Magister Scientiae (Medical Bioscience) - MSc(MBS) / Background: It is well known that some genetic factors and dietary factors, such as excessive salt intake and excessive caloric intake (resulting in obesity) are risk factors for hypertension. Fifty percent of all hypertensive patients are also insulin resistant. Both hypertension and insulin resistance are again risk factors for other cardiovascular diseases such as atherosclerosis and heart failure. The nature of the association between hypertension and insulin resistance has not been clearly elucidated. Spontaneously hypertensive rats are the ideal models to study the aspects of the relationships between hypertension and insulin resistance. Models of high-fat feeding induce obesity,hypertension and insulin resistance and are thus used extensively to study hypertension because these models closely mimic some of the renal and cardiovascular changes found in human hypertensive patients. The present study was initiated to evaluate if insulin resistance will develop within 6 weeks in a model of high-fat diet induced hypertension and if so, to determine whether captopril will affect the presence of insulin resistance.This model should in future be used to study vascular reactivity to phenylephrine (PHE),acetylcholine (ACH) and sodium nitroprusside (SNP) in hypertensive animals in theabsence or presence of insulin resistance and in normotensive insulin resistant animals. Methods: In a series of experiments, rats were divided into four groups that received different treatments: (i) laboratory pellets, (ii) high-fat diet, (iii) high-fat diet plus captopril and (iv) high-fat diet plus vehicle. Body weight was measured weekly for 6 weeks. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured every week during the 6-weeks feeding period by the tail cuff method using a two channel computerized non-invasive system from Kent Scientific Corporation, USA.m Intraperitonealy glucose tolerance tests (IPGTTs) were performed at week 3 and week 6.After 6 weeks, and after an overnight fast, the plasma lipid profile was determined using a portable CardiochekTM blood test system. Fasting plasma insulin was determined using an immunoenzymatic assay for the in vitro quantitative measurement of rat insulin (INS) in serum and plasma. Insulin sensitivity was estimated by the quantitative insulin sensitivity check index (QUICKI) using the fasting plasma insulin and fasting glucose levels. After week 6 on the high-fat diet, thoracic aortae from the control and high-fat fed(HFD) animals were excised and vascular response to PHE, ACH and SNP were assessed in intact and denuded endothelium.Result: High-fat feeding did not cause a significant increase in body weight. High-fat feeding significantly increased systolic blood pressure from 125±2.1 mmHg in control animals to 155±5.9 mmHg in the HFD group (P < 0.05) and 158±5.6 mmHg in the HFDV group (P < 0.05). Diastolic blood pressure was increased from 86±2.8 mmHg in the control group to 117±2.5 mmHg in the HFD group (P < 0.05) and 113±3.4 mmHg in the HFDV group (P < 0.05). Visceral fat was increased from 0.8±0.1g in the control group to 3.1±0.6 g in the HFD group and 3.8±0.6 g in the HFDV group. IPGTTs performed at weeks 3 and 6 respectively did not differ significantly from the control group as evidenced from the AUC’s at weeks 3 and 6 respectively. High-fat feeding had no significant effects on blood cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) values or and fasting plasma insulin levels. The KCl induced contraction in both aortic rings with intact and denuded endothelium did not differ significantly between the control and HFD groups (P= 0.4 and 0.8) respectively. The contraction mediated by KCl in aortic rings with intact and denuded endothelium from the control or HFD groups also did not differ significantly(control: intact vs denuded, P = 0.2; HFD: intact vs denuded, P = 1). Dose responsecurves(1-10 μM) to PHE indicated slightly stronger contractions in the high-fat fed animals at submaximal doses tested. The maximum contraction achieved was however the same (94±19% and 99±2.6% relative to KCl induced contraction, in the control and HFD group respectively, P<0.05). Relaxation responses to ACH and SNP represent preliminary data.Conclusion: These data suggest that 6 weeks of high-fat feeding induces hypertension but does not produce obesity, dyslipidemia and insulin resistance. However, this model may be useful in studying vascular reactivity in hypertension in the absence of insulin resistance.

Le métabolisme lipidique est stimulé lors de l'exercice musculaire. La contribution énergétique des lipides s'accentue pendant l'exercice d'endurance d'intensité modérée de longue durée (40% à 60% de VO2max). Outre les acides gras circulants, les réserves de lipides intramyocellulaires (IMCL) sont sensées être utilisées pendant des performances dépassant 4 heures. Devant le manque de preuves expérimentales jusqu'à ce jour, une 1ere étude a été entreprise sur 10 sportifs (40 ± 6 ans) lors d'une course de 24h. Les résultats obtenus sur le muscle vaste externe ont montré une baisse significative d'IMCL de 56% et 45% dans les fibres de type I et IIA respectivement, alors que le glycogène n'a diminué que dans les fibres I. Ces données indiquent un catabolisme d'IMCL plus efficace que celui du glycogène dans les fibres rapides lors de l'exercice d'ultra endurance, dont le mécanisme reste à déterminer. IMCL s'accumule lors du vieillissement ou de l'obésité et peut constituer un risque de résistance à l'insuline (RI). Un entraînement combiné en endurance (EE) et en résistance (ER) de 14 semaines a été mené sur des sujets âgés (73 ± 4 ans) et d'autres en surpoids (58 ± 5 ans). Dans les deux groupes IMCL a augmenté (p<0.05) dans le muscle vaste externe (après EE) mais est resté stable dans le muscle deltoïde (après ER) et s'est accompagné de l'augmentation (p<0.05) de la capacité enzymatique de la β-oxydation après EE. Les céramides musculaires, une classe de lipides impliquée dans RI, ont été diminués (p=0.052) par EE et non par ER. Ces résultats confirment que l'augmentation d'IMCL n'est pas un facteur de risque métabolique et que EE se traduit par une diminution des céramides et de RI / Lipid metabolism is involved during muscle exercise. Energetic contribution of lipids increases during long lasting endurance exercise of moderate intensity (40% à 60% of VO2max). As well as circulating free fatty acids, intramyocellular lipid storages (IMCL) are postulated to be used during performances longer than 4 hours. Due the the lack experimental evidences untill today, a first study was undertaken on 10 athletes (40 ± 6 yrs) during a 24h running. Results obtained on vastus lateralis muscle showed a significant 56% and 45% decrease of IMCL in type I and IIA fibres respectively while glycogen decreased only in type I fibres. These data indicate a more efficient catabolism of IMCL than those of glycogen in fast twitch fibres during ultra endurance exercise, of which mechanism remains to be explored. IMCL accumulates during ageing or overweighting and may constitute a risk of insulin resistance (IR). A combined 14 weeks endurance (ET) and resistance (RT) training was followed by older (73 ± 6 yrs) and overweighted (58 ± 5 yrs) subjects. In the two groups IMCL increased (p<0.05) in vastus lateralis muscle (after ET) but remained stable in deltoidus muscle (after RT) and was linked to an increase (p<0.05) of β-oxydation enzymatic capacity after ET. Muscle ceramides, a category of lipids implicated in IR, decreased (p=0.052) after ET and not after RT. These results confirm that increase in IMCL is not a metabolic risk factor and that ET induces a decrease of both ceramides and IR

Orientadores: Eliana Pereira de Araújo, Lício Augusto Velloso / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T10:03:16Z (GMT). No. of bitstreams: 1 Nunez_CarlaEvelynCoimbra_D.pdf: 6141570 bytes, checksum: e9e12c78d1c319b6c83160c2ebf9976b (MD5) Previous issue date: 2013 / Resumo: A obesidade e caracterizada pelo acumulo excessivo de gordura no organismo, podendo resultar em dano a saúde. Mudança socioeconômica, ocorrida nos últimos cinquenta anos tem contribuído para o aumento da prevalência da obesidade, a qual e hoje considerada um dos principais problemas de saúde publica no mundo. O acumulo progressivo de ácidos graxos no tecido adiposo, e eventualmente, em outros sítios anatômicos não especializados na estocagem de energia sob a forma de gordura como, por exemplo, o fígado e o músculo, e associado à ativação de uma resposta inflamatória subclinica que desempenha papel importante na indução da resistência a insulina. Esta, por sua vez, e considerada o mecanismo fisiopatogênico unificador de uma serie de doenças comumente associadas à obesidade, tais como o diabetes mellitus, a aterosclerose, a esteatohepatite nao-alcoolica, entre outros. A inflamação subclinica desempenha um papel central na indução da resistência a insulina em obesos. Atualmente o estresse de reticulo endoplasmático e a ativação da sinalização do TLR4 vêm sendo identificados como potenciais mecanismos ativadores da inflamação sub-clinica associada à obesidade. No ambiente intracelular a ativação dos sinais inflamatórios disparados por ambos, estresse de reticulo endoplasmático ou TLR4, podem associar-se, modulando ou sendo modulado por outros eventos. Um desses eventos e a autofagia que se caracteriza como um processo celular finamente regulado e desempenha um papel importante no controle de varias funções da célula, tais como, reciclagem de organelas, disponibilidade de nutrientes e diferenciação celular. Um estudo recente demonstrou a existência de aumento na atividade autofágica em tecido adiposo de pessoas obesas e propôs a associação causal entre autofagia e resistência a insulina. A redução da adiposidade e o mecanismo mais eficiente para reduzir à resistência a insulina em pessoas obesas. Entretanto, o impacto da redução de adiposidade sobre a regulação da autofagia no tecido adiposo não e conhecido. Neste estudo, a regulação da autofagia no tecido adiposo durante o emagrecimento foi observada em duas etapas distintas. Inicialmente, um modelo animal de obesidade induzida por dieta, submetido posteriormente, a restrição calórica de 40% durante quinze dias. Animais obesos alimentados ad libitum, apresentaram aumento dos marcadores de autofagia no tecido adiposo, o que foi revertido na restrição calórica. De forma diversa, a restrição ocasionou o aumento da autofagia nos animais magros. A reintrodução de alimentação ad libitum foi suficiente para reduzir a autofagia nos animais magros, mas não nos obesos, cuja supra-regulacao da autofagia foi mais uma vez observada. Na segunda parte do estudo, autofagia foi avaliada em fragmentos de tecido adiposo subcutâneo de pacientes obesos selecionados para cirurgia bariátrica colhidos no ato da cirurgia e apos um ano, aproximadamente. Foram incluídos no estudo nove pacientes obesos não-diabeticos e seis pacientes obesos diabéticos. Assim como no modelo animal, obesidade em humanos foi associada a um aumento dos marcadores de autofagia no tecido adiposo os quais foram reduzidos apos a perda de peso. Assim, na vigência da obesidade ocorre uma regulação anômala da autofagia, estando aumentada durante alimentação ad libitum e reduzindo-se com a restrição alimentar / Abstract: Obesity, defined as abnormal or excessive fat accumulation that may impair life quality, is one of the major public health problems in modern world. It results from an imbalance between food intake and energy expenditure leading to the progressive accumulation of fatty acids in the adipose tissue and in some tissues that are not specialized in energy storage, such as liver and muscle. Insulin resistance is one of the main outcomes of obesity and is regarded as the main mechanism connecting diseases that are commonly associated with obesity, such as, type 2 diabetes mellitus, atherosclerosis, and non-alcoholic steatohepatitis, among others. Subclinical inflammation plays a major role in the induction of insulin resistance in obesity. Recently, endoplasmic reticulum stress and the activation of TLR4 signaling have been identified as potential triggering mechanisms for obesity-associated subclinical inflammation. At the intracellular environment activation of inflammatory signaling triggered by either endoplasmic reticulum stress or TLR4 signaling can integrate and modulate or be modulated by other cellular events. One such event is autophagy which is a highly regulated process that plays an important role in the control of a wide range of cellular functions such as organelle recycling, nutrient availability and tissue differentiation. A recent study has shown an increased autophagic activity in the adipose tissue of obese subjects, and a role for autophagy in obesity associated insulin resistance was proposed. Body mass reduction is the most efficient approach to tackle insulin resistance in over-weight subjects; however, the impact of weight loss in adipose tissue autophagy is unknown. In this study we used a two-step approach to evaluate adipose tissue autophagy during body mass reduction. First, a mouse model of diet-induced obesity and diabetes was submitted to a fifteen-day, 40% caloric restriction. At base-line, markers of autophagy were increased in obese mice as compared to lean controls. Upon caloric restriction, autophagy increased in the lean mice, while decreasing in the obese mice. The reintroduction of ad libitum feeding was sufficient to rapidly reduce autophagy in the lean mice and increase autophagy in the obese mice. In the second part of the study, autophagy was evaluated in the subcutaneous adipose tissue of nine obese-non-diabetic and six obese-diabetic subjects undergoing bariatric surgery for body mass reduction. Specimens were collected during the surgery, and approximately one year later. As in the mouse model, human obesity was associated with increased autophagy and body mass reduction led to an attenuation of autophagy in the adipose tissue. Thus, while caloric restriction leads to increased autophagy in the adipose tissue in lean subjects, in obesity, autophagy is defectively regulated, being increased during ad libitum feeding and reduced upon caloric restriction / Doutorado / Clinica Medica / Doutora em Ciências

A NAFLD é uma das principais patologias de fígado. Estudos reportam o exercício físico como um dos principais alvos terapêuticos para esta doença. Verificamos se o treinamento melhora a resistência à insulina, inflamação e esteatose hepática causados pela dieta hiperlipídica (HF) e se o PPAR-alpha está envolvido neste processo. Animais selvagens C57BL6 (WT) e knockout para PPAR&alpha; (KO) foram alimentados com dieta padrão ou HF durante 12 semanas e treinados por 8 semana. Metade dos animais KO treinados receberam rosiglitazona. A dieta HF aumentou TAG hepático, e resistência periférica à insulina levando a NALFD. O treinamento foi eficiente em reduzir esses parâmetros em ambos genótipos. O desenvolvimento da NAFLD não foi associado à inflamação hepática, entretanto animais KO treinados apresentaram uma resposta inflamatória exacerbada, causada pela redução de PPAR&gamma;. Quando eles receberam rosi apresentaram melhora no quadro inflamatório hepático e na resistência à insulina. O exercício diminuiu os danos causados pela dieta HF independente do PPAR&alpha;; a ausência do PPAR&alpha; junto com exercício leva a queda na expressão de PPAR&gamma;, e a uma resposta inflamatória exacerbada, que é revertida pela administração da rosiglitazona. / NAFLD is one of the main liver diseases. Studies have shown the beneficial effects of exercise on reverse NAFLD. We verify whether exercise improve insulin resistance, liver inflammation and steatohepatitis caused by a high fat diet (HF) and whether PPAR&alpha; is involved in these actions. C57BL6 wild type (WT) and PPAR-&alpha; knockout (KO) mice were fed with a standard (SD) or HF during 12 weeks and trained on a treadmill during 8 weeks, half of KO trained animals received 15mg/kg/day of rosiglitazone. HF diet increased TAG in the liver and peripheral insulin resistance leading to NAFLD. Exercise reduced all this parameters in both animals genotype. NAFLD was not associated with inflammation, however KO mice when trained presented an inflammatory response that was caused by a decrease on PPAR&gamma;. When these mice were treated with rosiglitazone, they presented decrease on inflammatory cytokines as well as improvement on insulin sensitivity. Exercise improved the damage caused by a HF independently of PPAR&alpha; and the absence of PPAR&alpha; together with exercise leads to decrease on PPAR&gamma; expression and an inflammatory response, which was attenuated by rosiglitazone administration.

Objetivo: Valorar si existe asociación entre el hipotiroidismo subclínico y la resistencia a la insulina en pacientes no diabéticos de una clínica privada de Lima. Materiales y métodos: Se realizó un estudio observacional analítico de corte transversal. Se incluyeron como participantes a adultos que acudieron a consulta ambulatoria de endocrinología de una clínica privada de Lima en los años 2012 a 2018. Se excluyeron a los pacientes que previamente habían sido diagnosticados de diabetes o patologías tiroideas, que recibían alguna terapia con corticoides sistémicos o hipolipemiantes, o que estaban gestando. Las variables de estudio principales fueron hipotiroidismo subclínico y resistencia a la insulina, las cuales se trabajaron como variables categóricas. Para definir la variable hipotiroidismo subclínico se utilizaron los valores de TSH mayor o igual a 4.5 mU/L, y T4L en valores normales, mientras que la variable resistencia a la insulina se definió utilizando el valor de HOMA-IR mayor a 2.79. Asimismo, también se estudiaron otras variables como IMC, porcentaje de grasa corporal y sexo. Para valorar la asociación entre el hipotiroidismo subclínico y la resistencia a la insulina, se llevó a cabo un modelo lineal generalizado de la familia Poisson con varianza robusta crudo y ajustado. Se presentó como media de asociación a la razón de prevalencia (RP) con su intervalo de confianza al 95% Resultados: Se analizó 1389 pacientes, el promedio de edad fue 39.56 (13.54) años (SD) y el porcentaje de varones fue 25.87%. La prevalencia de HSC fue 13.17% (n=183) y la prevalencia de RI fue 40.4% en el grupo con HSC, y 46.2% en el grupo de eutiroideos. En el modelo de regresión crudo se encontró que los pacientes con HSC tenían un 12% menos probabilidades de tener RI en el análisis crudo (PR 0.876 IC 95%: 0.727-1.055) y 7% menos en el ajustado (PR 0.927, IC 95%: 0.792-1.084); no obstante, ninguno obtuvo una significancia estadística. Secundariamente, se encontró asociaciones entre RI con el IMC, porcentaje de grasa corporal y el sexo masculino. Conclusiones: En nuestra población de estudio no se encontró asociación entre hipotiroidismo subclínico y resistencia a la insulina. / Objective: To assess whether there is an association between subclinical hypothyroidism and insulin resistance in non-diabetic patients at a private clinic in Lima. Materials and methods: An analytical observational cross-sectional study was carried out. Participants were adults who attended the endocrinology outpatient clinic of a private clinic in Lima in the years 2012 to 2018. Patients who were previously diagnosed with diabetes or thyroid diseases, who received systemic corticosteroids, who took lipid-lowering drugs, were excluded. or that they were brewing. The main variables were subclinical hypothyroidism and insulin resistance, which were worked as categorical variables. To define the subclinical hypothyroidism variable, TSH values ​​greater than or equal to 4.5 mU / L and T4L were used in normal values, while the variable insulin resistance was defined using the HOMA-IR value greater than 2.79. Likewise, other variables such as BMI, percentage of body fat and sex were also studied. To assess the association between subclinical hypothyroidism and insulin resistance, a generalized linear model of the Poisson family with crude and adjusted robust variance was carried out. It was presented as an association measure to the prevalence ratio (PR) with its 95% confidence interval. Results: We analyzed 1389 patients, the average age was 39.56 (13.54) years (SD) and the % of men was 27.87%. The prevalence of HSC was 13.17% (n = 183) and the prevalence of IR was 40.4% in the HSC group and 46.2% in the non-HSC group. In the crude regression model, it was found that patients with HSC had a 12% lower probability of having IR in the crude analysis (PR 0.876 IC 95%: 0.727-1.055) and 7% less in the adjusted one (PR 0.927, IC 95%: 0.792-1.084); however, none obtained statistical significance. Secondarily, associations were found in IR with the IMC, percentage of body fat and male sex. Conclusions: In our study population there was no association between subclinical hypothyroidism and insulin resistance. / Tesis

L’hyperglycémie et l’insulino-résistance constituent les altérations métaboliques des patients septiques associées à un pronostic défavorable, à une augmentation des dysfonctions cardiovasculaires et à une augmentation de la mortalité. Si plusieurs études démontrent quel’insulinothérapie à forte dose diminue la mortalité et prévient la dysfonction multi-organes, elle est souvent controversée car responsable d’hypoglycémies délétères. La Protéine Tyrosine Phosphatase1B (PTP1B) est un régulateur négatif de la voie de signalisation dépendante de l’insuline et de la voie de production du NO.L’idée développée dans le laboratoire est que l’inhibition de la PTP1B pourrait constituer une cible thérapeutique potentielle dans le sepsis en améliorant la sensibilité à l’insuline et ainsi les conséquences sur la fonction endothéliale et cardiaque. Bien qu’il ait été montré que la délétion génétique en PTP1B diminue la dysfonction cardiovasculaire lors du sepsis, les effets de cette délétion sur le métabolisme glucidique dans l’amélioration de la dysfonction cardiovasculaire restent méconnus et constituent l’objectif de notre travail.Dans un modèle de sepsis induit par Ligature et Perforation Caecale, nous avons pu mettre en évidence que la délétion génétique totale de PTP1B limite l’insulino-résistance induite par le sepsis,améliore la voie de signalisation dépendante de l’AMPK et la translocation des GLUT-4 et diminue l’inflammation. Ces effets s’accompagnent d’une diminution de la dysfonction endothéliale induite parle sepsis et améliore la production de NO. La délétion génétique endothéliale de PTP1B permet quant à elle une amélioration significative de la fonction endothéliale et de la sensibilité à l’insuline et au glucose.Ces travaux ont donc permis de mettre en évidence l’effet bénéfique de la délétion génétique en PTP1B dans le sepsis par amélioration de la sensibilité à l’insuline et des conséquences sur la fonction endothéliale et cardiaque. / Hyperglycemia and insulin resistance are septic metabolic alterations associated with poorprognosis, increased cardiovascular dysfunction and mortality. Several studies have demonstrated thathigh-dose insulin therapy reduces mortality and prevents multi-organ dysfunction but is controversialbecause it is often associated with deleterious hypoglycemia. Protein Tyrosine Phosphatase 1B (PTP1B)is a negative regulator of both insulin signaling and NO production.The concept developed in our laboratory is that PTP1B inhibition could be a potentialtherapeutic target in sepsis by improving both insulin sensitivity and these consequences onendothelial and cardiac function. PTP1B genetic deletion has been shown to decrease cardiovasculardysfunction in sepsis but the effects of this deletion on carbohydrate metabolism in the improvementof cardiovascular dysfunction remain unknown and constitute the objective of our work.In a sepsis model induced by Ligature and Caecal Perforation, we have demonstrated that thetotal PTP1B genetic deletion limits insulin resistance induced by sepsis, improves the AMPK signalingpathway, the GLUT-4 translocation and reduces inflammation. These effects are followed by decreasedendothelial dysfunction induced by sepsis and improves NO production. The endothelial PTP1B geneticdeletion, significantly improves endothelial function, insulin and glucose sensitivity.This work demonstrate the beneficial effect of the PTP1B genetic deletion in the sepsis byimprovement of the insulin sensibility and these consequences on endothelial and cardiac function.

Le syndrome métabolique est caractérisé par un ensemble de perturbations métabolique. Il inclut la dyslipidémie, l’obésité abdominale, la résistance à l’insuline et l’hypertension. L’association de ces facteurs de risques est liée à une augmentation du risque de développer un diabète de type-2. Les acides gras polyinsaturés de la famille des oméga-3 ont plusieurs effets biologiques et modulent les facteurs de risques du syndrome métabolique par l’intermédiaire de multiples mécanismes. Cependant, leurs impacts sur la résistance à l’insuline et le diabète de type 2 sont encore inconnus.Au cours de ce travail, nous avons étudié l’effet d’Odontella aurita (OA), une microalgue riche en EPA (AGPI oméga-3) et antioxydants, sur la prévention de l’obésité et la résistance à l’insuline induites par un régime riche en acides gras saturés High-Fat (HF). En effet, nous avons montré que le régime HF soumis aux rats pendant 8 semaines conduit à une résistance à l’insuline qui se caractérise par une augmentation de l'insulinémie ainsi qu'à une diminution de l'expression protéique du récepteur de l'insuline. De plus, le régime HF provoque une diminution de la sensibilité du récepteur à l'insuline en inhibant son activité tyrosine kinase. Le régime HF conduit également à une augmentation de l'expression du récepteur TLR4, qui joue un rôle dans l'induction de la résistance à l'insuline par l'intermédiaire de l'activation des voies proinflammatoires par la résistine et le LPS. En effet, l'augmentation de l'expression de TLR4 est associée avec l'activation des MAPK proinflammatoires JNK et P38. Cependant, l'enrichissement du régime HF avec la microalgue normalise l'insulinémie et les niveaux d'expression du récepteur à l'insuline. Son activité tyrosine kinase est aussi restaurée. Et d'une manière intéressante, la supplémentation du régime HF avec la microalgue conduit à une réduction de l'expression du récepteur TLR4 ainsi qu'une inhibition des voies proinflammatoires prévenant ainsi la résistance hépatique à l’insuline.Le récepteur TLR4 et l’activation des voies pro-inflammatoires jouent un rôle important dans l’induction de la résistance à l’insuline. Afin d'explorer les mécanismes moléculaires impliqués dans la régulation de l’expression de TLR4 et déterminer les voies proinflammatoires impliquées dans l'induction de la résistance à l'insuline par les acides gras saturés, ainsi que la mise en évidence des mécanismes insulino-sensibilisateurs des AGPI oméga-3, nous avons utilisé les cellules SH-SY5Y (cellules de neuroblastome humain). En effet, les cellules SHSY5Y ont été exposées pendant 4h à l’acide palmitique (PA, acide gras saturé) ou au DHA (oméga-3) puis traitées avec la résistine. Tout d'abord, nous avons analysé l'effet de la résistine, le PA et le DHA sur les marqueurs de l'inflammation. Seule la résistine est capable d'activer NFkB et augmenter la phosphorylation d’Akt et de p38 MAPK. Toutefois, le prétraitement avec le PA augmente l'expression des cytokines inflammatoires (IL-6 et TNF-a), similaire à la résistine. D’une manière intéressante, le prétraitement au DHA supprime l’effet d PA et la résistine et prévient l’augmentation de l’expression d'IL-6 et TNF-α. Nous avons ensuite étudié la possibilité d'un effet synergique entre la résistine et le PA sur l’expression de TLR4. En effet, le prétraitement avec le PA augmente l'expression de TLR4 alors que le prétraitement au DHA n'a aucun effet. Nous avons montré aussi que le prétraitement au PA potentialise les effets de la résistine. En effet la résistine est le ligand de TLR4, et le PA, en augmentant l’expression de TLR4 favorise et amplifie les effets de la résistine.En conclusion, ces résultats montrent que les acides gras polyinsaturés oméga-3 préviennent l'inflammation et la résistance à l'insuline induite par les acides gras saturés via la régulation de la voie de signalisation de TLR4 empêchant ainsi l’installation du diabète de type 2 et du syndrome métabolique. / The metabolic syndrome is characterized by dyslipidemia, insulin resistance, abdominal obesity and hypertension, which are related to an elevated risk for type 2 diabetes mellitus. Omega-3 polyunsaturated fatty acids have extensive biological effects and modulate the risk factors for metabolic syndrome via multiple mechanisms. However their impact on insulin resistance and type 2 diabetes are still unknown.In the current study, we report that Odontella aurita, a microalga rich in the omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA), prevents High saturated fat diet induced insulin resistance and inflammation in the liver of Wistar rats. High fat diet (HFD), given for 8 weeks, increased plasma insulin levels associated with the down-regulation of insulin receptor (IR) and the impairment of insulin-dependent IR phosphorylation. Furthermore, HFD increased toll-like receptor 4 (TLR4) expressions. Indeed, we have recently reported that TLR4 is implicated in resistin-induced inflammation and insulin resistance in the hypothalamus (Benomar et al, 2013). We also show that TLR4 up-regulation is concomitant with the activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38). Importantly, Odontella aurita enriched HFD (HFOA, 12%) normalized body weight and plasma insulin levels, and restores IR expression at both protein and mRNA levels. In addition, HFAO improves insulin responsiveness as estimated by in vitro phosphorylation of hepatic plasma membrane IR. Furthermore, HFOA decreased TLR4 expression and JNK /p38 phosphorylation. In conclusion, we demonstrate, for the first time to our knowledge, that omega-3 fatty acids brought by Ondontella aurita overcomes HFD-induced insulin resistance through the inhibition of TLR4/JNK/p38 MAP kinase signaling pathways.To further explore the molecular process underlying the activation of TLR4 by fatty acids, we aim to decipher the mechanisms implicated in the regulation of TLR4 expression. For this purpose, human neuroblastoma cells (SHSY-5Y) were exposed during 4h to either palmitic acid (a saturated fatty acid) or the omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA). Cells were then treated with resistin. Firstly we analyzed the effect of resistin, palmitic acid and DHA on inflammation markers. We show that only resistin was able to activate NF-κB and to increase the phosphorylation of Akt and p38 MAPK. However, palmitic acid pretreatment increases the expression of inflammatory cytokines (IL-6 and TNF-α), similar to resistin. Interestingly, DHA pretreatment suppresses palmitic acid and resistin induced up-regulation of IL-6 and TNF-α. Secondly, we studied the possible synergistic interaction between resistin and palmitic acid on TLR-4 expression. We show that palmitic acid pretreatment increases TLR4 expression, at both protein and mRNA levels, while DHA pretreatment had no effect. Importantly, palmitic acid pretreatment potentiates resistin effects. In conclusion, we show for the first time, to our knowledge, that palmitic acid induces TLR4 expression and this leads to the amplification of resistin effects promoting then insulin resistance at the neuronal level.Taken together, these results demonstrate that omega-3 fatty acids prevent saturated fat-induced inflammation and insulin resistance through resistin/TLR4 signaling thereby preventing insulin resistance.

Die subklinische Klauenrehe ist eine weltweit vorkommende, multifaktorielle und bei Rindern vor allem nach der Abkalbung gehäuft auftretende Erkrankung. Als prädisponierender Faktor für weitere, z. T. sehr schmerzhafte Klauenerkrankungen wie Sohlengeschwüre oder White-Line-Disease besitzt sie nicht nur eine hohe tierschutzrelevante, sondern auch eine große wirtschaftliche Bedeutung. Die Ätiologie und Pathogenese der Klauenrehe sind bis heute nicht hinreichend geklärt. In neuerer Zeit konzentrieren sich die Forschungen auf den peripartalen Zeitraum und den damit verbundenen metabolischen und hormonellen Einflüssen auf das Klauengewebe. In dieser Arbeit wird der Frage nachgegangen, inwieweit eine erhöhte Fettmobilisation bei Kühen in der negativen Energiebilanz nach der Abkalbung sowie eine verstärkte Lipolyse bei antepartal verfetteten Kühen zur Entstehung der subklinischen Klauenrehe beitragen. Gleichzeitig soll die Frage beantwortet werden, welche Rolle die postpartale Insulinresistenz und die dadurch verminderte Glucoseaufnahme ins periphere Gewebe bei der Ausbildung der subklinischen Klauenrehe spielen. Außerdem soll untersucht werden, inwiefern sich systemische Einflüsse auf die verstärkte Ausbildung der Sohlenhämorrhagien nach der Abkalbung nachweisen lassen und ob diese anhand ausgewählter Blutparameter vorhersehbar sind. Für die Untersuchung wurden 30 primi- und 44 multipare Milchkühe aus drei Betrieben mit Laufstallhaltung ausgewählt. Die Sohlenflächen aller acht Hauptklauen eines Tieres wurden nach funktionellem Klauenschnitt 1 Woche und 8 Wochen p.p. fotografiert, in fünf Zonen eingeteilt und anhand Anzahl, Größe und Schweregrad der sichtbaren Läsionen beurteilt. Zur weiteren Differenzierung wurden verschiedene Klauenscores gebildet. 2 - 3 Wochen vor der Abkalbung sowie 1 Woche, 4 und 8 Wochen p.p. wurden Blutproben entnommen und die Konzentrationen der Freien Fettsäuren (FFS), ß-Hydroxy-Butyrat (BHB), Glucose, Insulin und Haptoglobin bestimmt. Die IGF-1-Konzentration wurde zur weiteren Einschätzung der Energiebilanz bzw. Energieaufnahme und der Körperkondition gemessen. Die Insulinresistenz wurde anhand basaler Insulin- und Glucose-Konzentrationen bestimmt. Des Weiteren wurde vor und nach der Abkalbungdie Rückenfettdicke sonographisch gemessen und die peripartale Fettmobilisierung über die Rückenfettdickenänderung errechnet. Nahezu alle untersuchten Tiere (96 %) zeigten für die subklinische Klauenrehe typische Veränderungen. Bei über der Hälfte der Kühe waren 8 Wochen p.p. an allen vier Gliedmaßen Sohlenhämorrhagien vorhanden. Es konnten signifikante Korrelationen der Klauenscoreparameter sowohl zwischen den einzelnen Zonen als auch zwischen den Hinter- und Vordergliedmaßen gefunden werden. 8 Wochen p.p. war eine signifikante Verschlechterung der Klauengesundheit im Gegensatz zur ersten Woche p.p. zu verzeichnen. Da diese Hämorrhagien erst nach zwei Monaten an der Fußungsfläche sichtbar werden, sind die ursächlichen Veränderungen in der Lederhaut zum Zeitpunkt der Abkalbung entstanden. Dabei waren die lateralen Klauen der Hintergliedmaße an der Rusterholzstelle am stärksten betroffen. Ein Einfluss auf die Klauengesundheit durch die Fütterung, die Bodenbeschaffenheit der Haltungssysteme, altersbedingt sowie durch peripartale Erkrankungen konnte nicht gefunden werden. Ungefähr zwei Drittel der untersuchten Kühe hatten nach der Abkalbung eine negative Energiebilanz. Sie zeigten signifikant weniger Läsionen an den Sohlenflächen als Kühe mit positiver Energiebilanz. Eine übermäßige Rückenfettmobilisierung führte in diesen Untersuchungen nicht zu einer Verschlechterung der Klauengesundheit. Antepartal unterkonditionierte Tiere mit wenig Fettmobilisierung waren sogar stärker von Klauenläsionen betroffen als normal- oder überkonditionierte Kühe. Bei Tieren mit einem peripartalen Abfall der IGF-1-Konzentration waren signifikant mehr Veränderungen an der Sohlenfläche nachzuweisen. Die IGF-1-Konzentration korrelierte dabei aber, im Gegensatz zu vielen Angaben in der Literatur, hochsignifikant negativ mit der Energiebilanz und zeigte keinen Bezug zur RFD, so dass fraglich ist, ob dieser Parameter alleine überhaupt geeignet wäre, eine Aussage über die Energiebilanz oder die Ausbildung einer subklinischen Klauenrehe zu treffen. Bei insulinresistenten Kühen waren eine signifikante Erhöhung der Klauenläsionen sowie ein signifikanter Abfall der IGF-1-Konzentration zu verzeichnen. Die Bestimmung der Insulinresistenz anhand basaler Blutglucose- und Insulin-Konzentrationen bei Kühen nach der Abkalbung erwies sich jedoch als äußerst fragwürdig. Signifikante Korrelationen zwischen den Konzentrationen der einzelnen Blutparameter und den Klauenscoreparametern bestanden, bis auf die FFSKonzentration 2 - 3 Wochen a.p., ausschließlich 1 Woche p.p.. Allerdings blieben die Korrelationen insgesamt relativ niedrig. Die IGF-1-Konzentration korrelierte am häufigsten sowie am engsten, Insulin und Haptoglobin korrelierten zu keinem Zeitpunkt mit den Klauenscoreparametern. Es kann davon ausgegangen werden, dass es sich bei den gefundenen Hämorrhagien an der Sohlenfläche um die Ausbildung einer subklinischen Klauenrehe aufgrund systemischer Einflüsse im peripartalen Zeitraum handelt. Eine negative Energiebilanz sowie die antepartale Verfettung der Milchkühe stellen nach den vorliegenden Untersuchungen keinen Risikofaktor dar. In dieser Arbeit konnte nicht bestätigt werden, dass Lipidmobilisation oder mangelnde Glucoseversorgung nach der Abkalbung eine Rolle bei der Entstehung der subklinischen Klauenrehe spielen.

Zusammenfassung: Effekte der L-Carnitinsupplementierung auf das metabolische Profil adipöser und insulinre- sistenter Ponys im Verlaufe einer mehrwöchigen Körpergewichtsreduktion Author: Uta Schmengler Institut für Tierernährung, Ernährungsschäden und Diätetik, Veterinärmedizinische Fakultät, Universität Leipzig Eingereicht im September 2012 76 S., 16 Abb., 23 Tab., 169 Lit., Anhang Einleitung: Das ”Equine Metabolische Syndrom” ist gekennzeichnet durch eine regionale oder generalisierte Adipositas, eine periphere Insulinresistenz sowie akute oder chronische Hufreheschübe. Die Ursache ist in einer bedarfsübersteigenden, hochkalorischen Fütterung und einem relativen Bewegungsmangel zu suchen, wobei auch der genetischen Prädisposition spezieller Rassen eine gewisse Bedeutung zukommt. Ziel dieser Studie war die Untersuchung der Effekte einer L-Carnitinsupplementierung in Kombination mit einer restriktiven Füt- terung und täglicher moderater Bewegung auf Körpermasseverlust, Insulinsensitivität und ausgewählte Parameter des Energiestoffwechsels adipöser und insulinresistenter Ponys. Material und Methoden: Für die placebokontrollierte Doppelblindstudie wurden 16 adipöse Ponys per Losverfahren in zwei Gruppen (N=8) eingeteilt. Zu Versuchsbeginn wiesen die Ponys einen mittleren Body Condition Score von 8,0±2,0 (Skala 1-9) und einen mittleren Cresty Neck Score von 4,0±1,0 (Skala 0-5) auf. Während des 14-wöchigen Körpermassere- duktionsprogramms wurden die Ponys restriktiv gefüttert mit 1 - 1,2 kg Heu/100 kg KM/d. Zusätzlich erhielten 8 Ponys eine L-Carnitin-Zulage (1,3 g/100 kg KM/2d) und 8 Tiere ein Placebo in Form einer Kieselsäureverbindung (1,3 g/100 kg KM/ 2d). Die Ergänzungen wur- den in einem Gemisch aus Grünmehl (50 g/2d) und Mineralfutter verabreicht. Über die 14-wöchige Versuchszeit wurde ein Bewegungsprogramm an sechs Tagen in der Woche durch- geführt, das 25 Minuten Schritt und 15 Minuten Trab beinhaltete. Zu Versuchsbeginn und nach Versuchsende wurde mit beiden Versuchsgruppen ein Frequently sampled intravenous glucose tolerance test (FSIGTT) zur Überprüfung der Insulinsensitivität durchgeführt. Über die gesamte Versuchszeit wurden wöchentlich Blutproben gewonnen zur Bestimmung der ba- salen Serum-Insulinaktivität und Plasma-Glucosekonzentration sowie der Konzentration der Freien Fettsäuren (FFS), Triacylglyceride (TAG), Harnstoff und Betahydroxybutyrat (BHB) im Serum. Die Körpermasseverluste wurden über wöchentliche Wägungen sowie Ermittlung von BCS und CNS kontrolliert. Die statistische Überprüfung wurde anhand parametrischer (ANOVA) und nicht-parametrischer Tests (Wilcoxon signed rank test) durchgeführt, die Kal- kulation der Insulinsensitivität erfolgte über das Minimalmodell anhand eines Computerpro- gramms (MINMOD). Ergebnisse: Im Mittel verloren die Ponys über den Versuchszeitraum von 14 Wochen 1- 3% ihrer Körpermasse pro Woche (Zeit: p < 0, 01, Behandlung: p=0,79), was einem totalen Körpermasseverlust von 14,3±% entsprach. Der BCS reduzierte sich in beiden Versuchs- gruppen um eine Differenz von 3 Einheiten, der CNS verringerte sich in der Carnitingrup- pe (GC ) um eine Differenz von 1,4 und in der Placebogruppe (GP ) um eine Differenz von 1,9 Einheiten. Der Körpermasseverlust war von einer signifikanten Verbesserung der Insu- linsensitivität (Zeit p < 0, 01, Behandlung: p=0,39) begleitet. Die Kalkulation der Insulin- sensitivität im Minimalmodell zeigte eine signifikante Erhöhung der SI-Werte am Versuch- sende in beiden Versuchsgruppen (Beginn Studie GC : 0,76±0,88 l/min/μU*10−4 und GP : 1,61±1,31 l/min/μU*10−4 ; Ende Studie GC : 5,45±0,81 l/min/μU*10−4 und GP : 6,08±2,98 l/min/μU*10−4 ). Signifikante, zeitabhängige Veränderungen wurden auch für die metabo- lischen Parameter beobachtet: Plasma-Glucose und Serum-Insulin reagierten mit einem si- gnifikanten Abfall (Glucose GC : 4,5±0,32 mmol/l vs. 4,21±0,61 mmol/l und Glucose GP : 4,34±0,62 mmol/l vs. 3,86±0,34 mmol/l; Insulin GC : 23,71±32,77 μU/ml vs. 3,67±3,94 μU/ml und GP : 13,55±12,67 μU/ml vs. 1,01±1,09 μU/ml). Dabei kam es zu einem signi- fikanten Anstieg des Serum-Harnstoffs (GC : 3,47±0,73 mmol/l vs. 4,31±1,06 mmol/l und GP : 3,71±0,79 mmol/l vs. 4,9±1,23 mmol/l) sowie der Serum-FFS (GC : 157±95 μmol/l vs. 731±138 μmol/l und GP : 113±63 μmol/l vs. 686±142 μmol/l) und Serum-TAG (GC : 0,53±0,28 mmol/l vs. 0,94±0,61 mmol/l und GP : 0,45±0,23 mmol/l vs. 0,64±0,25 mmol/l). Bezüglich der L-Carnitinsupplementierung wurden keine weiteren Effekte verzeichnet. Schlussfolgerungen: Die restriktive Energiezufuhr von 7 MJ DE/100 kg KM entspre- chend einer Heuzulage von 1 kg/100 kg KM führte zu KM-Verlusten von 1-3 %. Eine Kör- permassereduktion zeigte deutliche Auswirkungen auf den Glucose- und Lipidmetabolismus und führte zu einer signifikanten Verbesserung der Insulinsensitivität, wohingegen die L- Carnitinsupplementierung keine weiteren Effekte auf den Glucosestoffwechsel herbeiführte. Eine bedarfsdeckende Eigensynthese von L-Carnitin ist beim Pony offensichtlich auch im Zu- stand der Insulinresistenz gewährleistet und reicht aus um die obligatorischen Funktionen L-Carnitins im Energiestoffwechsel zu erfüllen. / Summary: The effects of L-carnitine supplementation on body weight losses and metabolic profile in obese and insulin resistant ponies during a several weeks lasting bodyweight reduction pro- gramme Author: Uta Schmengler Institute of Animal Nutrition, Nutrition Diseases and Dietetics, Faculty of Veterinary Medi- cine, University of Leipzig Submitted in September 2012 76 p., 16 fig., 23 tab., 169 ref., appendix Introduction: Insulin resistance, local or general adiposity and the predisposition towards acute or chronical laminitis are components of the equine metabolic syndrome. Contributing factors for this syndrome are the intake and the quality of a high caloric feed by a lack of physical exersice. Howewer, the genetically predisposition of so called ”easy keepers” seems to play a role in pathogenesis. The objective of this study was to investigate the effects of L- carnitine supplementation in combination with a body weight reduction programme (BWRP) on body weight (BW) losses, insulin sensitivity and selected metabolic parameters in obese and insulin resistant ponies. Material und methods: 16 obese ponies (mean BCS = 8.0±2.0, mean CNS = 4.0±1.0) were assigned to a randomized double blind, placebo-controlled study. The ponies werde di- vided into two equal groups (N=8). During a 14 weeks lasting BWRP the ponies were fed 1.0-1.2 kg hay/100 kg BW daily. Additionally, 8 ponies were supplemented with L-carnitine (1.3g/100 kg BW) and 8 ponies were supplemented with a placebo (1.3g/100 kg BW). The supplements were offered in a mixture of 50 g grass meal and 50 g of a commercial mineral mixture, twice a day. During BWRP ponies were exercised a low-intensity protocol 6 days a week (daily 25 min walk and 15 min trot across the countryside). A frequently sampled intravenous glucose tolerance test (FSIGTT) was undertaken in order to assess insulin sen- sitivity at the beginning and the end of the study. Routine blood samples were collected for analysis of plasma glucose, serum insulin, free fatty acids (FFA), triglycerides (TG), urea and beta-hydroxybutyrate (BHB). Ponies were weighed weekly after 12 h of feed restriction by using an electronic scale for large animals. BCS and CNS were recorded weekly by the same 2 observers throughout the study. The statistical analysis was performed by parametric and non-parametric tests (ANOVA and Wilcoxon ranked test). The minimal modell calcu- lation of insulin sensitivity (SI) from FSIGTT was calculated by the computer programme (MINMOD). Results: Ponies lost 1-3% BW per week over the BWRP (time P<0.01, L-carnitine supple- mentation P=0.79), meaning a total body weight loss of 14.3%. BCS decreased in both groups with a difference of three points and CNS was reduced with a difference of 1.4-1.9 points. BW losses were accompanied by a significant improvement in insulin sensitivity (Time: P<0.01, L-carnitine supplementation: P=0.39). The calculation for SI-values by the minimalmodell showed a significant increase in L-carnitine group (GC ) and placebo group (GP ) in the end of the study. (GC : 0.76±0.88 L/min/μU*10−4 to 5.45±0.81 L/min/μU*10−4 , GP : 1.61±1.31 L/min/μU*10−4 to 6.08±2.98 L/min/μU*10−4 ). Significant time related decreases were observed for plasma glucose (GC : 4.5±0.32 mmol/L to 4.21±0.61 mmol/L, GP : 4.34±0.62 mmol/L to 3.86±0.34 mmol/L) and serum insulin (GC : 23.71±32.77 μU/mL to 3.67±3.94 μU/mL, GP : 13.55±12.67 μU/mL to 1.01±1.09 μU/mL). A significant increase was observed for serum urea (GC : 3.47±0.73 mmol/L to 4.31±1.06 mmol/L, GP : 3.71±0.79 mmol/L to 4.9±1.23 mmol/L), FFA (GC : 157±95 μmol/L to 731±138 μmol/L und GP : 113±63 μmol/L to 686±142 μmol/L) and TG (GC : 0.53±0.28 mmol/L to 0.94±0.61 mmol/L, GP : 0.45±0.23 mmol/L to 0.64±0.25 mmol/L) during BWRP. There was no further improvement in metabolic responses by L-carnitine supplementation. Conclusions: Energy intake of 7 MJ DE/100 kg BW leads to bodyweight losses of 1- 3%, herby improving insulin sensitivity and glucose metabolism. L-carnitine supplementation does not further improve glucose or fat metabolism, suggesting that endogenous L-carnitine synthesis was sufficient to facilitate energy metabolism in obese and insulin resistant ponies.

Adipose tissue is one of the most dynamic tissues in the body and is vital for metabolic homeostasis. In the case of excess nutrient uptake, adipose tissue expands to store excess energy in the form of lipids, and in the case of reduced nutrient intake, adipose tissue can shrink and release this energy. Adipocytes are most functional when the balance between these two processes is intact. To understand the molecular mechanisms that drive insulin resistance or conversely preserve the metabolically healthy state in obese individuals, our laboratory performed a screen for differentially regulated adipocyte genes in insulin resistant versus insulin sensitive subjects who had been matched for BMI. From this screen, we identified the type II transmembrane protein tenomodulin (TNMD), which had been previously implicated in glucose tolerance in gene association studies. TNMD was upregulated in omental fat samples isolated from the insulin resistant patient group compared to insulin sensitive individuals. TNMD was predominantly expressed in primary adipocytes compared to the stromal vascular fraction from this adipose tissue. Furthermore, TNMD expression was greatly increased in human preadipocytes by differentiation, and silencing TNMD blocked adipogenic gene induction and adipogenesis, suggesting its role in adipose tissue expansion. Upon high fat diet feeding, transgenic mice overexpressing Tnmd specifically in adipose tissue developed increased epididymal adipose tissue (eWAT) mass without a difference in mean cell size, consistent with elevated in vitro adipogenesis. Moreover, preadipocytes isolated from transgenic epididymal adipose tissue demonstrated higher BrdU incorporation than control littermates, suggesting elevated preadipocyte proliferation. In TNMD overexpressing mice, lipogenic genes PPARG, FASN, SREBP1c and ACLY were upregulated in eWAT as was UCP-1 in brown fat, while liver triglyceride content was reduced. Transgenic animals displayed improved systemic insulin sensitivity, as demonstrated by decreased inflammation and collagen accumulation and increased Akt phosphorylation in eWAT. Thus, the data we present here suggest that TNMD plays a protective role during visceral adipose tissue expansion by promoting adipogenesis and inhibiting inflammation and tissue fibrosis.