Obesity is widely considered a primary risk factor for type 2 diabetes (T2D). However, less is known about the early adaptive responses to short-term periods of high-fat energy excess (HFEE). Previous reports detailing whole-body adaptation to fat and energy oversupply are equivocal, perhaps, in part, owing to use of different experimental protocols, varying durations of dietary manipulation and participant cohorts with individuals of varying characteristics. In addition to use of different dietary protocols between studies, alterations in functional end-point measures due to the type of dietary fat consumed warrants consideration. Daily n-3 PUFA intake, commonly obtained from pelagic fish oil (FO) consumption, has been shown to positively associate with insulin sensitivity in epidemiological studies and thus may be a useful dietary strategy for slowing insulin resistance development. Chapter 2 of this thesis extends previous literature by demonstrating that 6 d HFEE (150 % habitual energy intake; 60 % of energy from fat) does not clearly alter whole- body insulin sensitivity, irrespective of FO consumption. However, investigation of metabolism at the tissue level, as presented in Chapter 3 of this thesis, offers insight into a potential tissue-specific level of regulation that precedes whole-body regulation. Skeletal muscle insulin signalling protein (e.g. protein kinase B (PKB)) activity, levels of certain ceramide species, and AMPK α2 activity were altered following HFEE and may explain the early maladaptive responses to short-term HFEE. Moreover, FO intake as 10 % of total fats mediated some of these molecular responses, including PKB and AMPK α2 activity, reflecting possible functional effects of FO at the subcellular level. Regulation of these metabolic / molecular responses at both the tissue and whole- body level can be explained, in part, by genetic predisposition, environmental influence and more recently epigenetics, including microRNAs (miRNAs). In Chapter 4, we characterised the plasma and skeletal muscle miRNA responses to HFEE and oral glucose ingestion. We demonstrate transient changes in levels of certain miRNAs following oral glucose ingestion in both tissue types and in response to HFEE in skeletal muscle. However, no significant correlations between basal plasma and skeletal muscle miRNA levels were observed, suggesting that our candidate plasma miRNAs may be co-ordinating functional changes in other tissue types. Plasma miR- 145-5p and skeletal muscle miR-204-5p predicted a significant proportion of the variance in mean whole-body insulin sensitivity change in response to HFEE. These data indicate that these miRNAs may be useful biomarkers of insulin resistance development following HFEE. A constraint of this thesis is that all conclusions are made within the context of statistically unaltered insulin sensitivity. Therefore, future investigations of diet-induced maladaptation should consider establishing a time course of insulin resistance development in response to HFEE, or use different study populations. Populations that are more susceptible to T2D development, e.g., overweight, sedentary individuals would be of particular interest. These data would aid development of a working model of diet-induced insulin resistance that has more direct application to T2D progression and extends the data presented herein.

Identifying the relationship between fat oxidation and insulin resistance (IR) may provide vital clues to the mechanisms behind the development of metabolic disease in prepubertal children. The purpose of this study was to examine the association of fat oxidation with insulin resistance (IR) and insulin sensitivity (SI) in prepubertal children. A total of 34 prepubertal 7-9 year olds (18 females, 16 males, 13 non-Caucasian, 21 Caucasian, 8.0±0.8 years, 36.5±12.1 kg) were observed. Subjects participated in indirect calorimetry to obtain respiratory quotient (RQ) and a blood test to obtain fasting insulin and glucose to calculate IR by homeostatic model assessment (HOMA). A subset (n=16) participated in Frequently Sampled Intravenous Glucose Tolerance Testing (FSIGTT) to obtain insulin sensitivity. Pearson correlations between RQ and IR and RQ and SI were performed. Partial correlations with respect to physical activity, breastfeeding, and birth weight were also performed. A general linear model was used to examine RQ with IR, and separately SI with respect to physical activity, breastfeeding, birth weight, race and sex. Respiratory quotient and IR were significantly associated when adjusted for physical activity, sex and race and breastfeeding, sex and race. In regards to birth weight, RQ and IR were significantly associated when adjusted for breastfeeding, birth weight, and race, but not when breastfeeding was removed from the model. The results of this study suggest lack of physical activity and breastfeeding may be the most influential risk for factors in the development of IR via a mechanism of impaired fat oxidation. Further research is needed to examine the role of physical activity, breastfeeding, and birth weight on fat oxidation and the development of insulin resistance in prepubertal children, however, the results of this study support the promotion of physical activity, breastfeeding, and good maternal nutrition.

A influência da obesidade e da resistência à insulina (induzidas em ratos por injeção de glutamato monossódico em neonatos) sobre o desenvolvimento tumoral (5x105 células do tumor de Walker-256) e os efeitos da metformina (300mg/kg, v.o., 15 dias) nessa condição foram investigados. Na 16ª semana de vida, inocularam-se as células e iniciou-se o tratamento. Após 15 dias de tratamento, caracterizou-se a obesidade e a analisou-se o crescimento tumoral. O desenvolvimento tumoral e a caquexia foram maiores nos obesos. A metformina reduziu o desenvolvimento do tumor, mas não a caquexia. Apesar da metformina não ter melhorado a sensibilidade à insulina, corrigiu a dislipidemia, reduziu a peroxidação lipídica e as gorduras periepididimal e retroperitoneal. Conclui-se que a obesidade aumenta o desenvolvimento tumoral e que a metformina é eficaz em diminui-lo. O mecanismo envolvido parece não depender da melhora da sensibilidade à insulina / The influence of obesity and insulin resistance (induced in rats by monosodium glutamate in neonates) on tumor development (5x105 Walker-256 tumor cells) and the effect of metformin (300mg/kg, by gavage, for 15 d) on it. On the 16th week, tumor cells were subcutaneously injected and the treatment started. On the 18th week, the obesity was characterized and the tumor was evaluated. The tumor development and the cachexia were higher in obese rats. The tumor development was reduced by metformin, but not cachexia. Although metformin did not improve insulin sensitivity it did correct the dislypidemia, reduced the periepididimal and retroperitoneal adipose tissues and lipid peroxidation. In conclusion obesity increases tumor development and metformin is able to reduce it. The reduction occurred independently of the correction of insulin resistance

O aumento da prevalência do diabete melito do tipo 2 (DM2) é intenso e implica ampla busca pela prevenção e tratamento da doença. Estudos têm mostrado a participação do zinco na síntese, secreção e via de sinalização da insulina e sobre o controle glicêmico. Este trabalho objetivou analisar o mecanismo de ação do zinco no controle da secreção de insulina e no controle glicêmico, de modo a entender se a suplementação com o zinco previne ou retarda a manifestação do DM2. O projeto foi aprovado pela CEUA-ICB (USP). Camundongos machos C57BL/6 foram divididos em 4 grupos experimentais: dieta controle (NFD); dieta controle suplementada com ZnCl2 (NFDZ); dieta hiperlipídica (HFD); e dieta hiperlipídica suplementada com ZnCl2 (HFDZ). A massa corporal, a ingestão de ração e água e a glicemia foram acompanhados semanalmente. Testes intraperitoneais de tolerância à glicose (ipGTT) e à insulina (ipITT) foram realizados na 14ª semana de tratamento. Completado as 15 semanas de tratamento a glicemia, a insulinemia e a zincemia foram analisadas, sendo aplicados os testes de HOMA-IR e HOMA-&#946;. Em ilhotas pancreáticas isoladas foi analisada a secreção estática de insulina em diferentes concentrações de glicose. O teste de captação e metabolismo de glicose foi feito no músculo sóleo e a análise do conteúdo das proteínas AKT e GSK3-&#946; foi feita no músculo sóleo e no fígado. Os dados (média±SEM) foram analisados por Two-way ANOVA com pós-teste Bonferoni ou por teste t de Student (P 0,05). A suplementação com zinco melhorou a disfunção glicêmica induzida por dieta hiperlipídica, sem no entanto afetar a resistência à insulina ou a secreção de insulina por ilhotas isoladas. / The increase in prevalence of type 2 diabetes mellitus (DM2) is intense and implies broad quest for prevention and treatment of disease. Studies have shown the participation of zinc in the synthesis, secretion and signaling pathway of insulin and the glucose control. This study aimed to analyze the mechanism of action of zinc in the control of insulin secretion and glucose control in order to understand whether supplementation with zinc prevents or delays the manifestation of DM2. The project was approved by CEUA-ICB (USP). Male mice C57BL/6 were divided en 4 groups: control diet (NFD); control diet supplemented with ZnCl2 (NFDZ); high fat diet (HFD); and high fat diet supplemented with ZnCl2 (HFDZ). Body weight, feed intake and water and the glucose levels were monitored weekly. Intraperitoneal glucose tolerance test (ipGTT) and insulin (ipITT) were performed at the 14th week of treatment. Completing the 15 weeks of the treatment glycemia, insulinemia and zincemia were analyzed, being applied HOMA-IR and HOMA-&#946; tests. In isolated islets was assessed the static insulin secretion at different glucose concentrations. The uptake and glucose metabolism test was done in the soleus muscle and the content analysis of the AKT and GSK3-&#946; protein was made in the soleus muscle and liver. The data (mean ± SEM) were analyzed by two-way ANOVA with Bonferoni post-test or Student\'s t test (P < 0,05). Zinc supplementation improves glucose dysfunction induced by high fat diet, without nonetheless affecting insulin resistance and insulin secretion by isolated islets.

Introdução: O exercício físico como parte do tratamento do diabetes mellitus gestacional (DMG) pode ajudar na manutenção das concentrações da glicemia de jejum. Objetivos: Conduzir uma revisão sistemática, com metanálise de ensaios clínicos randomizados, para avaliar o efeito do exercício supervisionado e estruturado ou o efeito do aconselhamento de atividade física, em mulheres com DMG, e comparar ao pré-natal usual para o controle da glicemia. Métodos: Os estudos elegíveis foram identificados a partir das bases de dados MEDLINE, EMBASE, Web of Science, Scopus e SportDiscus até 4 de Junho de 2015. Os dados foram extraídos de ensaios clínicos randomizados que compararam o pré-natal usual ao pré-natal usual somado ao exercício supervisionado e estruturado (pelo menos uma vez na semana) ou ao aconselhamento de atividade física, pelas quais os valores de glicemia de jejum pré e pós-intervenção estavam disponíveis. A metanálise de efeitos randômicos foi conduzida para a diferença entre as médias pós-intervenção da glicemia de jejum. Resultados: Foram encontradas 664 publicações, nas quais 82 foram avaliadas pela elegibilidade e oito foram incluídas na análise final. O efeito total do exercício nas concentrações absolutas da glicemia de jejum não foi significativamente diferente (P = 0,11) comparado ao pré-natal usual. Entretanto, o aconselhamento de atividade física comparado ao pré-natal usual demonstrou uma redução significativa nas concentrações da glicemia de jejum (diferença da média ponderada -3,88 mg/dL, 95% CI-7,33 a -0,42; I2, 48%; P para heterogeneidade < 0,15). Conclusão: O exercício supervisionado ou o aconselhamento de atividade física em mulheres com DMG não foi significativamente diferente comparado ao pré-natal usual nas concentrações de glicemia de jejum. Visto que o pré-natal usual inclui algum tipo de recomendação de atividade física, estes resultados não são surpreendentes. O aconselhamento de atividade física com o pré-natal usual inclui modificações da dieta que podem motivar as mulheres com DMG a serem mais ativas e aderentes ao aconselhamento nutricional, enquanto que o exercício estruturado pode ser mais difícil de atingir. / Background: Exercise as part of the treatment for gestational diabetes mellitus (GDM) may help maintain fasting glucose concentrations. Objective: A systematic review with meta-analysis was performed to evaluate the effect of weekly-supervised exercise or physical activity (PA) counseling in GDM women compared to standard care (SC) on glycemic control. Methods: Eligible trials were identified from MEDLINE, EMBASE, Web of Science, Scopus and SportDiscus up to 4 June 2015. Data were retrieved from randomized controlled trials comparing SC with SC plus weekly-supervised (at least once a week) prenatal exercise or PA counseling for which fasting blood glucose (FBG) values pre and post intervention were available. Random-effects meta-analysis was conducted for mean difference in FBG post exercise intervention. Results: Our search yielded 664 publications of which 82 were assessed for eligibility. Eight were analyzed and all were included in the meta-analysis. The overall effect of exercise on absolute FBG concentrations was not different (P=0.11) compared to SC. However, PA counseling versus SC showed a significant reduction in the absolute FBG concentrations (weighted mean difference -3.88 mg/dL, 95% CI-7.33 to -0.42; I2, 48%; P for heterogeneity<0.15). Conclusions: Supervised exercise or PA counseling in GDM women was not significantly different compared to SC on FBG concentrations. Since SC includes some type of PA recommendation, these results are not surprising. PA counseling with SC including dietary modifications may help motivate GDM women to be more active and adherent to nutrition advice, while structured exercise may be more difficult to achieve.

L’Insulin Degrading Enzyme (IDE) est une protéase dont les mécanismes de fonctionnements ne sont pas encore complètement élucidés.Dans ce but, nous avons identifié un homologue d’IDE chez la levure Schizosaccharomyces pombe (S. pombe) : iph (Insulinase Pombe Homologue), et mis en évidence un lien entre Iph et la voie TOR (Target of Rapamycin) lors d’un stressprotéotoxique.La voie TOR comme les voies de vieillissement et de réponse au stress sont régulées par la présence de nutriments dans le milieu. Dans cette optique, nous nous sommes intéressés chez S. Pombe à la durée de vie chronologique qui, dans une souche sauvage, augmente lors d’une restriction en glucose.Cependant, les levures invalidées pour iph perdent cette capacité, et présentent une DVC identique indépendamment de la concentration du glucose dans le milieu.L’ensemble de ces résultats a permis de démontrer que la protéine Iph régule négativement la voie TOR, qui elle même favorise la survie lors d’un stress protéotoxique et le vieillissement des cellules. / Insulin Degrading Enzyme (IDE) is a 110 kDa protease whose function is not completely elucidated.To this aim, we have identified a homologue of IDE in the yeast Schizosaccharomyces pombe (S. pombe) : iph (Insulinase Pombe Homologue), and we have pointed out a link between Iph and the TOR (Target of Rapamycin) pathway during proteotoxic stress.The TOR pathway- like the pathways of ageing and the stress response- are regulated by the presence of nutrients in the environment.Knowing this, we were interested in S. Pombe chronological life span that increases in the case of glucose restriction in the wildtype strain. However, the yeast cells deleted for iph loose this capacity and show a lifespan chronology that is identical independently of the glucose concentration in the environment.Taking together these results show that the protein Iph regulates negatively the TOR pathway, which by oneself favours the survival during proteotoxic stress and ageing of the cells.

廣泛的區域內和跨民族的轉錄變化反映了人類的適應和自然選擇。基因表達是轉化基因組信息為功能基因產品 - 蛋白質的主要機制。異常基因的表達和疾病的發病機制有關。基因組革命提供了獨特的機會為複雜的人類轉錄組進行全面的研究。轉錄分析需要複雜的生物信息學方法。在技術角度，一個實證模型用了哺乳動物基因組中內含子長度幾何尾分佈的定律準確地確定剪接交界處和非唯一映射讀取的位置。這種方法在處理非唯一映射讀取比BWA更好。這方法還比其他工具檢測出更多已經實驗證實的剪接交界處。核糖核酸測序首先用於北京漢人和西歐之間的表達表型與的轉錄變化的詳盡研究。民族的具體剪接交界處被發現。此外，民族的具體特點體現在相對異構體的豐度差。最後，這分子表型剪接頻譜的變化在不同種族之間的不同表明了另一個描繪種族多樣性的方法，核糖核酸測序還被用於探索的一種複雜的疾病：二型糖尿病的分子異常。二型糖尿病表現在廣泛不同的基因表達。（1）這研究證實先前公佈的全基因組關聯研究;（2）改善策劃不佳的位點和（3）發現新型2型糖尿病相關的基因。本研究通過整合各種改變的信號，並在一個高度可信的基因 - 基因相互作用網絡進行解釋，增強表達異常在2型糖尿病的認識。在更廣泛的69×79的情況下，對照組的結果進行了驗證。本研究增強表達異常在2型糖尿病的認識。 / Extensive intra- and inter- ethnic transcriptome variation reflects human adaptation and natural selection. Gene expression is the primary mechanism that translates genome information into functional gene product that lead to physiological phenotypes. Aberrant gene expression has been associated to the pathogenesis of diseases. The genome revolution has offered unique opportunity for a comprehensive interrogation of the complexity of human transcriptome. Analysis of transcriptome using RNA-Seq requires sophisticated bioinformatics approach. In a technical perspective, an empirical model based on the geometric-tail distribution of intron lengths in mammalian genome was developed to accurately determine splice junctions from junction reads and locations of non-uniquely mapped reads. Such method handles non-uniquely mapped reads better than BWA. The method can also detect more experimentally confirmed splice junction than other tools. Expressional phenotyping was employed to explore global transcriptomic variation between Beijing Han Chinese and Western European. In addition to inter-ethnic variations in gene expression, ethnic specific splice juctions were found. Further, ethnic specific trait manifests in differential relative isoform abundance. Lastly, such spectrum of variations was different between different ethnic groups, suggesting alternative splicing as another molecular phenotype that delineates ethnic diversity. Expressional phenotyping was then used in a case-control study to explore the molecular abnormalities of a complex disease: Type 2 Diabetes (T2DM). T2DM manifested in wide-spread repression of gene expression. The study (1) confirmed previously reported Genome-wide Association Study (GWAS) loci; (2) curated poorly characteriezed GWAS loci and (3) discovered novel T2DM associated genes. By integrating various alteration signals and interpretation performed in a highly confident gene-gene interaction network, this study augmented the understanding of expressed abnormalities in T2DM. The results were validated in a broader 69 x 79 case-control group. / Detailed summary in vernacular field only. / Li, Jing Woei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 118-130). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.v / 中文擇要 --- p.vi / Thesis/Assessment Committee --- p.ix / Acknowledgement --- p.ix / List of figures --- p.x / List of tables --- p.xii / List of Abbreviations --- p.xiii / Scientific contributions --- p.xv / List of Publication(s) related to this thesis --- p.xvi / Conference presentations --- p.xvii / Chapter Chapter 1: --- Introduction and Literature Reviews --- p.1 / Chapter 1.1 --- The variable human transcriptome --- p.1 / Chapter 1.2 --- Significance of variation in gene expression and transcript variants --- p.2 / Chapter 1.3 --- Transcriptomic study in a technological perspective --- p.8 / Chapter 1.3.1 --- Microarray: Probing what was designed to be probed --- p.8 / Chapter 1.3.2 --- RNA-Seq: the ab initio decoder of biological sequences --- p.9 / Chapter 1.4 --- Analysis of RNA-Seq data --- p.10 / Chapter 1.4.1 --- The bioinformatics challenges prevail --- p.10 / Chapter 1.4.2 --- Identifying changes in gene expression --- p.16 / Chapter 1.4.3 --- Identifying splice site, quantification of isoform level expression --- p.17 / Chapter 1.5 --- Conclusion --- p.19 / Chapter 1.6 --- Aims of this study --- p.20 / Chapter 1.6.1 --- Splice junction determination --- p.20 / Chapter 1.6.2 --- Expressional phenotyping in ethnical context --- p.20 / Chapter 1.6.3 --- Expressional phenotyping in a disease context --- p.20 / Chapter Chapter 2: --- Detection of splicing events --- p.21 / Chapter 2.1 --- Abstract --- p.21 / Chapter 2.2 --- Introduction --- p.22 / Chapter 2.3 --- Methods and workflow --- p.25 / Chapter 2.4 --- Algorithm --- p.29 / Chapter 2.5 --- Geometric-tail distribution --- p.32 / Chapter 2.6 --- Insert-size distribution --- p.33 / Chapter 2.7 --- Multiread analysis --- p.34 / Chapter 2.7.1 --- GT model probably places multiread more accurately than BWA --- p.35 / Chapter 2.8 --- Splice-site comparison --- p.37 / Chapter 2.8.1 --- GT model discovers more experimentally confirmed splice junction --- p.37 / Chapter 2.8.2 --- GT model is highly accurate --- p.39 / Chapter 2.9 --- Discussion --- p.40 / Chapter 2.10 --- Limitation --- p.40 / Chapter Chapter 3: --- Transcriptomic variation in a ethnicity context --- p.41 / Chapter 3.1 --- Abstract --- p.41 / Chapter 3.2 --- Introduction --- p.42 / Chapter 3.3 --- Materials and Methods --- p.46 / Chapter 3.3.1 --- HapMap lymphoblastoid cell-lines --- p.46 / Chapter 3.3.2 --- Sequenced samples --- p.48 / Chapter 3.3.3 --- Paired-end RNA-Seq, dataset and reads processing --- p.48 / Chapter 3.3.4 --- Genome reference and annotation --- p.49 / Chapter 3.3.5 --- Strategies for reads mapping --- p.49 / Chapter 3.3.6 --- Pathway and Gene Ontology analysis --- p.50 / Chapter 3.3.7 --- Differential gene expression analysis --- p.50 / Chapter 3.3.8 --- Ethnic specific splice junction --- p.51 / Chapter 3.3.9 --- Junction sites saturation analysis --- p.51 / Chapter 3.3.10 --- Ethnical novel transcribed regions --- p.52 / Chapter 3.3.11 --- Isoform dynamics and meta-analysis --- p.53 / Chapter 3.4 --- Result --- p.54 / Chapter 3.4.1 --- Paired-end RNA-Seq --- p.54 / Chapter 3.4.2 --- Differential gene expression and meta-analysis --- p.56 / Chapter 3.4.3 --- Ethnic specific splice junction is rare --- p.58 / Chapter 3.4.4 --- Saturation of discovery of highly confident annotated junctions --- p.59 / Chapter 3.4.5 --- Novel transcribed regions --- p.62 / Chapter 3.4.6 --- Isoform dynamics and meta-analysis --- p.63 / Chapter 3.5 --- Discussion --- p.66 / Chapter 3.6 --- Limitations --- p.67 / Chapter 3.6.1 --- HapMap LCLs may not reflect the entire spectrum of natural variation --- p.67 / Chapter 3.6.2 --- Sequencing depth and the usefulness of published dataset --- p.67 / Chapter 3.6.3 --- Knowledge gap in understanding of the human genome --- p.69 / Chapter Chapter 4: --- Transcriptomic investigation of complex disease: Type 2 Diabetes --- p.70 / Chapter 4.1 --- Abstract --- p.70 / Chapter 4.2 --- Introduction --- p.72 / Chapter 4.3 --- Materials and Methods --- p.75 / Chapter 4.3.1 --- Subjects --- p.75 / Chapter 4.3.2 --- Strand-specific RNA-Seq Library Construction --- p.77 / Chapter 4.3.3 --- Genome annotation sequencing reads processing --- p.81 / Chapter 4.3.4 --- Reads mapping for expression analysis --- p.82 / Chapter 4.3.5 --- Differential Gene expression analysis --- p.82 / Chapter 4.3.6 --- GWAS candidate genes --- p.83 / Chapter 4.3.7 --- Individual network, pathway and Gene Ontology analysis --- p.83 / Chapter 4.3.8 --- Alternative Splicing Variation --- p.83 / Chapter 4.3.9 --- Reads mapping and processing for expressed genomic variants discovery --- p.84 / Chapter 4.3.10 --- Expressed and functional genomic variants --- p.85 / Chapter 4.3.11 --- Screening for gene fusion --- p.86 / Chapter 4.3.12 --- Sense and Antisense analysis --- p.86 / Chapter 4.3.13 --- Integrated multi-level T2DM alternations gene interaction network --- p.87 / Chapter 4.3.14 --- Validation of selected genes --- p.87 / Chapter 4.4 --- Results --- p.88 / Chapter 4.4.1 --- High quality strand-specific pair-ended RNA-Seq facilitated downstream analyses --- p.88 / Chapter 4.4.2 --- Definition of significance --- p.91 / Chapter 4.4.3 --- Wide-spread repressed gene expression in T2DM --- p.91 / Chapter 4.4.4 --- Confirmation and curation of T2DM GWAS loci by RNA-Seq --- p.92 / Chapter 4.4.5 --- Global expression alteration on T2DM associated genes --- p.97 / Chapter 4.4.6 --- Alteration of relative splicing isoforms variations and T2DM specific isoforms --- p.100 / Chapter 4.4.7 --- Rare and deleterious SNPs --- p.100 / Chapter 4.4.8 --- Absence of alteration in Sense/Antisense ratio and expressed fusion gene --- p.101 / Chapter 4.4.9 --- T2DM manifests a broad spectrum of expressed abnormalities --- p.101 / Chapter 4.4.10 --- Pathway-based integration of multiple levels of alteration expanded the T2DM network --- p.103 / Chapter 4.4.11 --- Validation of selected genes --- p.107 / Chapter 4.5 --- Discussion --- p.108 / Chapter Chapter 5: --- Conclusions and future perspectives --- p.115 / Chapter 5.1 --- Conclusions --- p.115 / Chapter 5.2 --- Future perspective --- p.115 / Chapter 5.2.1 --- Splicing detection --- p.115 / Chapter 5.2.2 --- Studies related to ethnicity --- p.116 / Chapter 5.2.3 --- Complex diseases --- p.116 / References --- p.118 / Appendix --- p.131

A compreensão dos mecanismos que regulam o endométrio e suas implicações clínicas podem contribuir para melhorar as taxas de implantação do embrião humano. Apesar de muitas proteínas e moléculas influenciarem a receptividade endometrial, sua contribuição coordenada para o processo de implantação do embrião ainda é pouco compreendida. Dentre a complexa rede que guia este processo em direção à preparação de um endométrio receptivo se encontram as citocinas, das quais a interleucina-11 (IL-11) e o fator inibidor de leucemia (LIF) desempenham papel essencial. Estudos demonstram que a interrupção das vias de sinalização celular dessas citocinas prejudica ou mesmo impede a implantação, implicando diretamente na fertilidade feminina. Além disso, a hiperinsulinemia afeta negativamente a fertilidade da mulher. Dentro desse contexto, a metformina, fármaco antidiabético, pode exercer efeitos positivos sobre a expressão da IL-11 e LIF, revertendo o possível prejuízo do excesso de insulina sobre a secreção dessas citocinas. Para avaliar esse efeito, utilizou-se um modelo de cultura primária de células estromais de endométrio humano expostas aos hormônios sexuais femininos estrogênio e progesterona, divididas em grupos: controle, metformina, insulina, e associação insulina e metformina. Utilizando RT-qPCR e ensaio imunoenzimático de ELISA, foram avaliadas a expressão gênica e proteica, respectivamente, das duas citocinas. Não foram observadas diferenças entre os grupos. O ensaio de MTT para avaliar a proliferação celular permitiu a verificação da ação antiproliferativa da metformina sobre o grupo hiperinsulinêmico. Embora as hipóteses formuladas nesse estudo encontrem forte sustentação na literatura, no modelo proposto não foi possível encontrar diferenças na expressão da IL-11 e LIF. Dada a complexa regulação de todos os fatores considerados nessa pesquisa e suas múltiplas inter-relações, mais estudos são necessários para esclarecer os mecanismos que orquestram essa complexa rede. / The understanding of the endometrium regulation and its clinical implications can help to improve implantation rates of the human embryo. Although many proteins and molecules influence the endometrial receptivity, their coordinated contribution to embryo implantation process is still poorly understood. Among the complex pathways involved in this process toward the preparation to a receptive endometrium are the cytokines, including interleukin -11 (IL- 11) and leukemia inhibitory factor (LIF) that play an essential role. It has been shown that disruption of cellular signaling pathways of these cytokines impairs or even prevents implantation, direct implications on fertility. Moreover, the hyperinsulinemia can negatively affected women's fertility. Within this context, metformin, an antidiabetic drug, may exert positive effects on the expression of IL-11 and LIF, reversing the possible effects insulin excess. To evaluate this effect, a model of primary culture of human endometrial stromal cells exposed to female sex hormones estrogen and progesterone was used. Cells were divided in groups: control, metformin, insulin, association insulin and metformin. From qRT-PCR and ELISA immunoenzymatic assay gene expression and protein, respectively, of the two cytokines were evaluated. No differences were observed between groups. Additionally, the assay to evaluate cell proliferation MTT found the important antiproliferative action of metformin on hyperinsulinemic group. In the proposed model could not find differences in the expression of IL-11 and LIF. Given the complex regulation of all factors considered in this study and their multiple interrelationships, more studies are required to unravel the mechanisms that orchestrate this complex network.

Em machos, tanto o envelhecimento quanto a obesidade induzida por dieta hiperlipídica (DHL) apresentam defeitos na ação insulínica, no entanto há carência de informações sobre fêmeas. Avaliamos a sensibilidade à insulina, tolerância à glicose, o padrão estral e a expressão de proteínas da via insulínica em hipotálamo e músculo de fêmeas C57BL6. Camundongos com 10 meses de vida e com 4 meses alimentados com DHL apresentaram aumento de coxins gordurosos, resistência à insulina e intolerância à glicose em relação a seus controles. A DHL induziu redução no número de corpos lúteos, precedido de alteração do padrão estral; aumento da expressão do IR e PI3K no hipotálamo e de PI3K/AKT1 e IL-6 em músculo gastrocnêmio. No hipotálamo dos animais com 10 meses foi detectada redução na expressão do IR e TNF alpha. Confirmamos a associação entre obesidade, resistência à insulina e intolerância a glicose induzidas por dieta hiperlipídica e envelhecimento em fêmeas. Além disso, nestes modelos foi detectada modificação na expressão do IR hipotalâmico de modo distinto. / In males, aging and obesity induced by high-fat diet (HFD) show defects in insulin action, but there is a lack of information about females. We evaluated the insulin sensitivity, glucose tolerance, estrous cycle and proteins expression of the intracellular insulin pathway in the hypothalamus and skeletal muscle of female C57BL6. Ten month-old mice and 4 mo HFD fed mice had increased fat pads, insulin resistance and glucose intolerance. The HFD fed group showed impaired estrous cycle pattern followed by reduced number of corpora lutea. There were also, increased IR and PI3K protein levels in the hypothalamus and increased PI3K/AKT1 and IL-6 proteins in skeletal muscle. In hypothalamus of 10 month-old mice there was a decreased IR and TNF alpha proteins level. Thus, our data confirm the association between obesity, insulin resistance and glucose intolerance induced by HFD and aging in female mice. Moreover, there were distinct change proteins in expression of the hypothalamic IR in both animal models.

Bardet Biedl Syndrome (BBS) displays heterogeneity in the genes involved and clinical features. Mutations in 19 genes have been associated with BBS. Eight BBS proteins (BBS1, 2, 4, 5, 7, 8, 9 and 18) form the BBSome. Assembly of the BBSome is mediated by three BBS proteins (BBS6, 10, 12) in a complex with the CCT/Tric chaperonins. The BBSome is involved in formation and maintenance of primary cilia and vesicle trafficking. The clinical features of BBS include obesity, degenerative retinopathy, polydactyly, renal dysfunction, hypogonadism, and learning disability. Diabetes mellitus is commonly associated with BBS, but the mechanisms remain unknown. Our objective was to understand the molecular mechanism of BBS-associated diabetes. The role of BBS in insulin receptor (IR) signaling in Bbs4-/-mice was tested by preventing obesity using calorie restriction. These studies demonstrate the genetic defect in BBS directly contributes to the diabetes phenotype independently from the obesity phenotype. Emerging evidence implicating neuronal mechanisms in various BBS phenotypes led us to test the possibility that loss of Bbs1 in the central nervous system (CNS) disrupts glucose homeostasis. We found that deletion of the Bbs1 gene throughout the CNS or in specific hypothalamic neurons leads to hyperglycemia, glucose intolerance and insulin resistance. Our data demonstrate the critical role of neuronal Bbs1 in the regulation of glucose in an insulin-independent manner. Finally, the IR was found to interact with BBS proteins. The loss of BBSome proteins leads to a specific reduction in the amount of IR at the cell surface. The results demonstrate that BBSome proteins are required to maintain adequate levels of IR at the cell surface. The role of BBS proteins in transporting IR has not been previously described. Loss of the BBSome appears to be a novel mechanism of insulin resistance.