Peptides (v.52, #C)

Women are at greater risk for major depression, PTSD, and other anxiety disorders. ERβ-selective agonists for the treatment of these disorders are the focus of pharmacologic development and clinical testing. Estradiol and its metabolites contribute to the neuroprotective effects of this steroid class, particularly in men, due to local conversion of testosterone to estiradiol in key brain regions which are predisposed to neurodegenerative diseases. We have used young adult female Sprague–Dawley rats to assess the role of TRH and TRH-like peptides, with the general structure pGlu-X-Pro-NH2 where “X” can be any amino acid residue, as mediators of the neurobiochemical effects of estradiol. The neuroprotective TRH and TRH-like peptides are coreleased with excitotoxic glutamate by glutamatergic neurons which contribute importantly to the regulation of the estrus cycle. The levels of TRH and TRH-like peptides during proestrus and/or estrus in the 12 brain regions analyzed were significantly decreased (due to accelerated release) 106 times but increased only 25 times when compared to the corresponding levels during diestrus days 1 and 2. These changes, listed by brain region in the order of decreasing number of significant decreases (↓) and/or increases (↑), were: striatum (20↓,1↑), medulla oblongata (16↓,2↑), amygdala (14↓,1↑), cerebellum (13↓,1↑), hypothalamus (12↓,1↑), entorhinal cortex (6↓,6↑), posterior cingulate (10↓,1↑), frontal cortex (3↓,5↑), nucleus accumbens (5↓,3↑), hippocampus (5↓,2↑), anterior cingulate (2↓,1↑), and piriform cortex (1↑). In peripheral tissues the corresponding changes were: ovaries (23↓), uterus (16↓,1↑), adrenals (11↓,3↑), and pancreas (1↓,6↑). We conclude that these peptides may be downstream mediators of some of the therapeutic effects of estrogen.
Keywords: Thyrotropin-releasing hormone; Estrus cycle; Limbic system; Depression treatment;

The rapid degradation of porcine glucagon-like peptide-2 (pGLP-2) by the enzyme dipeptidyl peptidase-IV (DPP-IV) is the main impediment in the development of pGLP-2 as a potential therapeutic agent for intestinal dysfunction and damage. In this study, one mono-modified Lys30-polyethylene glycol (PEG)-pGLP-2 was prepared using mPEG-succinimidyl propionate. To determine the optimized condition for PEGylation, the reactions were monitored by RP-HPLC and MALDI-TOF-MS. Stability was tested in purified DPP-IV in vitro. In vivo, the protective effects for colonic injury were measured in dextran sulfate sodium (DSS)-induced colitis in mice. The monoPEGylated products reached the maximum yield at 4:1 ratio of mPEG5k-SPA to pGLP-2. An effective method of successfully separating PEGylated pGLP-2 from mPEG-SPA5kD using CM Sepharose Fast Flow resin was established. The half-life of Lys30-PEG-pGLP-2 was 16-fold longer than that of pGLP-2 in DPP-IV. The DSS mice exhibited marked weight loss), which was significantly reduced by Lys30-PEG-pGLP-2 therapy. DSS treatment significantly increased colonic damage score, which was significantly reduced by administration of Lys30-PEG-pGLP-2 in DSS-mice. DSS-induced colitis clearly induced Myeloperoxidase activity in the colon, which was significantly reduced by treatments with 3% DSS-pGLP-2 or 3% DSS-PEG-pGLP-2. These results showed that site-specific Lys30-PEG-GLP-2 was resistant to degradation and reduced the severity of colonic injury in murine colitis. The enhanced biological potency of this product highlighted its potential as a therapeutic agent for intestinal diseases.
Keywords: PEGylation; Porcine glucagon-like peptide-2; Ion-exchange chromatography; Dextran sulfate sodium; Colitis;

Relationship between human cord blood adropin levels and fetal growth by Xiu Qiu; Jian-Rong He; Ming-Guang Zhao; Ya-Shu Kuang; Shu-Qin Xu; Hui-Zhu Zhang; Shun-Ping Hu; Jun Chen; Hui-Min Xia (19-22).
Adropin is a recently identified peptide and participates in the regulation of energy homeostasis and vascular function. The aim of this study was to examine the relationships between human cord blood adropin levels and fetal growth. A total of 159 newborns [preterm delivery (PTD), n  = 72; term delivery, n  = 87] were recruited. Adropin levels in cord blood were determined using enzyme-linked immunosorbent assay kits. Clinical information on fetal growth was collected. Adropin levels in PTD babies (median, 2028; 25th–75th, 1413–2484 pg/ml) were lower than those in term delivery babies (median, 2305; 25th–75th, 1960–2684 pg/ml, P  = 0.01). Birth weight and length z score, Ponderal index, placental length, breadth, thickness, surface area, volume and density were not significantly correlated to adropin concentrations in term delivery group. However, we found adropin concentrations were significantly correlated to gestational age at birth (Spearman's correlation coefficient = 0.35, P  < 0.01) and placental weight (Spearman's correlation coefficient = 0.24, P  = 0.04) in PTD group. We also found that boys had lower adropin levels than girls in PTD group (P  = 0.01). When the analysis was extended to the whole group (PTD and term deliveries combined), the results were similar to those for PTD group alone. After adjusting for maternal age and newborn's sex, every 100 pg/ml increase of adropin concentration was significantly associated with a decreased risk of PTD (odds ratio, 0.95; 95% confidence interval, 0.91–0.99). Our study showed that cord blood adropin levels were positively correlated with gestational age and placental weight but not with other fetal growth parameters.
Keywords: Adropin; Gestational age; Cord blood; Fetal growth;

Ghrelin protects human umbilical vein endothelial cells against high glucose-induced apoptosis via mTOR/P70S6K signaling pathway by Jianhua Zhu; Chenghong Zheng; Jie Chen; Jing Luo; Bintao Su; Yan Huang; Wen Su; Zixi Li; Tianpen Cui (23-28).
Ghrelin exhibits its biological effect through binding to the growth hormone secretagogue 1a receptor (GHS-R1a). Recently, it has been reported that ghrelin has an anti-apoptotic effect in several cell types. However, the molecule mechanisms underlying the anti-apoptotic effect of ghrelin remain poorly understood. In this study, we investigated the intracellular mechanisms responsible for anti-apoptotic effect of ghrelin on human umbilical vein endothelial cells (HUVEC). Treatment of HUVEC with ghrelin inhibited high glucose-induced cell apoptosis. Ghrelin stimulated the rapid phosphorylation of mammalian target of rapamycin (mTOR), P70S6K and S6. The GHS-R1a-specific antagonist [D-Lys3]-GHRP-6 abolished the anti-apoptotic effect and inhibited the activation of mTOR, P70S6K, S6 induced by ghrelin. Pretreatment of cells with specific inhibitor of mTOR blocked the anti-apoptotic effect of ghrelin. In addition, ghrelin protected HUVECs against high glucose induced apoptosis by increasing Bcl-2/Bax ratio. Taken together, our results demonstrate that ghrelin produces a protective effect on HUVECs through activating GHS-R1a and mTOR/P70S6K signaling pathway mediates the effect of ghrelin. These observations suggest that ghrelin may act as a survival factor in preventing HUVECs apoptosis caused by high glucose.
Keywords: Ghrelin; Apoptosis; mTOR/P70S6K; GHS-R1a;

Orexin as an input of circadian system in goldfish: Effects on clock gene expression and locomotor activity rhythms by Laura G. Nisembaum; Nuria de Pedro; María J. Delgado; Aída Sánchez-Bretaño; Esther Isorna (29-37).
Orexins are neuropeptides mainly known for regulating feeding behavior and sleep–wakefulness cycle in vertebrates. Daily variations of orexin-A expression have been reported in fish, with the highest levels preceding feeding time. However, it is unknown if such variations could be related with daily rhythms of clock genes, which form the molecular core of circadian oscillators. The aim of the present study was to identify the possible role of orexin as an input element of the goldfish circadian system. It was investigated the effects of orexin-A (10 ng/g bw) intracerebroventricular injections on the expression of clock genes, NPY and ghrelin, as well as on daily locomotor activity rhythms. Goldfish held under 12L:12D photoperiod and injected at midday with orexin or saline, were sacrificed at 1 and 3 h post-injection. The analysis of genes expression by qReal Time PCR showed an increment of Per genes in hypothalamus and foregut at 3 h post-injection, but not in hindgut and liver. The gBmal1a expression remained unaltered in all the studied tissues. Orexin induced NPY in the hypothalamus and ghrelin in the foregut. Locomotor activity was studied in fish daily injected with orexin for several consecutive days under different experimental conditions. Orexin synchronized locomotor activity in goldfish maintained in 24L and fasting conditions. Present results support a cross-talking between orexin-A and other feeding regulators at central and peripheral level, and suggest, for the first time, a role of this peptide as an input of the circadian system in fish.
Keywords: Orexin-A; Clock gene; Locomotor activity; Food anticipatory activity; Hypothalamus; Foregut;

Intact neural system of the portal vein is important for maintaining normal glucose metabolism by regulating glucagon-like peptide-1 and insulin sensitivity by Vo Nguyen Trung; Hiroshi Yamamoto; Tsuyoshi Yamaguchi; Satoshi Murata; Yoshinari Aimi; Atsukazu Kuwahara; Tohru Tani (38-43).
The portal neural system may have an important role on the regulation of glucose homeostasis since activation of the gut–brain–liver neurocircuit by nutrient sensing in the proximal intestine reduces hepatic glucose production through enhanced liver insulin sensitivity. Although there have been many studies investigating the role of portal neural system, surgical denervation of the sole portal vein has not been reported to date. The aim of this study was to clarify the role of the portal neural system on the regulation of glucose homeostasis and food intake in the physiological condition. Surgical denervation of portal vein (DV) was performed in 10 male 12 week-old Wistar rats. The control was a sham operation (SO). One week after surgery, food intake and body weight were monitored; an oral glucose tolerance test (OGTT) was performed; and glucagon-like peptide-1 (GLP-1) and insulin levels during OGTT were assayed. In addition, insulinogenic index, homeostatic model assessment, and Matsuda index were calculated. All rats regained the preoperative body weight at one week after surgery. There was no significant difference in food intake between DV and SO rats. DV rats exhibited increased blood glucose levels associated with decreased insulin sensitivity but increased GLP-1 and insulin secretion during OGTT. In summary, in the physiological state, loss of the portal neural system leads to decreased insulin sensitivity and increased blood glucose levels but does not affect food intake. These data indicate that an intact portal neural system is important for maintaining normal glucose metabolism.
Keywords: Portal vein; GLP-1; Insulin sensitivity; Glucose tolerance;

Plasma adrenomedullin levels are associated with long-term outcomes of acute ischemic stroke by Hao Zhang; Bo Tang; Cong-Guo Yin; Yan Chen; Qing-Lian Meng; Lin Jiang; Wei-Ping Wang; Guo-Zhong Niu (44-48).
Plasma adrenomedullin concentration has been found to be enhanced in ischemic stroke. Up to now, little is known about the association of plasma adrenomedullin concentration with clinical outcomes of ischemic stroke. This study recruited 138 patients with ischemic stroke and 138 healthy volunteers. Unfavorable outcome was defined as modified Rankin Scale score >2 at 3 months. Plasma adrenomedullin concentrations were determined by enzyme-linked immunosorbent assay. Plasma adrenomedullin concentrations were statistically significantly higher in patients than in healthy individuals (79.9 ± 27.3 pg/mL vs. 36.8 ± 10.4 pg/mL; P  < 0.001). 3-Month mortality was 20.3% (28/138) and sixty-six patients (47.8%) had unfavorable outcome in 3 months. A logistic regression analysis identified plasma adrenomedullin concentration as an independent predictor of 3-month mortality (odds ratio, 1.211; 95% confidence interval, 1.101–1.582; P  = 0.004) and unfavorable outcome (odds ratio, 1.193; 95% confidence interval, 1.082–1.447; P  = 0.006). Receiver operating characteristic curve analysis showed that plasma adrenomedullin concentration predicted 3-month mortality (area under curve, 0.806; 95% confidence interval, 0.730–0.868) and unfavorable outcome (area under curve, 0.816; 95% confidence interval, 0.742–0.877) with the high predictive value. Its predictive performance was similar to that of National Institutes of Health Stroke Scale score (P  = 0.694 or 0.206). Its combined use with National Institutes of Health Stroke Scale score did not improve the predictive value (P  = 0.236 or 0.590). Thus, adrenomedullin may aid to predict long-term clinical outcomes of patients with ischemic stroke.
Keywords: Ischemic stroke; Outcome; Adrenomedullin;

A P2 and P3 substrate specificity comparison between the Murray Valley encephalitis and West Nile virus NS2B/NS3 protease using C-terminal agmatine dipeptides by Melgious Jin Yan Ang; Zhitao Li; Huichang Annie Lim; Fui Mee Ng; Siew Wen Then; John Liang Kuan Wee; Joma Joy; Jeffrey Hill; C.S. Brian Chia (49-52).
The Murray Valley encephalitis virus (MVEV) and the West Nile virus (WNV) are mosquito-borne single-stranded RNA Flaviviruses responsible for many cases of viral encephalitis and deaths worldwide. The former is endemic in north Australia and Papua New Guinea while the latter has spread to different parts of the world and was responsible for a recent North American outbreak in 2012, resulting in 243 fatalities. There is currently no approved vaccines or drugs against MVEV and WNV viral infections. A plausible drug target is the viral non-structural NS2B/NS3 protease due to its role in viral replication. This trypsin-like serine protease recognizes and cleaves viral polyproteins at the C-terminal end of an arginine residue, opening an avenue for the development of peptide-based antivirals. This communication compares the P2 and P3 residue preferences of the MVEV and WNV NS2B/NS3 proteases using a series of C-terminal agmatine dipeptides. Our results revealed that both viral enzymes were highly specific toward lysines at the P2 and P3 positions, suggesting that a peptidomimetic viral protease inhibitor developed against one virus should also be active against the other.

Endothelin-1 (ET-1) is a short chained peptide primarily of endothelial origin. Concentrations of this peptide are increased in subjects with hypertension, primary pulmonary hypertension and myocardial infarction, however its short half-life makes quantification difficult. The C-terminal of proET-1 (CTproET-1) is stoichiometrically secreted with its bioactive peptide and would be a valid method of measuring the active peptide as it has a stable half-life and is less resistant to proteolytic cleavage. The objective of this study was to understand the factors (clinical, echocardiographic and biochemical) that specifically influence plasma CTproET-1 in healthy subjects. 518 healthy volunteers were recruited from a screening study. Plasma CTproET-1 concentrations were quantified using a novel immunoluminometric sandwich assay. In multivariate analyses, age (P  < 0.001), diastolic BP (P  = 0.007), LA size (P  = 0.001) and eGFR (P  < 0.001) were independently predictive of plasma CTproET-1 levels in the healthy subjects. Therefore the interpretation of plasma CTproET-1 levels in such individuals should take into account these variables to avoid potential confounding.
Keywords: Endothelin-1; Healthy population; C-terminal proEndothelin-1; Renal disease; Age; Diastolic blood pressure;

Obestatin and cardiovascular health by Xue-Jia Su; Rui-Xin Dong; Yan-Peng Li; Shu-Guang Yang; Zhao-Feng Li (58-60).
Obestatin, encoded by the same gene as ghrelin, was first described as a physiological opponent of ghrelin through an interaction with the orphan receptor GPR39. However, the effects of obestatin were not totally contrary to the effects of ghrelin in cardiovascular regulations based on the recent studies. We summarize here the current evidences surrounding the cardiovascular actions of obestatin, and the possible implications of obestatin as a therapeutic agent in common conditions such as hypertension and heart failure.
Keywords: Obestatin; Blood pressure; Heart failure; Cardiorenal syndrome; Cardiac muscle cell;

Antimicrobial and anti-biofilm effect of Bac8c on major bacteria associated with dental caries and Streptococcus mutans biofilms by Yonglin Ding; Wei Wang; Meng Fan; Zhongchun Tong; Rong Kuang; WenKai Jiang; Longxing Ni (61-67).
Dental caries is a common oral bacterial infectious disease. Its prevention and treatment requires control of the causative pathogens within dental plaque, especially Streptococcus mutans (S. mutans). Antimicrobial peptides (AMPs), one of the promising substitutes for conventional antibiotics, have been widely tested and used for controlling bacterial infections. The present study focuses on evaluating the potential of the novel AMPs cyclic bactenecin and its derivatives against bacteria associated with dental caries. The results indicate that Bac8c displayed highest activity against the bacteria tested, whereas both cyclic and linear bactenecin had weak antimicrobial activity. The cytotoxicity assay showed that Bac8c did not cause detectable toxicity at concentrations of 32–128 μg/ml for 5 min or 32–64 μg/ml for 60 min. S. mutans and Lactobacillus fermenti treated with Bac8c showed variable effects on bacterial structure via scanning electron microscopy and transmission electron microscopy. There appeared to be a large amount of extracellular debris and obvious holes on the cell surface, as well as loss of cell wall and nucleoid condensation. The BioFlux system was employed to generate S. mutans biofilms under a controlled flow, which more closely resemble the formation process of natural biofilms. Bac8c remarkably reduced the viability of cells in biofilms formed in the BioFlux system. This phenomenon was further analyzed and verified by real-time PCR results of a significant suppression of the genes involved in S. mutans biofilm formation. Taken together, this study suggests that Bac8c has a potential clinical application in preventing and treating dental caries.
Keywords: Antimicrobial peptides; Bac8c; Dental caries; Streptococcus mutans; Biofilms;

Cardiac, skeletal muscle and serum irisin responses to with or without water exercise in young and old male rats: Cardiac muscle produces more irisin than skeletal muscle by Suna Aydin; Tuncay Kuloglu; Suleyman Aydin; Mehmet Nesimi Eren; Ahmet Celik; Musa Yilmaz; Mehmet Kalayci; İbrahim Sahin; Orhan Gungor; Ali Gurel; Murat Ogeturk; Ozlem Dabak (68-73).
Irisin converts white adipose tissue (WAT) into brown adipose tissue (BAT), as regulated by energy expenditure. The relationship between irisin concentrations after exercise in rats compared humans after exercise remains controversial. We therefore: (1) measured irisin expression in cardiac and skeletal muscle, liver, kidney, peripheral nerve sheath and skin tissues, as also serum irisin level in 10 week-old rats without exercise, and (2) measured tissue supernatant irisin levels in cardiac and skeletal muscle, and in response to exercise in young and old rats to establishing which tissues produced most irisin. Young (12 months) and old rats (24 months) with or without 10 min exercise (water floating) and healthy 10 week-old Sprague-Dawley rats without exercise were used. Irisin was absent from sections of skeletal muscle of unexercised rats, the only part being stained being the perimysium. In contrast, cardiac muscle tissue, peripheral myelin sheath, liver, kidneys, and skin dermis and hypodermis were strongly immunoreactivity. No irisin was seen in skeletal muscle of unexercised young and old rats, but a slight amount was detected after exercise. Strong immunoreactivity occurred in cardiac muscle of young and old rats with or without exercise, notably in pericardial connective tissue. Serum irisin increased after exercise, being higher in younger than older rats. Irisin in tissue supernatants (cardiac and skeletal muscle) was high with or without exercise. High supernatant irisin could come from connective tissues around skeletal muscle, especially nerve sheaths located within it. Skeletal muscle is probably not a main irisin source.
Keywords: Irisin; Cardiac and skeletal muscle; Skin; Nerve sheath; Liver; Kidneys;

Enhanced activity of an angiotensin-(1–7) neuropeptidase in glucocorticoid-induced fetal programming by Allyson C. Marshall; Hossam A. Shaltout; Nancy T. Pirro; James C. Rose; Debra I. Diz; Mark C. Chappell (74-81).
We previously identified angiotensin converting enzyme (ACE) and an endopeptidase activity that degraded angiotensin-(1–7) [Ang-(1–7)] to Ang-(1–5) and Ang-(1–4), respectively, in the cerebrospinal fluid (CSF) of 6-month old male sheep. The present study undertook a more comprehensive analysis of the CSF peptidase that converts Ang-(1–7) to Ang-(1–4) in control and in utero betamethasone-exposed sheep (BMX). Characterization of the Ang-(1–7) peptidase revealed that the thiol agents 4-aminophenylmercuric acetate (APMA) and p-chloromercuribenzoic acid (PCMB), as well as the metallo-chelators o-phenanthroline and EDTA essentially abolished the enzyme activity. Additional inhibitors for serine, aspartyl, and cysteine proteases, as well as selective inhibitors against the endopeptidases neprilysin, neurolysin, prolyl and thimet oligopeptidases did not attenuate enzymatic activity. Competition studies against the peptidase revealed similar IC50s for Ang-(1–7) (5 μM) and Ang II (3 μM), but lower values for Ala1-Ang-(1–7) and Ang-(2–7) of 1.8 and 2.0 μM, respectively. In contrast, bradykinin exhibited a 6-fold higher IC50 (32 μM) than Ang-(1–7) while neurotensin was a poor competitor. Mean arterial pressure (78 ± 1 vs. 94 ± 2 mmHg, N  = 4–5, P  < 0.01) and Ang-(1–7) peptidase activity (14.2 ± 1 vs 32 ± 1.5 fmol/min/ml CSF, N  = 5, P  < 0.01) were higher in the BMX group, and enzyme activity inversely correlated with Ang-(1–7) content in CSF. Lower Ang-(1–7) expression in brain is linked to baroreflex impairment in hypertension and aging, thus, increased activity of an Ang-(1–7) peptidase may contribute to lower CSF Ang-(1–7) levels, elevated blood pressure and impaired reflex function in this model of fetal programming.
Keywords: Renin angiotensin system; Ang-(1–7); Cerebrospinal fluid; Peptidase;

We previously showed that R-954 (AcOrn[Oic2,(αMe)Phe5,dβNal7,Ile8]desArg9-bradykinin) is a potent, selective and stable peptide antagonist of the inducible GPCR kinin B1 receptor. This compound shows potential applications for the treatment of several diseases, including cancer and neurological disturbances of diabetes. To enable clinical translation, more information regarding its pharmacological, pharmacokinetics (PK) and toxicological properties at preclinical stage is warranted. This was the principal objective of the present study. Herein, specificity of R-954 was characterized in binding studies on 133 human molecular targets to reveal minor cross-reactivities against the angiotensin AT2 and the bombesin receptors (110- and 330-fold lower affinity than for B1R, respectively). The pharmacokinetic of R-954 was studied in both normal and streptozotocin-diabetic anaesthetized rats providing half-lives of 1.9–2.7 h. R-954 does not appear to be metabolized in the rat circulation and in several rat tissue homogenates, as the kidney, lung and liver. It appears to be excreted as parent drug in the bile (21%) and in urine. A preliminary toxicological profile of R-954 was obtained in rats under various administration routes. R-954 appears to be well tolerated. Overall, these results indicate that R-954 exhibits favorable preclinical pharmacological/PK characteristics and encouraging safety profiles, suitable for early studies in humans.
Keywords: B1 receptor; Kinins; Peptidic antagonist; Pharmacokinetics; Toxicology; Rats;

Effect of ghrelin on chronic liver injury and fibrogenesis in male rats: Possible role of nitric oxide by Nashwa N. Kabil; Hanan A. Seddiek; Nadia A. Yassin; Maha M. Gamal-Eldin (90-97).
Recent studies have revealed that ghrelin may be an antioxidant and anti-inflammatory agent in many organs, however its role in chronic liver injury (CLI) remains unclear. The role of nitric oxide (NO) in CLI is controversial as evidence suggests that NO is either a primary mediator of liver cell injury or exhibits a protective effect against injurious stimuli. Recent evidence demonstrated that the therapeutic potential for ghrelin was through eNOS activation and increase in NO production. However, its role on NO production in the liver has not been previously investigated. The aim of this study was to investigate the role of ghrelin in treatment of CLI, and whether this action is mediated through NO. Forty male rats were divided into four groups: Group I: Control; Group II: chronic liver injury (CLI); Group III: CLI + Ghrelin; and Group IV: CLI + Ghrelin +  l-NAME. Liver enzymes and tumor necrosis factor alpha (TNF-α), were measured to assess hepatocellular injury. Liver tissue collagen content, malondialdehyde (MDA), gene expression of Bax, Bcl-2, and eNOS were assessed to determine the mechanism of ghrelin action. Results showed that ghrelin decreased serum liver enzymes and TNF-α levels. Ghrelin also reduced liver tissue collagen, MDA, and Bax gene expression, and increased Bcl-2 and eNOS gene expression. The effects on TNF-α, collagen, MDA, Bax, and eNOS were partially reversed in Group IV, suggesting that ghrelin's action could be through modulation of NO levels. Therefore, ghrelin's hepatoprotective effect is partially mediated by NO release.
Keywords: Liver injury; Fibrosis; Thioacetamide; Ghrelin; Nitric oxide;

Intracerebroventricular administration of novel glucagon-like peptide suppresses food intake in chicks by Kazuhisa Honda; Takaoki Saneyasu; Takuya Yamaguchi; Tomohiko Shimatani; Koji Aoki; Kiwako Nakanishi; Hiroshi Kamisoyama (98-103).
Glucagon-related peptides such as glucagon, glucagon-like peptide-1, and oxyntomodulin suppress food intake in mammals and birds. Recently, novel glucagon-like peptide (GCGL) was identified from chicken brain, and a comparatively high mRNA expression level of GCGL was detected in the hypothalamus. A number of studies suggest that the hypothalamus plays a critical role in the regulation of food intake in mammals and birds. In the present study, we investigated whether GCGL is involved in the central regulation of food intake in chicks. Male 8-day-old chicks (Gallus gallus) were used in all experiments. Intracerebroventricular administration of GCGL in chicks significantly suppressed food intake. Plasma glucose level was significantly decreased by GCGL, whereas plasma corticosterone level was not affected. Central administration of a corticotrophin-releasing factor (CRF) receptor antagonist, α-helical CRF, attenuated GCGL-suppressed food intake. It seems likely that CRF receptor is involved in the GCGL-induced anorexigenic pathway. All our findings suggest that GCGL functions as an anorexigenic peptide in the central nervous system of chicks.
Keywords: Appetite; Chicken; Food intake; Glucagon; Corticosterone;

Synthesis of new potent agonistic analogs of growth hormone-releasing hormone (GHRH) and evaluation of their endocrine and cardiac activities by Renzhi Cai; Andrew V. Schally; Tengjiao Cui; Luca Szalontay; Gabor Halmos; Wei Sha; Magdolna Kovacs; Miklos Jaszberenyi; Jinlin He; Ferenc G. Rick; Petra Popovics; Rosemeire Kanashiro-Takeuchi; Joshua M. Hare; Norman L. Block; Marta Zarandi (104-112).
In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on cardiomyocytes, pancreatic islets and wound healing, three series of new analogs of GHRH(1–29) have been synthesized and evaluated biologically in an endeavor to produce more potent compounds. “Agmatine analogs”, MR-356 (N-Me-Tyr1-JI-38), MR-361(N-Me-Tyr1, D-Ala2-JI-38) and MR-367(N-Me-Tyr1, D-Ala2, Asn8-JI-38), in which Dat in JI-38 is replaced by N-Me-Tyr1, showed improved relative potencies on GH release upon subcutaneous administration in vivo and binding in vitro. Modification with N-Me-Tyr1 and Arg29-NHCH3 as in MR-403 (N-Me-Tyr1, D-Ala2, Arg29-NHCH3-JI-38), MR-406 (N-Me-Tyr1, Arg29-NHCH3-JI-38) and MR-409 (N-Me-Tyr1, D-Ala2, Asn8, Arg29-NHCH3-JI-38), and MR-410 (N-Me-Tyr1, D-Ala2, Thr8, Arg29-NHCH3-JI-38) resulted in dramatically increased endocrine activities. These appear to be the most potent GHRH agonistic analogs so far developed. Analogs with Apa30-NH2 such as MR-326 (N-Me-Tyr1, D-Ala2, Arg29, Apa30-NH2-JI-38), and with Gab30-NH2, as MR-502 (D-Ala2, 5F-Phe6, Ser28, Arg29,Gab30-NH2-JI-38) also exhibited much higher potency than JI-38 upon i.v. administration. The relationship between the GH-releasing potency and the analog structure is discussed. Fourteen GHRH agonists with the highest endocrine potencies were subjected to cardiologic tests. MR-409 and MR-356 exhibited higher potency than JI-38 in activating myocardial repair in rats with induced myocardial infarction. As the previous class of analogs, exemplified by JI-38, had shown promising results in multiple fields including cardiology, diabetes and wound healing, our new, more potent, GHRH agonists should manifest additional efficacy for possible medical applications.
Keywords: hGHRH agonist; hGHRH(1–29); s.c. administration; Cardioprotection;

Little is known about ghrelin's effects on intestinal epithelial cells even though it is known to be a mitogen for a variety of other cell types. Because ghrelin is released in close proximity to the proliferative compartment of the intestinal tract, we hypothesized that ghrelin may have potent pro-proliferative effect on intestinal epithelial cells as well. To test this hypothesis, we characterized the effects of ghrelin on FHs74Int and Caco-2 intestinal epithelial cell lines in vitro. We found that ghrelin has potent dose dependent proliferative effects in both cell lines through a yet to be characterized G protein coupled growth hormone secretagogue receptor (GHS-R) subtype. Consistent with above findings, cell cycle flowcytometric analyses demonstrated that ghrelin shifts cells from the G1 to S phase and thereby promotes cell cycle progression. Further characterization of subcellular events, suggested that ghrelin mediates its pro-proliferative effect through Adenylate cyclase (AC)-independent epidermal growth factor receptor (EGFR) trans-activation and PI3K-Akt phosphorylation. Both these pathways converge to stimulate MAPK, ERK 1/2 downstream. The role of ghrelin in states where intestinal mucosal injury and rapid mucosal repair occur warrants further investigation.
Keywords: Ghrelin; Intestinal cells; Proliferation; Signaling;

Bradykinin-related peptides (BRPs) from skin secretions of three genera of phyllomedusine leaf frogs and their comparative pharmacological effects on mammalian smooth muscles by Yingchun Jiang; Xinping Xi; Lilin Ge; Nan Yang; Xiaojuan Hou; Jie Ma; Chengbang Ma; Yuxin Wu; Xiaoxiao Guo; Renjie Li; Mei Zhou; Lei Wang; Tianbao Chen; Chris Shaw (122-133).
While bradykinin has been identified in the skin secretions from several species of amphibian, bradykinin-related peptides (BRPs) are more common constituents. These peptides display a plethora of primary structural variations from the type peptide which include single or multiple amino acid substitutions, N- and/or C-terminal extensions and post-translational modifications such as proline hydroxylation and tyrosine sulfation. Such modified peptides have been reported in species from many families, including Bombinatoridae, Hylidae and Ranidae. The spectrum of these peptides in a given species is thought to be reflective of its predator profile from different vertebrate taxa. Here we report the isolation of BRPs and parallel molecular cloning of their respective biosynthetic precursor-encoding cDNAs from the skin secretions of the Mexican leaf frog (Pachymedusa dacnicolor), the Central American red-eyed leaf frog (Agalychnis callidryas) and the South American orange-legged leaf frog (Phyllomedusa hypochondrialis). Additionally, the eight different BRPs identified were chemically synthesized and screened for bioactivity using four different mammalian smooth muscle preparations and their effects and rank potencies were found to be radically different in these with some acting preferentially through bradykinin B1-type receptors and others through B2-type receptors.
Keywords: Amphibian; Skin; Peptides; Molecular cloning; Bradykinin; Smooth muscle;

Glucagon-like peptide-1 (GLP-1) has been proved to have effects of anti-hyperglycemia and β-cell preservation. However, it is still unclear whether there are differences between early and late GLP-1 intervention in type 2 diabetes mellitus (T2DM). We divided the mice into 5 groups: early treated group (n  = 7, 8-week old, fasting glucose > 10 mmol/l), late treated group (n  = 7, 10-week old, fasting glucose > 20 mmol/l), early control group (n  = 7), late control group (n  = 7) and wild type group (n  = 7). Treated group was injected with liraglutide (a GLP-1 analog) 300 μg/kg bid for 4 weeks, while control group was given saline at the same time. The results showed that compared with control group, food intake and body weight gain were reduced in both early and late treated group (p  < 0.05), and there was no significance between the two treated groups. Early liraglutide intervention showed better improvements in glucose control, acute insulin response to glucose (AIRg) and disposition index (before vs. after treatment, AIRg 1.01 ± 0.53 vs. 2.98 ± 0.63, disposition index 10.81 ± 0.89 vs. 27.4 ± 2.15) than late intervention (AIRg 0.99 ± 0.02 vs. 1.41 ± 0.32, disposition index 3.47 ± 0.38 vs. 6.43 ± 1.62, p  = 0.001). The histopathology of the pancreas showed the estimated β-cell mass (BCM) was increased more in early treated group than that in late one (0.03 vs. 0.01 g). Expressions of the proliferation related genes PDX-1, MafA and GLP-1 receptor (GLP-1R) in early treated group were 1.81, 2.57 and 1.59 times as much as that in late treated group. In conclusion, early liraglutide intervention was better in glucose control, β-cell function improvement and β-cell mass preservation.
Keywords: T2DM; β-Cell; Liraglutide; Early intervention;

The association between the levels of N-terminal pro-brain natriuretic peptide (NT-proBNP) and the severity of coronary artery disease (CAD) diagnosed by coronary angiography and other approaches has been investigated. The clinical application of NT-proBNP is restricted by the drawbacks of these techniques now available in screening out patients who need intensive or conservative treatment. Fractional flow reserve (FFR) is superior to coronary angiography and other functional indicators. Accordingly, we designed to investigate the association between NT-proBNP and myocardial ischemia from the perspective of anatomy and physiology in patients with unstable angina and preserved left ventricular function. Plasma samples were collected from 110 patients and NT-proBNP levels were measured by radioimmunoassay. The severity of coronary artery stenosis in patients was measured by coronary angiography and FFR. Stenosis ≥50% in the left main artery or stenosis of 70%, and fractional flow reserve (FFR) ≤0.80 in one or more coronary branches with diameter ≥2 mm were defined as “positive”, which require revascularization. NT-proBNP levels increased progressively between patients with negative and positive angiographic results (p  < 0.05), and between FFR-negative and FFR-positive patients (p  < 0.05). A significant correlation was observed between log NT-proBNP and log GS (GS = Gensini score, p  < 0.001). NT-proBNP level serves as a predictor of positive results of angiographic stenosis and FFR, with the area under the receiver operating characteristic curve being 0.697 and 0.787, respectively. NT-proBNP levels are correlated with the severity of anatomic coronary obstruction and inducible myocardial ischemia, but NT-proBNP per se is insufficient to identify clinically significant angiographic and physiological stenoses.
Keywords: N-terminal pro-brain natriuretic peptide; Coronary artery disease; Coronary angiography; Fractional flow reserve;

Characterization and dietary regulation of oligopeptide transporter (PepT1) in different ploidy fishes by Zhen Liu; Yi Zhou; Shaojun Liu; Qiong Zhao; Junchang Feng; Shuangqing Lu; Gang Xiong; Dizhi Xie; Jianshe Zhang; Yun Liu (149-156).
The oligopeptide transporter (PepT1) is located on the brush-border membrane of the intestinal epithelium which has been regarded as a mediator of protein absorption. Here, we cloned and characterized PepT1 genes from diploid (red crucian carp), triploid and tetraploid fish. Then, the PepT1 expression pattern in different tissues and embryogenesis were assayed. Meanwhile, using real-time PCR and western blotting, we showed the expression profiles of diets with different protein levels, protein sources and additives (sodium butyrate) in triploids. The cDNAs of the three different ploidy fishes have a high sequence similarity of PepT1 among vertebrates. PepT1 mRNA expression was also developmentally regulated and showed the strongest expression around the 2-cell and 4-cell stage in all three kinds of fishes. The maternal transcripts were first detected in eggs and dropped from blastula stage to muscle contraction stage. Tissue expression studies showed higher expression of PepT1 genes in the intestines of fishes compared with other tissues. In adults, triploids showed significantly higher expression levels of PepT1 in the intestines of the three kinds of ploidy fishes during breeding season and non-breeding season. In addition, high or low protein level diets both promote PepT1 expression in the intestine. We also confirmed that fish meal showed a significant increase in PepT1 expression than soybean meal in triploid intestines. Furthermore, sodium butyrate additives induce PepT1 expression that may be mediated by CDX2 and CREB. This research provides a new insight into protein absorption and its regulation in triploid fish.
Keywords: Peptide transporter 1; Red crucian carp; Triploid; Tetraploid; Dietary protein level; Sodium butyrate;