Orientador: José Antonio Rocha Gontijo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T06:22:30Z (GMT). No. of bitstreams: 1 Possignolo_LuizFernando_M.pdf: 4767580 bytes, checksum: b843458e4c7049fc29741e4aca9f918d (MD5) Previous issue date: 2012 / Resumo: Devido ao maior consumo de alimentos ricos em gordura e um estilo de vida mais sedentário, houve um aumento na incidência de desordens metabólicas relacionadas ao metabolismo lipídico como a resistência à insulina, dislipidemias, e sua associação com doenças cardiovasculares. A alimentação materna desequilibrada, durante a gestação e lactação, pode predispor a prole à doenças durante a vida adulta. Alguns transportadores como o Scavenger Receptor class B type I (SR-BI) e ATP-binding cassette transporter A1 (ABCA1) são descritos como responsáveis pela captação de colesterol e transporte deste a partir de lipoproteínas, principalmente no transporte reverso de colesterol, sendo denominados receptores antiaterogênicos podendo modificar seu padrão de expressão frente a uma dieta hiperlipídica. Ratas Wistar receberam dieta hiperlipídica (DHL) ou dieta padrão (CTL) desde o desmame, até a lactação. Após o desmame a prole de machos recebeu a dieta padrão até a 16ª semana de vida. Nas mães quanto e na prole foram analisados os seguintes parâmetros: ingestão alimentar, ganho ponderal, perfil lipídico e glicídico. Na prole foi estudada a expressão e localização de ABCA1 e SR-BI na placenta, no rim, fígado, e intestino delgado em animais com 17 dias pré-natal (E17), 12 dias pós-natal (PN12d) com 8 e 16 semanas pós-natal (PN8s e PN16s). As mães DHL apresentaram: 1) maior ingestão calórica com menor ganho ponderal; 2) aumento glicêmico associado à menor produção de insulina nos três períodos estudados e, 3) aumento nos níveis séricos de triglicérides em M8s. A prole de mães DHL apresentaram menor massa corporal desde E17 até PN8s, sem que tenha havido diferenças ponderais e na ingestão de ração. PN8s e PN16S apresentaram menor captação tissular de glicose associada à hiperinsulinemia, e aumento nos níveis séricos de triglicérides com PN16S. Não houve alterações nos níveis de colesterol e HDLcolesterol. Não foi observada alteração na expressão de SR-BI e ABCA1 no intestino delgado, placenta enquanto no fígado houve uma queda tempo-dependente para ambos transportadores. No rim da prole DHL aos PN12d e PN16s observou-se maior expressão de ABCA1. Este estudo mostrou que o consumo materno crônico de uma dieta hiperlipídica causa alterações metabólicas nas mães e predispõe a prole, a modificações no metabolismo lipídico e glicídico, além de elevação da expressão de ABCA1 no rim / Abstract: Due to the abundance and accessibility to foods high in fat and a more sedentary lifestyle, there is an increased incidence of metabolic disorders related to lipid metabolism such as insulin resistance, dyslipidemia, and a high correlation with cardiovascular disease. The unbalanced maternal diet during pregnancy and lactation predisposes the offspring to diseases during adult life. Some carriers such as scavenger receptor class B type I (SR-BI) and ATP-binding cassette transporter A1 (ABCA1) are described as responsible for raising cholesterol and transporting it to and from lipoproteins, due the participation in the reverse cholesterol transport, these receptors are called antiatherogenic and are subject to change its pattern of expression when exposed to a high fat diet. Female Wistar rats were fed a diet (HFD) or a standard chow (CTL) from weaning, during pregnancy and lactation. After weaning the male offspring was exposed to a standard chow until the 16th week of life. Both the dams and the offsprings food intake were monitored, weight gain, lipid profile and glucose level. It was analyzed in the offspring the expression and localization of ABCA1 and SR-BI in the placenta, kidney, liver, and small intestine in animals at 17º prenatal day (E17), 12 days post-natal (PN12d), 8 and 16 postnatal weeks (PN8s PN16s respectively). DHL dams had a higher intake of calories in the diet, but the weight was smaller, they had higher blood glucose due to decreased production of insulin in the pre-pregnancy (m8s), during pregnancy (M17g) and lactation (M15l) and a higher triglyceride level in m8s. The offspring of dam fed a high fat diet had lower weight since E17 until PN8s, with no differences in weight gain and food intake. PN8s and PN16s had lower glucose uptake and hyperinsulinemia, and high triglycerides with PN16s, no changes were observed in cholesterol and HDL-cholesterol levels. There were no changes in the expression of SR-BI and ABCA1 in the small intestine, placenta and liver, however there was a decrease over the age for both receptors, and kidney of the offspring and DHL PN12d PN16s showed higher expression of ABCA1. The present study showed that chronic consumption of high fat diet causes metabolic changes in dams and predisposes offspring to changes in lipid and glucose metabolism of the offspring, increasing the expression of ABCA1 in the kidney / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

Orientador: Marcos Antonio Tambascia / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T11:51:28Z (GMT). No. of bitstreams: 1 Comucci_EleonoraBeltrame_M.pdf: 3925973 bytes, checksum: d7cf8e386ee2039949574917c2eba506 (MD5) Previous issue date: 2012 / Resumo: Objetivos: avaliar os níveis séricos de RBP4 em pacientes obesas diabéticas em comparação a pacientes não diabéticas na pré-menopausa. Avaliar a correlação da concentração sérica de RBP4 com marcadores bem estabelecidos de RI e SM. Comparar entre os grupos a porcentagem de gordura corporal (%GC) e espessura da camada íntima-média carotídea (EIMC), verificando a influência do DM2 nos grupos. Métodos: foram avaliadas mulheres (n = 139) divididas em três grupos: Grupo 1 (Controles; n = 45); Grupo 2 (Obesas; n = 53); Grupo 3 (Obesas DM2; n = 41), denominados G1, G2 e G3. Foram avaliados parâmetros bioquímicos, antropométricos e de composição corporal. Resultados: houve diferença estatística significante entre os grupos nos parâmetros relativos à caracterização da amostra quanto aos perfis esperados da síndrome metabólica, DM2 e obesidade. Porém, os níveis de RBP4 não se comportaram como esperado se comparado aos primeiros estudos à respeito da proteína carreadora do retinol. Conclusões: A RPB4 não se mostrou um bom marcador de RI e risco cardiovascular, pois entre os grupos sua distribuição não foi uniforme. De acordo com a análise dos dados obtivemos em G1 correlações positivas da RBP4 com IMC (p<0,05), hemoglobina glicada (p<0,05), insulinemia de jejum (p<0,05); em G2 com hemoglobina glicada (p<0,01); G3 com hemoglobina glicada (p<0,05), glicemia de jejum (p<0,01), HOMA-IR (p<0,01) / Abstract: Objectives: to evaluate the levels of serum RBP4 in premenopausal obese diabetic patients compared with nondiabetic patients. To evaluate the correlation between serum RBP4 with well-established markers of IR and MS. Compare between groups the percentage of body fat (% BF) and thickness of carotid intima-media (CIMT), checking the influence of T2DM into groups. Methods: We evaluated 139 women which were divided into 3 groups: Group 1 (Control, n = 45), Group 2 (obese, n = 53) and group 3 (obese T2DM, n = 41), known as G1, G2 and G3. We assessed biochemical, anthropometric and body composition parameters. Results: There was no statistically significant difference between groups in the parameters for sample characterization regarding the expected profiles of metabolic syndrome, type 2 diabetes and obesity. However, RBP4 levels did not behave as expected when compared to the first studies about the retinol-binding protein. Conclusions: The RPB4 is not a good marker of IR and cardiovascular risk, because distribution between the groups was not uniform. According to data analysis, we obtained in G1 positive correlations of RBP4 with BMI (p <0.05), and glycated hemoglobin (p <0.05), fasting insulin (p <0.05); in G2 with glycated hemoglobin (p <0.01); G3 with glycated hemoglobin (p <0.05), fasting glucose (p <0.01), HOMA-IR (p <0.01) / Mestrado / Clinica Medica / Mestre em Clinica Medica

L’huile de palme est l’huile végétale la plus consommée au monde. Du fait de sa teneur élevée en acides gras saturés (AGS), notamment en acide palmitique, cette huile est considérée par certains auteurs comme potentiellement nocive pour la santé. Cette étude avait pour objectif de comparer les effets de l’huile de palme (rouge ou oléine), à l’huile d’olive (réputée bonne pour la santé) et aux saindoux (riche en AGS), sur la santé. Pour réaliser cette étude, 40 rats mâles Wistar ont été répartis en 5 groupes de 8 rats chacun : 1 groupe contrôle et 4 groupes nourris par des régimes obésogènes contenant respectivement de l’huile de palme rouge, de l’oléine de palme, de l’huile d’olive ou du saindoux. Après 12 semaines de régime, les rats ont été sacrifiés et les tissus prélevés. Les examens réalisés sur les tissus ont montré que l’huile de palme (rouge ou oléine) induit un statut antioxydant et un profil lipidique superposables à ceux de l’huile d’olive. Tous les régimes obésogènes ont favorisé la prise de poids, l’altération de la fonction mitochondriale et la perturbation du métabolisme glucidique par l’induction d’une insulino-résistance. Il ressort de cette étude que l’huile d’olive est plus délétère pour le foie que l’huile de palme (rouge ou oléine) et le saindoux. Hormis l’huile de palme rouge, l’oléine de palme, l’huile d’olive et le saindoux influencent négativement les tissus adipeux. Les études menées sur l’aorte ont montré que les effets vasculaires de l’huile de palme sont moins délétères pour l’aorte que le saindoux et l’huile d’olive.Les résultats de cette étude indiquent que globalement, l’huile de palme (rouge ou oléine) n’a pas d’effets délétères supérieurs à ceux de l’huile d’olive concernant les organes qui ont été étudiés / Palm oil is the most consumed vegetable oil in the world. Because of its high content of saturated fatty acids (SFA), particularly palmitic acid, this oil is considered by some authors as potentially harmful to health. The aim of this study was to compare the effects of palm oil (red or olein), olive oil (considered good for health) and lard (rich in SFA), on health. To do this, 40 male Wistar rats were divided into 5 groups of 8 rats each: 1 control group et 4 groups fed by high fat diet (HFD) containing respectively red palm oil, palm olein, olive oil or lard. After 12 weeks of diet, the rats were sacrificed and the tissues removed. Tissue tests have shown that palm oil (red or olein) induces an antioxidant status and a lipid profile superimposed on those of olive oil. All HFD contributed to weight gain, impaired mitochondrial function, and disturbance of carbohydrate metabolism by the induction of insulin resistance. The study shows that olive oil is more deleterious to the liver than palm oil (red or olein) and lard. Apart from red palm oil, palm olein, olive oil and lard negatively influence adipose tissue. Studies on the aorta have shown that the vascular effects of palm oil are less deleterious to the aorta than lard and olive oil.Overall, the results of this study show that harmfull effects of palm oil (red or olein) were not worse than that of olive oil on organ that were analyzed

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Type 2 diabetes mellitus (DM2) is a multifactorial disease mainly characterized by metabolic disorders related to insulin. Hyperglycemia is one of the causes of excessive generation of reactive oxygen species (ROS) leading to oxidative stress. In addition, DM2 is associated with increased inflammatory response. There is an intensive search for novel therapeutic drugs because the synthetic drugs currently used have side-effects and have their effectiveness reduced along the time of use. The jaboticaba (Myrciaria jaboticaba Vell Berg) peel powder (JAB) exhibit high antioxidant capacity and anti-inflammatory action due to the presence of polyphenols. The aim of this study was to evaluate the potential of JAB to prevent biochemical changes, oxidative stress, inflammatory response and pancreatic damage in a DM2 model induced by high-fat diet and a singular injection of streptozotocin (STZ; 35 mg/kg) in Wistar rats. After DM2 induction, instead of drinking water, the animals received vehicle (water containing 0.5% carboxymethyl cellulose) or JAB at 2.7 (JAB-I), 5.4 (JAB-II) or 10.8 (JAB-III) g/L of drinking water (equivalent to 0.07, 0.14 and 0.28 g anthocyanins/L, respectively). After 8 weeks of treatment animals were killed to evaluate the glycemia, insulinemia, oxidative stress and inflammatory markers were evaluated in the serum, besides antioxidant enzymes activities were evaluated in the blood and the histological changes in pancreatic tissue. Increased glycemia and fructosamine levels, insulin resistance and epididymal fat were observerd in the DM2 rats beyond decreased number and area of pancreatic islets. All JAB doses prevented the increase in glycemia over time, whereas JAB-III also reduced the end glycemia, fructosamine levels and the insulin resistance. JAB-I and JAB-II prevented body weight gain over the experiment, whereas JAB-I also decreased the amount of epididymal fat. Furthermore, JAB-I and JAB-III increased the area of pancreatic islets of diabetic rats. In addition, JAB-II and JAB-III treatment reduced the levels of oxidized LDL, whereas JAB-II treatment also reduced protein oxidation in diabetic rats. JAB treatment also prevented the decrease in the activities of glutathione peroxidase, catalase and thioredoxin reductase in the diabetic rats but did not change the decrease in the activity of superoxide dismutase. Only JAB-III treatment prevented the diabetes-induced increase in the inflammatory markers (IL-6, IL-1 and TNF-α). These findings suggest that JAB could have a beneficial effect against diabetes, reducing the hyperglycemia, the insulin resistance, oxidative stress and inflammatory response, besides decreasing pancreatic damage. / O diabetes mellitus tipo 2 (DM2) é uma doença multifatorial caracterizada principalmente por desordens metabólicas relacionadas à insulina. A hiperglicemia é uma das causas da geração excessiva de espécies reativas de oxigênio (EROs) que leva ao estresse oxidativo. Além disso, o DM2 está associado a aumento da resposta inflamatória. Há uma intensa busca por novos medicamentos terapêuticos, pois os medicamentos sintéticos utilizados atualmente apresentam efeitos adversos e tem sua eficácia reduzida ao longo do tempo de uso. O pó de casca de jabuticaba (JAB) (Myrciaria jaboticaba (Vell.) Berg.) apresenta alta capacidade antioxidante e ação anti-inflamatória devido à presença de polifenóis. O objetivo deste trabalho foi avaliar o potencial do JAB em prevenir alterações bioquímicas, estresse oxidativo resposta inflamatória e dano pancreático em modelo de DM2 induzido por dieta hipercalórica e administração única de estreptozotocina (STZ, 35 mg/kg) em ratos Wistar. Após indução do DM2, em vez de água potável, os animais receberam veículo (água acrescida de 0,5% de carboximetilcelulose) ou JAB em 2,7 (JAB-I), 5,4 (JAB-II) e 10,8 (JAB-III) g/L (equivalente a 0,07, 0,14 and 0,28 g antocianinas/L, respectivamente). Após 8 semanas de tratamento os animais foram mortos para avaliar a glicemia, insulinemia, estresse oxidativo e marcadores inflamatórios avaliados no soro, além da atividade das enzimas antioxidantes avaliadas no sangue total e as alterações histológicas no tecido pancreático. O aumento da glicemia e dos níveis de frutosamina, resistência à insulina e gordura epididimal foram observados em ratos com DM2, além da redução do número e área das ilhotas pancreáticas. Todas as doses do JAB preveniram o aumento da glicemia ao longo do tempo, enquanto que JAB-III também reduziu os níveis de glicemia final, frutosamina e a resistência a insulina. JAB-I e JAB-II preveniram o ganho de peso corporal ao longo do experimento, enquanto que JAB-I também reduziu a quantidade de gordura epididimal. Além disso, JAB-I e JAB-III aumentaram a área das ilhotas pancreáticas dos ratos diabéticos. Adicionalmente, os tratamentos JAB-II e JAB-III reduziram os níveis de LDL oxidada, enquanto que o tratamento JAB-II também reduziu a oxidação proteica em ratos diabéticos. O tratamento com JAB também preveniu a redução nas atividades das enzimas glutationa peroxidase, catalase e tiorredoxina redutase nos ratos diabéticos, mas não alterou a redução na atividade da superóxido dismutase. Apenas o tratamento JAB-III preveniu o aumento dos marcadores inflamatórios (IL-6, IL-1 e TNF-α) induzidos no diabetes. Estes resultados sugerem que JAB poderia ter um efeito benéfico contra o diabetes, reduzindo a hiperglicemia, a resistência à insulina, estresse oxidativo e resposta inflamatória, além de diminuir o dano pancreático.

Abstract Abdominal obesity in adults is associated with insulin resistance. The purpose of the present study was to examine insulin resistance-associated abnormalities during military service as well as the effect of smoking on these abnormalities. In 1995 we invited all the 1268 servicemen attending military service in the Ostrobothnian Brigade and in 1997 all the 106 servicemen in the First Signal Company for measurements at the beginning of their service. Metabolic syndrome was defined: hyperinsulinemia (fasting insulin ≥ 13.0  mU/l) and dyslipidemia (triglycerides ≥ 1.7 mmol/l and/or total cholesterol/HDL cholesterol > 5) at the same time. Metabolic syndrome was present in 10% of the servicemen who had a body mass index > 27 kg/m2. Metabolic syndrome was present in 1% of all servicemen. None of the servicemen of normal weight had metabolic syndrome. Smoking increased the prevalence of metabolic syndrome sixfold. The mean weight of servicemen in the Ostrobothnian Brigade increased by 4,6 kg during the service. In the First Signal Company, weight did not change during the service. Total cholesterol, HDL and LDL cholesterol increased in both groups. Two out of three servicemen had increased their exercise activity and their consumption of doughnuts/confectionary during the service. Consumption frequence of doughnuts increased by 166%. Apparently, the changes in the diet had a major effect on lipids, and the beneficial effect of increased exercise could not compensate for it. Smokers had lower levels of adiponectin than non-smokers at the beginning of the service. During the service, the levels of adiponectin decreased. The decrease of adiponectin was related to a decrease in QUICKI but not to smoking. Thus, the decreased adioponectin level was due to a decrease of insulin sensitivity caused by diet. Overweighted servicemen should decrease their weight. I suggest that preventive health education should be pointed to overweighted servicemen. The aim should be to decrease obesity and to abandon smoking. By these means we could reduce or prevent arteriosclerosis and diabetes in the future. This is a common challenge for primary health care in Finland and the Finnish military forces. / Tiivistelmä Aikuisten keskivartalolihavuuteen liittyy insuliiniresistenssiä ja metabolista oireyhtymää, jotka ennakoivat tulevaa diabetestä ja valtimonkovetustautia. Nuorten ylipainoisuus on huomattavasti yleistymässä. Useat nuoret tupakoivat. Tässä väitöskirjatyössä tutkittiin insuliiniresistenssiin liittyviä kardiovaskulaarisia riskitekijöitä varusmiespalvelun aikana ja tupakoinnin yhteyttä näihin riskitekijöihin. Tutkimusaineiston muodostivat kaikki 1 268 Pohjan Prikaatissa vuonna 1995 ja kaikki 106 1. Viestikomppaniassa tammikuussa 1997 palvelunsa aloittanutta varusmiestä. Metabolinen oireyhtymä (MBO) määriteltiin siten, että varusmiehellä oli yhtä aikaa hyperinsulinemia (paastoinsuliini ≥ 13,0 mU/l) ja dyslipidemia (triglyseridipitoisuus ≥ 1,7 mmol/l ja/tai kokonaiskolesteroli/HDL-kolesteroli > 5). Näin määritelty MBO löytyi 10 %:lta niistä varusmiehistä, joiden painoindeksi oli yli 27 kg/m2. MBO esiintyi 1 %:lla kaikista varusmiehistä. Kenelläkään normaalipainoisella ei havaittu metabolista oireyhtymää. Painoindeksi yli 27 kg/m2 esiintyi 11 %:lla varusmiehistä. Tupakointi lisäsi MBO:n esiintymisriskiä kuusinkertaisesti. Pohjan Prikaatin varusmiesten paino lisääntyi 4,6 kg, mutta 1. Viestikomppanian varusmiesten paino ei muuttunut varusmiespalvelun aikana. Kuitenkin kokonaiskolesterolin, HDL- ja LDL-kolesterolin pitoisuudet lisääntyivät molemmilla. Ravinto- ja liikuntakyselytutkimus osoitti, että kahdella kolmesta varusmiehestä liikunnan määrä ja munkkien/kahvileipien käyttö lisääntyi varusmiespalvelun aikana. Munkkien käyttökerrat lisääntyivät 166 %. Ilmeisesti ruokavalion muutoksen aiheuttama "rasvarasitus" ja sen haitallinen vaikutus lipideihin oli niin merkittävä, että se mitätöi lisääntyneen liikunnan tuoman hyödyn. Tupakoivien adiponektiinipitoisuudet olivat varusmiespalvelun alussa matalampia kuin tupakoimattomien. Adiponektiinipitoisuudet vähenivät varusmiespalvelun aikana tupakoivilla ja tupakoimattomilla. QUICKI-indeksin pieneneminen oli itsenäinen selittäjä adiponektiinipitoisuuden muutokselle. Siten adiponektiinipitoisuuden vähenemisen syy oli ruokavalion aiheuttama insuliiniherkkyyden heikkeneminen varusmiespalvelun aikana ja se ei riippunut tupakoinnista. Ylipainoiset kutsuntaikäiset nuoret miehet ja varusmiehet tulisi saada laihtumaan. Suosittelen juuri heille suunnattua täsmäehkäisyä, joka tähtää painonpudotukseen ja tupakoimattomuuteen, sillä ylipainolla ja tupakoinnilla on selvä haitallinen vaikutus insuliiniresistenssiin liittyviin kardiovaskulaarisiin riskitekijöihin jo nuoruusvuosien aikana. Puuttumalla ajoissa vaaratekijöihin valtimonkovetustaudin ja diabeteksen kehittymistä voidaan hidastaa tai estää. Tämä on yhteinen haaste suomalaiselle perusterveydenhuollolle ja puolustusvoimille.

Abstract Gestational diabetes (GDM) indicates increased risk for diabetes and the metabolic syndrome in women. Research on prenatal exposure to GDM as a risk factor for metabolic diseases is conflicting. Overweight (body mass index ≥ 25 kg/m2) is a strong risk factor for GDM and metabolic diseases; however, there are few published previous studies distinguishing the separate effects of overweight and GDM on the later risk for metabolic diseases in women and their children. The present study evaluated pre-pregnancy overweight and GDM as determinants of long-term risk for diabetes and hypertension in women, and the metabolic consequences of prenatal exposures to maternal pre-pregnancy overweight and different types of maternal diabetes in children. The results are based on prospective, clinical data from Oulu University Hospital (n = 63 mothers and their children), and the Northern Finland Birth Cohort 1986 (NFBC 1986, n = 9,362 mothers and their 9,479 children). Compared to normal-weight mothers with normal glucose tolerance in pregnancy, the NFBC 1986 mothers with simultaneous pre-pregnancy overweight and GDM had strikingly high risks for developing diabetes (hazard ratio, HR 47.2; 95% confidence interval 25.5–87.4) and hypertension (HR 9.2 [6.1–13.9]) twenty years after delivery. The risks for these diseases were elevated in mothers with pre-pregnancy overweight even when they had normal glucose tolerance during pregnancy (HR diabetes 12.6 [7.4–21.6], HR hypertension 2.9 [2.1–3.9]). GDM per se indicated increased risk only for diabetes (HR 10.6 [4.2–27.0]). In the cohort from Oulu University Hospital, increased fasting insulin concentration (P = 0.04), first phase insulin response (P = 0.03), and HOMA-B (P = 0.008) were already observed at pre-school age in the offspring of mothers with Type 1 diabetes compared with offspring of mothers with GDM. In the NFBC 1986 offspring, the prevalence of metabolic syndrome was 2.4% at age 16 years, using the International Diabetes Federation pediatric definition. Abdominal obesity, a waist girth over half one’s length, defined approximately 85% of the adolescents with metabolic syndrome. The risks for overweight and abdominal obesity were high in those with prenatal exposure to both maternal pre-pregnancy overweight and GDM (odds ratio for overweight 4.1 [1.9–8.6], for abdominal obesity 3.8 [1.7–8.8]). In children of normal-weight women, prenatal exposure to GDM was not associated with increased risk of these outcomes. Based on this study, preventing and reducing overweight in fertile age seems to be a key target for preventing metabolic diseases in women and their children.

L’obésité est une pathologie multifactorielle qui contribue au développement de l’insulinorésistance (l’IR). De nos jours, même si de nombreux travaux ont permis de répondre à plusieurs questions relatives à la pathogenèse de l’IR, les mécanismes cellulaires et moléculaires qui sous-tendent la pathogenèse de l’IR ne sont pas encore complètement élucidés chez l’Homme. Deux hypothèses principales se sont dégagées de ces travaux.D’une part, certaines études suggèrent que le défaut primaire à l’origine de l’IR se trouverait au niveau du tissu adipeux, qui en libérant dans la circulation systémique des adipokines, des cytokines inflammatoires et des acides gras contribue au développement d’une inflammation et d’une hyperlipidémie systémique. Ces deux paramètres exercent des effets délétères sur la sensibilité à l’insuline des autres organes comme le muscle et le foie.D’autre part, le muscle étant l’organe gluco-régulateur par excellence en situation postprandiale, d’autres études le place au cœur de la physiopathologie de l’IR.Malgré ces controverses, aucune étude n’a à ce jour exploré simultanément l’inflammation et l’IR systémique /tissulaire dans le muscle et le tissu adipeux chez un même sujet.Dans ce contexte, l’objectif de ce travail de thèse était de mieux comprendre les mécanismes impliqués dans la mise en place de l’IR. Il s’inscrit dans le cadre d’un projet de recherche translationnel, qui compare dans une cohorte de femmes ménopausées, des sujets minces à des sujets obèses de grade I insulinosensibles (OIS) et insulinorésistants (OIR). Plusieurs paramètres systémiques et tissulaires (lipotoxicité, inflammation et activation du récepteur toll like receptor 4 (TLR4)) impliqués dans la physiopathologie de l’IR ont été analysés chez ces sujets.Les résultats de cette étude mettent en avant l’importance de l’activation des voies de l’immunité innée dans la régulation de la sensibilité à l’insuline. Ainsi, alors qu’aucune inflammation systémique n’est détectée, on observe une activation différentielle des voies de signalisation du récepteur TLR4 de l’immunité innée entre le tissu musculaire et le tissu adipeux des sujets OIR. La voie MyD88-dépendante qui est une voie pro-inflammatoire, est activée dans le muscle squelettique de ces sujets et est associée à une IR au sein de ce tissu. A l’inverse, la voie TRIF-dépendante qui est une voie anti-inflammatoire est activée au sein du tissu adipeux et permet le maintien de la sensibilité à l’insuline. Ce maintien de la réponse à l’insuline se fait grâce l’induction du système interféron et de l’enzyme anti-oxydante manganèse superoxyde dismutase (MnSOD).Dans ce travail, nous montrons d’une part, que le défaut de réponse à l’insuline musculaire se trouve au cœur de la physiopathologie de l’IR chez les sujets obèses de grade I. D’autre part, ces résultats mettent en exergue l’importance de la balance de régulation des voies de l’immunité innée « MyD88 » et « TRIF » dans le maintien de la sensibilité à l’insuline. / Obesity is a multifactorial disease promoting the development of insulin resistance (IR). Nowadays, cellular and molecular mechanisms underlying IR pathogenesis in humans are not fully understood although many studies have been attempted to improve our knowledge. Tow hypotheses arose from these researches.On one hand, several studies have led to the idea that a chronic inflammatory state combined with adipose tissue (AT) dysfunction could be the so-called “central mechanism” leading to IR. AT dysfunction is associated with increase in the release of inflammatory adipokines/cytokines and free fatty acid (FFA), leading to systemic inflammation and lipid overload. These latter parameters would have deleterious effects on diverse organs such as muscles and liver by affecting their insulin sensitivityOn the other hand, skeletal muscle (SM) is responsible for 80% of glucose uptake and metabolism in postprandial state and muscle failure in this function is often considered as the first defect causing IR.Interestingly, despite these controversies, in human, insulin sensitivity and the onset of inflammation have so far never been investigated simultaneously at systemic level and locally in SM and AT in the same individual.In this context, the aim of this thesis was to better understand the mechanisms involved in IR development in grade I obese subject. It is based on a translational research project that compares in a cohort of postmenopausal women, lean subjects with grade I obese insulin-sensitive (OIS) and insulin-resistant (OIR) subjects. Several systemic and tissue parameters (lipotoxicity, inflammation and toll like receptor 4 (TLR4) activation), involved in the IR pathophysiology were analyzed in these subjects.Our results highlight the importance of innate immunity pathways activation in the regulation of insulin sensitivity. Thus, while no inflammation was detected at the systemic level, there is a differential activation of innate immune TLR4 signaling pathways between muscle and AT of OIR subjects. The MyD88-dependent pathway, a pro-inflammatory pathway, is activated in their SM and is associated with IR in this tissue. Conversely, the TRIF-dependent pathway which is an anti-inflammatory pathway is activated in the AT thus maintaining insulin sensitivity in this tissue. This is supported by the induction of interferon system and the antioxidant enzyme manganese superoxide dismutase (MnSOD).In this work, we show that SM defect is the central mechanism which determines IR in grade I obese subject. We have also highlighted the importance of regulation of the balance between the two innate immunity pathways "MyD88" and "TRIF" in maintaining insulin sensitivity.

The ability of insulin to stimulate glucose uptake into muscle and adipose tissue is central to the maintenance of whole-body glucose homeostasis. Deregulation of insulin action manifests itself as insulin resistance, a key component of type 2 diabetes. Insulin resistance is also observed in HIV patients receiving protease inhibitors. An agent that can reversibly induce an insulin-resistant state would be a very useful tool in developing model systems that mimic the pathogenesis of type 2 diabetes. Insulin resistance can arise from defects in insulin signal transduction, changes in the expression of proteins or genes that are targets of insulin action, cross talk from other hormonal systems or metabolic abnormalities. Deterioration of the insulin-receptor-signalling pathway at different levels leading to decreased levels of signalling pathway intermediates and/or decreased activation through phosphorylation accounts for the evolution from an insulin-resistant state to type 2 diabetes. In addition, defects in GLUT4 glucose transporter translocation are observed, further fuelling impairments in skeletal muscle glucose uptake. Levels of insulin-induced GLUT4 translocation in the skeletal muscle of type 2 diabetic patients are typically reduced by 90%. Many cellular pathways & their intermediates are in some way or another linked to insulin signalling. This study focused on three of these namely the PI3-kinase/Akt pathway, the Mitogen Activated Protein Kinase (MAPK) cascade and the AMP Kinase pathway, with successful monitoring of the PI3-K pathway. Investigations involved observing and evaluating the effects of various compounds as well as an indigenous medicinal plant, Sutherlandia frutescens on the activities of key insulin signalling pathway intermediates within the three fore mentioned pathways including Akt, AMPK and MEK1/2 as well as membrane surface GLUT4 levels. Scientific research has in the past leant heavily on Western blotting as the method of choice for gaining vital information relating to signal transduction pathways, however for research into cellular mechanisms the negatives of this method outweigh the positives. The drawbacks include a need for large amount of cells, multiple washing steps which may be disadvantageous to any weak and transient interactions as well as lysing of cells which may interfere with the maintenance of the subcellular localisation of a specific signalling event. Based on these, the need for a better method in terms of speed & reliability to monitor phosphorylation states of signal transduction pathway intermediates & GLUT4 translocation was evident and was one VII of the main aims & successes of this study. The method created used the mouse muscle cell line C2C12 in conjunction with the quick, sensitive method of flow cytometry which allowed us to monitor these processes in these cells through immune-labelling. Adherent cell cultures such as the C2C12 cell line pose the problem of possible damage to plasma membrane receptors (including insulin receptors) during harvesting to obtain a cell suspension for flow cytometry. We however used C2C12 mouse myocytes to optimize a method yielding insulin responsive cells in suspension that were successfully used for flow cytometry after immunelabelling of insulin signalling intermediates. Insulin (0.1μM) significantly raised the levels of both P-Akt and GLUT4 above basal levels. This effect was shown to be dose dependent. At a concentration of 50μg/ml, Sutherlandia frutescens was able to act as an insulin-mimetic in terms of its ability to increase P-Akt levels, GLUT4 translocation and glucose utilisation in an acute manner. These increases could be reduced with the addition of wortmannin, a PI3-K inhibitor. Therefore, these results suggest the mechanism of the plant extract’s insulin-like activity may be in part due to the activation of the insulin signalling pathway leading to GLUT4 translocation, which involves the phosphorylation of insulin receptor- and subsequent PI3-K activity, leading to P-Akt activity. These results provide further evidence of this plant extract’s anti-diabetic potential. The effect of Sutherlandia frutescens on insulin secretion, calcium signalling and proliferation in INS-1 rat pancreatic cells was also investigated and it was found to increase the activities of all of these processes. However no change in the levels of GLUT2 glucose transporter was seen. Ritonavir is prescribed by the South African Department of Health in co-formulation with other protease inhibitors within its second regime in the treatment of HIV and AIDS. Using C2C12 cells, ritonavir decreased glucose uptake acutely and had no effect on GLUT4 translocation however surprisingly increased P-Akt levels. In conclusion, it was found that Sutherlandia frutescens has antidiabetic benefits, diverse in nature depending on tissue type as well as length of time administered. The establishment of novel flow cytometry techniques to assess antidiabetic properties using in vitro cell culture was achieved. These methods will be useful in the future for the assessment of insulin sensitivity and in the identification of novel compounds that stimulate the insulin signalling pathways.

Notre étude a montré l’implication de la déficience de PPARα dans la modulation de latranscription des gènes de l’insuline et de l’inflammation des adipocytes chez les sourisadultes C57BL/6J (WT) et PPARα-null. A jeun, les souris PPARα-null sont hypoglycémiquespar rapport aux animaux témoins WT. La concentration en insuline et l’expression de sesARNm pancréatiques, par rapport aux animaux témoins, sont diminuées chez les sourisPPARα-null, suggérant que la suppression du gène de PPARα contribuait à la faibletranscription de ces gènes. De plus, la suppression du gène de PPARα aboutit à la diminutiondes facteurs de transcription des gènes de l’insuline comme Pdx-1, Nkx6.1 et MafA. En outre,la capacité pancréatique fonctionnelle est aussi détériorée par la suppression du gène dePPARα puisque le pancréas des souris PPARα-null exprime de faibles taux de Glut2 et deglucokinase. Les souris PPARα-null expriment des taux élevés d’adiponectine et de leptinecomparées aux souris témoins. Dans les tissus adipeux, les souris PPARα-null présentent uneaugmentation de l’expression de CD14 et CD68 généralement exprimés par les macrophages.La suppression du gène de PPARα diminue, au niveau des adipocytes, l’expression de MCP-1, TNFα, IL-1β, IL-6 et RANTES, alors que l’expression de TLR-2 et de TLR-4 (récepteurspro-inflammatoires) était élevée dans les tissus adipeux. Ces résultats suggèrent qu’encondition normale, la déficience en PPARα, chez les souris est impliquée dans la modulationde la transcription des gènes de l’insuline et le statut inflammatoire du tissu adipeux.En outre, l'invalidation du gène de PPARα dans les cellules T a abouti àl'augmentation de T-bet et la diminution de GATA-3 tant aux niveaux de la protéine que del’ARNm. Comme prévu, l’acide Docosahexaénoïque (DHA) a exercé non seulement un effetinhibiteur sur la prolifération des cellules T, mais aussi a diminué la sécrétion d’IFN-γ etstimulé la sécrétion de l’IL-4 dans les deux types cellulaires. Le DHA a aussi diminué T-bet etaugmenté GATA-3 tant au niveau de la transcription qu’au niveau de la protéine. Quoique lescellules T des souris PPARα-null ont exprimé un plus fort niveau de phosphorylation de p38MAP kinase que les cellules T de WT, le DHA a diminué la phosphorylation des MAPkinases (p38 et ERK1/2) dans tous les deux les types cellulaires. Les inhibiteurspharmacologiques des MAP kinases ont aussi diminué T-bet et augmenté GATA-3 dans lescellules T. Ces résultats démontrent que le DHA, via son action sur les MAP kinases, modulel'expression des facteurs de transcription. Ces résultats expliquent aussi le mécanisme d'actionde cet acide gras sur la différenciation des cellules T dans la maladie et la santé / We assessed, in this study, the effects of PPARα deficiency on the expression of mRNAencoding for insulin gene transcription factors in pancreatic β-cells along with thoseimplicated in inflammation in adipose tissues. On fasting, the adult PPARα-null mice werehypoglycemic. Serum insulin concentrations and its pancreatic mRNA transcripts weredownregulated in PPARα-null mice, suggesting that PPARα gene deletion contributes to lowinsulin gene transcription. The PPARα gene deletion downregulates the mRNA expression ofinsulin gene transcription factors, i.e., Pdx-1, Nkx6.1 and MafA. Besides, the pancreaticfunction was diminished by PPARα deficiency as PPARα-null mice expressed low pancreaticGlut2 and glucokinase mRNA. PPARα-null mice also expressed high adiponectin and leptinmRNA levels compared to wild type animals. Adipose tissues of PPARα-null mice exhibitedupregulation of CD14 and CD68 mRNA, generally expressed by macrophages. PPAR-a genedeletion downregulates the adipocyte mRNA of certain pro inflammatory agents, like MCP-1,TNF-a, IL-1b, IL-6, and RANTES, though pro-inflammatory TLR-2 and TLR-4 mRNAswere upregulated in the adipose tissues. Our results suggest that PPAR-a deficiency, in mice,is implicated in the modulation of insulin gene transcription and inflammatory status inadipose tissues.The another part of the study was conducted on CD4+ T-cells, isolated from wild type(WT) and PPARα-null mice, in order to assess the mechanismof action of docosahexaenoicacid (DHA), an n-3 fatty acid, in the modulation of two transcription factors, i.e., T-bet andGATA-3, implicated in T-cell differentiation towards, respectively, TH1 and TH2 phenotype.The T-cells from PPARα-null mice secreted higher IFN-γ and lower IL-4 concentrations thanWT T-cells. Furthermore, the deletion of PPAR-α gene in T-cells resulted in the upregulationof T-bet and downregulation of GATA-3 both at mRNA and protein levels. DHA exerted notonly an inhibitory effect on T-cell proliferation, but also diminished IFN-γ and stimulated IL-4 secretions in both cell types. DHA also downregulated T-bet and upregulated GATA-3 bothat transcription and protein levels. Though the T-cells from PPARα-null mice expressedhigher p38 phosphorylation than WT T-cells, DHA diminished the MAP kinasephosphorylation (p38 and ERK1/2) in both the cell types. The pharmacological inhibitors ofMAP kinases also downregulated T-bet and upregulated GATA-3 in T-cells. Altogether, theseresults demonstrate that DHA, via its action on MAP kinases, modulates the expression oftranscription factors. These results also explain the mechanism of action of this fatty acid onT-cell differentiation in disease and health

Introdução: O PYY é um peptídeo regulador da saciedade produzido pelas células intestinais em resposta à chegada intraluminal de nutrientes. Objetivos: O presente estudo objetivou avaliar o efeito de sobrecargas agudas de gorduras saturadas (SAT) e monoinsaturadas (MUFA) na secreção aguda de PYY em ratos Wistar normais e após insulinorresistência induzida por dieta hiperlipídica. Métodos: Em experimento controlado, ratos Wistar foram submetidos a uma dieta altamente gordurosa (HFD) (55% de gordura) por 19 semanas (n=15) ou à dieta normal (GC) pelo mesmo tempo (ração ad libitum) (n=15). Ao final de 14 semanas foi realizado um experimento cross-over onde foi avaliada a resposta secretória de PYY sérico nos tempos basal e 60 minutos após sobrecarga oral lipídica isovolumétrica, por meio de gavagem, ajustadas para o peso, administrada de forma aleatória, em dias diferentes, constituídas por ácidos graxos saturados (SAT-banha de porco) ou monoinsaturados (MUFA-óleo de oliva) ou água (CONT). Diferenças entre médias e grupos foram avaliadas por meio de ANOVA de medidas repetidas e associação por regressão linear simples. Resultados: Em relação ao PYY, no grupo com dieta normal, ambas sobrecargas MUFA e SAT elevaram a resposta secretória de PYY significativamente em relação aos seus respectivos basais: MUFA-Basal 2,18 (± 0,24) vs. MUFA-60min 2,30 (± 0,26) pg/ml e SAT-basal 2,21 (± 0.25) pg/ml vs. SAT- 60min 2,29 (± 0,22) pg/ml ANOVA múltiplas entradas p= 0,019 intragrupos; entretanto, sem diferença entre grupos MUFA e SAT (ANOVA múltiplas entradas entre-grupos p= 0,314). No grupo HFD por outro lado, a sobrecarga SAT reduziu o PYY: SAT-basal 2,16 (± 0.21) pg/ml vs. SAT-60min 2,11 (± 0,30) pg/ml (p= 0,01,intragrupos) enquanto a sobrecarga MUFA manteve o mesmo aumento MUFAbasal 2,15pg/ml vs. MUFA-60min 2,22 (± 0.22) pg/ml. p=0,019 (intragrupos). A administração de água (CONT) também reduziu o PYY em relação ao basal, tanto com na dieta normal (p= 0,0091) como na dieta (HFD) (p= 0,0091), mas sem diferença entre os grupos (p= 0,7433). Conclusão: Em ratos Wistar, as sobrecargas lipídicas, tanto de MUFA como de gordura saturadas, aumentam agudamente a secreção de PYY. Entretanto, em ratos Wistar tornados insulinorresistentes através de uma dieta altamente rica em gordura saturada, a mesma sobrecarga de gordura saturada perde a capacidade de estimular os níveis de PYY, enquanto à resposta ao MUFA segue preservada. Esta resposta paradoxal a gorduras saturadas poderia representar um dano celular causado pela insulinorresistência ao tecido intestinal interferindo no aparato secretor de PYY em resposta a este nutriente. Estudos no tecido intestinal precisam ser realizados para identificar possíveis fatores envolvidos e suas implicações no controle da saciedade pelo PYY em indivíduos insulinorresistentes. / Background: PYY is a gut peptide released by L-cells from the intestine after a meal. Objective: The present study aimed to evaluate the effect of acute overloads of saturated fatty acids (SAT) and monounsaturated fatty acids (MUFA) on PYY release in normal and diet induced insulin resistant wistar rats. Methods: a nineteen weeks experiment was conducted with 30 wistar rats that were allocated into two groups: high fat diet (HFD group) (n=15) with diet composition of 55% of lard and 45% standard chow and control group (CG) (n=15). Both groups received water and food ad libitum. Later a cross-over experiment was conducted to evaluate PYY secretory response 60 minutes after two different lipid overloads (SAT-lard; MUFAolive oil) and water (CONT), adjusted by weight, all isovolumetric and lipids were isocaloric, randomly administered in different days. Mean differences were analyzed by repeated measures ANOVA and association by simple linear regression. Results: Both MUFA and SAT overloads significantly increased PYY release in the CG in comparison with baselines: MUFA-Baseline 2,18±0,24 vs. MUFA-60min 2,30±0,26pg/ml and SAT-baseline 2,21±0.25pg/ml vs. SAT-60min 2,29±0,22 pg/ml ANOVA multiple entry p=0,019 intra-group, however without difference between MUFA and SAT (ANOVA multiple entry inter-group p=0,314). In the other hand, HFD SAT overload significantly decreased PYY release: SAT-baseline 2,16±0.21 pg/ml vs. SAT-60min 2,11±0,30 pg/ml (p=0,01,intra-group) while MUFA overload was able to keep the increase on PYY release MUFA-baseline 2,15pg/ml vs. MUFA-60min 2,22±0.22 pg/ml. p=0,019 (intra-group). Water overload (CONT) also reduced PYY release in comparison with baseline in both CG (p=0,0091) and HFD (p=0,0091), without difference between them (p= 0,7433). Conclusion: MUFA and SAT overloads increase PYY release after 60 minutes in normal wistar rats. However, when became high fat diet induced insulin resistant the SAT overload looses the capacity to stimulate PYY release, while MUFA response keeps preserved. This paradoxal finding to saturated fatty acids could indicate a cellular damage caused by insulin resistance in the intestinal tissue which compromises PYY secretory apparatus in response to this nutrient. Studies in the intestinal tissue must be conducted in order to identify possible factors involved and its implications in satiety signals PYY mediated in insulin resistance individuals.