No description available.

Thesis (Ph. D.)--Duke University, 2008. / Includes bibliographical references.

AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable aß? heterotrimer comprising a catalytic a subunit and two non-catalytic subunits, ß and ?. The ß subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here I show that the conserved C-terminal 85-residue sequence of the ß subunit, ß1(186-270), is sufficient to form an active AMP-dependent heterotrimer a1ß1(186-270)?1, whereas the 25-residue ß1 C-terminal (246-270) sequence is sufficient to bind ?1, ?2, or ?3 but not the a subunit. Within this sequence (246-270), two residues were essential for ß? association based on Ala scanning mutagenesis. / Substitution of ß1 Tyr-267 for Ala precludes ß? but not aß association suggesting independent binding requirements. Substitution of Tyr-267 for Phe or His but not Ala or Ser can rescue ß? binding. Substitution of Thr-263 for Ala also resulted in decreased ß? but not aß association. Truncation of the a subunit reveals that ß1 binding requires the a1(313-473) sequence while the remainder of the a C-terminus is required for ? binding. The conserved C-terminal 85-residue sequence of the ß subunit (90% between ß1 and ß2) is the primary a? binding sequence responsible for the formation of the AMPK aß? heterotrimer. The ? subunits contain four repeat CBS sequences with variable N-terminal extensions and the ?1 isoform is N-terminally acetylated. The ?2 subunit can be multiply phosphorylated by protein kinase C (PKC) in vitro, with Ser-32 identified as a minor site. A detailed understanding of the structure and regulation of AMPK will enable rational drug design for treatment of such linked diseases as obesity, insulin resistance and type 2 diabetes.

AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable aß? heterotrimer comprising a catalytic a subunit and two non-catalytic subunits, ß and ?. The ß subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here I show that the conserved C-terminal 85-residue sequence of the ß subunit, ß1(186-270), is sufficient to form an active AMP-dependent heterotrimer a1ß1(186-270)?1, whereas the 25-residue ß1 C-terminal (246-270) sequence is sufficient to bind ?1, ?2, or ?3 but not the a subunit. Within this sequence (246-270), two residues were essential for ß? association based on Ala scanning mutagenesis. / Substitution of ß1 Tyr-267 for Ala precludes ß? but not aß association suggesting independent binding requirements. Substitution of Tyr-267 for Phe or His but not Ala or Ser can rescue ß? binding. Substitution of Thr-263 for Ala also resulted in decreased ß? but not aß association. Truncation of the a subunit reveals that ß1 binding requires the a1(313-473) sequence while the remainder of the a C-terminus is required for ? binding. The conserved C-terminal 85-residue sequence of the ß subunit (90% between ß1 and ß2) is the primary a? binding sequence responsible for the formation of the AMPK aß? heterotrimer. The ? subunits contain four repeat CBS sequences with variable N-terminal extensions and the ?1 isoform is N-terminally acetylated. The ?2 subunit can be multiply phosphorylated by protein kinase C (PKC) in vitro, with Ser-32 identified as a minor site. A detailed understanding of the structure and regulation of AMPK will enable rational drug design for treatment of such linked diseases as obesity, insulin resistance and type 2 diabetes.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Obesity, one of the most serious health problems of the 21st century, often occurs as a result of an imbalance between energy intake and energy expenditure. Dietary lipids play an important role in the development of obesity, partly because they represent the richest source of energy amongst all macronutrients. It is, however, not only the amount of consumed lipids, but also the composition of fatty acids, which strongly influences health effects of a particular diet. Saturated fatty acids (SFA) are generally considered as unhealthy due to their pro-inflammatory and lipotoxic properties, while monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) represent a healthier alternative, as they are more readily oxidized and do not disrupt biochemical properties of cellular membranes. Amongst PUFA, PUFA of n-3 series (Omega-3) represent an utterly unique class of lipids that have been documented to protect against cardiovascular disease and dyslipidemia in men and improve insulin sensitivity and glucose tolerance primarily in animal models of obesity. Some molecular mechanisms of Omega-3 action have been already uncovered, such as the modification of biological membranes composition, activation of various transcription factors and membrane receptors, and their role as precursors for...

Vaspin expression is increased in white adipose tissue (WAT) of diet-induced obese mice and rats and is supposed to compensate HFD-induced inflammatory processes and insulin resistance in adipose tissue by counteracting pro-inflammatory gene expression in obesity. Multiple studies have also demonstrated strong anti-inflammatory effects in vascular and skin cells. Here, we used vaspin treated 3T3-L1 murine adipocytes as well as 3T3-L1 cells with stable vaspin expression to investigate the effect of exogenous and endogenous vaspin on inflammatory processes and insulin signaling in adipocytes. Our stably transfected cells secreted significant amounts of vaspin which was in the physiological range of ∼0.5 ng/ml in cell supernatants. Adipocyte differentiation was not affected by vaspin as expression of adipogenic marker genes as well as lipid accumulation after full differentiation was similar to control cells. We found that IL-1β induced expression and secretion of pro-inflammatory cytokines, such as IL-6, MCP1 and TNFα was significantly blunted in vaspin expressing 3T3-L1 cells. Treatment of 3T3-L1 cells with exogenous vaspin resulted in reduced cytokine-induced activation of the intracellular and pro-inflammatory NFκB signaling cascades (IKKα/β, IκB and NFκB). Moreover, endogenous vaspin positively affected insulin signaling by increasing insulin-stimulated phosphorylation of the key mediator protein kinase B (AKT). Together, we demonstrate anti-inflammatory effects of vaspin in 3T3-L1 adipocytes as well as increased insulin signaling by endogenous expression or exogenous treatment. The results provide evidence for potent anti-inflammatory action of vaspin not only in vascular cells but also in adipose tissue.

Nos últimos anos, o corpo de evidência científica a respeito da relação entre as doenças periodontais e desordens metabólicas - como a obesidade e a síndrome metabólica - vem aumentando consideravelmente. Entretanto, estudos avaliando os mecanismos biológicos que permeiam esta associação ainda são escassos. A presente tese teve como objetivo principal avaliar, em ratos Wistar, o efeito da obesidade e da síndrome metabólica (MetS) sobre a patogênese da perda óssea alveolar. Nesse sentido, foram produzidos 4 artigos científicos, sendo 2 revisões de literatura e 2 estudos experimentais. A primeira revisão de literatura buscou avaliar a literatura disponível acerca da relação MetS e doença periodontal. Pode-se observar que poucos estudos foram localizados e, dentre aqueles avaliados, a maioria tratouse de estudos de natureza epidemiológica transversal. Não foram localizados estudos de natureza longitudinal e apenas um estudo de intervenção piloto foi avaliado. A análise desta evidência permitiu concluir que o tópico é de interesse, porém ainda não foi explorado na perspectiva dos fatores de risco. Para o segundo e terceiro artigos, foi utilizado um modelo animal que reproduz as características de obesidade (artigo 2) e MetS (artigo 3). Para o segundo artigo, foram utilizados 28 ratos Wistar machos divididos em 2 grupos experimentais. O grupo controle recebeu ração padrão e água e o grupo teste além da ração padrão/água foi exposto a uma dieta de cafeteria (CAF) para indução de obesidade. Foi definido um ponto de corte para ocorrência de doença periodontal, sendo essa considerada positiva quando da presença de perda óssea alveolar ≥0.51mm em fotografias padronizadas. Diferenças significativas para o peso e índice de Lee foram observadas no grupo exposto a dieta CAF indicando presença de obesidade neste grupo. Observou-se que os animais obesos apresentaram significativamente mais sítios positivos para doença periodontal. Portanto, pode se concluir que a obesidade aumenta a perda óssea alveolar espontânea em ratos Wistar. Para o terceiro artigo, 60 ratos Wistar foram divididos em 4 grupos: Controle (ração padrão e água), Doença Periodontal (DP) (ração padrão e ligadura no semana 12), MetS (dieta CAF por 17 semanas) e MetS+DP (ligadura na semana 12 e dieta CAF por 17 semanas). Dois animais foram perdidos ao longo do período experimental por razões não relacionadas ao protocolo do estudo. Diferenças significativas foram observadas para o peso, índice de Lee colesterol total e triglicerídeos, indicando presença de MetS. Foram realizadas análises do percentual de perda óssea alveolar através de micro-tomografias e os níveis séricos de IL1- e TNF- foram avaliados por ELISA. Observou-se que os animais portadores de MetS exibiram maiores percentuais de perda óssea alveolar induzida e espontânea. Não foram observadas diferenças significativas para os níveis séricos das citocinas avaliadas. Nesse sentido, pode ser concluído que a presença de MetS modula a resposta do hospedeiro, provavelmente pela quebra da homeostase do periodonto. O artigo 4 tratou-se de uma revisão de literatura com caráter informativo alertando aos profissionais da odontologia a responsabilidade da transcender as abordagens odontológicas tradicionais colocando a profissão como parte importante e integrantes da equipe de saúde. A partir dos resultados obtidos nos artigos constituintes da presente tese e frente às reflexões realizadas, pode-se concluir que a doença periodontal é influenciada por desordens metabólicas. Recomenda-se, portanto, a inclusão de questionamentos e análises dessas condições e suas potenciais inter-relações pelos profissionais da saúde / between periodontal diseases and metabolic disorders – like obesity and metabolic syndrome – has considerably increased. However, studies evaluating the biological mechanisms related to this association are still scarce. The present thesis had as main objective to evaluate, in Wistar rats, the effect of obesity and metabolic syndrome (MetS) on the pathogenesis of alveolar bone loss. In this respect, 4 scientific articles were written, being 2 literature reviews and 2 experimental studies. The first literature review evaluated the available literature relating MetS and periodontal disease. It can be observed that few studies were retrieved and, among thos evaluated, the majority were of cross-sectional epidemiological nature. Longitudinal studies were not found and only one pilot intervention study was evaluated. The analysis of this evidence lead to the conclusion that the topic is of interest, however still has not been explored in the perspective of risk factors. For the second and third articles, an animal model was used, that mimics obesity (article 2 and MetS (article 3). For the second article, 28 male Wistar rats were divided in 2 experimental groups. The control group received chow and water and the test group, besides the standard chow/water was exposed to cafeteria diet (CAF) in order to induce obesity. A cut-off point was defined for the occurrence of periodontal disease, being this considered positive when alveolar bone loss ≥0.51mm was present in standardized pictures. Significant differences for body weight and Lee Index were observed in the group exposed to CAF diet, indicating obesity. Obese animals presented significantly more sites positive for periodontal disease. Thus, it may be concluded that obesity increases spontaneous alveolar bone loss in Wistar rats. For the third article, 60 male Wistar rats were divided in 4 groups: Control (standard chow and water), Periodontal disease (PD) (standard rat chow and ligature on week 12, MetS (CAF diet for 17 weeks and MetS+PD (ligature on week 12 and CAF diet for 17 weeks). Two animals were lost during the experimental period for reasons not related to the study protocol. Significant differences were observed in body weight, Lee Index, total cholesterol and triglycerides, indicating the presence of MetS. Analyses of percent alveolar bone loss were performed by microcomputer tomography and the serum levels of IL1- and TNF- were performed by ELISA. Animals with MetS exhibited higher percentage of spontaneous and induced alveolar bone loss. No significant differences were observed for the serum levels of the evaluated cytokines. Thus, it may be concluded that the presence of MetS modulates the host response, probably breaking the periodontium homeostasis. Article 4 is a literature review with informative characteristics alerting the dental professionals about the responsibility of going beyond the traditional dental practices, making the profession an important component of the health team. Taking into consideration the obtained results in the articles in this thesis, and the thoughts coming from them, it may be concluded that periodontal disease is influenced by metabolic disorders. It is recommended that questions and analyzes are made about these conditions and their potential interrelationships by the health personnel.

Obesity-associated metabolic disorders are amongst the most prevalent causes of death worldwide. Understanding how obesity leads to the development of the Metabolic Syndrome (MetS) and cardiovascular disease (CVD) will enable the development of novel therapies that dissociate obesity from its cardiometabolic complications. Our laboratory views the functional capacity of white adipose tissue (WAT), the organ designed for safe lipid storage, as a key factor in the development of MetS and CVD. At a genetically-defined stage of the aberrant WAT expansion that occurs during obesity, adipocytes undergo a functional failure, resulting in an impaired control of serum free fatty acid (FFA) concentration. In such setting, FFAs and their metabolic derivatives accumulate in other organs, where they cause lipotoxicity, leading to the development of insulin resistance and CVD. We therefore aim to understand the pathophysiological mechanisms that induce adipocyte dysfunction. The past two decades of research have established the immune system as an important regulator of WAT function. The number of adipose tissue macrophages (ATMs), the most abundant immune cell type in WAT, increases during obesity, resulting in WAT inflammation. Multiple genetic and pharmacological intervention studies of murine models of obesity have assigned a causal link between ATM pro-inflammatory activation and WAT dysfunction. However, while the propagation of inflammation in ATMs during obesity has been extensively studied, factors triggering ATM inflammatory activation are less clear. Recently, our lab has observed lipid accumulation in the ATMs isolated from obese mice. Lipid-laden ATMs were pro-inflammatory, leading us to hypothesise that aberrant lipid build-up in macrophages triggers WAT inflammation during obesity. This thesis expands on the initial findings from our lab and describes two novel mechanisms that potentially contribute to lipid-induced inflammatory activation of ATMs. In chapter 3, the role of de novo phosphatidylcholine (PC) synthesis pathway during lipotoxicity in macrophages is addressed. The first part of the chapter demonstrates that lipotoxic environment increased de novo PC synthesis rate in bone marrow-derived macrophages (BMDMs) and ATMs, and that loss of rate-limiting enzyme in de novo PC synthesis pathway, CTP:phosphocholine cytidylyltransferase a (CCTa) diminished saturated FFA-induced inflammation in BMDMs. In the second part, I show that macrophage-specific CCTa deletion did not impact on the development of WAT inflammation or systemic insulin resistance, but had a minor benefitial effect on hepatic gene transcription during obesity. Chapter 4 develops on recent observations of interactions between sympathetic nerves and macrophages in WAT. In the first part of the chapter, I demonstrate that stimulating B2-adrenergic receptor (B2AR), the main receptor for sympathetic neurotransmitter norepinephrine in macrophages, enhanced intracellular triglyceride storage by up-regulating diacylglycerol O-acyltransferase 1 (Dgat1) gene expression in BMDMs. The second part of the chapter shows that macrophage-specific B2AR deletion did not modulate systemic glucose and lipid metabolism during obesity, but mice lacking B2ARs in macrophages demonstrated augmented hepatic glucose production on a chow diet. Furthermore, systemic B2AR blockade or macrophage-specific B2AR deletion in mice did not affect the thermogenic response to cold exposure. Chapter 5 includes the characterisation of B2AR stimulation-induced changes to the global cellular proteome of BMDMs, and a subsequent validation of the role of candidate transcription factors in regulating B2AR agonism-induced gene expression in BMDMs.

Nos últimos anos, o corpo de evidência científica a respeito da relação entre as doenças periodontais e desordens metabólicas - como a obesidade e a síndrome metabólica - vem aumentando consideravelmente. Entretanto, estudos avaliando os mecanismos biológicos que permeiam esta associação ainda são escassos. A presente tese teve como objetivo principal avaliar, em ratos Wistar, o efeito da obesidade e da síndrome metabólica (MetS) sobre a patogênese da perda óssea alveolar. Nesse sentido, foram produzidos 4 artigos científicos, sendo 2 revisões de literatura e 2 estudos experimentais. A primeira revisão de literatura buscou avaliar a literatura disponível acerca da relação MetS e doença periodontal. Pode-se observar que poucos estudos foram localizados e, dentre aqueles avaliados, a maioria tratouse de estudos de natureza epidemiológica transversal. Não foram localizados estudos de natureza longitudinal e apenas um estudo de intervenção piloto foi avaliado. A análise desta evidência permitiu concluir que o tópico é de interesse, porém ainda não foi explorado na perspectiva dos fatores de risco. Para o segundo e terceiro artigos, foi utilizado um modelo animal que reproduz as características de obesidade (artigo 2) e MetS (artigo 3). Para o segundo artigo, foram utilizados 28 ratos Wistar machos divididos em 2 grupos experimentais. O grupo controle recebeu ração padrão e água e o grupo teste além da ração padrão/água foi exposto a uma dieta de cafeteria (CAF) para indução de obesidade. Foi definido um ponto de corte para ocorrência de doença periodontal, sendo essa considerada positiva quando da presença de perda óssea alveolar ≥0.51mm em fotografias padronizadas. Diferenças significativas para o peso e índice de Lee foram observadas no grupo exposto a dieta CAF indicando presença de obesidade neste grupo. Observou-se que os animais obesos apresentaram significativamente mais sítios positivos para doença periodontal. Portanto, pode se concluir que a obesidade aumenta a perda óssea alveolar espontânea em ratos Wistar. Para o terceiro artigo, 60 ratos Wistar foram divididos em 4 grupos: Controle (ração padrão e água), Doença Periodontal (DP) (ração padrão e ligadura no semana 12), MetS (dieta CAF por 17 semanas) e MetS+DP (ligadura na semana 12 e dieta CAF por 17 semanas). Dois animais foram perdidos ao longo do período experimental por razões não relacionadas ao protocolo do estudo. Diferenças significativas foram observadas para o peso, índice de Lee colesterol total e triglicerídeos, indicando presença de MetS. Foram realizadas análises do percentual de perda óssea alveolar através de micro-tomografias e os níveis séricos de IL1- e TNF- foram avaliados por ELISA. Observou-se que os animais portadores de MetS exibiram maiores percentuais de perda óssea alveolar induzida e espontânea. Não foram observadas diferenças significativas para os níveis séricos das citocinas avaliadas. Nesse sentido, pode ser concluído que a presença de MetS modula a resposta do hospedeiro, provavelmente pela quebra da homeostase do periodonto. O artigo 4 tratou-se de uma revisão de literatura com caráter informativo alertando aos profissionais da odontologia a responsabilidade da transcender as abordagens odontológicas tradicionais colocando a profissão como parte importante e integrantes da equipe de saúde. A partir dos resultados obtidos nos artigos constituintes da presente tese e frente às reflexões realizadas, pode-se concluir que a doença periodontal é influenciada por desordens metabólicas. Recomenda-se, portanto, a inclusão de questionamentos e análises dessas condições e suas potenciais inter-relações pelos profissionais da saúde / between periodontal diseases and metabolic disorders – like obesity and metabolic syndrome – has considerably increased. However, studies evaluating the biological mechanisms related to this association are still scarce. The present thesis had as main objective to evaluate, in Wistar rats, the effect of obesity and metabolic syndrome (MetS) on the pathogenesis of alveolar bone loss. In this respect, 4 scientific articles were written, being 2 literature reviews and 2 experimental studies. The first literature review evaluated the available literature relating MetS and periodontal disease. It can be observed that few studies were retrieved and, among thos evaluated, the majority were of cross-sectional epidemiological nature. Longitudinal studies were not found and only one pilot intervention study was evaluated. The analysis of this evidence lead to the conclusion that the topic is of interest, however still has not been explored in the perspective of risk factors. For the second and third articles, an animal model was used, that mimics obesity (article 2 and MetS (article 3). For the second article, 28 male Wistar rats were divided in 2 experimental groups. The control group received chow and water and the test group, besides the standard chow/water was exposed to cafeteria diet (CAF) in order to induce obesity. A cut-off point was defined for the occurrence of periodontal disease, being this considered positive when alveolar bone loss ≥0.51mm was present in standardized pictures. Significant differences for body weight and Lee Index were observed in the group exposed to CAF diet, indicating obesity. Obese animals presented significantly more sites positive for periodontal disease. Thus, it may be concluded that obesity increases spontaneous alveolar bone loss in Wistar rats. For the third article, 60 male Wistar rats were divided in 4 groups: Control (standard chow and water), Periodontal disease (PD) (standard rat chow and ligature on week 12, MetS (CAF diet for 17 weeks and MetS+PD (ligature on week 12 and CAF diet for 17 weeks). Two animals were lost during the experimental period for reasons not related to the study protocol. Significant differences were observed in body weight, Lee Index, total cholesterol and triglycerides, indicating the presence of MetS. Analyses of percent alveolar bone loss were performed by microcomputer tomography and the serum levels of IL1- and TNF- were performed by ELISA. Animals with MetS exhibited higher percentage of spontaneous and induced alveolar bone loss. No significant differences were observed for the serum levels of the evaluated cytokines. Thus, it may be concluded that the presence of MetS modulates the host response, probably breaking the periodontium homeostasis. Article 4 is a literature review with informative characteristics alerting the dental professionals about the responsibility of going beyond the traditional dental practices, making the profession an important component of the health team. Taking into consideration the obtained results in the articles in this thesis, and the thoughts coming from them, it may be concluded that periodontal disease is influenced by metabolic disorders. It is recommended that questions and analyzes are made about these conditions and their potential interrelationships by the health personnel.