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To investigate interactive effects of dietary fatty acid composition and energy restriction on body lipid metabolism and its regulation, rats were fed for 10 weeks diets varying in fat type and energy intake level. Energy deficiency was achieved by removing carbohydrate from the diets while keeping fat and other nutrient intakes constant. Tissue fatty acid deposition was influenced by the interaction between the dietary fat source and body energy balance. Less total fatty acids were deposited in livers of the ad libitum beef tallow-fed animals than the other fat feedings. However, such difference no longer existed when energy intake was restricted. Similarly, less energy supply eliminated the higher docosahexaenoic acid and lower arachidonic acid contents associated with the fish oil feeding in hepatocyte membrane phosphatidylchohne, phosphatidylserine and sphingomyelin. Tissue lipogenesis was also examined as a function of the interaction of dietary fatty acid composition and energy restriction. Comparable absolute cholesterol synthesis rates were observed in livers of the food restricted animals fed different types of dietary fat, although the synthesis rates were different among the dietary fat groups fed ad libitum. Energy restriction increased the triglyceride-fatty acid synthesis rates in the intestine of the fish and safflower oil-fed groups, but not in that of the olive oil- and beef tallow-fed animals. Plasma leptin concentrations were 60% higher in the ad libitum-fed fish and safflower oil groups as compared with those in the beef tallow diet group, despite smaller perirenal fat mass and fat cell size in the fish oil-fed animals. Energy restriction decreased plasma leptin levels of the fish and safflower oil-fed rats, but not those in the beef tallow-fed animals. Taken together, these results demonstrate that the structural, functional and regulating aspects of tissue lipid metabolism were influenced by an interaction between dietary fatty acid composit

Orientador: Lucia Nassi Castilho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T17:01:42Z (GMT). No. of bitstreams: 1 Vasconcelos_EdilmaMariadeAlbuquerque_D.pdf: 990594 bytes, checksum: ec1dc188f8613c4270257732116a5e8b (MD5) Previous issue date: 2007 / Resumo: Atualmente, a aterosclerose é definida como uma doença crônica inflamatória caracterizada por elevado estresse oxidativo gerado por células da parede vascular. Neste estudo foi avaliada, em monócitos humanos, a contribuição de espécies reativas de oxigênio (EROs) mitocondrial nos processos do estresse oxidativo e inflamatório de pacientes dislipidêmicos. Vinte e nove indivíduos hiperlipidêmicos primários, sendo catorze portadores de hipercolesterolemia e quinze com hiperlipidemia mista (ambos sem uso de fármacos) e dezoito indivíduos normocolesterolêmicos, não tabagistas foram selecionados para este estudo. A geração de EROs e o potencial de membrana mitocondrial foram determinados em células mononucleares intactas, por meio de citometria de fluxo e a concentração plasmática de LDL oxidada (LDL-ox) por meio de ELISA. Apenas os monócitos isolados de indivíduos hipercolesterolêmicos apresentaram maior geração de EROs, porém ambos os grupos apresentaram concentração sérica de colesterol da LDL, LDL-ox e apolipoproteína B maiores do que o grupo-controle. Presença de LDL pequenas e densas também foi encontrada nos hiperlipidêmicos. Nos pacientes com hiperlipidemia mista igualmente foram detectadas, adicionalmente, concentrações significativamente maiores de insulina, triglicérides, colesterol de VLDL e ácidos graxos livres. Correlações positivas e significativas entre a geração de EROs mitocondrial com as concentrações séricas de LDL-oxidada e da proteína quimiotática para monócito-1 (MCP-1), foram encontradas. Esses resultados sugerem que a geração de EROs mitocondrial por monócitos circulantes em pacientes hiperlipidêmicos primários contribui para o estresse oxidativo e, portanto, para o desenvolvimento da aterosclerose, patologia comum nestes indivíduos. A hiperlipidemia está também acompanhada da elevação de marcadores inflamatórios séricos (ácido úrico e contagem global dos leucócitos). A correlação negativa encontrada entre a concentração sérica da interleucina 6 (IL-6) e da proteína induzida pelo IFN-? 10 (IP-10), nos pacientes hiperlipidêmicos, sugere que a IL-6 possa estar agindo como uma citocina antiinflamatória / Abstract: Actually, atherosclerosis is characterized like an inflammatory chronic disease due to a high oxidative stress state generated by vascular wall cells. In present study, we investigated the contribution of reactive oxygen species (ROS) generation by circulating monocyte mitochondria to the oxidative stress frequently observed in high risk hyperlipidemic subjects. Twenty-nine primary hyperlipidemic subjects (14 hypercholesterolemic and 15 combined hyperlipidemic) and 18 normolipidemic individuals, without any drug treatment and non-smokers, were enrolled in this study. The mitochondrial monocyte ROS generation and the mitochondrial electrical transmembrane potential in intact polymorphonuclear cells were estimated by flow cytometry and plasma oxidized LDL (ox-LDL) was measured by ELISA. The hypercholesterolemic, but not the combined hypelipidemic subjects presented higher levels of monocyte ROS generation when compared to control group. Both hyperlipidemic subjects presented elevated plasma levels of total LDL, oxidized LDL and apolipoprotein B. Combined hyperlipidemic individuals presented additionally increased levels of small dense LDL particles, insulin, triglycerides, VLDL-cholesterol and free fatty acids. Furthermore, highly significants positives correlations between monocyte ROS generation with oxidized LDL and monocyte chemotactic protein 1 (MCP-1) levels, were found. These data suggest that mitochondrial ROS production by circulating monocytes from primary hyperlipidemic subjects can contribute to the oxidative stress and hence to atherosclerosis, disease frequently found in this group. The hyperlipidemia is also associated with high levels of inflammatory markers (uric acid and leukocyte global count). A significant negative correlation between interleukin-6 (IL-6) and interferon-inducible protein 10 (IP-10) levels found in hyperlipidemic individuals may suggest an antinflamatory propertie of citokine IL-6 / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

A periodontite é uma doença bucal infecto-inflamatória resultante da quebra da homeostase entre biofilme dentário e o hospedeiro. Diversos estudos tem mostrado que a obesidade e o sobrepeso são importantes fatores de risco para o desenvolvimento da doença periodontal. O tecido adiposo representa um reservatório de mediadores inflamatórios. A chemerin é uma adipocina secretada pelo tecido adiposo e atua em numerosos processos fisiológicos, como metabolismo, proliferação e diferenciação celular. Levando em consideração que alterações de adiposidade, observadas por medidas antropométricas, estão relacionadas com maior incidência de periodontite, e que chemerin, uma citocina produzida por adipócitos, está envolvida na resposta inflamatória, nota-se uma lacuna na literatura que correlacione o papel de chemerin na progressão da doença periodontal. Portanto, o presente projeto tem como objetivo avaliar a participação da adipocina chemerin no desenvolvimento da doença periodontal em camundongos hiperlipidêmicos e avaliar o efeito desta adipocina sobre a diferenciação e ativação de células ósseas. No estudo in vivo, os animais foram submetidos ao modelo experimental de hiperlipidemia e/ou periodontite e divididos em 4 grupos: Grupo I: camundongos controle. Grupo II: camundongos controle infectados pela Porphyromonas gingivalis (Pg). Grupo III: camundongos hiperlipidêmicos não infectados. Grupo IV: camundongos hiperlipidêmicos infectados pela Pg. As amostras foram coletadas depois de 15, 30 e 60 dias. No estudo in vivo, o modelo de dieta hiperlipidêmica adotado foi eficaz em aumentar os níveis circulantes de colesterol e chemerin, embora não tenha alterado o peso dos animais. Observa-se ainda maior adiposidade corporal mostrada pelo aumento de peso dos tecidos adiposos retroperitoneal e epididimal dos animais hiperlipidêmicos. Na análise morfométrica foi observada uma maior perda óssea no grupo hiperlipidêmico quando comparado ao controle e essa perda óssea foi semelhante ao observado no animal infectado por Pg. Foi observado que a expressão de chemerin na gengiva e plasma se correlacionam com marcadores de osteoclastos no tecido gengival e com a reabsorção alveolar. No estudo in vitro, foi observado que chemerin leva à maior formação de depósitos mineralizados em cultura de osteoblastos e maior reabsorção óssea em cultura de osteoclastos. Conclui-se que a hiperlipidemia provoca reabsorção óssea alveolar semelhante ao observado com infecção oral com P. gingivalis. Chemerin participa da reabsorção óssea alveolar visto que os níveis desta adipocina na gengiva e plasma se correlacionam com marcadores de osteoclastos no tecido gengival e com a reabsorção alveolar. Chemerin aumenta atividade de osteoblastos e osteoclastos in vitro. / Periodontitis is an infectious inflammatory oral disease resultant from the breaking of homeostasis between biofilm and the host. Several studies have shown that overweight and obesity are major risk factors for the development of periodontal disease. Adipose tissue is a reservoir of inflammatory mediators. The chemerin is an adipokine secreted by adipose tissue and acts in numerous physiological processes, such as metabolism, proliferation and differentiation. Considering that adiposity changes, observed by anthropometric measurements, are related to higher incidence of periodontitis, and chemerin, a cytokine produced by adipocytes, is involved in the inflammatory response, there is a gap in the literature that correlates the paper chemerin in the progression of periodontal disease. Therefore, this project aims to evaluate the participation of chemerin adipokine in the development of periodontal disease in hyperlipidemic mice and evaluate the effect of this adipokine on the differentiation and activation of bone cells. In the in vivo study, the animals underwent to the experimental model of hyperlipidemia and / or periodontitis and divided into 4 groups: Group I: control mice. Group II: control mice infected with Porphyromonas gingivalis (Pg). Group III: hyperlipidemic mice not infected. Group IV: hyperlipidemic mice infected by Pg. Samples were collected after 15, 30 and 60 days. In the in vivo study, the model of hyperlipidemic diet adopted was effective in increasing circulating levels of cholesterol and chemerin, although it has not changed the weight of the animals. Greater body adiposity shown by the increased weight of the retroperitoneal and epididymal adipose tissues of hyperlipidemic animals was observed. In morphometric analysis, we observed an increased bone loss in hyperlipidemic group compared to the control and that this bone loss was similar to the observed in animals infected with Pg. It was observed that the gum chemerin expression and plasma correlate with markers of osteoclast in the gingival tissue and alveolar resorption. In the in vitro study, it was observed that chemerin leads to increased formation of mineralized deposits in cultured osteoblasts and increased bone resorption in osteoclast culture. It concludes that hyperlipidemia causes alveolar bone resorption similar to that observed on oral infection with P. gingivalis. Chemerin participates in alveolar bone resorption, at the levels of this adipokine gum and plasma correlate with osteoclast markers in gingival tissue and alveolar resorption. Chemerin increases osteoblast activity and osteoclasts in vitro.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(農学) / 甲第18319号 / 農博第2044号 / 新制||農||1021(附属図書館) / 学位論文||H26||N4826(農学部図書室) / 31177 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 河田 照雄, 教授 伏木 亨, 教授 金本 龍平 / 学位規則第4条第1項該当

The relationship between hyperlipidemia and sensorineural hearing loss remains obscure. In this study, we elucidate for the first time the cochlear morphological and auditory alterations and their relationships with hyperlipidemia, atherosclerosis, and endothelial dysfunction in apolipoprotein-E knockout (ApoE-KO) mice. Ten-week-old ApoE-KO mice were fed either atherosclerotic diet (1.25% cholesterol) or normal diet. Wild type mice (C57BL/6J) served as normal controls. Fourteen weeks later, marked hyperlipidemia, atherosclerosis, endothelial dysfunction, and hearing impairment, especially in the high frequencies, had developed in ApoE-KO mice as compared with C57BL/6J mice (P < 0.001). A high positive correlation between hearing loss and the extent of atherosclerosis and plasma total cholesterol levels was found. Hearing loss, especially at high frequencies, was detected in all ApoE-KO mice. Hair cell loss mainly at the base turn, thickening of vascular intima, and lumen stenosis of the spiral modiolar artery (SMA) in cochlea were also found; these histological changes were exacerbated by the atherosclerotic diet. Furthermore, endothelial nitric oxide synthase (eNOS) in aortic wall and cochlea was distinctly reduced in ApoE-KO mice. These results demonstrate that hyperlipidemia and atherosclerosis can induce alterations in cochlear morphology and function. The stenosis of SMA, which may cause cochlear ischemia and hypoxia, endothelial dysfunction, and low eNOS activity, may contribute to hearing loss.

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A nonsynonymous, loss of function variant (rs58542926, E167K) located in the gene encoding TM6SF2 was identified in multiple genetic association studies as significantly correlating with increased risk for non-alcoholic fatty liver disease (NAFLD) and decreased risk for hyperlipidemia. Given the pivotal role that lipoproteins play at the juncture of these two conditions, researchers hypothesize that the ER-membrane spanning TM6SF2 protein regulates the degree of lipidation of VLDL particles synthesized in the liver. However, not all published data supports this theory and contradictions regarding many aspects of the mechanistic function of TM6SF2 remain. The inconsistencies observed in the literature are in part due to drawbacks of the models used to study TM6SF2 activity; thus, there is an obvious need for an improved hepatocyte model to better understand how TM6SF2 impacts lipid metabolism.To address this need, we present an optimized protocol for the differentiation of inducible pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs), created in collaboration with the Leong Lab. We provide extensive validation of HLC maturity and hepatic functionality, including prolonged albumin secretion, evidence of membrane polarity, and cytochrome P450 induction. We also demonstrate that HLCs express proteins essential for lipoprotein metabolism, secrete authentic VLDL particles, and respond to metabolic perturbations, supporting their value for modeling hepatosteatosis and VLDL metabolism in vitro. To investigate the effect of TM6SF2 variant expression on hepatic lipid metabolism, we produced HLCs derived from 4 homozygous TM6SF2-carrier individuals (KK) and 4 age- and sex-matched unaffected siblings (EE). We describe the variability in differentiation efficiency that we observed in our sibling-matched HLC model and present the gene editing strategy we developed using CRISPR/Cas9 technology and transgene-induced expression to create isogenic iPSCs differing only in their TM6SF2 genotype [EE, KK, or knockout (KO)]. After extensive confirmation of successful gene editing, we explore the effect of TM6SF2 on lipid metabolism in the edited iPSCs. RNA-sequencing and qPCR validation reveal that the Sterol Regulatory Element Binding Protein 2 (SREBP2)-mediated transcriptional program regulating cholesterol synthesis is significantly increased in TM6SF2 KO iPSCs. However, lipidomics analysis and de novo lipogenesis functional assays show that free cholesterol (FC) levels are unchanged. In TM6SF2 KO iPSCs, we further show a reduction in the activities of Acyl-Coenzyme A: Cholesterol Acyltransferase 1 (ACAT1) and Phosphatidylserine Synthase 1 (PSS1), two enzymes that display optimal function when specifically localized to cholesterol enriched ER lipid raft-like domains. Our findings suggest that TM6SF2 may impact cholesterol localization within ER subdomains, which regulate expression levels of cholesterol synthesis genes and activities of ER lipid-raft associated enzymes. In summary, we present here methodological approaches for generating multiple cell culture models in which to investigate the function of TM6SF2, as well as novel evidence supporting a role for TM6SF2 in iPSC cholesterol metabolism.