Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Introduction: A westernized diet rich in saturated fats and sugars, together with a sedentary lifestyle, has contributed to the dramatic increase in obesity during the last decade (Zimmett et al, 2001; Wild et al, 2004). Obesity is associated with dyslipidemia and insulin resistance which are major risk factors for the development of type 2 diabetes (T2D) (Zimmet et al, 2001, Kahn et al, 2006; Schröder et al, 2007). High-fat feeding in rodents induces symptoms similar to the human metabolic syndrome without progression to T2D (Woods et al, 2002; Weir and Bonner-Weir, 2007). The addition of fructose to a high-fat diet exacerbates the insulin resistance and leads to impaired pancreatic function of insulin secretion and glucose intolerance (Basciano et al, 2005; Stanhope et al, 2009). Aims: The aim of this study was to establish the effect of a high-fat and sucrose/fructose diet on glucose metabolism, the development of insulin resistance and β-cell dynamics. Methods: Weanling Wistar rats were randomized into two study groups; study one over an experimental period for three months and study two for twelve months. Each study consisted of a control group that received standard rat chow and water; and two experimental groups receiving either a high-fat diet and water (HFD) or a café diet consisting of HFD with the addition of 15% sucrose/fructose (CFD). Fasting glucose and insulin concentrations, intravenous glucose tolerance test (IVGTT), glucose stimulated insulin secretion rates and 2-deoxy-[3H]-D-glucose uptake in muscle, liver and fat were measured. The pancreata were harvested for immunohistochemical labeling of β-cells (insulin), α-cells (glucagon), GLUT2 (glucose transport) and MIB5 (proliferation). Samples of the pancreata were also collected for electron microscopy. Results and discussion: Feeding Wistar rats a CFD induced obesity, insulin resistance and glucose intolerance. By twelve months the rats had an impaired glucose response with increased IVGTT peak values, area under the curve (AUC) values and glucose clearance rates. Concomitantly, the glucose stimulated insulin secretion rate (GS-ISR) was attenuated. Stimulated glucose disposal as measured by 2-deoxy-[3H]-D-glucose uptake was reduced in muscle and adipose tissue at three months. By twelve months, due to the age of the rats, stimulated glucose uptake declined compared to three months with no difference between groups. After three months the diets had no observable effect on the islets using light microscopy. However, by twelve months morphological changes were observed in both the HFD and CFD groups. In the HFD group large hypertrophied irregular islets with fibrous changes were observed. In the CFD group these morphological changes were more prominent with fibrous segregation and disruption of the normal endocrine arrangement. In addition, the presence of inflammatory cells within the affected islets is consistent with T2D. Conclusion: High-fat diet fed to Wistar rats induced obesity, abdominal adiposity and insulin resistance. The addition of sucrose/fructose to a high-fat diet exacerbated the insulin resistance and resulted in glucose intolerance and mild hyperglycemia. Morphological changes in the large islets were observed which are consistent with the development of T2D. / AFRIKAANSE OPSOMMING: Inleiding: ‘n Verwesterde dieët, ryk aan versadigde vette en suikers tesame met 'n passiewe lewenstyl, het bygedra tot die dramatiese verhoging in vetsug gedurende die laaste dekade (Zimmett et al, 2001; Wild et al, 2004). Vetsug word met dislipidemie en insulienweerstandigheid geassosieer wat hoof risikofaktore is vir die ontwikkeling van tipe 2 diabetes (T2D) (Zimmet et al, 2001; Kahn et al, 2006; Schröder et al, 2007). Hoë-vet voeding in knaagdiere induseer simptome soortgelyk aan menslike metaboliese sindroom sonder die ontwikkeling van T2D (Woods et al, 2002; Weir and Bonner-Weir, 2007). Die byvoeging van fruktose tot 'n hoë-vet dieët vererger insulienweerstandigheid en lei tot verswakte pankreas funksie, insuliensekresie en glukoseintoleransie (Basciano et al, 2005; Stanhope et al, 2009). Doelwitte: Die doelwitte van die studie was om die effek van hoë-vet en sukrose/fruktose voeding op glukosemetabolisme, die ontwikkeling van insulienweerstandigheid en β-sel dinamika te bepaal. Metodes: Gespeende Wistar rotte was in twee groepe gerandomiseer; studie een oor ʼn tydperk van drie maande en studie twee oor ʼn tydperk van twaalf maande onderskeidelik. Elke studie het 'n kontrole groep met standaard rot kos en water (control); en twee experimentele diëte wat of ʼn hoë-vet dieët en water (HFD) of 'n kafeedieët groep wat die HFD met die byvoeging van 15% sukrose/fruktose in hul drink water (CFD) ontvang. Fastende glukose en insulien, binneaarse glukose toleransie toets (IVGTT), glukose gestimuleerde insulien sekresie tempo en 2-deoxi-[3H]-D-glukose opname in spier, lewer en vet is gebruik om die effek van die dieët op glukosemetabolisme te bepaal. Die pankreata is uitgehaal vir immunohistochemiese identifisering van β-selle (insulien), α-selle (glukagoon), GLUT2 (glukose transport) en MIB5 (proliferasie). Monsters van die pankreata was ook vir elektronmikroskopie versamel. Resultate en bespreking: Voeding van ʼn CFD aan Wistar rotte induseer vetsug, insulienweerstandigheid en glukose-intoleransie Teen twaalf maande toon die rotte 'n verswakte respons tot glukose met verhoogde IVGTT piekwaardes, AUC waardes en glukose opruimingswaardes. Terselfdetyd is die glukose gestimuleerde insuliensekresie tempo (GS-ISR) ook verswak. Gestimuleerde glukose opruiming, soos deur 2-deoxi-[3H]-D-glukose opname bepaal, was verlaag in spier en vetweefsel teen drie maande. Teen twaalf maande, weens die ouderdom van die rotte, is die gestimuleerde glukose opname verlaag in vergelyking met drie maande sonder 'n verskil tussen groepe. Na drie maande kon geen sigbare morfologiese verskille met ligmikroskopie tussen die diëte waargeneem word nie. Teen twaalf maande is morfologiese verskille waargeneem in beide die HFD en die CFD groepe. In die HFD groep is groot hipertrofiese onreëlmatige eilande met fibrotiese verandering waargeneem. In die CFD groep was die morfologiese verandering meer gevorder met fibrotiese onderverdeling en ontwrigting van die normale endokriene rangskikking. Die teenwoordigheid van inflammatoriese selle in die geaffekteerde eilande is verenigbaar met T2D. Afleiding: Die voer van 'n hoë-vet dieët aan Wistar rotte veroorsaak vetsug, abdominale adipositeit en insulienweerstandigheid. Die byvoeging van sukrose/ fruktose tot die hoë-vet dieët vererger die insulienweerstandigheid en veroorsaak glukoseintoleransie en matige hiperglukemie. Morfologiese veranderings in die groter eilande was verenigbaar met T2D.

Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Introduction: Athrixia phylicoides is an aromatic, indigenous shrub with high antioxidant content and numerous indigenous medicinal properties inferred by ingestion of an herbal brew of the plant. Commercialization of “bush tea” (derived from A. phylicoides) holds economic and developmental potential for indigenous communities provided the safety and efficacy of the herbal tea is established. Recently A. phylicoides has been shown by McGaw et al. (2007) to have similar antioxidant activity to Rooibos tea, and a unique, new flavonol (i.e. a polyphenolic antioxidant plant metabolite) 5-hydroxy-6,7,8,3′,4′,5′-hexamethoxyflavon-3-ol, unique to A. phylicoides, was isolated by Mashimbye et al. in 2006. With changes in the socio-economic climate and a new trend in merging Western lifestyle with traditional practices, new interest has been shown in herbal/natural remedies. Study Aim: The aim of this study was to firstly, determine the in vitro effect of A. phylicoides aqueous extract on glucose metabolism in cell lines that mimic the three key organs implicated in glucose homeostasis. Secondly, the study aimed to determine the potential ex vivo antioxidant and anti-inflammatory effect of the extract in pancreatic β-cells and peripheral mononuclear cells respectively. Methods: Leaves and fine twigs of A. phylicoides were processed into an aqueous extract. C2C12, Chang and 3T3-L1 cells were cultured under standard conditions and acutely exposed to increasing concentrations of extract and water vehicle, as well as 1 μM insulin and metformin as positive controls. Glucose uptake from 8 mM glucose culture media was determined using a fluorimetric oxidase method. Radioactive 14C-glucose oxidation to 14CO2 and determination of glycogen content of cells were used to assess the fate of intracellular glucose. RT-PCR was used to assess the extract effect on insulin-signalling gene expression. The antioxidative effect of A. phylicoides extract in pancreatic β-cells isolated from Wistar rats was determined by measuring nitric oxide (NO) production in response to hyperglycemic conditions. NO was labelled with diaminofluorocein diacetate and fluorescence was measured using flow cytometry. Insulin secretion of pancreatic β- cells was measured using radio-immuno assay. The anti-oxidative effect of the extract in lipopolysaccharide-stimulated peripheral mononuclear cells isolated from Wistar rats was determined by measuring the production of TNF-α using an ELISA kit. Results: C2C12 myocytes showed maximal increased glucose uptake at the 0.05 μg/μl extract concentration (228.3% ± 66.2, p<0.001). In Chang cells, A. phylicoides extract maximally increased the amount of glucose taken up at the 0.05 μg/μl concentration (134.5% ± 2.5, p<0.05). In 3T3-L1 cells, the extract maximally increased the amount of glucose taken up at the 0.025 μg/μl concentration (143.5% ± 10.3, p<0.001). An extract-induced increase in insulin receptor and glucose transporter four expression was seen in C2C12 myocytes. The oxidation of 14C-glucose to 14CO2 by C2C12 myocytes was maximally increased following acute exposure to the extract at 0.1 μg/μl (2919.3 fmol/1x10^6 cells ± 428, p<0.01). The oxidation of 14C-glucose to 14CO2 by Chang cells was maximally increased following acute exposure to extract at 0.1 μg/μl (4476.7 fmol/1x10^6 cells ± 1620, p<0.05); as seen in the C2C12 cells. A. phylicoides extract increased glycogen storage at all three concentrations tested in Chang cells, but maximally at the 0.025 μg/μl concentration (13.6 μg/1x10^6 cells ± 0.7, p<0.05). A. phylicoides extract did not have any measurable effect on the oxidative status of β-cells or the anti-inflammatory status of peripheral mononuclear cells. The extract did show an increase in first phase insulin secretion of β-cells in hyperglycemic conditions, although it was not significant. Conclusion: Athrixia phylicoides aqueous extract stimulates in vitro glucose uptake and metabolism in an insulin-mimetic manner, suggesting that this extract could potentially be beneficial to type two diabetics as an adjunct therapy. / AFRIKAANSE OPSOMMING: Inleiding: Athrixia phylicoides is 'n aromatiese, inheemse struik met 'n hoë antioksidant inhoud. Vele tradisionele medisinale eienskappe is gekoppel aan die ingestie van 'n kruie brousel van die plant, wat ook bekend as “bostee” is. Kommersialisering van “bostee” hou ekonomiese en ontwikkelings potensiaal in vir inheemse gemeenskappe mits die veiligheid en effektiwiteit van die kruietee bevestig kan word. McGaw et al. (2007) het onlangs bevind dat A. phylicoides se antioksidant aktiwiteit vergelykbaar is met die van rooibostee. 'n Unieke nuwe flavonol ('n polifenoliese antioksidant plant metaboliet) 5-hydroksie-6,7,8,3′,4′,5′-hexamethoksieflavon-3-ol, eie aan A. phylicoides, is deur Mashimbye et al. in 2006 geïsoleer. Met veranderings in die sosio-ekonomiese klimaat en 'n nuwe tendens om die westerse lewenstyl met tradisionele gebruike aan te vul word nuwe belangstelling in kruie/natuurlike rate ondervind. Studie Doelwitte: Die doelwitte van hierdie studie was eerstens om die in vitro effek van A. phylicoides waterekstrak op die glukosemetabolisme van drie sellyne wat die sleutel organe naboots wat glukosehomeostase beheer, te bepaal. Tweedens, is die potensiële ex vivo antioksidant en anti-inflammatoriese effek van die ekstrak op pankreatiese β-selle en perifere mononuklêere-selle onderskeidelik ondersoek. Metodes: n Waterige ekstrak is van die blare en fyn takkies van A. phylicoides berei. C2C12, Chang and 3T3-L1 selle is gekultuur onder standaard kondisies en akuut blootgestel aan stygende ekstrakkonsentrasies. Water het as kontrole gedien, met 1 μM insulien en metformien as positiewe kontroles. Glukose opname vanuit 8 mM glukose kultuurmedia is bepaal deur 'n fluorimetriese oksidase metode. Radioaktiewe 14C-glukose-oksidasie na 14CO2 en die bepaling van die glukogeen inhoud van selle is gebruik om die lot van intrasellulêre glukose te bepaal. RT-PKR is gebruik om die effek van die ekstrak op die insulien-seinpad geen-uitdrukking te ondersoek. Die antioksidant effek van A. phylicoides ekstrak in pankreatiese β-selle geïsoleer van Wistar rotte, is bepaal deur stikstofoksied (NO) produksie na aanleiding van hiperglukemiese kondisies. NO is met diaminofluorosien diasetaat gemerk en die fluoresensie gemeet deur vloeisitometrie. Insulien afskeiding deur die pankreatiese β-selle is deur radio-immuno metode bepaal. Die anti-oksidatiewe effek van die ekstrak op lipopolisakkaried-gestimuleerde perifere mononuklêere-selle afkomstig van Wistar rotte is bepaal deur die meting van TNF-α produksie met 'n ELISA kit. Resultate: C2C12 miosiete het 'n maksimale toename in glukoseopname by 'n 0.05 μg/μl ekstrakkonsentrasie (228.3% ± 66.2, p<0.001) gehad. Dieselfde ekstrakkonsentrasie het maksimale toename in glukoseopname in Chang selle (134.5% ± 2.5, p<0.05 getoon. In 3T3-L1 selle is maksimale toename in die glukoseopname by 'n konsentrasie van 0.025 μg/μl (143.5% ± 10.3, p<0.001) bereik. 'n Ekstrak-geinduseerde verhoging in die insulienreseptor en glukosetransporter vier ekspressie is in C2C12 miosiete waargeneem. Die oksidasie van 14C-glukose na 14CO2 deur C2C12 miosiete is maksimaal verhoog deur akute blootstelling aan die ekstrak by 'n konsentrasie van 0.1 μg/μl (2919.3 fmol/1x10^6 cells ± 428, p<0.01). Die oksidasie van 14C-glukose na 14CO2 deur Chang selle was maksimaal verhoog deur akute blootstelling aan die ekstrak by 'n konsentrasie van 0.1 μg/μl (4476.7 fmol/1x10^6 cells ± 1620, p<0.05) soos gevind in die C2C12 selle. Die ekstrak het glukogeenstoring verhoog teen al drie die konsentrasies waarteen getoets is in Chang selle, maar 'n maksimale effek is gevind by 'n konsentrasie van 0.025 (13.6 μg/1x10^6 cells ± 0.7, p<0.05). A. phylicoides ekstrak het nie 'n meetbare effek op die oksidatiewe status van β-selle of die anti-inflammatoriese status van perifere mononuklêere-selle gehad nie. Die ekstrak het wel 'n verhoging in die eerstefase insuliensekresie van β-selle in hyperglukemiese kondisies gehad, alhoewel die verhoging nie statisties betekenisvol was nie. Afleiding: Athrixia phylicoides waterekstrak stimuleer in vitro glukoseopname en metabolisme in 'n insulin-mimetiese manier, wat beteken dat die ekstrak potensiëel voordele vir tipe twee diabete kan inhou as aanvullingsterapie.

Les oxydants infusés avec la nutrition parentéral (NP) néonatale induisent une modification du métabolisme des lipides et du glucose, donnant lieu à l’âge adulte à un phénotype de carence énergétique (faible poids, baisse de l’activité physique). L’hypothèse qu’une diète précoce riche en glucose prévient ces symptômes plus tard dans la vie, fut évalué chez le cobaye par un ANOVA en plan factoriel complet à deux facteurs (p < 0:05) : NP du jour 3 à 7, suivit d’une nourriture régulière (chow) (NP+) vs. chow à partir du 3ième jour (NP-), combiné avec une eau de consommation enrichie en glucose (G+) ou non (G-) à partir de la 3ième semaine. Les paramètres suivant ont été mesurés à l’âge de 9 semaine: taux de croissance, activité physique, activité de phosphofructokinase-1 et glucokinase (GK), niveau hépatique de glucose-6-phosphate (G6P), glycogène, pyruvate et potentiel redox du glutathion, poids du foie, glycémie, tolérance au glucose, concentrations hépatiques et plasmatiques en triacylglycérides (TG) et cholestérol. Le groupe G+ (vs. G-) avait un taux de croissance plus bas, une activité de GK et une concentration en G6P plus élevée, et un potentiel redox plus bas (moins oxydé). Le niveau plasmatique de TG était moins élevé dans le groupe NP+ (vs. NP-). Les traitements n’eurent aucun effet sur les autres paramètres. Ces résultats suggèrent qu’indépendamment de la NP, une alimentation riche en glucose stimule la glycolyse et déplace l’état redox vers un statut plus réduit, mais ne surmonte pas les effets de la NP sur le phénotype physique de carence énergétique. / Neonatal exposure to oxidant molecules from total parenteral nutrition (TPN) alters future lipid and glucose metabolism, resulting in an energy deficient phenotype characterized by lower body weight and physical activity. Using a guinea-pig model, the hypothesis that early diet supplementation with glucose could overcome such symptoms at week 9 of age was tested in a two-factor full-factorial ANOVA design (p<0:05): TPN day 3-7, chow thereafter (TPN+) vs: chow from day 3 (TPN-), combined with glucose-enriched diet from week 3 (G+) vs: plain chow throughout (G-). The growth rate, physical activity, phosphofructose kinase-1 and glucose kinase (GK) activities, glucose-6-phosphate (G6P), glycogen and pyruvate concentrations, relative liver weight, fasting blood glucose, glucose tolerance, hepatic and plasma triacylglyceride and cholesterol levels, individual glutathione levels and GSH/GSSG-based redox potential were determined at 9 weeks. Glucose supplementation (vs: the lack thereof) resulted in a lower growth rate, higher GK activity, and higher G6P concentration at week 9. Plasma triacylglycerides at week 9 were lower in TPN+ (vs: TPN-) subjects. Hepatic GSH=GSSG-derived redox potential shifted to a more reduced state in G+ (vs: G-) subjects. No other parameters showed significant differences. Independently of TPN, an early glucose-rich diet stimulated the glycolysis pathway, shifted the redox potential towards a more reduced status ; however, it did not overcome the effects of TPN on future physical and metabolic phenotype.

L’insuffisance rénale chronique (IRC) se définit par un défaut de filtration glomérulaire et est associée à plusieurs désordres. La perturbation de l’homéostasie glucidique en fait partie. L’homéostasie glucidique est contrôlée principalement par l’insuline, soit l’hormone sécrétée en réponse au glucose par les cellules bêta-pancréatiques contenues dans les îlots de Langerhans. La préservation de la fonction de la cellule bêta est essentielle au maintien de l’homéostasie glucidique. Il a été démontré que la sécrétion de l'insuline est altérée au cours l'IRC, cependant les mécanismes demeurent peu connus. Au cours de l’IRC, l’accumulation chronique de toxines urémiques pourrait contribuer à la défaillance de la cellule bêta. L’urée est une toxine urémique majeure et sa toxicité a été récemment rapportée dans plusieurs tissus. Le but de ce mémoire était donc de vérifier le rôle de l’urée dans la dysfonction de la cellule bêta-pancréatique au cours de l’IRC. Nous avons démontré que l’exposition des îlots de souris à des concentrations pathologiques d’urée entraîne une diminution de la sécrétion d’insuline via l’augmentation du stress oxydant et des O-glycosylations. Ce défaut est dû à une perturbation du métabolisme intracellulaire du glucose. Entre autres, nous avons observé une baisse de la glycolyse associée à la réduction de l’activité enzymatique de la phosphofructokinase-1. Ces résultats démontrent un effet toxique direct de l’urée sur la sécrétion d’insuline et permettent de mieux comprendre le mécanisme de dysfonction de la cellule bêta-pancréatique au cours de l’IRC. / Chronic kidney disease (CKD) is defined as a glomerular filtration defect and is associated with many disorders. Impaired glucose homeostasis is one of them. Glucose homeostasis is maintained in part by insulin, which is the hormone secreted by the pancreatic beta cells from the islets of Langerhans in response to glucose. The preservation of beta cell function is essential to maintain glucose homeostasis. It has been demonstrated that insulin secretion is altered during CKD; however, the underlying mechanisms remain unknown. In CKD, chronic accumulation of uremic toxins could contribute to beta cell dysfunction. Urea is a major uremic toxin and its toxicity has been recently reported in many tissues. The purpose of this master project was to ascertain the role of urea in pancreatic beta cell dysfunction during CKD. We have demonstrated that exposure of mouse islets to pathological concentrations of urea leads to diminution of insulin secretion via an increase in oxidative stress and O-glycosylation. This defect is due to disturbed intracellular glucose metabolism. Among others, we have observed a reduction in glycolysis associated with a decrease in the activity of phosphofructokinase-1. These results demonstrate a direct toxic effect of urea on insulin secretion and contribute to a better understanding of mechanisms of pancreatic beta cell dysfunction during CKD.

O desenvolvimento gradual e recente de uma epidemia mundial de obesidade alavancou sobremaneira o estudo dessa condição e de suas comorbidades metabólicas. No âmbito fisiopatológico, múltiplos estudos demonstraram a expressão aumentada de mediadores inflamatórios no tecido adiposo de animais e humanos obesos, o acúmulo local de macrófagos, e um papel central da inflamação no desequilíbrio da homeostase metabólica local e sistêmica na obesidade. A definição de um papel ativo dos macrófagos, e portanto da imunidade inata, na rede inflamatória do tecido adiposo, evocou a hipótese de que, similarmente a outras condições inflamatórias crônicas como a aterosclerose, a obesidade também contaria com a importante participação de elementos da imunidade adaptativa, como as células T e suas citocinas, em sua fisiopatologia. Com base nessas considerações, os objetivos principais desse estudo foram: 1) avaliar a presença das células T e o papel do interferon-gama (IFNy), clássica citocina T-helper 1 (ou Th1), na inflamação do tecido adiposo; e 2) estudar mecanismos de acúmulo das células T no tecido adiposo na obesidade, particularmente a participação do receptor CXCR3 nesse processo. Experimentos de citometria de fluxo mostraram que o tecido adiposo visceral de camundongos C57BL/6 obesos após consumo de dieta rica em gorduras apresentou maior número de macrófagos e também de células T, CD4+ e CD8+, em comparação a controles que receberam dieta pobre em gorduras. A expressão de I-Ab, marcador do complexo de histocompatibilidade principal classe II (MHC II) murino, também foi maior no tecido adiposo dos animais obesos, sugerindo a presença local da atividade de apresentação de antígeno com consequente ativação das células T. Quando estimuladas in vitro, células T derivadas do tecido adiposo de camundongos obesos produziram mais IFNy do que aquelas isoladas de controles, novamente sugerindo a ativação dessas células em um contexto de obesidade. Na análise das possíveis funções do IFNy no tecido adiposo, a estimulação da linhagem de células 3T3-L1 diferenciadas em adipócitos com IFNy recombinante resultou na produção aumentada de quimiocinas de macrófagos, como a proteína quimiotática de monócito (MCP-1), e de quimiocinas de células T, como a proteína 10 induzida por IFNy (IP-10) e monocina induzida por IFNy (MIG). A estimulação de adipócitos com o sobrenadante de células Th1 cultivadas in vitro, com abundante concentração de IFNy, também levou à produção aumentada de IP-10. Em análise mais ampla, através de microarray, dos possíveis efeitos do IFNy na expressão gênica de adipócitos, o tratamento dessas células com 100 U/ml de IFNy resultou na expressão aumentada de diversas quimiocinas e seus receptores em comparação ao grupo tratado com placebo. Similarmente à estimulação de células isoladas com IFNy, a incubação de tecido adiposo ex vivo de camundongos com essa citocina também resultou em secreção aumentada de IP-10, MIG e fator de necrose tumoral alfa (TNFy). A investigação do papel do IFNy na inflamação do tecido adiposo in vivo envolveu camundongos com deficiência de IFNy e controles, ambos os grupos submetidos a dieta rica em gorduras (obesos) ou pobre em gorduras (não obesos). Camundongos obesos deficientes em IFNy apresentaram expressão reduzida de mRNA de genes inflamatórios como TNFalfa e MCP-1 no tecido adiposo; acúmulo local reduzido de macrófagos; e melhor tolerância à glicose em comparação aos controles sob mesma dieta. Animais com deficiência de apolipoproteína E (ApoE) e também do receptor de IFNy também apresentaram em seu tecido adiposo a expressão reduzida de mRNA de genes inflamatórios, particularmente relacionados às células T, como IP-10, MIG, e o receptor CXCR3, em comparação aos controles com deficiência única de ApoE. Resultados in vitro e in vivo sugerem conjuntamente um importante papel do IFNy, e portanto, das células T e da imunidade adaptativa, na rede inflamatória do tecido adiposo na obesidade, com consequente impacto metabólico sistêmico. A presença de células T ativadas no tecido adiposo e seu acúmulo diferencial na obesidade motivaram também a pesquisa de potenciais mecanismos quimiotáticos reguladores desse processo. CXCR3, receptor das quimiocinas de células T, IP-10, MIG e quimiocina alfa de células T IFNy-induzida (I-TAC), é expresso preferencialmente em células T ativadas, e detém papel central na migração dessas células em outras condições inflamatórias crônicas, como a aterosclerose. Em camundongos com deficiência de CXCR3 e que receberam dieta rica em gorduras por 8 ou 16 semanas, o tecido adiposo apresentou significativamente menos células T, incluindo as células CD4+ e CD8+, em comparação a controles submetidos a mesma dieta. Os números similares de células T e outras populações de leucócitos no baço e sangue periférico dos animais deficientes em CXCR3 e controles fortalecem o conceito de um efeito do CXCR3 sobre o acúmulo de células T no tecido adiposo, independentemente do número de células circulantes e periféricas. Os camundongos deficientes em CXCR3 apresentaram também maior tolerância à glicose e expressão reduzida de mRNA de mediadores inflamatórios em seu tecido adiposo em comparação aos controles após 8 semanas de dieta rica em gorduras. No entanto, a diferença na tolerância à glicose entre os dois grupos tornou-se não significativa após 16 semanas de dieta gordurosa, coincidindo com redução substancial na expressão de mRNA de mediadores anti-inflamatórios (como interleucina-10 [IL-10] e Arginase 1), e número reduzido de células T regulatórias no tecido adiposo de camundongo s deficientes em CXCR3 em relação a controles. Esses resultados sugerem que o CXCR3 é capaz de regular o acúmulo de células T de diferentes subtipos, com perfil proinflamatório ou anti-inflamatório. Em conclusão, nossos resultados revelam um importante papel da citocina Th1 IFNy na rede inflamatória do tecido adiposo na obesidade em camundongos, sugerindo a participação fundamental das células T e portanto, da imunidade adaptativa nesse cenário. Além disso, o receptor CXCR3 contribui significativamente para o acúmulo das células T, incluindo as células T regulatórias, no tecido adiposo desses animais / The gradual and recent development of a worldwide epidemic of obesity greatly leveraged the study of this condition and its metabolic comorbidities. In the pathophysiologic context, multiple studies have demonstrated increased expression of inflammatory mediators in adipose tissue of obese animals and humans, the local macrophage accumulation, and a central role of inflammation in the imbalance of local or systemic metabolic homeostasis in obesity. The concept of an active role of macrophages and thus of innate immunity in the inflammatory network of adipose tissue, suggested the hypothesis that, similar to other chronic inflammatory conditions such as atherosclerosis, obesity also count on the participation of important elements of adaptive immunity such as T cells and their cytokines in its pathophysiology. Based on these considerations, the main objectives of this study were: 1) to evaluate the presence of T cells and the role of interferon-gamma (IFNy), classic T-helper 1 (Th1) cytokine, in adipose tissue inflammation, and 2) to study mechanisms of T cell accumulation in adipose tissue in the context of obesity, particularly the involvement of CXCR3 receptor in this process. Flow cytometry experiments showed that the visceral fat tissue of C57BL/6 obese mice fed a high fat diet showed a greater number of macrophages and also T cells, including CD4+ and CD8+ cells, compared to controls fed a low-fat diet. The expression of I-Ab, murine marker of class II major histocompatibility complex (MHC II), was also higher in adipose tissue of obese animals, suggesting the presence of local antigen presentation and consequent T cell activation. When stimulated in vitro, T cells derived from adipose tissue of obese mice produced more IFNy than those isolated from controls, again suggesting the activation of these cells in the context of obesity. In the analysis of possible functions of IFNy in adipose tissue, stimulation of 3T3 -L1 cells differentiated into adipocytes with recombinant IFNy resulted in enhanced production of macrophage chemokines, such as monocyte chemotactic protein-1 (MCP-1) and T-cell chemokines, such as interferon gamma-induced protein 10 (IP-10) and monokine induced by gamma interferon (MIG). The stimulation of adipocytes with the supernatant of in vitro cultured Th1 cells, with abundant levels of IFNy, has also led to increased IP-10 production. In a broader analysis, by microarray, of the possible effects of IFNy on adipocyte gene expression, treatment of these cells with 100 U/ml of IFNy resulted in increased expression of chemokines and their receptors in comparison to the placebo group. Similarly to the stimulation of isolated cells with IFNy, incubation of ex vivo adipose tissue with this cytokine also resulted in increased IP-10, MIG and tumor necrosis factor alpha (TNFalpha) secretion

INTRODUÇÃO: O consumo mundial de açúcar triplicou nos últimos 50 anos e a sua ingesta abusiva é responsável pela resistência periférica à insulina, que origina a síndrome metabólica - obesidade, diabetes melito, hipertensão arterial e doenças coronarianas . Motivados pelo elevado número de pacientes que nos procuram com queixas vestibulares associadas aos distúrbios de metabolismo da glicose (DMG) resolvemos avaliar de forma objetiva, a influência dos DMG nas disfunções labirínticas e o efeito da dieta restritiva de carboidratos como forma de tratamento. OBJETIVO: Observar o impacto da dieta fracionada e restritiva de carboidratos na qualidade de vida dos pacientes portadores de distúrbios do equilíbrio corporal e DMG por meio da posturografia dinâmica computadorizada (PDC), do disability index (DI) e da escala análogo-visual (EAV). CASUÍSTICA E METODOLOGIA: Este estudo foi desenhado como um ensaio clínico prospectivo controlado randomizado realizado no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. A amostra foi constituída de 51 pacientes divididos em dois grupos: Grupo Dieta (GD): indivíduos tratados com comprimidos de placebo e dieta fracionada com restrição de glicose, Grupo Controle (GC): receberam apenas placebo. Os pacientes realizaram PDC, DI e EAV no primeiro e trigésimo dias do estudo. RESULTADOS: A amostra mostrou-se homogêna quando comparados os grupos e observou-se melhora, estatísticamente comprovada nas condições posturográficas avaliadas quando comparados GD e GC. Observou-se ainda melhora clínica do GD na análise da EAV. CONCLUSÃO: A dieta fracionada e restritiva de carboidratos mostrou-se eficaz no tratamento da nossa amostra de pacientes portadores de disfunções vestibulares associadas a DMG. / INTRODUCTION: World sugar consumption has tripled in the last 50 years and its abusive ingestion is responsible for peripheral insulin resistance, which leads to metabolic syndrome - obesity, diabetes mellitus, hypertension and coronary heart disease. Because of the high number of patients with vestibular complaints and with glucose metabolism disorders (GMD) we decided to objectively evaluate the effect of glucose restrictive and fractionated diet as a option of treatment in these patients. OBJECTIVE: To evaluate the impact of the glucose restrictive and fractionated diet on the Computerized Dynamic Posturography (CDP), disability index (DI) and the visual analogue scale (VAS) in patients with balance disorders and disorders of glucose metabolism. SAMPLES AND METHODOLOGY: Randomized controlled trial. Sample of 51 patients divided into two groups: Diet Group (DG) treated with placebo pills and glucose restrictive and fractionated diet and Control Group (CG) with only placebo. The individuals performed CDP, DI and VAS at first and thirtieth days of study. RESULTS: The sample groups were homogeneous before the study. There were significant improvement of DG on CDP conditions 4, 5, 6 and composite score. There was, also, significant improvement of VAS analysis on DG after intervention. CONCLUSION: The glucose restrictive and fractionated diet was effective in the treatment of patients with vestibular dysfunction associated with glucose metabolism disorders

Les oxydants infusés avec la nutrition parentéral (NP) néonatale induisent une modification du métabolisme des lipides et du glucose, donnant lieu à l’âge adulte à un phénotype de carence énergétique (faible poids, baisse de l’activité physique). L’hypothèse qu’une diète précoce riche en glucose prévient ces symptômes plus tard dans la vie, fut évalué chez le cobaye par un ANOVA en plan factoriel complet à deux facteurs (p < 0:05) : NP du jour 3 à 7, suivit d’une nourriture régulière (chow) (NP+) vs. chow à partir du 3ième jour (NP-), combiné avec une eau de consommation enrichie en glucose (G+) ou non (G-) à partir de la 3ième semaine. Les paramètres suivant ont été mesurés à l’âge de 9 semaine: taux de croissance, activité physique, activité de phosphofructokinase-1 et glucokinase (GK), niveau hépatique de glucose-6-phosphate (G6P), glycogène, pyruvate et potentiel redox du glutathion, poids du foie, glycémie, tolérance au glucose, concentrations hépatiques et plasmatiques en triacylglycérides (TG) et cholestérol. Le groupe G+ (vs. G-) avait un taux de croissance plus bas, une activité de GK et une concentration en G6P plus élevée, et un potentiel redox plus bas (moins oxydé). Le niveau plasmatique de TG était moins élevé dans le groupe NP+ (vs. NP-). Les traitements n’eurent aucun effet sur les autres paramètres. Ces résultats suggèrent qu’indépendamment de la NP, une alimentation riche en glucose stimule la glycolyse et déplace l’état redox vers un statut plus réduit, mais ne surmonte pas les effets de la NP sur le phénotype physique de carence énergétique. / Neonatal exposure to oxidant molecules from total parenteral nutrition (TPN) alters future lipid and glucose metabolism, resulting in an energy deficient phenotype characterized by lower body weight and physical activity. Using a guinea-pig model, the hypothesis that early diet supplementation with glucose could overcome such symptoms at week 9 of age was tested in a two-factor full-factorial ANOVA design (p<0:05): TPN day 3-7, chow thereafter (TPN+) vs: chow from day 3 (TPN-), combined with glucose-enriched diet from week 3 (G+) vs: plain chow throughout (G-). The growth rate, physical activity, phosphofructose kinase-1 and glucose kinase (GK) activities, glucose-6-phosphate (G6P), glycogen and pyruvate concentrations, relative liver weight, fasting blood glucose, glucose tolerance, hepatic and plasma triacylglyceride and cholesterol levels, individual glutathione levels and GSH/GSSG-based redox potential were determined at 9 weeks. Glucose supplementation (vs: the lack thereof) resulted in a lower growth rate, higher GK activity, and higher G6P concentration at week 9. Plasma triacylglycerides at week 9 were lower in TPN+ (vs: TPN-) subjects. Hepatic GSH=GSSG-derived redox potential shifted to a more reduced state in G+ (vs: G-) subjects. No other parameters showed significant differences. Independently of TPN, an early glucose-rich diet stimulated the glycolysis pathway, shifted the redox potential towards a more reduced status ; however, it did not overcome the effects of TPN on future physical and metabolic phenotype.

INTRODUÇÃO: O consumo mundial de açúcar triplicou nos últimos 50 anos e a sua ingesta abusiva é responsável pela resistência periférica à insulina, que origina a síndrome metabólica - obesidade, diabetes melito, hipertensão arterial e doenças coronarianas . Motivados pelo elevado número de pacientes que nos procuram com queixas vestibulares associadas aos distúrbios de metabolismo da glicose (DMG) resolvemos avaliar de forma objetiva, a influência dos DMG nas disfunções labirínticas e o efeito da dieta restritiva de carboidratos como forma de tratamento. OBJETIVO: Observar o impacto da dieta fracionada e restritiva de carboidratos na qualidade de vida dos pacientes portadores de distúrbios do equilíbrio corporal e DMG por meio da posturografia dinâmica computadorizada (PDC), do disability index (DI) e da escala análogo-visual (EAV). CASUÍSTICA E METODOLOGIA: Este estudo foi desenhado como um ensaio clínico prospectivo controlado randomizado realizado no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. A amostra foi constituída de 51 pacientes divididos em dois grupos: Grupo Dieta (GD): indivíduos tratados com comprimidos de placebo e dieta fracionada com restrição de glicose, Grupo Controle (GC): receberam apenas placebo. Os pacientes realizaram PDC, DI e EAV no primeiro e trigésimo dias do estudo. RESULTADOS: A amostra mostrou-se homogêna quando comparados os grupos e observou-se melhora, estatísticamente comprovada nas condições posturográficas avaliadas quando comparados GD e GC. Observou-se ainda melhora clínica do GD na análise da EAV. CONCLUSÃO: A dieta fracionada e restritiva de carboidratos mostrou-se eficaz no tratamento da nossa amostra de pacientes portadores de disfunções vestibulares associadas a DMG. / INTRODUCTION: World sugar consumption has tripled in the last 50 years and its abusive ingestion is responsible for peripheral insulin resistance, which leads to metabolic syndrome - obesity, diabetes mellitus, hypertension and coronary heart disease. Because of the high number of patients with vestibular complaints and with glucose metabolism disorders (GMD) we decided to objectively evaluate the effect of glucose restrictive and fractionated diet as a option of treatment in these patients. OBJECTIVE: To evaluate the impact of the glucose restrictive and fractionated diet on the Computerized Dynamic Posturography (CDP), disability index (DI) and the visual analogue scale (VAS) in patients with balance disorders and disorders of glucose metabolism. SAMPLES AND METHODOLOGY: Randomized controlled trial. Sample of 51 patients divided into two groups: Diet Group (DG) treated with placebo pills and glucose restrictive and fractionated diet and Control Group (CG) with only placebo. The individuals performed CDP, DI and VAS at first and thirtieth days of study. RESULTS: The sample groups were homogeneous before the study. There were significant improvement of DG on CDP conditions 4, 5, 6 and composite score. There was, also, significant improvement of VAS analysis on DG after intervention. CONCLUSION: The glucose restrictive and fractionated diet was effective in the treatment of patients with vestibular dysfunction associated with glucose metabolism disorders

Abstract Uterine leiomyomas are the most common benign tumours in females. They are myometrial neoplasms, may present single or multiple, and may be located in various sites of the uterus. Leiomyomas distort the uterine cavity and the uterus itself, causing abnormal vaginal bleeding, reduced fertility and also pelvic pressure and pain symptoms. The aim of this study was to elaborate current knowledge on familial uterine leiomyomas and to explore the possible association between uterine leiomyoma and cardiovascular disease risk factors, and also the association between leiomyomas and endometriosis. The natural history of familial uterine leiomyoma study showed significant differences between familial and non-familial leiomyoma cases, familial cases having more severe clinical characteristics. They presented with multiple uterine leiomyomas and were more often symptomatic. They were also diagnosed at a younger age. The prevalence study on uterine leiomyomas and endometriosis offered confirmation of an association between the diseases. Uterine leiomyomas and endometriosis seem to decrease female fertility independently of each other. Uterine leiomyomas related to the hereditary leiomyomatosis and renal cell cancer (HLRCC) tumour syndrome were studied in regard to their clinical characteristics and immunophenotype. The study provided evidence that women with HLRCC may be identified through distinct leiomyoma clinical characteristics, and routine-use IHC of CD34 and Bcl-2. Distinguishing these leiomyoma cases from sporadic ones may identify families affected by fumarate hydratase (fumarase, FH) mutation. Uterine leiomyoma and cardiovascular disease risk factors were studied in The Northern Finland Birth Cohort 1966 (NFBC1966). The study showed an association between leiomyomas and raised cardiovascular disease risk factors, serum lipids and metabolic syndrome in particular. These findings may suggest that there are shared predisposing factors underlying both uterine leiomyomas and adverse metabolic and cardiac disease risks, or that metabolic factors have a role in biological mechanisms underlying leiomyoma development. This study provides novel information on clinical characteristics of familial uterine leiomyomas and on the immunophenotype of HLRCC-related leiomyomas. The study also offers significant confirmation of associations between uterine leiomyomas and both endometriosis and several CVD risk factors. / Tiivistelmä Kohdun leiomyoomat ovat naisten yleisin hyvänlaatuinen kasvain. Ne ovat myometriumin neoplastisia muutoksia ja ne ilmenevät joko yksittäisinä tai monilukuisina, ja ne voivat sijaita missä tahansa kohdun myometriumia. Leiomyoomat muuttavat kohdun ja kohtuontelon säännöllistä muotoa. Lisäksi ne aiheuttavat vuotohäiriöitä, alentunutta hedelmällisyyttä, ja lantion alueen painetta ja kipua. Tämän tutkimuksen tavoitteena oli laajentaa nykyistä tietämystä suvuittain esiintyvistä kohdun leiomyoomista ja selvittää mahdollista leiomyoomien ja kardiovaskulaaritautiriskin assosiaatiota, ja lisäksi selvittää leiomyoomien ja endometrioosin assosiaatiota. Suvuittain esiintyvien kohdun leiomyoomien taudinkulkua selvittävässä tutkimuksessa osoitettiin merkittäviä eroja suvuittain ja ei-suvuittain esiintyvien leiomyoomien välillä. Suvuittain esiintyvien leiomyoomien kliininen taudinkuva oli vaikeampi, leiomyoomia oli kohdussa useampia ja ne aiheuttivat useammin oireita ja lisäksi ne diagnosoitiin nuoremmalla iällä. Kohdun leiomyoomien ja endometrioosin yleisyyttä selvittävä tutkimus antoi lisävahvistusta sille havainnolle, että nämä taudit assosioivat keskenään. Tutkimustuloksen mukaan leiomyoomat ja endometrioosi vähentävät naisen hedelmällisyyttä toisistaan riippumatta. Perinnöllinen kohdun leiomyomatoosi ja munuaissyöpä (hereditary leiomyomatosis and renal cell cancer, HLRCC) -kasvainoireyhtymään liittyvän kohdun leiomyoomia selvittävän tutkimuksen tuloksien mukaan HLRCC-naisten kohdun leiomyoomien kliiniset ominaisuudet poikkeavat satunnaisesti esiintyvien leiomyoomien ominaisuuksista. Naisella HLRCC voitaisiinkin tunnistaa näiden poikkeavien ominaisuuksien perusteella, sekä immunohistokemiallisilla värjäyksillä CD34 ja Bcl-2. Fumaraattihydrataasi (fumaraasi, FH) -geenin mutaatiota kantava suku voitaisiin siten tunnistaa yksittäisen HLRCC leiomyoomatapauksen avulla. Pohjois-Suomen syntymäkohortti 1966 (Northern Finland Birth Cohort 1966, NFBC1966) tutkittiin kohdun leiomyoomia ja kardiovaskulaarisairauden riskitekijöitä. Tutkimustuloksien perusteella kohdun leiomyoomat assosioivat koholla olevien kardiovaskulaarisairauden riskien kanssa, erityisesti seerumin lipidien ja metabolisen syndrooman suhteen. Näiden tutkimustulosten perusteella voidaan esittää, että leiomyoomien ja terveydelle epäedullisen metabolian ja kardiovaskulaaritaudin riskien taustalla on mahdollisesti joitain yhteisiä altistavia tekijöitä, tai että metabolisilla tekijöillä on rooli kohdun leiomyoomien tautimekanismissa. Tämä tutkimus on tuottanut uutta tietoa suvuittain esiintyvien kohdun leiomyoomien kliinisestä taudinkuvasta ja HLRCC:n liittyvien leiomyoomien immunofenotyypistä. Lisäksi tämä tutkimus esittää lisävahvistusta kohdun leiomyoomien ja endometrioosin assosiaatiolle sekä useille kardiovaskulaaririskitekijöille.

Gestational diabetes (GDM) is a disorder of glucose metabolism arising for the first time in pregnancy and spontaneously receding after birth. The issue of GDM is very topical since, according to the latest update of diagnostic criteria, up to 17% of pregnant women is threatened by this disorder. The incidence of GDM correlates with the increasing prevalence of overweight/obesity and metabolic syndrome. It is proved that women who have had gestational diabetes have an enormously increased risk of developing type 2 diabetes mellitus (DM2T). The risk accosiated with a gestational diabetes pregnancy stretches beyond the host, and can affect the fetus both directly (e. g. macrosomia development), and epigenetically (increases susceptibility to obesity, DM2T development or cardiovascular disease). Significant influence on the development of GDM (or DM2T) is a body composition that is directly related to lifestyle (nutritional intake and physical activity) and genetic role i salso involved. Early intervention may help delay the risk of developing DM2T and other metabolic complications. In this diploma thesis we monitored metabolic profiles of glucose and lipids and body composition based on anthropometric examination and questionnaires of nutritional income and physical activity. For the complex...