G-protein coupled receptors (GPCRs) have been shown to act as part of GPCR associated protein complexes (GAPCs) which are required to appropriately transduce downstream signaling pathways leading to specific cellular actions. I hypothesize that there are distinct molecular effectors that couple to the glucagon superfamily of B-class GPCRs (glucagon, GLP-1, GLP-2, GIP receptors) to effect the myriad of reported actions in numerous target cells including regulation of insulin secretion, intestinal growth and appetite suppression. GLP-1R, GIPR, GLP-2R and GCGR were screened using a newly developed membrane-based split-ubiquitin yeast two-hybrid (MYTH) system to reveal 181 novel candidate protein interactors associated with signal transduction, transport, metabolism and cell survival. Each candidate was validated using yeast two-hybrid prey retransformation tests and by co-purification to confirm coupling to each receptors. The present work is the first demonstration of a split-ubiquitin interaction screen using in situ membrane expressed GPCRs of the secretin-like B class.

G-protein coupled receptors (GPCRs) have been shown to act as part of GPCR associated protein complexes (GAPCs) which are required to appropriately transduce downstream signaling pathways leading to specific cellular actions. I hypothesize that there are distinct molecular effectors that couple to the glucagon superfamily of B-class GPCRs (glucagon, GLP-1, GLP-2, GIP receptors) to effect the myriad of reported actions in numerous target cells including regulation of insulin secretion, intestinal growth and appetite suppression. GLP-1R, GIPR, GLP-2R and GCGR were screened using a newly developed membrane-based split-ubiquitin yeast two-hybrid (MYTH) system to reveal 181 novel candidate protein interactors associated with signal transduction, transport, metabolism and cell survival. Each candidate was validated using yeast two-hybrid prey retransformation tests and by co-purification to confirm coupling to each receptors. The present work is the first demonstration of a split-ubiquitin interaction screen using in situ membrane expressed GPCRs of the secretin-like B class.

The development of a bioartificial pancreas (BAP) has the potential to substantially improve the treatment of insulin-dependent diabetes. Composed of insulin-secreting cells encapsulated in a hydrogel material, a BAP may provide superior glycemic regulation compared with conventional exogenous insulin-delivery therapies. Towards this goal, β- cells or islets encapsulated in alginate microcapsules remain a promising approach. Due to the limited supply of human islets, alternative cell sources are under investigation for incorporation into a BAP, including porcine islets and β- cell lines. Several challenges remain to clinical implementation, including loss of islet or β- cell function and viability following transplantation and host response to the transplanted microcapsules. The objective of this work was to evaluate strategies to improve a BAP by supporting the function and survival of encapsulated islets and β -cells. Towards this goal, two areas were explored: 1) the provision of pro-survival and insulinotropic factors, namely, CXCL12 and GLP-1 (or a GLP-1 analog, Exendin-4), to encapsulated islets and β-cells and 2) modification of the alginate microcapsule to confer long-term resistance to host cell adhesion. To achieve the first objective, methods to deliver both pro-survival and insulinotropic factors to a BAP were developed and their effects on encapsulated β-cells and porcine islets were studied, both in vitro and in vivo. Results demonstrate that delivery of pro-survival and insulinotropic factors is a promising strategy to prolong the survival and function of a BAP. To reduce host cell adhesion to the microcapsule, we employed covalent conjugation of PEG to the surface of alginate-PLL capsules to replace the un-crosslinked layer of alginate used in traditional alginate-PLL-alginate (APA) microcapsules. Results demonstrate that while PEGylation of alginate-PLL microcapsules initially reduced host cell adhesion over 2 weeks in vivo compared with APA capsules, the PEG coating did not provide long-term protection over 3 months. Taken together, these studies represent a multipronged approach towards improving the duration of BAP function, with the ultimate goal of advancing this technology to the clinic.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13429号 / 論医博第2233号 / 新制||医||1053(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 長船 健二, 教授 妹尾 浩, 教授 川口 義弥 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Diabetes Mellitus has continued to drastically affect the health of the world and many complications can prove fatal. As long as this metabolic disease persist, research discoveries will need to continue to be made so that patient outcomes and healthcare are dramatically enhanced. In recent years, GLP-1 has been the topic of conversation for diabetes research, due to its promising effects in promoting insulin sensitivity. Furthermore, bile acids and their receptors (TGR5 & FXR) have shown promise in their actions in the regulation of GLP-1, and thus glucose homeostasis. Here we have shown the detection and increased expression of TGR5 and GLP-1, and decreased expression of FXR in diabetic mouse intestinal mucosa tissues. We have also shown the detection and increased expression of these receptors in STC-1 cells. More importantly we have linked the connection of increased glucose concentration (hyperglycemia) to increased TGR5 activation to increased GLP-1 release, thus leading to increased insulin sensitivity and altered diabetic outcomes.

In response to ingestion of nutrients, enteroendocrine L cells secrete the incretin hormone, glucagon-like peptide-1 (GLP-1), to enhance glucose-dependent insulin release. Therapies related to GLP-1 are approved for type 2 diabetes. The GLP-1 receptor (GLP-1R) is expressed in cells of the gastrointestinal tract and elsewhere. In pancreatic beta cells, GLP-1R are coupled to the Gs/cAMP/PKA pathway. The expression and function of GLP-1R in gastrointestinal smooth muscle are not known. Aim. To test the hypothesis that GLP-1 regulates smooth muscle function by acting on GLP-1R expressed on smooth muscle. Methods. Smooth muscle cells (SMC) were isolated and cultured. Expression of GLP-1R mRNA was measured by RT-PCR. Expression of GLP-1R protein was measured by western blot. The effect of GLP-1 (7-36) amide on Gαs activation, cAMP formation, and PKA activity was examined in cultured SMC. The effect of GLP-1 on basal activity and on acetylcholine-induced contraction was measured in intact colon via organ bath. Results. Amplification of GLP-1R mRNA suggested expression of GLP-1R mRNA in mucosal and non-mucosal colon cells, which was confirmed in pure SMC cultures. Similar patterns of protein expression were obtained with western blot. Addition of GLP-1 caused relaxation of phasic activity and agonist-induced tonic contractions in intact colon, suggesting a role of smooth muscle Gs-coupled GLP-1R in mediating relaxation. In SMC, GLP-1 (7-36) amide activated Gas, increased cAMP levels, and stimulated PKA activity. Conclusion. Colonic SMC express GLP-1R, and GLP-1 inhibits both basal and acetylcholine-induced contraction. The GLP1-R is coupled to the heterotrimeric G protein, Gas.

Insulin and glucagon are released in pulses from pancreatic β- and α-cells, respectively. Both cell types are electrically excitable, and elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) due to depolarization with voltage-dependent entry of the cation is the main trigger of hormone secretion. Store-operated Ca2+ entry  (SOCE) also contributes to the [Ca2+]i elevation and this process has been suggested to be particularly important for glucagon secretion. cAMP is another important messenger that amplifies Ca2+-triggered secretion of both hormones, but little is known about cAMP dynamics in islet cells. In type-2 diabetes, there is deteriorated β-cell function associated with elevated concentrations of fatty acids, but the underlying mechanisms are largely unknown. To clarify the processes that regulate insulin and glucagon secretion, cAMP signalling and the store-operated pathway were investigated in β- and α-cells, primarily within their natural environment in intact mouse and human islets of Langerhans. Fluorescent biosensors and total internal reflection microscopy were used to investigate signalling specifically at the plasma membrane (PM). Adrenaline increased and decreased the sub-PM cAMP concentration ([cAMP]pm) in immuno-identified α-cells and β-cells, respectively, which facilitated cell identification. Glucagon elicited [cAMP]pm oscillations in α- and β-cells, demonstrating both auto- and paracrine effects of the hormone. Whereas glucagon-like peptide 1 (GLP-1) consistently elevated [cAMP]pm in β-cells, only few α-cells responded, indicating that GLP-1 regulates glucagon secretion without changes of α-cell [cAMP]pm. Both α- and β-cells responded to glucose with pronounced oscillations of [cAMP]pm that were partially Ca2+-dependent and synchronized among islet β-cells. The glucose-induced cAMP formation was mediated by plasma membrane-bound adenylyl cyclases. Several phosphodiesterases (PDEs), including the PDE1, -3, -4, and -8 families, were required for shaping the [cAMP]pm signals and pulsatile insulin secretion. Prolonged exposure of islets to the fatty acid palmitate deteriorated glucose-stimulated insulin secretion with loss of pulsatility. This defect was associated with impaired cAMP generation, while [Ca2+]i signalling was essentially unaffected. Stromal interacting molecule 1 (STIM1) is critical for activation of SOCE by sensing the Ca2+ concentration in the endoplasmic reticulum (ER). ER Ca2+ depletion caused STIM1 aggregation, co-clustering with the PM Ca2+ channel protein Orai1 and SOCE activation. Glucose, which inhibits SOCE by filling the ER with Ca2+, reversed the PM association of STIM1. Consistent with a role of the store-operated pathway in glucagon secretion, this effect was maximal at the low glucose concentrations that inhibit glucagon release, whereas considerably higher concentrations were required in β-cells. Adrenaline induced STIM1 translocation to the PM in α-cells and the reverse process in β-cells, partially reflecting the opposite effects of adrenaline on cAMP in the two cell types. However, cAMP-induced STIM1 aggregates did not co-cluster with Orai1 or activate SOCE, indicating that STIM1 translocation can occur independently of Orai1 clustering and SOCE.

Introdução: A prevalência de doenças crônicas tem crescido em decorrência do aumento da obesidade nos últimos anos. Peptídeos secretados pelo trato gastrointestinal, como o peptídeo semelhante ao glucagon 1 (GLP-1) e o peptídeo YY (PYY), exercem papel fundamental no controle da ingestão alimentar, pois levam informações acerca dos nutrientes ingeridos até o sistema nervoso central, possuindo efeitos anorexígenos e/ou orexígenos. Tanto GLP-1 quanto PYY são produzidos pelas células-L do íleo distal e cólon, sendo liberados após a ingestão alimentar. Além do efeito incretina produzido pelo GLP-1, ambos os peptídeos apresentam implicações no controle do apetite, o qual reflete na redução do peso corpóreo. Objetivos: Demonstrar um aumento na secreção de GLP-1 e PYY após sobrecarga oral de diferentes tipos de lipídios, comparados à controle negativo (água) e positivo (glicose). Métodos: Foi realizado um estudo experimental controlado em ratos Wistar, distribuídos em 4 grupos de acordo com a sobrecarga oral: grupo MUFA (óleo de oliva); grupo SAT (banha suína); grupo GLUC (glicose) e grupo CONT (água), foram avaliadas as concentrações séricas de GLP-1 ativo e PYY3-36 nos tempos: 0, 15, 30, 60 e 120 minutos. As sobrecargas foram isovolumétricas e isocalóricas, com exceção do grupo controle. Resultados: Houve pico de secreção do GLP-1 pós-sobrecarga no grupo MUFA no ponto 120’ vs CONT e GLUC (p≤0,001) e no ponto 30’ quando comparado ao seu baseline. Também observou-se pico de secreção de PYY no grupo MUFA vs CONT no ponto 30’ (p=0,015); no ponto 60’ vs CONT e GLUC (p=0,019) e no ponto 120’ vs CONT e SAT (p=0,02). A carga secretada do PYY foi maior no MUFA quando comparada ao CONT (p=0,04). Verificou-se forte correlação entre os níveis basais e AUC’s do GLP-1 e PYY (r=0,57; (p= 0,02); r=0,39; (p≤0,001)). A proporção GLP-1/PYY apresentou um coeficiente médio de 3,77 (±2,04). O grupo MUFA evidenciou níveis menores de glicose e maiores de insulina no ponto 15’, enquanto SAT mostrou níveis maiores de glicose e menores de insulina neste ponto, porém, sem diferença significativa. Conclusão: A sobrecarga oral de fontes de ácidos graxos monoinsaturados promoveu um pico de secreção do GLP-1 de forma rápida e no que diz respeito ao PYY, o pico foi mais sustentado. Estudos adicionais são necessários, a fim de se avalir o efeito de fontes distintas de lipídeos da dieta sobre a secreção destes peptídeos e seus efeitos na saciedade. / Background: The increased prevalence of chronic diseases has risen due to obesity. Gut peptides, such as glucagon-like peptide 1 (GLP-1) and peptide YY (PYY), play an important role controlling food intake in response to a meal. GLP-1 exerts the known incretin effect stimulating the release of insulin in a glucosedependent manner. Besides its insulinotropic effects, it is well established that GLP-1 slows gastric emptying, and also inhibits inappropriate glucagon release, additionally improves satiety. Both PYY and GLP-1 are produced by L cells of the distal ileum and colon. Objective: Demonstrate an increased secretion of PYY and GLP-1 after oral overload of different types of lipids, compared to negative (water) and positive (glucose) control. Methods: We conducted a controlled experimental study in Wistar rats, divided into 4 groups according to oral overload: MUFA group (olive oil), SAT group (lard:), carbohydrates group (glucose) and CONT group (water), It was evaluated the serum concentration of active GLP-1 and PYY3-36 in the times: 0, 15, 30, 60 and 120 minutes. Overloads were isovolumetric and isocaloric, but the control group. Results: It was verified a higher peak secretion of GLP-1 in MUFA group at 120' after overload vs. CONT and carbohydrates (p ≤ 0.001) and at 30' when compared to its baseline (p=0,01). It was shown a higher secretion peak of GLP-1 after MUFA overload at time 120’ vs CONT e GLUC (p≤0,001) and at time 30’ when compared to its baseline. It was also verified a PYY release peak in MUFA vs CONT at time 30’ (p=0,015); 60’ vs CONT e GLUC (p=0,019) and 120’ vs CONT e SAT (p=0,02). PYY release load presented higher in MUFA group when compared to CONT (p=0,04). A strong correlation was seen between baseline PYY and GLP-1 (r=0,57; p= 0,02) as their AUC’s (r=0,39; p≤0,001). GLP-1/PYY proportion release presented a mean coefficient of 3, 77(±2,04).Conclusion: monounsaturated fatty acids promoted a release peak of GLP-1 in a faster manner and concerning PYY this peak was more sustained. Further studies are necessary to evaluate whether distinct diet fatty acids can ameliorate these gut peptides release and their role on satiety.

Introdução: A prevalência de doenças crônicas tem crescido em decorrência do aumento da obesidade nos últimos anos. Peptídeos secretados pelo trato gastrointestinal, como o peptídeo semelhante ao glucagon 1 (GLP-1) e o peptídeo YY (PYY), exercem papel fundamental no controle da ingestão alimentar, pois levam informações acerca dos nutrientes ingeridos até o sistema nervoso central, possuindo efeitos anorexígenos e/ou orexígenos. Tanto GLP-1 quanto PYY são produzidos pelas células-L do íleo distal e cólon, sendo liberados após a ingestão alimentar. Além do efeito incretina produzido pelo GLP-1, ambos os peptídeos apresentam implicações no controle do apetite, o qual reflete na redução do peso corpóreo. Objetivos: Demonstrar um aumento na secreção de GLP-1 e PYY após sobrecarga oral de diferentes tipos de lipídios, comparados à controle negativo (água) e positivo (glicose). Métodos: Foi realizado um estudo experimental controlado em ratos Wistar, distribuídos em 4 grupos de acordo com a sobrecarga oral: grupo MUFA (óleo de oliva); grupo SAT (banha suína); grupo GLUC (glicose) e grupo CONT (água), foram avaliadas as concentrações séricas de GLP-1 ativo e PYY3-36 nos tempos: 0, 15, 30, 60 e 120 minutos. As sobrecargas foram isovolumétricas e isocalóricas, com exceção do grupo controle. Resultados: Houve pico de secreção do GLP-1 pós-sobrecarga no grupo MUFA no ponto 120’ vs CONT e GLUC (p≤0,001) e no ponto 30’ quando comparado ao seu baseline. Também observou-se pico de secreção de PYY no grupo MUFA vs CONT no ponto 30’ (p=0,015); no ponto 60’ vs CONT e GLUC (p=0,019) e no ponto 120’ vs CONT e SAT (p=0,02). A carga secretada do PYY foi maior no MUFA quando comparada ao CONT (p=0,04). Verificou-se forte correlação entre os níveis basais e AUC’s do GLP-1 e PYY (r=0,57; (p= 0,02); r=0,39; (p≤0,001)). A proporção GLP-1/PYY apresentou um coeficiente médio de 3,77 (±2,04). O grupo MUFA evidenciou níveis menores de glicose e maiores de insulina no ponto 15’, enquanto SAT mostrou níveis maiores de glicose e menores de insulina neste ponto, porém, sem diferença significativa. Conclusão: A sobrecarga oral de fontes de ácidos graxos monoinsaturados promoveu um pico de secreção do GLP-1 de forma rápida e no que diz respeito ao PYY, o pico foi mais sustentado. Estudos adicionais são necessários, a fim de se avalir o efeito de fontes distintas de lipídeos da dieta sobre a secreção destes peptídeos e seus efeitos na saciedade. / Background: The increased prevalence of chronic diseases has risen due to obesity. Gut peptides, such as glucagon-like peptide 1 (GLP-1) and peptide YY (PYY), play an important role controlling food intake in response to a meal. GLP-1 exerts the known incretin effect stimulating the release of insulin in a glucosedependent manner. Besides its insulinotropic effects, it is well established that GLP-1 slows gastric emptying, and also inhibits inappropriate glucagon release, additionally improves satiety. Both PYY and GLP-1 are produced by L cells of the distal ileum and colon. Objective: Demonstrate an increased secretion of PYY and GLP-1 after oral overload of different types of lipids, compared to negative (water) and positive (glucose) control. Methods: We conducted a controlled experimental study in Wistar rats, divided into 4 groups according to oral overload: MUFA group (olive oil), SAT group (lard:), carbohydrates group (glucose) and CONT group (water), It was evaluated the serum concentration of active GLP-1 and PYY3-36 in the times: 0, 15, 30, 60 and 120 minutes. Overloads were isovolumetric and isocaloric, but the control group. Results: It was verified a higher peak secretion of GLP-1 in MUFA group at 120' after overload vs. CONT and carbohydrates (p ≤ 0.001) and at 30' when compared to its baseline (p=0,01). It was shown a higher secretion peak of GLP-1 after MUFA overload at time 120’ vs CONT e GLUC (p≤0,001) and at time 30’ when compared to its baseline. It was also verified a PYY release peak in MUFA vs CONT at time 30’ (p=0,015); 60’ vs CONT e GLUC (p=0,019) and 120’ vs CONT e SAT (p=0,02). PYY release load presented higher in MUFA group when compared to CONT (p=0,04). A strong correlation was seen between baseline PYY and GLP-1 (r=0,57; p= 0,02) as their AUC’s (r=0,39; p≤0,001). GLP-1/PYY proportion release presented a mean coefficient of 3, 77(±2,04).Conclusion: monounsaturated fatty acids promoted a release peak of GLP-1 in a faster manner and concerning PYY this peak was more sustained. Further studies are necessary to evaluate whether distinct diet fatty acids can ameliorate these gut peptides release and their role on satiety.

Introdução: A prevalência de doenças crônicas tem crescido em decorrência do aumento da obesidade nos últimos anos. Peptídeos secretados pelo trato gastrointestinal, como o peptídeo semelhante ao glucagon 1 (GLP-1) e o peptídeo YY (PYY), exercem papel fundamental no controle da ingestão alimentar, pois levam informações acerca dos nutrientes ingeridos até o sistema nervoso central, possuindo efeitos anorexígenos e/ou orexígenos. Tanto GLP-1 quanto PYY são produzidos pelas células-L do íleo distal e cólon, sendo liberados após a ingestão alimentar. Além do efeito incretina produzido pelo GLP-1, ambos os peptídeos apresentam implicações no controle do apetite, o qual reflete na redução do peso corpóreo. Objetivos: Demonstrar um aumento na secreção de GLP-1 e PYY após sobrecarga oral de diferentes tipos de lipídios, comparados à controle negativo (água) e positivo (glicose). Métodos: Foi realizado um estudo experimental controlado em ratos Wistar, distribuídos em 4 grupos de acordo com a sobrecarga oral: grupo MUFA (óleo de oliva); grupo SAT (banha suína); grupo GLUC (glicose) e grupo CONT (água), foram avaliadas as concentrações séricas de GLP-1 ativo e PYY3-36 nos tempos: 0, 15, 30, 60 e 120 minutos. As sobrecargas foram isovolumétricas e isocalóricas, com exceção do grupo controle. Resultados: Houve pico de secreção do GLP-1 pós-sobrecarga no grupo MUFA no ponto 120’ vs CONT e GLUC (p≤0,001) e no ponto 30’ quando comparado ao seu baseline. Também observou-se pico de secreção de PYY no grupo MUFA vs CONT no ponto 30’ (p=0,015); no ponto 60’ vs CONT e GLUC (p=0,019) e no ponto 120’ vs CONT e SAT (p=0,02). A carga secretada do PYY foi maior no MUFA quando comparada ao CONT (p=0,04). Verificou-se forte correlação entre os níveis basais e AUC’s do GLP-1 e PYY (r=0,57; (p= 0,02); r=0,39; (p≤0,001)). A proporção GLP-1/PYY apresentou um coeficiente médio de 3,77 (±2,04). O grupo MUFA evidenciou níveis menores de glicose e maiores de insulina no ponto 15’, enquanto SAT mostrou níveis maiores de glicose e menores de insulina neste ponto, porém, sem diferença significativa. Conclusão: A sobrecarga oral de fontes de ácidos graxos monoinsaturados promoveu um pico de secreção do GLP-1 de forma rápida e no que diz respeito ao PYY, o pico foi mais sustentado. Estudos adicionais são necessários, a fim de se avalir o efeito de fontes distintas de lipídeos da dieta sobre a secreção destes peptídeos e seus efeitos na saciedade. / Background: The increased prevalence of chronic diseases has risen due to obesity. Gut peptides, such as glucagon-like peptide 1 (GLP-1) and peptide YY (PYY), play an important role controlling food intake in response to a meal. GLP-1 exerts the known incretin effect stimulating the release of insulin in a glucosedependent manner. Besides its insulinotropic effects, it is well established that GLP-1 slows gastric emptying, and also inhibits inappropriate glucagon release, additionally improves satiety. Both PYY and GLP-1 are produced by L cells of the distal ileum and colon. Objective: Demonstrate an increased secretion of PYY and GLP-1 after oral overload of different types of lipids, compared to negative (water) and positive (glucose) control. Methods: We conducted a controlled experimental study in Wistar rats, divided into 4 groups according to oral overload: MUFA group (olive oil), SAT group (lard:), carbohydrates group (glucose) and CONT group (water), It was evaluated the serum concentration of active GLP-1 and PYY3-36 in the times: 0, 15, 30, 60 and 120 minutes. Overloads were isovolumetric and isocaloric, but the control group. Results: It was verified a higher peak secretion of GLP-1 in MUFA group at 120' after overload vs. CONT and carbohydrates (p ≤ 0.001) and at 30' when compared to its baseline (p=0,01). It was shown a higher secretion peak of GLP-1 after MUFA overload at time 120’ vs CONT e GLUC (p≤0,001) and at time 30’ when compared to its baseline. It was also verified a PYY release peak in MUFA vs CONT at time 30’ (p=0,015); 60’ vs CONT e GLUC (p=0,019) and 120’ vs CONT e SAT (p=0,02). PYY release load presented higher in MUFA group when compared to CONT (p=0,04). A strong correlation was seen between baseline PYY and GLP-1 (r=0,57; p= 0,02) as their AUC’s (r=0,39; p≤0,001). GLP-1/PYY proportion release presented a mean coefficient of 3, 77(±2,04).Conclusion: monounsaturated fatty acids promoted a release peak of GLP-1 in a faster manner and concerning PYY this peak was more sustained. Further studies are necessary to evaluate whether distinct diet fatty acids can ameliorate these gut peptides release and their role on satiety.