Orientador: Licio Augusto Velloso / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T14:54:51Z (GMT). No. of bitstreams: 1 Dragano_NathaliaRomanelliVicente_D.pdf: 1805761 bytes, checksum: fe734ef29b5f10bfd7ff5e19b729fec0 (MD5) Previous issue date: 2015 / Resumo: A recente caracterização da atividade anti-inflamatória atípica exercida pelo receptor de ácidos graxos poli-insaturados de cadeia longa GPR120 tem despertado grande interesse sobre esta classe de receptores, como potenciais alvos para o tratamento da obesidade e distúrbios metabólicos relacionados. Até o momento, a maioria dos estudos realizados tem explorado principalmente os benefícios metabólicos potenciais de uma ativação sistêmica dos receptores GPR120 e GPR40; no entanto, estudos recentes demonstraram que o hipotálamo é afetado logo durante as fases iniciais do desenvolvimento da obesidade desempenhando um papel crucial na patogênese desta doença e de suas comorbidades. Neste trabalho, nós avaliamos a expressão e potenciais ações terapêuticas do GPR120 e GPR40 no hipotálamo de camundongos obesos. Nós observamos que ambos os receptores são expressos no hipotálamo, sendo o GPR120 principalmente presente em células da microglia e o GRP40 preferencialmente expresso em neurônios orexigênicos NPY. Após o tratamento intracerebroventricular com GW9508, um agonista não específico destes receptores, foi observado uma redução da eficiência energética e da expressão de genes inflamatórios no hipotálamo de camundongos obesos. O silenciamento hipotalâmico do GPR120, por meio de lentivírus, aboliu o efeito anti-inflamatório induzido por GW9508 e resultou em maior eficiência energética nos animais tratados. Entretanto, o tratamento intracerebroventricular com os agonistas específicos do GPR120 e GPR40, TUG1197 e TUG905, respectivamente, resultaram em efeitos mais moderados sobre a homeostase energética e inflamação induzida por HFD do que aqueles observados com o GW9508. O TUG1197 atuou reduzindo a inflamação induzida por HFD por meio da redução da expressão das citocinas pró-inflamatórias, TNF? e IL1?, e aumento da expressão das interleucinas anti-inflamatórias, IL10 e IL6. O TUG905 promoveu a redução do peso corporal e da expressão do neuropeptídeo anorexigênico POMC. Em conclusão nosso trabalho demosntrou que os receptores GPR120 e GPR40 de forma coordenada no hipotálamo de roedores reduzindo a eficiência energética e regulando a inflamação associada à obesidade. A ativação de ambos os receptores no hipotálamo levou a melhores desfechos metabólicos se comparada com a ativação de qualquer um dos receptores isoladamente / Abstract: The recent characterization of the atypical anti-inflammatory activity exerted by the polyunsaturated fatty acid receptor GPR120 has raised the interest on this class of receptors as potential targets for the treatment of obesity and related disorders. Most studies performed to date have explored the potential metabolic benefits of a systemic activation of GPR120 and GPR40; however, it is currently known that the hypothalamus is affected during the early stages of obesity and plays an important role in the pathogenesis of this disease and its comorbidities. Here, we evaluate the expression and potential therapeutic actions of hypothalamic GPR120 and GPR40 in experimental diet-induced obesity. We show that both receptors are expressed in the hypothalamus; GPR120 is mostly present in microglia, whereas GRP40 is preferentially expressed in NPY neurons. Upon intracerebroventricular treatment, GW9508, a non-specific agonist of both receptors, reduced energy efficiency and the expression of inflammatory genes in the hypothalamus. Reducing GPR120 hypothalamic expression using a lentivirus approach resulted in the loss of the anti-inflammatory effect of GW9508 and increased energy efficiency. The intracerebroventricular treatment with GPR120 and GPR40 specific agonists, TUG1197 and TUG905, respectively, resulted in milder effects than those produced by GW9508. TUG1197 acted predominantly controlling inflammation reducing the expression of TNF? and IL1? while increasing IL10 and IL6. Conversely, TUG905 acted reducing body mass and increasing the expression of POMC. We conclude that GPR120 and GPR40 act in concert in the hypothalamus to reduce energy efficiency and regulate the inflammation associated with obesity. The combined activation of both receptors in the hypothalamus results in better metabolic outcomes than the isolated activation of either alone / Doutorado / Fisiopatologia Médica / Doutora em Ciências

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

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Prevalence of type 2 diabetes mellitus (T2DM) is still rising and even so in the juvenile population. Obesity is highly associated with increased risk for developing T2DM. The development has been related to elevated fasting concentrations of the pancreatic islet hormones insulin and glucagon as well as to an increase in plasma lipids that occurs during obesity. Specifically, research has indicated that chronic exposure to high levels of saturated free fatty acids cause dysfunction in islet alpha- and beta-cells. Fatty acids can affect islet cells by various mechanisms one of which is the G-protein coupled receptor FFAR1/GPR40. The role of the receptor in the effects of fatty acids on pancreatic islet-cell function is not clear. The aim of this thesis was to clarify the role of FFAR1 in how fatty acids, and more specifically the long-chain saturated fatty acid palmitate, affect insulin and glucagon secretion. In children and adolescents with obesity elevated fasting levels of insulin and glucagon were positively correlated with lipid parameters. Specifically, plasma triglycerides and free fatty acids were positively correlated with insulin and glucagon at fasting as well as with visceral adipose tissue volume. Elevated glucagon levels at fasting were associated with worsening of glucose tolerance in the same population. In in vitro studies of isolated human islets palmitate stimulated basal insulin and glucagon secretion as well as mitochondrial respiration at fasting glucose levels. The effect was mediated by FFAR1 and fatty acid beta-oxidation. At higher glucose concentrations the receptor was involved in the potentiation of insulin secretion from isolated human islets and insulin-secreting MIN6 cells. Furthermore, we found that the effects of palmitate on hormone secretion were associated with enhanced mitochondrial respiration mediated by FFAR1 Gαq signaling and PKC activity as well as increased intracellular metabolism induced by the fatty acid. When islets were exposed to palmitate for long time periods and in the presence of FFAR1 antagonist, normalized insulin and glucagon secretion during culture and insulin response to glucose after culture were observed. In MIN6 cells chronic palmitate treatment increased mitochondrial uncoupling irrespective of FFAR1 involvement. However, FFAR1 antagonism during palmitate exposure resulted in elevated respiration and reduced apoptosis. In conclusion, children and adolescents with obesity have elevated fasting concentrations of insulin and glucagon that correlate with free fatty acids and fatty acid sources. High glucagon levels are linked to worsening of glucose tolerance in these subjects. In vitro the combination or synergy of FFAR1 activation and intracellular metabolism caused by palmitate is decisive for both the short-term enhancement effects and the negative chronic effects on insulin and glucagon secretion.

La relation entre l’obésité et le cancer, bien qu’établie par des études épidémiologiques, est peu connue. Pourtant, environ 25 % des cancers pourraient y être attribuables. Parmi les cancers reliés à l’obésité, les cancers du côlon, du sein chez les femmes ménopausées et de la prostate sont les plus fréquents. Des études sur modèles animaux ont suggéré une association positive entre une diète riche en gras et le développement du cancer mammaire et de la prostate. Nous avons étudié les mécanismes moléculaires par lesquels les acides gras influencent le devenir de lignées de cellules cancéreuses du sein et de la prostate. Ces travaux ont montré que les acides gras insaturés, dont l’oléate, induisent la prolifération cellulaire tandis que les acides gras saturés, dont le palmitate, diminuent la prolifération. Un traitement à l’oléate stimule la formation de gouttelettes lipidiques dans le cytoplasme des cellules de cancer du sein MDA-MB-231 et de la prostate DU145 alors qu’un traitement au palmitate entraîne l’apoptose. Le mécanisme d’action de l’oléate sur la prolifération a été étudié de façon plus approfondie. L’utilisation d’inhibiteurs pharmacologiques nous a permis de déterminer que l’effet prolifératif de l’oléate implique la voie PI3K/Akt, la voie ERK1/2 et l’activation d’un ou de plusieurs récepteur(s) couplé(s) aux protéines G (GPCR). L’oléate induit la phosphorylation rapide des protéines Akt et ERK1/2 dans les cellules de cancer du sein MDA-MB-231 et de la prostate DU145. Au cours des dernières années, deux GPCRs ont été identifiés comme étant activables par des acides gras à moyennes et à longues chaînes, GPR40 et GPR120. GPR40 étant exprimé dans plusieurs lignées cellulaires de cancer du sein et de la prostate contrairement à l’expression de GPR120 qui était inexistante dans la plupart des lignées, nous avons étudié l’implication de GPR40 dans l’effet prolifératif de l’oléate. Ces deux récepteurs n’étant pas exprimés dans les cellules épithéliales mammaires humaines en culture primaire, ces cellules ne répondent pas aux effets de l’oléate sur la prolifération et l’activation des voies de signalisation. L’activation des voies Akt et ERK1/2 par l’oléate dans les cellules MDA-MB-231 et DU145 est potentialisée par la surexpression du récepteur GPR40 et inhibée par l’utilisation d’un siRNA dirigé contre ce récepteur. Cependant, la prolifération induite par l’oléate ne semble pas affectée par la présence d’un siRNA dirigé contre GPR40. L’oléate étant un acide gras, il est capable d’entrer librement dans les cellules et une partie de ses effets sur la prolifération pourrait être attribuée à sa métabolisation. Un agoniste de GPR40, le GW9508, est en mesure d’activer GPR40 sans toutefois entrer dans les cellules ni activer le métabolisme de l’oléate. Le GW9508 stimule la phosphorylation des protéines Akt et ERK1/2 dans les cellules du cancer du sein MDA-MB-231 et de la prostate DU145, mais il n’est pas en mesure d’induire la prolifération cellulaire comme le fait l’oléate. Ces résultats nous permettent de mieux comprendre le mécanisme d’action de l’oléate sur les cellules de cancer du sein et de la prostate. L’oléate induit la signalisation de GPR40 qui est impliquée dans l’activation rapide des voies de signalisation Akt et ERK1/2. De son côté, l’effet prolifératif induit par l’oléate s’effectue par un mécanisme GPR40-indépendant, possiblement lié au métabolisme de l’oléate. / The relationship between obesity and cancer, although established by epidemiological studies, remains relatively unknown. However, about 25 % of cancers could be attributed to obesity. Among cancers that are affected by obesity, colon cancer, post-menopausal breast cancer and prostate cancer are the more frequent. Studies on animal models have suggested a positive association between high fat diets and de development of mammary and prostate cancer. We have studied the molecular mechanisms by which fatty acids influence breast and prostate cancer cells fate. This work has shown that unsaturated fatty acids, including oleate, can induce cellular proliferation while saturated fatty acids, including palmitate, reduce proliferation. An oleate treatment stimulates lipid droplets formation in the cytoplasm of breast cancer cells MDA-MB-231 and prostate cancer cells DU145 while a palmitate treatment induces apoptosis. The action mechanism of oleate on proliferation was studied more closely. Using pharmacological inhibitors, we determine that oleate-induced cell proliferation involves PI3K/Akt signaling pathway, ERK1/2 signaling pathway and the activation of one or many G protein coupled receptor(s) (GPCR). Oleate induces rapid Akt and ERK1/2 phosphorylation in breast cancer cells MDA-MB-231 and prostate cancer cells DU145. In the last few years, two GPCRs were identified as being activated by medium and long chain fatty acids, GPR40 and GPR120. GPR40 being expressed in many breast and prostate cancer cell lines while GPR120 expression was null in most cell lines tested, we studied the role of GPR40 in oleate-induced proliferation. Human epithelial mammary cells in primary culture did not express GPR40 nor GPR120 and failed to respond to oleate-induced cell proliferation or activation of signaling pathways. Akt and ERK1/2 signaling pathways activation by oleate in MDA-MB-231 and DU145 cells is potentiated by GPR40 over-expression and inhibited by the use of an siRNA directed against that receptor. However, oleate-induced cell proliferation does not seem to be affected by the presence of the siRNA directed against GPR40. Oleate being a fatty acid, it can enter cells freely by crossing the plasma membrane and part of its effects on proliferation could be attributed to its metabolism. A GPR40 agonist, GW9508, is able to activate GPR40 without entering the cells nor activating oleate’s metabolism. GW9508 stimulates Akt and ERK1/2 phosphorylation in breast cancer cells MDA-MB-231 and prostate cancer cells DU145, but does not induce cell proliferation as does oleate. These results help us to understand the action mechanism of oleate in breast and prostate cancer cells. Oleate induces GPR40 signalization which is involved in the rapid Akt and ERK1/2 signaling pathways activation. On the other hand, oleate-induced cell proliferation is carried out by a GPR40-independent mechanism, possibly linked to oleate’s metabolism.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Linhagens secretoras de insulina, como a BRIN-BD11, são utilizadas para o estudo do mecanismo de secreção de insulina estimulado pela glicose (GSIS). Essas células expressam o GPR40, que quando ativado por ácidos graxos (AG), potencializam a GSIS. Considerando que as espécies reativas de oxigênio (EROs) podem estar relacionadas com a secreção de insulina mediada por AG, este estudo tem como foco avaliar se há envolvimento do GPR40 na GSIS e na produção de EROs, via NADPH oxidase (NADPHox), em células BRIN-DB11. A ativação do GPR40 foi feita com agonista GW9508 (GW). A secreção de insulina (SI), o conteúdo de superóxido (CS) e ativação da NADPHox foram analisados em resposta a glicose na presença ou ausência de GW (20mM). O envolvimento da NADPHox no CS induzido pelo GW foi analisado após inibição da NADPHox. O aumento da concentração de glicose paralela com aumento da SI, causa redução no CS. A presença do GW potencializa a SI em altas concentrações de glicose, aumenta o CS, e aumenta a translocação da subunidade p47phox do plasma para a membrana. Com a inibição da NADPHox o efeito do GW não ocorre. Os dados confirmam um efeito positivo do GPR40 na secreção de insulina estimulada pela glicose. O GW ativa a NADPHox resultando em um aumento do CS nas células BRIN-BD11. / BRIN-BD11, an Insulin secreting cell line, are used to study the mechanisms of glucose-stimulated insulin secretion (GSIS). These cells express GPR40, which is activated by fatty acids (FA) and potentiates GSIS. Considering that reactive oxygen species (ROS) is a signal that modulates insulin secretion (IS) induced by FA, this study aimed to investigate the involvement of GPR40 in ROS production, via NADPH oxidase (NADPHox), and in GSIS. To activate the GPR40 its agonist, GW9508 (GW), was used. IS and superoxide production (SP) were analyzed in response to increasing glucose concentrations (IGC), with or without GW (20mM). The activation of NADPHox was analyzed. The involvement of NADPHox on SP induced by GW was also evaluated using a small interference RNA or using the NADPHox inhibitor, VAS2870 (12mM). The presence of GW potentiated the IS at high glucose concentrations, together with an increased effect on SC. This increase was paralleled by an increase in the translocation of p47phox to the plasma membrane. Moreover, the inhibition of NADPHox abolished the GW9508 effect. These results show a positive effect of the GPR40 activation in GSIS. Additionally, GW9508 activates NADPHox resulting in an increase in the SC in BRIN-BD11 cells.

La relation entre l’obésité et le cancer, bien qu’établie par des études épidémiologiques, est peu connue. Pourtant, environ 25 % des cancers pourraient y être attribuables. Parmi les cancers reliés à l’obésité, les cancers du côlon, du sein chez les femmes ménopausées et de la prostate sont les plus fréquents. Des études sur modèles animaux ont suggéré une association positive entre une diète riche en gras et le développement du cancer mammaire et de la prostate. Nous avons étudié les mécanismes moléculaires par lesquels les acides gras influencent le devenir de lignées de cellules cancéreuses du sein et de la prostate. Ces travaux ont montré que les acides gras insaturés, dont l’oléate, induisent la prolifération cellulaire tandis que les acides gras saturés, dont le palmitate, diminuent la prolifération. Un traitement à l’oléate stimule la formation de gouttelettes lipidiques dans le cytoplasme des cellules de cancer du sein MDA-MB-231 et de la prostate DU145 alors qu’un traitement au palmitate entraîne l’apoptose. Le mécanisme d’action de l’oléate sur la prolifération a été étudié de façon plus approfondie. L’utilisation d’inhibiteurs pharmacologiques nous a permis de déterminer que l’effet prolifératif de l’oléate implique la voie PI3K/Akt, la voie ERK1/2 et l’activation d’un ou de plusieurs récepteur(s) couplé(s) aux protéines G (GPCR). L’oléate induit la phosphorylation rapide des protéines Akt et ERK1/2 dans les cellules de cancer du sein MDA-MB-231 et de la prostate DU145. Au cours des dernières années, deux GPCRs ont été identifiés comme étant activables par des acides gras à moyennes et à longues chaînes, GPR40 et GPR120. GPR40 étant exprimé dans plusieurs lignées cellulaires de cancer du sein et de la prostate contrairement à l’expression de GPR120 qui était inexistante dans la plupart des lignées, nous avons étudié l’implication de GPR40 dans l’effet prolifératif de l’oléate. Ces deux récepteurs n’étant pas exprimés dans les cellules épithéliales mammaires humaines en culture primaire, ces cellules ne répondent pas aux effets de l’oléate sur la prolifération et l’activation des voies de signalisation. L’activation des voies Akt et ERK1/2 par l’oléate dans les cellules MDA-MB-231 et DU145 est potentialisée par la surexpression du récepteur GPR40 et inhibée par l’utilisation d’un siRNA dirigé contre ce récepteur. Cependant, la prolifération induite par l’oléate ne semble pas affectée par la présence d’un siRNA dirigé contre GPR40. L’oléate étant un acide gras, il est capable d’entrer librement dans les cellules et une partie de ses effets sur la prolifération pourrait être attribuée à sa métabolisation. Un agoniste de GPR40, le GW9508, est en mesure d’activer GPR40 sans toutefois entrer dans les cellules ni activer le métabolisme de l’oléate. Le GW9508 stimule la phosphorylation des protéines Akt et ERK1/2 dans les cellules du cancer du sein MDA-MB-231 et de la prostate DU145, mais il n’est pas en mesure d’induire la prolifération cellulaire comme le fait l’oléate. Ces résultats nous permettent de mieux comprendre le mécanisme d’action de l’oléate sur les cellules de cancer du sein et de la prostate. L’oléate induit la signalisation de GPR40 qui est impliquée dans l’activation rapide des voies de signalisation Akt et ERK1/2. De son côté, l’effet prolifératif induit par l’oléate s’effectue par un mécanisme GPR40-indépendant, possiblement lié au métabolisme de l’oléate. / The relationship between obesity and cancer, although established by epidemiological studies, remains relatively unknown. However, about 25 % of cancers could be attributed to obesity. Among cancers that are affected by obesity, colon cancer, post-menopausal breast cancer and prostate cancer are the more frequent. Studies on animal models have suggested a positive association between high fat diets and de development of mammary and prostate cancer. We have studied the molecular mechanisms by which fatty acids influence breast and prostate cancer cells fate. This work has shown that unsaturated fatty acids, including oleate, can induce cellular proliferation while saturated fatty acids, including palmitate, reduce proliferation. An oleate treatment stimulates lipid droplets formation in the cytoplasm of breast cancer cells MDA-MB-231 and prostate cancer cells DU145 while a palmitate treatment induces apoptosis. The action mechanism of oleate on proliferation was studied more closely. Using pharmacological inhibitors, we determine that oleate-induced cell proliferation involves PI3K/Akt signaling pathway, ERK1/2 signaling pathway and the activation of one or many G protein coupled receptor(s) (GPCR). Oleate induces rapid Akt and ERK1/2 phosphorylation in breast cancer cells MDA-MB-231 and prostate cancer cells DU145. In the last few years, two GPCRs were identified as being activated by medium and long chain fatty acids, GPR40 and GPR120. GPR40 being expressed in many breast and prostate cancer cell lines while GPR120 expression was null in most cell lines tested, we studied the role of GPR40 in oleate-induced proliferation. Human epithelial mammary cells in primary culture did not express GPR40 nor GPR120 and failed to respond to oleate-induced cell proliferation or activation of signaling pathways. Akt and ERK1/2 signaling pathways activation by oleate in MDA-MB-231 and DU145 cells is potentiated by GPR40 over-expression and inhibited by the use of an siRNA directed against that receptor. However, oleate-induced cell proliferation does not seem to be affected by the presence of the siRNA directed against GPR40. Oleate being a fatty acid, it can enter cells freely by crossing the plasma membrane and part of its effects on proliferation could be attributed to its metabolism. A GPR40 agonist, GW9508, is able to activate GPR40 without entering the cells nor activating oleate’s metabolism. GW9508 stimulates Akt and ERK1/2 phosphorylation in breast cancer cells MDA-MB-231 and prostate cancer cells DU145, but does not induce cell proliferation as does oleate. These results help us to understand the action mechanism of oleate in breast and prostate cancer cells. Oleate induces GPR40 signalization which is involved in the rapid Akt and ERK1/2 signaling pathways activation. On the other hand, oleate-induced cell proliferation is carried out by a GPR40-independent mechanism, possibly linked to oleate’s metabolism.

Linhagens secretoras de insulina, como a BRIN-BD11, são utilizadas para o estudo do mecanismo de secreção de insulina estimulado pela glicose (GSIS). Essas células expressam o GPR40, que quando ativado por ácidos graxos (AG), potencializam a GSIS. Considerando que as espécies reativas de oxigênio (EROs) podem estar relacionadas com a secreção de insulina mediada por AG, este estudo tem como foco avaliar se há envolvimento do GPR40 na GSIS e na produção de EROs, via NADPH oxidase (NADPHox), em células BRIN-DB11. A ativação do GPR40 foi feita com agonista GW9508 (GW). A secreção de insulina (SI), o conteúdo de superóxido (CS) e ativação da NADPHox foram analisados em resposta a glicose na presença ou ausência de GW (20mM). O envolvimento da NADPHox no CS induzido pelo GW foi analisado após inibição da NADPHox. O aumento da concentração de glicose paralela com aumento da SI, causa redução no CS. A presença do GW potencializa a SI em altas concentrações de glicose, aumenta o CS, e aumenta a translocação da subunidade p47phox do plasma para a membrana. Com a inibição da NADPHox o efeito do GW não ocorre. Os dados confirmam um efeito positivo do GPR40 na secreção de insulina estimulada pela glicose. O GW ativa a NADPHox resultando em um aumento do CS nas células BRIN-BD11. / BRIN-BD11, an Insulin secreting cell line, are used to study the mechanisms of glucose-stimulated insulin secretion (GSIS). These cells express GPR40, which is activated by fatty acids (FA) and potentiates GSIS. Considering that reactive oxygen species (ROS) is a signal that modulates insulin secretion (IS) induced by FA, this study aimed to investigate the involvement of GPR40 in ROS production, via NADPH oxidase (NADPHox), and in GSIS. To activate the GPR40 its agonist, GW9508 (GW), was used. IS and superoxide production (SP) were analyzed in response to increasing glucose concentrations (IGC), with or without GW (20mM). The activation of NADPHox was analyzed. The involvement of NADPHox on SP induced by GW was also evaluated using a small interference RNA or using the NADPHox inhibitor, VAS2870 (12mM). The presence of GW potentiated the IS at high glucose concentrations, together with an increased effect on SC. This increase was paralleled by an increase in the translocation of p47phox to the plasma membrane. Moreover, the inhibition of NADPHox abolished the GW9508 effect. These results show a positive effect of the GPR40 activation in GSIS. Additionally, GW9508 activates NADPHox resulting in an increase in the SC in BRIN-BD11 cells.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22360号 / 医博第4601号 / 新制||医||1043(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 岩田 想, 教授 松原 和夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM