Embora seja bem estabelecido que a exposição a baixas temperaturas resulte em ativação simpática e hiperglicemia, os mecanismos moleculares envolvidos na regulação neural da produção hepática de glicose ainda não são bem conhecidos. Portanto, o objetivo do presente estudo foi investigar o papel da inervação simpática na regulação de genes e enzimas chaves da neoglicogênese hepática, em roedores expostos agudamente ao frio. Para isso, foram utilizados dois modelos experimentais de desnervação simpática: a simpatectomia química em camundongos induzida pelo tratamento com 6-hidroxidopamina (100 mg.kg-1.dia-1; 1º, 2º e 7º dia de vida pós-natal; i.p). e a desnervação local dos nervos hepáticos por fenol (95%) em ratos. A exposição de camundongos ao frio (4ºC), durante 1, 3 e/ou 6h, induziu hiperglicemia, hipotermia, depleção do conteúdo de glicogênio hepático e ativação da neoglicogênese, estimada pela elevada expressão gênica e atividade das enzimas glicose-6-fosfatase e fosfoenolpiruvato carboxiquinase (PEPCK). Em paralelo, verificou-se que a exposição ao frio induziu um drástico aumento na expressão do RNAm do Nr4a1 e do PGC1-?, dois genes-alvos de CREB que participam do complexo de ativação transcricional dos genes que codificam as enzimas neoglicogênicas estudadas. A simpatectomia não afetou a depleção dos estoques de glicogênio hepático induzida pelo frio, mas reduziu o aumento do conteúdo hepático de noradrenalina e AMPc e acentuou a hipotermia, bem como preveniu a hiperglicemia, sendo este efeito associado ao bloqueio da expressão gênica e atividade da glicose-6-fosfatase e da PEPCK e do RNAm do Nr4a1, em todos os tempos estudados. Resultados bastante semelhantes foram observados em ratos submetidos à lesão dos nervos hepáticos com fenol e expostos ao frio durante 24h. Tanto a adrenalectomia como a adrenodemedulação (remoção da medula da adrenal) não alteraram o efeito estimulatório do frio na atividade e expressão gênica das enzimas neoglicogênicas de camundongos. Porém, em ratos expostos ao frio, a adrenodemedulação bloqueou aIII hiperglicemia e reduziu parcialmente a hiperexpressão dos genes da glicose-6-fosfatase e PEPCK. Estes resultados mostram a importância fisiológica da inervação simpática do fígado de roedores na estimulação do programa gênico da neoglicogênese, durante o estresse térmico agudo. / Although it is well established that exposure to low temperatures leads to sympathetic activation and hyperglycemia, the molecular mechanisms related to the neural regulation of hepatic glucose production remain elusive. Therefore, the goal of the present study was to investigate the role of sympathetic innervation in the regulation of genes and key enzymes of hepatic gluconeogenesis in liver of rodents exposed to acute cold. For this, two experimental models of sympathetic denervation were used: the chemical sympathectomy in mice induced by 6-hydroxydopamine treatment (100 mg.kg-1.day-1; 1º, 2º and 7ºdays of neonatal life; i.p.) and local denervation of hepatic nerves by phenol (95%) in rats. Exposure of mice to cold (4° C) for 1, 3 and / or 6 h induced hyperglycemia, hypothermia, depletion of hepatic glycogen content and activation of gluconeogenesis, estimated by high gene expression and activity of the enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase (PEPCK). In parallel, it was found that exposure to cold induced a dramatic increase in Nr4a1 and PGC1-? mRNA expression, two CREB target genes that participate in the transcriptional activation complex of the genes encoding the gluconeogenesis enzymes studied. The sympathectomy did not affect the depletion of cold-induced hepatic glycogen stores but reduced the hepatic content of noradrenaline and cAMP and increased hypothermia, as well as prevented hyperglycemia, and this effect was associated with the blockade of gene expression and enzymatic activity of glucose-6-phosphatase and PEPCK and Nr4a1 mRNA levels, at all time intervals investigated. Similar results were observed in rats submitted to hepatic nerve damage with phenol and exposed to cold for 24h. Either adrenalectomy or adrenodemedullation (surgical removal of the adrenal medulla) did not alter the stimulatory effect of cold on the activity and gene expression of the gluconeogenesis enzymes of mice. However, in cold-exposed rats, adrenodemedullation blocked hyperglycemia and partially reduced overexpression of the glucose-6-phosphatase and PEPCK mRNA.V These results show the physiological role of the hepatic sympathetic innervation in the transcriptional program associated with gluconeogenesis, during acute thermal stress.

Orientador: Gabriel Forato Anhê / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T03:29:53Z (GMT). No. of bitstreams: 1 Kinote_AndrezzaPinheiroBezerradeMenezes_D.pdf: 2061057 bytes, checksum: ec05cb4119a4301a63d35bb8a38a59cc (MD5) Previous issue date: 2014 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The complete abstract is available with the full electronic document / Doutorado / Farmacologia / Doutora em Farmacologia

Diabetes Mellitus (DM) is one of the most common metabolic disorders in the world with an estimated prevalence of over 415 million patients. Heart failure (HF) is the most common cardiovascular complication of diabetes. The prevalence of diabetes in patients with HF is reported at approximately 30%. However, the molecular mechanisms that contribute to the development of heart failure in diabetic patients remain uncertain. To study this, a genetic mouse model of diabetes (GENA348) with a point mutation in the glucokinase gene was used. Glucokinase is a glucose sensor that controls insulin release. This mutation in the glucokinase is similar to that found in Maturity Onset Diabetes of the Young Type 2 (MODY2) in humans. Our group has previously shown that GENA348 mice exhibit a diabetic phenotype. At 6 months, the mice developed a diabetic cardiomyopathy analogous to that seen in clinical practice with the development of cardiac hypertrophy and diastolic dysfunction, which progressed to dilatation of the left ventricle and systolic dysfunction at 12 months. The aim of the project was to examine the molecular and pathophysiological mechanisms that contribute to development of this cardiac phenotype in diabetic GENA348 mice in the setting of hypertension and at baseline. To study the mice under hypertensive stress conditions, 6 month old-GENA348 HO and WT mice were infused with angiotensin II (ANG II) via minipump. After ANG II treatment, HO and WT GENA348 mice showed a significantly greater increase in systolic and diastolic blood pressure compared to untreated controls. It was evident that ANG II treatment resulted in cardiac hypertrophy with the same level observed in both HO and WT mice. The diastolic function was generally preserved in the WT and HO mice following the ANG II treatment. Our data indicates that the HO mice have had a blunted hypertrophic response to the hypertension induced by ANG II. At baseline, two hypothesis-generating methods were used. Firstly, gas chromatography-mass spectrometry (GC-MS) and ultra performance liquid chromatography-mass spectrometry (UPLC-MS) were used on 12-month-old GENA348 mice heart and serum samples. Secondly, diabetes PCR array plates were used on 6- and 12-month-old GENA348 mice heart samples. For the GCMS and UPLC-MS, there were 43 differences in metabolites from tissue samples and 93 from serum samples. The main altered metabolites from tissue samples were sugars and fatty acids. However, fatty acids, phospholipids and sphingolipids were the main altered metabolites from serum samples. After the validation of the array plates the most apparent observation was that only two up-regulated genes, Phosphoenolpyruvate carboxykinase 1 (Pck1)and Glucose-6-Phosphatase, Catalytic Subunit (G6pc) showed a comparable pattern as the array results. Pck1 and G6pc are the main enzymes that play a key role in gluconeogenesis regulation. We also looked at the expression level of one of the main transcriptional regulators of gluconeogenesis, Forkhead boxprotein O1 (FoxO1). It was found that the expression was altered at 12 months. In conclusion, it was clear that hyperglycaemia altered gene expression and the metabolites profiles in 12 month old HO mice, with evident alterations detected in genes involved in the metabolic regulation of the heart. In addition, this study may provide preliminary insight into pathophysiological alterations in the cardiac metabolism that may contribute to the development of diabetic cardiomyopathy.

Trypanosoma brucei est un parasite protozoaire responsable de la trypanosomiase humaine africaine. Il présente un cycle de vie complexe alternant entre des hôtes mammifères et un vecteur insecte, la mouche tsé-tsé. Au cours de ce cycle, il rencontre des environnements radicalement distincts auxquels il s’adapte en régulant son métabolisme. Nous avons étudié le métabolisme intermédiaire et énergétique de la forme procyclique évoluant dans le tractus digestif de l’insecte vecteur. Dans cet environnement dépourvu de glucose, la néoglucogenèse est cruciale pour la croissance et la survie des parasites car elle permet la synthèse d’hexoses phosphates et en particulier du glucose 6-phosphate qui alimente plusieurs voies de biosynthèse essentielles. Nos travaux confirment ce flux néoglucogénique alimenté par la proline mais aussi par le glycérol. Nous montrons que le glycérol est une source de carbone efficacement métabolisée et préférentiellement utilisée par la forme procyclique à défaut de la proline et même du glucose pour alimenter son métabolisme intermédiaire. Cette situation qu in’a jamais été décrite auparavant met en évidence la répression du glycérol sur le métabolisme du glucose. Nous montrons également que l’enzyme fructose 1,6-biphosphatase(FBPase), spécifique de la néoglucogenèse, n’est pas essentielle à la survie du parasite en conditions dépourvues de glucose indiquant qu’il existe une alternative à cette enzyme.Toutefois, FBPase joue un rôle important dans la virulence de T. brucei dans l’insecte.De plus, nous avons mis en évidence une autre stratégie d’adaptation de T. brucei basée sur des réarrangements génomiques qui peuvent mener à la synthèse de gènes chimères. / Trypanosoma brucei is a protozoan parasite responsible for human African trypanosomiasis. His complex life cycle alternates between mammalian hosts and the insect vector, the tsetsefly. During this cycle, the parasite encounters dissimilar environments and adapts to the sechanging conditions by regulating his metabolism. We have studied intermediate and energetic metabolism of the procyclic form living in the midgut of the insect vector. In this glucose-depleted environment, gluconeogenesis is crucial for growth and viability of the parasites. Indeed, it allows the synthesis of hexoses phosphates and in particular glucose 6-phosphate which feeds several essential biosynthetic pathways. Our work has confirmed the existence of a gluconeogenic flux fed by proline and glycerol. We have shown that glycerol is an efficiently metabolized carbon source and is preferentially used by the procyclic form rather than proline or even glucose. This situation never described before highlights glycerol repression on glucose metabolism. We have also showed that the enzyme fructose 1,6-biphosphatase (FBPase), specific of the gluconeogenesis, is not essential for the viability ofthe parasite in glucose-depleted conditions, suggesting that there is an alternative to this enzyme. However, FBPase plays an important role for virulence of T. brucei in the insect. Moreover, we have showed another adaptation strategy developed by T. brucei which is basedo n genomic rearrangements leading to the synthesis of chimeric genes.

E2F1, un régulateur crucial du métabolisme dans les cellules normales et cancéreuses.Résumé: Le facteur de transcription E2F1 est largement décrit pour son implication dans le contrôle du cycle cellulaire. Notre laboratoire et d'autres ont montré qu'il jouait également un rôle majeur dans le contrôle de l'homéostasie du glucose et des lipides. Dans les travaux présentés dans cette thèse, nous avons démontré ici en utilisant les souris invalidées pour E2F1 (souris E2f1-/-), qu'il joue un rôle dans le mécanisme de sécrétion d'insuline, dans la lipogenèse et la gluconéogenèse hépatique et dans le contrôle du métabolisme oxydatif. Dans la cellules β pancréatique, E2F1 contrôle la sécrétion d'insuline via le contrôle de l'expression de Kir6.2. Nous avons également montré l'implication de E2F1 dans la régulation de l'expression de gènes oxydatifs dans le TAB et le muscle. De plus, l'étude du foie de ces souris a permis de montrer le rôle de E2F1 dans le contrôle de la lipogenèse et la néoglucogenèse. E2F1 semble en effet capable de réguler l'expression d'un gène clé de la lipogenèse, Fas et de la G6Pase, un gène impliqué dans la production hépatique de glucose, en coopérant avec le facteur de transcription foxo-1. Enfin, nous avons observé que la diminution de la néoglucogenèse en absence de E2F1 empêche la formation de métastases pulmonaires. Ces différents résultats démontrent que E2F1 est un régulateur clé du métabolisme, et que la modulation de son activité pourrait avoir des conséquences majeures dans le développement de maladies comme le diabète, l'obésité, les myopathies et le cancer.Mots clés: E2F1, sécrétion d'insuline, métabolisme oxydatif, lipogenèse, gluconéogenèse, cancer. / E2F1, a crucial regulator of metabolism in normal and cancer cells. Abstract: E2F1 is a key transcription factor involved in the control of the cell cycle. We and others have previously demonstrated a a major role for E2F1 in the control of glucose and lipid homeostasis. In this thesis, we showed bu using E2F1 null mice, that E2F1 plays a major role in the control of insulin secretion, oxidative metabolism, lipogenesis and gluconeogenesis. E2F1 controls insulin secretion through the modulation of Kir6.2 expression. Moreover, we demonstrated that E2F1 controls the expression of oxidative genes in BAT and muscle. In addition, we observed that E2F1 is involved in the control of lipogenesis and gluconeogenesis in the liver. E2F1regulates the expression of key lipogenic genes, such as Fas, and G6Pase, a gene involved in hepatic glucose production, through cooperation with foxo-1. Finally, we observed that the inhibition of gluconeogenesis upon E2f1 genetic ablation impaired the formation of lung metastases. These different results show that E2F1 is a key regulator of metabolism, and that modulating its activity could have High outcomes on diseases such as diabetes, obesity, muscular distrophies or cancers.Key words: E2F1, insulin secretion, oxidative metabolism, lipogenesis, gluocneogenesis, cancer.

Orientador: Gabriel Forato Anhê / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T19:10:52Z (GMT). No. of bitstreams: 1 Barbosa_AnaPauladeLima_D.pdf: 1915868 bytes, checksum: d146b07dfcd99b2676c519233cddf2f2 (MD5) Previous issue date: 2014 / Resumo: O Diabetes Mellitus tipo II (DMT2) é uma doença caracterizada pela diminuição da sensibilidade à insulina e consequente prejuízo na homeostasia glicêmica. Estudos recentes mostraram que a melatonina, um hormônio produzido pela glândula pineal, melhora a tolerância à glicose e a resistência à insulina, diminui a expressão de proteínas ligadas à gliconeogênese hepática (G6Pase e PEPCK) e reduz a apoptose de células beta pancreáticas in vivo. Entretanto, ainda não estava claro se a melatonina conseguia, de fato, reverter in situ as ações lipotóxicas dos ácidos graxos saturados em células beta pancreáticas e na musculatura esquelética, e se podia diminuir a expressão dos genes reguladores da gliconeogênese hepática. Para responder a esse questionamento, utilizamos culturas celulares de insulinoma (MIN6), de músculo esquelético (miotubos de L6) e de hepatoma (HepG2). Os resultados mostraram que a melatonina reverte a resistência à insulina gerada por palmitato em miotubos de L6, mas somente em passagens baixas, não diminui a apoptose em MIN6 e não altera a expressão do G6pc, o gene que codifica a G6Pase, em HepG2 / Abstract: Type II Diabetes Mellitus (T2DM) is characterized by decrease of insulin sensitivity, resulting impairment glucose homeostasis. Recent studies have shown that melatonin, a hormone produced by the pineal gland, improves glucose tolerance and insulin resistance, reduce the expression of hepatic gluconeogenesis proteins (G6Pase and PEPCK) and diminish pancreatic beta cell apoptosis in vivo. However, it was unclear if melatonin could reverse the lipotoxic actions of saturated fatty acid in pancreatic beta cells and skeletal muscle, and if melatonin could decrease the expression of regulatory genes of hepatic gluconeogenesis in situ. To answer this question, we used cell cultures of insulinoma (MIN6), skeletal muscle (L6 myotubes) and hepatoma (HepG2). The results showed that melatonin reverses palmitate insulin resistance in L6 myotubes, but only at low passages, melatonin does not decrease apoptosis in MIN6 and does not alter the expression of G6pc, the encoding gene of G6Pase, in culture cell of HepG2 / Doutorado / Farmacologia / Doutora em Farmacologia

A oligopeptidase neurolina (E.C.3.4.24.16; nln ) foi identificado pela primeira vez em membranas sinápticas de cérebro de ratos como sendo capaz de participar no metabolismo de peptídeos bioativos, como neurotensina e bradicinina. Recentemente, foi sugerido que a ausência de Nln pode melhorar a sensibilidade a insulina. Aqui, nós mostrado que camundongos knockout para Nln (KO) são mais tolrerantes à glicose, sensíveis à insulina e apresentam maior gliconeogênese. Os animais KO apresentou um aumento na expressão de mRNA de vários genes relacionados com a gliconeogênese no fígado. A semiquantificação de peptídeos intracelulares revelou um aumento em peptídeos intracelulares específicos no gastrocnêmio e tecido adiposo epididimal, que estão envolvidos com o aumento da tolerância a glicose e maior sensibilidade à insulina nos animais KO. Esses resultados sugerem fortemente a nova possibilidade de que Nln é uma enzima chave no metabolismo energético e pode ser um novo alvo terapêutico para melhorar a captação de glicose e sensibilidade a insulina. / The oligopeptidase neurolysin (EC 3.4.24.16; Nln) was first identified in rat brain synaptic membranes and shown to ubiquitously participate in the catabolism of bioactive peptides such as neurotensin and bradykinin. Recently, it was suggested that Nln reduction could improve insulin sensitivity. Here, we have shown that Nln knockout mice (KO) have increased glucose tolerance, insulin sensitivity and gluconeogenesis. KO mice have increased liver mRNA for several genes related to gluconeogenesis. Isotopic label semi-quantitative peptidomic analysis suggests increase in specific intracellular peptides in gastrocnemius and epididymal adipose tissue, which likely is involved with the increased glucose tolerance and insulin sensitivity in the KO mice. These results suggest the exciting new possibility that Nln is a key enzyme for energy metabolism and could be a novel therapeutic target to improve glucose uptake and insulin sensitivity.

Hypoxia is a significant low oxygen state that has complex and diverse impacts on organisms. In aerobes, various adaptive responses to hypoxia are observed that vary depending on the level of oxygen depletion and previous adaptation, hence the continued attention to hypoxia as an important abiotic stressor. Adaptive responses to hypoxia are primarily governed by the hypoxia-inducible factors (HIFs), which activate downstream genetic pathways responsible for oxygen transport and metabolic plasticity. In aquatic habitats, oxygen availability can vary greatly over time and space. Therefore, aquatic organisms’ adaptation to hypoxia is likely pervasive, especially in genotypes originating from waterbodies prone to hypoxia. Here we report the transcriptional response to severe hypoxia in the freshwater crustacean Daphnia magna. We observe improved survival in media containing elevated calcium ion (Ca2+) concentrations. Additionally, we observe changes in lactate and pyruvate concentrations within tissues. To elucidate the transcriptome basis of these effects, we examine transcripts with known gene ontologies indicating roles in Ca2+ homeostasis and signaling, and in pyruvate metabolism, including gluconeogenesis (GNG). We observe the up-regulation of numerous transcripts encoding GNG pathway enzymes, including the rate-limiting enzyme phosphoenolpyruvate carboxykinase (PEPCK-C) and fructose-1,6-bisphosphatase (FBP). In contrast, no transcripts involved in Ca2+ homeostasis or signaling showed any significant differential expression. Some GNG transcripts are more up-regulated in clones from permanent waterbodies not prone to hypoxia, inconsistent with the hypothesis about its protective effects. One exception is the FBP transcript, which has been identified to be up-regulated in some hypoxia-tolerant aquatic organisms.

京都大学 / 0048 / 新制・論文博士 / 博士(工学) / 乙第13016号 / 論工博第4141号 / 新制||工||1650(附属図書館) / 32944 / (主査)教授 跡見 晴幸, 教授 森 泰生, 教授 梅田 眞郷 / 学位規則第4条第2項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Rainbow trout are an important salmonid species whose poor utilization of dietary carbohydrates spurred research investigating molecular and physiological components of its glucoregulation. Among the environmental factors described to exert robust changes in glucose metabolism in rainbow trout, nutrition and social stress are among the most studied: Diets exceeding 20% of carbohydrates and chronic social stress induce hyperglycemia in adult and juvenile rainbow trout, respectively. Common to both responses is a contribution of hepatic de novo gluconeogenesis, which has been described to evade repression in response to high dietary carbohydrate content and to be stimulated in subordinate rainbow trout. Compared to previous studies investigating the regulation of hepatic gluconeogenesis at the molecular level, the recent publication of the annotated rainbow trout genome has opened novel possibilities to investigate paralogue-specific and posttranscriptional regulation of gluconeogenesis. In this thesis, I identify and describe the regulation of the novel phosphoenolpyruvate carboxykinase paralogue pck2b in rainbow trout and identify specific miRNA candidates predicted to contribute to gene paralogue-specific regulation of gluconeogenesis in nutritional and social contexts using small RNA next generation sequencing, real-time RT-PCR and in silico target prediction approaches. In nutritional and social status experiments, in silico predicted targets of differentially expressed hepatic miRNAs are enriched for gluconeogenesis regulation, suggesting a posttranscriptional component in regulating gluconeogenic transcript abundance. Differentially expressed hepatic miRNAs in both experiments comprise evolutionarily conserved and teleost-specific miRNAs, and are indicative of both environmental factor-specific and common regulation of gluconeogenesis transcripts in rainbow trout liver. Together this work provides novel comparative insight into hepatic miRNA-dependent glucoregulation and identifies several specific candidate miRNAs for future functional validation in hepatic glucoregulation in rainbow trout.