Didier Guillochon (969-977).
A peptic hemoglobin hydrolysate was fractioned by a semi-preparative reversed-phase HPLC and some fractions have an antibacterial activity against four bacteria strains: Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis. These fractions were analyzed by ESI/MS and ESI/MS/MS, in order to characterize the peptides in these fractions. Each fraction contains at least three peptides and some fractions contain five peptides. All these fractions were purified several times by HPLC to obtain pure peptides. Thirty antibacterial peptides were identified. From the isolated antibacterial peptides, 24 peptides were derived from the α chains of hemoglobin and 6 peptides were derived from the β chains of hemoglobin. The lowest concentration of these peptides (minimum inhibitory concentration (MIC)) necessary to completely inhibit the growth of four bacteria strain was determined. The cell population of all of the tested bacteria species decreased by at least 97% after a 24-h incubation with any of the peptides at the minimum inhibitory concentration.
Keywords: Bovine hemoglobin; Antibacterial peptides; Pepsin; Hydrolysate; Active peptides;

Characterization of anti-Legionella activity of warnericin RK and delta-lysin I from Staphylococcus warneri by Julien Verdon; Jean-Marc Berjeaud; Christian Lacombe; Yann Héchard (978-984).
Legionella pneumophila, the causative agent of Legionnaires’ disease, is a waterborne bacteria. It can multiply in man-made water systems and infect people who inhale contaminated droplets. We have previously reported a Staphylococcus warneri strain that display an anti-Legionella activity. In this work, we characterized three anti-Legionella peptides that are produced by S. warneri. One peptide, warnericin RK, is original, while the two others are delta-lysin I and delta-lysin II, whose genes were previously described. Due to high sequence similarity of the two delta-lysins, further characterization was performed only on delta-lysin I. Warnericin RK and delta-lysin I displayed the same antibacterial spectrum, which is almost restricted to the Legionella genus. Also, both peptides have a hemolytic activity. These results led to the hypothesis that warnericin RK and delta-lysin I share a similar mode of action, and that Legionella should have a specific feature that may explain the high specificity of these antibacterial peptides.
Keywords: Warnericin RK; Delta-lysin; Legionella pneumophila; Staphyloccocus warneri; Antimicrobial peptide;

Cone snails are predatory gastropod mollusks distributed in all tropical marine habitats with a highly sophisticated defense strategy using small peptides in their venoms. Here, we report the discovery and initial characterization of the V-superfamily conotoxins. A novel conotoxin vi15a was purified from the venom of a worm-hunting species Conus virgo. The sequence of vi15a was determined to have a unique arrangement of cysteine residues (C-C-CC-C-C-C-C), which defines the new V-superfamily conotoxins. The cDNA of vi15a was cloned with RACE method. Its unique signal peptide sequence led to the cloning of another V-superfamily conotoxin, Vt15.1, from Conus vitulinus. These results, as well as the existence of Lt15.1 from Conus litteratus and ca15a from Conus caracteristicus with the same cysteine pattern, suggest that V-superfamily might be a large and diverse group of peptides widely distributed in different Conus species. Like other eight Cys-containing toxins, V-superfamily conotoxins might also adopt an “ICK+1” disulfide bond connectivity. The identification of this novel class of conotoxins will certainly improve our understanding of the structure diversity of disulfide rich toxins.
Keywords: Conotoxin; V-superfamily; Cysteine framework; Diversity;

New potent antimicrobial peptides from the venom of Polistinae wasps and their analogs by Václav Čeřovský; Jiřina Slaninová; Vladimír Fučík; Hana Hulačová; Lenka Borovičková; Rudolf Ježek; Lucie Bednárová (992-1003).
Four new peptides of the mastoparan family, characterized recently in the venom of three neotropical social wasps collected in the Dominican Republic, Polistes major major, Polistes dorsalis dorsalis and Mischocyttarus phthisicus were synthesized and tested for antimicrobial potency against Bacillus subtilis, Staphylococcus aureus, Escherichia coli (E.c.) and Pseudomonas aeruginosa, and for hemolytic and mast cells degranulation activities. As these peptides posses strong antimicrobial activity (minimal inhibitory concentration (MIC) values against Bacillus subtillis and E.c. in the range of 5–40 μM), we prepared 40 of their analogs to correlate biological activities, especially antimicrobial, with the net positive charge, hydrophobicity, amphipathicity, peptide length, amino acid substitutions at different positions of the peptide chain, N-terminal acylation and C-terminal deamidation. Circular dichroism spectra of the peptides measured in the presence of trifluoroethanol or SDS showed that the peptides might adopt α-helical conformation in such anisotropic environments.
Keywords: Antimicrobial peptides; Analogs; Wasp venom; Circular dichroism; Hemolytic activity; Mast cell degranulation; Amphipathicity;

The melanocortin peptides α-MSH, Lys-Pro-Val and Lys-Pro-d-Val are known to be potent anti-inflammatory agents; however their role as antibacterial peptides is less clear. The aim of this study was to determine whether these peptides displayed antibacterial properties, and specifically whether the Lys-Pro-d-Val tripeptide was more potent than Lys-Pro-Val, consistent with their anti-inflammatory actions. α-MSH, Ac-Lys-Pro-d-Val-NH2 and Ac-Lys-Pro-Val-NH2 were found to be antibacterial against both Gram-positive and Gram-negative bacteria (Staphylococcus aureus and Escherichia coli) over a broad range of concentrations compared to a control peptide, Ac-Ala-Ala-Ala-NH2. However, the relative potency of α-MSH, Ac-Lys-Pro-d-Val-NH2, Ac-Lys-Pro-Val-NH2 did not differ. Furthermore, it was found that the cationic charge on the lysine residue was not required for activity as a variant peptide Ac-Ala-Pro-d-Val-NH2 was also antibacterial. We therefore describe a novel X-Pro-d/l-Val peptide sequence with similarity to the short melanocortin peptides, which possess antibacterial activity. The combined anti-inflammatory and antibacterial action of such peptides may also have potential value therapeutically.
Keywords: Melanocortin peptides; Anti-microbial peptides; Staphylococcus aureus; Escherichia coli;

Cyclic analogs of α-melanocyte-stimulating hormone (αMSH) with high agonist potency and selectivity at human melanocortin receptor 1b by Maria A. Bednarek; Tanya MacNeil; Rui Tang; Tung M. Fong; M. Angeles Cabello; Marta Maroto; Ana Teran (1010-1017).
Keywords: Melanocortin; Melanocortin receptor; Agonist; Human melanocortin receptor 1; hMC1R; Binding affinity; cAMP accumulation assay; MTII;

Peripheral obestatin has no effect on feeding behavior and brain Fos expression in rodents by Peter Kobelt; Anna-Sophia Wisser; Andreas Stengel; Miriam Goebel; Norbert Bannert; Guillaume Gourcerol; Tobias Inhoff; Steffen Noetzel; Bertram Wiedenmann; Burghard F. Klapp; Yvette Taché; Hubert Mönnikes (1018-1027).
Obestatin is produced in the stomach from proghrelin by post-translational cleavage. The initial report claimed anorexigenic effects of obestatin in mice. Contrasting studies indicated no effect of obestatin on food intake (FI). We investigated influences of metabolic state (fed/fasted), environmental factors (dark/light phase) and brain Fos response to intraperitoneal (ip) obestatin in rats, and used the protocol from the original study assessing obestatin effects in mice. FI was determined in male rats injected ip before onset of dark or light phase, with obestatin (1 or 5 μmol/kg), CCK8S (3.5 nmol/kg) or 0.15 M NaCl, after fasting (16 h, n  = 8/group) or ad libitum (n  = 10–14/group) food intake. Fos expression in hypothalamic and brainstem nuclei was examined in freely fed rats 90 min after obestatin (5 μmol/kg), CCK8S (1.75 nmol/kg) or 0.15 M NaCl (n  = 4/group). Additionally, fasted mice were injected ip with obestatin (1 μmol/kg) or urocortin 1 (2 nmol/kg) 15 min before food presentation. No effect on FI was observed after obestatin administration during the light and dark phase under both metabolic conditions while CCK8S reduced FI irrespectively of the conditions. The number of Fos positive neurons was not modified by obestatin while CCK8S increased Fos expression in selective brain nuclei. Obestatin did not influence the refeeding response to a fast in mice, while urocortin was effective. Therefore, peripheral obestatin has no effect on FI under various experimental conditions and did not induce Fos in relevant central neuronal circuitries modulating feeding in rodents.
Keywords: Obestatin; Food intake; CCK; Urocortin 1; Rats; Mice; Dark phase; Light phase;

Central and peripheral administration of amylin induces energy expenditure in anesthetized rats by Toshimasa Osaka; Ayako Tsukamoto; Yu Koyama; Shuji Inoue (1028-1035).
Amylin is a peptide hormone that is co-released with insulin from pancreatic β-cells following a meal. Intracerebroventricular (icv) administration of amylin (1–100 pmol), or an amylin agonist, salmon calcitonin, elicited dose-dependent thermogenic, tachycardic, and hyperthermic responses in urethane-anesthetized rats. Intravenous (iv) administration of higher doses of amylin (100 pmol–20 nmol) also induced similar responses, although the amplitudes of these responses were significantly smaller than those elicited by icv administration, suggesting the primary action of amylin to be in the brain. However, the iv administration of amylin induced the responses slightly faster than the icv injection, the former responses occurring <4 min and the latter, at 8–10 min, after the administration. The iv but not the icv injection of amylin increased the respiratory exchange ratio transiently (<20 min), though the thermogenic response lasted for a longer period after both injections, indicating a shift from mixed fuel to predominantly carbohydrate utilization in the initial phase of thermogenesis induced by the iv injection of amylin. The differences in substrate utilization and latency of the responses suggest that the actions of amylin include partly different targets when administered centrally and peripherally. Moreover, pretreatment with a β-adrenergic blocker, propranolol (5 mg kg−1, iv), blocked all responses elicited by either icv or iv administration of amylin, whereas ablation of the area postrema in the hindbrain did not influence the effects of icv-administered amylin. These results suggest the involvement of amylin in postprandial energy expenditure, mediated by peripheral β-adrenoceptors.
Keywords: Energy homeostasis; Postprandial thermogenesis; Salmon calcitonin; Area postrema; β-Adrenoceptor;

GIP receptor antagonism with (Pro3)GIP protects against obesity, insulin resistance, glucose intolerance and associated disturbances in mice fed high-fat diet. Furthermore, cannabinoid CB1 receptor antagonism with AM251 reduces appetite and body weight gain in mice. The present study has examined and compared the effects of chronic daily administrations of (Pro3)GIP (25 nmol/kg body weight), AM251 (6 mg/kg body weight) and a combination of both drugs in high-fat fed mice. Daily i.p. injection of (Pro3)GIP, AM251 or combined drug administration over 22 days significantly (P  < 0.05 to <0.01) decreased body weight compared with saline-treated controls. This was associated with a significant (P  < 0.05 to <0.01) reduction of food intake in mice treated with AM251. Plasma glucose levels and glucose tolerance were significantly (P  < 0.05) lowered by 22 days (Pro3)GIP, AM251 or combined drug treatment. These changes were accompanied by a significant (P  < 0.05) improvement of insulin sensitivity in all treatment groups. In contrast, AM251 lacked effects on glucose tolerance, metabolic response to feeding and insulin sensitivity in high-fat mice when administered acutely. These data indicate that chemical blockade of GIP- or CB1-receptor signaling using (Pro3)GIP or AM251, respectively provides an effective means of countering obesity and related abnormalities induced by consumption of high-fat energy-rich diet. AM251 lacks acute effects on glucose homeostasis and there was no evidence of a synergistic effect of combined treatment with (Pro3)GIP.
Keywords: Gastric inhibitory polypeptide (GIP); CB1 receptor; AM251; Food intake; Glucose homeostasis; High-fat feeding;

Transport of μ-opioid receptor agonists and antagonist peptides across Caco-2 monolayer by Małgorzata Iwan; Beata Jarmołowska; Krzysztof Bielikowicz; Elzbieta Kostyra; Henryk Kostyra; Maciej Kaczmarski (1042-1047).
Milk is the source of β-casomorphins – biologically active peptides with opioid activity – which are suspected to play various roles in the human body. The local influence of exogenous opioid peptides on gastrointestinal functions has been widely reported. After passing the gut barrier, β-casomorphins may affect the functions of immunological system, as well as dopaminergic, serotoninergic and GABA-ergic systems in brain, regulate the opioid receptor development and elicit behavioral effects. However, possibilities and mechanisms of the intestinal transport of β-casomorphins in human body in vivo have not been reported so far. In our research, the transepithelial transport of μ-opioid receptor agonists – human β-casomorphin-5 and 7(BCM5, BCM7) and antagonist – lactoferroxin A (LCF A) have been investigated using Caco-2 monolayer. In order to determine the pathway of investigated peptide transport across Caco-2 monolayer, two directions of the transport (apical to basolateral and basolateral to apical) have been studied. All investigated peptides were transported across the human intestinal cell line Caco-2 and the curves of cumulative amount of transported peptides in time were linear in each case. In addition, the hydrolysis of β-casomorphins during 60 min of experiment by dipeptidyl peptidase IV was observed. The data suggest the possibility of transport of opioid peptides derived from food across human intestinal mucosa.
Keywords: Casomorphin; Opioid peptide; Caco-2; Peptide transport; Transepithelial transport;

Differential cardiovascular effects of synthetic peptides derived from endomorphin-1 in anesthetized rats by Hongmei Liu; Yang Yang; Ruihua Xin; Xing Liu; Yiming Cao; Jingman Ni; Rui Wang (1048-1056).
Previously, five synthetic peptides derived from endomorphin-1 (Tyr1-Pro2-Trp3-Phe4-NH2, EM-1), including Tyr-d-Ala-Trp-p-Cl-Phe-NH2 (HDAPC), Tyr-d-Ala-Trp-Phe-NH2 (HDADC), N α-amidino-Tyr-d-Ala-Trp-p-Cl-Phe-NH2 (GDAPC), N α-amidino-Tyr-d-Ala-Trp-Phe-NH2 (GDADC) and N α-amidino-Tyr-d-Pro-Gly-Trp-p-Cl-Phe-NH2 (GBDPC), were described to elicit analgesia by subcutaneous administration with enhanced metabolic stabilities. To further our knowledge of the influences of particular modification on the pharmacological activities of EM-1, the present study was undertaken to investigate cardiovascular effects of these peptides in anesthetized rats by intravenous injection. Our results showed that the four d-Ala-containing peptides decreased the systemic arterial pressure (SAP) and heart rate (HR) through a naloxone-sensitive mechanism. Different patterns, potencies and durations of cardiovascular effects were observed among these peptides. When compared to EM-1, the hemodynamic responses to these four tetrapeptides were significantly lower in magnitude but much longer in duration. Surprisingly, intravenous administration of the only pentapeptide GBDPC produced fairly prolonged hypertensive and tachycardiac effects, which was naloxone-insensitive, thus providing evidence that changes in the primary structure of a peptide can profoundly affect its pharmacological activity. Comparisons of the cardiovascular effects between these peptides showed that each modification introduced into EM-1, including N-amidination, chloro-halogenation and unnatural amino acid substitution, played a role in the influence on the cardiovascular regulation of these peptides.
Keywords: Endomorphin-1; Systemic arterial pressure; Heart rate; Intravenous injection; μ-Opioid receptor;

Regulation of endothelin-1 release from human endothelial cells by sex steroids and angiotensin-II by Lachlan J. Pearson; Timothy G. Yandle; M. Gary Nicholls; John J. Evans (1057-1061).
It is well documented that there are gender differences in the incidence and patterns of cardiovascular disease; males have a higher incidence of cardiovascular disease than premenopausal women. We have therefore investigated whether the sex hormones, estradiol and testosterone, could directly influence the secretion of vascular peptides from human aortic endothelial cells (HAEC). Previously we have shown that testosterone can increase the number of HAECs that secrete adrenomedullin. In this study we investigated sex hormone regulation of endothelin-1 in HAEC. Several studies have observed a reduction in endothelin-1 secretion from endothelial cells in the presence of estradiol, the effect being more marked for stimulated cells. Studies on the actions of testosterone are much fewer and inconclusive. In this study we observed that estradiol did not change the number of cells secreting endothelin-1 during 4 h incubation under basal conditions but decreased the number of secreting cells stimulated with angiotensin-II. Testosterone induced an increase in the number of cells secreting endothelin-1 (p  = 0.03). Complementary incubations revealed that testosterone up-regulated endothelin-1 mRNA at 1–3 h (p  < 0.05). These results, together with our previous observations, indicate that angiotensin-II, testosterone and estradiol have parallel effects on the production of endothelin-1 as on adrenomedullin in HAEC. We conclude that there is potential for coordinated modulation by sex steroids and angiotensin-II of vasoactive peptide production in human endothelial cells.
Keywords: Endothelin; Testosterone; Estradiol; Angiotensin; Endothelial cells; Vascular activity;

The antihypertensive effect of peptides: A novel alternative to drugs? by Fang Hong; Luo Ming; Sheng Yi; Li Zhanxia; Wu Yongquan; Liu Chi (1062-1071).
Many types of bioactive peptides that inhibit angiotensin I, angiotensin I converting enzyme (ACE) and Ang II type 1 receptor (AT1) in the cardiovascular system contribute to the prevention and treatment of hypertension. These inhibitory peptides are derived from many food proteins or artificial synthetic products. Further research examining the bioavailability of ACE inhibitory peptides will lead to the development of more effective ACE inhibitory peptides and foods. Our research also demonstrates that ACE inhibitory peptide LAP may lower blood pressure with no adverse effects.
Keywords: Hypertension; Bioactive peptides; Renin-angiotensin system;