Peptides (v.90, #C)

Atrial natriuretic peptide: A novel mediator for TGF-β1-induced epithelial-mesenchymal transition in 16HBE-14o and A549 cells by Shuyuan Chu; Xiufeng Zhang; Yabing Sun; Yuanyuan Yu; Yaxi Liang; Ming Jiang; Jianwei Huang; Libing Ma (1-9).
Atrial natriuretic peptide (ANP) is increasingly expressed on airway and inhibits pulmonary arterial remodeling. However, the role of ANP in remodeling of respiratory system is still unclear. The role of ANP on airway remodeling and the possible mechanism was explored in this study. Both human bronchial epithelial 16HBE-14o cells and alveolar epithelial A549 cells were stimulated by TGF-β1, ANP, cGMP inhibitor, PKG inhibitor, and cGMP analogue. The expressions of epithelial markers, mesenchymal markers, and Smad3 were assessed by quantitative real-time PCR and western blotting. Immunohistochemical staining was employed to assess Smad3 expression once it was silenced by siRNA in 16HBE-14o or A549 cells. Our results showed that the mRNA and protein expressions of E-Cadherin were decreased, whereas α-SMA expressions were increased after induction by TGF-β1 in 16HBE-14o and A549 cells. The E-Cadherin expressions were increased and α-SMA expressions were decreased after ANP stimulation. Inhibition of cGMP or PKG decreased E-Cadherin expression but increased α-SMA expression, which could be reversed by cGMP analogue. Moreover, the phosphorylated Smad3 expression was consistent with α-SMA expression. After smad3 was silenced, Smad3 was mostly expressed in cytoplasm instead of nucleus as non-silenced cells during epithelial-mesenchymal transition (EMT). In conclusion, ANP inhibits TGF-β1-induced EMT in 16HBE-14o and A549 cells through cGMP/PKG signaling, by which it targets TGF-β1/Smad3 via attenuating phosphorylation of Smad3. These findings suggest the potential of ANP in the treatment on pulmonary diseases with airway remodeling.
Keywords: Atrial natriuretic peptide; Epithelial-mesenchymal transition; Bronchial epithelial cells; Alveolar epithelial cells;

Angiotensin-(1–7) [Ang-(1–7)] exhibits blood pressure lowering actions, inhibits cell growth, and reduces tissue inflammation and fibrosis which may functionally antagonize an activated Ang II-AT1 receptor axis. Since the vascular actions of Ang-(1–7) and the associated receptor/signaling pathways vary in different vascular beds, the current study established the vasorelaxant properties of the heptapeptide in the renal artery of male Wistar male rats. Ang-(1–7) produced an endothelium-dependent vasodilator relaxation of isolated renal artery segments pre-contracted by a sub-maximal concentration of phenylephrine (PE) (3 × 10−7  M). Ang-(1–7) induced vasodilation of the rat renal artery with an ED50 of 3 ± 1 nM and a maximal response of 42 ± 5% (N = 10). The two antagonists (10−5  M each) for the AT7/Mas receptor (MasR) [D-Pro7]-Ang-(1–7) and [D-Ala7]-Ang-(1–7) significantly reduced the maximal response to 12 ± 1% and 18 ± 3%, respectively. Surprisingly, the AT2R receptor antagonist PD123319, the AT1R antagonist losartan and B2R antagonist HOE140 (10−6  M each) also significantly reduced Ang-(1–7)-induced relaxation to 12 ± 2%, 22 ± 3% and 14 ± 7%, respectively. Removal of the endothelium or addition of the soluble guanylate cyclase (sGC) inhibitor ODQ (10−5  M) essentially abolished the vasorelaxant response to Ang-(1–7) (10 ± 4% and 10 ± 2%, P < 0.05). Finally, the NOS inhibitor LNAME (10−4  M) reduced the response to 13 ± 2% (p < 0.05), but the cyclooxygenase inhibitor indomethacin failed to block the Ang-(1–7) response. We conclude that Ang-(1–7) exhibits potent vasorelaxant actions in the isolated renal artery that are dependent on an intact endothelium and the apparent stimulation of a NO-sGC pathway. Moreover, Ang-(1–7)-dependent vasorelaxation was sensitive to antagonists against the AT7/Mas, AT1, AT2 and B2 receptor subtypes.
Keywords: Angiotensin; Kidney; Renal artery; Endothelium; c-GMP,;

Intraplaque angiogenesis associates with the instability of atherosclerotic plaques. In the present study, we investigated the effects of ghrelin on intraplaque angiogenesis and plaque instability in a rabbit model of atherosclerosis. The rabbits were randomly divided into three groups, namely, the control group, atherosclerotic model group, and ghrelin-treated group, with treatments lasting for 4 weeks. We found that the thickness ratio of the intima to media in rabbits of the ghrelin-treated group was significantly lower than that in rabbits of the atherosclerotic model group. The number of neovessels and the levels of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) decreased dramatically in rabbits of the ghrelin-treated group compared to those of the atherosclerotic model group. Ghrelin significantly decreased the plaque content of macrophages, matrix metalloproteinase (MMP)-2, and MMP-9, in a rabbit model of atherosclerosis. In addition, the level of the pro-inflammatory factor monocyte chemoattractant protein (MCP)-1 was significantly lower in rabbits of the ghrelin-treated group than in rabbits of the atherosclerotic model group. In summary, ghrelin can inhibit intraplaque angiogenesis and promote plaque stability by down-regulating VEGF and VEGFR2 expression, inhibiting the plaque content of macrophages, and reducing MCP-1 expression at an advanced stage of atherosclerosis in rabbits.
Keywords: Ghrelin; Vulnerable plaque; Macrophage; MMP-2; MMP-9;

Irisin is a peptide cleaved from the fibronectin type III domain containing protein 5 (FNDC5) gene that is secreted predominantly by muscle cells but also by other tissues including brain and intestine. In mammals, irisin has been shown to have thermogenic actions via the modulation of uncoupling proteins (UCPs) and to affect feeding and energy homeostasis via actions in brain, adipose tissue, liver, muscle and gastrointestinal tract. To examine the role of irisin on feeding and metabolism in fish, the effects of peripheral (intraperitoneal) injections of irisin on feeding behavior, glucose levels and the mRNA expressions of appetite regulators (cocaine and amphetamine regulated transcript CART, agouti related protein AgRP, orexin), UCPs and lipoprotein lipase LPL and brain factors (brain-derived neurotrophic factor , BDNF and tyrosine hydroxylase TH) were assessed in brain, white muscle and intestine. Irisin injections (100 ng/g) induced a decrease in food intake and increases in brain orexin, CART1 and CART2, UCP2, BDNF, muscle UCP2 and intestine LPL mRNA expressions but did not affect blood glucose levels, brain AgRP, TH, UCP1, UCP3 and LPL or muscle UCP1, UCP3 and LPL expressions. A partial goldfish FNDC5 cDNA was isolated and the expressions of FDNC5, UCPs, LPL and BDNF were also compared between fed and fasted fish. Fasting induced decreases FNDC5 mRNA expression in the brain and intestine, but not in muscle. Fasting also induced increases in brain BDNF and LPL expressions and increases in UCP1, UCP2, UCP3 and LPL expressions in muscle. Our result suggest that irisin is an anorexigenic factor in fish and its actions might be in part mediated by appetite-regulating factors such as CART and orexins as well as UCP2 and brain factors such as BDNF.
Keywords: Irisin; FNDC5; Orexin; Cart; BDNF; UCP; Goldfish; Food intake; mRNA expression;

Obestatin improves oxidative brain damage and memory dysfunction in rats induced with an epileptic seizure by Türkan Koyuncuoğlu; Caner Vızdıklar; Doğan Üren; Hakan Yılmaz; Çağan Yıldırım; Sefa Semih Atal; Dilek Akakın; Elif Kervancıoğlu Demirci; Meral Yüksel; Berrak Ç. Yeğen (37-47).
Obestatin was shown to alleviate renal, gastrointestinal and haemorrhage-induced brain injury in rats. In order to investigate the neuroprotective effects of obestatin on seizure-induced oxidative brain injury, an epileptic seizure was induced with a single intraperitoneal (i.p.) dose of pentylenetetrazole (PTZ, 45 mg/kg) in male Wistar rats. Thirty minutes before the PTZ injection, rats were treated with either saline or obestatin (1 μg/kg, i.p.). Seizure was video-taped and then evaluated by using Racine’s scoring (0–5). For the assessment of memory function, passive-avoidance test was performed before seizure induction, which was repeated on the 3rd day of seizure. The rats were decapitated at the 24th or 72nd hour of seizures and brain tissues were obtained for histopathological examination and for measuring levels of malondialdehyde (MDA), glutathione (GSH), reactive oxygen radicals and myeloperoxidase (MPO) activity. Obestatin treatment reduced the average seizure score, decreased the occurrence and duration of generalized tonic-clonic seizures, presenting with a shorter latency to their onset. Increased lipid peroxidation and enhanced generation of oxygen-derived radicals detected at the post-seizure 72nd h were suppressed by the consecutive treatments of obestatin, but no changes were observed by the single obestatin treatment in the 24-h seizure group. Neuronal damage and increased GFAP immunoreactivity, observed in the hippocampal areas and cortex of PTZ-induced rats were alleviated in 3-day obestatin-treated PTZ group. PTZ-induced memory dysfunction was significantly improved in obestatin-treated PTZ group as compared to saline-treated rats. The present data indicate that obestatin ameliorated the severity of PTZ-induced seizures, improved memory dysfunction and reduced neuronal damage by limiting oxidative damage.
Keywords: Seizure; Oxidative stress; Obestatin; Memory dysfunction;

Inhibitory effects of dynorphin 3-14 on the lipopolysaccharide-induced toll-like receptor 4 signalling pathway by Siti Sarah Fazalul Rahiman; Michael Morgan; Paul Gray; Paul Nicholas Shaw; Peter John Cabot (48-54).
Dynorphin 1-17 (DYN 1-17) is biotransformed rapidly to a range of fragments in rodent inflamed tissue with dynorphin 3-14 (DYN 3-14) being the most stable and prevalent. DYN 1-17 has been shown previously to be involved in the regulation of inflammatory response following tissue injury, in which the biotransformation fragments of DYN 1-17 may possess similar features. This study investigated the effects of DYN 3-14 on lipopolysaccharide (LPS)-induced nuclear factor-kappaB/p65 (NF-κB/p65) nuclear translocation and the release of pro-inflammatory cytokines interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in differentiated THP-1 cells. Treatment with DYN 3-14 (10 nM) resulted in 35% inhibition of the LPS-induced nuclear translocation of NF-κB/p65. Furthermore, DYN 3-14 modulated both IL-1β and TNF-α release; inhibiting IL-1β and paradoxically augmenting TNF-α release in a concentration-independent manner. A number of opioids have been implicated in the modulation of the toll-like receptor 4 (TLR4), highlighting the complexity of their immunomodulatory effects. To determine whether DYN 3-14 modulates TLR4, HEK-Blue™hTLR4 cells were stimulated with LPS in the presence of DYN 3-14. DYN 3-14 (10 μM) inhibited TLR4 activation in a concentration-dependent fashion by suppressing the LPS signals around 300-fold lower than LPS-RS, a potent TLR4 antagonist. These findings indicate that DYN 3-14 is a potential TLR4 antagonist that alters cellular signaling in response to LPS and cytokine release, implicating a role for biotransformed endogenous opioid peptides in immunomodulation.
Keywords: Dynorphin; TLR4; LPS; NF-κ; B/p65; IL-1β; TNF-α;

There is a well-documented association between cyclic changes to food intake and the changing ovarian hormone levels of the reproductive cycle in female mammals. Limited research on appetite-controlling gastrointestinal peptides has taken place in females, simply because regular reproductive changes in steroid hormones present additional experimental factors to account for. This study focussed directly on the roles that gastrointestinal-secreted peptides may have in these reported, naturally occurring, changes to food intake during the rodent estrous cycle and aimed to determine whether peripheral changes occurred in the anorexigenic (appetite-reducing) hormones peptide-YY (PYY) and glucagon-like peptide-1 (GLP-1) in female Wistar rats (32–44 weeks of age). Total forms of each peptide were measured in matched fed and fasted plasma and descending colon tissue samples for each animal during the dark (feeding) phase. PYY concentrations did not significantly change between defined cycle stages, in either plasma or tissue samples. GLP-1 concentrations in fed plasma and descending colon tissue were significantly increased during proestrus, just prior to a significant reduction in fasted stomach contents at estrus, suggesting increased satiety and reduced food intake at this stage of the cycle. Increased proestrus GLP-1 concentrations could contribute to the reported reduction in food intake during estrus and may also have biological importance in providing the optimal nutritional and metabolic environment for gametes at the potential point of conception. Additional analysis of the findings demonstrated significant interactions of ovarian cycle stage and fed/fasted status with age on GLP-1, but not PYY plasma concentrations. Slightly older females had reduced fed plasma GLP-1 suggesting that a relaxation of regulatory control of this incretin hormone may also take place with increasing age in reproductively competent females.
Keywords: Appetite; Estrous cycle; Gut peptides; GLP-1; Ovarian cycle; PYY; Satiety;

Association between circulating angiotensin-converting enzyme 2 and cardiac remodeling in hypertensive patients by Shichao Li; Zhijun Wang; Xiuhong Yang; Bo Hu; Yuling Huang; Sujing Fan (63-68).
Angiotensin-converting enzyme 2 (ACE2) plays a vital role in the pathogenesis of hypertension-induced cardiac remodeling and exhibits cardioprotective properties in hypertensive animal models. Evidence that ACE2 is an important regulator of hypertensive cardiac remodeling in humans has not been addressed directly yet.A total of 161 patients with essential hypertension and 47 age- and sex-matched normotensive healthy subjects were consecutively recruited. Serum concentration levels of ACE2 were determined by enzyme-linked immunosorbent assay. Cardiac structural and functional parameters were measured by echocardiography.Serum ACE2 concentrations were higher in hypertensive patients compared to healthy subjects (170.31 [83.50–707.12] pg/ml in patients versus 59.28 [39.71–81.81] pg/ml in healthy subjects, P <  0.001). After adjustment for confounders, including age, sex, body mass index, snoring, smoking, duration of hypertension, comorbidities, medication use, mean arterial pressure and N-terminal pro-brain natriuretic peptide, serum ACE2 concentrations were positively correlated with left atrial diameter, left ventricular end-diastolic diameter and left ventricular mass in hypertensive patients. Moreover, multiple regression analyses adjusting for covariates revealed that serum ACE2 concentrations were also independently associated with left ventricular ejection fraction and late diastolic filling velocities of the mitral inflow.This study reveals an elevated serum concentration of ACE2 and independent associations between serum ACE2 and echocardiographic parameters in hypertensive patients.
Keywords: Angiotensin-converting enzyme 2; Renin-angiotensin system; Cardiac remodeling; Hypertensive heart disease; Hypertension;

Exendin-4 inhibits structural remodeling and improves Ca2+ homeostasis in rats with heart failure via the GLP-1 receptor through the eNOS/cGMP/PKG pathway by Jingjing Chen; Dandan Wang; Fangai Wang; Shaobo Shi; Yuting Chen; Bo Yang; Yanhong Tang; Congxin Huang (69-77).
The glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 is a long-acting analog of GLP-1, which stimulates insulin secretion and is clinically used in the treatment of type 2 diabetes. Previous studies have demonstrated that GLP-1 agonists and analogs serve as cardioprotective factors in various conditions. Disturbances in calcium cycling are characteristic of heart failure (HF); therefore, the aim of this study was to investigate the effect of exendin-4 (a GLP-1 mimetic) on the regulation of calcium handling and to identify the underlying mechanisms in an HF rat model after myocardial infarction (MI). Rats underwent surgical ligation of the left anterior descending coronary artery or sham surgery prior to infusion with vehicle, exendin-4, or exendin-4 and exendin9-39 for 4 weeks. Exendin-4 treatment decreased MI size, suppressed chamber dilation, myocyte hypertrophy, and fibrosis and improved in vivo heart function in the rats subjected to MI. Exendin-4 resulted in an increase in circulating GLP-1 and GLP-1R in ventricular tissues. Additionally, exendin-4 activated the eNOS/cGMP/PKG signaling pathway and inhibited the Ca2+/calmodulin-dependent kinase II (CaMKII) pathways. Myocytes isolated from exendin-4-treated hearts displayed higher Ca2+ transients, higher sarcoplasmic reticulum Ca2+ content, and higher l-type Ca2+ current densities than MI hearts. Exendin-4 treatment restored the protein expression of sarcoplasmic reticulum Ca2+ uptake ATPase (SERCA2a), phosphorylated phospholamban (PLB) and Cav1.2 and decreased the levels of phosphorylated ryanodine receptor (RyR). Moreover, the favorable effects of exendin-4 were significantly inhibited by exendin9-39 (a GLP-1 receptor antagonist). Exendin-4 treatment of an HF rat model after MI inhibited cardiac and cardiomyocytes progressive remodeling. In addition, Ca2+ handling and its molecular modulation were also improved by exendin-4 treatment. The beneficial effects of exendin-4 on cardiac remodeling may be mediated through activation of the eNOS/cGMP/PKG pathway.
Keywords: Exendin-4, Heart Failure, Calmodulin-Dependent Kinase II, eNOS/cGMP/PKG Pathway, Calcium Handing;

Gastric bypass in the pig increases GIP levels and decreases active GLP-1 levels by Andreas Lindqvist; Mikael Ekelund; Stefan Pierzynowski; Leif Groop; Jan Hedenbro; Nils Wierup (78-82).
Gastric bypass surgery results in remission of type 2 diabetes in the majority of patients. The incretin hormones glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) have been implicated in the observed remission. Most knowledge so far has been generated in obese subjects. To isolate the surgical effects of gastric bypass on metabolism and hormone responses from the confounding influence of obesity, T2D, or food intake, we performed gastric bypass in lean pigs, using sham-operated and pair-fed pigs as controls. Thus, pigs were subjected to Roux-en-Y gastric bypass (RYGB) or sham surgery and oral glucose tolerance tests (OGTT). RYGB pigs and sham pigs exhibited similar basal and 120-min glucose levels in response to the OGTT. However, RYGB pigs had approximately 1.6-fold higher 30-min glucose (p  < 0.01). Early insulin release (EIR) was enhanced approximately 3.5-fold in the RYGB pigs (p  < 0.01). Furthermore, GIP release, both acute and sustained release (p  < 0.001 and p  < 0.01, respectively), was increased approximately 2.5-fold and 1.4-fold, respectively, in RYGB pigs. Although total GLP-1 release increased approximately 2.1-fold after RYGB (p  < 0.001), active GLP-1 was 33% lower (p  < 0.01). Interestingly basal DPP4-activity was approximately 3.2-fold higher in RYGB pigs (p  < 0.001). In conclusion, RYGB in lean pigs increases the response of GIP, total GLP-1, and insulin, but reduces levels of active GLP-1 in response to an oral glucose load. These data challenge the role of active GLP-1 as a contributor to remission from diabetes after RYGB.
Keywords: Gastric Bypass; GIP; GLP-1; Insulin; Bariatric surgery; Pig model;

Niemann-Pick C1 like-1 (NPC1L1) mediates cholesterol absorption at the apical membrane of enterocytes through a yet unknown mechanism. Bean, pea, and lentil proteins are naturally hydrolyzed during digestion to produce peptides. The potential for pulse peptides to have high binding affinities for NPC1L1 has not been determined. In this study , in silico binding affinities and interactions were determined between the N-terminal domain of NPC1L1 and 14 pulse peptides (5≥ amino acids) derived through pepsin-pancreatin digestion. Peptides were docked in triplicate to the N-terminal domain using docking program AutoDock Vina, and results were compared to those of ezetimibe, a prescribed NPC1L1 inhibitor. Three black bean peptides (−7.2 to −7.0 kcal/mol) and the cowpea bean dipeptide Lys-Asp (−7.0 kcal/mol) had higher binding affinities than ezetimibe (−6.6 kcal/mol) for the N-terminal domain of NPC1L1. Lentil and pea peptides studied did not have high binding affinities. The common bean peptide Tyr-Ala-Ala-Ala-Thr (−7.2 kcal/mol), which can be produced from black or navy bean proteins, had the highest binding affinity. Ezetimibe and peptides with high binding affinities for the N-terminal domain are expected to interact at different locations of the N-terminal domain. All high affinity black bean peptides are expected to have van der Waals interactions with SER130, PHE136, and LEU236 and a conventional hydrogen bond with GLU238 of NPC1L1. Due to their high affinity for the N-terminal domain of NPC1L1, black and cowpea bean peptides produced in the digestive track have the potential to disrupt interactions between NPC1L1 and membrane proteins that lead to cholesterol absorption.
Keywords: NPC1L1; Bean peptides; Cholesterol absorption; Black beans; AutoDock Vina;

Endothelin causes transactivation of the EGFR and HER2 in non-small cell lung cancer cells by Terry W. Moody; Irene Ramos-Alvarez; Paula Moreno; Samuel A. Mantey; Lisa Ridnour; David Wink; Robert T. Jensen (90-99).
Endothelin (ET)-1 is an important peptide in cancer progression stimulating cellular proliferation, tumor angiogenesis and metastasis. ET-1 binds with high affinity to the ETA receptor (R) and ETBR on cancer cells. High levels of tumor ET-1 and ETAR are associated with poor survival of lung cancer patients. Here the effects of ET-1 on epidermal growth factor (EGF)R and HER2 transactivation were investigated using non-small cell lung cancer (NSCLC) cells. ETAR mRNA was present in all 10 NSCLC cell lines examined. Addition of ET-1 to NCI-H838 or H1975 cells increased EGFR, HER2 and ERK tyrosine phosphorylation within 2 min. The increase in EGFR and HER2 transactivation caused by ET-1 addition to NSCLC cells was inhibited by lapatinib (EGFR and HER2 tyrosine kinase inhibitor (TKI)), gefitinib (EGFR TKI), ZD4054 or BQ-123 (ETAR antagonist), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor) or Tiron (superoxide scavenger). ET-1 addition to NSCLC cells increased cytosolic Ca2+ and reactive oxygen species. ET-1 increased NSCLC clonal growth, whereas BQ123, ZD4054, lapatinib or gefitinib inhibited proliferation. The results indicate that ET-1 may regulate NSCLC cellular proliferation in an EGFR- and HER2-dependent manner.
Keywords: Endothelin; Lung cancer; Transactivation; EGFR; HER2;

Molecular characterization and functional analyses of a diapause hormone receptor-like gene in parthenogenetic Artemia by Hui-Li Ye; Dong-Rui Li; Jin-Shu Yang; Dian-Fu Chen; Stephanie De Vos; Marnik Vuylsteke; Patrick Sorgeloos; Gilbert Van Stappen; Peter Bossier; Hiromichi Nagasawa; Wei-Jun Yang (100-110).
In arthropods, mature females under certain conditions produce and release encysted gastrula embryos that enter diapause, a state of obligate dormancy. The process is presumably regulated by diapause hormone (DH) and diapause hormone receptor (DHR) that were identified in the silkworm, Bombyx mori and other insects. However, the molecular structure and function of DHR in crustaceans remains unknown. Here, a DHR-like gene from parthenogenetic Artemia (Ar-DHR) was isolated and sequenced. The cDNA sequence consists of 1410 bp with a 1260-bp open reading frame encoding a protein consisting of 420 amino acid residues. The results of real-time PCR (qRT-PCR) and Western blot analysis showed that the mRNA and protein of Ar-DHR were mainly expressed at the diapause stage. Furthermore, we found that Ar-DHR was located on the cell membrane of the pre-diapause cyst but in the cytoplasm of the diapause cyst by analysis of immunofluorescence. In vivo knockdown of Ar-DHR by RNA interference (RNAi) and antiserum neutralization consistently inhibited diapause cysts formation. The results indicated that Ar-DHR plays an important role in the induction and maintenance of embryonic diapause in Artemia. Thus, our findings provide an insight into the regulation of diapause formation in Artemia and the function of Ar-DHR.
Keywords: Diapause; Diapause hormone (DH); Diapause hormone receptor (DHR); Artemia; RNAi; Antiserum neutralization;