Dissertations / Theses on the topic 'Rosiglitazone'

L’obésité abdominale est un facteur de risque des maladies cardiovasculaires et englobe le tissu adipeux viscéral (TAV) et le tissu adipeux sous-cutané (TASC). Une augmentation de TAV est associée à une diminution de la variabilité de la fréquence cardiaque, représentant l’équilibre du système nerveux autonome, ainsi qu’à une augmentation des risques d’évènements cardiovasculaires. Par contre, l’impact d’une augmentation du TASC sur la variabilité de la fréquence cardiaque n’est pas documenté. La rosiglitazone, un antidiabétique oral, entraine une augmentation du poids corporel préférentiellement au niveau du TASC et de la rétention hydrosodée. Suite au traitement à la rosiglitazone pendant 12 mois, nous avons observé une augmentation du TASC. Cette augmentation de TASC n’influençait pas la variabilité de la fréquence cardiaque chez des patients diabétiques de type 2 avec pontage aorto-coronarien. Ces observations renforcent le fait que la prise de TASC n’est pas délétère pour la variabilité de la fréquence cardiaque contrairement au TAV.
Abdominal obesity is a risk factor for cardiovascular disease, which includes visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT). An increase in VAT is associated with decreased heart rate variability, representing the balance of the cardiac autonomic nervous system, as well as an increased risk of cardiovascular events. Also, the effect of an increase in SAT on heart rate variability is not documented. Rosiglitazone, an oral antidiabetic, induce an increase in body weight preferentially by increasing body water and SAT. Following treatment with rosiglitazone for 12 months, we observed an increase in SAT. This increase in SAT did not influence heart rate variability in patients with type 2 diabetes after coronary artery bypass surgery. These data suggest that an increase in SAT has no impact on heart rate variability in contrast to VAT.

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

It has long been known that in addition to disruptions in glucose homeostasis, individuals with insulin resistance have a breakdown in lipid dynamics, often manifested by elevated levels of circulating fatty acids (FA) together with accumulation of lipids in insulin-sensitive tissues, including skeletal muscle. However, little is known about how common therapies used to treat insulin resistant individuals (such as Rosiglitazone and exercise training) improve skeletal muscle lipid and glucose metabolism. Thus, the primary aim of the studies undertaken for this thesis was to enhance our understanding of the mechanisms by which Rosiglitazone and exercise training improve skeletal muscle lipid metabolism and insulin sensitivity in two distinct models of insulin resistance. The first investigation determined the effect of chronic Rosiglitazone treatment on the accumulation of lipid metabolites and enzymatic regulators of lipid metabolism in the skeletal muscle of obese Zucker rats. The observation that Rosiglitazone treatment exacerbated the accumulation of muscle ceramide and diacylglycerol in skeletal muscle, while improving glucose tolerance led to the conclusion that this insulin sensitising drug improves insulin sensitivity by mechanisms other than reduction of fatty acid metabolites in this tissue. Accordingly, the second investigation sought to identify an alternative mechanism by which Rosiglitazone treatment may improve skeletal muscle insulin sensitivity. It was found that Rosiglitazone restored AMP-activated protein kinase (AMPK) á2 activity in the skeletal muscle of obese Zucker rats, providing a potential peroxisome proliferator activated receptor (PPAR) ã-independent mechanism by which this drug may mediate its insulinsensitising actions. The final experiment undertaken for this thesis determined the independent and interactive effects on Rosiglitazone and exercise training on various aspects of skeletal muscle glucose and lipid metabolism in a model of diet-induced insulin resistance, the high-fat fed rat. Exercise training, but not Rosiglitazone treatment restored skeletal muscle insulin sensitivity in high-fat fed rats. Improvements in insulin sensitivity with exercise training were associated with increased FA oxidation, increased AMPK activity and a normalisation of the expression of the Akt substrate, AS160. In contrast, Rosiglitazone treatment was associated with increased FA uptake and decreased insulin-stimulated glucose uptake in skeletal muscle. Exercise prevented the accumulation of skeletal muscle lipids in Rosiglitazone-treated animals when the two treatments were combined. In summary, the results from the studies undertaken for this thesis provide novel information regarding the mechanisms by which two insulinsensitising therapies, exercise training and Rosiglitazone treatment, act to improve glucose and lipid metabolism in skeletal muscle.It has long been known that in addition to disruptions in glucose homeostasis, individuals with insulin resistance have a breakdown in lipid dynamics, often manifested by elevated levels of circulating fatty acids (FA) together with accumulation of lipids in insulin-sensitive tissues, including skeletal muscle. However, little is known about how common therapies used to treat insulin resistant individuals (such as Rosiglitazone and exercise training) improve skeletal muscle lipid and glucose metabolism. Thus, the primary aim of the studies undertaken for this thesis was to enhance our understanding of the mechanisms by which Rosiglitazone and exercise training improve skeletal muscle lipid metabolism and insulin sensitivity in two distinct models of insulin resistance. The first investigation determined the effect of chronic Rosiglitazone treatment on the accumulation of lipid metabolites and enzymatic regulators of lipid metabolism in the skeletal muscle of obese Zucker rats. The observation that Rosiglitazone treatment exacerbated the accumulation of muscle ceramide and diacylglycerol in skeletal muscle, while improving glucose tolerance led to the conclusion that this insulin sensitising drug improves insulin sensitivity by mechanisms other than reduction of fatty acid metabolites in this tissue. Accordingly, the second investigation sought to identify an alternative mechanism by which Rosiglitazone treatment may improve skeletal muscle insulin sensitivity. It was found that Rosiglitazone restored AMP-activated protein kinase (AMPK) á2 activity in the skeletal muscle of obese Zucker rats, providing a potential peroxisome proliferator activated receptor (PPAR) ã-independent mechanism by which this drug may mediate its insulinsensitising actions. The final experiment undertaken for this thesis determined the independent and interactive effects on Rosiglitazone and exercise training on various aspects of skeletal muscle glucose and lipid metabolism in a model of diet-induced insulin resistance, the high-fat fed rat. Exercise training, but not Rosiglitazone treatment restored skeletal muscle insulin sensitivity in high-fat fed rats. Improvements in insulin sensitivity with exercise training were associated with increased FA oxidation, increased AMPK activity and a normalisation of the expression of the Akt substrate, AS160. In contrast, Rosiglitazone treatment was associated with increased FA uptake and decreased insulin-stimulated glucose uptake in skeletal muscle. Exercise prevented the accumulation of skeletal muscle lipids in Rosiglitazone-treated animals when the two treatments were combined. In summary, the results from the studies undertaken for this thesis provide novel information regarding the mechanisms by which two insulinsensitising therapies, exercise training and Rosiglitazone treatment, act to improve glucose and lipid metabolism in skeletal muscle.

Estudos de metabolismo in vitro possuem o intuito de caracterizar e quantificar possíveis metabólitos, elucidar as vias metabólicas e sugerir modelos a serem seguidos para a realização de estudos in vivo. Com o intuito de estudar o metabolismo in vitro não estereosseletivo da rosiglitazona (RSG) empregando fração microssomal de fígado de ratos,foi desenvolvida uma metodologia por cromatografia líquida de alta eficiência (HPLC) com detecção UV em 245 nm, para analisar a RSG e seus principais metabólitos, p-hidroxi rosiglitazona (?-OH-R) e N-desmetil rosiglitazona (N-Dm-R). Os analitos foram separados em fase reversa, utilizando uma coluna X-Terra MS C-18 (partículas de 3,5 ?m) e fase móvel composta por água:acetonitrila:ácido acético (85:15:0,5, v/v/v), na vazão de 1 mL min-1. Matrizes biológicas contém um grande excesso de proteínas, lipídeos e outros materiais endógenos que interferem na análise de fármacos e metabólitos, tornando necessário um procedimento adequado de preparação das amostras antes da análise cromatográfica. A microextração em fase líquida com membrana cilíndrica oca (HF-LPME) é uma técnica promissora para a preparação de amostras em estudos de metabolismo in vitro, pois, além de promover o clean-up, promove também o enriquecimento dos analitos na amostra. A HF-LPME foi aplicada pela primeira vez para a extração simultânea desse fármaco e seus metabólitos. O sistema de três fases foi escolhido como o mais apropriado, empregando uma solução de ácido clorídrico como fase aceptora e 1-octanol como solvente orgânico. A otimização dos demais parâmetros foi realizada através de planejamento fatorial fracionário. O método foi validado e foi linear no intervalo de 50-6000 ng mL-1, apresentando limites de quantificação de 50 ng mL-1 e recuperações acima de 47 % para a RSG e seus metabólitos (?-OH-R e N-Dm-R). O método validado foi empregado em um estudo de metabolismo in vitro com fração microssomal de fígado de ratos. Nesse estudo, foi possível estimar as constantes de Michaelis-Menten (Km) e a velocidade inicial máxima (Vmax). N-Dm-R e ?-OH-R apresentaram valores de Vmax de 87,30 ± 8,04 e 51,64 ± 12,25 ?mol min-1 mg proteína-1, respectivamente, enquanto que os valores de Km foram de 58,14 ± 11,85 e 77,84 ± 36,77 mmol L-1, respectivamente. Outros metabólitos foram observados nos cromatogramas e a identificação foi feita por espectrometria de massas: ?rto-hidroxi-rosiglitazona e N-desmetil-hidroxi-rosiglitazona. A RSG é comercializada como uma mistura racêmica, apesar de possuir sua atividade antidiabética relacionada essencialmente com o enantiômero (S). O centro quiral desse fármaco possui um grupo carbonila, por isso, o enantiômero (R) pode se converter no enantiômero (S) ou vice-versa, via tautomerismo cetoenólico. Dados da literatura indicavam que essa racemização poderia ser lenta o suficiente para possibilitar o estudo dos enantiômeros isoladamente. Entretanto, até o momento não há dados sobre a disposição cinética ii e metabolismo enantiosseletivos desse fármaco. Sendo assim, propôs-se o desenvolvimento de metodologias analíticas para estudar a racemização da RSG e seus metabólitos e avaliar a possibilidade de estudar seu metabolismo in vitro de forma estereosseletiva. O método foi desenvolvido empregando HPLC com detecção em 245 nm. A separação dos enantiômeros do fármaco e metabólitos, também quirais, foi obtida empregando uma coluna Chiralcel OJ-H e fase móvel constituída por metanol:etanol (90:10; v/v), na vazão de 0,3 mL min-1. O estudo de racemização mostrou que o fármaco e seus metabólitos são racemizados nas condições em que o estudo de metabolismo é conduzido. Finalmente, para estudar o metabolismo in vitro da pioglitazona (PGZ), foi desenvolvido um método para análise desse fármaco e de seus principais metabólitos, a hidroxi-pioglitazona (M-IV) e a ceto-pioglitazona (M-III) empregando a eletroforese capilar (CE). As análises foram realizadas em capilar de sílica de 50 ?m de diâmetro interno e com comprimento efetivo de 40 cm, utilizando tampão fosfato de sódio 50 mmol L-1, pH 2,5, detecção em 190 nm, tensão de 30 kV e temperatura do capilar de 35 °C. A HF-LPME também foi empregada para a preparação das amostras. O sistema de três fases foi escolhido, empregando solução de ácido clorídrico como fase aceptora e o 1-octanol como solvente orgânico. A otimização dos demais parâmetros foi realizada através de planejamento fatorial fracionário. O método validado foi linear no intervalo de 200 - 25000 ng mL-1 para PGZ e 200 - 2000 ng mL-1 para os metabólitos, apresentando limites de quantificação de 200 ng mL-1 e recuperações acima de 19 % para a RSG e seus metabólitos M-IV e M-III. O método validado foi empregado em um estudo de metabolismo in vitro contendo fração microssomal de fígado de ratos, mas nesse estudo, não foi possível observar a formação dos metabólitos. Entretanto, esse método pode ser usado em outros modelos de metabolismo in vitro (microssomas humanos), nos quais se observa a formação desses metabólitos em concentrações maiores.
In vitro metabolism studies have been used to characterize and to quantify possible metabolites, to elucidate metabolic pathways and to suggest models to perform in vivo studies. So the purpose of this study was to evaluate the in vitro rosiglitazone (RSG) metabolism employing microsomal fraction obtained from rat livers. A high- performance liquid chromatography (HPLC) method with UV detection at 245 nm was developed to analyze RSG and the main metabolites, p-hydroxy rosiglitazone (?-OH-R) e N-desmethyl rosiglitazone (N-Dm-R). The analytes were separated under reversed phase conditions, using a X-Terra MS C-18 column (3.5 ?m particle size) and a mobile phase consisting of water:acetonitrile:acetic acid (85:15:0.5, v/v/v), at a flow rate of 1 mL min-1. Biological matrices contain a large excess of proteins, lipids and other endogenous compounds that interfere in the analysis of drugs and metabolites. So, a suitable sample preparation technique is required. Hollow-fiber liquid phase microextraction (HF-LPME) is a promising technique for the preparation of biological samples. Besides the clean-up, analytes enrichment is also achieved. HF-LPME for the simultaneous analysis of RSG and its main metabolites is described for the first time. The three-phase extraction was performed using hydrochloride acid solution as acceptor phase and 1-octanol as organic solvent. The other parameters were optimized by fractional factorial design. The method was validated and it was linear over the concentration range of 50-6000 ng mL-1, with quantification limits of 50 ng mL-1 and recoveries above 47 %. The validated method was used to estimate Michaelis-Menten (Km) constant and maximum initial velocity (Vmax). N-Dm-R e ?-OH-R showed Vmax values of 87.30 ± 8.04 and 51.64 ± 12.25 ?mol min-1 mg protein-1, respectively, while the Kmvalues were 58.14 ± 11.85 e 77.84 ± 36.77 mmol L-1, respectively. Other possible metabolites were observed in the chromatograms and they were identified by mass spectrometry: ?rtho-hydroxy-rosiglitazone e N-desmethyl-hydroxy-rosiglitazone. RSG is marketed as a racemic mixture although the antidiabetic activity is related essentially to the (S)-enantiomer. The chiral center has a carbonyl group, therefore the (R)-enantiomer could be transformed to the (S)-enantiomer or vice-versa by keto-enolic tautomerism. The literature indicates that this racemization is slow enough to allow the evaluation of the properties of the isolated enantiomers. However, there is no information in the literature about enantioselective RSG kinetic disposition and metabolism. Considering this facts, an analytical procedure was developed to study the racemization of RSG and its metabolites under different conditions and to determine if the enantioselective metabolism would be performed. The method was developed by HPLC with detection at 245 nm. The chiral separation of RSG and metabolites was achieved on a Chiralcel OJ-H column, with the mobile phase consisting of methanol:ethanol (90:10,v/v). The results obtained showed that the racemization occurs under the conditions used in in vitro iv metabolism studies. Finally, to study the in vitro metabolism of pioglitazone (PGZ), another method was developed by capillary electrophoresis (CE) to determine this drug and its main metabolites, hydroxy-pioglitazone (M-IV) and keto-pioglitazone (M-III). The analyses were conducted using a fused silica capillary (50 ?m inner diameter and 40 cm effective length), sodium phosphate buffer 50 mmol L-1, pH 2.5, detection at 190 nm, voltage of 30 kV and capillary temperature of 35°C. HF-LPME was also used for sample preparation with hydrochloride acid solution as acceptor phase and 1-octanol as organic solvent. The other parameters were optimized by fractional factorial design. The method was validated showing to be linear in the concentration range of 200 - 25000 ng mL-1 for PGZ and 200 - 2000 ng mL-1 for the metabolites. Quantification limits were 200 ng mL-1 for all analytes and the recoveries were higher than 19%. The validated method was used to study the in vitro metabolism of PGZ by rat liver microsomal fraction, but it was not possible to observe the formation of the metabolites in this study. However this method could be used in other in vitro metabolism models (human microssomes), in which higher concentrations of these metabolites are observed. Keywords:

Thiazolidinediones such as rosiglitazone are used in the treatment of Type-2 Diabetes, and are ligands for peroxisome proliferator-activated receptor-gamma (PPARγ), a ligand-activated transcription factor that regulates expression of genes involved in glucose and lipid metabolism. However, rosiglitazone is known to exert PPARγ-independent effects alongside its classical receptor-dependent effects. This study investigated the PPARγ-independent effects of rosiglitazone on intracellular calcium (Ca2+i) signalling in cultured monocytic and vascular smooth muscle cells Rosiglitazone rapidly (5-30min) inhibited the Ca2+ sequestration activity of the ER-resident Ca2+ pump enzyme SERCA2b in a dose-dependent manner (IC50~2μM). 10μM Rosiglitazone triggered rapid increases in [Ca2+]i; however, restoration to basal levels occurred within 72h. Consequently, cell viability was not adversely affected by rosiglitazone treatment. Initiation of the unfolded protein response (UPR) was identified as the mechanism underpinning rosiglitazone's Ca2+ homeostatic restorative properties. Rosiglitazone induced alternate splicing of the UPR transcription factor XBP-1, which led to increased mRNA and protein expression of SERCA2b (shown via bioinformatics analysis to be a UPR target gene), and increased ER Ca2+-ATPase activity in rosiglitazone-treated cells. In tissue (rabbit aortic ring) samples, 10μM rosiglitazone induced transient (within 1h) non-significant losses in vasorelaxatory sensitivity to sodium nitroprusside, but extended treatment (4-24h) increased sensitivity beyond that of vehicle-treated samples. Thus, at cell and tissue levels, this data suggests that rosiglitazone initially causes increased [Ca2+]i due to inhibition of SERCA2b, but extended incubation induces - via upregulation of UPR target genes - the restoration of Ca2+ homeostasis, and possibly even improvements in function in some contexts. Clearly, the data presented in this in vitro study must be treated tentatively, and more research is needed before these potentially beneficial effects can be firmly identified as being clinically significant. Nevertheless, the data obtained here may constitute preliminary evidence that, alongside its PPARy-dependent effects, rosiglitazone may exert functional improvements on the vasculature.

Introduction: Animal models of joint contracture induced by capsular injury and prolonged immobilization are used to elucidate the mechanisms of arthrofibrosis. Clinically, patients with joint contracture commonly undergo surgical capsular release. Peroxisome proliferator-activated receptor gamma (PPARγ) agonists (such as rosiglitazone) hold promise as antifibrogenic agents that may be used as an adjunct to capsular release.

Methods: A surgically induced capsular contracture was created in thirty skeletally mature female rabbits. Twenty rabbits underwent a limited capsular release, which consisted of elevation of the posterior capsule through a lateral femoral incision under tension. Ten of these received rosiglitazone, while ten received placebo. Ten rabbits had a similar sham incision, without elevation of the capsule or joint extension (control group). Flexion contracture was measured using intraoperative fluoroscopy. All animals were allowed free cage activity for 16 weeks following this procedure. Joint contracture was measured using a custom device.

Results: All animals survived both operations without operative complications. At the time of surgical release or sham surgery, the average flexion contracture was 129° ± 11° in the control group versus 30° ± 8° in the release group (p=0.0002). Following sixteen weeks of remobilization, the animals that had a capsular release had significantly decreased flexion contractures compared to the control animals: 37° ± 14° and 49° ± 13° respectively (p=0.035). Following sixteen weeks of remobilization, the difference in flexion contracture between the rosiglitazone and capsular release groups was not statistically significant (rosiglitazone 33° ± 11°; control, 37° ± 14°; p = 0.39). Several gene products were significantly affected by rosiglitazone administration.

Discussion: In this animal model, a limited capsular release decreased flexion contracture immediately after surgery as well as following sixteen weeks of remobilization. This resembles clinical experience. Rosiglitazone did not prevent flexion contracture in this model, but did modulate gene expression in the joint capsule.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro
Este trabalho teve o objetivo de estudar o efeito de medicamentos com diferentes ações agonista PPAR (rosiglitazona, fenofibrato e bezafibrato) sobre o perfil lipídico, glicídico e alterações na massa corporal e morfologia do tecido adiposo e pancreático em modelo de diabetes e sobrepeso induzido por dieta. Camundongos C57BL/6 (2 meses de idade) foram alimentados com dieta padrão (SC, n=10) ou dieta hiperlipídica rica em sacarose (HFHS, n=40) por 6 semanas. Logo após, os animais HFHS foram subdividos em: HFHS não tratado e HFHS tratado com rosiglitazona (HFHS-Ro), fenofibrato (HFHS-Fe) ou bezafibrato (HFHS-Bz) (5 semanas). Os camundongos alimentados com dieta HFHS apresentaram maior glicemia e insulina de jejum (+33% e +138%, respectivamente), intolerância à glicose, resistência à insulina, aumento da massa corporal (MC) (+20%) e adiposidade, hipertrofia de adipócitos e redução da imunocoloração para adiponectina no tecido adiposo. No pâncreas houve aumento da massa (+28%), acúmulo de gordura (+700%), hipertrofia da ilhota (+38%) e redução da imunocoloração para GLUT-2 (-60%). A rosiglitazona diminuiu a glicemia e insulina de jejum, porém induziu o ganho de MC e hipertrofia cardíaca. O fenofibrato estabilizou a MC, enquanto o bezafibrato levou a perda de MC. Apenas o bezafibrato impediu a hipertrofia da ilhota. A imunocoloração para GLUT-2 foi aumentada por todos os medicamentos, e não houve alterações na imunocoloração para o PPARα. Sinais morfológicos de pancreatite foram vistos no grupo HFHS-Fe, apesar dos níveis normais de amilase e lipase séricos. A rosiglitazona exacerbou a infiltração intrapancreática de gordura (+75% vs. HFHS), e o bezafibrato aumento a imunocoloração para o PPARβ/δ nas ilhotas pancreáticas. Em conclusão, o bezafibrato apresentou um efeito mais amplo sobre as alterações metabólicas, morfológicas e biométricas decorrentes da dieta HFHS, sugerindo que a inibição das três isoformas do PPAR seria melhor do que a inibição de apenas uma isoforma. A rosiglitazona exacerbou o ganho de MC, a infiltração de gordura no pâncreas e induziu hipertrofia cardíaca, assim, é necessário cautela ao prescrever este medicamento a um paciente obeso.
This work aimed to evaluate the effect of peroxisome proliferator-activated receptor (PPAR) agonists (rosiglitazone, fenofibrate and bezafibrate) on lipid and glucose metabolism, body mass, and adipose and pancreatic tissue morphology in a model of diet-induced type 2 diabetes and overweight in mice. Two-month-old male C57BL/6 mice were fed a standard chow (SC, n=10) or a high-fat high-sucrose chow (HFHS, n=40) for 6 weeks, and then HFHS-fed mice were subdivided by treatment: untreated HFHS and HFHS treated with rosiglitazone (HFHS-Ro), fenofibrate (HFHS-Fe), or bezafibrate (HFHS-Bz) (5 weeks on medication). HFHS-fed mice have altered fasting glucose (+33%) and insulin (+138%), GI, IR, increased body mass (+20%) and fat pad weight, adipocyte hypertrophy, and decreased adiponectin immunostain. They also presented increased pancreatic (+28%) mass, intrapancreatic fat (+700%), islet hypertrophy (+38%), and decreased GLUT-2 immunostain (-60%). Rosiglitazone reduced fasting glucose and insulin but induced weight gain and heart hypertrophy. Fenofibrate impaired body mass gain, while bezafibrate induced weight loss. Only bezafibrate impaired islet hypertrophy. GLUT-2 immunostain was improved by all treatments, and there were no alterations in PPAR-α stain. There were morphological signs of pancreatitis in fenofibrate-treated mice, although there was no alteration in serum amylase and lipase. Rosiglitazone exacerbated pancreatic fat infiltration (+75% vs. HFHS group), and bezafibrate increased PPAR-β expression in pancreatic islets. In conclusion, bezafibrate showed a wider range of action on metabolic, morphologic, and biometric alterations due to HFHS intake, suggesting that inhibiting the three PPAR isoforms is better than inhibiting each isoform alone. Rosiglitazone exacerbated body mass gain, pancreatic fat infiltration and induced heart hypertrophy as well, thus, precaution has to be taken in prescribing rosiglitazone to obese patients.

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

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O objetivo deste trabalho é avaliar as anormalidades da esclera, coroide e retina de coelhos induzidas pela dieta hipercolesterolêmica, além da possibilidade de prevenção dessas anormalidades com administração sistêmica de rosiglitazona. Para isto, 54 coelhos new zealand foram distribuídos em quatro grupos: grupo-controle (GC) recebeu dieta normal; grupo 1 recebeu dieta hipercolesterolêmica; grupo 2 recebeu dieta hipercolesterolêmica associada à administração diária de 3 mg de rosiglitazona a partir do 14º dia do início do experimento; e grupo 3 recebeu dieta hipercolesterolêmica associada à administração diária de 3 mg de rosiglitazona desde o início do experimento. Os coelhos foram pesados e submetidos à dosagem sérica de colesterol total, triglicerídeos, high density lipoprotein (HDL) colesterol e glicemia de jejum no início do experimento, no 14º dia e no momento da eutanásia (42º dia). A esclera e coroide foram submetidas à análise histológica e histomorfométrica. A retina foi submetida à análise imuno-histoquímica com o anticorpo monoclonal anticalretinina (CR) e anticorpo anti-glial fibrillary acidic protein (GFAP). Quando positivo para o marcador anticalretinina, duas análises quantitativas foram realizadas. Na primeira, foram contadas todas as células ganglionares imunorreativas. Na segunda, todas as células e elementos celulares imunorreativos foram avaliados pelo exame de morfometria de cores. Os dados foram analisados pelo teste nãoparamétrico de Kruskal-Wallis e teste de Shapiro-Wilks-Testand. Valores abaixo de 0,05 foram considerados estatisticamente significantes. Os resultados referentes ao peso demonstraram significativo aumento nos grupos 1 e 3 em relação ao GC no 14º dia (p<0,009), enquanto no 42º dia os grupos 1, 2 e 3 apresentaram representativamente mais peso que o GC (p<0,023). Quanto às variáveis laboratoriais, destacaram-se o aumento significativo da glicose e colesterol total de G1 em relação ao controle (p<0,001), assim como o acentuado aumento da HDL no G3 em relação aos demais grupos (p<0,001), no 14º dia. A HDL manteve-se expressivamente elevada no G3 em relação aos demais grupos no momento da eutanásia (p<0,001). À análise histomorfométrica da esclera e coroide obteve-se normalidade do GC. Por outro lado, o G1 mostrou marcante aumento da espessura da esclera e coroide em relação ao GC (p=0,008), enquanto que no G3 houve espessamento de esclera e coroide menor que no G1 (p=0,048). Elevado número de histiócitos foi observado na parede escleral do grupo submetido à dieta hipercolesterolêmica (G1), seguido de forma decrescente por G2, G3 e GC. A análise imuno-histoquímica da retina com o anticorpo monoclonal anticalretinina ressaltou número mais alto de células ganglionares imunorreativas no G1 que no G3 (p=0,002). O exame de morfometria de cores revelou significativa imunorreatividade das células e elementos celulares do G1 em relação aos outros grupos (p<0,001). Nesta análise evidenciou-se também acentuada imunorreatividade das células e elementos celulares de G2 e G3 em relação ao GC (p≤0,002). GFAP foi negativo em todos os grupos. Neste modelo, os achados permitem concluir que a hipercolesterolemia provoca anormalidades precoces histomorfométricas e imuno-histoquímicas do complexo esclerocoriorretiniano; e a ativação dos receptores do PPAR gama-ocular, a partir da dieta oral de rosiglitazona, foi efetiva em atenuar tais anormalidades nessas estruturas.
The purpose of this study is to evaluate scleral, choroid and retinal abnormalities in rabbits induced by a hypercholesterolemic diet and the prevention of these abnormalities after oral administration of rosiglitazone in rabbits. Fifty-four new zealand rabbits were divided into four groups: the control group (CG) was fed a normal diet; group 1 G1), a hypercholesterolemic diet; group 2 (G2) a hypercholesterolemic diet associated with daily administration of 3 mg of rosiglitazone from day 14 after the beginning of the diet; and group 3 G3), a hypercholesterolemic diet associated with daily administration of 3 mg of rosiglitazone since the beginning of the experiment. The rabbits were weighed and underwent the following examinations: seric dosages of total cholesterol, triglycerides, cholesterol HDL, and fasting glycemia at the beginning of the experiment, on the 14th day and on the 42nd, the euthanasia day. The sclera and choroid underwent histologic and histomorphometric analyses and the retina underwent immunohistochemical analysis with anti-calretinin (CR) and anti-glial fibrillary acidic protein (GFAP) antibody. When positive for the anti-calretinin marker, two quantitative analyses were performed. In the first analysis, all immunoreactive ganglion cells were counted. In the second analysis, all immunoreactive cells and cell elements were studied with the color morphometry method. The data were evaluated using the nonparametric Kruskal-Wallis and the Shapiro – Wilk tests. Values of p<0.05 were considered statistically significant. The results obtained showed a significant weight increase in Groups 1 and 3 in relation to CG on Day 14 (p<0.009). Additionally, a significant weight increase was observed in G1, G2 and G3 in relation to CG on Day 42 (p<0.023). The lab results showed a significant increase in glucose and total cholesterol in G1 in relation to CG (p<0.001) on Day 14, as well as a significant HDL increase in G3, when compared with the other groups (p<0.001) on Day 14. HDL in G3 was significantly high when compared to the other groups, on the euthanasia day (p<0.001). The results obtained regarding weight showed a significant increase in Groups 1, 2 and 3 in relation to CG on Day 14 (p<0.01) and Day 42 (p<0.02). The lab results showed a significant increase in glucose and total cholesterol in Groups 1, 2 and 3 in relation to CG (p<0.01) on Day 14, as well as a significant increase in HDL in G3 when compared with the other groups, on euthanasia day (p<0.01). The histomorphometric analysis of CG sclera and choroid presented normal results. Conversely, G1 showed a significant increase in sclera and choroid thickness in relation to CG (p= 0,008), whereas G3 showed thickness lower than in G1 (p=0,048). A larger number of histiocytes were observed on the scleral wall of the group that was fed the hypercholesterolemic diet (G1), followed, in a descending order, by groups 2 and 3, and the control group. The immunohistochemical analysis of the retina with the anti-calretinin monoclonal antibody showed that G1 presented a larger number of immunoreactive ganglion cells than G3 (p = 0.002). The color morphometry showed significant immunoreactivity of G1 cells and cell elements when compared with the other groups (p<0.001). A significant immunoreactivity of G2 and G3 cells and cell elements in relation to CG was also observed (p<0.002). GFAP results were negative in all groups. The findings of this proposed study model suggest that hypercholesterolemia induces early histomorphometric and immunohistochemical abnormalities in the sclerochorioretinal complex and that the activation of PPAR gamma in ocular cells attenuated these abnormalities with the administration of the oral rosiglitazone diet.
TEDE
BV UNIFESP: Teses e dissertações

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O presente estudo tem como objetivo avaliar, em pacientes com a síndrome dos ovários policísticos, os efeitos clínicos, endócrinos e metabólicos da rosiglitazona, antes e após doze semanas de tratamento. Foram avaliados, além do padrão menstrual e do hiperandrogenismo, o perfil hormonal (FSH, LH, 17 beta-estradiol, testosterona total e livre, 17 hidroxiprogesterona, sulfato de dehidroepiandrosterona), os níveis séricos de IGF-1, IGFBP-3, SHBG (globulina ligadora de hormônios sexuais), as repercussões no risco cardiovascular (circunferência abdominal, HDL-colesterol, triglicérides, pressão arterial sistêmica, glicemia e insulina), o fluxo sangüíneo dos ovários pela ultrassonografia transvaginal e os aspectos histomorfológicos do endométrio após o tratamento. O estudo foi prospectivo, randomizado, duplo-cego e controlado com placebo. Foram incluídas 33 pacientes, subdivididas em dois grupos: 1) Grupo Placebo (XZ), com 17 pacientes e 2) Grupo Rosiglitazona (ZX), com 16 pacientes, que fizeram uso de uma cápsula de 4 mg, por via oral, duas vezes ao dia, por 12 semanas. Concluímos que o tratamento com rosiglitazona por três meses determinou: 1) melhora do padrão menstrual e dos sintomas e sinais clínicos relacionados com o hiperandrogenismo; 2) redução dos níveis de testosterona livre, androstenediona e do fator de crescimento insulinóide tipo 1; 3) elevação dos índices da proteína carreadora de esteróides sexuais (SHBG); 4) melhora da resistência insulínica, evidenciada pela sobrecarga glicêmica de 2 horas; 5) redução do número de mulheres com síndrome metabólica e 6) predomínio de endométrio secretor. Não houve variação significante do volume e do fluxo ovariano após o tratamento com rosiglitazona. Sugere-se que a rosiglitazona constitui alternativa para a correção da resistência insulínica, diminuindo a ocorrência de síndrome metabólica. Além disso, melhora o padrão menstrual e o hiperandrogenismo.
The objectives of the present study are to evaluate the clinical, endocrine and metabolic effects of the rosiglitazone in patients with polycystic ovarian syndrome before and after twelve weeks of treatment. It was be evaluated, besides menstrual pattern and the hyperandrogenism, the hormonal profile (FSH, LH, 17 B-estradiol, total and free testosterone, 17 hydroxiprogesterone, dehudroepiandrosterone sulphate) and the seric levels of IGF-1, IGFBP-3, SHBG (globulin that links sexual hormones); examine the repercussions of cardiovascular risk (abdominal circumference, HDLs cholesterol, triglycerides, systemic arterial pressure, glycemy and insulin); verify thru transvaginal ultrasonography, the ovary blood flow and the endometrial histomorphologic aspects after treatment. The study was prospective, randomized, doubleblinded, using placebo as control. 33 patients were included and divided in two groups 1) Placebo Group (XZ), with 17 patients and 2) Rosiglitazone Group (ZX), with 16 patients, who used a capsule with 4mg, oral way, twice a day, for 12 weeks. We concluded that the treatment with rosiglitazone during three months determined: 1) improved the menstrual pattern and the symptoms and clinic signs related with hyperandrogenism; 2) a decrease in androstenedione, free testosterone and type 1 growth factor insulinoid values; 3) increase of the sexual steroids carry protein (SHBG) index; 4) improvement of the insulinic resistance, shown by the 2 hours glycemic overload; 5) a number reduction of women with metabolic syndrome and 6) the endometrium secrecy pattern got predominant. No significance variation in ovarian volume and flow were found after treatment with rosiglitazone. It’s suggested that rosiglitazone is a alternative way to correct insulinic resistance, decreasing the occurrence of the metabolic syndrome. Besides, it increases the menstrual pattern and hyperandrogenism.
TEDE
BV UNIFESP: Teses e dissertações

Tese (doutorado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2017.
Texto parcialmente disponibilizado pelo autor. Conteúdo restrito: Apêndices.
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A identificação e caracterização de moléculas capazes de modular a resposta inflamatória pulpar, facilitando ou mesmo promovendo o reparo dentinário oferecem imenso potencial para aumentar o sucesso na manutenção da vitalidade do dente. Neste sentido, os receptores gama e beta/delta ativados por proliferadores peroxissomais (PPARγ e PPAR β/δ), por apresentarem efeitos anti-inflamatórios, configuram-se como alvos farmacológicos potenciais. Enquanto a expressão do PPAR β/δ e sua participação na fisiopatologia pulpar ainda não foram exploradas, a capacidade do PPARγ em modular alguns aspectos inflamatórios em células da polpa já foi demonstrada. Porém, aspectos celulares relacionados com o reparo dentinário não foram avaliados. Assim, como objetivo da primeira parte deste trabalho, investigou-se o efeito da rosiglitazona (RSG), um agonista total do PPARγ sobre a viabilidade (MTT), apoptose (anexina-V FITC /iodeto de propídeo), migração (scratch) e diferenciação osteo/odontogênica de células da polpa dentária humana (vermelho de alizarina S e expressão gênica). Os resultados mostraram que RSG não interferiu na viabilidade, na apoptose ou na migração de células pulpares. Quanto a diferenciação, os resultados com coloração indicaram que RSG estimulou formação precoce de matriz mineralizada e a análise da expressão gênica revelou um aumento significativo da expressão de osteopontina nos grupos tratados com RSG. Como objetivos da segunda parte, a expressão do PPAR β/δ por células pulpares foi verificada (RT-PCRq e imunofluorescência). Posteriormente, essas células foram tratadas com LPS (Escherichia coli) ou H2O2 e o potencial do GW0742, agonista total do PPAR β/δ, em modular a inflamação foi explorado, por meio de análise da expressão gênica de citocinas e metaloproteinases (RT-PCRq), por análise da atividade de gelatinases (zimografia) e por ensaio de quimiotaxia com cocultura em transwell, sendo macrófagos murinos imortalizados RAW 264.7 as células submetidas a atração quimiotática por células pulpares. Células RAW 264.7 também foram estimuladas com LPS e a expressão gênica de citocinas inflamatórias foi analisada (RT-PCRq). Por fim, o efeito do GW0742 sobre o reparo começou a ser investigado neste estudo, por meio da análise da migração de células pulpares (scracth). Os resultados mostraram expressão de PPAR β/δ (transcrito e proteína). O tratamento com GW0742 reprimiu a expressão do RNAm de IL6, IL1β , MCP1 e MMP1 em células pulpares estimuladas com LPS ou H2O2, e de Il6 e Tnfα em células RAW 264.7 estimuladas com LPS. GW0742 reduziu a atividade de MMP2 e de MMP9, em células pulpares estimuladas com 2μg/mL ou 10 μg/mL de LSP, respectivamente. Além disso, o tratamento com GW0742 reduziu significativamente a capacidade de células pulpares estimuladas com LPS em recrutar macrófagos RAW 264.7. Por fim, GW0742 não interferiu na migração de células pulpares. Os resultados da primeira parte deste trabalho sugerem que RSG parece não alterar eventos celulares relacionados com o reparo dentinário. Quanto a segunda parte, esta indica que GW0742 possa modular a resposta inflamatória pulpar, sugerindo um efeito anti-inflamatório do receptor PPAR β/δ ativado.
Identification and characterization of molecules able to dampen inflammatory events within the dental pulp, facilitating or even promoting dentinal repair offer immense potential to increase the success in maintaining the tooth vitality. Due to anti-inflammatory properties, the peroxisome proliferator-activated receptors gamma and beta/dela (PPARγ and PPAR β/δ) could be envisioned as putative therapeutic targets. While neither PPAR β/δ expression nor its participation within the pulp pathophysiology were explored, the ability of PPARγ to modulate some inflammatory aspects in pulp cells has been demonstrated previously. However, cellular aspects related to dentine repair were not assessed. Thus, the aim of the first part of this study was to evaluate the effects of rosiglitazone (RSG), a PPARγ full agonist, on human dental pulp cells (hDPCs) viability (MTT), apoptosis (annexin-V FITC and propidium iodide), migration (scratch) and osteo/odontogenic differentiation (alizarin red and RT-qPCR). RSG did not affect cell viability, apoptosis/necrosis or cell migration. Whereas, alizarin red S showed that RSG stimulated early formation of mineralized matrix. Gene expression analysis revealed a significant increase in osteopontina mRNA expression at the RSG-treated groups. As the aim of the second part of this study, hDPCs PPAR β/δ expression was verified (RT-qPCR; immunofluorescence). Thereafter, hDPCs were treated with LPS or H2O2, and the anti-inflammatory ability of GW0742, a PPAR β/δ full agonist, was assessed by cytokine and metalloproteinases mRNA expression assay (RT-qPCR), gelatinase activity assay (zymography), and chemotaxis assay with transwell co-culture. In this system, an immortalized murine macrophage cell line, RAW 264.7, was attracted by hDPCs. In addition, RAW 264.7 cells were treated with LPS and GW0742, and cytokines mRNA expression was assessed (RT-qPCR). Finally, the effect of GW0742 on pulp repair was initiated through hDPCs migration assay (scracth). The results showed hDPCs PPAR β/δ expression (transcript and protein). GW0742 repressed IL6, IL1β, MCP1 and MMP1 gene expression in LPS/H2O2 treated hDPCs. Il6 and Tnfα mRNA expression was repressed by GW0742 in LPS-stimulated RAW 264.7 cells. GW0742 reduced MMP2 and MMP9 activity in pulp cells stimulated with 2μg / mL or 10μg / mL of LSP, respectively. In addition, treatment with GW0742 significantly reduced the ability of LPS-stimulated pulp cells to recruit RAW 264.7 macrophages. Finally, GW0742 did not impair hDPCs migration. The results of the first part of this study suggested that RSG appear does not impair cellular events related with dentin repair. The second part indicates that GW0742 can modulate pulp inflammatory response, suggesting an anti-inflammatory effect of activated PPARβ/δ receptor.

Introdução: A nefrotoxicidade dos antiretrovirais constituem atualmente fator importante na morbidade e mortalidade de pacientes com HIV. O tenofovir DF (TDF) se enquadra em um dos antiretrovirais mais lesivos ao rim. Conhecer seu mecanismo de nefrotoxicidade e estudar medidas protetoras podem melhorar seu uso clínico. Material e Métodos: Ratos foram tratados durante 30 dias com uma de duas doses de TDF (50 ou 300mg/Kg de dieta), sendo que um grupo teve adicionado em sua dieta maleato de rosiglitazona (RSG) na dose de 92mg/Kg de dieta nos últimos 15 dias. Após esse período, os ratos foram colocados em gaiola metabólica e sacrificados. Foram estudados parâmetros bioquímicos, fluxo sanguíneo renal e os rins extraídos para expressão semiquantitativa dos transportadores epiteliais tubulares. Resultados: Os animais que receberam TDF em dose alta apresentaram insuficiência renal severa acompanhada de redução da expressão da oxido-nítrico sintase endotelial e vasoconstricção renal intensa. Todos esses parâmetros foram parcialmente revertidos pela administração de RSG. Baixas doses de TDF não causou alteração significativa do ritmo de filtração glomerular, porém induziu fosfatúria, acidose tubular proximal, poliúria e redução da capacidade de concentração urinária. Essas alterações foram associadas a redução da expressão de alguns transportadores epiteliais (cotransportador sódio-fosforo, contratransportador sódio-hidrogênio tipo 3 e aquaporina tipo 2). Não foi caracterizado síndrome de Fanconi, pois não houve proteinúria ou glicosúria. O tratamento com RSG reverteu todos os parâmetros de nefrotoxicidade estudados, normalizando as alterações bioquímicas urinárias e a expressão dos transportadores de membrana. Conclusões: Os achados desses experimentos tem potencial aplicação clínica em pacientes com nefrotoxicidade induzida pelo TDF, especialmente naqueles com hipofosfatemia e/ou redução do ritmo de filtração glomerular.
Objective: To characterize the mechanisms of tenofovir disoproxil fumarate (TDF)- induced nephrotoxicity and the protective effects of rosiglitazone (RSG), a peroxisome proliferator-activated receptor-y agonist. Methods: Rats were treated for 30 days with one of two TDF doses (50 or 300 mg/kg of food), to which RSG (92 mg/kg of food) was added for the last 15 days. Biochemical parameters were measured, and renal tissue was extracted for immunoblotting. Results: Mean daily ingestion was comparable among all the treated groups. Highdose TDF induced severe renal failure accompanied by reduced expression of endothelial nitric-oxide synthase and intense renal vasoconstriction. All of these features were ameliorated by RSG administration. Low-dose TDF did not alter the glomerular filtration rate but induced significant phosphaturia, proximal tubular acidosis and polyuria, as well as reducing urinary concentrating ability. These alterations were caused by specific downregulation of the sodium-phosphorus cotransporter, sodium/hydrogen exchanger 3 and aquaporin 2. No Fanconi\'s syndrome was identified (proteinuria was normal and there was no glycosuria). Treatment with RSG reversed TDF-induced tubular nephrotoxicity, normalizing urinary biochemical parameters and membrane transporter protein expression. Conclusion: These findings have potential clinical applications in patients presenting with TFV-induced nephrotoxicity, especially in those presenting with hypophosphatemia or a reduction in glomerular filtration rate.

Tese (doutorado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Prorgama de Pós-Graduação em Ciências da Saúde, 2015.
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A prevalência de obesidade, associada à resistência insulínica e diabetes tipo 2 vem aumentando nas últimas décadas. As tiazolidinadionas (TZDs) são agonistas do receptor gama ativado por proliferadores peroxissomais (PPARγ) com eficiente ação sensibilizadora da insulina. Contudo, o uso das TZDs está associado a efeitos adversos como o ganho de peso, retenção de líquido, edema, insuficiência cardíaca congestiva e perda óssea. GQ-16 é um agonista parcial e específico do PPARγ que melhorou a tolerância à glicose e a sensibilidade à insulina de camundongos submetidos a dieta hiperlipidica de forma semelhante à rosiglitazona, mas, ao contrário dessa, não induziu ganho de peso ou edema. Considerando que um dos principais alvos farmacológicos das TZDs é o tecido adiposo, o objetivo desse estudo foi determinar o efeito de GQ-16 no transcriptoma de adipócitos 3T3-L1 maduros e comparar sua ação com a da rosiglitazona. A análise realizada pela técnica de microarranjo revelou que esses ligantes modificam a expressão gênica de forma diferenciada. Enquanto a rosiglitazona modificou a expressão de 1156 genes, GQ-16 regulou a expressão de apenas 89. Dos 544 genes regulados positivamente pela rosiglitazona (47%), somente 22 (4,2%) foram regulados pelo GQ-16. No mesmo sentido, dos 612 genes regulados negativamente pela rosiglitazona (53%), apenas 24 (4%) foram reprimidos pelo GQ-16. Assim, 46 genes foram regulados simultaneamente pela rosiglitazona e GQ-16, ou seja, uma concordância de somente 4%. Além disso, GQ-16 regulou a expressão de 43 genes (18 ativados e 25 reprimidos) não controlados pela rosiglitazona. A maior semelhança entre o GQ-16 e a rosiglitazona foi sobre a repressão de genes relacionados à resposta inflamatória como o Dcn, Vcam1, Orm2 e Lox, por exemplo. A comparação do GQ-16 com outros agonistas parciais de PPARγ (MRL24 e SR1664) revelou efeitos similares, tanto sobre os genes ativados quanto os reprimidos. Nós propomos que o reduzido efeito sobre genes relacionados à adipogênese e a habilidade do GQ-16 em reprimir eficientemente alguns genes responsivos ao PPARγ relacionados ao processo inflamatório, podem contribuir para o efeito sistêmico sobre a sensibilidade à insulina sem ganho de peso. Novos estudos são necessários para examinar se as similaridades entre a regulação transcricional do GQ-16 e da rosiglitazona estariam envolvidos no efeito terapêutico do GQ-16.
The prevalence of obesity associated with increased insulin resistance and type 2 diabetes has been increasing in the last decades. Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists that improve insulin resistance. However, TZDs have well-established side effects such as weight gain, fluid retention and edema, congestive heart failure and bone loss. GQ-16 is a specific PPARγ partial agonist that improves glucose tolerance and insulin sensitivity in a murine model of obesity and diabetes similarly to rosiglitazone, but does so without inducing weight gain or edema. Since one of the main pharmacological targets of TZDs is adipocyte tissue, the aim of this study was to determine how GQ- 16 influences PPARγ activity at the transcriptome wide level on PPARγ-dependent gene expression in mature 3T3-L1 adipocytes and also to compared these effects with rosiglitazone. Microarray analysis revealed these ligands modify gene expression in a different manner. Rosiglitazone changed the expression of 1156 genes whereas GQ-16 only changed the expression of 89 genes. Of the 544 genes upregulated by rosiglitazone (47%), only 22 (4.2%) were also regulated by GQ-16. Similarly, of the 612 genes repressed by rosiglitazone (53%), only 24 (4%) were by GQ-16. Therefore, just 46 genes were regulated by both ligands. Furthermore, GQ- 16 regulated the expression of 43 genes (18 activated and 25 repressed) that were not controlled by rosiglitazone. Whereas GQ-16 showed weak effects upon most rosiglitazone regulated genes, a subset of weakly rosiglitazone induced and strongly rosiglitazone repressed genes displayed disproportionately stronger responses to GQ-16. The greatest similarity between GQ-16 and rosiglitazone was upon repressed genes related to inflammatory response such as Dcn, Vcam, Orm2 and Lox, for example. Comparison of GQ-16 with other PPARγ partial agonists (MRL24 and SR1664) revealed similar effects on transcriptional repression. We propose that the ability of GQ-16 displays a continuum of partial agonist effects and that the ability of GQ-16 to efficiently repress some PPARγ responsive genes may partly explain systemic effects on insulin sensitivity without weight gain. Further studies are necessary to examine whether the similarities between transcriptional regulation of GQ-16 and rosiglitazone were involved in the therapeutic effect of GQ-16.

Orientador: Marcos Antonio Tambascia
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: A principal causa de mortalidade entre os portadores de Diabetes tipo 2 é a doença cardiovascular. Estudos têm cada vez mais procurado alterações inerentes ao diabetes tipo 2 que justifiquem a maior incidência de doença cardiovascular nesse grupo. A presença de resistência à insulina, redução de adiponectina, aumento de PCR, disfunção endotelial e aumento de PAI-1 são candidatos possivelmente relacionados a esse aumento. A redução da resistência à insulina com uso de tiazolidinedionas, entre elas a rosiglitazona, tem potencial de reduzir o risco cardiovascular em diabéticos tipo 2, uma vez que altera citocinas relacionadas a risco cardiovascular de forma positiva. O objetivo desse estudo é avaliar o efeito clínico e laboratorial (sensibilidade à insulina, função de célula ß, lípides, PCR, adiponectina, resistina e PAI-1) e o efeito sobre a espessura da íntima-média carotídea da administração, por 12 semanas, de 8mg de rosiglitazona ao dia, em pacientes diabéticos tipo 2 virgens de tratamento anti-diabético, atendidos no Ambulatório de Diabetes Mellitus tipo 2, do Hospital das Clínicas da Faculdade de Ciências Médicas da UNICAMP. Os pacientes foram submetidos a uma avaliação inicial com dosagem de glicemia, hemoglobina glicada, insulinemia, colesterol total, HDL, LDL, triglicérides, ácidos graxos livres, AST, ALT, adiponectina, resistina, PAI-1, PCR, ácido úrico e fibrinogênio, após jejum de 12 horas. A sensibilidade à insulina e a função de célula ß foram avaliadas pela fórmula matemática do HOMA e a espessura da íntima média carotídea foi avaliada pelo ultrassom doppler. Os pacientes iniciavam o uso de Rosiglitazona na dose de 8 mg/dia dividida em duas tomadas diárias. Após 12 semanas de tratamento todas as avaliações foram novamente realizadas. Para a análise estatística foi realizado o teste de Wilcoxon para estudar as variações pré e pós Rosiglitazona e o coeficiente de correlação de Spearman. O nível de significância adotado foi de 5 % (p<0,05). Dos 15 pacientes inicialmente incluídos, 13 completaram o tratamento. Houve redução estatisticamente significante dos níveis de PCR, ácido úrico e aumento de adiponectina. Houve redução de HOMA IR e resistina, não estatisticamente significante e aumento do HOMA ß A análise das correlações possíveis mostrou relação inversa entre HOMA ß e ácidos graxos livres. Não houve alteração significante da espessura da íntima-média carotídea. O tratamento do Diabetes Mellitus com rosiglitazona tem potencial de reduzir o risco cardiovascular à medida que reduz marcadores de risco, como a PCR, e aumenta a adiponectina. Apesar de não ter sido estatisticamente significante, possivelmente devido ao tamanho da amostra, houve redução de 25% do valor médio da resistina, sugerindo uma relação entre resistina e resistência à insulina, controversa na literatura. Além da melhora da sensibilidade à insulina houve notável aumento do HOMA ß mostrando melhora da função da célula ß. Esse dado sugere que o tratamento com rosiglitazona desacelera a progressão da doença. A relação entre o aumento do HOMA ß e a redução dos ácidos graxos livres fala a favor da melhora da lipotoxicidade como um dos fatores de melhora da função da célula ß. Mais estudos populacionais de longa duração são necessários para comprovar o efeito da rosiglitazona sobre eventos cardiovasculares
Abstract: Cardiovascular disease is the major mortality cause among diabetic patients. Most studies are trying to find disturbances typical of diabetes that could explain the grater incidence of cardiovascular disease in this group. Insulin resistance, adiponectin reduction, CRP elevation, endothelial dysfunction and PAI-1 elevation are candidates possibly related to this prevalence. Reducing insulin resistance with thiazolidinediones, including rosiglitazone, probably reduces cardiovascular risk among type 2 diabetic patients once it alters cytokines related to cardiovascular risk in a positive manner. The aim of this study is to evaluate clinical and laboratorial effects (insulin sensitivity, lipids profile, ß-cell function, CRP, adiponectin, resistin and PAI-1) and the effects on carotid intima media thickness of 12 weeks use of rosiglitazone 4 mg BID for type 2 diabetic drug naïve patients currently assisted at Hospital das Clínicas da Faculdade de Ciências Médicas da UNICAMP Type 2 diabetes out-clinics. At the first visit we evaluated glycemia, glicated hemoglobin, insulin, total cholesterol, LDL, HDL, triglycerides, AST, ALT, free fatty acids, uric acid, PAI-1, fibrinogen, CRP, adiponectin and resistin after a twelve hours fasting. Insulin sensitivity and ß cell function were estimated using the HOMA model and intima media thickness was evaluated by a Doppler ultrasound. Patients started using Rosiglitazone 4 mg BID and after 12 weeks the same parameters were evaluated again. The statistical analyses used Wilcoxon test to study variations before and after rosiglitazone treatment and Spearman correlation coefficient. We considered p<0,05 as statistical significant. From the 15 patients included, 13 completed treatment. We observed a statistically significant reduction on CRP and acid uric levels and an adiponectin levels elevation. Non statistical significant HOMA IR and resistin reductions and HOMA ß improvement occurred. Correlations analyses showed negative correlation between HOMA ß and free fatty acids. It was observed no change in intima media thickness. Treating type 2 diabetes mellitus with rosiglitazone has a potencial to reduce cardiovascular risk once it reduces cardiovascular risk markers as CRP and increases adiponectin. Although is was no statistically significant, possibly due to sample size, there was a 25% reduction in medium resistin levels, suggesting relation between resistin and insulin resistance, still unproved in the literature. Besides the improvement in insulin sensitivity there was a notable increase in HOMA ß showing improvement in ßcell function. This data suggests that rosiglitazone treatment slows disease progression. Correlation between HOMA ß improvement and free fatty acid agrees with improvement in lipotoxicity as one factor that leeds to improvement in ß cell function. We need more long term epidemiological studies to attest rosiglitazone effect in cardiovascular events
Mestrado
Clinica Medica
Mestre em Clinica Medica

Mestrado em Biomedicina Farmacêutica
Este estudo teve como objetivos a recolha e análise dos alertas de segurança a medicamentos publicados em Portugal, bem como avaliar o impacto dos alertas de segurança para medicamentos contendo rosiglitazona como substância ativa nas vendas de antidiabéticos em Portugal. Foi conduzida uma pesquisa dos alertas de segurança publicados pelo INFARMED, I.P. e pela EMA, complementada pela análise dos alertas publicados nos Boletins de Farmacovigilância. Para o estudo do impacto dos alertas da rosiglitazona desenhou-se um modelo de análise de regressão segmentada, usada para avaliar as diferenças ocorridas após cada um dos quatro alertas de segurança relacionados com a substância ativa rosiglitazona. Observou-se que dos 106 alertas selecionados, 43 são mencionados nos Boletins de Farmacovigilância. Entre 2000 e 2012, manteve-se a tendência do tipo de medida de segurança mais frequente, com o maior número de alertas relacionado com a alteração do Resumo das Características do Medicamento e Folheto Informativo e 17 alertas levaram à retirada do medicamento do mercado, temporária ou definitivamente, de forma voluntária ou não. O impacto dos alertas de segurança a medicamentos contendo rosiglitazona como substância ativa, foi de aumento de 32,9% (0,202 DID, p <0,001) nas vendas após o primeiro alerta em janeiro de 2006, tendência não repetida após os alertas subsequentes. Após os alertas de segurança de janeiro de 2006 e janeiro de 2008, as vendas descreveram uma tendência decrescente a longo prazo, com uma diminuição de 3,75% (-0.023 DID, p> 0,05) e 0,24% (-0,001 DID, p> 0,05), respetivamente. É importante para os profissionais de saúde e para os doentes terem acesso rápido e claro à informação de segurança relacionada com os medicamentos. A avaliação dos alertas de segurança é importante para aceder à relação benefício-risco de um medicamento e melhorar a sua utilização pela população.
This study aimed the collection and analysis of safety alerts published in Portugal, as well as assessing the impact of rosiglitazone safety alerts in the sales of oral antidiabetics in Portugal. We conducted a search of safety alerts published by INFARMED, I.P. and EMA, and complemented with analysis of Pharmacovigilance Bulletins. To study the impact of the warnings of rosiglitazone it was drawn up a model of segmented regression analysis, used to assess differences occurred after each of the four safety alerts related to rosiglitazone. It was observed that from the 106 selected alerts, 43 are mentioned in Pharmacovigilance Bulletins. Between 2000 and 2012, the trend remained with the change in the SPC and PL as the most frequent safety measure taken and 17 safety alerts leading to withdrawal, either temporarily or permanently, voluntarily or not. The impact of rosiglitazone safety alert, was an increase of 32.9 % (0.202 DID, p<0.001) in sales after the first alert in January 2006, a trend not repeated after subsequent alerts. After safety alerts in January 2006 and January 2008, sales of rosiglitazone describe a long-term downward trend, with a decrease of 3.75 % (DID -0.023, p>0.05) and 0.24 % (DID -0.001, p>0.05), respectively. It was observed that safety alerts conditioned the life cycle of rosiglitazone and the effects of safety alerts are immediately felt in the sales of other oral antidiabetic agents. It is important for healthcare professionals and patients have a quick and clear safety information related to drugs. The evaluation of safety alerts is important to access the benefit-risk ratio of a drug and to improve the use of medication, improving patient safety.

El presente trabajo desarrolla la validación prospectiva de una forma farmacéutica sólida cuyos principios activos contenidos son Rosiglitazona y Glimepiride, de tal modo que se demostró y estableció evidencia documentada que el proceso de análisis cumple de manera robusta y repetitiva lo que estaba previsto basado protocolo planificado, llamado protocolo de validación, obtenido de la secuencia de desarrollo del nuevo producto y de la metodología de análisis. En la parte inicial de este trabajo se procede a recopilar toda la información bibliografica necesaria para luego proceder con los primeros análisis exploratorios. Inicialmente con la intención de cuantificar ambos principios activos mediante un solo sistema cromatográfico, no obteniendo resultados esperados, seguidamente se procedió a probar sistemas cromatográficos diferentes para cada principio activo obteniendo los resultados adecuados para obtener la metodología analítica correcta. Una vez establecidas las condiciones cromatográficas finales con los parámetros definidos para el trabajo, se ejecuta el protocolo de validación del método desarrollado para lo cual se cuenta con el diseño experimental y los procedimientos estadísticos, concluyendo que la metodología analítica propuesta es lineal, exacta, y selectiva, cumpliendo así con los parámetros de validación establecidos en las obras oficiales; por lo cual el método validado es confiable y puede ser empleado en los análisis de rutina.
-- The present work develops the Prospective Validation of a Solid Pharmaceutical Form, which drogs are Rosiglitazone and Glimepiride, in such a way that it was demonstrated and established documented evidence that the process of analysis fulfills in a robust and repetitive way what there was foreseen based planned protocol, so-called Protocol of Validation, obtained of the sequence of development of the new product and of the methodology of analysis. At the first part of this work, all the bibliographical necessary information is proceeded to compile then to proceed with the first exploratory analyses. Initially with the intention of quantifying both active beginning by means of only one system cromatographic, not obtaining awaited results, then it was proceeded to prove in different system cromatographics by the every active beginning obtaining the results adapted to obtain the analytical correct methodology. As soon as the last conditions cromatographic were established with the parameters defined for the work, there is executed the Protocol of Validation the method developed for which is counted by the experimental design and the statistical procedures, concluding that the analytical proposed methodology is linear, exact, and selective, expiring this way with the parameters of ratification established in the official works; for which the validated method is reliable and can be used in the analyses of routine.
Tesis

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2012.
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Estudos recentes sugerem que o receptor gama ativado por proliferadores peroxissomais (PPAR) e seu agonista, a rosiglitazona, podem modular a expressão de moléculas relacionadas com eventos imuno-inflamatórios em células da polpa dentária humana, indicando, assim, a possibilidade de se utilizar agonistas deste receptor nuclear (RN) no controle da inflamação pulpar. Entretanto, eventos igualmente importantes no processo de reparo do complexo dentino-pulpar, tais como proliferação e expressão de genes envolvidos com a formação de dentina terciária, não foram abordados nesses estudos. Diante disso, a proposta do presente trabalho foi investigar o efeito da rosiglitazona sobre a proliferação celular e sobre a expressão do TGF1, em modelo experimental de cultura primária de células da polpa dentária humana. Para cumprir com os objetivos propostos, foram estabelecidas culturas primárias de polpas obtidas de terceiros molares inclusos e hígidos, com a raiz ainda em formação, extraídos por indicações clínicas. Os efeitos da rosiglitazona sobre a proliferação celular foram avaliados por meio de ensaio de 3incorporação de timidina marcada radioativamente com trítio ([H]-timidina) e os efeitos sobre a expressão do gene que codifica o TGF1 foram avaliados por meio da técnica de amplificação por reação em cadeia da polimerase em tempo real/quantitativa (RT-PCRq). Os resultados dos ensaios de proliferação demonstraram que o tratamento com rosiglitazona durante 24 horas diminuiu a proliferação das células em cultura, enquanto que o tratamento durante 72 horas não alterou a proliferação celular. Os resultados dos ensaios de expressão gênica sugeriram que o tratamento com rosiglitazona durante 7 dias não alterou a expressão do gene que codifica o TGF1, no entanto, após 14 dias de tratamento com o ligante, sua expressão aumentou. Em conjunto os resultados dos ensaios de proliferação e de expressão gênica sugerem que a rosiglitazona possa estar envolvida com a ativação de vias de diferenciação ou de vias apoptóticas. No entanto, investigações adicionais são necessárias para melhor esclarecer o papel do PPAR na fisiopatologia pulpar. ______________________________________________________________________________ ABSTRACT
Several studies have suggested that peroxisome proliferator-activated receptor gamma (PPARγ) and his agonist, rosiglitazone, can regulate the expression of immune-inflammatory related molecules in human dental pulp cells. The authors have raised the possibility of using PPAR agonists as an option in pulpal inflammation treatment. However, others events involved in the dental-pulp complex repair process, such as cell proliferation and gene expression of molecules related with the tertiary dentin formation, were not assessed in these studies. Therefore, the aim of this study was to investigate the effect of rosiglitazone on cell proliferation and on the TGF1 gene expression in primary human dental pulp cells culture. In order to perform the purposes of the present study, primary cultures were established from pulp tissue obtained from non-erupted, caries-free third molars, which were extracted due to therapeutic reasons. Effects of rosiglitazone on cell proliferation were 3evaluated by tritium labeled thymidine incorporation assay ([H]-thymidine) and effects on TGF1 gene expression were evaluated by quantitative polymerase chain reaction (RT-PCRq). The proliferation assays showed that treatment with rosiglitazone for 24 hours decreased cell proliferation, while after 72 hours treatment no changes in cell proliferation where observed when compared with controls. Gene expression results suggested that treatment with rosiglitazone for 7 days did not modify TGF1 gene expression, however, after 14 days ligand treatment, an increase in the expression was observed. Proliferation and TGF1 gene expression results suggest that rosiglitazone might be involved in differentiation or apoptotic pathways activation in the primary pulp cell cultures studied. However, further investigations are required to better understand the role of PPAR in dental pulp pathophysiology.

碩士
國立中興大學
化學系所
104
Thiazolidinedione drugs (TZDs) are the only current antidiabetic agents that function primarily by increasing insulin sensitivity. TZDs were first reported as insulin-sensitizing drugs in the early 1980s, but their mechanism remained a mystery until the mid-1990s, when they were found to be ligands for the nuclear receptor transcription factor PPARγ. Molecular compounds such as the clinically available rosiglitazone and pioglitazone have been designed to target PPARγ and for the oral treatment of type 2 Diabetes. However, in addition to attenuating inflammation and promoting insulin sensitivity, these compounds are often associated with severe side effects including weight gain, edema, bone fracture, and congestive heart failure, which have severely limited their clinical application. A rosiglitazone delivery strategy composing of polymeric emulsion particles and injectable gel system was established. In this study, Sprague Dawley® rats were used to evaluate the physiological reactions, but all the combinations of hydrogels with rosiglitazone emulsion particles show the unwanted foreign body reaction in a certain degree.

Sustained nitroglycerin (GTN) therapy impairs endothelial function in healthy volunteers and patients with cardiovascular disease, caused by an increase in vascular oxidative stress. This study aims to estimate the effect of rosiglitazone on vascular endothelial function in healthy volunteers continuously dosed to transdermal GTN (0.6mg/hr) for 7 days. To assess endothelial function, forearm blood flow was measured by venous occlusion strain-gauge plethysmography in response to intra-brachial infusions of acetylcholine. GTN-treated subjects experienced significant attenuation of endothelium-dependent responses to acetylcholine (p<0.05; compared to placebo), but was reversed with vitamin C (p=ns; compared to placebo). Endothelium-dependent responses to acetylcholine were blunted in groups randomized to rosiglitazone alone (p<0.05; compared to placebo) and rosiglitazone + GTN (p<0.05 compared to placebo). Interestingly, this effect was not modified by vitamin C. In conclusion, rosiglitazone impairs endothelial function and concurrent therapy with rosiglitazone does not attenuate the adverse effects of transdermal GTN on the vasculature.

碩士
輔仁大學
生命科學系碩士班
106
英文摘要 Diabetes is an increasingly common chronic disease worldwide, and it has been confirmed in trampolines that thiazolidinediones can improve insulin sensitivity in patients with insulin resistance, but have been found to have side effects like hepatotoxicity. ENERGI is a novel AMPK activator discovered in our laboratory. It is a water-soluble small molecular compound. By increasing the activity of activated AMPK, it can inhibit the activity of transcription factors in fat cells and the differentiation of adipocytes. The aim of this research is to explore the synergistic effect of ENERGI and Rosiglitazone. After the cultured cells 3T3-L1 were differentiated for 9 days, ENERGI and Rosiglitazone were added and western blot analysis was conducted. The results showed that the expression of PPAR-γ and C/EBPα increased after ENERGI being added. There was no distinct difference when Leptin was with Rosiglitazone. The mechanism of ENERGI being added and promoted being increased awaits further discussions. In addition, the results of Oil Red O staining showed that ENERGI could promote the accumulation of oil droplets in fat cells, and the efficacy was better when Rosiglitazone and ENERGI were added. It shows that the Synergism of ENERGI and Rosiglitazone can reduce the use of Rosiglitazone. It is also assumed that the side effects of Rosiglitazone and toxicity can be reduced and effects cab be multiplied.

碩士
臺北醫學大學
生物醫學技術研究所
91
Rosiglitazone is a peroxisome proliferator-activated receptor-gamma (PPAR-g) agonist that has been shown to halt mesangium expansion in experimental models of type 2 diabetes mellitus. In the present study, rat mesangial cells were treated with rosiglitazone and its molecular mechanisms coupling growth arrest were examined. Treatment of rat mesangial cells with rosiglitazone resulted in reduction of viable cells and inhibition of [3H] thymidine incorporation. Treatment with rosiglitazone increased the expression of CDK inhibitor, p21CIP-1, which was associated with cell cycle arrest. Rosiglitazone increased PDK-1 and protein kinase B/Akt (PKB/Akt) Serine473 phosphorylation and Akt kinase activity in rat mesangial cells, and these responses were inhibited with the pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3-K), LY294002 or wortmannin. Rosiglitazone increased the expression of smooth muscle a-actin (SMA), a differentiation marker for mesangial cells. LY294002 or wortmannin pretreated cells failed to blocked SMA expression suggesting SMA expression is not mediated through PI3-K dependent pathway. The expression of SMA was inhibited by genistein (a protein tyrosine kinase inhibitor), by PD98059 (a MEK inhibitor), suggesting protein tyrosine kinase-MEK-mitogen-activated protein kinase pathway mediates the differentiation of renal glomerular mesangial cells. These data demonstrate a role for PPAR-g in mediating a molecular mechanism coupling growth arrest and mesangial cell differentiation.