No description available.

No description available.

Cholecystokinin (CCK) is a gut peptide involved in the regulation of energy homeostasis by duodenal lipids via a neuronal network. However, it is unknown whether CCK also regulates glucose homeostasis through a neuronal network. Using an in vivo rat model, we demonstrated that duodenal CCK-8 (biologically active form of CCK) can lower glucose production through the activation of a gut-brain-liver axis via CCK-A receptors, and this glucose-regulatory effect is physiologically relevant. Since duodenal lipids can also lower glucose production through a gut-brain-liver axis, we verified that this duodenal-lipid effect is mediated by CCK-A receptor activation. Lastly, in rats fed on a high-fat diet for three days, duodenal CCK failed to suppress glucose production, suggesting a state of CCK-resistance. In summary, these findings revealed that intestinal CCK can regulate glucose homeostasis through a neuronal network and suggest that intestinal CCK resistance may contribute to hyperglycemia in response to high-fat feeding.

Cholecystokinin (CCK) is a gut peptide involved in the regulation of energy homeostasis by duodenal lipids via a neuronal network. However, it is unknown whether CCK also regulates glucose homeostasis through a neuronal network. Using an in vivo rat model, we demonstrated that duodenal CCK-8 (biologically active form of CCK) can lower glucose production through the activation of a gut-brain-liver axis via CCK-A receptors, and this glucose-regulatory effect is physiologically relevant. Since duodenal lipids can also lower glucose production through a gut-brain-liver axis, we verified that this duodenal-lipid effect is mediated by CCK-A receptor activation. Lastly, in rats fed on a high-fat diet for three days, duodenal CCK failed to suppress glucose production, suggesting a state of CCK-resistance. In summary, these findings revealed that intestinal CCK can regulate glucose homeostasis through a neuronal network and suggest that intestinal CCK resistance may contribute to hyperglycemia in response to high-fat feeding.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21340号 / 医博第4398号 / 新制||医||1031(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 岩井 一宏, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Thesis advisor: Barbara E. Wolfe / The core defining features of bulimia nervosa (BN) are repeated binge eating episodes and compensatory purging behavior. The biobehavioral aspects of binge eating are complex and not fully understood. One area of recent interest is the role of the satiety-signaling peptide cholecystokinin (CCK). Previous research observed a blunted postprandial plasma CCK response in those with BN, therefore suggesting this may be a cause, consequence, or maintenance factor in binge eating. It is unknown whether this altered response is due to a state versus trait phenomenon, thus having implications in the development of clinical treatment strategies. To examine the nature of this altered response, this study assessed whether CCK normalizes following remission from BN (RBN). This biobehavioral study utilized a comparative design to prospectively evaluate the biological CCK response and the corresponding behavioral ratings of satiety and other eating-related sensations in individuals with BN (n=10), RBN (n =14), and healthy controls (CON, n=13). CCK and behavioral ratings were assessed at baseline, +15, +30, and +60 minutes following the ingestion of a standardized liquid test meal. The BN group's CCK response was blunted and approached significance (p =.052) when compared to the RBN and CON groups. As predicted the RBN and CON groups' CCK response did not significantly differ. This finding supports the premise that CCK may normalize following abstinence from binge and purge (vomit) episodes and that this is a state versus trait related phenomenon. A significant positive relationship between CCK response and ratings of satiety occurred in the RBN group only (r=.59, p<.05). A new and unanticipated finding in the BN group was a significant relationship (r=.86, p < .01, two-tailed) between their CCK response and urge to vomit. A greater urge to vomit was reported by those individuals who had increased CCK response. Therefore, it is unknown whether the normalization of CCK functioning is a protective or liability factor in the stabilization and recovery process. Replication studies utilizing a larger sample size are needed to understand the role of CCK in recovery and the subsequent development of novel treatment strategies for those suffering with BN. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Connell School of Nursing. / Discipline: Nursing.

This research examines the hypothesis that the satiety effect of cholecystokinin octapeptide (CCK-8) is mediated by changes in gastric emptying. A method for collection of gastric emptying data, the double sampling procedure, is developed and validated for use in the rat. The double sampling technique permits repeated measurements of liquid gastric volume and thus describes the time course of emptying within a single experimental session. Further, the method allows determination of the amount of gastric secretion, volume emptied into the intestines, and amount of gastric load remaining in the stomach. Experiments are presented which: i) demonstrate the utility of the technique; ii) validate its accuracy in determining gastric volume; iii) indicate the stability of measurements obtained with this procedure; and iv) provide a procedure for quantitative evaluation of data obtained with this technique. Using the double sampling procedure, the ability of CCK-8 to delay gastric emptying and to influence feeding are then compared under similar experimental conditions. The effect of CCK-8 on gastric emptying is assessed in 6 hr deprived rats receiving 10 ml intragastric test loads of either .15M saline or 15% sucrose. Intraperitoneal (ip) injections of CCK-8 in doses of 1.4-22.4 ug/kg produce a dose-dependent retardation of gastric emptying of both saline and nutrient. Lower doses of CCK-8, 0.01 and 0.1 ug/kg, have no effect on gastric emptying. The effect of CCK-8 on feeding is assessed in rats tested under the same experimental conditions used in the gastric emptying studies. Doses of CCK-8 capable of retarding gastric emptying also suppress eating in a dose-dependent manner. These findings provide necessary correlational support for the hypothesis that satiety produced by CCK-8 is mediated by inhibition of gastric emptying. However, a further quantitative analysis of the correspondence of the gastric emptying and feeding effects of CCK-8 suggest that retardation of emptying may not account for the entire satiety effect of the peptide. The next set of studies provide direct tests of whether changes in gastric emptying mediate CCK-induced satiety. If gastric emptying plays a significant role in the satiety produced by CCK-8 then: i) the effects of CCK-8 on emptying and feeding should share similar kinetics, and ii) peptides that inhibit emptying should also inhibit feeding. I show that CCK-8 (5.6 ug/kg) injected coincident with introduction of an intragastric load or presentation of a test meal produces a rapid inhibition of both emptying and feeding. In contrast, the identical dose of CCK-8 administered 15 min before testing causes no inhibition of emptying, even though the peptide retains its ability to produce satiety. I also test the abilities of the peptides pentagastrin (100 ug/kg), bombesin (8 & 16 ug/kg) and secretin (2.86, 14.3 & 28.6 ug/kg) to reduce food intake and inhibit gastric emptying. Pentagastrin does not affect food intake or gastric emptying. Bombesin causes a small transient delay in emptying but a large and sustained suppression of eating. High dose secretin (14.3 ug/kg) causes no significant reduction of food intake, even though this dose of secretin inhibits emptying to the same degree as 1.4 ug/kg CCK-8, which does reduce intake. Thus, although CCK-8 does influence the rate of gastric emptying, the present results indicate that the inhibition of emptying by CCK is neither necessary nor sufficient to explain its satiety effect. / Thesis / Doctor of Philosophy (PhD)

Ce travail s'inscrit dans le cadre général de l'étude des mécanismes d'intégration des informations viscérales. Nous avons étudié la dépression synaptique à long terme (DLT) dans le noyau du tractus solitaire et sa modulation par le statut nutritionnel Dans la première étude, nous avons caractérisé une DLT au niveau du NTS. Cette DLT, déclenchée par la stimulation des afférences viscérales à basse fréquence, est exprimée au niveau de l’élément présynaptique. Elle met en jeu une libération d'endocannabinoïdes qui en agissant au niveau de l’élément présynaptique réduisent la probabilité de libération de glutamate. De manière surprenante l’élément postsynaptique ne semble jouer aucun rôle dans cette DLT. Elle nécessite une activation séquentielle des récepteurs NMDA, la libération d'anandamide et l'activation des récepteurs aux cannabinoïdes de type 1 (CB1) et l’activité de l’élément présynaptique. Nos résultats suggèrent que cette DLT pourrait être entièrement organisée dans le compartiment présynaptique des afférences viscérales. Dans une deuxième partie du travail, nous nous sommes intéressés à la modulation de cette DLT dépendante des endocannabinoïdes (DLT-eCBs) par le statut nutritionnel. La privation de nourriture pendant 24 h empêche l'induction de la DLT-eCBs par la stimulation des afférences viscérales. Ces effets sont mimés par l'activation des récepteurs à la ghréline au niveau du NTS. Une re-nutrition pendant 3h restaure la DLT-eCBs via l'action périphérique de la Cholécystokinine (CCK) et l'activation de la voie ERK. Au total ces travaux soulignent la forte plasticité des afférences viscérales en fonction du statut nutritionnel. / This work joins within the framework of studies about the mechanisms of integration of the visceral informations. We studied long term synaptic depression in the nucleus of tractus solitarii (NTS) and it's modulation during changes in the nutritional status. In the first study, we characterized a long-term synaptic depression (LTD) in the NTS. This LTD, triggered by low frequency stimulation of visceral afferents is expressed at the presynaptic level. It involves release of endocannabinoids that would eventually reduce glutamate release probability. Surprisingly the postsynaptic element seems to play no role in this LTD. It requires sequential activation of NMDA receptors, the release of anandamide and activation of the cannabinoids type 1 receptors (CB1) and presynaptic activation. Our results suggest that this LTD could be entirely organized at the presynaptic compartment of visceral afferents. In the second part of this work, we were interested on the modulation of this endocannabinoïds dependent long-term depression (eCBs-LTD) by the nutritional status. Food deprivation during 24 h prevents the induction of eCBs-LTD by the stimulation of visceral afferents. These effects are mimicked by the activation of ghrelin receptors in the NTS. 3 h refeeding restores the eCBs-LTD via peripheral action of cholecystokinin (CCK) and the activation of the ERK pathway. Altogether, this work emphasizes the high plasticity of visceral afferents and its regulation by the nutritional status.

O objetivo do presente trabalho foi propor e estabelecer um novo método de preparação de peptídeos protegidos &#945;-esterificados. Para tal, investigamos inicialmente o efeito da presença de HAc, Ca(OAc)2 e mistura de ambos na eficiência de metanólise de Ac-Ala-Gly-X-KOR (onde, X= Gly, Ala ou Phe) em DCM. Surpreendemente, as reações assistidas por Ca2+ forneceram os produtos esterificados desejados com os mais altos rendimentos. Com base nestes dados comparamos as eficiências das reações de metanólise de Ac-Ala-Gly-X-KOR [onde, X= Lys(2-Cl-Z), Phe, Ala ou Gly] asssistidas pelo íon cálcio. Estas se mostraram dependentes da natureza do resíduo C-terminal ligado à resina, pois os rendimentos de desligamento peptídeo da KOR obtidos para as reações de 6 horas apresentaram a seguinte ordem: Lys(2-Cl-Z)< Phe <Ala < Gly. Após 48 h, os rendimentos foram superiores a 68%. A possibilidade de preparar Ac-Ala-Gly-X-OEt [onde, X= Lys(2-Cl-Z) ou Ala] usando o mesmo procedimento foi também investigada. Ac-Ala-Gly-Ala-OEt e Ac-Ala-Gly-Lys(2-Cl-Z)-OEt foram produzidos com 69 e 24% de rendimento em 72h a 37ºC, respectivamente. Quando DCM foi substituído por DMSO ou 25%DMSO/tolueno a 55ºC, houve um aumento significativo nos rendimentos de etanólise da Ala-Gly-Lys(2-Cl-Z)-KOR. Benzólise e tiólise de Ac-Ala-Gly-X-OEt (onde, X= Lys(2-Cl-Z)ou Ala] foram testadas. As análises de aminoácidos das peptidil-KOR residuais mostraram que o desligamento do peptídeo da KOR ocorreu em certa extensão, mas infelizmente nas condições de análise por RP-HPLC empregadas não fomos capazes de detectar os produtos formados. Metanólises adicionais foram realizadas em presença de Ca(OAc)2 empregando peptidil-KOR relacionados ao hormônio colecistocinina: Ac-lle-Ser(Bzl)-Asp(OBzl)-KOR(1), Ac-lle-Ser(Bzl)-Asp(OcHex)-KOR (2) e Ac-lle-Ser(Bzl)-Asp(OBzl)-Arg(Mtr)-KOR (3). Os rendimentos de desligamento dos peptídeos 1, 2 e 3 foram 54, 68 e 84%, respectivamente. Não foram detectadas nos meios reacionais quantidades significativas de produtos secundários resultantes de rearranjos ou de transesterificação simultânea do éster presente na cadeia lateral do resíduo de ácido aspártico. Estes resultados evidenciaram que a assistência do Ca2+ é seletiva e o método aplicável a peptídeos contendo arginina e ácido aspártico protegidos ligados à resina de Kaiser. O efeito do solvente foi investigado realizando-se metanólises de Ac-lle-Ser(Bzl)-Asp(OcHex)-KOR em DCM, DMSO e CHCl3 em presença de Ca(OAc)2. Em 72 horas as metanólises realizadas em DMSO foram quantitativas, enquanto que em DCM ou CHCl3 estas apresentaram rendimentos de ~63%. Em seguida, examinamos a capacidade do Eu3+ em assistir a reação de metanólise de peptidil-KOR em DCM. Os rendimentos de desligamento do peptídeo foram semelhantes ou ligeiramente superiores aos obtidos em presença de Ca2+. Estudos adicionais demonstraram que não é necessário excesso molar de Ca2+ ou Eu3+ para o aumento da eficiência das metanólises. Metanólise e etanólise de Ac-lle-Ser(Bzl)-Asp(OcHex)-KOR em DCM e DMSO em presença de Ca(OAc)2, EuCl3 e TbCl3 também foram realizadas. As metanólises em DCM em presença de Ca2+, Eu3+ ou Tb3+ forneceram o produto desejado com 62, 95 ou 95% de rendimento, respectivamente. Em DMSO, a reação assistida por Ca2+ foi quantitativa, mas nenhum produto foi obtido em presença de Eu3+ ou Tb3+. As etanólises em DCM assistidas por estes ións lantanídicos foram também mais eficientes do que as mediadas por Ca2+ (28, 17 e 5%, respectivamente). Em DMSO, observou-se a formação do éster etílico apenas para a reação assistida por Ca2+. O rendimento de desligamento do peptídeo da KOR foi de 80%, mas observou-se a presença de um produto secundário no peptídeo &#945;-esterificado bruto. Novamente, nenhum produto foi obtido nas reações assistidas por Eu3+ ou Tb3+ em DMSO. Estudos adicionais demonstraram que Ca(OAc)2 pode também assistir a aminólise de Ac-lle-Ser(Bzl)-Asp(OcHex)-KOR por Arg(HCl)OMe ou Arg(HCl)OEt em DMF. Os rendimentos de desligamento do peptídeo da KOR foram superiores a 70%, sendo os ésteres desejados os produtos majoritários formados nos meios reacionais (47 e 68% do total, respectivamente). Na ausência do íon metálico, os rendimentos foram de apenas 10-13%. Reações comparativas evidenciaram que a aminólise assistida por Ca2+ é equivalente (ou ligeiramente inferior em termos de seletividade) àquelas descritas na literatura catalisadas pelo ácido acético. Tentativas de aminólise em DMSO, NMP e DMSO/tolueno (1:1, v:v) na presença de Ca2+, Eu3+ e Tb3+ também foram feitas. Os resultados demonstraram que apenas o Ca2+ foi capaz de assistir a maioria delas. Isto ocorreu provavelmente devido a interações entre o DMSO e os lantanídeos. Uma análise preliminar de Ac-Ala-Gly-Lys(2-Cl-Z)-KOR e de Ac-lle-Ser(Bzl)-Asp(OcHex)-KOR através de HR-MAS na presença e ausência de Ca2+ foi feita com o intuito de investigar as interações estabelecidas entre o íon metálico e os peptídeos sob o ponto de vista estrutural. Os resultados obtidos sugeriram que a presença do Ca2+ diminue a mobilidade da cadeia peptídica, o que sugere complexação. Uma complexação tomaria a ligação peptídeo-KOR mais susceptível ao ataque nucleofílico do álcool ou aminoácido esterificado. Medidas de inchamento de peptidil-KOR em diferentes solventes puros e nas misturas utilizadas nos estudos descritos acima demonstraram que a solvatação da peptidil-KOR não é o fator determinante na eficiência destas reações. Por outro lado, estudos qualitativos sugeriram, que a solubilidade dos sais de cálcio, európio e térbio seja crucial. Concluindo, os dados obtidos até o momento indicam que a metanólise, etanólise e aminólise de peptidil-KOR assistidas pelos íons Ca2+ consistem em uma alternativa nova e atraente para preparar ésteres &#945;-metílicos e &#945;-etílicos de N&#945;-acil-peptídeos totalmente protegidos ou desprotegidos. As reações foram realizadas em pH aparente neutro e se mostraram muito seletivas à ligação oxima. / The ultimate goal of the present work was to propose and stablish a new method to prepare a-esterified protected peptides. At first, we examined the effect of HAc, Ca(OAc)2 and mixtures of both on the efficiency of methanolysis of Ac-Ala-Gly-X-KOR (where, X= Gly, Ala or Phe) in DCM. Surprisingly, the reactions carried-out in the presence of Ca(OAc)2 gave the corresponding esterified peptides with the highest yields. Based on these data, we compared the efficiencies of methanolysis of Ac-Ala-Gly-X-KOR [where, X= Lys(2-Cl-Z), Phe, Ala or Gly] assisted by the calcium ion. They showed to be dependent on the nature of the C-terminal residue linked to the solid support since the yields found for 6-hour reactions were: Lys(2-Cl-Z)< Phe <Ala < Gly. After 48 h of reaction, yields were all higher than 68%. The possibility of obtaining Ac-Ala-Gly-X-OEt [where, X= Lys(2-Cl-Z) or Ala] using the same procedure was then investigated. In 72 hours at 37°C Ac-Ala-Gly-Ala-OEt and Ac-Ala-Gly-Lys(2-Cl-Z)-OEt were produced with yields of 69% and 24%, respectively. When DCM was replaced by DMSO or 25%DMSO/toluene at 55°C, the percentages of peptide release from Ala-Gly-Lys(2-Cl-Z)-KOR were 44% in 72h and 66% in 36h, respectively. Benzolysis and thiolysis of Ac-Ala-Gly-X-OEt [where, X= Lys(2-C-Z) or Ala] were tested as well. Despite the fact that amino acid analysis of the remaining peptidyl-KOR showed that the peptide detachment from the resin had occurred to some extent, we were not able to detect by analytical RP-HPLC the products formed in the reaction media. We also studied the methanolyses of some peptidyl-resins related to the peptide hormone cholecystokinin: Ac-lle-Ser(Bzl)-Asp(OBzl)-KOR (1), Ac-lle-Ser(Bzl)-Asp(OcHex)-KOR (2) and Ac-lle-Ser(Bzl)-Asp(OBzl)-Arg(Mtr)-KOR (3). The yields of peptide detachment from 1, 2 or 3 were 54, 68 and 84%, respectively. No significant amounts of byproducts resulting from possible rearrangements or simultaneous transesterification of the ester present at the lateral side chain of aspartic acid were detected in the reaction media. Such results allowed us to conclude that the procedure proposed was quite selective and applicable to peptides containing protected arginine or aspartic acid attached to Kaiser oxime resin. The effect of the solvent on the methanolysis efficiency was then investigated. For that, reactions containing Ac-lle-Ser(Bzl)-Asp(OcHex)-KOR and Ca(OAc)2 suspended in DCM, DMSO and CHCl3 were carried-out. That in DMSO was quantitative while those in DCM or CHCl3 yielded the esterified peptide with 63% in 72 h. The ability of Eu3+ to assist methanolysis of peptidyl-KOR in DCM was demonstrated since peptide detachment yields for some reactions were similar or slightly superior to those assisted by Ca2+. Additional studies showed that molar excess of Ca2+ or Eu3+ is not required for the enhancement of methanolysis efficiency. Comparative methanolyses and ethanolyses of Ac-lle-Ser(Bzl)Asp(OcHex)-KOR assisted by Ca2+, Eu3+ and Tb3+ in different solvents were also studied. Methanolysis in DCM in the presence of Ca2+, Eu3+ or Tb3+ supplied the desired product with yields of 62, 95 or 95%, respectively. In DMSO, the reaction assisted by Ca2+ was quantitative, but no product was formed in presence of Eu3+ or Tb3+. Ethanolysis in DCM assisted by Eu3+ or Tb3+ was also more efficient than that performed in presence of Ca2+ (28, 17 and 5%, respectively). However, in DMSO the desired &#945;-ethyl ester was formed only in the presence of Ca2+. We were able to show that Ca(OAc)2 can also assist the aminolysis of Ac-lle-Ser(Bzl)-Asp(OcHex)-KOR in DMF by Arg(HCl)OMe or Arg(HCl)-OEt. The peptide detachment yields were higher than 70%, being the desired esters the major products formed in the reaction medium. In the absence of the metal ion, yields were only 10-13%. Further studies evidenced that aminolysis assisted by Ca2+ is equivalent (or slightly inferior in terms of selectivity) to those described in the literature that uses HAc as catalyst. Attempts of aminolysis in DMSO, NMP, DMSO/toluene (1:1, v/v) containing Ca2+, Eu3+ and Tb3+ showed that only Ca2+ was able to assist the formation of the esterified peptide in all these solvents. This is probably due to DMSO interaction with the lanthanide ions. Preliminary analyses of two peptidyl-KOR by HR-MAS in the presence and absence of Ca2+ were performed in order to obtain structural information that could help us to determine the interactions established between the metal ion and peptides. The results found indicated that the presence of Ca2+ makes peptide chain less mobile, which suggests complexation. A complexation could make the peptide-resin oxime bond more susceptible to nucleophilic attacks, favoring solvolysis and aminolysis. Swelling measurements of Ac-Ala-Gly-Lys(2-Cl-Z)-KOR and Ac-lle-Ser(Bzl)-Asp(OcHex)-KOR in pure solvents and in the mixtures employed showed that the solvation of peptidyl-KOR is not the factor that determines the efficiency of these reactions. On the other hand, qualitative studies suggested that the solubility of the calcium, europium and terbium salts could be crucial. Hence, the data presented here indicate that methanolysis, ethanolysis and aminolysis of peptidyl-KOR assisted by Ca2+ provide a new alternative and attractive way to prepare fully protected or unprotected N&#945;-acyl-peptides &#945;-methyl and &#945;-ethyl esters. The reactions were performed at neutral pH and showed to be very selective to oxime bond.