RESUMO
The blood brain barrier (BBB) forms a stringent barrier that protects the brain from components in the circulation that could interfere with neuronal function. At the same time, the BBB enables selective transport of critical nutrients and other chemicals to the brain. Beyond these functions, another recently recognized function is even less characterized, specifically the role of the BBB in modulating behavior by affecting neuronal function in a sex-dependent manner. Notably, signaling in the adult Drosophila BBB is required for normal male courtship behavior. Courtship regulation also relies on male-specific molecules in the BBB. Our previous studies have demonstrated that adult feminization of these cells in males significantly lowered courtship. Here, we conducted microarray analysis of BBB cells isolated from males and females. Findings revealed that these cells contain male- and female-enriched transcripts, respectively. Among these transcripts, nuclear receptor Hr46/Hr3 was identified as a male-enriched BBB transcript. Hr46/Hr3 is best known for its essential roles in the ecdysone response during development and metamorphosis. In this study, we demonstrate that Hr46/Hr3 is specifically required in the BBB cells for courtship behavior in mature males. The protein is localized in the nuclei of sub-perineurial glial cells (SPG), indicating that it might act as a transcriptional regulator. These data provide a catalogue of sexually dimorphic BBB transcripts and demonstrate a physiological adult role for the nuclear receptor Hr46/Hr3 in the regulation of male courtship, a novel function that is independent of its developmental role.
Assuntos
Barreira Hematoencefálica/metabolismo , Corte , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Drosophila melanogaster/genética , Ecdisona/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Caracteres Sexuais , Comportamento Sexual Animal/fisiologiaRESUMO
High-protein diet (HPD) curtails obesity and/or fat mass, but it is unknown whether it reverses neuroinflammation or alters glucose levels, CCK sensitivity, and gut microbiome in rats fed a Western diet (WD)-induced obesity (DIO). Male rats fed a WD (high fat and sugar) for 12 wk were switched to a HPD for 6 wk. Body composition, food intake, meal pattern, sensitivity to intraperitoneal CCK-8S, blood glucose, brain signaling, and cecal microbiota were assessed. When compared with a normal diet, WD increased body weight (9.3%) and fat mass (73.4%). CCK-8S (1.8 or 5.2 nmol/kg) did not alter food intake and meal pattern in DIO rats. Switching to a HPD for 6 wk reduced fat mass (15.7%) with a nonsignificantly reduced body weight gain, normalized blood glucose, and decreased feeding after CCK-8S. DIO rats on the WD or switched to a HPD showed comparable microbial diversity. However, in HPD versus WD rats, there was enrichment of 114 operational taxonomic units (OTUs) and depletion of 188 OTUs. Of those, Akkermansia muciniphila (enriched on a HPD), an unclassified Clostridiales, a member of the RF39 order, and a Phascolarctobacterium were significantly associated with fat mass. The WD increased cytokine expression in the hypothalamus and dorsal medulla that was unchanged by switching to HPD. These data indicate that HPD reduces body fat and restores glucose homeostasis and CCK sensitivity, while not modifying brain inflammation. In addition, expansion of cecal Akkermansia muciniphila correlated to fat mass loss may represent a potential peripheral mechanism of HPD beneficial effects.
Assuntos
Encéfalo/efeitos dos fármacos , Ceco/efeitos dos fármacos , Colecistocinina/farmacologia , Proteínas Alimentares/farmacologia , Encefalite/metabolismo , Microbiota/efeitos dos fármacos , Obesidade/microbiologia , Animais , Glicemia/metabolismo , Composição Corporal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Encéfalo/metabolismo , Ceco/metabolismo , Ceco/microbiologia , Citocinas/metabolismo , Dieta Ocidental , Ingestão de Alimentos/efeitos dos fármacos , Masculino , Obesidade/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The site(s) of action that control meal size and intermeal interval (IMI) length by cholecystokinin-58 (CCK-58), the only detectable endocrine form of CCK in the rat, are not known. To test the hypothesis that the gastrointestinal tract may contain such sites, we infused low doses of CCK-58 (0.01, 0.05, 0.15 and 0.25nmol/kg) into the celiac artery (CA, supplying stomach and upper duodenum), the cranial mesenteric artery (CMA, supplying small and most of the large intestines), the femoral artery (FA, control) and the portal vein (PV, draining the gastrointestinal tract) prior to the onset of the dark cycle in freely fed male rats. We measured the first meal size (chow), second meal size, IMI and satiety ratio (SR, IMI/meal size). We found that (1) all doses of CCK-58 given in the CA and the highest dose given in the CMA reduced the first meal size, (2) all doses of CCK-58 given in the CA reduced the second meal size, (3) a CCK-58 dose of 0.15nmol/kg given in the CA and 0.15 and 0.25nmol/kg given in the CMA prolonged the IMI, (4) CCK-58 (0.05, 0.15, 0.25nmol/kg) given in the CA and 0.25nmol/kg given in the CMA increased the SR, and (5) CCK-58 given in the FA and PV had no effect on the meal size or intermeal interval. These results support our hypothesis that the gastrointestinal tract contains sites of action that regulate meal size and IMI length via CCK-58. The stomach and upper duodenum may contain sites regulating meal size, whereas the small intestine and part of the large intestine may contain sites regulating the IMI.
Assuntos
Artéria Celíaca/metabolismo , Colagogos e Coleréticos/farmacologia , Colecistocinina/farmacologia , Comportamento Alimentar/fisiologia , Artéria Mesentérica Superior/metabolismo , Resposta de Saciedade/fisiologia , Animais , Colagogos e Coleréticos/administração & dosagem , Colecistocinina/administração & dosagem , Artéria Femoral/metabolismo , Masculino , Veia Porta/metabolismo , Ratos , Ratos Sprague-Dawley , Resposta de Saciedade/efeitos dos fármacosRESUMO
Microisolation techniques utilizing several reverse phase high performance liquid chromatography (HPLC) steps have resulted in the purification of two rat gastrin releasing peptide (GRP) forms suitable for microsequence and mass spectral analysis. The sequence of the larger form is APVSTGAGGGTVLAKMYPRGSHWAVGHLM-amide and the smaller form is GSHWAVGHLM-amide which is the carboxyl terminal decapeptide of the larger peptide. The peptides were synthesized and their feeding patterns e.g. first meal size (MS), intermeal interval (IMI) and satiety ratio (SR, IMI/MS) were determined in overnight food-, but not water deprived, male Sprague Dawley rats. The peptides were administered in the femoral vein (0, 0.21, 0.41 and 1.03 nmol/kg) immediately before presenting the rats with a 10% sucrose solution. We found that (1) GRP-10 (all doses) and GRP-29 (0.41 nmol/kg) reduced first MS, (2) both peptides prolonged IMI length and (3) both peptides increased the SR to similar extents. In conclusion, GRP-10 and GRP-29 are the two endogenous forms of GRP in the rat intestine and they reduce short term feeding to similar extents when administered intravenously.
Assuntos
Comportamento Alimentar/efeitos dos fármacos , Peptídeo Liberador de Gastrina/química , Peptídeo Liberador de Gastrina/farmacologia , Análise Serial de Proteínas , Animais , Cromatografia Líquida de Alta Pressão , Peptídeo Liberador de Gastrina/administração & dosagem , Peptídeo Liberador de Gastrina/análise , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
It has been accepted for decades that "all forms of cholecystokinin (CCK) have equal bioactivity," despite accumulating evidence to the contrary. To challenge this concept, we compared two feeding responses, meal size (MS, 10% sucrose) and intermeal interval (IMI), in response to CCK-58, which is the major endocrine form of CCK, and CCK-8, which is the most abundantly utilized form. Doses (0, 0.1, 0.5, 0.75, 1, 3 and 5 nmol/kg) were administered intraperitoneally over a 210-min test to Sprague Dawley rats that had been food-deprived overnight. We found that (1) all doses of CCK-58, except the lowest dose, and all doses of CCK-8, except the lowest two doses, reduced food intake more than vehicle did; (2) at two doses, 0.75 and 3 nmol/kg, CCK-58 increased the IMI, while CCK-8 failed to alter this feeding response; and (3) CCK-58, at all but the lowest two doses, increased the satiety ratio (IMI between first and second meals (min) divided by first MS (ml)) relative to vehicle, while CCK-8 did not affect this value. These findings demonstrate that the only circulating form of CCK in rats, CCK-58, prolongs the IMI more than CCK-8, the peptide generally utilized in feeding studies. Taken together, these results add to a growing list of functions where CCK-8 and CCK-58 express qualitatively different bioactivities. In conclusion, the hypothesis that "all forms of cholecystokinin (CCK) have equal bioactivity" is not supported.
Assuntos
Depressores do Apetite/farmacologia , Estimulantes do Apetite/farmacologia , Colecistocinina/farmacologia , Sincalida/farmacologia , Animais , Apetite/efeitos dos fármacos , Ingestão de Energia , Comportamento Alimentar/efeitos dos fármacos , Masculino , Ratos Sprague-DawleyRESUMO
Reduction of food intake by exogenous cholecystokinin (CCK) has been demonstrated primarily for its short molecular form, CCK-8. Mounting evidence, however, implicates CCK-58 as a major physiologically active CCK form, with different neural and exocrine response profiles than CCK-8. In three studies, we compared meal-pattern effects of intraperitoneal injections CCK-8 vs. CCK-58 in undeprived male Sprague-Dawley rats consuming sweetened condensed milk. In study 1, rats (N=10) received CCK-8, CCK-58 (0.45, 0.9, 1.8 and 3.6 nmol/kg) or vehicle before a 4-h test-food presentation. At most doses, both CCK-8 and CCK-58 similarly reduced meal size relative to vehicle. Meal-size reduction prompted a compensatory shortening of the intermeal interval (IMI) after CCK-8, but not after CCK-58, which uniquely increased the satiety ratio (IMI/size of the preceding meal). In the second study, lick patterns were monitored after administration of 0.9 nmol/kg CCK-58, CCK-8 or vehicle. Lick cluster size, lick efficiency and interlick-interval distribution remained unaltered compared to vehicle, implying natural satiation, rather than illness, following both CCK forms. In study 3, threshold satiating doses of the two CCK forms were given at 5 and 30 min after meal termination, respectively. CCK 58, but not CCK-8 increased the intermeal interval and satiety ratio compared to vehicle. In conclusion, while CCK 58 and CCK-8 both stimulate satiation, thereby reducing meal size, CCK-58 consistently exerts a satiety effect, prolonging IMI. Given the physiological prominence of CCK-58, these results suggest that CCK's role in food intake regulation may require re-examination.
Assuntos
Colecistocinina/farmacologia , Fragmentos de Peptídeos/farmacologia , Saciação/efeitos dos fármacos , Resposta de Saciedade/efeitos dos fármacos , Animais , Colecistocinina/administração & dosagem , Relação Dose-Resposta a Droga , Masculino , Refeições , Fragmentos de Peptídeos/administração & dosagem , Ratos Sprague-DawleyRESUMO
To investigate the mechanism by which 5-aminoimidazole-4-carboxamide-1-ß-riboside (AICAr) induces apoptosis in multiple myeloma cells, we conducted an unbiased metabolomics screen. AICAr had selective effects on nucleotide metabolism, resulting in an increase in purine metabolites and a decrease in pyrimidine metabolites. The most striking abnormality was a 26-fold increase in orotate associated with a decrease in uridine monophosphate (UMP) levels, indicating an inhibition of UMP synthetase (UMPS), the last enzyme in the de novo pyrimidine biosynthetic pathway, which produces UMP from orotate and 5-phosphoribosyl-α-pyrophosphate (PRPP). As all pyrimidine nucleotides can be synthesized from UMP, this suggested that the decrease in UMP would lead to pyrimidine starvation as a possible cause of AICAr-induced apoptosis. Exogenous pyrimidines uridine, cytidine, and thymidine, but not purines adenosine or guanosine, rescued multiple myeloma cells from AICAr-induced apoptosis, supporting this notion. In contrast, exogenous uridine had no protective effect on apoptosis resulting from bortezomib, melphalan, or metformin. Rescue resulting from thymidine add-back indicated apoptosis was induced by limiting DNA synthesis rather than RNA synthesis. DNA replicative stress was identified by associated H2A.X phosphorylation in AICAr-treated cells, which was also prevented by uridine add-back. Although phosphorylation of AICAr by adenosine kinase was required to induce multiple myeloma cell death, apoptosis was not associated with AMP-activated kinase activation or mTORC1 inhibition. A possible explanation for inhibition of UMP synthase activity by AICAr was a depression in cellular levels of PRPP, a substrate of UMP synthase. These data identify pyrimidine biosynthesis as a potential molecular target for future therapeutics in multiple myeloma cells.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Antineoplásicos/farmacologia , Mieloma Múltiplo/metabolismo , Pirimidinas/metabolismo , Ribonucleosídeos/farmacologia , Apoptose/efeitos dos fármacos , Humanos , Metabolômica/métodos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Pirimidinas/biossínteseRESUMO
Various molecular forms of CCK reduce food intake in rats. Although CCK-8 is the most studied form, we reported that CCK-58 is the only detectable endocrine peptide form in rats. We investigated the dark-phase rat chow intake pattern following injection of CCK-8 and CCK-58. Ad libitum-fed male Sprague-Dawley rats were intraperitoneally injected with CCK-8, CCK-58 (0.6, 1.8, and 5.2 nmol/kg), or vehicle. Food intake pattern was assessed during the dark phase using an automated weighing system that allowed continuous undisturbed monitoring of physiological eating behavior. Both CCK-8 and CCK-58 dose dependently reduced 1-h, dark-phase food intake, with an equimolar dose of 1.8 nmol being similarly effective (-49% and -44%). CCK-58 increased the latency to the first meal, whereas CCK-8 did not. The intermeal interval was reduced after CCK-8 (1.8 nmol/kg, -41%) but not after CCK-58. At this dose, CCK-8 increased the satiety ratio by 80% and CCK-58 by 160%, respectively, compared with vehicle. When behavior was assessed manually, CCK-8 reduced locomotor activity (-31%), whereas grooming behavior was increased (+59%). CCK-58 affected neither grooming nor locomotor activity. In conclusion, reduction of food intake by CCK-8 and CCK-58 is achieved by differential modulation of food intake microstructure and behavior. These data highlight the importance of studying the molecular forms of peptides that exist in vivo in tissue and circulation of the animal being studied.
Assuntos
Colecistocinina/administração & dosagem , Ritmo Circadiano/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Sincalida/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Asseio Animal/efeitos dos fármacos , Injeções Intraperitoneais , Masculino , Atividade Motora/efeitos dos fármacos , Fotoperíodo , Ratos , Ratos Sprague-Dawley , Tempo de Reação/efeitos dos fármacos , Resposta de Saciedade/efeitos dos fármacos , Fatores de TempoRESUMO
The unpredictable nature of peptide binding to surfaces requires optimization of experimental containers to be used. To demonstrate the variable recoveries of peptides from multiple surfaces commonly employed in peptide research, we tested the recovery of radiolabeled (125)I endocrine peptides under different conditions and provide guidelines for determining the surfaces to use for other peptides. (125)I-labeled peptides (ghrelin, sulfated cholecystokinin-8, corticotropin-releasing factor, glucagon-like peptide-1 [GLP-1], insulin, leptin, nesfatin-1, and peptide YY), representing a wide spectrum in net charge, size, end group, and modification, were incubated for 48 h in glass and plastic tubes untreated or coated with siliconizing fluid. Best surfaces were chosen and peptides were incubated with bovine serum albumin (BSA, 1%) with or without subsequent lyophilization. Recovery of (125)I-labeled peptides was determined by gamma counting. Important differences in (125)I-labeled peptide binding capacities to various types of surfaces exist. Siliconization decreased, whereas the addition of BSA improved recovery from surfaces tested. Lyophilizing solutions containing (125)I-labeled peptides and BSA in the tubes best suited for individual peptides rendered more than 89% recovery for all peptides. Ghrelin specifically displaced (125)I-ghrelin from borosilicate glass, whereas GLP-1 and Fmoc-arginine did not. Choosing the appropriate experimental container avoids unpredictable peptide loss that results in inaccurate measurements and false conclusions.
Assuntos
Peptídeos/análise , Radioimunoensaio/instrumentação , Adsorção , Sequência de Aminoácidos , Animais , Liofilização , Grelina/análise , Vidro/química , Radioisótopos do Iodo/análise , Dados de Sequência Molecular , Plásticos/química , Silício/química , Propriedades de SuperfícieRESUMO
The gut hormone peptide YY(3-36)-amide [PYY(3-36)-NH(2)] is significantly more potent than PYY(1-36)-NH(2) in reducing food intake in rats and humans. Other Gly-extended and Ser(13)-phosphorylated PYY forms have been detected or predicted based upon known cellular processes of PYY synthesis and modification. Here we compared the effects of 3-h IV infusion of PYY(1-36)-NH(2), PYY(3-36)-NH(2), PYY(1-36)-Gly-OH, PYY(3-36)-Gly-OH, Ser(13)(PO(3))-PYY(1-36)-NH(2), Ser(13)(PO(3))-PYY(3-36)-NH(2), Ser(13)(PO(3))-PYY(1-36)-Gly-OH, and Ser(13)(PO(3))-PYY(3-36)-Gly-OH during the early dark period on food intake in freely feeding rats. PYY(3-36)-NH(2) and Ser(13)(PO(3))-PYY(3-36)-NH(2) reduced food intake similarly at 50 pmol/kg/min, while only PYY(3-36)-NH(2) reduced food intake at 15 pmol/kg/min. PYY(1-36)-NH(2) and Ser(13)(PO(3))-PYY(1-36)-NH(2) reduced food intake similarly at 50 and 150 pmol/kg/min. In contrast, PYY(1-36)-Gly-OH, PYY(3-36)-Gly-OH, Ser(13)(PO(3))-PYY(3-36)-Gly-OH, and Ser(13)(PO(3))-PYY(1-36)-Gly-OH had no effect on food intake at doses of 50 or 150 pmol/kg/min. Taken together, these results indicate that (i) PYY(3-36)-NH(2) is significantly more potent than PYY(1-36)-NH(2) in reducing food intake, (ii) Gly-extended forms of PYY are significantly less potent than non-extended forms, and (iii) Ser(13)-phosphorylation of PYY(3-36)-NH(2) decreases the anorexigenic potency PYY(3-36)-NH(2), but not PYY(1-36)-NH(2). Thus, PYY(3-36)-NH(2) appears to be the most potent PYY form for reducing food intake in rats.
Assuntos
Comportamento Alimentar/efeitos dos fármacos , Glicina/química , Peptídeo YY/farmacologia , Serina/química , Animais , Peptídeo YY/química , Fosforilação , RatosRESUMO
BACKGROUND: Corticotropin-releasing hormone (CRH) is expressed in the brain, immune cells and the gut, where gene expression is upregulated by lipopolysaccharide (LPS) 6 h after injection. Whether these changes are reflected by increased circulating levels of CRH and adrenocorticotropic hormone (ACTH) is unknown. METHODS: LPS (100 µg/kg) was injected intraperitoneally in conscious rats, and blood processed for CRH using the new RAPID (reduced temperatures, acidification, protease inhibition, isotopic exogenous controls and dilution) method compared with EDTA blood with or without plasma methanol extraction. Hormone levels were measured by commercial radioimmunoassay. RESULTS: The RAPID method improved blood recovery of ¹²5I-CRH in vitro compared to EDTA only added to the blood without or with methanol extraction (90.8 ± 2.0 vs. 66.9 ± 2.6 and 47.5 ± 2.0%, respectively; p < 0.001 vs. RAPID). Basal CRH levels from blood processed by the RAPID method were 28.9 ± 2.8 pg/ml, and by other methods below the radioimmunoassay detection limit (<10 pg/ml). At 6 h after LPS, CRH plasma levels increased significantly by 2.9 times, and in the proximal colon tended to decrease (-27.6 ± 5.7%; p > 0.05), while circulating levels were unchanged at 3 or 4 h. ACTH levels rose compared to control rats (135.3 ± 13.8 vs. 101.4 ± 6.0 pg/ml; p < 0.05) 30 min after the increase in CRH, while at 3 or 6 h after LPS, the levels were not changed. CONCLUSION: Intraperitoneal LPS induces a delayed rise in plasma CRH levels associated with an elevation in ACTH plasma levels 30 min later, suggesting that under conditions of immune challenge, CRH of peripheral origin may also contribute to pituitary activation, as detected using the RAPID method of blood processing, which improves CRH recovery.
Assuntos
Preservação de Sangue/métodos , Hormônio Liberador da Corticotropina/sangue , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Hormônio Adrenocorticotrópico/sangue , Animais , Expressão Gênica , Injeções Intraperitoneais , Masculino , Hipófise/metabolismo , Radioimunoensaio , Ratos , Ratos Sprague-DawleyRESUMO
We measured molecular forms of PYY in the distal half of rat small intestine using a new method for tissue extraction, three sequential reverse phase chromatography steps, and PYY radioimmunoassay and mass spectrometry to measure their levels. The extraction method called RAPID, developed to minimize artifactual degradation of PYY during tissue extraction and sample preparation, uses Reduced temperature, Acidified buffer, Peptidase inhibitors, Isotopically enriched mass spectrometry standards, and Dilution to inhibit and monitor endogenous peptide degradation during tissue processing. Synthetic peptides [PYY(1-36)-NH(2), PYY(3-36)-NH(2), PYY(1-36)-Gly-OH, and PYY(3-36)-Gly-OH] selectively enriched with (13)C(3)-alanine were added as internal standards to the extraction buffer. By collecting mass spectra rather than multiple-reaction-monitoring (MRM) profiles, we simultaneously screen for any PYY forms that were present in the immunoreactive fractions. PYY(1-36)-NH(2), PYY(3-36)-NH(2), PYY(1-36)-Gly-OH, and PYY(3-36)-Gly-OH were identified and quantified at 64.3±4.5, 6.1±0.9, 0.9±0.1, and <0.3pmol/g of tissue, respectively (n=3). Thus, we found that in rat distal small intestine proPYY is processed to PYY(1-36)-NH(2) with little conversion to PYY(3-36)-NH(2). These data suggest that production of PYY(3-36)-NH(2) (a form with greater potency than PYY(1-36)-NH(2) for inhibition of feeding and gastric emptying) occurs after the peptide leaves its cell of synthesis by enzymatic action in the circulation.
Assuntos
Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Peptídeo YY/metabolismo , Animais , Masculino , Espectrometria de Massas , Radioimunoensaio , Ratos , Ratos Sprague-DawleyRESUMO
Bacterial lipopolysaccharide (LPS) in rodents is an established model for studying innate immune responses to gram-negative bacteria and mimicking symptoms of infections including reduced food intake associated with decreased circulating total ghrelin levels. The ghrelin-acylating enzyme, ghrelin-O-acyltransferase (GOAT) involved in the formation of acyl ghrelin (AG) was recently identified. We investigated changes in circulating AG, desacyl ghrelin (DG) and GOAT induced by intraperitoneal LPS (100 microg/kg) and associated changes in food intake. Plasma AG and total ghrelin were assessed by radioimmunoassay, GOAT protein by Western blot and mRNA by RT-qPCR. DG was derived from total minus AG. Plasma AG and DG were decreased at 2, 5 and 7 h (p<0.01) post-injection compared to vehicle and recovered at 24 h. At 2 h there was a significantly greater decrease of AG (-53%) than DG (-28%) resulting in a decreased AG/DG ratio (1:5, p<0.01), which thereafter returned to pre-injection values (1:3). This altered ratio was associated with a 38% decrease in plasma GOAT protein compared to vehicle (p<0.001), whereas gastric GOAT protein was slightly increased by 10% (p<0.05). GOAT mRNA expression was unchanged. Food intake was reduced by 58% measured during the 1.5-2 h period post-LPS injection. Decreased plasma AG and DG preceded the rise in rectal temperature and blood glucose that peaked at 7 h. These data indicate that LPS induces a long-lasting reduction of AG and DG levels that may have a bearing with the decrease in food intake. The faster drop in AG than DG within 2 h is associated with reduced circulating GOAT.
Assuntos
Aciltransferases/sangue , Aciltransferases/metabolismo , Regulação para Baixo , Grelina/sangue , Lipopolissacarídeos/toxicidade , Reação de Fase Aguda/fisiopatologia , Aciltransferases/genética , Animais , Regulação do Apetite , Glicemia/análise , Western Blotting , Temperatura Corporal , Mucosa Gástrica/metabolismo , Infecções por Helicobacter/fisiopatologia , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
The correct identification of circulating molecular forms and measurement of peptide levels in blood entails that the endocrine peptide being studied is stable and recovered in good yields during blood processing. However, it is not clear whether this is achieved in studies using standard blood processing. Therefore, we compared peptide concentration and form of 12 (125)I-labeled peptides using the standard procedure (EDTA-blood on ice) and a new method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls, and Dilution (RAPID). During standard processing there was at least 80% loss for calcitonin-gene-related peptide and cholecystokinin-58 (CCK-58) and more than 35% loss for amylin, insulin, peptide YY forms (PYY((1-36)) and PYY((3-36))), and somatostatin-28. In contrast, the RAPID method significantly improved the recovery for 11 of 12 peptides (P < 0.05) and eliminated the breakdown of endocrine peptides occurring after standard processing as reflected in radically changed molecular forms for CCK-58, gastrin-releasing peptide, somatostatin-28, and ghrelin. For endogenous ghrelin, this led to an acyl/total ghrelin ratio of 1:5 instead of 1:19 by the standard method. These results show that the RAPID method enables accurate assessment of circulating gut peptide concentrations and forms such as CCK-58, acylated ghrelin, and somatostatin-28. Therefore, the RAPID method represents an efficacious means to detect circulating variations in peptide concentrations and form relevant to the understanding of physiological function of endocrine peptides.
Assuntos
Técnicas de Química Analítica/métodos , Colecistocinina/sangue , Peptídeo Liberador de Gastrina/sangue , Grelina/sangue , Somatostatina-28/sangue , Animais , Masculino , RatosRESUMO
The gastrointestinal hormone CCK exists in various molecular forms, with differences in bioactivity between the well-characterized CCK-8 and larger CCK-58 previously reported. We have compared the effects of these peptides on cytosolic calcium concentration ([Ca(2+)](c)), mitochondrial metabolism, enzyme secretion, and cell fate in murine isolated pancreatic acinar cells using fluorescence confocal microscopy and patch-clamp electrophysiology. CCK-58 (1-10 pM) induced transient, oscillatory increases of [Ca(2+)](c), which showed apical to basolateral progression and were associated with a rise of mitochondrial NAD(P)H. CCK-58 (10 pM) induced zymogen exocytosis in isolated cells and amylase secretion from isolated cells and whole tissues. Hyperstimulation with supraphysiological CCK-58 (5 nM) induced a single large increase of [Ca(2+)](c) that declined to a plateau, which remained above the basal level 20 min after application and was dependent on external Ca(2+) entry. In cells dispersed from the same tissues, CCK-8 induced similar patterns of responses to those of CCK-58, with oscillatory increases of [Ca(2+)](c) at lower (pM) concentrations and sustained responses at 5 nM. CCK-58 and CCK-8 exhibited similar profiles of action on cell death, with increases in necrosis at high CCK-58 and CCK-8 (10 nM) that were not significantly different between peptides. The present experiments indicate that CCK-8 and CCK-58 have essentially identical actions on the acinar cell at high and low agonist concentrations, suggesting an action via the same receptor and that the differences observed in an intact rat model may result from indirect effects of the peptides. Our data strengthen the argument that CCK-58 is an important physiological form of this gastrointestinal hormone.
Assuntos
Sinalização do Cálcio , Diferenciação Celular , Colecistocinina/metabolismo , Precursores Enzimáticos/metabolismo , Pâncreas Exócrino/metabolismo , Fragmentos de Peptídeos/metabolismo , Amilases/metabolismo , Animais , Exocitose , Humanos , Potenciais da Membrana , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Necrose , Pâncreas Exócrino/enzimologia , Pâncreas Exócrino/patologia , Técnicas de Patch-Clamp , Fatores de TempoRESUMO
OBJECTIVES: To define the role of protein kinase C delta (PKC delta) in acinar cell responses to the hormone cholecystokinin-8 (CCK) using isoform-specific inhibitors and a previously unreported genetic deletion model. METHODS: Pancreatic acinar cells were isolated from (1) rat, and pretreated with a PKC delta-specific inhibitor or (2) PKC delta-deficient and wild type mice. Isolated cells were stimulated with CCK (0.001-100 nmol/L) and cell responses were measured. RESULTS: The PKC delta inhibitor did not affect stimulated amylase secretion from rat pancreatic acinar cells. Cholecystokinin-8 stimulation induced a typical biphasic dose-response curve for amylase secretion in acinar cells isolated from both PKC delta(-/-) and wild type mice, with maximal stimulation at 10-pmol/L CCK. Cholecystokinin-8 (100 nmol/L) induced zymogen and nuclear factor kappaB activation in both PKC delta(-/-) and wild type mice, although it was up to 50% less in PKC delta(-/-). CONCLUSIONS: In contrast to previous studies, this study has used specific and complementary approaches to examine PKC delta-mediated acinar cell responses. We could not confirm that it mediates amylase release but corroborated its role in the early stages of acute pancreatitis.
Assuntos
Colecistocinina/farmacologia , Pâncreas/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proteína Quinase C-delta/metabolismo , Acetofenonas/farmacologia , Amilases/metabolismo , Animais , Benzopiranos/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Immunoblotting , Indóis/farmacologia , Masculino , Maleimidas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Pâncreas/citologia , Pâncreas/metabolismo , Proteína Quinase C-delta/antagonistas & inibidores , Proteína Quinase C-delta/genética , Ratos , Ratos Sprague-Dawley , Tripsinogênio/metabolismoRESUMO
Cholecystokinin, like many peptide hormones, is present as multiple molecular forms. CCK-58 has been identified as the dominant form in the circulation, whereas most of the studies of CCK-receptor interactions have been performed with CCK-8. Despite both sharing the pharmacophoric region of CCK, representing its carboxy terminal heptapeptide amide, studies in vivo have demonstrated biological diversity of action of the two peptides, with CCK-58, but not CCK-8, stimulating pancreatic fluid secretion and lengthening the interval between meals. Here, we have directly studied the ability of these two CCK peptides to bind to the type 1 CCK receptor and to stimulate it to elicit an intracellular calcium response. The calcium response relative to receptor occupation was identical for CCK-58 and CCK-8, with the longer peptide binding with approximately fivefold lower affinity. We also examined the ability of the two peptides to elicit receptor internalization using morphological techniques and to disrupt the constitutive oligomerization of the CCK receptor using receptor bioluminescence resonance energy transfer. Here, both full agonist peptides had similar effects on these regulatory processes. These data suggest that both molecular forms of CCK act at the CCK1 receptor quite similarly and elicit similar regulatory processes for that receptor, suggesting that the differences in biological activity observed in vivo most likely reflect differences in the clearance and/or metabolism of these long and short forms of CCK peptides.
Assuntos
Sinalização do Cálcio , Colecistocinina/metabolismo , Receptores da Colecistocinina/metabolismo , Animais , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Endocitose , Transferência Ressonante de Energia de Fluorescência , Humanos , Luciferases de Renilla , Proteínas Luminescentes , Ligação Proteica , Ratos , Receptores da Colecistocinina/genética , Proteínas Recombinantes de Fusão/metabolismo , Sincalida/análogos & derivados , Sincalida/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
UNLABELLED: It has been shown in the rat that endogenous cholecystokinin (CCK), released in response to the non-nutrient trypsin inhibitor camostat, reduces food intake at meals and increases Fos-like immunoreactivity (Fos-LI; a marker for neuronal activation) in the dorsal vagal complex (DVC) of the hindbrain but not the myenteric plexus of the duodenum and jejunum. Experiment 1: We examined Fos-LI in the myenteric and the submucosal plexuses of the gut in response to orogastric gavage of camostat in rats. As we reported previously, camostat failed to increase Fos-LI in the myenteric plexus. We show here that camostat increased Fos-LI in the submucosal plexus of the duodenum and jejunum. Camostat also increased Fos-LI in the DVC. Experiment 2: Pretreatment with devazepide, a specific CCK(1) receptor antagonist abolished camostat-induced Fos-LI in the submucosal plexus and the DVC. Experiment 3: Bilateral subdiaphragmatic vagotomy reduced camostat-induced Fos-LI in the submucosal plexus approximately 40% and abolished it in the DVC. CONCLUSIONS: Activation of the submucosal plexus by cholecystokinin at the CCK(1) receptor accompanies stimulation of the dorsal vagal complex of the hindbrain and inhibition of food intake. Unlike the submucosal plexus, activation of the myenteric plexus is not necessary for cholecystokinin's influence on the dorsal vagal complex and food intake. The lack of activation in the myenteric plexus after camostat stimulation, in contrast to nutrient releasers of CCK such as oleate, suggests that intestinal stimulants can either release different amounts of CCK or cause release of CCK from I cells with different molecular forms of CCK. This would suggest that CCK-8 is released by camostat and is not able to travel to the myenteric plexus while a more stable form of CCK such as CCK-58 can travel to this site that is further away from the I cell.
Assuntos
Ingestão de Alimentos/efeitos dos fármacos , Gabexato/análogos & derivados , Trato Gastrointestinal/efeitos dos fármacos , Plexo Mientérico/metabolismo , Plexo Submucoso/efeitos dos fármacos , Animais , Colecistocinina/farmacologia , Devazepida/farmacologia , Ésteres , Gabexato/farmacologia , Trato Gastrointestinal/metabolismo , Guanidinas , Antagonistas de Hormônios/farmacologia , Modelos Biológicos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores da Colecistocinina/fisiologia , Núcleo Solitário/efeitos dos fármacos , Plexo Submucoso/metabolismo , Nervo Vago/efeitos dos fármacosRESUMO
JMV-180 ( 1) and CCK-8(s) are high affinity ligands at the CCK 1 receptor that have similar and different actions via this receptor. Here we calculate the tertiary structure of 1 or CCK-8(s) in the presence of dodecylphosphocholine micelles at pH 5.0 and 35 degrees C from 2D (1)H NMR data recorded at 600 MHz. The NMR derived 3D structures of 1 and CCK-8(s) share a common type I beta-turn around residues Nle3/M3 and G4 and diverge from each other structurally at the N- and C-termini. The fluorescence and circular dichroism spectral properties of these peptides are consistent with their NMR derived structures. The structures determined in the presence of DPC micelles are compared to available models of 1 or CCK-8(s) bound to the CCK 1 receptor. For CCK and 1, these comparisons show that DPC micelle associated structures duplicate some important aspects of the models calculated from cross-linking derived constraints at the CCK 1 receptor.
Assuntos
Micelas , Modelos Moleculares , Receptor de Colecistocinina A/química , Receptor de Colecistocinina A/metabolismo , Sincalida/química , Dicroísmo Circular , Imageamento Tridimensional , Espectroscopia de Ressonância Magnética , Ligação Proteica , Estrutura Terciária de Proteína , Sincalida/análogos & derivados , Espectrometria de FluorescênciaRESUMO
BACKGROUND & AIMS: Cholecystokinin (CCK) has been thought to act only indirectly on human pancreatic acinar cells via vagal nerve stimulation, rather than by direct CCK receptor activation as on rodent pancreatic acinar cells. We tested whether CCK (CCK-8 and human CCK-58) can act directly on human pancreatic acinar cells. METHODS: Human acinar cells were freshly isolated from pancreatic transection line samples, loaded with Fluo4-AM or quinacrine, and examined for Ca(2+), metabolic and secretory responses to CCK-8, human CCK-58, or acetylcholine with confocal microscopy. RESULTS: CCK-8 and human CCK-58 at physiologic concentrations (1-20 pmol/L) elicited rapid, robust, oscillatory increases of the cytosolic Ca(2+) ion concentration, showing apical to basal progression, in acinar cells from 14 patients with unobstructed pancreata. The cytosolic Ca(2+) ion concentration increases were followed by increases in mitochondrial adenosine triphosphate production and secretion. CCK-elicited Ca(2+) signals and exocytosis were not inhibited by atropine (1 mumol/L) or tetrodotoxin (100 nmol/L), showing that CCK was unlikely to have acted via neurotransmitter release. CCK-elicited Ca(2+) signals were inhibited reversibly by caffeine (5-20 mmol/L), indicating involvement of intracellular inositol trisphosphate receptor Ca(2+) release channels. Acetylcholine (50 nmol/L) elicited similar Ca(2+) signals. CONCLUSIONS: CCK at physiologic concentrations in the presence of atropine and tetrodotoxin elicits cytosolic Ca(2+) signaling, activates mitochondrial function, and stimulates enzyme secretion in isolated human pancreatic acinar cells. We conclude that CCK acts directly on acinar cells in the human pancreas.