RESUMEN

Cholecystokinin (CCK) is widely distributed in the gastrointestinal tract and central nervous system, regulating a range of physiological functions by activating its receptors (CCK1R and CCK2R). Compared to those in mammals, the CCK gene and its receptors have already been cloned in various birds, such as chickens. However, knowledge regarding their functionality and tissue expression is limited. In this study, we examined the expression of CCK and its 2 receptors in chicken tissues. In addition, the functionality of the 2 receptors was investigated. Using 3 cell-based luciferase reporter systems and western blots, we demonstrated that chicken (c-) CCK1R could be potently activated by cCCK-8S but not cCCK-4, whereas cCCK2R could be activated by cCCK-8S and cCCK-4 with similar efficiency. Using RNA-sequencing, we revealed that cCCK is abundantly expressed in the testis, ileum, and several brain regions (cerebrum, midbrain, cerebellum, hindbrain, and hypothalamus). The abundant expression of CCK in the hypothalamus was further supported by immunofluorescence. In addition, cCCK1R is highly expressed in the pancreas and moderately expressed in various intestinal regions (ileum, cecum, and rectum) and the pituitary gland, whereas cCCK2R expression is primarily restricted to the brain. Our data reveal the differential specificities of CCK receptors for various CCK peptides. In combination with the differential tissue distribution of CCK and its receptors, the present study helps to understanding the physiological functions of CCK/CCKRs in birds.

Asunto(s)

Pollos , Colecistoquinina , Masculino , Animales , Colecistoquinina/genética , Colecistoquinina/metabolismo , Pollos/genética , Pollos/metabolismo , Receptores de Colecistoquinina/genética , Receptores de Colecistoquinina/metabolismo , Intestinos , Íleon/metabolismo , Mamíferos/metabolismo

RESUMEN

BACKGROUND: The molecular characterization of thyroid nodules in cytological samples has so far been focused on discriminating between benign and malignant forms in a purely diagnostic setting. The evidence on the impact of molecular biomarkers to determine the risk of aggressiveness in cytologically "neoplastic" lesions is limited to genomic alterations (such as BRAF and TERT mutations). The aim of our study was to assess the preoperative role of microRNAs (miRNAs) in predicting the nodal status of patients with papillary thyroid cancer. METHODS: A pilot series of histological samples of papillary thyroid carcinoma with (6 cases) or without (6 cases) lymph node metastases, matched for other major clinical and pathological features, was analyzed for global miRNA expression in a screening phase. A set of miRNAs was then validated in a series of 63 consecutive cytological samples of papillary carcinomas: 48 pN-negative and 15 pN-positive at histology. RESULTS: Unsupervised cluster analysis segregated surgical pN-negative and pN-positive samples, except for 1 case. The 45 differentially expressed miRNAs in pN-positive versus pN-negative cases were predicted to regulate a wide range of cellular pathways, enriched for Wnt, gonadotropin-releasing hormone receptor, and cerulein/cholecystokinin receptor signaling. In agreement with their profiles in surgical samples, 4 miRNAs of the 10 selected for validation (miR-154-3p, miR-299-5p, miR-376a-3p, and miR-302E) had a significant differential expression in cytological samples of papillary carcinoma with lymph node metastases and predicted the positive nodal status with a relatively good performance. CONCLUSIONS: MiRNA profiling is a potential promising strategy to define papillary carcinoma aggressiveness in the preoperative setting.

Asunto(s)

Carcinoma Papilar , MicroARNs , Neoplasias de la Tiroides , Biomarcadores de Tumor/genética , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Carcinoma Papilar/cirugía , Ceruletida/genética , Ceruletida/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis Linfática/genética , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Receptores de Colecistoquinina/genética , Receptores de Colecistoquinina/metabolismo , Receptores LHRH/genética , Receptores LHRH/metabolismo , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/cirugía , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/cirugía

RESUMEN

Gastrin and cholecystokinin peptides bind a common G-protein coupled receptor, cholecystokinin receptor B (CCKBR) whilst cholecystokinin receptor A (CCKAR) is preferentially bound by CCK. Gastrin and cholecystokinin mediate signalling from the gastrointestinal tract to regulate appetite and digestive function. In this study, expression of the cholecystokinin/gastrin family and distribution of their receptors expression was measured to understand the target organs for the peptides and how expression responds to changes in food intake. We confirmed the restricted expression of gastrin in the antrum and the abundant expression of cholecystokinin in the hypothalamus. The expression of gastrin in the antrum was significantly elevated in broiler breeders when released from feed restriction. CCKBR was most abundant in the hypothalamus and proventriculus. CCKAR was most abundant in the pancreas and crop, more than tenfold greater than the gastrointestinal tract. Cholecystokinin expression in the pancreas increased after removal of food restriction. CCKAR in the gastrointestinal tract peaks around the distal ileum, distal to the peak of cholecystokinin expression. There was virtually no cholecystokinin expression in the caecum but CCKAR expression was high. The CCKAR expression in the crop was unexpected, supporting a role of cholecystokinin in mediating crop emptying which was supported by the observation of in-vitro contraction after cholecystokinin administration. The response to changes in food intake and the expression pattern of the cholecystokinin/gastrin family and their receptors will stimulate and inform new hypotheses on their role in growth in poultry.

Asunto(s)

Colecistoquinina , Receptores de Colecistoquinina , Animales , Pollos/metabolismo , Gastrinas/metabolismo , Receptor de Colecistoquinina B/genética , Receptores de Colecistoquinina/genética , Receptores de Colecistoquinina/metabolismo

RESUMEN

Cholecystokinin 1 receptor (CCK1R) is activated photodynamically. For this to happen in situ, genetically encoded protein photosensitizers (GEPP) may be tagged to natively expressed CCK1R, but how to best tag GEPP has not been examined. Therefore, GEPP (miniSOG or KillerRed) was tagged to CCK1R and light-driven photodynamic CCK1R activation was monitored by Fura-2 fluorescent calcium imaging, to screen for optimized tagging patterns. Blue light-emitting diode irradiation of CHO-K1 cells expressing miniSOG fused to N- or C-terminus of CCK1R was found to both trigger persistent calcium oscillations-a hallmark of permanent photodynamic CCK1R activation. Photodynamic CCK1R activation was accomplished also with miniSOG fused to N-terminus of CCK1R via linker (GlySerGly)4 or 8 , but not linker (GSG)12 or an internal ribosomal entry site insert. KillerRed fused to N- or C-terminus of CCK1R after white light irradiation resulted in similar activation of in-frame CCK1R. Photodynamic CCK1R activation in miniSOG-CCK1R-CHO-K1 cells was blocked by singlet oxygen (1 O2 ) quencher uric acid or Trolox C, corroborating the role of 1 O2 as the reactive intermediate. It is concluded that photodynamic CCK1R activation can be achieved either with direct GEPP fusion to CCK1R or fusion via a short linker, fusion via long linkers might serve as the internal control.

Asunto(s)

Fármacos Fotosensibilizantes , Receptores de Colecistoquinina , Calcio , Colecistoquinina , Fura-2 , Fármacos Fotosensibilizantes/metabolismo , Fármacos Fotosensibilizantes/farmacología , Proteínas , Receptores de Colecistoquinina/genética , Receptores de Colecistoquinina/metabolismo , Oxígeno Singlete/metabolismo , Ácido Úrico

RESUMEN

Cholecystokinin receptors, CCKAR and CCKBR, are important neurointestinal peptide hormone receptors and play a vital role in food intake and appetite regulation. Here, we report three crystal structures of the human CCKAR in complex with different ligands, including one peptide agonist and two small-molecule antagonists, as well as two cryo-electron microscopy structures of CCKBR-gastrin in complex with Gi2 and Gq, respectively. These structures reveal the recognition pattern of different ligand types and the molecular basis of peptide selectivity in the cholecystokinin receptor family. By comparing receptor structures in different conformational states, a stepwise activation process of cholecystokinin receptors is proposed. Combined with pharmacological data, our results provide atomic details for differential ligand recognition and receptor activation mechanisms. These insights will facilitate the discovery of potential therapeutics targeting cholecystokinin receptors.

Asunto(s)

Devazepida/química , Receptores de Colecistoquinina/química , Secuencia de Aminoácidos , Microscopía por Crioelectrón , Cristalización , Humanos , Ácidos Indolacéticos/química , Ligandos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Receptores de Colecistoquinina/genética , Relación Estructura-Actividad , Tiazoles/química

RESUMEN

Combined activation of GLP-1 and CCK1 receptors has potential to synergistically augment the appetite-suppressive and glucose homeostatic actions of the individual parent peptides. In the current study, pancreatic beta-cell benefits of combined GLP-1 and CCK1 receptor upregulation were established, before characterising bioactivity and antidiabetic efficacy of an acylated dual-acting GLP-1/CCK hybrid peptide, namely [Lys12Pal]Ex-4/CCK. Both exendin-4 and CCK exhibited (p<0.001) proliferative and anti-apoptotic effects in BRIN BD11 beta-cells. Proliferative benefits were significantly (p<0.01) augmented by combined peptide treatment when compared to either parent peptide alone. These effects were linked to increases (p<0.001) in GLUT2 and glucokinase beta-cell gene expression, with decreased (p<0.05-p<0.001) expression of NFκB and BAX. [Lys12Pal]Ex-4/CCK exhibited prominent insulinotropic actions in vitro, coupled with beneficial (p<0.001) satiety and glucose homeostatic effects in the mice, with bioactivity evident 24 h after administration. Following twice daily injection of [Lys12Pal]Ex-4/CCK for 28 days in diabetic high fat fed (HFF) mice with streptozotocin (STZ)-induced compromised beta-cells, there were clear reductions (p<0.05-p<0.001) in energy intake and body weight. Circulating glucose was returned to lean control concentrations, with associated increases (p<0.001) in plasma and pancreatic insulin levels. Glucose tolerance and insulin secretory responsiveness were significantly (p<0.05-p<0.001) improved by hybrid peptide therapy. In keeping with this, evaluation of pancreatic histology revealed restoration of normal islet alpha- to beta-cell ratios and reduction (p<0.01) in centralised islet glucagon staining. Improvements in pancreatic islet morphology were associated with increased (p<0.05) proliferation and reduced (p<0.001) apoptosis of beta-cells. Together, these data highlight the effectiveness of sustained dual GLP-1 and CCK1 receptor activation by [Lys12Pal]Ex-4/CCK for the treatment of obesity-related diabetes.

Asunto(s)

Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Exenatida/farmacología , Péptido 1 Similar al Glucagón/metabolismo , Obesidad/fisiopatología , Fragmentos de Péptidos/farmacología , Receptores de Colecistoquinina/metabolismo , Animales , Biomarcadores/sangre , Glucemia/análisis , Peso Corporal , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Dieta Alta en Grasa , Péptido 1 Similar al Glucagón/genética , Hipoglucemiantes/farmacología , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Colecistoquinina/genética , Regulación hacia Arriba

RESUMEN

Hypertensive nephropathy (HN) is a common cause of end-stage renal disease with renal fibrosis; chronic kidney disease is associated with elevated serum gastrin. However, the relationship between gastrin and renal fibrosis in HN is still unknown. We, now, report that mice with angiotensin II (Ang II)-induced HN had increased renal cholecystokinin receptor B (CCKBR) expression. Knockout of CCKBR in mice aggravated, while long-term subcutaneous infusion of gastrin ameliorated the renal injury and interstitial fibrosis in HN and unilateral ureteral obstruction (UUO). The protective effects of gastrin on renal fibrosis can be independent of its regulation of blood pressure, because in UUO, gastrin decreased renal fibrosis without affecting blood pressure. Gastrin treatment decreased Ang II-induced renal tubule cell apoptosis, reversed Ang II-mediated inhibition of macrophage efferocytosis, and reduced renal inflammation. A screening of the regulatory factors of efferocytosis showed involvement of peroxisome proliferator-activated receptor α (PPAR-α). Knockdown of PPAR-α by shRNA blocked the anti-fibrotic effect of gastrin in vitro in mouse renal proximal tubule cells and macrophages. Immunofluorescence microscopy, Western blotting, luciferase reporter, and Cut&tag-qPCR analyses showed that CCKBR may be a transcription factor of PPAR-α, because gastrin treatment induced CCKBR translocation from cytosol to nucleus, binding to the PPAR-α promoter region, and increasing PPAR-α gene transcription. In conclusion, gastrin protects against HN by normalizing blood pressure, decreasing renal tubule cell apoptosis, and increasing macrophage efferocytosis. Gastrin-mediated CCKBR nuclear translocation may make it act as a transcription factor of PPAR-α, which is a novel signaling pathway. Gastrin may be a new potential drug for HN therapy.

Asunto(s)

Gastrinas/farmacología , Hipertensión Renal/fisiopatología , Nefritis/fisiopatología , PPAR alfa/metabolismo , Receptores de Colecistoquinina/metabolismo , Angiotensina II/administración & dosificación , Animales , Apoptosis , Fibrosis , Humanos , Hipertensión/complicaciones , Células Jurkat , Túbulos Renales Proximales/patología , Ratones , Ratones Noqueados , PPAR alfa/genética , Fagocitosis , ARN Interferente Pequeño , Receptores de Colecistoquinina/genética , Transducción de Señal/efectos de los fármacos , Obstrucción Ureteral/fisiopatología

RESUMEN

Cholecystokinin 1 receptor (CCK1R) is activated by singlet oxygen (1O2) generated in photodynamic action with sulphonated aluminum phthalocyanine (SALPC) or genetically encoded protein photosensitizer (GEPP) KillerRed or mini singlet oxygen generator (miniSOG). A large number of GEPP with varied 1O2 quantum yields have appeared recently; therefore, in the present work, the efficacy of different GEPP to photodynamically activate CCK1R was examined, as monitored by Fura-2 calcium imaging. KillerRed, miniSOG, miniSOG2, singlet oxygen protein photosensitizer (SOPP), flavin-binding fluorescent protein from Methylobacterium radiotolerans with point mutation C71G (Mr4511C71G), and flavin-binding fluorescent protein from Dinoroseobacter shibae (DsFbFP) were expressed at the plasma membrane (PM) in AR4-2J cells, which express endogenous CCK1R. Light irradiation (KillerRed: white light 85.3 mW‧cm-2, 4' and all others: LED 450 nm, 85 mW·cm-2, 1.5') of GEPPPM-expressing AR4-2J was found to all trigger persistent calcium oscillations, a hallmark of permanent photodynamic CCK1R activation; DsFbFP was the least effective, due to poor expression. miniSOG was targeted to PM, mitochondria (MT) or lysosomes (LS) in AR4-2J in parallel experiments; LED light irradiation was found to all induce persistent calcium oscillations. In miniSOGPM-AR4-2J cells, light emitting diode (LED) light irradiation-induced calcium oscillations were readily inhibited by CCK1R antagonist devazepide 2 nM; miniSOGMT-AR4-2J cells were less susceptible, but miniSOGLS-AR4-2J cells were not inhibited. In conclusion, different GEPPPM could all photodynamically activate CCK1R. Intracellular GEPP photodynamic action may prove particularly suited to study intracellular GPCR.

Asunto(s)

Proteínas Bacterianas , Proteínas Luminiscentes , Methylobacterium/genética , Fármacos Fotosensibilizantes/metabolismo , Receptores de Colecistoquinina , Rhodobacteraceae/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Línea Celular , Humanos , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Receptores de Colecistoquinina/biosíntesis , Receptores de Colecistoquinina/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética

RESUMEN

In contrast to reversible activation by agonist, cholecystokinin 1 receptor (CCK1R) is permanently activated by singlet oxygen generated in photodynamic action, with sulphonated aluminium phthalocyanine or genetically encoded mini singlet oxygen generator (miniSOG) as photosensitizer. In these works, a halogen light source was used to power photodynamic action. For possible in vivo application of photodynamic CCK1R physiology, bearing a cumbersome light-delivery device connected to an external light source by experimental animals might interfere with their behavior. Therefore, in the present work, the possibility of bioluminescence-driven miniSOG photodynamic CCK1R activation was examined, as monitored by Fura-2 calcium imaging. In parallel experiments, it was found that, after plasma membrane (PM)-localized expression of miniSOGPM in AR4-2J cells, light irradiation with blue light-emitting diode (LED) (450 nm, 85 mW·cm-2, 1.5 min) induced persistent calcium oscillations that were blocked by CCK1R antagonist devazepide 2 nM. NanoLuc was expressed bicistronically with miniSOGPM via an internal ribosome entry site (IRES) sequence (pminiSOGPM-IRES-NanoLuc). The resultant miniSOGPM-IRES-NanoLuc-AR4-2J cells were found to generate strong bioluminescence upon addition of NanoLuc substrate coelenterazine. Strikingly, coelenterazine 5 microM was found to trigger long-lasting calcium oscillations (a hallmark for permanent CCK1R activation) in perifused miniSOGPM-IRES-NanoLuc-AR4-2J cells. These data indicate that NanoLuc bioluminescence can drive miniSOGPM photodynamic CCK1R activation, laying the foundation for its future in vivo applications.

Asunto(s)

Proteínas Luminiscentes/metabolismo , Receptores de Colecistoquinina/metabolismo , Oxígeno Singlete/metabolismo , Animales , Técnicas Biosensibles/métodos , Señalización del Calcio , Línea Celular Tumoral , Sitios Internos de Entrada al Ribosoma , Luz , Luminiscencia , Proteínas Luminiscentes/genética , Ingeniería de Proteínas/métodos , Ratas , Receptores de Colecistoquinina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

BACKGROUND: Nutrient-mediated release of cholecystokinin and glucagon-like peptide-1 (GLP-1) regulates gastric emptying (GE) via duodenogastric feedback mechanisms; GLP-1 also regulates postprandial insulin secretion. Some patients with functional upper gastrointestinal symptoms have impaired glucose tolerance during enteral dextrose infusion. Our hypothesis was that variants in CCK, GLP-1, and TCF7L2 (transcription factor 7-like 2 locus), which is associated with greatest genetic risk for development of type 2 diabetes mellitus, are associated with GE and independently with glucose tolerance. Our aims were to evaluate the associations between these GE, glucose tolerance, and these single nucleotide polymorphisms (SNPs). METHODS: Genetic variants, scintigraphic GE of solids, plasma glucose, insulin, and GLP-1 during enteral dextrose infusion (75gm over 2 hours) were measured. GE and enteral dextrose infusion were, respectively, evaluated in 44 (27 controls and 17 patients with functional dyspepsia or nausea) and 42 (28 controls, 14 patients) participants; of these, 51 participants consented to assessment of SNPs. Four functional SNPs were studied: rs6923761 and rs1042044 at GLP-1 receptor, rs7903146 (TCF7L2), and rs1800857 (CCK receptor). KEY RESULTS: Gastric emptying was normal in 38, rapid in 4, and delayed in two participants; 38 had normal, and four had impaired glucose tolerance. The T allele at rs7903146 (TCF7L2) was non-significantly associated (P = .14) with faster GE. The associations between SNPs and demographic variables, GE thalf , glucose tolerance and plasma GLP1 levels were not significant. CONCLUSIONS & INFERENCES: There is a trend toward an association between faster GE and the diabetes-associated allele at rs7903146 in TCF7L2. However, these SNPs were not associated with plasma glucose or GLP1 concentrations during enteral dextrose infusion.

Asunto(s)

Vaciamiento Gástrico/genética , Péptido 1 Similar al Glucagón/genética , Intolerancia a la Glucosa/genética , Receptores de Colecistoquinina/genética , Proteína 2 Similar al Factor de Transcripción 7/genética , Adulto , Femenino , Enfermedades Gastrointestinales/genética , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple

RESUMEN

We investigated expression of cholecystokinin (CCK) in humans and mice, and the bitter taste receptor TAS2R14 in the human placenta. Because CCK and gastrin activate the CCKBR receptor, we also explored placental gastrin expression. Finally, we investigated calcium signaling by CCK and TAS2R14. By RT-PCR, we found CCK/Cck and GAST/Gast mRNA expression in both normal human and mouse placentas, as well as in human trophoblast cell lines (TCL). Although both Cckar and -br mRNA were expressed in the mouse placenta, only CCKBR mRNA was detected in the human placenta and TCL. mRNA expression for TAS2R14 was also observed in the human placenta and TCL. Using immunohistochemistry, CCK protein was localized to the syncytiotrophoblast (ST) and extravillous trophoblast (EVT) in the human term placenta, and to trophoblast glycogen cells in mouse and human placentas. Gastrin and TAS2R14 proteins were also observed in ST and EVT of the human placenta. Both sulfated and nonsulfated CCK elicited a comparable rise in intracellular calcium in TCL, consistent with CCKBR expression. Three TAS2R14 agonists, flufenamic acid, chlorhexidine, and diphenhydramine, also evoked rises in intracellular calcium in TCL. These results establish CCK, gastrin, and their receptor(s) in both human and mouse placentas, and TAS2R14 in the human placenta. Both CCK and TAS2R14 agonists increased intracellular calcium in human TCL. Although the roles of these ligands and receptors, and their potential cross talk in normal and pathological placentas, are currently unknown, this study opens new avenues for placental research.

Asunto(s)

Colecistoquinina/metabolismo , Gastrinas/metabolismo , Receptor de Colecistoquinina B/metabolismo , Receptores de Colecistoquinina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Trofoblastos/metabolismo , Animales , Señalización del Calcio , Línea Celular , Colecistoquinina/genética , Colecistoquinina/farmacología , Femenino , Gastrinas/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor de Colecistoquinina B/genética , Receptores de Colecistoquinina/agonistas , Receptores de Colecistoquinina/genética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/efectos de los fármacos , Trofoblastos/efectos de los fármacos

RESUMEN

OBJECTIVE: This study evaluated the effect of a protein, the isolated Trypsin Inhibitor (TTI) from Tamarindus indica L. seed, as a CCK secretagogue and its action upon food intake and leptin in obese Wistar rats. METHODS: Three groups of obese rats were fed 10 days one of the following diets: Standard diet (Labina®) + water; High Glycemic Index and Load (HGLI) diet + water or HGLI diet + TTI. Lean animals were fed the standard diet for the 10 days. Food intake, zoometric measurements, plasma CCK, plasma leptin, relative mRNA expression of intestinal CCK-related genes, and expression of the ob gene in subcutaneous adipose tissue were assessed. RESULTS: TTI decreased food intake but did not increase plasma CCK in obese animals. On the other hand, TTI treatment decreased CCK-1R gene expression in obese animals compared with the obese group with no treatment (p = 0.027). Obese animals treated with TTI presented lower plasma leptin than the non-treated obese animals. CONCLUSION: We suggest that TTI by decreasing plasma leptin may improve CCK action, regardless of its increase in plasma from obese rats, since food intake was lowest.

Asunto(s)

Depresores del Apetito/farmacología , Ingestión de Alimentos/efectos de los fármacos , Leptina/sangre , Obesidad , Proteínas de Vegetales Comestibles/farmacología , Receptores de Colecistoquinina/genética , Tamarindus/química , Animales , Depresores del Apetito/aislamiento & purificación , Depresores del Apetito/uso terapéutico , Regulación hacia Abajo/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Masculino , Obesidad/sangre , Obesidad/tratamiento farmacológico , Obesidad/genética , Proteínas de Vegetales Comestibles/aislamiento & purificación , Ratas , Ratas Wistar , Receptores de Colecistoquinina/metabolismo , Respuesta de Saciedad/efectos de los fármacos , Semillas/química

RESUMEN

The gastrointestinal peptide cholecystokinin (CCK) is released from the duodenum in response to dietary fat to aid in digestion, and plasma CCK levels are elevated with the consumption of high-fat diets. CCK is also a trophic peptide for the pancreas and has also been shown to stimulate growth of pancreatic cancer. In the current investigation, we studied the influence of a diet high in saturated fat on the growth of pancreatic cancer in syngeneic murine models before the mice became obese to exclude the confounding factors associated with obesity. The high-fat diet significantly increased growth and metastasis of pancreatic cancer compared with the control diet, and the stimulatory effect was blocked by the CCK-receptor antagonist proglumide. We then selectively knocked out the CCK receptor on the pancreatic cancer cells using clustered regularly interspaced short palindromic repeats technology and showed that without CCK-receptors, dietary fat was unable to stimulate cancer growth. We next demonstrated that dietary fat failed to influence pancreatic cancer xenograft growth in genetically engineered CCK peptide knockout mice. The tumor-associated fibrosis that is so prevalent in the pancreatic cancer microenvironment was significantly decreased with CCK-receptor antagonist therapy because fibroblasts also have CCK receptors. The CCK-receptor antagonist proglumide also altered tumor metalloprotease expression and increased tumor suppressor genes by a PCR array. Our studies confirm that a diet high in saturated fat promotes growth of pancreatic cancer and the action is mediated by the CCK-receptor pathway. NEW & NOTEWORTHY Diets high in long-chain saturated fats promote growth of pancreatic cancer independent of obesity. The mechanism through which dietary fat promotes cancer is mediated through the cholecystokinin (CCK) receptor pathway. Therapy with a CCK-receptor antagonist altered the tumor microenvironment by reducing fibrosis, increasing cluster of differentiation 8+ lymphocytes, increasing tumor suppressor genes, and thus decreasing metastases. Use of CCK-receptor antagonist therapy with standard chemotherapy for pancreatic cancer may improve response by altering the tumor microenvironment.

Asunto(s)

Grasas de la Dieta/efectos adversos , Neoplasias Pancreáticas/etiología , Receptores de Colecistoquinina/metabolismo , Microambiente Tumoral , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Femenino , Fibrosis , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias Pancreáticas/tratamiento farmacológico , Proglumida/farmacología , Proglumida/uso terapéutico , Receptores de Colecistoquinina/antagonistas & inhibidores , Receptores de Colecistoquinina/genética

RESUMEN

The study examined implication of tributyrin, a metabolic precursor in small intestinal microflora, in stimulation of electrical activity of duodenum and jejunum. Enteral administration of tributyrin enhanced electrical activity of both examined structures in small intestine with elimination of the rest periods. The stimulatory effect was manifested in a prolongation of the periods of irregular activity. The up-regulating effects of tributyrin on electrical activity in upper segments of small intestine are mediated via the cholecystokinin receptors being also associated with activation of cholinergic and blockade of nitrinergic signal transduction pathways.

Asunto(s)

Duodeno/efectos de los fármacos , Fármacos Gastrointestinales/farmacología , Yeyuno/efectos de los fármacos , Complejo Mioeléctrico Migratorio/efectos de los fármacos , Triglicéridos/farmacología , Animales , Animales no Consanguíneos , Atropina/farmacología , Duodeno/metabolismo , Electrodos Implantados , Electromiografía , Nutrición Enteral , Microbioma Gastrointestinal/fisiología , Expresión Génica , Yeyuno/metabolismo , Complejo Mioeléctrico Migratorio/fisiología , Nitroglicerina/farmacología , Octreótido/farmacología , Ratas , Receptores de Colecistoquinina/agonistas , Receptores de Colecistoquinina/genética , Receptores de Colecistoquinina/metabolismo

RESUMEN

By performing two high-content small molecule screens on dextran sodium sulfate- and trinitrobenzene sulfonic acid-induced zebrafish enterocolitis models of inflammatory bowel disease, we have identified novel anti-inflammatory drugs from the John Hopkins Clinical Compound Library that suppress neutrophilic inflammation. Live imaging of neutrophil distribution was used to assess the level of acute inflammation and concurrently screen for off-target drug effects. Supporting the validity of our screening strategy, most of the anti-inflammatory drug hits were known antibiotics or anti-inflammatory agents. Novel hits included cholecystokinin (CCK) and dopamine receptor agonists. Using a pharmacological approach, we show that while CCK and dopamine receptor agonists alleviate enterocolitis-associated inflammation, receptor antagonists exacerbate inflammation in zebrafish. This work highlights the utility of small molecule screening in zebrafish enterocolitis models as a tool to identify novel bioactive molecules capable of modulating acute inflammation.

Asunto(s)

Antiinflamatorios/farmacología , Colitis Ulcerosa/tratamiento farmacológico , Enfermedad de Crohn/tratamiento farmacológico , Disbiosis/tratamiento farmacológico , Ensayos Analíticos de Alto Rendimiento , Factores Inmunológicos/farmacología , Animales , Animales Modificados Genéticamente , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/patología , Enfermedad de Crohn/inducido químicamente , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Agonistas de Dopamina/farmacología , Disbiosis/inducido químicamente , Disbiosis/inmunología , Disbiosis/patología , Embrión no Mamífero , Expresión Génica , Humanos , Intestinos/efectos de los fármacos , Intestinos/inmunología , Intestinos/patología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/patología , Receptores de Colecistoquinina/agonistas , Receptores de Colecistoquinina/genética , Receptores de Colecistoquinina/inmunología , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/inmunología , Bibliotecas de Moléculas Pequeñas/farmacología , Ácido Trinitrobencenosulfónico , Pez Cebra

RESUMEN

The ability to design agonists that target peptide signaling is a strategy to delineate underlying mechanisms and influence biology. A sequence that uniquely characterizes a peptide provides a distinct site to generate novel agonists. Drosophila melanogaster sulfakinin encodes non-sulfated drosulfakinin I (nsDSK I; FDDYGHMRF-NH2) and nsDSK II (GGDDQFDDYGHMRF-NH2). Drosulfakinin is typical of sulfakinin precursors, which are conserved throughout invertebrates. Non-sulfated DSK II is structurally related to DSK I, however, it contains a unique 5-residue N-terminal extension; drosulfakinins signal through G-protein coupled receptors, DSK-R1 and DSK-R2. Drosulfakinin II distinctly influences adult and larval gut motility and larval locomotion; yet, its structure-activity relationship was unreported. We hypothesized substitution of an N-terminal extension residue may alter nsDSK II activity. By targeting the extension we identified, not unexpectedly, analogs mimicking nsDSK II, yet, surprisingly, we also discovered novel agonists with increased (super) and opposite (protean) effects. We determined [A3] nsDSK II increased larval gut contractility rather than, like nsDSK II, decrease it. [N4] nsDSK II impacted larval locomotion, although nsDSK II was inactive. In adult gut, [A1] nsDSK II, [A2] nsDSKII, and [A3] nsDSK II mimicked nsDSK II, and [A4] nsDSK II and [A5] nsDSK II were more potent; [N3] nsDSK II and [N4] nsDSK II mimicked nsDSK II. This study reports nsDSK II signals through DSK-R2 to influence gut motility and locomotion, identifying a novel role for the N-terminal extension in sulfakinin biology and receptor activation; it also led to the discovery of nsDSK II structural analogs that act as super and protean agonists.

Asunto(s)

Proteínas de Drosophila/genética , Locomoción/genética , Péptidos/agonistas , Péptidos/genética , Receptores Acoplados a Proteínas G/genética , Secuencia de Aminoácidos/genética , Animales , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Péptidos y Proteínas de Señalización Intercelular , Larva/efectos de los fármacos , Larva/genética , Locomoción/efectos de los fármacos , Neuropéptidos/química , Neuropéptidos/genética , Oligopéptidos/química , Oligopéptidos/genética , Péptidos/química , Péptidos/farmacología , Receptores de Colecistoquinina/química , Receptores de Colecistoquinina/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad

RESUMEN

Cholecystokinin (CCK) regulates appetite and reduces food intake by activating the type 1 CCK receptor (CCK1R). Attempts to develop CCK1R agonists for obesity have yielded active agents that have not reached clinical practice. Here we discuss why, along with new strategies to target CCK1R more effectively. We examine signaling events and the possibility of developing agents that exhibit ligand-directed bias, to dissociate satiety activity from undesirable side effects. Potential allosteric sites of modulation are also discussed, along with desired properties of a positive allosteric modulator (PAM) without intrinsic agonist action as another strategy to treat obesity. These new types of CCK1R-active drugs could be useful as standalone agents or as part of a rational drug combination for management of obesity.

Asunto(s)

Receptores de Colecistoquinina/metabolismo , Regulación Alostérica/genética , Regulación Alostérica/fisiología , Animales , Colecistoquinina/metabolismo , Humanos , Obesidad/genética , Obesidad/metabolismo , Receptor de Colecistoquinina A/agonistas , Receptor de Colecistoquinina A/metabolismo , Receptores de Colecistoquinina/agonistas , Receptores de Colecistoquinina/genética

RESUMEN

AIM: Cholecystokinin (CCK) and gastrin (Gs) are a well known trophic factor for the gastrointestinal tract and their trophic effects are shown mainly toward pancreas and stomach, respectively. Though, the exact characterization of CCK and Gs receptors subtype (cholecystokinin type A receptor [CCKAR] and cholecystokinin type B receptor/gastrin receptor [CCKBR/GR]) in stomach cancer (SC) and pancreatic cancer (PC) is still controversial and necessities further validation. SUBJECTS AND METHODS: CCKAR and CCKBR/GR expression was analyzed by immunohistochemistry in 55 SC, 25 benign gastric diseases (BGDs), 38 PC (including periampullary carcinoma), and 10 normal pancreatic tissue. The results were statistically correlated with the patient's clinical history to observe the prognostic significance if any. RESULT: CCKAR expression was detected in 18.2% of SC, 20% of BGD, 65.8% of PC, and 30.0% of normal pancreas tissue samples. The CCKBR/GR expression was detected in 58.2% of SC, 48.0% of BGD, 18.4% of PC, and 60.0% of normal pancreas tissue samples. CCKBR/GR expression was significantly high in well and moderately differentiated SC samples as compared to poorly differentiated samples. CONCLUSION: Our study showed significantly higher expression of CCKAR and down regulation of CCKBR in PC as compared to control while CCKBR/GR was detected in majority of SC samples. Thus, our study suggests that CCK and Gs receptors may have diagnostic and therapeutic implications. However, study need to be validated in significantly bigger sample size and need to be replicated in different cohorts.

Asunto(s)

Biomarcadores de Tumor/biosíntesis , Neoplasias Pancreáticas/genética , Receptor de Colecistoquinina B/biosíntesis , Receptores de Colecistoquinina/biosíntesis , Neoplasias Gástricas/genética , Adulto , Anciano , Biomarcadores de Tumor/genética , Colecistoquinina/genética , Femenino , Gastrinas/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Páncreas/patología , Neoplasias Pancreáticas/patología , Receptor de Colecistoquinina B/genética , Receptores de Colecistoquinina/genética , Estómago/patología , Neoplasias Gástricas/patología

RESUMEN

Nucleotide-binding oligomerization domain 1 (NOD1) fulfills important host-defense functions via its responses to a variety of gut pathogens. Recently, however, we showed that in acute pancreatitis caused by administration of cholecystokinin receptor (CCKR) agonist (cerulein) NOD1 also has a role in inflammation via its responses to gut commensal organisms. In the present study, we explored the long-term outcome of such NOD1 responsiveness in a new model of chronic pancreatitis induced by repeated administration of low doses of cerulein in combination with NOD1 ligand. We found that the development of chronic pancreatitis in this model requires intact NOD1 and type I IFN signaling and that such signaling mediates a macrophage-mediated inflammatory response that supports interleukin (IL)-33 production by acinar cells. The IL-33, in turn, has a necessary role in the induction of IL-13 and TGF-ß1, factors causing the fibrotic reaction characteristic of chronic pancreatitis. Interestingly, the Th2 effects of IL-33 were attenuated by the concomitant type I IFN response since the inflammation was marked by clear increases in IFN-γ and TNF-α production but only marginal increases in IL-4 production. These studies establish chronic pancreatitis as an IL-33-dependent inflammation resulting from synergistic interactions between the NOD1 and CCKR signaling pathways.

Asunto(s)

Ceruletida/administración & dosificación , Ácido Diaminopimélico/análogos & derivados , Interleucina-33/inmunología , Proteína Adaptadora de Señalización NOD1/inmunología , Pancreatitis Crónica/inmunología , Receptores de Colecistoquinina/inmunología , Células Acinares/efectos de los fármacos , Células Acinares/inmunología , Células Acinares/patología , Animales , Ácido Diaminopimélico/administración & dosificación , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-13/genética , Interleucina-13/inmunología , Interleucina-33/genética , Interleucina-4/genética , Interleucina-4/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Adaptadora de Señalización NOD1/deficiencia , Proteína Adaptadora de Señalización NOD1/genética , Pancreatitis Crónica/inducido químicamente , Pancreatitis Crónica/genética , Pancreatitis Crónica/patología , Receptores de Colecistoquinina/genética , Transducción de Señal , Células Th2/efectos de los fármacos , Células Th2/inmunología , Células Th2/patología , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología

RESUMEN

BACKGROUND: Cholecystokinin (CCK) receptors are overexpressed in numerous human cancers, such as pancreatic, colon and gastric cancers. Previous studies have shown that the specific receptor-binding property of CCK for CCK receptors (CCKRs) can be exploited to produce immunotoxins (ITs) that target cancer cells overexpressing CCK receptors. PURPOSE: Construct a new IT-targeting CCKR-overexpressing colon cancers. METHODS: To construct the CCKR-targeted IT, a reverse CCK8 peptide was fused with a modified 38-kDa truncated form of the Pseudomonas exotoxin (PE38KDEL). An efficient immunoaffinity purification procedure was used to produce a PE38-based IT. Several analyses, including CCK8 competition and indirect immunofluorescence assays, were performed to confirm the interaction between rCCK8 and CCKR. After cytotoxic assays on several cell lines, the anti-tumor activity of the new IT was detected in nude mice. RESULTS: The rCCK8PE38 IT showed specific cytotoxicity for two colon cancer cell lines and one gastric cancer cell line. After purification, 18-26 mg of pure rCCK8PE38 per 1 L of culture was obtained. Purified rCCK8PE38 showed high cytotoxicity in colon cancer cell lines with IC50 values of 0.8-3.5 ng/mL. The results of the CCK8 competition and indirect immunofluorescence assays showed that rCCK8 had a specific interaction with CCKR. Nude mice inoculated with HCT-8 tumor xenografts were treated with rCCK8PE38, which efficiently decreased the tumor size in those mice. CONCLUSIONS AND DISCUSSION: All of these data suggest that rCCK8PE38 has potential as a new immunotherapy agent. Furthermore, the results of this study further support the high value of the immunoaffinity method for IT purification procedures.

Asunto(s)

Colecistoquinina/administración & dosificación , Neoplasias del Colon/terapia , Inmunotoxinas/administración & dosificación , Fragmentos de Péptidos/administración & dosificación , Receptores de Colecistoquinina/metabolismo , Animales , Línea Celular Tumoral , Colecistoquinina/farmacología , Neoplasias del Colon/inmunología , Exotoxinas/administración & dosificación , Exotoxinas/farmacología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Inmunoterapia/métodos , Inmunotoxinas/farmacología , Ratones , Ratones Desnudos , Fragmentos de Péptidos/farmacología , Pseudomonas/metabolismo , Receptores de Colecistoquinina/genética , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/terapia , Ensayos Antitumor por Modelo de Xenoinjerto