RESUMEN
Development of optimal therapeutics for disease states that can be associated with increased membrane cholesterol requires better molecular understanding of lipid modulation of the drug target. Type 1 cholecystokinin receptor (CCK1R) agonist actions are affected by increased membrane cholesterol, enhancing ligand binding and reducing calcium signaling, while agonist actions of the closely related CCK2R are not. In this work, we identified a set of chimeric human CCK1R/CCK2R mutations that exchange the cholesterol sensitivity of these 2 receptors, providing powerful tools when expressed in CHO and HEK-293 model cell lines to explore mechanisms. Static, low energy, high-resolution structures of the mutant CCK1R constructs, stabilized in complex with G protein, were not substantially different, suggesting that alterations to receptor dynamics were key to altered function. We reveal that cholesterol-dependent dynamic changes in the conformation of the helical bundle of CCK receptors affects both ligand binding at the extracellular surface and G protein coupling at the cytosolic surface, as well as their interrelationships involved in stimulus-response coupling. This provides an ideal setting for potential allosteric modulators to correct the negative impact of membrane cholesterol on CCK1R.
Asunto(s)
Colesterol , Proteínas de Unión al GTP , Unión Proteica , Receptor de Colecistoquinina A , Receptor de Colecistoquinina B , Animales , Humanos , Células CHO , Colesterol/metabolismo , Cricetulus , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Células HEK293 , Ligandos , Mutación , Conformación Proteica , Receptor de Colecistoquinina A/metabolismo , Receptor de Colecistoquinina A/genética , Receptor de Colecistoquinina B/metabolismo , Receptor de Colecistoquinina B/genéticaRESUMEN
Obesity increases asthma prevalence and severity. However, the underlying mechanisms are poorly understood, and consequently, therapeutic options for asthma patients with obesity remain limited. Here we report that cholecystokinin-a metabolic hormone best known for its role in signaling satiation and fat metabolism-is increased in the lungs of obese mice and that pharmacological blockade of cholecystokinin A receptor signaling reduces obesity-associated airway hyperresponsiveness. Activation of cholecystokinin A receptor by the hormone induces contraction of airway smooth muscle cells. In vivo, cholecystokinin level is elevated in the lungs of both genetically and diet-induced obese mice. Importantly, intranasal administration of cholecystokinin A receptor antagonists (proglumide and devazepide) suppresses the airway hyperresponsiveness in the obese mice. Together, our results reveal an unexpected role for cholecystokinin in the lung and support the repurposing of cholecystokinin A receptor antagonists as a potential therapy for asthma patients with obesity.
Asunto(s)
Asma , Hipersensibilidad Respiratoria , Animales , Ratones , Asma/tratamiento farmacológico , Asma/metabolismo , Colecistoquinina/metabolismo , Pulmón/metabolismo , Ratones Obesos , Obesidad/complicaciones , Obesidad/metabolismo , Receptor de Colecistoquinina A/genética , Receptor de Colecistoquinina A/metabolismo , Hipersensibilidad Respiratoria/tratamiento farmacológico , Hipersensibilidad Respiratoria/metabolismoRESUMEN
Cholecystokinin (CCK) is a peptide hormone mainly secreted by small intestinal endocrine I-cells and functions as a regulator of gallbladder contraction, gastric emptying, gastrointestinal (GI) motility, and satiety. The cellular effects of CCK in these peripheral tissues are predominantly mediated via CCK-A receptors which are found in smooth muscles, enteric neurons, and vagal afferent neurons in humans and animal models. Although various functions of CCK have been reported to be neurally mediated, it can also stimulate contraction via the CCK receptor on the smooth muscle. However, the entire underlying neural and cellular mechanisms involved in CCK-induced GI contractions are not clearly understood. Here, we first determined the cDNA and amino acid sequences of CCK and CCK-A receptor along with the distributions of cck mRNA and CCK-producing cells in house musk shrew (Suncus murinus, the laboratory strain named as suncus) and examined the mechanism of CCK-induced contraction in the GI tract. Mature suncus CCK-8 was identical to other mammalian species tested here, and suncus CCK-A receptor presented high nucleotide and amino acid homology with that of human, dog, mouse, and rat, respectively. Suncus CCK mRNA and CCK-producing cells were found mainly in small intestine and colon. In the organ bath study, CCK-8 induced dose-dependent contractions in the suncus stomach, duodenum, and jejunum, and these contractions were inhibited by atropine and CCK-A receptor antagonist. These results suggest that CCK-8-induced contraction is mediated in the myenteric cholinergic neural network and that CCK-A receptor is partly responsible for CCK-8-induced contractions. This study indicates that suncus is a useful animal model to study the functions of CCK involved in GI motility.
Asunto(s)
Colecistoquinina , Receptor de Colecistoquinina A , Musarañas , Animales , Colecistoquinina/genética , Clonación Molecular , Perros , Motilidad Gastrointestinal , Humanos , Ratones , Contracción Muscular , ARN Mensajero/genética , Ratas , Receptor de Colecistoquinina A/genética , Musarañas/genética , Sincalida/farmacologíaRESUMEN
Cholecystokinin A receptor (CCKAR) is a key receptor mediating satiety. Previous studies found that decreased expression of CCKAR attenuated satiety, and thus contributed to the high-growth of broiler chickens. The objective of this study is to map sequence variants associated with the growth of chickens in the CCKAR. The CCKAR and upstream 1.4 kb genomic sequences were resequenced to find out all sequence variants using 35 Lueyang black-boned chickens (LBC). Haplotypes were reconstructed using the PHASE program. Linkage disequilibrium between variants was analyzed using the Haploview software. Associations of 33 tag SNPs that captured 89% of all variants with body weight of LBC (n = 675) at 16 (BW16), 20 (BW20) weeks of age and the onset (BWOEP) of egg production were tested using linear mixed models. A total of 126 SNPs were found and formed 41 haplotypes in 35 resequenced samples. Average length of haplotype blocks is 129 bp, indicating that LBC maintains low linkage disequilibrium at the CCKAR locus. Eleven of 33 tag SNPs were significantly associated with BW16, but not with BW20 and BWOEP. These significantly associated variants were most (8/11) distributed in a 2 kb region (chr4:73206169-73208244) around the Exon3. They together with 33 captured variants potentially disrupted binding sites of 471 transcription factors. Twelve variants can disrupt appetite (FOXO1) or lipid metabolism-related TF (AR and C/EBP) motifs. This study recognized chr4:73206169-73208244 as a key region harboring functional variants affecting the growth of chickens.
Asunto(s)
Pollos , Polimorfismo de Nucleótido Simple , Animales , Peso Corporal , Pollos/genética , Haplotipos , Desequilibrio de Ligamiento , Receptor de Colecistoquinina A/genéticaRESUMEN
BACKGROUND: This study aimed to explore genetic polymorphisms of the CCKAR gene and their relationship with the growth and development of Qinchuan cattle which could be used as molecular markers for the improvement of the breeding of Qinchuan cattle. RESULTS: Here, we have identified seven single nucleotide polymorphisms (SNPs) at loci g. 1463 C>G; g. 1532 T>A; g. 1570 G>A; g. 1594 C>A; g. 1640 T>C; g. 1677 G>C; and g. 1735 C>T in the coding region of the bovine CCKAR gene. The frequencies identified on allelic and genotypic characteristics have shown that all seven SNPs diverged from the Hardy-Weinberg-Equilibrium. The SNP2, SNP3, SNP6 and SNP7 had the lowest polymorphism information content values, and remaining SNPs were found to be moderate (0.25 < PIC < 0.50). The genotype CG in SNP1 at loci g.1463 C>G had the greatest association with WH, HW, CD and CCF, while the genotype TA at the very same loci was associated with BFT, ULA and IMF content in Qinchuan cattle. The CCKAR gene expression level in adipose tissue, small intestine, liver and skeleton muscle was found to be higher, whereas, the expression level of mRNA in organs of other digestive system including reticulum, abomasum and omasum was moderate. Some expression of CCKAR mRNA was found in the large intestine, kidney and rumen. CONCLUSIONS: In summary, our finding suggested that the CCKAR gene could be used as a potential candidate for the improvement of carcass quality and body measurements of Qinchuan cattle.
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Animales , Bovinos , Bovinos/genética , Receptor de Colecistoquinina A/genética , Variación Genética , Desequilibrio de Ligamiento , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , Sistema Digestivo , Ganado , Técnicas de Genotipaje , Frecuencia de los Genes , Productos de la CarneRESUMEN
In 1973, Gibbs, Young, and Smith showed that exogenous cholecystokinin (CCK) administration reduces food intake in rats. This initial report has led to thousands of studies investigating the physiological role of CCK in regulating feeding behavior. CCK is released from enteroendocrine I cells present along the gastrointestinal (GI) tract. CCK binding to its receptor CCK1R leads to vagal afferent activation providing post-ingestive feedback to the hindbrain. Vagal afferent neurons' (VAN) sensitivity to CCK is modulated by energy status while CCK signaling regulates gene expression of other feeding related signals and receptors expressed by VAN. In addition to its satiation effects, CCK acts all along the GI tract to optimize digestion and nutrient absorption. Diet-induced obesity (DIO) is characterized by reduced sensitivity to CCK and every part of the CCK system is negatively affected by chronic intake of energy-dense foods. EEC have recently been shown to adapt to diet, CCK1R is affected by dietary fats consumption, and the VAN phenotypic flexibility is lost in DIO. Altered endocannabinoid tone, changes in gut microbiota composition, and chronic inflammation are currently being explored as potential mechanisms for diet driven loss in CCK signaling. This review discusses our current understanding of how CCK controls food intake in conditions of leanness and how control is lost in chronic energy excess and obesity, potentially perpetuating excessive intake.
Asunto(s)
Colecistoquinina/genética , Ingestión de Alimentos/genética , Obesidad/genética , Receptor de Colecistoquinina A/genética , Animales , Colecistoquinina/biosíntesis , Colecistoquinina/farmacología , Dieta Alta en Grasa/efectos adversos , Grasas de la Dieta/efectos adversos , Conducta Alimentaria/efectos de los fármacos , Conducta Alimentaria/fisiología , Tracto Gastrointestinal/metabolismo , Humanos , Neuronas Aferentes/metabolismo , Neuronas Aferentes/patología , Obesidad/etiología , Obesidad/fisiopatología , Obesidad/terapia , RatasRESUMEN
The aim of this study was to elucidate the role and the pathways used by bile acid receptor TGR5 in transmitting satiety signals. We showed TGR5 colocalized with cholecystokinin type A (CCK-A) receptors in a subpopulation of rat nodose ganglia (NG) neurons. Intra-arterial injection of deoxycholic acid (DCA) dose-dependently increased firing rate in NG while a subthreshold dose of DCA and CCK-8 increased firing rates synergistically. TGR5-specific agonist oleanolic acid induced NG neuronal firing in a dose-dependent manner. However, the same units did not respond to GW4064, a nuclear receptor-specific agonist. Quantity of DCA-activated neurons in the hypothalamus was determined by c-Fos expression. Combining DCA and CCK-8 caused a 4-fold increase in c-Fos activation. In the arcuate nucleus, c-Fos-positive neurons coexpressed cocaine and amphetamine regulated transcript and proopiomelanocortin. DCA-induced c-Fos expression was eliminated following truncal vagotomy or silencing of TGR5 in the NG. Feeding studies showed intravenous injection of 1 µg/kg of DCA reduced food intake by 12% ± 3%, 24% ± 5%, and 32% ± 6% in the first 3 hours, respectively. Silencing of TGR5 or CCK-A receptor in the NG enhanced spontaneous feeding by 18% ± 2% and 13.5% ± 2.4%, respectively. When both TGR5 and CCK-A receptor were silenced, spontaneous feeding was enhanced by 37% ± 4% in the first 3 hours, suggesting that bile acid may have a physiological role in regulating satiety. Working in concert with CCK, bile acid synergistically enhanced satiety signals to reduce spontaneous feeding.
Asunto(s)
Ácidos y Sales Biliares/farmacología , Ácido Desoxicólico/farmacología , Neuronas/efectos de los fármacos , Receptor de Colecistoquinina A/genética , Receptores Acoplados a Proteínas G/genética , Vías Aferentes/efectos de los fármacos , Animales , Ácidos y Sales Biliares/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Isoxazoles/farmacología , Leptina/genética , Neuronas/patología , Ganglio Nudoso/efectos de los fármacos , Ratas , Receptor de Colecistoquinina A/antagonistas & inhibidores , Respuesta de Saciedad/efectos de los fármacos , Respuesta de Saciedad/fisiología , Nervio Vago/efectos de los fármacos , Nervio Vago/patologíaRESUMEN
Genistein is an isoflavone abundant in soybean and infants are exposed to high levels of genistein in soy-based formula. It is known that genistein mediates estrogen receptor (ER) signaling, and exposure during neonatal development could cause acute and long term endocrine effects. We assayed genistein's impact on the neonatal mouse pituitary gland because it is an endocrine signaling hub and is sensitive to endocrine disruption during critical periods. Pituitary explant cultures, which actively proliferate and differentiate, were exposed to 0.06⯵M-36⯵M genistein and assayed for mRNA and protein changes. Genistein induced mRNA expression of the ERα regulated gene, Cckar, to the same magnitude as estradiol (E2) but with less potency. Interestingly, 36⯵M genistein strongly inhibited pituitary proliferation, measured by a reduction in mKi67 mRNA and phospho-Histone H3 immunostaining. Examining cell cycle dynamics, we found that 36⯵M genistein decreased Ccnb1 (Cyclin B1) mRNA; while mRNA for the cyclin dependent kinase inhibitor Cdkn1a (p21) was upregulated, correlated with an apparent increase in p21 immunostained cells. Strikingly, we observed a robust onset of cellular senescence, permanent cell cycle exit, in 36⯵M genistein treated pituitaries by increased senescence activated ß-galactosidase staining. We also found that 36⯵M genistein decreased Bcl2 mRNA levels, a gene protective against apoptosis. Taken together these data suggest that genistein exposure during the neonatal period could initiate senescence and halt proliferation during a time when the proper numbers of endocrine cells are being established for mature gland function.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Genisteína/farmacología , Hipófisis/efectos de los fármacos , Animales , Animales Recién Nacidos , Proteínas de Ciclo Celular/genética , Femenino , Antígeno Ki-67/genética , Masculino , Ratones , Hipófisis/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptor de Colecistoquinina A/genéticaRESUMEN
Psoriasis is a multigene and multifactorial skin disease with heterogeneous genetic inheritance. Mental disorders participate in the development of psoriasis as predisposing factors; a correlation of dermatological diseases with pathological anxiety and stress was shown. Meanwhile, there are no studies describing molecular mechanisms of the linkages between psycho-emotional disorders and skin diseases. The aim of this study is to find the associations between SNP in genes COMT (rs4680), DBH (rs141116007), CCKAR (rs1800857) and CCKBR (rs1805002), and psoriasis. Patients were selected according to the 10th revision of International Classification of Diseases (L-40). The sample size was 88 patients. The size of the control sample (population control) was 365 people. Genotyping was performed using PCR-RFLP and real-time PCR. Statistical analysis was performed using WinPepi software. Identification of complex genotypes was performed by the Monte Carlo method using APSampler 3.6.1 algorithm. Among the studied genes, only GA genotype of COMT gene is significantly associated with psoriasis [χ2 = 19.163 (p = 1.3E-5), F (p) = 1.2E-5, OR 3.47 (CI 99% = 1.61-7.91)]. At the moment, the functional significance of this phenomenon is difficult to explain.
Asunto(s)
Trastornos de Ansiedad/epidemiología , Catecol O-Metiltransferasa/genética , Dopamina beta-Hidroxilasa/inmunología , Genotipo , Psoriasis/genética , Receptor de Colecistoquinina A/genética , Receptor de Colecistoquinina B/genética , Algoritmos , Biología Computacional , Dopamina beta-Hidroxilasa/genética , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Polimorfismo de Nucleótido Simple , Psoriasis/epidemiología , Federación de RusiaRESUMEN
AIM: Cholecystokinin (CCK) participates in the storage of dietary triglycerides in white adipose tissue (WAT). Our goal was to characterize, both in subcutaneous (Sc-WAT) and visceral WAT (Vis-WAT), the functional expression of the two known CCK receptors, CCK-1 (CCK-1R) and CCK-2 (CCK-2R), as well as of CCK. MAIN METHODS: Gene and protein expression was assessed in different cell types of rat and human WAT by means of RT-PCR and western-blot, respectively. The functionality of CCK-Rs was tested by quantifying protein kinase B (Akt) phosphorylation after treatment of pre-adipocytes with the bioactive fragment of CCK, CCK-8. The CCK receptor subtype involved in Akt phosphorylation was investigated by using selective CCK-1R (SR-27,897) and CCK-2R antagonists (L-365,260). KEY FINDINGS: In rats, CCK-1R (Cckar) and CCK-2R (Cckbr) gene expression was detected in the two types of WAT analyzed as well as in isolated adipocytes, mesenchymal stem cells and pre-adipocytes. CCK-1R and CCK-2R proteins were identified in adipocytes and, to a minor extent, in pre-adipocytes. In addition, CCK-2R were detected in subcutaneous mesenchymal stem cells. Gene expression of the CCK precursor preproCCK as well as CCK immunoreactivity were also found in Sc-WAT and Vis-WAT. In human WAT, CCK gene expression as well as CCK-2Rs and CCK were also identified. CCK-8 evoked Akt phosphorylation in rat pre-adipocytes, and this effect was antagonized by SR-27,897 and L-365,260. SIGNIFICANCE: Our data show that both human and rat WAT express a complete CCK system, and suggest that CCK may have an autocrine/paracrine role in regulating adipose tissue biology.
Asunto(s)
Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/fisiología , Colecistoquinina/metabolismo , Colecistoquinina/fisiología , Adipocitos/metabolismo , Animales , Benzodiazepinonas/farmacología , Regulación de la Expresión Génica/genética , Silenciador del Gen , Humanos , Ácidos Indolacéticos/farmacología , Masculino , Células Madre Mesenquimatosas/metabolismo , Proteína Oncogénica v-akt/genética , Proteína Oncogénica v-akt/metabolismo , Compuestos de Fenilurea/farmacología , Fosforilación , Ratas , Ratas Wistar , Receptor de Colecistoquinina A/antagonistas & inhibidores , Receptor de Colecistoquinina A/biosíntesis , Receptor de Colecistoquinina A/genética , Receptor de Colecistoquinina B/antagonistas & inhibidores , Receptor de Colecistoquinina B/biosíntesis , Receptor de Colecistoquinina B/genética , Tiazoles/farmacologíaRESUMEN
The orexin (OX1R) and cholecystokinin A (CCK1R) receptors play opposing roles in the migration of the human colon cancer cell line HT-29, and may be involved in the pathogenesis and pathophysiology of cancer cell invasion and metastasis. OX1R and CCK1R belong to family A of the G-protein-coupled receptors (GPCRs), but the detailed mechanisms underlying their functions in solid tumor development remain unclear. In this study, we investigated whether these two receptors heterodimerize, and the results revealed novel signal transduction mechanisms. Bioluminescence and Förster resonance energy transfer, as well as proximity ligation assays, demonstrated that OX1R and CCK1R heterodimerize in HEK293 and HT-29 cells, and that peptides corresponding to transmembrane domain 5 of OX1R impaired heterodimer formation. Stimulation of OX1R and CCK1R heterodimers with both orexin-A and CCK decreased the activation of Gαq, Gαi2, Gα12, and Gα13 and the migration of HT-29 cells in comparison with stimulation with orexin-A or CCK alone, but did not alter GPCR interactions with ß-arrestins. These results suggest that OX1R and CCK1R heterodimerization plays an anti-migratory role in human colon cancer cells.
Asunto(s)
Movimiento Celular , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Receptores de Orexina/metabolismo , Multimerización de Proteína , Receptor de Colecistoquinina A/metabolismo , Transducción de Señal , Células HEK293 , Células HT29 , Humanos , Receptores de Orexina/genética , Unión Proteica , Dominios Proteicos , Receptor de Colecistoquinina A/genética , beta-Arrestinas/metabolismoRESUMEN
The CCK(1) receptor is a G-protein-coupled receptor activated by the sulfated forms of cholecystokinin (CCK), a gastrin-like peptide released in the gastrointestinal tract and mammal brain. A substantial body of research supports the hypothesis that CCK(1)r stimulates gallbladder contraction and pancreatic secretion in the gut, as well as satiety in brain. However, this receptor may also fulfill relevant roles in behavior, thanks to its widespread distribution in the brain. The strategic location of CCK(1)r in mesolimbic structures and specific hypothalamic and brainstem nuclei lead to complex interactions with neurotransmitters like dopamine, serotonin, and glutamate, as well as hypothalamic hormones and neuropeptides. The activity of CCK(1)r maintains adequate levels of dopamine and regulates the activity of serotonin neurons of raphe nuclei, which makes CCK(1)r an interesting therapeutic target for the development of adjuvant treatments for schizophrenia, drug addiction, and mood disorders. Unexplored functions of CCK(1)r, like the transmission of interoceptive sensitivity in addition to the regulation of hypothalamic hormones and neurotransmitters affecting emotional states, well-being, and attachment behaviors, may open exciting roads of research. The absence of specific ligands for the CCK(1) receptor has complicated the study of its distribution in brain so that research about its impact on behavior has been published sporadically over the last 30 years. The present review reunites all this body of evidence in a comprehensive way to summarize our knowledge about the actual role of CCK in the neurobiology of mental illness.
Asunto(s)
Trastornos de Ansiedad/metabolismo , Encéfalo/metabolismo , Receptor de Colecistoquinina A/metabolismo , Animales , Encéfalo/citología , Encéfalo/fisiología , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/fisiología , Humanos , Receptor de Colecistoquinina A/genéticaRESUMEN
The natriuretic hormone CCK exhibits its gene transcripts in total kidney extracts. To test the possibility of CCK acting as an intrarenal mediator of sodium excretion, we examined mouse kidneys by 1) an in situ hybridization technique for CCK mRNA in animals fed a normal- or a high-sodium diet; 2) immuno-electron microscopy for the CCK peptide, 3) an in situ hybridization method and immunohistochemistry for the CCK-specific receptor CCKAR; 4) confocal image analysis of receptor-mediated Ca2+ responses in isolated renal tubules; and 5) metabolic cage experiments for the measurement of urinary sodium excretion in high-salt-fed mice either treated or untreated with the CCKAR antagonist lorglumide. Results showed the CCK gene to be expressed intensely in the inner medulla and moderately in the inner stripe of the outer medulla, with the expression in the latter being enhanced by high sodium intake. Immunoreactivity for the CCK peptide was localized to the rough endoplasmic reticulum of the medullary interstitial cells in corresponding renal regions, confirming it to be a secretory protein. Gene transcripts, protein products, and the functional activity for CCKAR were consistently localized to the late proximal tubule segments (S2 and S3) in the medullary rays, and the outer stripe of the outer medulla. Lorglumide significantly diminished natriuretic responses of mice to a dietary sodium load without altering the glomerular filtration rate. These findings suggest that the medullary interstitial cells respond to body fluid expansion by CCK release for feedback regulation of the late proximal tubular reabsorption.
Asunto(s)
Colecistoquinina/metabolismo , Médula Renal/metabolismo , Túbulos Renales Proximales/metabolismo , Natriuresis , Transducción de Señal , Sodio en la Dieta/administración & dosificación , Equilibrio Hidroelectrolítico , Animales , Calcio/metabolismo , Colecistoquinina/antagonistas & inhibidores , Colecistoquinina/genética , Retroalimentación Fisiológica , Antagonistas de Hormonas/farmacología , Inmunohistoquímica , Hibridación Fluorescente in Situ , Médula Renal/efectos de los fármacos , Médula Renal/ultraestructura , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/ultraestructura , Masculino , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Inmunoelectrónica , Natriuresis/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor de Colecistoquinina A/genética , Receptor de Colecistoquinina A/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Equilibrio Hidroelectrolítico/efectos de los fármacosRESUMEN
Food intake occurs in bouts or meals, and numerous meal-generated signals have been identified that act to limit the size of ongoing meals. Hormones such as cholecystokinin (CCK) are secreted from the intestine as ingested food is being processed, and in addition to aiding the digestive process, they provide a signal to the brain that contributes to satiation, limiting the size of the meal. The potency of CCK to elicit satiation is enhanced by elevated levels of adiposity signals such as insulin. In the present experiments we asked whether CCK and insulin interact at the level of the blood-brain barrier (BBB). We first isolated rat brain capillary endothelial cells that comprise the BBB and found that they express the mRNA for both the CCK1R and the insulin receptor, providing a basis for a possible interaction. We then administered insulin intraperitoneally to another group of rats and 15min later administered CCK-8 intraperitoneally to half of those rats. After another 15min, CSF and blood samples were obtained and assayed for immunoreactive insulin. Plasma insulin was comparably elevated above baseline in both the CCK-8 and control groups, indicating that the CCK had no effect on circulating insulin levels given these parameters. In contrast, rats administered CCK had CSF-insulin levels that were more than twice as high as those of control rats. We conclude that circulating CCK greatly facilitates the transport of insulin into the brain, likely by acting directly at the BBB. These findings imply that in circumstances in which the plasma levels of both CCK and insulin are elevated, such as during and soon after meals, satiation is likely to be due, in part, to this newly-discovered synergy between CCK and insulin.
Asunto(s)
Encéfalo/anatomía & histología , Insulina/metabolismo , Microvasos/efectos de los fármacos , Receptor de Colecistoquinina A/metabolismo , Sincalida/farmacología , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas In Vitro , Masculino , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Long-Evans , Receptor de Colecistoquinina A/genéticaRESUMEN
AIM: To investigate the mechanisms and effects of sphincter of Oddi (SO) motility on cholesterol gallbladder stone formation in guinea pigs. METHODS: Thirty-four adult male Hartley guinea pigs were divided randomly into two groups, the control group (n = 10) and the cholesterol gallstone group (n = 24), which was sequentially divided into four subgroups with six guinea pigs each according to time of sacrifice. The guinea pigs in the cholesterol gallstone group were fed a cholesterol lithogenic diet and sacrificed after 3, 6, 9, and 12 wk. SO manometry and recording of myoelectric activity were obtained by a multifunctional physiograph at each stage. Cholecystokinin-A receptor (CCKAR) expression levels in SO smooth muscle were detected by quantitative real-time PCR (qRT-PCR) and serum vasoactive intestinal peptide (VIP), gastrin, and cholecystokinin octapeptide (CCK-8) were detected by enzyme-linked immunosorbent assay at each stage in the process of cholesterol gallstone formation. RESULTS: The gallstone formation rate was 0%, 0%, 16.7%, and 83.3% in the 3, 6, 9, and 12 wk groups, respectively. The frequency of myoelectric activity in the 9 wk group, the amplitude of myoelectric activity in the 9 and 12 wk groups, and the amplitude and the frequency of SO in the 9 wk group were all significantly decreased compared to the control group. The SO basal pressure and common bile duct pressure increased markedly in the 12 wk group, and the CCKAR expression levels increased in the 6 and 12 wk groups compared to the control group. Serum VIP was elevated significantly in the 9 and 12 wk groups and gastrin decreased significantly in the 3 and 9 wk groups. There was no difference in serum CCK-8 between the groups. CONCLUSION: A cholesterol gallstone-causing diet can induce SO dysfunction. The increasing tension of the SO along with its decreasing activity may play an important role in cholesterol gallstone formation. Expression changes of CCKAR in SO smooth muscle and serum VIP and CCK-8 may be important causes of SO dysfunction.
Asunto(s)
Cálculos Biliares/fisiopatología , Disfunción del Esfínter de la Ampolla Hepatopancreática/fisiopatología , Esfínter de la Ampolla Hepatopancreática/fisiopatología , Animales , Colesterol , Modelos Animales de Enfermedad , Electromiografía , Ensayo de Inmunoadsorción Enzimática , Cálculos Biliares/genética , Cálculos Biliares/metabolismo , Gastrinas/genética , Gastrinas/metabolismo , Cobayas , Manometría , Músculo Liso/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor de Colecistoquinina A/genética , Receptor de Colecistoquinina A/metabolismo , Sincalida/genética , Sincalida/metabolismo , Esfínter de la Ampolla Hepatopancreática/metabolismo , Disfunción del Esfínter de la Ampolla Hepatopancreática/genética , Disfunción del Esfínter de la Ampolla Hepatopancreática/metabolismo , Péptido Intestinal Vasoactivo/genética , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
In the present study, we investigated expression pattern of Cholecystokinin type A receptor (CCKAR) in relation to its commonly studied polymorphism (rs1800857, T/C) in gallstone disease (GSD) patients and controls. A total of 502 subjects (272 GSD and 230 controls) were enrolled, and genotyping was performed by evaluating restriction fragments of PstI digested DNA. For analyzing expression pattern of CCKAR in relation to polymorphism, gallbladder tissue samples from 80 subjects (GSD-55; control-25) were studied. Expression of CCKAR mRNA was evaluated by reverse transcriptase-PCR and confirmed using real-time PCR. Protein expression was evaluated by enzyme-linked immunosorbent assay. We observed significantly (p < 0.0001) lower expression of CCKAR mRNA and protein in GSD tissues as compared with control. Significantly higher frequency of A1/A1 genotype (C/T transition) (p = 0.0005) was observed for GSD as compared with control. Expression of CCKAR protein was found to be significantly lower (p < 0.0001) in A1/A1 genotype as compared with other genotypes for GSD patients. Perhaps, this is the first report providing evidence of alteration in CCKAR expression in relation to its polymorphism elucidating the molecular pathway of the disease. Additional investigations with lager sample size are needed to confirm these findings.
Asunto(s)
Cálculos Biliares/genética , Polimorfismo de Nucleótido Simple , Receptor de Colecistoquinina A/genética , Receptor de Colecistoquinina A/metabolismo , Adulto , Susceptibilidad a Enfermedades , Regulación hacia Abajo , Femenino , Cálculos Biliares/metabolismo , Técnicas de Genotipaje , Humanos , India , Masculino , Persona de Mediana Edad , Distribución Tisular , Adulto JovenRESUMEN
BACKGROUND & AIMS: The type 1 cholecystokinin receptor (CCK1R) mediates the actions of CCK to support nutritional homeostasis, including post-cibal satiety. However, elevated levels of membrane cholesterol, such as have been observed in metabolic syndrome, interfere with CCK stimulus-activity coupling at the CCK1R, thereby disrupting this important servomechanism. We hypothesize that reversal of the negative impact of cholesterol on this receptor could be useful in the management of obesity. METHODS: We have studied the effects of ß-sitosterol, a phytosterol structurally related to cholesterol, on CCK receptor function. This included CCK binding and biological activity at wild type CCK1R and CCK2R, as well as at CCK1R in a high cholesterol environment, and at a CCK1R mutant, Y140A, which mimics the behavior of wild type receptor in high cholesterol. RESULTS: ß-sitosterol (100 µM and 10 µM) significantly improved the defective signaling of the CCK1R present in high cholesterol (p < 0.05), without affecting CCK binding affinity. This effect was absent at the CCK1R present in a normal cholesterol environment, as well as at the structurally-related CCK2R. Furthermore, the cholesterol-insensitive Y140A mutant of CCK1R was resistant to the effects of ß-sitosterol. CONCLUSION: These data suggest that ß-sitosterol affects CCK1R function in high cholesterol by competing with cholesterol at a receptor cholesterol-binding site and may shift its conformation toward normal. This phytosterol extends our understanding of the structure-activity relationships for developing a drug that can target the external surface of CCK1R. Since the concentrations of ß-sitosterol shown to be effective in this study are similar to serum levels of this compound achievable during oral administration, it may be worthwhile to study possible beneficial effects of ß-sitosterol in metabolic syndrome.
Asunto(s)
Hipercolesterolemia/tratamiento farmacológico , Receptor de Colecistoquinina A/metabolismo , Sitoesteroles/farmacología , Animales , Células CHO , Cricetulus , Regulación de la Expresión Génica , Hipolipemiantes/farmacología , Receptor de Colecistoquinina A/genética , Transducción de SeñalRESUMEN
BACKGROUND: A defect in gallbladder contraction function plays a key role in the pathogenesis of gallstones. The cholecystokinin-1 receptor (CCK-1R) antagonists have been extensively investigated for their therapeutic effects on gastrointestinal and metabolic diseases in animal studies and clinical trials. However, it is still unknown whether they have a potential effect on gallstone formation. DESIGN: To study whether the CCK-1R antagonists enhance cholelithogenesis, we investigated cholesterol crystallization, gallstone formation, hepatic lipid secretion, gallbladder emptying function and intestinal cholesterol absorption in male C57BL/6J mice treated by gavage with devazepide (4 mg/day/kg) or vehicle (as controls) twice per day and fed the lithogenic diet for 21 days. RESULTS: During 21 days of feeding, oral administration of devazepide significantly accelerated cholesterol crystallization and crystal growth to microlithiasis, with 40% of mice forming gallstones, whereas only agglomerated cholesterol monohydrate crystals were found in mice receiving vehicle. Compared to the vehicle group, fasting and postprandial residual gallbladder volumes in response to the high-fat meal were significantly larger in the devazepide group during cholelithogenesis, showing reduced gallbladder emptying and bile stasis. Moreover, devazepide significantly increased hepatic secretion of biliary cholesterol, but not phospholipids or bile salts. The percentage of intestinal cholesterol absorption was higher in devazepide-treated mice, increasing the bioavailability of chylomicron-derived cholesterol in the liver for biliary hypersecretion into bile. These abnormalities induced supersaturated bile and rapid cholesterol crystallization. CONCLUSIONS: The potent CCK-1R antagonist devazepide increases susceptibility to gallstone formation by impairing gallbladder emptying function, disrupting biliary cholesterol metabolism and enhancing intestinal cholesterol absorption in mice.
Asunto(s)
Colelitiasis/inducido químicamente , Colesterol/metabolismo , Devazepida/farmacología , Vaciamiento Vesicular/efectos de los fármacos , Vesícula Biliar/efectos de los fármacos , Antagonistas de Hormonas/farmacología , Intestinos/efectos de los fármacos , Receptor de Colecistoquinina A/antagonistas & inhibidores , Animales , Ácidos y Sales Biliares/metabolismo , Colelitiasis/metabolismo , Vesícula Biliar/metabolismo , Mucosa Intestinal/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Receptor de Colecistoquinina A/efectos de los fármacos , Receptor de Colecistoquinina A/genéticaRESUMEN
BACKGROUND: A polymorphism (C825T) in G-protein ß polypeptide-3 (GNß3) gene alters intracellular signal transduction, which may cause motor or sensory abnormalities in the gastrointestinal tract. Cholecystokinin-A receptor (CCK-AR) gene T/C polymorphism is associated with a defective splicing of the primary transcript of CCK-AR mRNA, which may modulate satiety signal and delay gastric emptying. Therefore, we evaluated the role of these polymorphisms in patients with functional dyspepsia (FD) as compared with healthy controls (HC). PATIENTS AND METHODS: A total of 237 consecutive patients with FD (Rome III) and 250 HC were genotyped for GNß3 C825T and CCK-AR T/C polymorphisms (PCR-RFLP). RESULTS: Patients with FD [173 (73%) men, age: 38 ± 12 years] were comparable with HC [195 (78%) men, age: 37 ± 12 years] with respect to age and sex. Out of 237 patients, 26 (11%) had epigastric pain syndrome (EPS), 55 (23.2%) had postprandial distress syndrome (PDS), and 156 (65.8%) had EPS-PDS overlap. Among 237 patients with FD, TT genotype (associated with increased intracellular signal transduction) of GNß3 C825T polymorphism was more common among patients than among HC [26 (11%) vs. 12 (4.8%), P=0.014; odds ratio (OR): 2.47, 95% confidence interval (CI): 1.2-5.1]. CC (variant) genotype of CCK-AR T/C polymorphism was infrequent among patients than among HC [19 (8%) vs. 46 (18.4%), P=0.001; OR: 0.36, 95% CI: 0.19-0.66]. However, these polymorphisms were comparable among patients with different subtypes of FD (P=0.80 and 0.44). CONCLUSION: TT genotype of GNß3 C825T is more common among patients with FD than among HC, suggesting that increased signal transduction associated with this genotype may be important in its pathophysiology. However, CCK-AR polymorphism is protective against FD.