Dissertations / Theses on the topic 'Insulin'

A angiotensina II (AngII) pode ativar as vias de sinalização das proteínas quinases ativadas por mitógenos (MAPKs). Investigamos o papel da obesidade e das MAPKs na resposta à AngII em ratos obesos por injeção de glutamato monossódico (Ob). Artérias mesentéricas de resistência com endotélio intacto e não as artérias sem endotélio, isoladas de Ob, respondem menos à AngII. As respostas à noradrenalina e ao cloreto de potássio estavam inalteradas. Aumento da expressão do receptor AT2 (AT2R), da óxido nítrico sintase (eNOS) e da ERK1/2 podem estar envolvidos na menor resposta pois a inibição do AT2R, da eNOS e da ERK1/2 corrigiram-na. A maior ativação da ERK1/2 nos Ob levou à maior ativação da eNOS e maior geração de NO, diminuindo a resposta à Ang II. Concluímos que na obesidade, a resposta contrátil à Ang II é menor, como possível mecanismo adaptativo frente ao aumento da ativação do sistema renina-angiotensina. Esse mecanismo envolve a participação do endotélio com maior liberação de NO, aumento do número de AT2R, e da fosforilação da eNOS e da ERK1/2.<br>Angiotensin II (AngII) can activate mitogen-activated protein kinases (MAPKs) pathways. We investigated the role of obesity and MAPKs in AngII response in monosodium glutamate-induced obese rats (Ob). Endothelium-intact but not endothelium-denuded mesenteric resistance arteries isolated from Ob exhibited a lower response to AngII. The response to nordrenaline and potassium chloride were unaltered. Increased expression of AT2 receptor, nitric oxide synthase (eNOS) and ERK1/2 might be involved in the reduced response since inhibition of AT2R, eNOS and ERK1/2 corrected it. Increased activation of ERK 1/2 in Ob might activate eNOS, generating more NO and vasodilation that contributed to reduce the contraction to AngII. We concluded that, in obesity, the lower response to AngII might be an adaptive mechanism against the increased activation of the renin-angiotensin system. This mechanism involves the participation of the endothelium through a greater release of NO, increased AT2R, eNOS and ERK1/2 expressions.

La diabetes mellitus es un serio desorden metabólicocrónico que se caracteriza por el incremento anormal de laglucosa en la sangre y por complicaciones vasculares yneurológicas. La diabetes mellitus es causada por una falta oun defecto en la acción de la insulina. Si bien actualmenteexisten varios medicamentos orales además de la insulina oanálogos de la insulina, ninguno de estos es el ideal.El vanadio es capaz de imitar los efectos mostrados por lainsulina tanto in vitro como in vivo y se estudia la posibilidadde usar compuestos de vanadio como agentes antidiabéticos. Eneste artículo se revisará la acción insulino-mimética del vanadioy sus posibles mecanismos en comparación con la insulina.<br>Diabetes mellitus is a serious chronic metabolic disordercharacterized by an increased plasma glucose concentrationand vascular and neurologic complications as well. Diabetesmellitus results from relative or absolute deficiency of insulinsecretion or insulin deficient action. Although there are anumber of oral antidiabetic agents besides insulin or insulinanalogues, none of them is optimal.Vanadium can mimic insulin effects in vitro and in vivoand the possibility of using vanadium compounds asantidiabetic agents is under study. This review will summarizethe insulin mimetic action of vanadium and its possiblemechanisms in comparison with insulin.

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.<br>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.

The structure of insulin molecule was determined by Dorothy Hodgkin in 1969. Subsequently, it has been established that insulin must rearrange upon binding to its receptor (Insulin Receptor – IR). However, all known structures of the hormone depict its storage or inactive form. It has been shown that some residues, key for IR binding, are buried inside the insulin molecule and must be exposed for an efficient insulin-IR complex formation. It has been postulated that the C-terminal region of the B-chain (~B20-B30) is dynamic in this process, and that the detachment of the B20-B30 β-strand leads to the activation of insulin. However, the understanding of the molecular basis of the insulin regulatory role is hindered by the lack of the structure of the insulin-IR complex; only 3-D description of the apo-form of the IR ectodomain is known. The very complex molecular biology behind expression and production of IR fragments also hampers progress in this field. In order to facilitate progress towards determination of the insulin-IR complex crystal structure this work delivered: (i) structural characterisation of highly-active insulin analogues for stable hormone-IR complexes, (ii) development of various attempts for an alternative production of L1 domain of human IR, (iii) structural characterisation of the role of residues B24 and B26 for insulin function, (iv) clarification of individual contributions of hydrogen bonds stabilising the insulin dimer, (v) understanding of the structural basis of different functionality of click-chemistry based novel insulin analogues. This work established that: (i) the structural signature of the highly active insulin analogues is new -turn at the Cterminus of the B-chain (the B26 turn) achieved by trans-to-cis isomerisation of the PheB25- TyrB26 peptide bond. This conformational change exposes residues responsible for IR binding, (ii) the production of the L1 domain in E. coli, instead of the usual mammalian expression system, is not feasible, (iii) the structural invariance of the PheB24 is fundamental to the formation of the insulin-IR complex. It acts as an anchoring and side-chain pivot for the B26 turn, (iv) removal of the NHB25-COA19 dimer interface hydrogen bond is sufficient for a complete disruption of the dimer, whilst the other four hydrogen bonds had a less marked effect in this process, (v) the formation of the B26 turn can be efficiently mimicked by click-chemistry based intra-B-chain crosslinks. These results provide a wealth of information about the active form of insulin, they provide important tools towards the first insulin-IR complex, and deliver novel insulin analogues.

Insulin and insulin-like growth factor I (IGF-I) are two related peptides with similar structure. They mediate their effects by binding to their respective receptor, the insulin receptor (IR) and the IGF-I receptor (IGF-IR) and induce intracellular signalling cascades resulting in metabolic or mitogenic effects. The relative abundance of IR and IGF-IR is of importance for the type of effect that is the outcome of the signal. There are few studies investigating the relative receptor abundance and its effects in human cells and tissues. In this thesis we wanted to study abundance and regulation of insulin and IGF-I receptors in different human cells and tissues and examine the effects of variations in insulin and IGF-I receptor abundance between different cells and tissues. We examined IR and IGF-IR gene and protein expression and the effects of insulin and IGF-I on receptor phosphorylation, DNA synthesis and glucose transport. Our results show that there is a large variation in the distribution of IR and IGF-IR in different human cells and tissues. Renal artery intima-media expressed predominantly IGF-IR while in liver IR was the predominant receptor type. Differentiation of human preadipocytes results in a change in relative expression of IGF-IR to IR. Mature adipocytes express almost 10-fold more IR than IGF-IR while preadipocytes express almost the same amounts of both receptors. Mature tissues, such as liver, skeletal muscle, myometrium and renal artery intima-media, express predominantly IR-B. Preadipocytes express IR-A and the expression of IR-B is induced during differentiation. We could show the presence of insulin/IGF-I hybrid receptors in preadipocytes but not in mature adipocytes. Cultured endothelial cells express mostly IGF-IR and insulin/IGF-I hybrid receptors and these cells respond mainly to IGF-I. Due to the large abundance of IR mature adipocytes are sensitive to insulin but insensitive to IGF-I whereas preadipocytes expressing equal amounts of both receptors respond to both insulin and IGF-I. Insulin and IGF-I are only partial agonists to each other’s receptors in human preadipocytes and adipocytes. The overall results indicate that differential expression of IGF-IR and IR is a key mechanism in regulation of growth and metabolism.

A series of studies is described in which specific and conventional insulin immunoassays, the hyperinsulinaemic clamp technique and forearm venous occlusion plethysmography with local intra-arterial infusions have been used to investigate: the effect of insulin assay specificity on the relationships among serum insulin concentrations, insulin sensitivity, and blood pressure in diabetic and non-diabetic subjects with and without essential hypertension (Chapter 5) the effect of sustained physiological activation of the renin-angiotensin system induced by moderate dietary sodium restriction on insulin sensitivity in patients with non-insulin-dependent diabetes mellitus (Chapter 6) the relationship between endothelial function and insulin sensitivity in healthy subjects (Chapter 7) Prior to these investigations, preliminary studies (Chapters 3 and 4) were performed in order to validate aspects of the clinical physiological techniques required for the measurement of blood flow and insulin sensitivity. The reproducibility of bilateral forearm venous occlusion plethysmography Studies using this technique to measure changes in forearm blood flow (FBF) during intra-arterial infusions of vasoactive substances often report changes in blood flow ratio (expressing responses in the intervention arm as a ratio of responses in the control arm) rather than absolute values for flow. However, unilateral measurements are reported by other investigators, and the possibility was considered that the method used for expressing responses might influence the conclusions reached. A reproducibility study was performed (Chapter 3) which demonstrated that the between-day intra-subject variability of bilateral forearm venous occlusion plethysmography (FBF ratios) was less than that of unilateral FBF measurements. The bilateral technique was used thereafter where possible.

Insulin resistance in metabolic syndrome subjects is profound in spite of muscle insulin receptor and insulin-responsive glucose transporter (GLUT4) expression being nearly normal. Insulin receptor tyrosine kinase phosphorylation of insulin receptor substrate-1 (IRS-1) at Tyr896 is a necessary step in insulin stimulation of translocation of GLUT4 to the cell surface. Serine phosphorylation of IRS-1 by some kinases diminishes insulin action in mice. We evaluated the phosphorylation status of muscle IRS-1 in 33 subjects with the metabolic syndrome and seventeen lean controls. Each underwent euglycemic insulin clamps and a thigh muscle biopsy before and after 8 weeks of either strength or endurance training. Muscle IRS-1 phosphorylation at six sites was quantified by immunoblots. Metabolic syndrome muscle IRS-1 had excess phosphorylation at Ser337 and Ser636 but not at Ser307, Ser789, or Ser1101. Ser337 is a target for phosphorylation by glycogen synthase kinase 3 (GSK3) and Ser636 is phosphorylated by c-Jun N-terminal kinase 1 (JNK1). Exercise training without weight loss did not change the IRS-1 serine phosphorylation. These data suggest that baseline hyperphosphorylation of at least two key serines within muscle IRS-1 diminishes the transmission of the insulin signal and thereby decreases the insulin-stimulated translocation of GLUT4. Excess fasting phosphorylation of muscle IRS-1 at Ser636 may be a major cause of the insulin resistance seen in obesity and might prevent improvement in insulin responsiveness when exercise training is not accompanied by weight loss.

Orientador: Doris Hissako Sumida<br>Banca: Nancy Alfieri Nunes<br>Banca: Cléa Adas Saliba Garbin<br>Banca: Doris Hissako Sumida<br>Resumo: Nos últimos anos tem ocorrido uma redução acentuada nos índices de cárie dentária em diversas regiões do planeta, fato que tem se atribuído ao consumo de produtos fluoretados. Entretanto, o flúor, quando ingerido em excesso, causa intoxicação crônica ou aguda, como a fluorose dentária e distúrbios na homeostase da glicose. As crianças se tornam foco de preocupação, principalmente às portadoras de diabetes mellitus (DM), pois geralmente ingerem grandes quantidades de dentifrício fluoretado durante a escovação, ultrapassando a dose preconizada como limite de ingestão diária de flúor de 0,05 a 0,07mg/F/kg de peso corpóreo. Este trabalho, que foi dividido em duas partes, pretende realizar uma breve revisão de literatura sobre os efeitos decorrentes da ingestão de NaF no metabolismo de carboidratos e avaliar os efeitos da ingestão do fluoreto na sensibilidade à insulina e na transdução do sinal insulínico. A primeira parte, baseada em artigos científicos publicados, procura discorrer sobre os efeitos da ingestão de flúor no metabolismo de carboidratos, na tolerância à glicose e no sinal insulínico, e algumas considerações sobre o diabetes mellitus e sobre as possíveis complicações que a ingestão de NaF pode ocasionar às crianças portadoras desta doença. Estes trabalhos demonstraram que o tratamento agudo ou prolongado com altas doses de fluoreto de sódio interfere na homeostase da glicose. Convém salientar que esta alteração é similar à observada em casos de diabetes mellitus. Além do mais, o flúor quando ingerido em excesso, também ocasiona diminuição da secreção de insulina, inibição da glicólise e depleção de glicogênio. Muitas dessas respostas sugerem que o NaF pode promover resistência à insulina. Portanto, a ingestão em excesso de NaF pode prejudicar a saúde, principalmente de crianças portadora de DM.<br>Abstract: Over the last few years there has been a significant reduction in the incidence of dental caries in several regions of the world. This has been attributed to the consumption of fluoridated products. However, excess of fluoride intake can cause chronic or acute intoxication, such as dental fluorosis and impaired glucose homeostasis. Concern is focused on children, especially those with diabetes mellitus, because children usually swallow large amounts of fluoridated dentifrice during tooth brushing, in excess of the maximum recommended daily fluoride dose of 0.05 to 0.07 mg/F/kg of body weight. This report, divided into two parts, intends to make a brief literature review about effects of NAF intake on glucose metabolism, and to determine the effects of this intake on insulin sensitivity and insulin signal transduction. The first part, based on published scientific articles, endeavors to describe the effects of NaF intake on glucose metabolism, glucose tolerance and insulin signal, and put forward considerations concerning diabetes mellitus (DM), and the possible complications that NaF intake could cause in children with DM. These reports demonstrated that the acute or chronic treatment with high sodium fluoride dose interferes in glucose homeostasis, resulting in conditions such as hyperglycemia. This alteration is similar to that observed in DM. Furthermore, NaF ingestion in high doses can produce abnormalities in insulin secretion, glycolysis inhibition, and glycogen depletion. Many of these evidences suggest that NaF can induce insulin resistance. Thus, excessive fluoride consumption could worsen health, particularly of diabetic children. Based on that fluoride can interfere in the glucose metabolism, it is important for the second part of this report to determine the acute effect of fluoride on insulin sensitivity and pp185 (IRS-1/IRS-2) phosphorylation in insulin sensitive tissues.<br>Mestre

Orientador: Mario Jose Abdalla Saad<br>Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas<br>Made available in DSpace on 2018-08-08T15:02:32Z (GMT). No. of bitstreams: 1 Zecchin_HenriqueGottardello_D.pdf: 4518336 bytes, checksum: db2ce3333dfb7dda86b0298470e924ca (MD5) Previous issue date: 2007<br>Resumo: A resistência seletiva à insulina através da via IRS/PI3-K/Akt/eNOS associada à ativação normal ou exacerbada da via de crescimento MAPK tem sido proposta como um possível elo entre situações de resistência à insulina e doença cardiovascular. Inicialmente demonstramos que animais com resistência à insulina e doença cardiovascular (o rato espontaneamente hipertenso, SHR) apresentam menor ativação da via IRS/PI3-K/Akt/eNOS e hiperativação/hiperexpressão da via da MAPK na aorta torácica, enquanto a ativação normal da via IRS/PI3-K/Akt/eNOS pode proteger o animal obeso, resistente à insulina e que não apresenta doença cardiovascular. Posteriormente, outras vias estimulatórias do crescimento celular, como a via JAK/STAT, foram estudadas no vaso de outro modelo animal de resistência à insulina e doença cardiovascular - o rato com obesidade induzida por dieta. Este modelo demonstrou que a redução da ativação da via PI3-K/Akt/eNOS ocorre em paralelo à hiperativação das vias da MAPK e JAK/STAT, e isso pode desempenhar função importante da patogênese da doença cardiovascular neste estado patológico. Em outro estudo foi demonstrado pela primeira vez que a acetilcolina pode ativar a eNOS de maneira cálcio-independente, através da via IRS/PI3-K/Akt utilizando para isso uma tirosina quinase intracelular, a JAK2. Em ratos com obesidade induzida por dieta, resistentes à insulina e com disfunção endotelial, foi demonstrado que há resistência na via da PI3-K/Akt/eNOS tanto em resposta à insulina quanto à acetilcolina, em decorrência de maior fosforilação inibitória do IRS-1 e da redução dos níveis teciduais da eNOS. Assim, o desequilíbrio entre a ativação reduzida da via IRS/PI3-K/Akt/eNOS e a maior ativação das vias de crescimento (MAPK e JAK/STAT) pode contribuir para o desenvolvimento de doença cardiovascular em estados de resistência à insulina<br>Abstract: The actions of acetylcholine on endothelium are mainly mediated through muscarinic receptors, which are members of the G protein-coupled receptor family. In the present study we show that acetylcholine induces rapid tyrosine phosphorylation and activation of Janus kinase 2 (JAK2) in rat aorta. Upon JAK2 activation, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) is detected. In addition, acetylcholine induces JAK2/IRS-1 and IRS-1/phosphatidylinositol (PI) 3-kinase associations, downstream activation of Akt/protein kinase B, endothelial cell-nitric oxide synthase (eNOS), and extracellular signal-regulated kinase (ERK1/2). The pharmacological blockade of JAK2 or PI 3-kinase reduced acetylcholine-stimulated eNOS phosphorylation, NOS activity and aorta relaxation. These data indicate a new signal transduction pathway for IRS-1/PI 3- kinase/Akt/eNOS activation and ERK1/2 by means of JAK2 tyrosine phosphorylation stimulated by acetylcholine in vessels. Moreover, we demonstrate that, in aorta of obese rats (high-fat diet), there is an impairment in insulin- and acetylcholinestimulated IRS-1/PI 3-kinase pathway, leading to reduced activation with lower protein levels of eNOS associated with a hyperactivated ERK/MAP kinase pathway. These results suggest that in aorta of obese rats, there is not only insulin resistance, but also acetylcholine resistance, probably mediated by a common signaling pathway that controls the activity and the protein levels of eNOS<br>Doutorado<br>Medicina Experimental<br>Doutor em Fisiopatologia Medica

Orientador: Mario Jose Abdalla Saad<br>Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas<br>Made available in DSpace on 2018-08-10T01:17:39Z (GMT). No. of bitstreams: 1 Lalli_CristinaAlba_D.pdf: 9125967 bytes, checksum: 5ab6be7c7ee8bef63c8acdec567c428b (MD5) Previous issue date: 2007<br>Resumo: A insulina é o principal hormônio anabólico, que atua através da ativação de transportadores, regulação de enzimas e expressão de genes que codificam enzimas envolvidas na captação e armazenamento de substratos. Para exercer suas ações, a insulina emprega duas vias principais de sinalização intracelular: a via da PI 3-quinase e a via da MAPK. A regulação da ação do hormônio se faz por meio de vários mecanismos. O modelo animal de dieta hiperlipídica apresenta alterações metabólicas e de sinalização semelhantes aos encontrados na resistência insulínica de humanos com obesidade induzida por dieta. O objetivo de nosso trabalho foi estudar em ratos alimentados com dieta hiperlipídica, etapas da sinalização insulínica e também algumas vias de regulação da sinalização, após o uso de duas drogas: a rosiglitazona, uma tiazolidinediona usada para o tratamento do diabetes melito tipo 2 como sensibilizadora de insulina e a lovastatina, droga inibidora da HMGCoA redutase, que diminui a síntese de colesterol, mas que também tem apresentado efeito de aumentar a sensibilidade à insulina, tanto em modelos animais como em humanos. Ratos alimentados com dieta hiperlipídica por quatro semanas e tratados na última semana com as drogas, isoladamente, foram submetidos à extração de tecidos hepático e muscular e os fragmentos obtidos foram submetidos à análise de concentração protéica ou de grau de fosforilação de proteínas através de técnicas de imunoprecipitação e immunoblotting. A sensibilidade à insulina foi avaliada pelo teste de tolerância à insulina e cálculo da constante de decaimento da glicose (Kitt). No estudo. da rosiglitazona, foi observada diminuição significativa da sensibilidade à insulina expressa por diminuição no Kitt, nos animais que receberam a dieta e recuperação da sensibilidade após o uso da droga. A fosforilação do IRS-l, associação do substrato com a enzima PI3K e a ativação da Akt no tecido hepático e muscular também se mostraram diminuídas pela dieta e recuperadas com o uso da rosiglitazona. O estudo da lovastatina demonstrou efeito positivo da droga sobre a sensibilidade insulínica, revertendo a resistência induzida pela dieta hiperlipídica, expressa por valor de Kitt semelhante ao dos animais controle. Na via de sinalização da PI3K: fosforilação do IR e do IRS-I, associação do IRS-l à PI3k e ativação da Akt, tanto no tecido hepático como muscular, a lovastatina reverteu as alterações da dieta, com recuperação a valores semelhantes aos do grupo controle. Também nas vias de regulação, a dieta induziu maior fosforilação do IR em serina, maior fosforila do IRS-I em serina307, maior atividade da JNK e da proteína fosfatase PTPIB e menor ativação do IKB, efeitos que podem explicar a resistência desse modelo. A lovastatina reverteu todas essas alterações. Concluindo, a rosiglitazona reverteu as alterações causadas pela dieta hiperlipídica sobre as etapas iniciais da sinalização insulínica na via da PI3K. A lovastatina recuperou as alterações induzidas pela dieta hiperlipídica na transmissão do sinal insulínico, agindo sobre as vias de regulação da sinalização: ação da PTPIB, fosforilação do IR e do IRS-l em serina induzidos pela JNK e PTPIB e ativação da via inflamatória<br>Abstract: Insulin is the major anabolic hormone and acts through transporter activation, enzymes regulation and gene expression. This hormone uses' two main signaling pathways: the PI3K pathway, involved in its metabolic effects and the MAPK pathway, responsible for cell growth and differentiation. There are many mechanisms of regulation of insulin signaling. The use of a high- fat diet is a known model of insulin resistance with metabolic and signaling changes similar to those of human insulin resistance syndrome observed in diet induced obesity. Rosiglitazone is an agent of the class of thiazolidinediones, insulin-sensitizing agents whose effects are mainly due to the activation of PP ARy and are used to treat type 2 diabetes. Lovastatin is one of a class of a class of cholesterol synthesis inhibitors; recent studies have shown that this agent might have relevant effects on insulin resistance in both animal models and humans. The aim of this study was to evaluate the effects of two different drugs independent1y, rosiglitazone and lovastatin, on insulin signaling in liver and muscle of rats fed on a hígh-fat diet. We used four week old male Wistar rats, fed on a high- fat diet during four weeks and treated with rosiglitazone or lovastatin during the last week, compared to rats fed on standard chow. Fragments of liver and muscle tis sues were extracted from anesthetized animaIs and protein concentrations and phosphorylation degree were studied through immunoprecipitation and immunoblotting techniques. Insulin sensitivity was evaluated by insulin tolerance test and calculation of the disappearance rate constant (KitD. We observed that high-fat fed diet rats presented a significant decrease in Kitt compared to control rats. The animaIs that were fed with the high-fat diet and were treated with either one ofthe drugs presented a reversal ofthis effect. In the study of rosiglitazone, the high-fat model demonstrated a decrease in the IRS-I phosphorylation, IRS-l/PI3K association and activation of Akt and rosiglitazone administration resulted in the reversion of all the effects in liver and muscle. In addition to the effect on insulin sensitivity, the use of lovastatin was also associated with an increase in insulin-induced IR tyrosine phosphorylation and, in parallel, a decrease in IR serine phosphorylation and association with PTPIB. Our data also show that lovastatin treatment was associated with an increase in the insulin-stimulated IR/IRS-l/PI3KJ Akt pathway in the liver and musc1e of high-fat fed rats, in parallel with a decrease in the inflammatory pathway (JNK and IKKI3/IKB/NFKB) related to insulin resistance. In conclusion, rosiglitazone and lovastatin improved the altera_s in insulin signaling pathways presented by the high-fat model of insulin resistance<br>Doutorado<br>Clinica Medica<br>Doutor em Clínica Médica

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<br>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

Insulin resistance is a disease state characterised by the reduced ability of insulin to exert its effects in peripheral tissues, skeletal muscle and adipose tissue. This condition has been associated with a number of other disease states including obesity and hypertension. The hypertensive Milan rat has previously been shown to be insulin resistant. Unlike any other hypertensive, insulin resistant model, the Milan rat has a normotensive, isogenic control which responds normally to insulin. As GLUT-4, the insulin-stimulated glucose transporter, had been implicated in insulin resistance I examined the levels of GLUT-4 present in the Milan rat. Results suggest that the insulin resistance experienced by this hypertensive strain may be due to a reduction in GLUT-4 within the intracellular membranes of skeletal muscle. This is due to the nature of insulin-stimulated glucose transport, which arises as a result of GLUT-4 translocatlng to the cell surface from an intracellular pool, and therefore increasing the rate of glucose uptake. Consequently, any reduction in intracellular GLUT-4may account for the insulin resistance observed. Further studies eXamined the stroke-prone spontaneously hypertensive rat, and the stroke-prone spontaneously hypertensive rat F2 generation. The F2 generation contains individuals that are extremely hypertensive and others which are normotensive. This was done to determine if the decrease in GLUT-4 observed in the hypertensive Milan rat correlated with increasing blood pressure. The results suggest that GLUT-4 levels in the stroke-prone spontaneously hypertensive rat are not altered by an increase in blood pressure. This result is in agreement with most studies on skeletal muscle GLUT-4, and highlights the complex nature of insulin resistance associated with hypertension. The concluding chapter discusses the effects of oestrogen, and derivatives, on insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Previous studies have shown that females taking steroid hormones, either by means of the contraceptive pill, of hormone replacement therapy, tend to suffer from insulin resistance. In 3T3-L1 adipocytes a 48 hour treatment with 30nM oestrogen significantly reduces insulin-stimulated glucose transport. This demonstrates that the cells have developed insulin resistance. However these cells do not have reduced GLUT-4 levels and the insulin resistance, induced by oestrogen, occurs by an as yet unknown mechanism.

In this study a chimeric receptor system was employed in which the extracellular domain of the TrkC receptor was fused to the intracellular portion of either the insulin (TIR) or IGF-1 (TIGFR) receptor. These chimeric receptors were expressed in separate populations of the skeletal muscle cell line L6. Initial analysis of individual downstream signalling components and assessment of cell proliferation, induced by TIR or TIGFR stimulation revealed little difference between the two chimeras. To more comprehensively screen for potential differences, microarray analysis was used to compare regulation of gene expression by the two chimeric receptors. This led to the identification of several differentially regulated genes.  Whilst it was initially hypothesised that skeletal muscle cells might yield several selectively insulin-sensitive genes, the majority of genes selectively regulated by one receptor were preferentially IGF-1 responsive, consistent with previous studies in other cell types. This perhaps reflects the more mitogenic effect of this ligand <i>in vivo</i>, manifest as an increased ability to regulate transcription <i>per se</i>. Of the differentially regulated genes, that encoding Fit-1m was found to be preferentially induced through activation of the TIGFR rather than the TIR. Further characterisation using real-time PCR established that induction of Fit-1 expression required an intact MAPK signalling pathway. Similar effects were observed when the regulation of Fit-1 expression by insulin and IGF-1 was examined. Subsequent work attempted to establish regions of promoter responsible for the preferential induction of Fit-1m expression by IGF-1. Despite defining promoter and putative enhancer regions which are important for Fit-1m transcription, no region was found which confers a response to stimulation with various ligands, including IGF-1. Rather, a high level of constitutive expression was driven by these DNA sequences, suggesting that an IGF-1 response inhibitory factor may control expression of this gene, binding outside the regions examined. Fit-1 joins an increasing list of genes preferentially regulated by the IGF-1R over the IR and provides and end point with which to analyse potential inherent differences in the signalling capacity of these two highly homologous receptors.

The descriptive study which explored the insulin dependent diabetics' and non-insulin dependent diabetics' perceptions of susceptibility to diabetic complications, severity of diabetes, and benefits of and barriers to preventive measures. The Wortell Diabetic Perception Scale was developed by the researcher for this study, and administered to a convenience sample of 71 subjects. The Subjects' age ranged from 22 to 80 years. There were 33 females and 38 males in the sample. Forty three percent of the diabetics were classified as insulin dependent diabetics and 57% as non-insulin dependent diabetics. Findings indicated that insulin dependent diabetics perceived diabetes to be significantly more severe than did non-insulin dependent diabetics. No significant difference was found to exist between the insulin dependent diabetics and non-insulin dependent diabetics with regards to perceived susceptibility to diabetic complications, and benefits of and barriers to preventive measures.

Insulin resistance is an increasing public health issue with the current literature, suggesting reduced sensitivity of insulin leads to adult onset diabetes and associated downstream pathologies that reduce life expectancy. The main objectives of this study were to evaluate the ability of the Oral Glucose Insulin Secretion Test (OGIST) to identify insulin resistance and examine differences in the insulin sensitivity based on gender, age, and ethnicity. This study was supported by the insulin resistance theory which focuses on the reduced ability of insulin to bind to the cellular insulin receptor, reducing the sensitivity of insulin. The OGIST lab results of a total of 250 patients, aged 18-65, were included in this study from a major city in the midwestern United States. Binomial logistic regression was used to evaluate the relationship between the dependent variables and the validation independent variables and analyze the possible differences seen in insulin, proinsulin, C-peptide, and HbA1c with age. The OGIST demonstrated the ability to identify elevated levels of insulin, proinsulin, and C-peptide at the end of the first phase insulin secretion to glucose. The results of this study demonstrated patients with insulin resistance exhibited a greater reduction in insulin production with age compared to those without insulin resistance. There were no changes observed between gender or ethnicity. The OGIST was the only test that demonstrated the ability to identify the individual's insulin sensitivity, β-cell function, and progression to diabetes. The ability of the OGIST to identify both insulin resistance and β-cell function can contribute to positive social change by encouraging further research for the early diagnosis and treatment of insulin resistance and the reduction in adult onset diabetes.

Orientador: Mario Jose Abdalla Saad<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas<br>Made available in DSpace on 2018-08-06T23:49:19Z (GMT). No. of bitstreams: 1 Lemos_ChristineMarinhode_M.pdf: 1925163 bytes, checksum: 3618c226fce7bf7ab354e89c68dcfa14 (MD5) Previous issue date: 2006<br>Resumo: Estudos recentes demonstraram que tratamentos prolongados com drogas inibidoras da atividade tirosina quinase (TKI) poderiam agir favoravelmente não só controlando crescimento e replicação celular, como também funções fisiológicas responsáveis por manter a homeostase da glicose. Porém, os efeitos diretos de PD 153035, um TKI, na regulação das etapas iniciais da ação da insulina não são conhecidos. A insulina, ao se ligar à subunidade a de seu receptor heterotetramérico, dá início a uma série de ações imediatas e tardias, metabólicas e promotoras de crescimento .Tais eventos ocorrem através da estimulação da subunidade ß transmembrana do receptor, que autofosforila e ativa a fosforilação de substratos endógenos intracelulares, conhecidos como substratos do receptor de insulina ou IRSs. Os principais substratos do receptor de insulina são o IRS-1 e IRS-2, que quando fosforilados em tirosina se ligam e ativam proteínas com porção SH2, como a PI 3-quinase. A ativação destas proteínas desencadeia a ativação de suas serinas-quinases importantes que são a AKT e as ERKs (1/2), que são essenciais, respectivamente, para os efeitos metabólicos e de controle gênico do homônio. No presente estudo investigamos o efeito do tratamento com PD 153035, por 7 dias, na sensibilidade e sinalização da insulina em fígado, musculo e tecido adiposo de ratos Wistar. Foi investigado o grau de fosforilação em tirosina do receptor de insulina, dos substratos 1 e 2 do receptor ( IRS-1 e IRS-2 ), a associação deles com a enzima PI 3-quinase, o grau de fosforilação em serina/treonina da AKT, a fosforilação das ERKs (1/2), da p70s6k, da AMPK, da JNK e I?Ba nos três tecidos. Nenhuma diferença nos níveis glicêmicos foi observado durante o GTT entre os grupos tratado com PD 153035 e controle. As taxas de desaparecimento de glicose plasmática estavam altas nos animais tratados. No fígado de ratos tratados com PD 153035, observamos um aumento da fosforilação em tirosina do IR, IRS-1 e do IRS-2 e um aumento da associação desses substratos com a PI 3-quinase. Porém uma diminuição significativa da fosforilação da AKT e da p70s6k foi observada, sem alteração no grau de fosforilação das ERKS (1/2). Não foi observado diferença significativa no grau de fosforilação da JNK, porém os animais tratados apresentaram uma redução significativa nos níveis de I?Ba . O grau de fosforilação da AMPK mostrou-se aumentado nos animais tratados. Quando estudamos o tecido muscular, após estímulo agudo com insulina observamos uma diminuição significativa no grau de fosforilação do receptor de insulina nos animais tratados com PD 153035. Entretanto, nesses animais, pudemos observar após estímulo agudo com insulina, que a fosforilção em tirosina do IRS-1 aumentou significativamente. O aumento da fosforilação do IRS-1 foi acompanhada pelo aumento na associação IRS-1/ PI 3-quinase e pelo aumento no grau de fosforilação da AKT e da p70s6k. a. O grau de fosforilação da AMPK mostrou-se aumentado nos animais tratados. Não foi observada alteração no grau de fosforilação das ERKs (1/2) neste tecido. Foi observado uma redução do grau de fosforilação das serinas quinases JNK e I??a no músculo dos animais tratados. Os animais que receberam tratamento crônico com PD 153035 por 7 dias apresentaram uma redução da adiposidade visceral, bem como uma perda de peso em relação ao grupo controle. Observamos nesses animais uma redução significativa no grau de fosforilação do IR e do IRS-1 no tecido adiposo. A associação IRS-1/PI 3-quinase mostrou uma redução significativa. A fosforilação da AKT e da P70s6k mostrou-se significativamente reduzida nos animais tratados. Entretanto observamos um aumento significativo da fosforilação do IRS-2 após estímulo insulínico agudo, porém acompanhado pela redução significativa na associação IRS-2/PI 3-quinase. Não observamos diferença nos níveis de fosforilação das ERKs. Observamos que o grau de fosforilação da proteína AMPK aumentou significativamente no ratos que receberam tratamento com PD 153035. Foi observado uma redução significativa do grau de fosforilação da JNK e I??a no tecido adiposo dos animais tratados. Sumariamente, o tratamento com PD 153035 por 7 dias aumentou a fosforilação em tirosina do IR, IRS-1 e IRS-2 no fígado, apesar da fosforilação da AKT apresentar-se reduzida neste tecido. No músculo dos animais tratados com PD 153035 observamos que a droga melhora a sinalização da insulina, provavelmente pela redução da atividade das serinas quinases JNK, IKKß e mTOR. No tecido adiposo a droga induziu resistência à insulina, acompanhada de redução no ganho de peso e redução da adiposidade visceral, possivelmente pelo aumento da secreção de adiponectina pelos adipócitos. Em conclusão, os resultados do nosso estudo demonstram que o tratamento com PD 153035 aumentou a sensibilidade à insulina, por aumento da adiponectina, aumento da AMPK em fígado, músculo e adiposo, e aumentada via IRS/PI3K/AKT em músculo<br>Abstract: It has been recently demonstrated that long-term treatment with some of tyrosine kinase inhibitor (TKI) drugs, might favorably act at steps in controlling not only cell growth and replication, but also physiological functions responsible for maintaining glucose homeostasis. However, the direct effects of PD 153035, a TKI, in the regulation of the early steps of insulin action are not known. Insulin initiates its growth and metabolic promoting effects by biding to its receptor at the plasma membrane, which has tyrosine-kinase activity, and is able to autophosphorylates and phosphorylates cytoplasmatic proteins called insulin receptor substrates (IRSs). The main substrates of insulin receptor are IRS-1 and IRS-2, which when phosphorylated in tyrosine bind and activate several proteins, including phosphatidylinositol (PI) 3-kinase. These initial steps lead to the activation of two serine/threonine kinases ¿ AKT and ERK family (1/2) of MAPK. In the present study, we investigated the effect of treatment with PD 153035, for 7 days, on insulin sensitivity and insulin signaling in liver, muscle and adipose tissue of Wistar rats. It was investigated the tyrosine phosphorylation of IR, IRS-1 and IRS-2, their association with PI 3-kinase, and Akt serine/threonie phosphorylation , ERKs (1/2) phosphorylation, p70s6k, AMPK, JNK and ???a phosphorylation, in the three tissues. No differences in fasting plasma glucose levels were observed in animals treated with PD 153035. Plasma glucose disappearance rates were higher in treated animals In the liver of rats treated with PD 153035, we observed an increased IR, IRS-1 and IRS-2 tyrosine phosphorilation and an increased association of these substracs with PI 3-quinase. However a significant decrease of AKT and of the p70s6k phosphorylation was observed too, without alteration in ERKs phosphorylation levels (1/2). No significant difference was observed in JNk phosphorylation levels, however ??? showed a reduced phosphorylation in the liver of these animals. AMPK showed a significant increased phosphorylation in treated animals. When we studied muscle, insulin-induced IR tyrosine phosphorylation showed significantly reduced in these animals, however treating rats with PD153035 significantly increased the insulin-induced IRS-1 phosphorylation in the muscle. The increased phosphorylation of IRS-1 was accompanied by increase in IRS-1/PI3-kinase association and Akt and p70s6k phosphorylation were higher in treated animals after insulin stimulation. AMPK showed a significant increased phosphorylation in treated animals. There was no significant changes in ERKs (/2) phosphorylation in this tissue. Reduced phosphorylation of serine-kinases as c-jun N terminal (Jnk) and I?kß was observed. The animals that received chronic treatment with PD 153035 for 7 days had presented a reduction in visceral fat mass, as well as a loss of weight, regarding the control group We observed in these animals a significant decreased IR and IRS-1 tyrosine phosphorylation in the adipose tissue. The association IRS-1/PI3-kinase showed a significant reduction. AKT and P70s6K phosphorylation showed significantly decreased in treated animals. However we observe a significant increased phosphorylarion of insulin-induced IRS-2 phosphorylation, but the association IRS-2/PI 3-kinase showed a significant reduction. We did not observe any difference in phosphorylation levels of ERKs (1/ 2). AMPK phosphorylarion increased significantly in animals that received treatment with PD 153035. Reduced phosphorylation of JNK and Ikkßa was observed in adipose tissue of treated animals. In summary, our results demonstrated that in 7 days of treatment with PD 153035 increased tyrosine phosphorylation of IR/IRS-1/IRS-2 in the liver were observed, in spite of AKT phosphorylation had decreased in this tissue. In muscle of animals treated with PD 153035 we observed that the drug had improved the insulin signalling, probably by reduction of the serine kinase activity JNK, ???ß and mTOR. In the adipose tissue, the drug induced insulin resistance, accompanied of visceral fat mass reduction as well as a loss of weight, probably due to an increased adiponectin secretion by fat cels. In conclusion, the results of our study demonstrate that the treatment with PD 153035 increased the insulin sensibility, by increased levels of adiponectin, increased AMPK in liver, muscle and adipose tissue, and increased IRS/PI3K/AKT pathway in muscle<br>Mestrado<br>Ciencias Basicas<br>Mestre em Clinica Medica

Introduction: Black African populations present with low insulin sensitivity (SI) and hyperinsulinemia, the latter due to high insulin secretion and reduced clearance. In addition, they exhibit lower levels of central and ectopic fat, compared to their white counterparts, contradicting the known correlates of SI typically reported in white populations. Moreover, in black African women hyperinsulinemia is in excess of the level needed to compensate for low SI, with a corresponding high disposition index (DI), a marker of insulin response that accounts for the level of SI. Further, obese, black African women have a high risk for type 2 diabetes (T2D), but the correlates of hyperinsulinemia have not been fully elucidated, especially the role of ectopic fat and body fat distribution. Exercise training is beneficial to improve SI and DI, however, whether these effects are mediated by changes in ectopic fat in skeletal muscle, liver and pancreatic depots is unknown. Accordingly, exercise training can be used as a model to assess the correlates of hyperinsulinemia and SI in cohorts at high risk for developing T2D, such as obese black African women. This thesis therefore aims to describe the correlates of hyperinsulinemia and SI and to evaluate the effect of exercise training on these components with emphasis on the role of body fat distribution and ectopic fat in mediating these changes. Methods: Firstly, a cross-sectional analysis of 45 obese (BMI 30-40 kg/m2 ) black South African women (age 20-35 years) without T2D was conducted. Thereafter the women were block randomized into an exercise training (n=23) or no exercise (control, n=22) group. The exercise training group participated in a 12-week combined aerobic and resistance training programme (40-60 min session, 4 days/week) supervised by a biokineticist. Pre and post-intervention testing included assessment of acute insulin response to glucose (AIRg), SI, DI (AIRg x SI), insulin secretion rate (ISR), hepatic insulin extraction (HIE) and peripheral insulin clearance (CLp) (frequently sampled intravenous glucose tolerance test); body fat mass and regional adiposity (dual-energy X-ray absorptiometry); hepatic, pancreatic and skeletal muscle fat and abdominal subcutaneous (aSAT) and visceral adipose tissue (VAT) (magnetic resonance imaging); intramyocellular (IMCL) and extramyocellular fat content (EMCL) (magnetic resonance spectroscopy). Results: The baseline results showed that a high DI was associated with low VAT (r0.565, p< 0.001), pancreatic fat, soleus IMCL and EMCL with VAT explaining most of the variance in DI (32%). SI was inversely associated with VAT (rho -0.417, p=0.007) and AIRg was inversely and HIE was positively associated with VAT-aSAT ratio (rho - 0.345, p=0.029 and rho 0.510, p=0.011, respectively). DI was positively associated with CLp (rho 0.528, p=0.006), while its components (SI and AIRg) were not. Results from the intervention showed that exercise training increased DI (median (interquartile range): 6.1 (3.6-7.1) to 6.5 (5.6-9.2) x103 arbitrary units, p=0.028), SI (2.0 (1.2-2.8) to 2.2 (1.5-3.7) (mU/l) -1 min -1 , p=0.005) and VO2peak (mean ± standard deviation: 24.9±2.42 to 27.6±3.39 ml/kg/min, p< 0.001), with no changes in control group. Exercise training decreased body weight (84.1±8.7 to 83.3±.9.7 kg, p=0.038) and gynoid fat mass (18.5±1.7 to 18.2±1.6%, p< 0.001). AIRg, ISR, HIE, CLp, aSAT, VAT and ectopic fat were unchanged after exercise training. However, the control group increased body weight and aSAT. The increase in SI and DI were not associated with changes in body composition, body fat distribution or ectopic fat. Conclusion: Novel results from our cross-sectional analysis showed that, in obese black South African women, DI was positively associated with peripheral insulin clearance, probably due to higher SI of peripheral tissue. Moreover, the most important correlate of a high DI was low VAT independent of ectopic fat accumulation in other sites. Further, we showed that low AIRg and high HIE correlated with a high VAT-aSAT ratio, while low SI was associated with high VAT. These associations require further exploration to determine direction of causality. Findings from our exercise intervention study extend on previous research by showing that moderate-to-high intensity combined aerobic and resistance exercise training increased SI and improved cardiovascular fitness, but insulin secretion, hepatic insulin clearance, ectopic and central fat depots did not change. Our results suggest that hyperinsulinemia may not occur solely as a compensatory mechanism for low SI and that ectopic and central fat might not be the primary correlates of insulin resistance in this cohort. Rather, intrinsic factors within muscle and adipose tissue may be putative mediators for observed improvements in the metabolic outcomes but will require further elucidation. Further research is required to confirm the causal role of VAT on low DI and to determine whether a long-term exercise training program and/or a low carbohydrate/glycemic index diet will reduce AIRg in those with hyperinsulinemia.

La prise en charge du diabète de type I se fait à l’heure actuelle par des injections pluriquotidiennes d’insuline ou par l’utilisation d’une pompe à insuline qui va mimer l’activité pancréatique. Dans ce contexte, les nanogels sensibles au glucose représentent des candidats à fort potentiel pour une délivrance contrôlée de l’insuline.La majorité des matériaux développés à ce jour ne présentent pas d’études en vue d’application in vivo et ce, pour diverses raisons telles que la non validation du caractère biocompatible et biorésorbable de la matrice polymère. Afin de répondre à ces deux critères, nous avons choisi de développer des nanogels à base de polysaccharides biocompatibles et biodégradables.Des travaux antérieurs au sein du laboratoire ont porté sur la conception d’hydrogels à base d’acide hyaluronique. Le polysaccharide a été fonctionnalisé avec des dérivés de l’acide phénylboronique (PBA) et du maltose. Ces modifications permettent dans des conditions physiologiques de générer des réticulations boronate-ester. Ces liaisons permettent d’induire une modification de la structure des hydrogels en réponse à divers stimuli tel que le pH ou l’addition de composés saccharidiques.Afin de faciliter l’administration de tels matériaux, nous avons étendu ce concept à la formation de nanogels. Des nanogels sensibles au pH et/ou à l’addition de saccharides ont pu être obtenus en conditions physiologiques grâce au choix judicieux des polysaccharides partenaires modifiés par le PBA et des molécules portant des fonctions diol. Ces nanogels sont capables de piéger l’insuline lors de leur formation avec une efficacité d’encapsulation allant de 45% à 80% et une capacité d’encapsulation de 10% à 60%. Les premiers tests ont montré un faible relargage de l’insulin par nos nanogels.Finalement, au vue de la sensibilité au pH de nos nanogels et l’environnement acide présent autour des tumeurs, leur utilisation pour le traitement du cancer a été étudié. Des analyses in vitro ont démontré une faible toxicité de nos gels sur les cellules cancéreuses. Les premières expériences in vivo ont montré la capacité des nanogels à circuler dans le sang<br>Type 1 diabetes management is currently done by multiple insulin injections or by the use of an insulin pump that will mimic pancreatic activity. In this context, glucose-sensitive nanogels represent high potential candidates for controlled delivery of insulin.The majority of materials developed so far are limited to biological in vitro studies, which is partly due to the non-biocompatibility and limited biodegradability of polymers used for the preparation of such materials. To fulfill these criteria, we proposed to develop nanogels based on biocompatible and biodegradable polysaccharides.Previous work in our laboratory focused on the design of boronate-crosslinked hydrogels based on hyaluronic acid. This polysaccharide was functionalized with derivatives of phenylboronic acid (PBA) and of maltose. The dynamic covalent boronate ester crosslinks between the polysaccharide chains enabled to induce a structural change of the hydrogel in response to various stimuli such as pH or addition of carbohydrate molecules.In order to facilitate administration of such materials, we extended the concept to the formation of nanogels. Sugar- and pH-sensitive nanogels could be successfully obtained in physiological conditions thanks to the judicious choice of the polysaccharide partners, bearing PBA moieties and diol-containing molecules.These nanogels can entrap insulin during their formation with an entrapment efficiency of 45% to 80% and a loading capacity ranging from 10% to 60%. Preliminary experiments indicated a low release of insulin from the nanogels.Finally, in view of the pH-sensitivity of these nanogels and the slight acidic pH of the tumor environment, we investigated their potential application for the treatment of cancer. In vitro experiment demonstrated a low toxicity of our nanogels on cancer cells. Preliminary in vivo experiments indicated that the nanogels can circulate in the bloodstream

O efeito de refeições ricas em carboidratos e lipídeos sobre a sensibilidade à insulina foi avaliado. Além disso, investigou-se o conteúdo da proteína GLUT4 em músculo esquelético e tecido adiposo branco. Ratos foram realimentados por 1, 2, 4 e 6 horas com: refeição balanceada (B); rica em carboidrato (C) e rica em lipídeo (L). O índice glicose/insulina revelou que C e L apresentavam resistência à insulina 2 horas após o início da ingestão. No teste de tolerância à insulina, uma redução (~47%) na sensibilidade à insulina foi observada em C após 2 e 4 horas de realimentação. O teste de tolerância à glicose confirmou a resistência à insulina em C e L após 2 horas de ingestão. Não houve alteração no conteúdo de GLUT4, nos momentos em que se verificou alteração na sensibilidade à insulina. Tais resultados indicam que, em ratos, refeições não balanceadas (alto teor de carboidrato ou alto teor de lipídeo), induzem menor sensibilidade à insulina 2 horas após o início da ingestão, e este fenômeno não envolve alterações no conteúdo de GLUT4 nos tecidos avaliados.<br>The effect of high carbohydrate and fat meals on the insulin sensitivity was evaluated. Furthermore, it was investigated the content of GLUT4 protein on the skeletal muscle and white adipose tissue. Rats were refed for 1, 2, 4 and 6 hours with: balanced meal (B); high carbohydrate meal (C) and high fat meal (L). The glucose/insulin index shows that C and L meals exhibited insulin resistance after 2 hours of ingestion. In the insulin tolerance test, a reduction (~47%) in the insulin sensitivity was observed in C group after 2 and 4 hours of refeeding. The glucose tolerance test confirmed the insulin resistance in C and L-groups after 2 hours of ingestion and such phenomena did not involve alterations in the GLUT4 content on both skeletal muscle or white adipose tissue.

L'insulino-résistance est un des mécanismes physiopathologiques associé au diabète de type II et à l'obésité. Améliorer la compréhension de ce mécanisme est nécessaire pour essayer de limiter l'extension de ces maladies considérées à l'heure actuelle comme de véritables épidémies. Une des causes de l'insulino-résistance réside dans l'accumulation intrahépatique et intramusculaire de lipides due à la rupture de la balance entre oxydation mitochondriale et lipogenèse de novo, perturbant la signalisation insulinique à l'origine de l'insulino-résistance. Connaître les facteurs induisant la modification de cette balance et la manière dont ceux-ci la déséquilibrent permettrait de prévenir l'insulino-résistance hépatique et musculaire. Nous avons souhaité approfondir, grâce à des études in vivo et in vitro, le rôle de l'acide nicotinique, agent hypolypémiant, des polyphénols, micronutriments présents dans les fruits et légumes et du fructose, molécule lipogénique. Ces composés agiraient comme modulateurs de la résistance à l'insuline et comme inducteurs du déséquilibre entre oxydation mitochondriale et lipogenèse de novo. Après avoir mis au point deux améliorations techniques (stabilité des solutions de [6,6-²H2]-glucose utilisées pour la mesure de la sensibilité à l'insuline et validation des mesures de dépenses énergétiques d'un nouveau calorimètre), nous avons montré que l'acide nicotinique est capable d'améliorer le profil lipidique plasmatique, tout en induisant une insulino-résistance hépatique dans une population d'hommes présentant une dyslipidémie mixte. Cette insulino-résistance, confirmée dans un modèle cellulaire d'hépatome humain, est reliée à l'inhibition d'une des enzymes « clés » de la lipogenèse de novo, la diacylglycérol acétyltransférase (DGAT). Cette inhibition enzymatique diminue les concentrations plasmatiques en triglycérides et induit une augmentation d'intermédiaires lipidiques à l'origine de l'insulino-résistance. Nous avons également mis en évidence le rôle insulinosensibilisateur d'un extrait de polyphénols de raisins rouges, lors d'un stress métabolique induit par la consommation de fructose dans une population de sujets à risque génétique de défaut d'oxydation mitochondriale. Cet effet insulino-sensibilisateur est dû à l'augmentation de l'activité et de la biogenèse mitochondriales réduisant l'effet lipogénique du fructose. Ces résultats montrent l'importance du lien entre rupture de la balance lipogenèse de novo oxydation lipidique et impact sur l'insulino-sensibilité. L'acide nicotinique est impliqué dans l'induction d'une insulino-résistance hépatique par inhibition de la DGAT, enzyme clé de la lipogenèse de novo. Les polyphénols sont impliqués dans l'augmentation de la sensibilité à l'insuline par amélioration de la fonctionnalité et de la biogenèse mitochondriales en réponse à une augmentation de la lipogenèse induite par le fructose

Insulin resistance is the major metabolic defect in obesity and Type 2 diabetes. Increased lipid accumulation is strongly associated with insulin resistance. A significant component of insulin resistance is thought to be a reduced ability of insulin to activate the cascade of phosphorylation events that lead to the metabolic effects of this hormone. The broad aims of this thesis were to examine the effect of high-fat diets containing different fat subtypes on in vivo insulin signalling, under conditions normally used to detect whole body insulin resistance, and to compare the effects of acute and chronic lipid oversupply on insulin signalling in vivo. Time-course and dose-response effects of insulin stimulation on site-specific phosphorylation of key signalling proteins were studied in rat tissues in vivo, to establish an appropriate experimental system to examine the onset of activation of the insulin signalling pathway. It was determined that short insulin infusions with concurrent glucose infusion, similar to the beginning of a euglycaemic-hyperinsulinaemic clamp, significantly increased the phosphorylation of major intermediates of the insulin signalling pathway in important tissues of insulin action (skeletal muscle [RQ], liver [LIV] and white adipose tissue [EPI]). These experiments provided a platform to study insulin signalling under the same conditions used to study lipid-induced insulin resistance. The provision of diets enriched in polyunsaturated or saturated fatty acids (FA) resulted in the corresponding enrichment of these fat subtypes in rat plasma and tissues. However, the effects on insulin signalling were essentially the same. Both fat diets induced defects in sitespecific phosphorylation of insulin receptor substrate (IRS)-1 and protein kinase B (PKB) in RQ and LIV, but not EPI. This suggests that the amount of fat in the diet, rather than enrichment in a particular fat subtype, had a greater impact on the development of signalling defects and that the response to high-fat feeding was tissue-specific. A 3hr elevation of circulating FA (using a lipid/heparin infusion), to a level that is relevant in clinical Type 2 diabetes, impaired insulin-stimulated PKB phosphorylation with no significant effect on IRS-1 phosphorylation. This suggests that there may be differences in the way acute and chronic exposure to increased FA impair insulin signalling. The phosphorylation defects observed in both chronic and acute studies did not seem to be associated with activation of major stress signalling pathways (JNK and NFkB), which have been suggested to have a role in lipidinduced insulin resistance. In conclusion, these studies demonstrate that impaired IRS-1 and PKB phosphorylation do have a role in the reduced insulin action observed with lipid oversupply in vivo, because the changes were detected under similar conditions as those used to determine whole body insulin resistance.

Hyperglycemia is prevalent in critical care, as patients experience stress-induced hyperglycemia, even with no history of diabetes. Hyperglycemia has a significant impact on patient mortality and other negative clinical outcomes such as severe infection, sepsis and septic shock. Tight glycemic control can significantly reduce these negative outcomes by reducing hyperglycemic episode, but achieving it remains clinically elusive, particularly with regard to what constitutes tight control and what protocols are optimal in terms of results and clinical effort. The model used in this thesis is validated using an independent data and readily be used for different clinical interventions. Moreover, this model also able to accurately predict clinical intervention outcomes given that the model prediction error is very small, which is better than any other reported model. In particular, model-based glycemic control methods is used to capture patient-specific physiological dynamics, such as insulin sensitivity, SI. To date, sepsis diagnosis has been a great challenge despite advancement in technologies and medical research. Critically, septic patients are often classified by practitioners according to their experience before standard test results can be assessed, as to avoid delay in treatment. Moreover, several scoring systems have also been widely used to represent sepsis condition and better standardization of sepsis definition across different centers. In this thesis, insulin sensitivity, SI, a model-based metric is used to identify sepsis condition based on the finding that SI represents metabolic condition of a patient. Additionally, several clinical and physiological variables obtained during patient’s stay in critical care are also investigated using mathematical computation and statistical analysis to identify relevant metric which can be accurately use for sepsis interventions. Even though information on SI, clinical and physiological variables of a patient are insufficient to determine the sepsis status, these informations have brought to a different perspective of diagnosing sepsis. Microcirculation dysfunction is very common in sepsis. Tracking of microcirculation state among septic patient enable better tracking of patient state particularly sepsis status. The tracking can potentially be done by using a pulse oximeter that can extract additional information related to oxygen extraction level. The processed signals are therefore represent relative absorption of oxyhemoglobin and reduced hemoglobin that can be used to assess microcirculation status. In addition, this thesis focus on the real challenge of early treatment of sepsis and sepsis diagnosis where several potential metabolic markers are investigated. Microcirculation conditions are assessed using a non-invasive method that is generally used in typical ICU settings. In particular, the concept and method used to assess microcirculation and metabolic conditions are developed in this thesis. Finally, the work presented in this thesis can act as a starting point for many other glycemic control problems in other environments. These areas include cardiac critical care and neonatal critical care that share most similarities to the environment studied in this thesis, to general diabetes where the population is growing exponentially world wide. Eventually, this added knowledge can lead clinical developments from protocol simulations to better clinical decision making.

Type 2 diabetes or adult onset diabetes, has been a global epidemic for the past two decades, and the number of new cases accelerates every year. Insulin resistance is one of the major factors behind this, wherein the insulin receptor, which signals to regulate glucose levels, based on the hormone insulin, loses its sensitivity. Obesity is one other major concern which is caused due to the improper balance between the caloric intake and the energy utilized. Gastric bypass surgeries (GBP) are performed to avert obesity. However, a beneficial side-effect is that the state of insulin resistance is reset to near baseline levels within a few days after the procedure. The reason behind this remains unexplained, with possible humoral effects, hypothesized to occur after the bariatric procedure. In this work, high-five insect cell line was utilized to recombinantly produce full length insulin receptors (IR). However commercially sourced IR ectodomains (eIR – soluble version of the full length IR with the completely extracellular α subunits along with extracellular and transmembrane regions of the β subunit), were used to study the interaction. Measuring the kinetics of IR-insulin interactions is critical to improving our understanding of this disease. In this study, a multiplex surface plasmon resonance (SPR) assay was developed for studying the interaction between insulin and the eIR. A scaffold approach was used in which anti-insulin receptor monoclonal antibody 83–7 (Abcam, Cambridge, UK) was first immobilized on the SPR sensorchip by amine coupling, followed by eIR capture. The multiplex SPR system (ProteOn XPR36TM, Bio-Rad Laboratories, Hercules, CA) enabled measurement of replicate interactions with a single, parallel set of analyte injections, whereas repeated regeneration of the scaffold between measurements caused variable loss of antibody activity. The main approach was to replicate the physiological IR-insulin interaction using this assay. It was also observed that insulin at higher concentrations tend to form dimers and hexamers in solution. This was tested using size exclusion chromatography analysis and proved to be true. Therefore an alternative analyte with the similar binding properties and affinity of insulin and at the same time with reduced self- association characteristics was explored. Lispro, the analogue of insulin with reduced self-association properties (generated by swapping of residue 28 and 29 with Lys and Pro respectively) was finally used to study the interaction with eIR. Interactions between recombinant human insulin with eIR-A (A isoform of the insulin receptor ectodomain) followed a two-site binding pattern (consistent with the literature), with a high-affinity site (dissociation constant KD1 = 38.1 ± 0.9 nM) and a low-affinity site (KD2 = 166.3 ± 7.3 nM). The predominantly monomeric insulin analogue Lispro had corresponding dissociation constants KD1 =73.2 ± 1.8 nM and KD2 =148.9 ± 6.1 nM, but the fit to kinetic data was improved when conformational change factor was included in which the high-affinity site was converted to the low-affinity site. Kinetics of interaction of insulin with eIR-A and eIR-B isoforms were then compared. eIR-A bound insulin with apparently higher affinity (with both the binding sites) when compared with eIR-B. This was again consistent with literature that IR-A had two-fold higher affinity for binding insulin than IR-B. The assay was further extended to study the effect of external factors such as glucose, visfatin on this interaction. Glucose (the main biomolecule which is regulated by the IR-insulin interaction) was tested, if it had any direct effect on the interaction. It was observed that glucose did not have any effect on eIR-insulin interactions. Visfatin, an adipocytokine which has been highly debated in literature for its insulin mimetic effects and IR binding properties, was then tested. The standard assay did not provide much insights as the reference channel immobilized with 83-7 monoclonal antibody to the receptor had much affinity for visfatin, leading to non-specific binding and negative responses. Therefore, in an alternative methodology was used - visfatin, Lispro and insulin were immobilized on separate channels along with bovine serum albumin immobilized on reference channel and eIR isoforms used as analyte to study the effect of visfatin on IR. This study showed that visfatin, a higher molecular weight protein compared to insulin, bound both the eIR isoforms. This is consistent with literature that visfatin binds IR at a site distinct from insulin, but the assay described here could not confirm the fact that it mimicked the signalling carried out by IR-insulin binding. Further studies are required to interpret the kinetics of visfatin-eIR interaction. To my knowledge, this is the first SPR assay developed to study eIR-insulin interactions in real-time. This could potentially be extended to study the interaction of insulin with full length insulin receptors and the effect of humoral and other external factors on the interaction, without the need for insulin labelling.

In a cross-sectional survey of coronary risk factors in subjects with recently diagnosed NIDDM I confirm the high prevalence of individual risk factors described by others, and show a broadly normal distribution pattern for the clustering of risk factors forming Reaven's Syndrome X, in contrast to that reported in other populations. I suggest that hyperinsulinaemia and hypertriglyceridaemia are the best markers of high coronary risk and note the association of obesity and other risk factors. In a study to examine the effects of the new oral hypoglycaemic drug A4166 in NIDDM I confirm its major effect as an insulin secretagogue but find limited effects on intermediary metabolism when assessed during and intravenous glucose tolerance test. Any enhancement of glucose clearance appears to be secondary to the increase in insulin secretion. If hypertriglyceridaemia exacerbates insulin resistance in NIDDM, then its treatment may reverse this process. In a randomised study comparing the fibrate drug gemfibrozil with placebo I show that reduction in serum triglyceride concentrations with gemfibrozil is associated with improved insulin sensitivity to non-esterified fatty acid (NEFA) and ketone metabolism but not to glucose metabolism, when assessed using a low-dose incremental insulin infusion technique. In established diabetes (NIDDM) the contribution of body fat to insulin resistance is less clear than among non-diabetic subjects. In a group of NIDDM subjects I show no consistent effects of body fat on insulin resistance using the low-dose incremental insulin infusion, and no relation between body fat indices and euglycaemic clamp measures of insulin sensitivity. From the insulin infusion data I show that fasting glucose concentration has the greatest impact on insulin resistance, suggesting that hyperglycaemia or perhaps glucose toxicity has an effect overwhelming that of other factors. Using the data from the body fat studies described, I compare measures of insulin resistance derived from insulin infusions, euglycamic clamps and homeostatic modelling (HOMA-R) in NIDDM, and discuss uses and limitations of these techniques. In a concluding chapter I attempt to summarise the findings from these studies and draw together a discussion of the results obtained in the context of the existing published literature.

The first event in insulin action is the binding of insulin to insulin-receptors embedded in the cell membrane. This study investigated how plasma membrane cholesterol influences this interaction, as examined in several experimental models including cultured cells, virus-like-particles and liver cell plasma membranes. The study is of relevance to a better understanding of diabetes at a molecular level. Novel results include the demonstration that alterations in membrane cholesterol content significantly reduce insulin binding and signalling.

CDK4 est une sérine/thréonine kinase qui est largement décrite pour son implication dans le contrôle du cycle cellulaire. Notre laboratoire et d'autres ont montré qu'elle jouait également un rôle majeur dans le contrôle de l'homéostasie du glucose (croissance des cellules β du pancréas et sécrétion d'insuline) et des lipides (adipogenèse). Nous avons montré au cours de cette thèse, par le biais de deux modèles de souris, invalidés pour CDK4 (Cdk4-/- ;cre/cre) ou exprimant un mutant hyperactif de la kinase (Cdk4R24C/R24C), qu'elle est un médiateur important de la voie de l'insuline et régule la lipogenèse et la lipolyse. Les souris Cdk4-/- ;cre/cre ont une diminution significative de la taille des adipocytes et du poids du WAT ou l'inverse est observé sur les souris Cdk4R24C/R24C. CDK4 est activée par l'insuline et va ainsi promouvoir le transport de glucose, la synthèse des lipides de novo et réprimer la lipolyse dans les adipocytes. De plus, nous avons démontré que dans l'adipocyte, cellule non proliférative, CDK4 et son partenaire la cycline D3 sont préférentiellement localisés dans le cytoplasme suggérant un rôle indépendant de leurs fonctions nucléaires. Nous avons identifié deux nouveaux substrats de CDK4 : IRS1 et IRS2. CDK4 phosphoryle IRS1 et IRS2 activant un rétrocontrôle positif permettant le maintien de l'action de l'insuline sur les adipocytes. Nos résultats prouvent un nouveau rôle de CDK4 sur la signalisation de l'insuline et sa fonction dans l'adipocyte. Par conséquent, la modulation de son activité pourrait avoir des conséquences majeures sur le mécanisme de résistance à l'insuline, une complication fréquente dans le développement de pathologies comme le diabète et l'obésité<br>CDK4 is a serine/threonine kinase mainly known by its involvement in the control of cell cycle progression. Our laboratory and other laboratories have previously shown a major role for CDK4 in the control of glucose homeostasis (pancreatic β-cell growth) and lipid homeostasis (adipogenesis). In this thesis, we showed that CDK4 is an insulin effector that controls lipogenesis and lipolysis in mature adipocytes. We used Cdk4-/- ;cre/cre mice and Cdk4R24C/R24C mice, carrying a hyperactive mutant Cdk4 allele, for this study. Cdk4-/ - ;cre/cre mice have a manifest adipose tissue phenotype with a significant decrease in body weight and WAT mass. On the other hand, Cdk4R24C/R24C mice show increased body weight and increased adiposity. Furthermore, we demonstrate that CDK4 is activated by insulin to promote glucose transport, lipogenesis and repress lipolysis in adipocytes. Interestingly, we showed that in mature quiescent adipocytes CDK4 and its partner, Cyclin D3, are preferentially localized in the cytoplasm, suggesting a role independent from their nuclear functions. We identified two novel substrates of CDK4: IRS1 and IRS2. CDK4 phosphorylates both IRS1 and IRS2 in order to sustain insulin signaling in adipocytes via a positive feed-back loop. To sum up, our results identify a new function of CDK4 on insulin signaling in adipocyte metabolism. Thus, the modulation of its activity could have consequences on insulin resistance, a common complication of obesity and diabetes