Dissertationen zum Thema „Insulin metabolism“

Dissertation (Ph.D.)--University of Toledo, 2009.<br>"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 57-61, p. 20-145.

Diabetes mellitus is not only a very serious disease, causing discomfort and pain to millions across the world, but with the aging of the population and the prevalence of a sedentary lifestyle, it is assuming the proportion of a real epidemic, becoming a public health and social emergency. In answer to this call, research on diabetes has been intensely carried out in the past decades and the knowledge and understanding of its etiology have been significantly improved. However, investigation is still ongoing, many important questions are still unanswered, and the causes eliciting the pathogenesis and progression of the diseases are not yet fully understood. During all these years of research, sophisticated tools have been developed to study the glucose-insulin metabolic system in vivo, and cope with the inaccessibility to direct measurement of some of the key phenomena underlying the glycemic control. Such tools, as complex test protocols and model-based approaches used to interpret the experimental data, have proven powerful weapons of investigation, but, considering the epidemic proportion of the disease, there is great demand for approaches that are less invasive, less expensive and therefore more suitable for large clinical studies. Mathematical and statistical techniques that collectively go under the name of “population approaches” have already been developed and are largely employed for pharmacokinetics and pharmacodynamics studies in drug development. However, in spite of their interesting potential, they have not found significant application yet in the context of metabolism research. Thus, investigation is required to probe the feasibility and relevance of such approaches in the study of diabetes. The research presented here addresses these issues, and is aimed at applying these sophisticated techniques to the modeling of glucose metabolism, first assessing the applicability of these approaches and tailoring them to the problem under investigation, then employing them to the analysis of data from population studies. First, a simulated but physiologically plausible dataset is created based on previous real data and employed as a benchmark to assess the applicability of population approaches to the Intra Venous Glucose Tolerance Test (IVGTT) minimal model of glucose disappearance. Various population algorithms have been proposed in the literature, therefore a thorough comparison of the available methodologies is performed, and a sparse data situation is replicated to test the robustness of these methods in such cases. The results select the First-Order Conditional Estimation as method of choice and show its robustness to poor sampling. Then, a larger real dataset is employed and analyzed with the same techniques, this time assessing the quality of the results with a Monte Carlo sampling approach to profile the likelihood function. Then the population model is optimized, to provide a base model for the following covariate analysis. In fact, at the time of the experiments, demographic data about the subjects has been collected, and the purpose of the covariate analysis is to determine whether some of these variables are significantly correlated with the model parameters and can be successfully used to explain part of the differences among the subjects. After a first exploratory regression analysis, different models are tested, integrating the most significant covariates directly as predictors into the model. In agreement with previous findings in literature, basal insulinemia, age and visceral abdominal fat are shown to be good predictors of insulin sensitivity and their introduction in the model is able to account for about a third of the between-subject variability of the values of this parameter. The use of covariates enhances the explanatory power of the model and opens the way for devising new lighter experimental protocols. One of the main benefits of the population approaches, in fact, consists in their ability to borrow information across the population and use it to improve the individual parameter estimates. As a result, the experimental protocols can be less demanding, both in invasiveness and economic cost, allowing in this way a broader use in large clinical studies. The results presented here, in fact, show that population approaches are very robust and able to cope with sparse data situations. In addition, the use of covariates in the model enhances even further the power of such techniques and makes them very appealing approaches to the study of glucose-insulin metabolism. In addition, a population approach is proposed to solve the problem of the estimation of the Disposition Index (DI) of glucose tolerance in a population. Since both insulin sensitivity and beta-cell response must be taken into account to assess the actual efficiency of the glucose disposal system, the DI was proposed to condense the information conveyed by both these parameters in a single value. Traditionally, approaches based on a geometrical fit are used to determine the value of DI in a population of subjects characterized by the same degree of glucose tolerance. However, all these methods rely on the assumption that all the subjects in the population share exactly the same value of DI and are therefore not able to account for the population variability, which is inevitably inherent to biological data. In this work, a NonLinear Mixed-Effects Approach is proposed to analyze the distribution of the insulin sensitivity and beta-cell response indices across a population, and then obtaining the information on the DI from the population features thus estimates. Comparisons on simulated datasets between the newly proposed method and its competitors prove that a proper model of the variability structure is essential to avoid severe bias in the estimates.<br>Il diabete mellito è non solo una patologia molto seria, che causa disagi e sofferenze a milioni di persone nel mondo, ma, anche a causa dell’affermarsi di uno stile di vita sedentario e dell’invecchiamento della popolazione, negli ultimi decenni ha raggiunto proporzioni epidemiche, diventando una vera e propria emergenza sanitaria e sociale. Per fronteggiare questo problema, molte risorse sono state dedicate all’attività di ricerca scientifica, che ha permesso una più profonda conoscenza dell’eziologia del diabete. Tuttavia, il diabete è a tutt’oggi ancora inguaribile e molte questioni rimangono aperte, fra cui la completa comprensione dei fattori che causano e fanno progredire la malattia. Anni di ricerca hanno permesso di sviluppare molti sofisticati strumenti per studiare il sistema metabolico glucosio-insulina in vivo e poter così fronteggiare il problema dell’inaccessibilità diretta di alcuni dei fenomeni chiave che controllano la glicemia. Tali strumenti, fra cui protocolli di studio e approcci basati su modello usati per interpretare i dati sperimentali, si sono rivelati armi molto potenti nelle mani dei ricercatori, ma le proporzioni epidemiche della malattia e il parziale cambiamento delle strategie e obiettivi della ricerca hanno sollevato l’esigenza di poter disporre di metodologie meno invasive, più economiche, e quindi più adatte ad essere applicate ad estesi studi clinici. Alcuni strumenti matematici e statistici che sono collettivamente conosciuti con il nome di “approcci di popolazione” sono già stati sviluppati e vengono largamente impiegati in studi di farmacocinetica e farmacodinamica, per lo sviluppo di farmaci. Tali approcci si prefiggono come obiettivo primario di stimare la distribuzione dei parametri di un modello all’interno di una popolazione e pertanto si avvalgono, per la stima individuale, delle informazioni disponibili sull’intero gruppo di soggetti. Sono particolarmente adatti a situazioni in cui il campionamento intensivo in un singolo soggetto non è possibile, e quando l’interesse del ricercatore è focalizzato sulla variabilità inter-individuale. Tuttavia, nonostante le loro interessanti potenzialità, gli approcci di popolazione non sono ancora apprezzati all’interno dell’ambiente di ricerca sulle malattie metaboliche, e la loro applicazione in tali studi è stata molto limitata. Pertanto è necessaria dell’attività di ricerca per saggiare l’effettiva fattibilità e rilevanza dell’utilizzo di tali approcci nello studio del diabete. La ricerca qui presentata risponde a queste esigenze, proponendosi come obiettivo l’applicazione di queste sofisticate tecniche ai modelli di metabolismo del glucosio, prima testandone la fattibilità e adattandole al problema in esame, e poi impiegandole nell’analisi di dati raccolti in studi di popolazione. Poiché in letteratura sono stati proposti molti diversi algoritmi, come primo passo, un dataset simulato è stato utilizzato per effettuare un confronto delle metodologie quando applicate al modello minimo del glucosio per il Test di Tolleranza IntraVenosa al Glucosio (IVGTT). First-Order Conditional Estimation (FOCE) si è rivelato come l’algoritmo più soddisfacente, in quanto ha fornito i risultati più accurati e robusti in caso di scarsità o rumorosità dei campioni. Successivamente, per validare i risultati trovati su dati reali, l’analisi è stata ripetuta su un dataset più esteso, relativo a 204 soggetti sani testati con IVGTT. Per poter saggiare la bontà delle soluzioni fornite dai vari algoritmi, è stato impiegato un sistema di stima della likelihood function basato su campionamento Monte Carlo. Questa analisi, non solo ha permesso di confermare la scelta di FOCE come metodo preferenziale, ma si è anche rivelata come un potente strumento per valutare la precisione delle stime dei parametri di popolazione. Successivamente, è stato messo a punto e ottimizzato un modello di popolazione, conservando nella matrice di covarianza solo i termini di correlazione fra i parametri SI-P2 e SG-VOL. Questo modello è servito come base per la successiva integrazione di covariate nel modello. Al momento dell’esecuzione degli esperimenti, infatti, sono stati raccolti alcuni dati sui pazienti, fra cui altezza, peso, sesso, età, glicemia e insulinemia basali, informazioni sul grasso corporeo. È stata effettuata una analisi per determinare quali fra queste variabili potessero essere usate per spiegare parte della variabilità nei valori dei parametri del modello minimo fra i diversi soggetti. Il risultato è un modello che integra queste informazioni direttamente nelle sue equazioni, mentre i coefficienti di regressione per ognuno dei predittori diventano veri e propri parametri del modello e il loro valore viene ottimizzato insieme agli altri parametri di popolazione. L’analisi effettuata ha trovato come buoni predittori per SI e P2 l’ età, l’insulinemia basale e il grasso addominale, che in ambo i parametri riescono a spiegare una buona fetta della variabilità inter-individuale. Sia l’impiego di metodologie di popolazione, sia l’introduzione delle covariate nel modello, permettono di aumentarne il potere predittivo, e sono in grado di usare informazioni indipendenti dai soli dati sperimentali. Questo permette di mettere a punto dei protocolli di studio meno invasivi, meno costosi, e pertanto più adatti ad un impiego su larga scala: ulteriore ricerca potrebbe avere come obiettivo l’ottimizzazione di una sampling schedule ridotta, che si avvantaggi dell’utilizzo degli approcci di popolazione. Ad ogni modo, il dataset utilizzato in questa analisi comprende solo soggetti sani, ed è quindi caratterizzato da una quantità limitata di variabilità di popolazione. Pertanto, sarebbe necessario ripetere l’analisi su altri dataset, per poter confermare questi risultati, in particolare sulle covariate. Inoltre, in una sezione successiva, un metodo di popolazione è stato applicato anche ad un altro problema diverso, la stima del Disposition Index (DI) del glucosio. Questo è un indice calcolato combinando sensitività e responsività all’insulina, che serve per testare l’effettiva efficacia del sistema di controllo della glicemia. Ci sono due versioni proposte per la formula, una semplificata, che consiste semplicemente nel prodotto (da cui il nome di Legge Iperbolica), e una con un parametro aggiuntivo ad esponente della sensitività all’insulina. Per poter calcolare il DI medio in una popolazione, e per poter saggiare quale delle due formule sia effettivamente più adatta, in letteratura si trovano alcuni approcci basati su un fit geometrico. Tuttavia, alcune approssimazioni sono utilizzate per semplificare il fit, e sono molte le questioni metodologiche spesso sottovalutate. Pertanto viene presentato qui un nuovo metodo Total Least Squares (TLS) che affronta il problema senza l’impiego di approssimazioni. Grazie ad alcune simulazioni, si è effettuato un paragone fra i vari metodi disponibili, e il nuovo algoritmo è risultato migliore rispetto ai predecessori. Tuttavia, tutti gli algoritmi basati su fit si fondano sull’ipotesi che i soggetti appartenenti alla popolazione abbiano lo stesso valore di DI, e l’unica fonte di incertezza nei dati sia dovuta alla stima degli indici di secrezione e sensitività. Questa ipotesi sembra una forte semplificazione e, in effetti, l’analisi di un dataset reale sembra confermare la presenza di variabilità di popolazione nei valori del DI. Ulteriori simulazioni hanno confermato che tutti metodi basati su fit, TLS compreso, falliscono quando la variabilità di popolazione è presente. Pertanto, è stato ideato un altro metodo basato su approcci di popolazione e, in particolare, su NonLinear Mixed-Effects Models (NLMEM), che è in grado di separare la variabilità nei dati, poiché fondato su ipotesi meno restrittive. Tale algoritmo stima i parametri della distribuzione di probabilità congiunta degli indici di secrezione e sensitività, e poi estrae le informazioni sul DI dalla matrice di covarianza. NLMEM si è rivelato equivalente a TLS quando non c’è variabilità di popolazione, ma di gran lunga più affidabile quando le ipotesi per il fit geometrico non sono rispettate, pertanto si è deciso di utilizzarlo sul dataset reale per testare la validità della legge iperbolica. Anche se una validazione su altri dataset è auspicabile per validare i risultati qui presentati, il modello con il parametro aggiuntivo sembra spiegare i dati in maniera più soddisfacente, e il valore del parametro sembra dipendere dalla coppia di parametri usata per la definizione del DI, più che dalla popolazione in esame (anziani piuttosto che giovani). Inoltre, nello studio qui proposto, il punto di partenza sono stati i valori degli indici di secrezione già calcolati, insieme con la loro precisione, grazie ad un metodo tradizionale; un approccio ancora più potente consisterebbe nell’utilizzare un modello di popolazione per stimare contemporaneamente sia gli indici di secrezione che sensitività, sia i parametri della loro distribuzione di popolazione, da cui ricavare le informazioni sul DI. Riassumendo, in questo lavoro si sono messi in luce i vantaggi dell’applicazione di approcci di popolazione nello studio nel diabete. Le potenzialità sono molte, dal miglioramento delle stime dei parametri individuali grazie all’uso dei prior di popolazione o di covariate e la relativa possibilità di mettere a punto protocolli di studio più leggeri, fino all’analisi di situazioni in cui la struttura gerarchica della variabilità è un aspetto cruciale.

Molts estudis previs sobre el paper de les cinases c-JUN N-terminal (JNK) no tenien en compte les diferències existents en l’activitat de cadascuna de les isoformes. Això va provocar que les propostes terapèutiques que regulaven les JNK de manera inespecífica es trobessin amb problemes importants. L’objectiu de la present tesi doctoral era ampliar els coneixements que es tenen actualment sobre el paper individual de les isoformes de les JNK i, avaluar qualsevol interès terapèutic que pugui derivar de la seva modulació per a l’epilèpsia del lòbul temporal i afectacions cognitives derivades del metabolisme. Estudis previs van demostrar que la inactivació genètica de la JNK1 tenia efectes neuroprotectors davant el dany citotòxic derivat de l’administració d’àcid kainic, un model d’epilèpsia del lòbul temporal. Per tant, es va posar a prova el potencial terapèutic de la Licochalcona A (LIC- A), un inhibidor de la JNK1. Els resultats van confirmar que quan els animals eren pretractats amb LIC-A, aquests quedaven protegits dels efectes de l’àcid kainic, tal i com ho demostrava l’absència de cèl·lules en degeneració ni de teixit escleròtic, així com una menor resposta neuroinflamatòria en astròcits i micròglia. A més a més, es van estudiar les conseqüències metabòliques d’una alimentació crònica amb una dieta rica en greixos (HFD). Els resultats demostraven que la dieta provocava l’aparició de resistència la insulina a escala central i perifèrica com a resultat d’estrès en les mitocòndries i el reticle endoplasmàtic, desregulacions de l’autofàgia, entre altres. Al final, això portava a l’aparició d’afectacions cognitives. Paral·lelament, es van avaluar els efectes de la inactivació genètica de la JNK2 i, es va determinar que afavoria l’aparició d’aquestes mateixes alteracions, especialment quan es combinava amb HFD. Per contra, la inactivació de JNK1 protegia de les conseqüències metabòliques d’una ingesta crònica de HFD, afavorint una major sensibilitat a la insulina, un menor pes corporal i una activitat mitocondrial més eficient. A més a més, aquests animals estaven protegits davant l’aparició de dèficits cognitius derivats d’alteracions metabòliques.<br>Many past reports on the c-JUN N-terminal Kinases (JNKs) did not take into account the existing differences in the activity of each of the isoforms. And so, therapeutic proposals that regulated the JNKs unspecifically encountered setbacks of import. The aim of the present thesis was to contribute to current understanding of the role of individual JNK isoforms in the development of pathology and, to appraise any therapeutic interest derived of their modulation for temporal lobe epilepsy and the metabolic- cognitive syndrome. Reported results demonstrated that the knock-out JNK1 had neuroprotective effects against excitotoxic damage derived of the administration of kainic acid, a model of temporal lobe epilepsy. Thus, Licochalcone A (LIC-A), a JNK1 inhibitor, was tested for its potential as a therapeutic agent. Results confirmed that when animals were pre-treated with LIC-A they were protected from the effects of kainic acid, as demonstrated by the absence of degenerating cells and sclerotic tissue, as well as lower neuroinflammatory responses in astrocytes and microglia. Additionally, the metabolic consequences of a chronic feeding of a fat- enriched diet (High fat diet; HFD) were also assessed. Data demonstrated that HFD caused the appearance of peripheral and central insulin resistance as a result of mitochondrial and endoplasmic stress, dysregulation of autophagy and other alterations. In the end, it led to the appearance of cognitive impairments. Parallelly, the effects of the ablation of JNK2 were evaluated and, it was determined that it favoured the appearance of these same alterations, especially when combined with HFD. On the contrary, the knockout of JNK1 protected against the metabolic consequences of a chronic feeding with HFD, showing improved sensibility to insulin, reduced body weight and more efficient mitochondrial activity. Moreover, these animals were protected against the appearance of metabolic-derived cognitive dysfunctions.<br>Muchos estudios previos sobre el papel de las quinasas c-JUN N- terminal (JNK) no tenían en cuenta las diferencias existentes en la actividad de cada una de las isoformas. Esto provocó que las propuestas terapéuticas que regulaban las JNK de forma inespecífica se encontraran con problemas importantes. El objetivo de la presente tesis doctoral era ampliar los conocimientos que se tienen actualmente sobre el papel individual de las isoformas de las JNK y, evaluar cualquier interés terapéutico que pueda derivar de su modulación para la epilepsia del lóbulo temporal y afectaciones cognitivas derivadas del metabolismo. Estudios previos demostraron que la inactivación genética de la JNK1 tenía efectos neuroprotectores ante el daño citotóxico derivado de la administración de ácido kaínico, un modelo de epilepsia del lóbulo temporal. Por tanto, se puso a prueba el potencial terapéutico de la Licochalcona A (LIC-A), un inhibidor de la JNK1. Los resultados confirmaron que cuando los animales eran pretratados con LIC-A, estos quedaban protegidos de los efectos del ácido kaínico, tal y como lo demostraba la ausencia de células en degeneración ni de tejido esclerótico, así como una menor respuesta neuroinflamatoria en astrocitos y microglía. Además, se estudiaron las consecuencias metabólicas de una alimentación crónica con una dieta rica en grasas (HFD). Los resultados demostraban que la dieta provocaba la aparición de resistencia a la insulina a escala central y periférica como resultado de estrés en las mitocondrias y el retículo endoplasmático, desregulaciones de la autofagia, entre otros. Al final, esto llevaba a la aparición de afectaciones cognitivas. Paralelamente, se evaluaron los efectos de la inactivación genética de la JNK2 y, se determinó que favorecía la aparición de estas mismas alteraciones, especialmente cuando se combinaba con HFD. Por lo contrario, la inactivación de JNK1 protegía ante las consecuencias metabólicas de una ingesta crónica de HFD, favoreciendo una mayor sensibilidad a la insulina, un menor peso corporal y una actividad mitocondrial más eficiente. Además, estos animales quedaban protegidos ante la aparición de déficits cognitivos derivados de alteraciones metabólicas.

Thesis (Ph.D.)--Medical University of Ohio, 2004.<br>"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Sonia M. Najjar. Includes abstract. Document formatted into pages: v, 217 p. Title from title page of PDF document. Bibliography: pages 158-216.

Background/Aims: In order to mediate a diverse range of growth and metabolic functions, insulin receptor substrate (IRS) proteins recruit a complex network of intracellular signalling molecules including PI3-kinase and the Grb2 adaptor protein. These studies proposed to answer the question to what extent are the different phenotypes observed in Irs1 and Irs2 knockout mice due to the loss of the whole protein or due to the loss of specific signalling interactions between IRS proteins and either p85 (the regulatory subunit of PI3K) or Grb2? Determining which interactions mediate which biological responses is of interest to our understanding of insulin resistance and its associated pathologies. Methods: The strategy utilised for these studies was to mutagenise the IRS proteins in vivo with specific signalling defects with the aim of disrupting IRS1 or IRS2 signalling via two major insulin mediated pathways, the PI3K-Akt axis and the Grb2-Ras-Raf-MAPK cascade. The phenotypes of the mice were assessed in five main areas: growth, glucose homeostasis, energy homeostasis, cognitive behaviour and fertility. Results: The IRS1-PI3K mutants presented with a severe growth-defect and acute insulin resistance, though through compensatory β-cell expansion maintained normal glycaemia. They were also lean with low circulating leptin levels and displayed a severe defect in learning and memory. IRS2-PI3K mutants were glucose intolerant and insulin resistant with a reduction in β-cell area that resulted in hyperglycaemia and onset of diabetes around 3-months of age. Additionally, the mice had increased fat mass and high circulating leptin levels. In contrast, the Grb2 mutants displayed normal metabolic phenotypes. Conclusion: The data presented here revealed a primary role for IRS signalling via PI3K in regulating metabolic functions. Both the IRS-PI3K mutants largely phenocopy the corresponding Irs knockout mice. In contrast, the Grb2 mutants appeared metabolically normal, suggesting a relatively minor role for IRS-Grb2 interactions in metabolic and growth control.

Protein phosphorylation dynamically integrates environmental and intracellular information to control virtually all biological processes. Challenges in determining the upstream regulation and downstream effects of phosphorylation obscures our functional understanding of cellular communication. In this thesis, I developed approaches to contextualise and prioritise phosphorylation events, focusing on the dynamic regulation of skeletal muscle metabolism by insulin and exercise. I measured phosphoproteomes of an in vitro panel of exercise-like stimuli to dissect upstream influences on exercise signalling. My integrative analysis with acute exercise phosphoproteomes revealed that emergent outcomes like protein secretion require concurrent kinase activation by distinct stimuli. To address the challenge of prioritising phosphosites with downstream functions most crucial to a biological phenomenon, I developed personalised phosphoproteomics. This experimental and computational framework identifies functionally enriched signalling by utilising human biological variance. To employ this method, I performed two independent phosphoproteomics studies investigating how exercise potentiates insulin sensitivity and how insulin resistance impacts metabolism in human skeletal muscle. My approach identified signalling on both known and previously unidentified sites on proteins intimately involved in glucose metabolism. This included a new co-operative relationship between mTOR and AMPK, for which I found a role in metabolic regulation. Collectively, these studies delineated skeletal muscle signalling responses and established personalised phosphoproteomics as a general approach to investigate the signal transduction underlying complex biology.

Thesis (Ph.D.)--Medical College of Ohio, 2004.<br>In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences. Major advisor: Sonia Najjar. Includes abstract. Document formatted into pages: v, 167 p. Bibliography: pages 117-165.

Insulin plays a vital role in whole-body metabolism and provides a major anabolic stimulus for cellular signaling pathways, including those involved in the metabolism of glucose and protein. Consequently, insulin dysregulation (ID) is known to alter molecular signal transduction in insulin-sensitive tissues such as skeletal muscle, thereby disrupting glucose metabolism and compromising protein synthetic capacity. Our first objective was to induce ID in healthy horses by administering dexamethasone (DEX), a potent glucocorticoid, for 21 days. We evaluated the effects on insulin-stimulated muscle protein signaling components involved in the mammalian target of rapamycin (mTOR) pathway. DEX-induced ID reduced insulin-stimulated activation of downstream (rpS6, 4EBP-1) mTOR signaling and increased atrogin-1 abundance, a marker for protein breakdown (P < 0.05). Additionally, 21 days of DEX elevated plasma amino acids levels in insulin-stimulated conditions, indicative of reduced uptake or increase release into circulation (P < 0.05). The second objective was to evaluate the short-term effects of DEX treatment in healthy horses. Plasma insulin, glucose and amino acid dynamics and activation of mTOR signaling pathways following an oral sugar test (OST) or intake of a high protein meal were evaluated before and after 7 days of DEX treatment, and after 7 days of no treatment. Seven days of DEX treatment increased basal levels of glucose, insulin and several amino acids (P < 0.05). Additionally horses treated with DEX had an exacerbated insulin response to the OST and consumption of the high protein meal in comparison to control horses (P < 0.05). The majority of blood metabolites returned to basal levels after 7 days of recovery from DEX treatment, indicating these effects were transient. Short-term DEX treatment decreased overall activation of mTOR and FoxO3 but increased total FoxO3 and IRS-1 abundance (P < 0.05). Postprandial activation of rpS6 was greater in horses treated with DEX for 7 days but was lower in those horses after 7 days of recovery from treatment (P < 0.05). Postprandial activation of ULK and AMPK tended to be greater in DEX treated horses (P < 0.1). Akt phosphorylation and mysotatin abundance were lower after the OST in DEX treated horses (P < 0.05). The final objective was to evaluate whether similar changes in postprandial metabolic responses would be seen in horses with naturally occurring ID. Plasma insulin, glucose and amino acid responses following ingestion of a high protein meal were determined in mature horses with equine metabolic syndrome (EMS). Horses with EMS had higher basal plasma insulin concentrations but lower levels of aspartate, glutamate, asparagine and plasma urea nitrogen in comparison to healthy controls (P < 0.05). Consumption of a high protein meal resulted in a 9-fold greater insulin response and higher postprandial levels of various amino acids (P < 0.05). Together this research indicates that ID affects whole body protein metabolism by altering cellular signaling pathways in healthy and diseased horses.

Orientador: Elinton Adami Chaim<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas<br>Made available in DSpace on 2018-08-23T03:30:35Z (GMT). No. of bitstreams: 1 Cazzo_Everton_M.pdf: 2625294 bytes, checksum: 2f57e795cce0f0c4c5a74d56bad92609 (MD5) Previous issue date: 2013<br>Resumo: A Síndrome Metabólica é um conjunto de fatores interconectados que elevam diretamente o risco de doenças cardiovasculares e diabetes mellitus tipo II. Estes fatores são alterações no metabolismo glicídico, elevação da pressão arterial, níveis elevados de triglicerídeos e reduzidos de lipoproteína de alta densidade, associados à obesidade, especialmente sua forma central ou abdominal. Apresenta prevalência crescente nas últimas décadas, levando a importantes consequências socioeconômicas. Sua fisiopatologia é complexa e ainda não foi totalmente esclarecida, porém sabe-se que possui como elemento principal a resistência insulínica. Em decorrência, observa-se a ocorrência de hiperinsulinemia, disfunção endotelial e inflamação. Seu tratamento clínico consiste em mudanças de estilo de vida associadas à terapia farmacológica concomitante de seus fatores individuais. Com a realização cada vez mais frequente de procedimentos cirúrgicos bariátricos, tem-se observado relevantes resultados advindos de tais operações no controle da síndrome. O objetivo deste estudo é avaliar o impacto do bypass gastrojejunal em Y de Roux sobre a síndrome metabólica, seus componentes individuais e as alterações bioquímicas observadas após a cirurgia. Foi realizada uma coorte histórica envolvendo 96 indivíduos com obesidade grau II/III previamente portadores de síndrome metabólica que foram submetidos à cirurgia há pelo menos 12 meses, avaliando os parâmetros clínicos e bioquímicos dos mesmos antes e após a cirurgia. Observaram-se índices relevantes de resolução pós-operatória da síndrome metabólica (88,5%), diabetes mellitus tipo II (90,6%), hipertensão arterial (85,6%) e dislipidemias (54,2%). A resolução da síndrome metabólica esteve estatisticamente associada aos seguintes fatores: controle glicêmico pós-operatório; glicemia, trigliceridemia e hemoglobinemia glicada pós-operatórias; homeostasis model assessment (HOMA) pós-operatório; número de classes anti-hipertensivas pré e pós-operatórias; percentual de perda de peso. O número de medicações anti-hipertensivas reduziu-se inclusive em indivíduos que não obtiveram resolução completa da hipertensão. Houve redução significativa nos níveis séricos de glicose, insulina, hemoglobina glicada, colesterol total, triglicerídeos e lipoproteína de baixa densidade, e elevação da lipoproteína de alta densidade. Observou-se decréscimo importante da resistência insulínica avaliada através do HOMA. Os índices de resolução de síndrome metabólica e comorbidades relacionadas encontradas neste estudo foram consistentes com achados prévios na literatura. A melhora no perfil glicêmico, insulínico e lipídico foi comparável à descrita em outros estudos, assim como a significativa redução da resistência insulínica avaliada pelo HOMA. Dentro do grupo analisado, o bypass gastrojejunal em Y de Roux apresentou resultados benéficos significativos, constituindo, portanto, uma importante ferramenta no tratamento da síndrome metabólica, propiciando elevados índices de resolução da mesma e de seus componentes individuais, bem como melhora geral do perfil bioquímico relativo à patologia<br>Abstract: Metabolic syndrome is defined as a set of interconnected factors that increase risk for cardiovascular disease and type II diabetes. These factors are changes on glycemic metabolism, high blood pressure, high serum triglyceride levels and low high density lipoprotein serum levels, associated with obesity, specially the central or abdominal type. Its prevalence has raised on later decades leading to important socioeconomical consequences. It has a complex pathophysiology that has not been completely understood yet but it is known that its main element is insulin resistance. As consequence it is observed hyperinsulinemia, endothelial disfunction and inflammation. Clinical management is based on lifestyle changes associated to drug treatment of its individual components. As surgical treatment of obesity has become more frequent lately it has been seen great results on metabolic syndrome control after bariatric procedures. This study aims to determine the impact of Roux-en-Y gastric bypass on metabolic syndrome and its individual components as well as biochemical changes upon glycemic metabolism observed after surgery. A historic cohort was carried out evaluating 96 subjects with grade II/III obesity previously diagnosed with metabolic syndrome who underwent surgery and whose postoperative follow-up was at least 12 months. Clinical and biochemical parameters were analyzed before and after surgery. This study has shown high resolution rates of metabolic syndrome (88,5%), diabetes (90,6%), hypertension (85,6%) and dyslipidemias (54,2%). Metabolic syndrome resolution was statistically linked to these factors: postoperative glycemic control; postoperative glycemia, hemoglobin A1c and triglyceridemia; postoperative homoestasis model assessment (HOMA); pre and postoperative number of anti-hypertensive classes; percent weight loss. Number of anti-hypertensive classes decreased even in subjects who do not achieve complete hypertension resolution. There was significant decrease on serum levels of glucose, insulin, hemoglobin A1c, total cholesterol, triglycerides and low density lipoprotein, and increase on high density lipoprotein. It was observed relevant decrease on insulin resistance assessed through HOMA. Resolution rates for metabolic syndrome and related comorbidities found on this study were comparable to previous findings on medical literature. Improvement on glycemic, insulinic and lipid profiles was also similar to previous findings, as well as significant decrease on insulin resistance as evaluated through HOMA. Within this group of subjects Roux-en-Y gastric bypass has led to significant positive results proving to be an important therapeutical tool on metabolic syndrome management since it can bring high resolution rates of the syndrome and its individual components as well as general improvement on the biochemical profile related to this pathology<br>Mestrado<br>Fisiopatologia Cirúrgica<br>Mestre em Ciências

Orientador: Patricia de Oliveira Prada<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Aplicadas<br>Made available in DSpace on 2018-08-25T21:25:58Z (GMT). No. of bitstreams: 1 Reis_SabrinaKaren_M.pdf: 2044889 bytes, checksum: 02eeb1437ecda9df9b2500ae61a282e8 (MD5) Previous issue date: 2014<br>Resumo: Obesidade está associada à inflamação crônica de baixo grau e ao desenvolvimento da resistência à insulina. A suplementação com L-glutamina tem sido utilizada para reduzir a inflamação em pacientes críticos. No entanto, os efeitos da suplementação deste aminoácido em doenças inflamatórias de baixo grau, como a obesidade, ainda não foram investigados. Por tanto o objetivo do presente estudo foi investigar se a suplementação oral com L-glutamina altera a massa corporal, circunferência da cintura, sensibilidade à insulina e os níveis séricos de hormônios relacionados ao metabolismo energético em indivíduos com sobrepeso e obesidade. Foi realizado um estudo clínico randomizado, simples cego com 67 voluntários adultos do Hospital Estadual de Sumaré (HES), classificados com sobrepeso (IMC 25 a 29,9 kg/m²) ou obesidade (IMC ? 30). Os indivíduos foram suplementados com 30 g/dia de glutamina ou alanina durante 14 dias. Os níveis séricos de insulina, leptina, adiponectina e GLP-1 foram avaliados no período pré e pós-suplementação, pelo método de ELISA. A glicose sérica foi avaliada pelo método da glicose oxidase. O índice de HOMA foi calculado. A massa corporal, o índice de massa corporal (IMC), a circunferência da cintura e o recordatório alimentar de 24 horas, também, foram avaliados pré e pós-suplementação. A suplementação oral com L-glutamina não alterou a massa corpórea e o IMC, no entanto reduziu a circunferência da cintura e os níveis séricos de leptina, sugerindo uma diminuição na massa adiposa. Não houve diferença significativa nos níveis séricos de glicose, adiponectina e GLP-1. Entretanto, houve uma redução nos níveis séricos de insulina e índice de HOMA, sugerindo uma redução na inflamação de baixo grau e uma melhora na sensibilidade à insulina. Os dados obtidos sugerem que a suplementação oral com L-glutamina em um curto período de tempo pode reduzir a massa adiposa de indivíduos com sobrepeso e obesidade. Essa redução refletiu nos níveis séricos de leptina e provavelmente nos níveis séricos de insulina, melhorando a sensibilidade à insulina. Por tanto, a suplementação com L-glutamina pode tornar-se uma abordagem terapêutica interessante para os indivíduos com sobrepeso e obesidade<br>Abstract: Obesity is associated with low-grade inflammation and insulin resistance. Glutamine supplementation has been used to reduce inflammation in critically ill patients. However, the effects of glutamine supplementation were not yet investigated in diseases with lowgrade inflammation such as obesity. Previous data showed that glutamine supplementation reduced inflammation, adipose mass and improved insulin sensitivity of obese rats. Thus, the aim of this study was to investigate whether oral glutamine supplementation alters body weight (BW), waist circumference (WC) and hormones levels and insulin sensitivity in overweight and obese humans. A randomized clinical trial, single blind with 67 adult volunteers from the State Hospital of Sumaré (HES), classified as being overweight (IMC 25 a 29,9 kg/m²) or obese (IMC ? 30) was performed. The subjects were supplemented with 30 g / day glutamine or alanine for 14 days. Serum levels of insulin, leptin, adiponectin and GLP-1 were evaluated before and after supplementation, by ELISA. Serum glucose was measured by glucose oxidase method. The HOMA index was calculated. The body weight, body mass index (BMI), waist circumference and 24-hour food record were also evaluated before and after supplementation. Glutamine supplementation did not change BW and BMI, however, reduced WC and serum leptin levels, suggesting a decrease in fat mass. Glutamine supplementation l did not alter serum adiponectin, GLP-1 and glucose levels. However, was reduced insulin levels and HOMA index, suggesting a reduction in low-grade inflammation associated with an improvement of insulin sensitivity. The data suggest that oral supplementation with L-glutamine in a short period of time can reduce fat mass in overweight and obese individuals. This reduction reflected in serum leptin levels and probably in serum insulin levels, improving insulin sensitivity. Thus, glutamine supplementation may become an interesting therapeutic approach for individuals with overweight and obesity<br>Mestrado<br>Nutrição<br>Mestra em Ciências da Nutrição e do Esporte e Metabolismo

O objetivo deste estudo foi determinar os efeitos da suplementação com maltodextrina, na substituição do amido na dieta de eqüinos avaliando a digestibilidade aparente total da matéria seca e nutriente e as diferenças nas respostas glicêmicas e insulinêmicas das dietas determinadas. Foram utilizados quatro eqüinos fêmeas com idade aproximada de 30 meses, peso médio de 400 kg, pertencentes ao rebanho da USP. Os animais foram imunizados contra tétano, vermifugados e pulverizados contra ectoparasitos. A dieta foi composta por 50% de concentrado e 50% de volumoso, formulada para atender as exigências de crescimento e mantença da categoria eqüina utilizada. As dietas continham 50% de amido no concentrado, substituídas em 33, 66% e 100% de maltodextrina. A colheita total de fezes foi feita em um período de 24 horas durante 03 (três) dias, com animais mantidos em baias, com piso de concreto, sem cama. No primeiro dia de colheita de fezes foram realizadas as coletas de sangue em tubos previamente preparados com estabilizantes, a partir da veia jugular, 30 minutos antes, 30 minutos, 90 minutos, 150 minutos, 210 minutos após o fornecimento do concentrado para dosagem de glicose e insulina. Não foi observado efeito linear ou quadrático para os coeficientes de digestibilidade aparente total da matéria seca e de nutriente, no entanto foi observado uma maior coeficiente de digestibilidade aparente total da ração com 100% de substituição do amido pela maltodextrina no concentrado, para a matéria seca, matéria orgânica, proteína bruta, fibra em detergente neutro e ácido. Em relação as respostas glicêmicas e insulinêmicas foi observado efeito quadrático para a glicose quando se analisou a área abaixo da curva desta variável. Da mesma forma foi observado um comportamento linear para a insulina. Quando se analisou a resposta das variáveis em função do tempo se observou um pico de glicose e insulina entre os tempo de 1,5 a 2 horas pós-alimentação, em relação a glicose a ração com 66% de substituição apresentou maior pico e a ração com 100% de substituição apresentou maior pico de insulina. O resultado do uso da maltodextrina sobre a fisiologia digestiva e o comportamento pós-brandial em eqüinos, permite sua inclusão na dieta dos animais.<br>The objective of this study was to determine the effects of supplementation with maltodextrin, the replacement of starch in the diet of horses by evaluating the apparent digestibility of dry matter and nutrient and differences in glycemic responses and insulinemic certain diets. There were four female horses aged approximately 30 months, average weight of 400 kg, belonging to the flock of USP. The animals were immunized against tetanus, wormed and pulverized against ectoparasites. The diet was composed by 50% concentrate and 50% forage diet formulated to meet the demands of growth and maintenance of category equine use. The diets contained 50% starch in the concentrate, replaced in 33, 66 and 100% of maltodextrin. The total collection of feces was made in a period of 24 hours over 03 (three) days, with animals kept in stalls with concrete floor, without bedding. On the first day of faeces were carried out blood samples in tubes previously prepared with stabilizers from the jugular vein 30 minutes before 30 minutes, 90 minutes, 150 minutes, 210 minutes after delivery of the concentrate to measure glucose and insulin. Were not observed effects linear or quadratic for the coefficients of apparent digestibility of dry matter and nutrient, however it was observed a higher coefficient of apparent digestibility of the ration with 100% replacement of starch by maltodextrin in concentrated, dry matter, organic matter, crude protein, neutral detergent fiber and acid. For the glycemic responses and insulin levels were observed quadratic effect for glucose when we analyzed the area under the curve of this variable. Similarly, it was observed a linear behavior for insulin. When analyzing their response as a function of time was observed a peak of glucose and insulin between the time of 1.5 to 2 hours post-feeding in relation to glucose diet with 66% replacement had a higher peak and cooperation with 100% replacement had a higher peak insulin. The results of maltodextrin use on digestive physiology and its post prandial behavior in equines allow its inclusion in animal diets.

Mammals and fish rely on hormones to regulate blood glucose levels. The two major glucose regulating hormones are insulin and glucagon. Literature on mammalian insulin and glucagon is quite extensive, however, there is limited information on how these hormones regulate blood glucose levels in fish. The material available for fish mostly pertains to changes in glucose concentration and gene expression of enzymes, but there is no information on the direct influence they have on glucose kinetics. Therefore, the main goal of my thesis is to measure the change in hepatic glucose production and glucose disposal when rainbow trout are administered insulin or glucagon. The beginning of my research focused on insulin. I hypothesized that rainbow trout respond to insulin by decreasing hepatic glucose production and increase glucose disposal, just like mammals. To test this, I infused insulin for 4 hours at 1.5 g insulin kg 1 min-1. I measured glucose disposal (Rd glucose), hepatic glucose production (Ra glucose), and blood glucose concentration. Following insulin administration the glucose fluxes decreased steadily (Rd glucose -37% and Ra glucose -43%). The decline in blood glucose levels follows the difference between Rd and Ra. These results explain why rainbow trout are unable to clear a glucose load to the same degree as mammals. The second major glucose hormone (glucagon) is what interested me for the second part of the research. The limited information on fish glucagon is even less than that of fish insulin. I speculated that trout respond to glucagon the same way mammals do (increase hepatic glucose production and show no affect on glucose disposal). To study the effects of glucagon on glucose fluxes, I tracked the changes in Ra and Rd glucose. The results showed glucose fluxes showed no siginificant difference from baseline in the first few hours, then steadily decreasing until the final time point reached values below baseline. Therefore, these experiments revealed that glucagon follows a similar pattern of effects in trout as mammals. However, the strength of the response to glucagon is different between trout and mammals. This thesis is the first to investigate the effects of insulin and glucagon on glucose kinetics in rainbow trout. I have concluded that rainbow trout have different responses to insulin and glucagon when compared to mammals. Furthermore, fish showing limited glucoregulatory capacity can be partially explained by their responses to insulin and glucagon.

Although feeding is important for optimal development and growth in insects, there are several points during the insect life cycle that are non-feeding: metamorphosis, pupation, and overwintering. Non-feeding periods also occur in response to internal cues, such as feedback from nutrient thresholds and immune responses being activated. Additionally, as an insect goes through different developmental stages, its nutritional requirements change in response to or in preparation for non-feeding periods. Most physiological responses like feeding are regulated through an interconnection of pathways, but how these networks change in response to different energy demands, such as immune challenges or changes in metabolism, is poorly understood. One significant pathway that is involved in regulating several physiological processes is the insulin signaling pathway. In my dissertation research, I tested hypotheses explaining the regulation of physiological processes during non-feeding periods in two agriculturally relevant insects, Manduca sexta and Megachile rotundata. First, I investigated how internal cues such as dietary lipid content and immune challenges cause non-feeding periods in M. sexta. Then, I investigated how insulin signaling regulates development during a non-feeding period like overwintering changes in M. rotundata. Since the insulin signaling (IIS) pathway is critical for development and growth, I focused on testing if this pathway plays a role in regulating non-feeding periods. My research showed that increased dietary lipid content causes a cessation of feeding, which suggests there is a possible lipid threshold that when reached, causes M. sexta to switch from lipid consumption for storage to lipid excretion. When looking at another cue like immune challenges, my results showed that during a bacterial infection, a Toll-mediated suppression of IIS pathway may be regulating feeding and causing a non-feeding period exhibited as sickness-induced anorexia. Lastly, my results also showed that the IIS pathway is suppressed in overwintering M. rotundata, and that this process can change in response to temperature. Overall, my dissertation research showed that the insulin signaling pathway and nutrient content play a vital role in regulating non-feeding periods. Investigating insulin signaling, lipid metabolism, and innate immunity in these species closes a gap in knowledge of invertebrate development.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária<br>A diabetes mellitus é um síndrome ou distúrbio na homeostase da glicose causada por uma deficiência de insulina ou por uma ação anormal desta no metabolismo de hidratos de carbono, proteínas e gordura. É a desordem endrocrino-metabólica mais comum na infância e adolescência, com consequências no desenvolvimento físico e emocional dos seus portadores. (Meyle et alii, 2001) O objetivo do presente trabalho é caracterizar o paciente diabético, reconhecendo as suas características particulares, limitações e possíveis complicações e relacioná-lo com o tratamento ortodôntico, referindo as alterações orais que um paciente diabético pode apresentar aquando do tratamento; para além disto, é também focada a importância da saúde periodontal para estes pacientes, pois esta, juntamente com o bom controlo metabólico da doença, permite a realização do tratamento ortodôntico em condições favoráveis. A realização desta Monografia insere-se no Mestrado Integrado em Medicina Dentária e tem como base um estudo de literatura científica nas áreas da Biologia, Anatomia, Medicina e Ortodontia, tendo sido utilizados diversos motores de busca para o efeito. Diabetes mellitus is a syndrome or disturb in the glucose homeostasis, caused by a insulin deficiency or by a abnormal action of this hormone in the carbohidrates, protein and fat metabolism. It’s the most common endocrine-metabolic disease in childhood and adolescence, which has several consequences in the physical and psychological development of those who have it. (Meyle et alii, 2001) The purpose of this work is to characterize the diabetic patient, recognizing its particular characteristics, limitations and possible complications, and relate it with and orthodontic treatment, presenting as well the oral alterations that this patients can present at the time of the orthodontic treatment; it is as well refered the importance of periodontal health to this patients because this, combined with a good metabolic control, allows the realization of the treatment in good conditions. This monography was undertaken within the Master in Dental Medicine and was based in a scientific literature study based os areas such as Biology, Anatomy, Medicine and Orthodontics.

Submitted by Biblioteca do Instituto Biomédico BIB (uffbib@gmail.com) on 2017-06-05T16:57:34Z No. of bitstreams: 1 Dissertação_mestrado-versão_final-_Jessika_Geisebel.pdf: 1194239 bytes, checksum: b59363ae595a444e379ef361c1b537c2 (MD5)<br>Made available in DSpace on 2017-06-05T16:57:34Z (GMT). No. of bitstreams: 1 Dissertação_mestrado-versão_final-_Jessika_Geisebel.pdf: 1194239 bytes, checksum: b59363ae595a444e379ef361c1b537c2 (MD5)<br>A programação metabólica ocorre quando insultos promovem alterações adaptativas nos períodos críticos do desenvolvimento e alteram as funções endócrino-metabólicas ao longa da vida. Estudos com alimentos conhecidos por trazerem benefícios à saúde além do valor nutritivo, mostraram que seu consumo por mães gestantes e/ou lactantes podem vir a ter desfechos negativos para a prole. Já está bem caracterizado que a ingestão de canela gera benefícios para homeostase energética. Entretanto, dados prévios obtidos pelo grupo mostraram que a suplementação com extrato aquoso de canela (400mg/kg peso corporal/dia) durante a lactação leva ao desenvolvimento de obesidade visceral, hiperleptinemia, hiperinsulinemia, associada a normoglicemia, na prole adulta aos 180 dias de vida. Com o objetivo de melhor compreender as alterações endócrino-metabólicas deste modelo, avaliamos a expressão proteica no fígado e músculo esquelético por Western blot, além de avaliarmos o conteúdo de triglicerídeo e glicogênio hepáticos através de ensaios colorimétricos. No fígado, vimos uma diminuição na fosforilação do IR, entretanto, observamos aumento na fosforilação do IRS1 e AKT. Quando avaliamos a sinalização de leptina, não observamos alteração na expressão do ObRB e SOCS3, no entanto, vimos um aumento na expressão da JAK2 e na fosforilação da STAT3, sugerindo que a ativação do IRS1/AKT possa decorrer da maior ativação da via da leptina. Apesar de não observarmos alteração da via gliconeogênica (PEPCK, G6Pase, GLUT2), vimos que a programação leva a um menor conteúdo de glicogênio hepático acompanhado por maior ativação da GSK3β. Observamos aumento do conteúdo de triglicerídeo hepático acompanhado por expressão normal do PPARα e uma interessante redução da expressão do SREBP1c. Concluímos que a programação por canela na lactação altera as principais ações da insulina no fígado, levando a uma menor síntese de glicogênio e acúmulo de gordura neste tecido, sem gerar alterações significativas no músculo esquelético.<br>Metabolic programming occurs when insults promotes adaptive changes in critic periods of development that alters the endocrine-metabolic functions in the offspring in medium and long terms. Studies with foods known to promote health benefits in addition to the nutritive value, shows that its consumption by pregnant and/or lactating females could induce negative outcomes to the offspring. It is well characterized that cinnamon intake promotes benefits to energy homeostasis. However, previous data obtained by our research group showed that supplementation with cinnamon water extract (400mg/kg body weight/day) during lactation leads to the development of visceral obesity, hyperleptinemia, hyperinsulinemia, associated to normoglycemia, in the adult offspring with 180 days old. To increase the knowledge about the endocrine-metabolic changes in this model, we evaluated the protein expression in liver and skeletal muscle by Western blot, and evaluated the hepatic content of triacylglycerol and glycogen using colorimetric assays. In liver, we observed a reduced IR phosphorylation; however, we observed increased phosphorylation of IRS1 and AKT. Concerning leptin signaling pathway, we did not observed changes in the ObRB and SOCS3 expression, but we observed an increased expression of JAK2 and phosphorylation of STAT3, suggesting that the activation of IRS1/AKT could be resulted by the increased activation of leptin signaling pathway. Although we observed no changes in the gluconeogenic pathway (PEPCK, G6Pase, GLUT2), we observed that the programmed group exhibit a lower hepatic glycogen content accompanied by increased activation of GSK3β. We observed increased hepatic triacylglycerol content accompanied by normal PPARα expression and an interesting reduction of SREBP1c expression. We conclude that the programming induced by cinnamon intake during lactation alters the main actions of insulin in liver; leading to lower glycogen synthesis and accumulation of fat in this tissue, without promote important changes in skeletal muscle.

In Drosophila, neuropeptides have regulatory roles in development, growth, metabolism and reproduction. This study focused on GABA and the neuropeptides Drosophila tachykinin (DTK), short neuropeptide F (sNPF), adipokinetic hormone (AKH), corazonin (CRZ) and Drosophila insulin-like peptides (DILPs) as possible regulators of metabolic stress responses and homeostasis. We showed that metabotropic GABAB receptors (GBRs) are expressed on brain insulin producing cells (IPCs), suggesting an inhibitory regulation of these cells by GABA. Knockdown of GBR on IPCs shortened lifespan and stress resistance, altered carbohydrate and lipid metabolism at stress (paper I). We showed that three different neuropeptides; DTK, sNPF and ITP, are co-expressed in five pairs of adult neurosecretory cells (paper II). ITP-knock down was not studied yet, but sNPF- and DTK-knock down flies showed decreased stress resistance at desiccation and starvation and decreased water levels at desiccation, suggesting that these peptides are involved in water homeostasis during stress conditions. sNPF was previously shown to affect feeding, growth and DILP expression via the IPCs, but it was not known which sNPF-expressing neurons are responsible for these actions. We could identify a specific set of bilateral neurons (DLPs) that co-express sNPF and corazonin that target the IPCs. We showed that these peptides co-released from DLPs regulate DILP transcription and probably release in the adult Drosophila brain and thus have roles in regulation of stress resistance and metabolism (paper III). AKH signaling was previously shown to affect hemolymph carbohydrate levels and lipid stores in Drosophila. Insulin (DILP) signaling and AKH signaling are suggested to have opposing effects on lipid and sugar metabolism in Drosophila. We studied the possible functional relationship between these two systems; do they mutually regulate each other?  Our results suggest action of DILPs via the Insulin Receptor on the IPCs and the AKH producing cells, but we could not provide evidence for AKH action on IPCs or AKH cells (paper IV).<br><p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Epub ahead of print. Paper 4: Manuscript.</p>

Inflammation plays a key role in the underlying pathogenesis of obesity and its associated health risks, with increased markers of inflammation evident in both liver and adipose tissue. In parallel, there is dysregulation of glucocorticoid metabolism in obesity, with increased adipose levels of the glucocorticoid-regenerating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) and increased hepatic levels of 5α-reductase type 1 (5αR1), which catalyses the reduction of glucocorticoids. Both the mechanisms and consequences of this glucocorticoid metabolism dysregulation remain unclear, however, there is evidence that it may be related to inflammation. In vitro studies have demonstrated that pro-inflammatory markers upregulate 11βHSD1 expression in adipocytes, potentially explaining increased expression of this enzyme in obesity. Previous work has also demonstrated that the glucocorticoid metabolites produced by 5αR1 lack the metabolic effects of the parent glucocorticoid, but retain its anti-inflammatory properties, indicating that increased expression of hepatic 5αR1 may serve to dampen down inflammation in the liver. The hypotheses addressed in this thesis are that in obesity, inflammation regulates adipose glucocorticoid metabolism through 11βHSD1, and that hepatic glucocorticoid metabolism regulates the inflammatory state of the liver through 5αR1. The role of inflammation in the regulation of 11βHSD1 was assessed in vivo in mice treated with the anti-inflammatory compound sodium salicylate (salicylate). In diet-induced obese mice, salicylate downregulated 11βHSD1 expression and activity selectively in visceral adipose tissue, alongside improved glucose tolerance, reduced plasma non-esterified fatty acids, and changes in adipose lipid metabolism. 11βHSD1-deficient mice fed a high-fat diet were resistant to the insulin sensitising effects of salicylate treatment. These results indicate a novel role for 11βHSD1 down-regulation in mediating the insulin sensitising effect of anti-inflammatory treatment. The mechanisms underpinning the anti-inflammatory properties of 5α-reduced glucocorticoids were explored in vitro and in vivo. In lipopolysaccharide-stimulated murine macrophages, both 5α-reduced glucocorticoid metabolites tested, namely 5α-dihydrocorticosterone (5αDHB) and 5α-tetrahydrocorticosterone (5αTHB), suppressed tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) release, although to a lesser extent than corticosterone (B). Similar to B, both 5αDHB and 5α THB suppressed phosphorylation of intra-cellular inflammatory signalling mitogen-activated protein kinases (MAPK) proteins c-Jun N-terminal kinase (JNK) and p38, as well as increasing protein expression of MAPK phosphatase-1 (MKP-1). Treatment of phorbol ester-stimulated HEK293 kidney cells with these 5α-metabolites revealed that 5αDHB suppressed nuclear factor κB (NFκB) and activator protein-1 (AP-1) activation to a similar extent to that of B, whilst 5αTHB increased activation of these pro-inflammatory transcription factors, indicating cell-specific effects of 5αTHB. In conclusion, reduced intra-adipose glucocorticoid regeneration by 11βHSD1 mediates the insulin sensitising effects of salicylate, suggesting that altered glucocorticoid metabolism may reflect altered intra-adipose inflammation in obesity. Furthermore, these data support the concept that this enzyme provides a therapeutic target in obesity-related metabolic disorders. 5α-reduced metabolites of glucocorticoids have similar anti-inflammatory properties to the parent glucocorticoid, indicating that the elevated hepatic levels of 5α-reductase in obesity may be a protective mechanism to limit the adverse metabolic effects of glucocorticoids upon the liver, but maintain the beneficial anti-inflammatory properties. These 5α-reduced glucocorticoid metabolites may provide a potential therapeutic treatment as selective glucocorticoid receptor modulators for inflammatory conditions.

A case control study comparing fasting lipid and apolipoprotein concentrations in NIDDM patients and normal control subjects matched for age, sex and body mass, reveals abnormalities of high density lipoproteins and apolipoprotein B containing lipoproteins despite hypoglycaemic therapy. HDL cholesterol concentration is reduced in these NIDDM patients, whilst apolipoprotein concentrations are similar to the controls. Therefore HDL particles may be cholesterol poor, possibly reflecting reduced reverse cholesterol transport. LDL cholesterol concentration may also be reduced in these patients whilst apolipoprotein B concentrations are similar to controls. Therefore cholesterol content of apo B containing lipoproteins may also be reduced in these patients, helped by their low fat high fibre diets and relatively good glycaemic control. The ratio of HDL cholesterol to non HDL cholesterol is reduced in the NIDDM patients suggesting continued atherosclerotic risk, but the apo AI:apo B ratio is normal. The relationship between these two risk ratios is altered in the NIDDM patients and they therefore require reassessment as risk indices in such patients. Simple assay systems reveal lipoprotein compositional abnormalities and may be useful in the management of NIDDM patients. Fasting lipoprotein abnormalities are also shown to be associated with macrovascular disease in treated NIDDM patients. An association is seen between the presence of macrovascular disease and increased serum triglyceride concentrations and a low apoB:non HDL cholesterol ratio suggesting relative cholesterol enrichment of apo B containing lipoproteins. Associations with macrovascular disease are also seen with a higher body mass, increased systolic blood pressure and smoking. In male NIDDM patients studied the presence of macrovascular disease is associated with a high HbA1 in addition to the above factors but not with BMI. In multivariate analysis in addition to the above factors a high non HDL cholesterol is associated with macrovascular disease in all patients or male patients alone, as is a low HDL cholesterol and high apo AI:HDL cholesterol ratio suggesting cholesterol poor HDL particles in male patients. Smoking habit and a high cholesterol content of apo B containing lipoproteins are independently associated with macrovascular disease in the whole group or in male patients alone. The precise lipoprotein subfraction abnormality underlying this abnormal ratio is unclear. Smoking combines with lipoprotein abnormalities to increase the risk of macrovascular disease in these patients. No other potential risk factors are associated with macrovascular disease in the patients studied. Lipoprotein concentration and composition changes postprandially. Following a normal mixed meal there is a similar rise in triglyceride rich lipoproteins and fall in LDL and HDL cholesterol content in treated NIDDM patients and normal subjects of similar age and body mass. The postprandial increase in HDL triglyceride seen in normal subjects is less apparent in NIDDM patients but their LDL and HDL are already slightly triglyceride rich in the fasted state. Thus the reductions in cholesterol in LDL and HDL in these patients enhances the triglyceride richness of their core, and thus may exacerbate the atherogenic nature of these particles. The rise in TRL is related to glycaemia. NIDDM patients with coronary artery disease have abnormal fasting lipoprotein concentration and composition but these abnormalities are accentuated in the postprandial state, when compared to similarly treated NIDDM patients with no detectable macrovascular disease. They have a greater postprandial increase in triglyceride rich lipoprotein triglyceride, and a greater reduction in HDL cholesterol despite similar fasting HDL cholesterol levels. HDL triglyceride is increased in the fasting and postprandial state in patients with coronary artery disease compared to those with no macrovascular disease. Despite differences in fasting LDL concentrations between these two groups postprandially changes in LDL are similar. Patients with coronary artery disease also have worse glycaemic control and higher insulinaemia. An association is therefore described between macrovascular disease and potentially atherogenic lipoprotein abnormalities both in the fasting state and postprandially in NIDDM patients treated with diet alone or diet and sulphonylureas.

Orientador: Patrícia de Oliveira Prada<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas<br>Made available in DSpace on 2018-08-19T14:40:09Z (GMT). No. of bitstreams: 1 Penteado_Erica_M.pdf: 2591549 bytes, checksum: 18851851d9a1a75cb2bd1ef2c4f7cb86 (MD5) Previous issue date: 2011<br>Resumo: O topiramato (TPM) é utilizado atualmente no tratamento da epilepsia e da enxaqueca por ser antagonista do receptor AMPA/KA e por aumentar o receptor de GABA, desencadeando a estabilização dos canais de sódio e cálcio. O efeito colateral mais conhecido dessa droga é a perda de peso, o aumento do gasto energético e da termogênese. Os hormônios anorexigênicos insulina e leptina regulam a atividade de populações distintas de neurônios que controlam o balanço energético via ativação da via IR/PI3K/Akt e OBR/JAK2/STAT3 respectivamente. Entretanto, desconhece-se se o efeito do TPM na perda de peso é decorrente de alterações da ação insulínica ou de leptina no hipotálamo. Assim, o objetivo do estudo é investigar se o tratamento com essa droga altera a sinalização da insulina e da leptina em hipotálamo de camundongos alimentados com dieta hiperlipídica (DH) ou ração padrão. Camundongos Swiss obesos ou com dieta padrão foram submetidos à cirurgia estereotáxica para inserção de cânula no ventrículo lateral. Após uma semana de recuperação, as cânulas foram testadas, e os animais foram tratados com 110mg/kg/dia de TPM, via gavagem, por sete dias, tendo sua ingestão alimentar e peso corpóreo monitorados diariamente. Observou-se que os animais em DH, tratados com TPM, possuem um menor ganho de peso e de ingestão alimentar do que seus controles, tratados com veículo. Não houve, porém, diferença quanto ao ganho de peso e ingestão alimentar dos animais tratados com a droga não obesos. Observou-se um aumento da termogênese nos animais obesos tratados com TPM pelo aumento da expressão da proteína UCP1, proteína desacopladora mitocondrial do tecido adiposo marrom, e também pelo aumento do consumo de O2 e produção de CO2, marcadores de termogênese. A nível molecular, os animais obesos apresentaram redução da fosforilação do receptor de insulina, IRS-1 e Akt induzidos por insulina e redução da fosforilação de OBR/JAK2/STAT3 induzida por leptina em hipotálamo. Entretanto, o tratamento com TPM reverteu este efeito, sugerindo que o tratamento com a droga induz uma melhora da ação/sinalização insulínica e de leptina no hipotálamo de camundongos obesos. Dessa maneira, sugere-se que o tratamento com TPM, pelo menos a curto prazo, melhora da ação e sinalização da insulina e leptina em hipotálamo, podendo ser um dos mecanismos pelos quais ocorre redução da ingestão alimentar e aumento do gasto energético nesses animais. Essa alteração de ingestão e de termogênese pode contribuir para a redução da adiposidade de camundongos obesos tratados com topiramato. Assim, essa droga parece ter um potencial terapêutico no tratamento da obesidade e da resistência à insulina<br>Abstract: Topiramate (TPM) is an anticonvulsant drug used for the treatment of epilepsy and prophylaxis of migraine. Weight loss is a frequently side effect reported in patients and animal models treated with TPM. In animal models topiramate may increase levels of hypothalamic corticotropic-releasing hormone (CRH), which is an anorexigenic neuropeptide and may decrease food intake. Some studies showed that topiramate may decrease energy storage, and thus increase energy expenditure and thermogenesis. However, the mechanisms by which TPM reduces body weight are not completely known so far. The hypothalamus is acknowledged as an important regulator of whole-body energy homeostasis by integrating nutrient and hormones signals from central and peripheral inputs. Anorexigenic hormones such as insulin and leptin regulate the activity of distinct neuron populations that control energy balance via IR/PI3K/Akt/Foxo1 pathway or OBR/JAK2/STAT3 pathway respectively. However, if topiramate alters hypothalamic insulin or leptin sensitivity is not known. Thus, in the present study, we asked whether topiramate treatment alters energy balance by altering insulin and leptin action/signaling in the hypothalamus from control and diet-induced obesity (DIO) mice. Our data provide evidence that short treatment with topiramate improves hypothalamic insulin and leptin signaling and action in obese mice. The improvement of hypothalamic insulin and leptin may reduce food ingestion and increase energy expenditure by increases in CRH and TRH mRNA expression. The lower food intake and higher energy expenditure induced by topiramate treatment may reduce obesity in mice on high fat feeding and may be an alternative therapy for obese treatment<br>Mestrado<br>Ciencias Basicas<br>Mestre em Clinica Medica

No músculo esquelético (ME) resistente à insulina, a disponibilidade elevada de ácidos graxos (AGs) livres observada na obesidade provoca alterações na função mitocondrial. Sendo os microRNAs (miRs) moléculas recentemente apontadas na regulação gênica de vias metabólicas, nosso objetivo foi investigar em ME de camundongos com resistência à insulina (RI) induzida por dieta hiperlipídica durante 8 semanas, tratados com fenofibrato (CF e HF) ou metilcelulose (veículo; grupos C e H) nas duas semanas antes do sacrifício, se os miRs-1a, 133a/b, 206, 208b, 499 e miR-223 participam da patogênese da RI. O quadro de RI foi induzido no grupo H e o fenofibrato reverteu parcialmente a RI (grupo HF) observada através das alterações em parâmetros metabólicos e enzimáticos, que parecem ser mediados pelo miR-1a regulando a proteína AMPK&#945;2. O aumento na transcrição de AMPK&#945;2 ativa processos catabólicos tais como a captação de glicose e oxidação de AGs, sendo considerada a principal enzima reguladora do metabolismo celular ao estimular a expressão de genes mitocondriais via PGC-1&#945;.<br>In skeletal muscle (SM) tissue, evidences suggest that the high availability of free fatty acids (FFAs) observed in obesity plays a central role in the development of insulin resistance (IR) by causing changes in mitochondrial function. Since microRNAs (miRs) are recently identified molecules acting as gene regulators of metabolic pathways, we aimed to investigate in SM of insulin resistant mice induced by 8 weeks of high-fat diet (HFD) feeding, treated with fenofibrate (CF and HF) or metilcelulose (vehicle, C and H) two weeks before euthanasia, if miRs-1a, 133a/b, 206, 208b, 499 and 223 are involved in IR pathogenesis. IR was induced after 8 weeks of HFD (H), and fenofibrate treatment (HF) partially reverted this condition by causing alterations on metabolic and enzymatic parameters, which seems to be mediated by miR-1a regulating AMPK&#945;2 protein. AMPK&#945;2 increased translation active catabolic processes such as glucose uptake and FFAs oxidation, being considered the main regulator of cell metabolism by stimulating mitochondrial genes expression via PGC-1&#945;.