Polycystisktovarialsyndrom (PCOS) förekommer hos 5-10% av alla kvinnor och är den vanligaste orsaken till anovulatorisk infertilitet. Andra delar av syndromet är hyperandrogena och metabola symtom. Infertilitet behandlas med klomifencitrat. Akne och hirsutism behandlas i första hand med kombinerade p-piller med östrogenprofil. Förhöjda blodsockernivåer, hypertoni, dyslipidemi och övriga komplikationer till syndromet behandlas farmakologiskt vid behov. Hyperandrogenism och insulinresistens tycks spela en huvudroll i uppkomsten av sjukdomen. Då metformin förbättrar insulinkänsligheten, och tros kunna påverka patofysiologin, har det föreslagits som en alternativ förstahandsbehandling. Denna litteraturstudie syftade till att undersöka vilket vetenskapligt underlag som finns för att ändra behandlingsrekommendationerna vid PCOS. De studier som jämfört resultatet av metformin och klomifencitrat vid anovulatorisk infertilitet visar att klomifencitrat mer effektivt framkallar ovulation och graviditet hos kvinnor med PCOS och övervikt, men att metformin är lika effektivt hos normalviktiga kvinnor. Medan metforminbehandling är en välbeprövad och säker behandling med få biverkningar har klomifencitrat allvarliga biverkningar i form av risk för flerbörd och ovarialthyperstimuleringssyndrom. De få studier som undersökt metformins påverkan på fostret finner inga belägg för teratogena effekter. Metformin har positiva effekter på hyperandrogena symtom vid PCOS, och man har inte kunnat se någon signifikant skillnad i effekt mellan metformin och p-piller då det gäller att minska akne och hirsutism. Dessutom finns det belägg för att metformin kan ha en positiv påverkan på BMI och blodtryck, förbättra lipidprofilen genom att sänka nivåerna av LDL kolesterol, samt minskar risken hos denna patientgrupp att utveckla typ 2 diabetes. Sammantaget kan dock sägas att det vetenskapliga underlaget ännu är för svagt för att man ska ändra den rådande behandlingsrekommendationen. Det finns behov av större, blindade studier där man jämför metforminbehandling med klomifencitrat och tittar på en kombination av faktorer och utfall kopplade till symtombilden vid PCOS.

La prévalence de l’obésité et du diabète de type 2 est en constante augmentation dans les pays industrialisés. Ces pathologies sont associées à des troubles de l’équilibre énergétique de l’organisme, ce qui se traduit au niveau hépatique par le développement de NAFLD (Non‐alcoholic Fatty Liver Disease). La NAFLD est engendrée par l’accumulation excessive de lipides dans les hépatocytes ou stéatose et est associée à la résistance à l’insuline. L’insulinorésistance hépatique est dite sélective car l’insuline n’est plus capable d’inhiber la production hépatique de glucose, contribuant à l’hyperglycémie, alors que la synthèse lipidique est exacerbée, induisant une stéatose hépatique. La stéatose peut évoluer en pathologies plus graves, telles que la fibrose, la cirrhose ou le cancer hépatocellulaire (CHC). L’objectif de ma thèse a été d’étudier les mécanismes moléculaires pouvant contribuer à la sélectivité de la résistance à l’insuline hépatique et à l’évolution de la stéatose simple vers le CHC. Dans un premier temps, nous avons étudié le rôle de l’adaptateur moléculaire Grb14, un inhibiteur de l’activité kinase du récepteur de l’insuline, dans la régulation du métabolisme hépatique chez la souris. L’invalidation de Grb14 dans le foie conduit d’une part à une activation de la signalisation de l’insuline et une amélioration de tolérance au glucose, et d’autre part à une diminution de la lipogenèse consécutive à l’inhibition du facteur lipogénique LXR via la voie Nrf2. L’invalidation de Grb14 dans le foie de souris obèses et diabétiques permet de ramener la glycémie et la stéatose hépatique à des valeurs similaires aux témoins. Ces données suggèrent que Grb14 est un nouvel acteur impliqué dans la sélectivité de la résistance à l’insuline du foie. La seconde étude a permis de montrer l’implication de la stéatose hépatique induite par un régime hypercalorique sur le développement de CHC. En effet, sur un fond génétique favorisant la carcinogenèse, un régime riche en graisse et en sucre contribuant à l’insulinorésistance hépatique accélère la cinétique d’apparition des tumeurs et augmente leur nombre. / The prevalence of metabolic diseases, including obesity and type 2 diabetes, are expanding in a worldwide epidemic way. These diseases are associated with metabolic disorders, resulting in the development of NAFLD (Non‐alcoholic Fatty Liver Disease) in the liver. NAFLD is generated by excessive accumulation of lipids in hepatocytes, and is associated with insulin resistance. In liver, insulin resistance leads to a blunted inhibitory action on hepatic glucose production, inducing hyperglycemia, whereas de novo lipogenesis, which is positively regulated by insulin, is paradoxically exacerbated, contributing to hepatic steatosis. Steatosis may also evolve into more serious diseases such as fibrosis, cirrhosis and hepatocellular carcinoma (HCC). The aim of my thesis was to study the molecular mechanisms that may contribute to the selectivity of hepatic insulin resistance and the development of HCC. Initially, we studied the role of the molecular adapter Grb14, an inhibitor of the insulin receptor kinase activity, in the regulation of hepatic metabolism in mice. Invalidation of Grb14 in the liver leads on the one hand to an activation of the insulin signaling and improved glucose tolerance, and on the other hand to a decrease of LXR activity resulting in lipogenesis inhibition. Invalidation of Grb14 in the liver of obese and diabetic mice restores blood glucose levels and hepatic steatosis similar to control values. These data suggest that Grb14 is a new player involved in the selectivity of the insulin resistance in the liver. The second study demonstrated the involvement of hepatic steatosis induced by a high‐calorie diet on the development of HCC. Indeed, on a genetic background favoring carcinogenesis, a diet rich in fat and sugar contributing to hepatic insulin resistance accelerates appearance of tumors and increases their number.

Abstract Most women with polycystic ovary syndrome (PCOS) exhibit features of metabolic syndrome, including insulin resistance, abdominal obesity, dyslipidaemia, glucose intolerance and low-grade chronic inflammation, reflected in elevated levels of serum C-reactive protein (CRP), placing these women at increased risk of cardiovascular disease and type 2 diabetes (type 2 DM). The aim of this study was to investigate the effects of two well-known insulin-lowering drugs used in the treatment of type 2 DM, metformin and rosiglitazone, on traditional cardiovascular risk factors and inflammation in women with PCOS. In addition, the impact of rosiglitazone was evaluated as regards clinical, endocrine and metabolic aspects of PCOS. Six-months of metformin treatment in women with PCOS had beneficial effects on levels of CRP, lipid profile and blood pressure, expressed as increased levels of high-density lipoprotein cholesterol (HDL-C), and decreased levels of triglycerides (TGs), decreased ratio of total cholesterol/HDL-C, decreased levels of CRP, and decreased systolic and diastolic blood pressures. Four-month treatment with rosiglitazone in a randomised, double-blind, placebo-controlled study in overweight women with PCOS resulted in significant improvements in menstrual cyclicity, hyperandrogenism, insulin resistance and hyperinsulinaemia. In addition, rosiglitazone decreased levels of markers of low-grade inflammation, CRP and white blood cell (WBC) count, and the liver function marker alanine aminotransferase (ALAT), while having neutral effects on levels of lipids, and blood pressure. In conclusion, metformin treatment, in accordance with the known beneficial metabolic effects of this drug, could be useful in the prevention of cardiovascular complications in women with PCOS. Rosiglitazone represents an alternative treatment for overweight anovulatory women with PCOS. It could be useful in the prevention of type 2 DM in overweight women with PCOS and for those suffering from possible side-effects related to metformin treatment. In addition, alleviation of inflammation and improvement of liver function during rosiglitazone treatment may indicate decreased future risks of cardiovascular diseases and non-alcoholic fatty liver disease (NAFLD).

Doctor of Philosophy / Department of Anatomy and Physiology / Thomas J. Barstow / The overall aims of this dissertation are to determine the incidence and magnitude of insulin resistance (IR) in a cohort of normoglycemic college-age subjects with a family history of type 2 diabetes, and to ascertain if there is early macro- and microvascular dysfunction relative to IR. Study 1 (Chapter 2) revealed a 7-fold range in IR in healthy college subjects concomitant with measures of insulin, both fasted and during an oral glucose tolerance test, but not related with any measure of plasma glucose. These results emphasize that early in the etiology of carbohydrate dysregulation, abnormalities first occur with regard to insulin sensitivity. Using brachial artery blood flow (BABF, Doppler fluxometry) and near-infrared spectroscopy (NIRS) (Chapter 3) we extended the understanding of the use of these non-invasive tools to assess forearm resting metabolic rate and to compare the parameters of both the NIRS oxy-hemoglobin signal, as a index of perfusion in the microcirculation, and BABF, as an independent measure of microvascular reactivity during post occlusive reactive hyperemia (PORH). Resting metabolic rate ranged ~ 2 fold (2.83-5.15 [Mu]MO[subscript2]/min/100g) similar to direct measures. Amplitude, but not kinetic parameters for NIRS variables correlated with comparable parameters for BABF, providing evidence for the possible utility of NIRS in examining microvascular reactivity. In study 3 (Chapter 4), utilizing our extended understanding of hemodynamics garnered from the results of study 2, we assessed the influence of IR on macro- and microvascular reactivity. We observed that i) the magnitude of IR was significantly correlated with attenuation of endothelium-dependent vasodilation of the brachial artery (P< .01) indicating the possibility of a reduced nitric oxide bioavailability and an enhanced atherogenic milieu. Additionally we found ii) BABF at rest and during reactive hyperemia to be strongly correlated with conductance (reduced downstream resistance—an indicator of microvascular control abnormalities) independent of forearm metabolic rate, and iii) parameters of BABF (microvascular response) were also strongly correlated with brachial artery vasoreactivity (macrovascular response). In conclusion, this body of work furthers our insight into the need for earlier identification of "disease" earlier in the progression to type 2 diabetes, and provides direction for future investigations into prevention / intervention to improve microvessel functionality and to slow the atherosclerotic process in larger vessels.

Doctor of Philosophy / Department of Biology / Stephen K. Chapes / Silvia Mora Fayos / My work focused on understanding adipocyte function and regulation because of the importance to diabetes. In addition to being a fat storage depot, adipose tissue is an endocrine tissue. Adiponectin and leptin are two adipokines that control insulin sensitivity and energy balance. In spite of their importance, there are still questions about their secretion. I hypothesized that leptin and adiponectin follow different secretory routes. I found adiponectin localized in Golgi and the trans Golgi Network, while leptin mostly localized in ER during basal metabolisms. Common requirements for their secretion were the presence of class III Arf proteins and an intact Golgi apparatus, since BFA treatment inhibited secretion of both adiponectin and leptin. I found that trafficking of adiponectin is dependent on GGA1 coated vesicles. Endosomal inactivation significantly reduced adiponectin, but not leptin, secretion in both 3T3L1 and isolated rat adipocytes. Also, adiponectin, but not leptin, secretion was reduced in cells expressing non- functional form of Rab11 and Rab5 proteins. However, secretion of leptin, but not adiponectin was inhibited in cells expressing mutants of Protein Kinase D1. These results suggest that leptin and adiponectin secretion involve distinct intracellular compartments and pathways. Insulin resistance is associated with macrophage infiltration into adipose tissue and elevated levels of IL-6, TNF-alpha and IL-1beta Therefore, the second part of my dissertation tested the hypothesis that the interaction of macrophages and adipocytes causes insulin resistance. To test this hypothesis, I co-cultured macrophages and adipocytes. I found that mouse elicited peritoneal macrophages significantly decreased insulin-stimulated GLUT4 translocation to the plasma membrane in a contact-independent manner. IL-6 was the most inhibitory cytokine in reducing GLUT4 translocation, GLUT4 expression, Akt phsphorylation and reducing adipocyte differentiation compared to TNF-alpha and IL-1beta. These data suggest that IL-6 is the most effective cytokine secreted by macrophages involved in insulin resistance. Lastly, I tested the impact of adipocytes on macrophage differentiation in vitro and in vivo. I found that C2D macrophages isolated from the peritoneal cavity had increased IL-6 transcript levels after co-culture with 3T3L1 adipocytes in vitro. After i.p. injection, C2D macrophages isolated from WAT increased expression of mature macrophage surface markers and transcript levels of proinflammatory cytokines compared to C2D cells in vitro. However, macrophages isolated from BAT expressed low levels of cytokines and macrophage surface markers.

Magister Artium (Sport, Recreation and Exercise Science) - MA(SRES) / Insulin resistance has shown to be a precursor to a number of lifestylerelated chronic diseases and abnormalities in adults and is affected by a number of factors including genetics, age, physical activity and acute exercise, diet, obesity, body fat distribution and medication. Physical activity has shown to have marked effects on improving sensitivity to insulin though various physiological mechanisms, and numerous correlation studies have identified a relationship between these two variables, suggesting the beneficial role of exercise on insulin resistance. This study aimed to identify a relationship between current levels of physical activity, physical fitness and insulin resistance in adults between the ages of 35 and 65 years of age residing in a northern suburb community in Cape Town. A total of 186 volunteers participated in this study ranging from healthy individuals to those with diagnosed chronic conditions. Insulin resistance (determined by the homeostasis model assessment of insulin resistance), physical activity (measured by the Global Physical Activity Questionnaire) and five health-related physical fitness tests were measured. The five components included body composition, determined by body mass index and waist circumference, the 3-minute cardiorespiratory step test, the handgrip muscle strength test, one-minute crunches for muscle endurance and the sit-and-reach flexibility test. Spearman correlation was used to identify the relationships between the homeostasis model assessment of insulin resistance, age, body composition and physical activity and fitness.Results showed that body mass index and waist circumference were the only two variables which produced significant correlations with the homeostasis model assessment of insulin resistance (p < 0.019). No physical activity or fitness data produced significant scores with the homeostasis model assessment of insulin resistance. Body mass index in men was the only significant predictor of HOMA-IR and explained 37% of the variance in insulin resistance, whereas in women, only waist circumference was related to HOMA-IR, but explained less than 16% of the variance. Associations between reported MET-minutes from the Global Physical Activity Questionnaire and the four fitness tests indicated significance with handgrip strength (&rho; = 0.17; p =0.039), one-minute crunches (&rho; = 0.18; p = 0.024) and sit-and-reach flexibility (&rho; = 0.17; 0.034). This study has shown that body composition is an important component in influencing insulin resistance therefore physical activity interventions should be targeted at increasing physical activity levels and reducing body weight. / South Africa

>Magister Scientiae - MSc / About 180 million people have been estimated to suffer from type 2 diabetes (T2DM) in 2006 and the annual death rate due to this disease was 3 million by that time. More than 400 medicinal plants used for the treatment of diabetes mellitus have been recorded, but only a small number of these plants have received scientific and medical evaluation to assess their efficacy. The most common plant used to treat diabetes mellitus is Hypoxis hemerocallidea (HH). The present study was undertaken to investigate the effects of Hypoxis hemerocallidea (HH) on T2DM in rats. Male Wistar rats weighing 200-250 g were used in this experiment. Hypoxis hemerocallidea (HH) corm was used as plant material in the experiment. The study was based on three parts, an acute diabetes study, chronic diabetes study and insulin secretion study. In the acute study, the rats were randomly divided into 2 groups (control and diabetes). The saline solution was added to different concentrations of HH corm to produce concentration of (50, 200, 400, 800 mg/ml). Diabetes was induced by intraperitoneal injections of STZ (65mg/kg). Two weeks after the injection (STZ 65 mg/kg), different concentrations of HHS was administered intraperitoneally after an overnight fast. The blood glucose levels were monitored in the diabetic and control rats at, 30, 60, 120, 180 and 240 minutes post injection. In the chronic study, the rats were randomly divided into 6 different groups (control, HFD, DM, DM-HH, DM-PTHH, and HH). Diabetes mellitus was then induced in the groups of diabetic rats by intraperitoneal injections of STZ (40 mg/kg) and rats were fed a high fat diet (HFD). The body weight of the rats were measured weekly for 7 weeks. An intraperitoneal glucose tolerance test (IPGTT) was performed at the end of week 6. At the end of week 7, rats were killed and serum sample were collected for determination of fatty acid and insulin. Liver and pancreatic tissue was collected for histological evaluation. In the insulin secretion study, Hypoxis hemerocallidea was tested for its effects on insulin secretion by pancreatic islet cells exposed to low (3mM) and high (20mM) glucose medium. Results of the acute study indicated that HHS at a dose 800 mg/ml decreased blood glucose levels fastest in both normal and diabetic rats reaching significance after 30 minutes and 60 minutes respectively and remained below the baseline value until 240 minutes. In the chronic study, it was illustrated that HH had no effect in normal rats on any of the parameters evaluated. Animals in the DM group gained weight the first two weeks, but thereafter began to lose weight. At the end of seven weeks the animals gained significantly less weight than the rest. Animals fed a HFD have more visceral fat compared to the control group. The visceral fat gain occurred in the absence of a significant increase in body weight. We found a markedly lower fasting glucose level in HH treated diabetic animals compared to untreated DM animals. At time zero the blood glucose level of the HFD group (5.8±0.5mmol/l) and the HH group (4.9±0.7mmol/l) were in the normal range, and were not significantly different (P > 0.05) from the control group (5.0±0.2mmol/l). After glucose load peak blood glucose levels was measured after 30 minutes in the control group (9.0±0.6mmol/l), the HFD group (9.8±0.4 mmol/l), the DM-HH group (21±5.7 mmol/l) and the DM-HHPT group (27.8±5.3 mmol/l). In the HH group the blood glucose level reached a peak at 60 minutes (7.6±0.6 mmol/l). In the DM group two peaks were recorded one after 10 minutes (27.2±7.1mmol/l) and another after 60 minutes (31±5.2 mmol/l). In the groups control, HFD, DMHH, DM-HHPT and HH groups the blood glucose level after 120 minutes were not significantly different from the time zero value. The blood glucose level after 120 minutes in the DM group (28.2±7.1 mmol/L) was significantly higher (P ≤ 0.01) than from the time zero value. Serum fatty acid levels were increased in all groups fed a high fat diet. The serum insulin levels in the HFD group (6.2 ± 0.76 μUI/ml protein; P ≤ 0.05 ), the DM group (2.0 ± 0.9 μUI/ml protein; P ≤ 0.001), the DMHH group (3.4 ± 0.7 μUI/ml protein; P ≤ 0.001) and the DM-HHPT group (3.0 ± 1.1 μUI/ml protein; P ≤ 0.001) were significantly lower than the control group. The β-cell function in the HFD group (62 ± 8 %; P ≤ 0.001), the DM group (3 ± 1 %; P ≤ 0.001), the DM-HH (11 ± 9 %; P ≤ 0.001) group and the DM-HHPT group (4 ± 2 %; P ≤ 0.001) were significantly lower than the control group. The histological observation of the liver and the pancreas in rats after 7 weeks on different dietary regimes showed some morphological changes within the liver and pancreas parenchyma of some rats. In the insulin secretion study, glucose stimulated insulin secretion in low (3mM) and high (2mM) glucose concentration. Furthermore, insulin secretion was significantly higher when the glucose concentration was increased from 3mM to 20 mM (1.10 ± 0.13 μUI/ml protein and 1.5 ± 0.17 mIU/mg protein respectively P≤ 0.01). In the presence of low HH (100 µg/ml), there was a marked increase in insulin secretion when exposure to high glucose compared to low glucose concentration, while in the presence of high HH (500 µg/ml), there was no significant different in insulin secretion in the presence of low or high glucose. In conclusion, the results of this experimental study indicate that a concentration 800 mg/kg of HHS produces maximal hypoglycaemic effect in fasted normal and diabetic rats. HH has an antidiabetic activity as it lowers serum glucose levels in T2DM rats and significantly increases glucose tolerance. It also increases body weight of diabetic rats. HH treatment was found to improve insulin secretion in pancreatic islet cells.

Introducción: Pocos son los estudios que analizan la relación entre el síndrome metabólico y la enfermedad nodular tiroidea, tema en el que existe un vacío de conocimiento. El objetivo de este estudio es determinar la asociación entre síndrome metabólico y enfermedad nodular tiroidea en un hospital de Lima, Perú. Materiales y métodos: Estudio longitudinal, prospectivo, analítico, observacional de casos y controles, realizado en el Hospital Nacional Edgardo Rebagliati Martins en Lima - Perú. Un total de 182 pacientes se separaron como casos a los pacientes en los que se encontrara por lo menos un nódulo tiroideo detectado por ultrasonografía mayor a 3 mm (n=91) y como controles a los pacientes en los cuales se excluyera la presencia del nódulo de las características descritas por la misma técnica diagnostica (n=91). Se evaluaron el nivel y la fuerza de asociación entre la presencia de síndrome metabólico y cada uno de sus componentes por separado con la presencia de enfermedad nodular tiroidea. Resultados: El análisis bivariado muestra asociación significativa entre la presencia de nódulo tiroideo y síndrome metabólico con un OR de 2.56 (IC: 95% 1.41 a 4.66, p < 0.05). Se evidenció que los niveles de HDL bajo y la glicemia basal alterada se encuentran asociadas significativamente con la presencia de nódulo tiroideo, independientemente de la presencia de síndrome metabólico con OR de 2.81 ( IC: 95% 1.54 a 5.12, p<0.05) y 2.05 (IC:95% 1.10 a 3.78, p<0.05) respectivamente. El análisis multivariado mantuvo la asociación entre nódulo tiroideo y el síndrome metabólico con un OR de 2.96 (IC: 95% 1,47 a 5,95 , p<0.05), así mismo con niveles de HDL bajo con un OR de 2.77 (IC:95 % 1,44 a 5,3, p<0.05) y con la glicemia basal alterada con un OR de 2,23 (IC:95% 1,14 a 4,34, p<0,05). Conclusiones: El Síndrome metabólico incrementa el riesgo de padecer enfermedad nodular tiroidea, específicamente la disminución de valores de HDL y la glicemia basal alterada fueron los factores en los que halló mayor asociación. / Introduction: Few studies analyses the relation between metabolic syndrome and thyroid nodular disease, subject in which there is a knowledge gap. The object of this study is to determinate the association between metabolic syndrome and thyroid nodular disease in a hospital in Lima, Peru. Materials and methods: A longitudinal, prospective, analytic, observational, case - control study, was performed “Hospital Nacional Edgardo Rebagliati Martins” in Lima- Peru. A total of 182 patients were separated as cases in which at least find a thyroid nodule detected by ultrasonography greater than 3 mm ( n = 91) and controls as patients in whom the presence of the node with the characteristics described was excluded by the same technique (n=91). The level and strength of association was evaluated between the presence of metabolic syndrome and each of its components by itself with the presence of thyroid nodular was evaluated. Results: Bivariate analysis shows significant association between the presence of thyroid nodule and metabolic syndrome with an OR of 2.56 (IC:95% 1.41 to 4.66, p < 0.05). Low levels of HDL and impaired fasting glucose are significant associated with the presence of thyroid nodule, independent of the presence of metabolic syndrome, with an OR of 2.81 (IC:95% 1.54 to 5.12, p<0.05) and 2.05 (IC: 95% 1.10 to 3.78, p<0.05) respectively. The multivariate analysis maintained the association between thyroid nodule and metabolic syndrome with an OR of 2.96 (IC: 95% 1,47 to 5,95 , p<0.05); like was the low levels of HDL with an OR of 2.77 ( IC: 95% 1,44 to 5,3, p<0.05) and with impaired fasting glucose with an OR of 2,23 ( IC 95% 1,14 to 4,34, p<0,05).Conclusions: Metabolic syndrome increases de risk of having thyroid nodule disease. Low HDL levels and impaired fasting glucose were the factors with more association.

L'obésité et le diabète de type 2 sont associés à la sédentarité et à une alimentation riche en graisses. En effet, les acides gras saturés s'accumulent dans les tissus non adipeux, comme les muscles squelettiques pour générer des lipides appelés céramides (CER). Mon projet de thèse s'est articulé en deux parties dont l'objectif est d'empêcher les CER d'agir. Nous avons montré que, selon la structure de la membrane plasmique, les CER altèrent la voie de signalisation insulinique en ciblant la PKB, protéine clef de la voie insulinique, via la voie PKC? dans les myotubes L6 et la voie PP2A dans les myotubes C2C12. Nous avons aussi mis en évidence que les CER altèrent la sensibilité à l'insuline via la voie PKC? dans les cellules musculaires humaines. Une fois les CER produits au niveau du réticulum endoplasmique (RE), ils sont transportés au Golgi par un transporteur CERT pour y être métabolisés en sphingomyéline (SM) et des études ont montré que la transformation des CER en SM pouvait être une étape cruciale pour empêcher les CER d'agir. Dans plusieurs modèles d'insulino-résistance musculaire, l'expression de CERT est diminuée et nous avons démontré l'importance du transport des céramides du RE vers le Golgi en inhibant artificiellement l'activité ou l'expression de CERT. A l'opposé, la surexpression de CERT améliore la sensibilité à l'insuline dans les cellules musculaires dans des conditions lipotoxiques. Nos résultats montrent que CERT joue un rôle crucial dans les mécanismes conduisant au développement de l'insulinorésistance musculaire puisque sa présence est essentielle pour le maintien d'un trafic normal des CER entre le RE et le golgi. / Obesity and type 2 diabetes are associated with a sedentary lifestyle and a diet rich in fat. Indeed, saturated fatty acids accumulate in non-adipose tissue such as skeletal muscle to generate lipids called ceramides (CER). My thesis project was divided into two parts with the objective to prevent CER to act. We have shown that, depending on the structure of the plasma membrane, CER alter the insulin signaling pathway by targeting PKB, a key insulin signalling protein, via a PKCζ pathway in L6 myotubes and a PP2A pathway in C2C12 myotubes. We also demonstrated that CER affect insulin sensitivity via the PKCζ pathway in human muscle cells. Once CER generated in the endoplasmic reticulum (ER), they are transported to the Golgi by a carrier called CERT to be metabolized into sphingomyelin (SM). Studies have shown that the transformation of CER into SM could be a crucial step to prevent CER to act. In several muscle insulin resistance models, expression of CERT is decreased and we demonstrated the importance of the transport of ceramide from the ER to the Golgi by inhibiting artificially the activity or the expression of CERT. In contrast, overexpression of CERT enhances insulin sensitivity in muscle cells in lipotoxiques conditions. Our results show that CERT plays a crucial role in mechanisms leading to the development of muscle insulin resistance since its presence is essential for maintaining normal traffic of CER between the ER and the Golgi.

Insulin resistance, a key risk factor for type 2 diabetes, can be defined as when cells fail to respond effectively to insulin. In striated muscle and fat, this manifests as impaired insulin-stimulated glucose uptake due to reduced plasma membrane insertion of the glucose transporter GLUT4. In cell culture models, insulin resistance induced by chronic exposure to insulin, endothelin-1 or glucosamine, is correlated with reduced immunoreactivity of the lipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in plasma membrane sheets. However, the reason for this decrease, and whether other factors that induce insulin resistance affect PtdIns(4,5)P2 levels, is unknown. Using L6 skeletal muscle myotubes and 3T3-L1 adipocytes, this project has investigated whether PtdIns(4,5)P2 levels are perturbed in insulin resistance induced by several factors, including exposure to insulin, oxidative stress, and treatment with tumour necrosis factor α, endothelin-1 or angiotensin II (Ang II).All these pre-treatments were found to abolish insulin-stimulated 3H 2-deoxy-glucose uptake, and significantly decrease PtdIns(4,5)P2 levels, measured in cell extracts by quantitative blotting using a PtdIns(4,5)P2-specific probe, developed from the PH domain of phospholipase C (PLC) δ. Importantly the ability of insulin to stimulate glucose uptake can be restored by replenishing PtdIns(4,5)P2 in L6 myotubes treated with insulin and Ang II. PtdIns(4,5)P2 levels are regulated by three families of proteins; PIP kinases, which synthesise it, phosphatases, which remove phosphate groups from the inositol headgroup, and PLCs, which hydrolyse it. Membrane preparations from Ang II- and insulin-induced insulin resistant L6 myotubes showed no differences in PtdIns(4,5)P2 production or dephosphorylation. However a significant increase in PLC activity was detected in membranes from insulin resistant cells and membrane localisation of PLCβ family members was increased in insulin resistant cells. Furthermore, studies using PLC inhibitors show a restoration of PtdIns(4,5)P2 levels in insulin resistant cells, leading to partial reversal of insulin resistance.This study therefore shows a causal link between decreased PtdIns(4,5)P2 levels and insulin resistance in L6 myotubes, and that PLCs are the reason for the PtdIns(4,5)P2 decrease in Ang II- and insulin-induced insulin resistance. PLCs, or their activation pathways, may thus be a novel target for combating insulin resistance, and preventing type 2 diabetes.