The long-range goal of this project is to investigate the protective effects of conjugated linoleic acid (CLA) against carcinogenesis. In this dissertation, we demonstrate the mechanisms of CLA action on cell cycle progression and repression of polycyclic aromatic hydrocarbon (PAH)-induced Cyclooxygenase-2 (COX-2) in breast and colon cancer cells. CLA reduced the expression of factors required for G1 to S-phase transition including cyclins D1 and E, and hyperphoshorylated retinoblastoma Rb protein. In contrast, the over-expression of mutant p53 (175Arg to His) in MCF-7 cells prevented the CLA-dependent accumulation of p21 and the reduction of cyclin E levels suggesting that the expression of wild-type p53 is required for CLA-mediated activation of the G1 restriction point. We also report, CLA reduced the expression of COX-2 promoter activity induced by the aromatic hydrocarbon receptor (AhR)-ligand benzo[a]pyrene (B[a]P). Mutagenesis or deletion of potential xenobiotic responsive elements (XREs) within the COX-2 promoter abrogated its ability to be induced by the high affinity AhR-ligand TCDD. In addition, promoter studies using a XRE-dependent CYP1A1 plasmid revealed CLA can inhibit PAH-induced AhR/XRE-driven genes. In both studies, the t10,c12-CLA isomer was more effective than c9,t11-CLA in inhibiting cell proliferation and AhR/XRE-dependent genes. Taken together, these data suggest that the anti-cancerous properties of CLA appear to be a function, at least in part, of the relative content of specific isomers and their 1) ability to elicit a p53 response that leads to the accumulation of pRb and cell growth arrest, and 2) ability to inhibit PAH-induced cyclooxygenase-2 promoter activity through an AhR-dependent mechanism.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2015-12-04T11:30:17Z No. of bitstreams: 1 marianamacedodealmeida.pdf: 4650594 bytes, checksum: 73217ac8cb6474bd22ccc88955919c7c (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2015-12-07T03:19:20Z (GMT) No. of bitstreams: 1 marianamacedodealmeida.pdf: 4650594 bytes, checksum: 73217ac8cb6474bd22ccc88955919c7c (MD5) / Made available in DSpace on 2015-12-07T03:19:20Z (GMT). No. of bitstreams: 1 marianamacedodealmeida.pdf: 4650594 bytes, checksum: 73217ac8cb6474bd22ccc88955919c7c (MD5) Previous issue date: 2015-03-04 / A prevalência de sobrepeso e obesidade está crescendo em taxa alarmante no mundo. Essas condições fornecem maior risco para doenças crônicas, incluindo diabetes tipo 2. Nesse contexto, o ácido linoléico conjugado (CLA) tem atraído considerável atenção, principalmente devido às suas propriedades antiobesidade e antidiabética. O presente estudo objetivou avaliar de forma independente os efeitos do CLA proveniente de fontes natural e sintética sobre o risco de obesidade, sensibilidade à insulina e perfil de lipídeos séricos. Cinquenta ratos Wistar machos foram atribuídos aos seguintes tratamentos dietéticos (n=10/ grupo), por 60 dias: Controle Normolipídico (CN): dieta contendo 4,0% de óleo de soja (OS); Manteiga Controle (MC): dieta contendo 21,7% de manteiga padrão e 2,3% de OS; Manteiga enriquecida com CLA (M-CLA): dieta contendo 21,7% de manteiga enriquecida com CLA cis-9, trans-11 e 2,3% de OS; Controle Hiperlipídico (CH): dieta contendo 24,0% de OS e CLA Sintético (S-CLA): dieta contendo 1,5% de mistura de isômeros de CLA (Luta-CLA 60) e 22,5% de OS. Os animais alimentados com M-CLA tiveram menores níveis de insulina em jejum do que aqueles alimentados com dieta MC, enquando os níveis de PPARγ no tecido adiposo foram aumentados no grupo M-CLA comparado com o grupo MC. Além disso, R-QUICK foi menor nos animais alimentados com MC do que naqueles alimentados com CN, enquanto nenhuma diferença no R-QUICK foi observada entre os grupos CN e M-CLA. Os níveis de colesterol HDL e triglicérides foram maiores nos ratos alimentados com CLA-M. Portanto, a manteiga alto CLA preveniu a hiperinsulinemia, aumentou o colesterol HDL e causou hipertrigliceridemia. A dieta S-CLA não teve efeito sobre a ingestão alimentar e composição alimentar. Os ratos alimentados com S-CLA apresentaram menores níveis da proteína PPARγ no tecido adiposo, hiperinsulinemia, hiperglicemia, índice de HOMA aumentado e maiores níveis de colesterol HDL. Portanto, cautela deve ser tomada antes que suplemetos sintéticos contendo CLA cis-9, trans-11 e CLA trans- 10, cis-12 sejam recomendados. / The prevalence of overweight and obesity is increasing at an alarming rate. These conditions are a major risk for chronic diseases, including type 2 diabetes. In this context, conjugated linoleic acid (CLA) has attracted considerable attention, mainly due to its anti-obesity and anti-diabetic properties. The present study aimed to independently evaluate the effects of CLA from natural and synthetic sources on obesity risk, insulin sensibility and profile of serum lipids. Fifty male Wistar rats were assigned to the following dietary treatments (n=10/group), for 60 days: Normolipidic Control (CN): diet containing 4.0% soybean oil (SO); Control Butter (MC): diet containing 21.7% standard butter and 2.3% SO; and CLA enriched Butter (M-CLA): diet containing 21.7% cis-9, trans-11 CLA-enriched butter and 2.3% SO; Hyperlipidic Control (CH), diet containing 24.0% SO; Synthetic CLA (S-CLA), diet containing 1.5% of an isomeric CLA mixture (Luta-CLA 60) and 22.5% SO. M-CLA-fed rats had lower serum insulin levels at fasting than those fed with the MC diet, while the PPARγ protein levels in adipose tissue was increased in M-CLA-fed rats compared to MCfed rats. Furthermore, R-QUICK was lower in MC than in CN group, while no differences in R-QUICK were observed among CN and M-CLA. Serum HDL cholesterol and triacylglycerol levels were higher in M-CLA-fed rat. Thus, feeding rats on a high-fat diet containing butter naturally enriched in cis-9, trans-11 CLA prevented hyperinsulinemia, increased HDL cholesterol levels and caused hypertriglyceridemia. The S-CLA diet had no effect on dietary intake and body composition. S-CLA-fed rats had lower levels of PPARγ protein in adipose tissue, hyperinsulinemia, hyperglycemia, increased HOMA index and higher levels of serum HDL cholesterol. Therefore, caution should be taken before synthetic supplements containing cis-9, trans-11 and trans-10, cis-12 CLA are recommended.

Approximately 200 million people worldwide, including 25 million in the United States, suffer from osteoporosis. The pathophysiology of osteoporosis suggests that prevention through dietary intervention can be one of the most important actions. Diet can also be a successful alternative for minimizing bone loss and the need for osteoporosis related drug therapy. Conjugated linoleic acid (CLA) is the collective term used to describe positional and geometric isomers of linoleic acid with two conjugated double bonds. CLA has been shown to be biologically active in many systems, yet CLA’s effect on bone mass is not clearly established. The purpose of this research is to investigate CLA’s isomeric effect and synergy with dietary calcium on bone mass. CLA was observed to increase the body ash, representative of bone mass, in male ICR mice after 4-weeks of supplementation. CLA was also administered to mouse bone marrow mesenchymal stem cells during osteoblastic differentiation. CLA increased protein expression related to osteogenesis as well as increasing the calcium concentration of the osteoblastic matrix. In summary, this research provides evidence for the ability of CLA to preserve bone mass.

Four Holstein cows with ruminal and duodenal cannulas were used to evaluate the effects of dietary fatty acylamides (canolamide) or abomasally infused conjugated linoleic acid on milk production and composition. In the first experiment, cows were fed diets with no supplemental fat(control), or the control diet supplemented at 3.3% of DM with canola oil, canolamide, or a mixture of equal amounts of canola oil and canolamide in a 4 x 4 Latin square. DMI and milk yield were decreased when cows were fed canolamide. Intake and duodenal flow of diet components and fatty acids were decreased by canolamide, but their apparent digestibilities were not affected by treatment. Fat supplementation decreased concentrations of fatty acids with 8 to 16 carbons and increased oleic acid in milk. In the second experiment, cows were infused abomasally with 100 g Linoleic acid (LA) or a mixture of 100 g LA plus 100 g conjugated linoleic acid (LA-CLA) for 24 h in a single crossover design. Infused CLA was a mixture of 70% cis-9, trans-11-18:2 and 30% trans-10, cis-12-18:2. Milk yield and DMI were not affected by treatment. Milk fat percentage and yield were decreased by LA-CLA. Concentration and yield of oleic and arachidonic acid and fatty acids with 6 to 16 carbons in milk were reduced by LA-CLA. Stearic acid and CLA concentrations in milk, however, were higher in response to LA-CLA. Infusion of LA-CLA led to increased (from 23 to 45%) concentration of unsaturated fatty acids with a concomitant decrease (from 70 to 42%) in saturated fatty acid concentration in milk fat. Feeding canolamide at 3.3% significantly decreased DMI and milk yield compared with canola oil or the mixture of canola oil and canolamide. However, results indicated that oleic acid and CLA concetration in milk fat can be increased proportionally to their flow into the small intestine. Utilization of these fatty acids for milk fat synthesis may cause a reduction in the amount of medium and short chain fatty acids synthesized de novo within the mammary gland. Furthermore, CLA appears to be a potent inhibitor of milk fat synthesis and desaturation of stearic and linoleic acid. / Master of Science

The key transcription factor sterol regulatory element binding protein-1 (SREBP1) plays a central role in milk fat synthesis. SREBP1 stimulates the transcription of genes encoding lipogenic enzymes. The overall objective of these studies was to investigate the mechanisms of SREBP1 regulation by nutrients. In the first study, chromatin immunoprecipitation (ChIP) accompanied with deep-sequencing was employed to investigate the potential sterol regulatory elements (SRE) in the promoter of SREBP1-target genes. The SRE in three known SREBP1-target genes SREBP1, fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) were first validated in a bovine mammary epithelial cell line (MacT) and in bovine mammary tissues. At least one or two SRE binding sites in 24 selected lipogenic genes were identified within 50,000 base pair to the 5'-transcription start site through ChIP-seq. The genes closest to the highest enriched peaks were involved in cell integrity, defense or signal transduction whereas lipogenic genes were not among the top enrichment leading to the questions about the success of the ChIP. The second study was conducted to determine the effect of t10, c12-conjugated linoleic acid (CLA) on insulin induced gene-1 (Insig1), an endoplasmic reticulum (ER) protein that anchors SREBP1 and prevents proteolytic activation of SREBP1. MacT cells were treated with increasing levels of t10, c12-CLA. High concentration of t10, c12-CLA inhibited Insig1 degradation therefore decreased SREBP1 maturation. Furthermore, immunoprecipitation (IP) confirmed that t10, c12-CLA reduced Insig1 proteasomal degradation by disrupting the interaction between Insig1 and UBX domain-containing protein 8 (Ubxd8), which is part of a degradation complex that removes Insig1 from the ER. In the third study, three potential regulators of SREBP1 activation and their pathways were investigated in insulin, t10, c12-CLA or glucose treated MacT cells. Insulin-induced mammalian target of rapamycin (mTOR) signaling stimulated lipogenesis via activation of SREBP1 and the stimulatory effect was based on the regulation on cAMP response element binding protein coactivator 2 (CRTC2) phosphorylation, Lipin1 translocation and glycogen synthase kinase-3 (GSK3)-dependent proteasomal degradation. t10, c12-CLA inhibited SREBP1 through AMP-activated protein kinase (AMPK) phosphorylation, a key protein kinase in energy homeostasis. Glucose stabilized the SREBP1 chaperone protein SCAP and facilitated SREBP1 activation. Overall, SREBP1 activation is under specific regulation of t10, c12-CLA and interacts with multiple major cellular signaling pathways in response to hormonal stimulation and nutrient availability. / Ph. D.

Foi realizado um experimento com objetivo de avaliar a adição de diferentes níveis de ácido linoleico conjugado (CLA) e vitamina E na dieta de frangos de corte. Foram utilizadas 900 aves, distribuídas em delineamento inteiramente casualizado em esquema fatorial 3x3, sendo 3 níveis de CLA (0,0%, 2,0% e 4,0%) e 3 níveis de vitamina E (0 mg, 250mg e 500 mg), totalizando 9 tratamentos com 4 repetições por tratamento e 25 aves por repetição. Avaliaram-se os parâmetros de desempenho, rendimento de carcaça, densitometria e resistência óssea das tíbias, valores de TBARS, perfil lipídico e colesterol, análise sensorial e maciez objetiva da carne de peito dos animais, abatidos aos 49 dias de idade. O programa de alimentação utilizado foi dividido em 3 fases de criação: inicial (1 - 21 dias), crescimento (22 - 42 dias) e abate (43 - 49 dias), sendo as dietas formuladas a base de milho e farelo de soja. Os resultados foram analisados com o auxílio do procedimento GLM do software estatístico SAS (2003). As médias para análise sensorial e resistência óssea foram comparadas pelo teste de Tukey ao nível de 5% de probabilidade. Os demais parâmetros foram analisados com o procedimento MIXED, e obtidas as respectivas equações de regressão. Em todo o período de criação, foram encontrados efeitos significativos nas interações entre CLA e vitamina E para os parâmetros consumo de ração e ganho de peso. A conversão alimentar não foi influenciada pelas dietas.Para rendimento de carcaça, foi encontrado efeito de CLA para rendimento de asas e gordura abdominal, e interação significativa entre vitamina E e CLA para rendimento de pernas. Não foram verificados efeitos significativos para os parâmetros de resistência óssea e maciez objetiva. Para densitometria óssea, foi observada interação significativa entre os níveis de vitamina E e CLA. Em relação à análise sensorial, a adição de CLA produziu diferenças significativas para sabor, firmeza e suculência. A inclusão de vitamina E gerou efeito sobre firmeza e suculência, não alterando o sabor da carne das aves. Foi encontrado efeito de CLA sobre o perfil de ácidos graxos e colesterol. Os ácidos graxos poliinsaturados aumentaram com a inclusão de CLA à dieta. A concentração de colesterol decaiu a medida do aumento de vitamina E, o mesmo ocorrendo com os níveis de 2 e 4% de CLA. Em relação aos valores de TBARS, foram verificadas interações significativas para CLA e vitamina E, mostrando que a oxidação lipídica é altamente dependente do conteúdo de lipídios presentes da carne, como também da concentração de vitamina E. Como conclusão, a suplementação de CLA e vitamina E influenciou o ganho de peso e consumo, contudo, sem causar efeitos sobre a conversão alimentar, proporcionou aumento na densidade óssea, elevação dos parâmetros de qualidade de carne, atributos sensoriais, influenciando a oxidação lipídica e concentração de colesterol. / A study was conducted with the aim to evaluate the addiction of dietary levels of conjugated linoleic acid and vitamin E for broilers. Nine hundreds broilers chicks were allocated in factorial model 3x3, 9 treatments on total with 4 replications. Parameters of the performance, carcass yield, bone densitometry, bone resistance, TBARS values, fatty acid composition, cholesterol, sensory analysis, tenderness objective analysis of breast meat of chicks 49 days old, were evaluated. The feed program was divided in three phases: initial, grower and finisher. Diets were formulated on corn and soybean meal basis. The obtained data were analyzed by the GLM procedure of the SAS (2003) software package, means for sensory analysis and bone resistance were compared by the test of Tukey at 5% probability level. The other parameters were analyzed with the MIXED procedure and obtained the respective equations. Throughout trial period were found effects for interaction between vitamin E and CLA for feed intake and weight gain. The feed conversion ratio was not influenced for dietary treatments. Were founded CLA effect for wings yield and abdominal fat deposition, and interaction between vitamin E and CLA for legs yield. There were not effects for bone resistance and objective tenderness. For bone densitometry, were obtained interaction between CLA and vitamin E levels. With regard to sensorial analysis the CLA addiction resulted in effects for taste, firmness and juiciness, and vitamin E for firmness and juiciness, but not altering the taste of meat. CLA effect was obtained in fatty acid composition and cholesterol. The polyunsaturated fatty acids increased with the CLA levels. The cholesterol concentration reduced with the increase of the vitamin E level, the same occur with the 2% and 4% CLA levels. About TBARS values, there were interactions between CLA and vitamin E, showing that lipid peroxidation is linked to meat lipid content and vitamin E concentration. In conclusion, the CLA and vitamin E supplementation collaborated with the feed intake and weight gain, although without difference in the feed conversion ratio, increased on bone density, improvement of meat quality parameters, sensory attributes, reduction on lipid peroxidation and cholesterol.

O presente trabalho teve como objetivo estudar os efeitos da suplementação de ácido linoléico conjugado (CLA), associada à redução do nível de proteína bruta (PB) da dieta de frangos de corte. No primeiro ensaio, o período experimental transcorreu dos 21 aos 41 dias, quando 1440 machos da linhagem Ross, com mesmo peso inicial, foram distribuídos em delineamento inteiramente casualizado, em esquema fatorial 3x3, sendo três níveis de CLA (0%, 0,5% e 1,0%) e três níveis de PB (19%, 17%, 15%). Houve redução do ganho de peso e aumento da conversão alimentar apenas em função do decréscimo de PB e ambos os fatores elevaram o índice econômico mensurado. Os rendimentos de carcaça inteira e peito diminuíram proporcionalmente a PB da dieta. Houve interação entre CLA e PB para o tamanho do fígado, ambos os fatores elevaram a gordura abdominal. Em relação aos parâmetros analisados em amostras de peito, o pH elevou-se proporcionalmente, em relação a PB, as intensidades de vermelho e amarelo foram modificadas ou pela PB, ou pelo CLA e para a luminosidade houve interação. Quanto ao perfil de ácidos graxos, a PB elevou somente o ácido esteárico e o CLA aumentou os ácidos graxos saturados e reduziu os monoinsaturados. Houve incorporação crescente dos isômeros de CLA, em função da maior inclusão do produto nas dietas com 19% ou 17% de PB. Nas dietas com 15% de PB, ocorreu um limite máximo para a incorporação do CLA. No segundo experimento, utilizando 144 aves Ross e mesmo delineamento e modelo experimental, houve efeito quadrático da PB para o balanço de nitrogênio e interação entre CLA e PB para a digestibilidade da PB dietária. / The objective of this research was to study the effects of the conjugated linoleic acid, known as CLA, associated with reduction of crude protein (CP) level in broiler diets. At first assay, the period of experiment was conducted from 21 to 41 days, when 1440 male broilers from Ross line, with the same weight initial, were allocated in accordance to a completely randomized design, with factorial model 3x3, corresponding in 3 CLA inclusions levels (0%, 0,5% and 1,0%) and 3 CPs levels (19%,17%,15%). Weight gain reduction and increased feed conversion were only related to decrease of CP inclusion and both of factors, CP and CLA inclusion levels, elevated the economic index parameters. Carcass and breast meat yields decreased due to the reduction of CP addition in diets. The interaction between CLA and CP factors modified in liver size, and both elevated the abdominal fat percents. In relation to parameters analyzed in breast samples, the pH elevated proportionally to CP inclusion, the redness and yellowness were modified by CP or CLA addition and interaction of both were detected to luminosity parameter. About fatty acid composition, the addition of CP only elevated the stearic acid and CLA inclusion improved the saturated fatty acid levels and reduced the monounsaturated fatty acid levels. An improvement on incorporation of CLA isomers was verified due to more inclusion of this product in 19% or 17% CPs diets. In the case of 15% CPs diets, the incorporation of CLA isomers had a maxim limit. In the second assay, using 144 Ross line broilers with the same design and model, a quadratic effect for nitrogen balance was verified due to CP diets and an effect of interaction between CLA and CP inclusion was found to digestibility of dietary CP.

A obesidade é atualmente um dos problemas públicos de saúde mais visível e negligenciado. Ainda, essa condição pode causar sérios problemas de saúde como doenças cardiovasculares, diabetes, hipertensão arterial e diversos problemas fisiológicos. Dietas alimentares e/ou o uso de fármacos tem demonstrado pouca eficiência (e grandes efeitos colaterais) em reduzir a incidência de sobrepeso e obesidade no mundo. Dessa forma outras estratégias são requeridas para auxiliar no controle desta epidemia global. Nesse contexto, alguns ácidos graxos específicos podem desempenhar um importante papel na regulação da expressão de genes que possuem a habilidade de modular metabolismo. O ácido linoléico conjugado (CLA, 18:2) e o ácido oléico (18:1) tem sido descritos com propriedades anti-obesidade: a respeito de CLA, são bem conhecidos os seus efeitos adipotróficos; quanto ao oléico, são bem conhecidos os seus efeitos anti-diabetes; outros efeitos desses ácidos graxos sobre o metabolismo corporal são desconhecidos ou controversos. Dessa forma, o objetivo deste trabalho foi avaliar os efeitos da suplementação da dieta de camundongos (C57bl6) com esses dois ácidos graxos, individualmente ou em conjunto, na modulação do metabolismo corporal e mitocondrial como uma possível estratégia de combate à obesidade. Foram analisados parâmetros bioquímicos, moleculares, fisiológicos, morfológicos e funcionais. Nossos resultados demonstram que o ácido graxo indutor de aumento do gasto energético corporal e mitocondrial hepático é o CLA e não o oléico; quando suplementados em conjunto, os efeitos metabólicos do CLA se sobrepõem aos do oléico, mantendo esses metabolismos elevados. O aumento de metabolismo mitocondrial está relacionado ao aumento de expressão/atividade de proteínas desacopladoras, as quais parecem ser controladas por espécies reativas de O2 mitocondriais. Demonstramos também que no tecido adiposo branco o ácido oléico não exerce efeitos atróficos como o CLA, os quais estão relacionados a inibição da expressão de PPAR1. Por outro lado, verificamos que a suplementação conjunta com ácido oléico previne efeitos adversos da suplementação da dieta com CLA, como a hipertrofia hepática e resistência a insulina. Concluímos dessa forma que a suplementação da dieta com CLA aumenta o metabolismo corporal e mitocondrial; a suplementação com ácido oléico em conjunto não potencializa os efeitos do CLA, porém previne seus efeitos adversos como a hipertrofia hepática e resistência a insulina. Em conjunto esses ácidos graxos constituem uma boa estratégia alimentar contra o ganho de peso corporal. / Obesity is one of the major public health problems. This condition can leads to the development of other complications such as cardiovascular disease, diabetes and hypertension. Diet and/or drug treatments have shown low efficiency (and major side effects) in reducing the incidence of overweight and obesity in worldwide. On the other hand, some specific fatty acids may play an important role regulating the expression of genes that modulate the metabolism. Conjugated linoleic acid (CLA, 18:2) and oleic acid (18:1) have been known for their anti-obesity properties: CLA is well described inducer of atrophy in adipose tissue, while oleic acid is known by its anti-diabetic effects; other effects of these fatty acids on body metabolism are unknown or controversial. Therefore, the aim of this study was to evaluate the effects of a murine diet supplemented with CLA and oleic acid, individually or together, in the modulation of body and mitochondrial metabolism. Biochemical, molecular, physiological, morphological and functional parameters were analyzed in mice. Our results show that CLA, and not oleic acid, is the fatty acid inductor of increase in body metabolism and liver mitochondrial energy expenditure; when supplemented together, the metabolic effects of CLA overlap the oleic effects, maintaining the high metabolism. Increased mitochondrial metabolism is associated with an increased expression/activity of uncoupling proteins, which appear to be controlled by mitochondrial oxygen reactive species. It was also demonstrated that, in white adipose tissue, oleic acid exerts no atrophic effects as compared to CLA, which are related to inhibition of PPAR1 expression. On the other hand, we demonstrated that the supplementation with oleic acid prevents adverse effects of dietary supplementation with CLA, such as liver hypertrophy and insulin resistance in mice. In conclusion, supplementing the murine diet with CLA increases body and mitochondrial metabolism and reduces the adiposity; in addition, supplementation with oleic acid does not potentialize the metabolic effects of CLA, but prevents its adverse effects. Together CLA and oleic in conjunction appears as a good dietary strategy against the excessive weight gain.

A obesidade é atualmente um dos problemas públicos de saúde mais visível e negligenciado. Ainda, essa condição pode causar sérios problemas de saúde como doenças cardiovasculares, diabetes, hipertensão arterial e diversos problemas fisiológicos. Dietas alimentares e/ou o uso de fármacos tem demonstrado pouca eficiência (e grandes efeitos colaterais) em reduzir a incidência de sobrepeso e obesidade no mundo. Dessa forma outras estratégias são requeridas para auxiliar no controle desta epidemia global. Nesse contexto, alguns ácidos graxos específicos podem desempenhar um importante papel na regulação da expressão de genes que possuem a habilidade de modular metabolismo. O ácido linoléico conjugado (CLA, 18:2) e o ácido oléico (18:1) tem sido descritos com propriedades anti-obesidade: a respeito de CLA, são bem conhecidos os seus efeitos adipotróficos; quanto ao oléico, são bem conhecidos os seus efeitos anti-diabetes; outros efeitos desses ácidos graxos sobre o metabolismo corporal são desconhecidos ou controversos. Dessa forma, o objetivo deste trabalho foi avaliar os efeitos da suplementação da dieta de camundongos (C57bl6) com esses dois ácidos graxos, individualmente ou em conjunto, na modulação do metabolismo corporal e mitocondrial como uma possível estratégia de combate à obesidade. Foram analisados parâmetros bioquímicos, moleculares, fisiológicos, morfológicos e funcionais. Nossos resultados demonstram que o ácido graxo indutor de aumento do gasto energético corporal e mitocondrial hepático é o CLA e não o oléico; quando suplementados em conjunto, os efeitos metabólicos do CLA se sobrepõem aos do oléico, mantendo esses metabolismos elevados. O aumento de metabolismo mitocondrial está relacionado ao aumento de expressão/atividade de proteínas desacopladoras, as quais parecem ser controladas por espécies reativas de O2 mitocondriais. Demonstramos também que no tecido adiposo branco o ácido oléico não exerce efeitos atróficos como o CLA, os quais estão relacionados a inibição da expressão de PPAR1. Por outro lado, verificamos que a suplementação conjunta com ácido oléico previne efeitos adversos da suplementação da dieta com CLA, como a hipertrofia hepática e resistência a insulina. Concluímos dessa forma que a suplementação da dieta com CLA aumenta o metabolismo corporal e mitocondrial; a suplementação com ácido oléico em conjunto não potencializa os efeitos do CLA, porém previne seus efeitos adversos como a hipertrofia hepática e resistência a insulina. Em conjunto esses ácidos graxos constituem uma boa estratégia alimentar contra o ganho de peso corporal. / Obesity is one of the major public health problems. This condition can leads to the development of other complications such as cardiovascular disease, diabetes and hypertension. Diet and/or drug treatments have shown low efficiency (and major side effects) in reducing the incidence of overweight and obesity in worldwide. On the other hand, some specific fatty acids may play an important role regulating the expression of genes that modulate the metabolism. Conjugated linoleic acid (CLA, 18:2) and oleic acid (18:1) have been known for their anti-obesity properties: CLA is well described inducer of atrophy in adipose tissue, while oleic acid is known by its anti-diabetic effects; other effects of these fatty acids on body metabolism are unknown or controversial. Therefore, the aim of this study was to evaluate the effects of a murine diet supplemented with CLA and oleic acid, individually or together, in the modulation of body and mitochondrial metabolism. Biochemical, molecular, physiological, morphological and functional parameters were analyzed in mice. Our results show that CLA, and not oleic acid, is the fatty acid inductor of increase in body metabolism and liver mitochondrial energy expenditure; when supplemented together, the metabolic effects of CLA overlap the oleic effects, maintaining the high metabolism. Increased mitochondrial metabolism is associated with an increased expression/activity of uncoupling proteins, which appear to be controlled by mitochondrial oxygen reactive species. It was also demonstrated that, in white adipose tissue, oleic acid exerts no atrophic effects as compared to CLA, which are related to inhibition of PPAR1 expression. On the other hand, we demonstrated that the supplementation with oleic acid prevents adverse effects of dietary supplementation with CLA, such as liver hypertrophy and insulin resistance in mice. In conclusion, supplementing the murine diet with CLA increases body and mitochondrial metabolism and reduces the adiposity; in addition, supplementation with oleic acid does not potentialize the metabolic effects of CLA, but prevents its adverse effects. Together CLA and oleic in conjunction appears as a good dietary strategy against the excessive weight gain.

Made available in DSpace on 2016-12-08T16:24:08Z (GMT). No. of bitstreams: 1 PGCA10MA063.pdf: 806269 bytes, checksum: d98ad588b52836b1be3fce9b0b39e24e (MD5) Previous issue date: 2010-12-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Embryo cryopreservation is an important tool that allows the storage of genetic material for long time, without loss of functional activity or genetic damage. Cryopreservation of in vivo-produced embryos is well established and provides similar results than those obtained after fresh embryos transfer. However, in vitro-produced (IVP) bovine embryos are easily damaged by conventional methods of cryopreservation, resulting in low survival rates after re-warming. Such sensitivity seems to be related to an excessive amount of lipid in the embryos cytoplasm during in vitro development. This might occur due to serum containing medium cultured embryos. Mechanical removal of lipid droplets can improve survival of early developmental stages embryos after cryopreservation. Nevertheless, delipidation method are laborious and time-consuming, besides altering embryo development after transfer to the recipients. Therefore, studies have searched for non invasive and less laborious techniques to reduce the amount of cytoplasmic lipids of bovine IVP embryos, thus improving their cryotolerance. Trans-10, cis-12 CLA, a conjugated isomer of linoleic can inhibit the fatty acid synthesis, thus decreasing the amount of lipid in several human and animal cells. It is known that lipid synthesis reduction involve down-regulation of mRNA expression of lipogenic enzymes associated with fat synthesis. Studies showed that addition of t10, c12 CLA to culture media, can reduce lipid content of bovine IVP embryos, improving their cryotolerance. Therefore, this study evaluated two CLA isomers on cryotolerance of IVP bovine embryos, as well as the enzymes acetyl-CoA carboxylase (ACCa), stearoyl-CoA desaturase (SCD1) and fatty acid synthase (FASN) mRNA expression of these embryos. In Experiment 1, embryos were cultured in media with increasing concentrations of trans-10, cis-12 CLA isomer: control group (0 μM), 50CLA group (50 μM), 100CLA group (100 μM) and 200CLA group (200 μM), looking for the higher concentration that does not negatively affect embryo development. In Experiment 2, zygotes were cultured in medium containing 100 μM (determined in Experiment 1) of different CLA isomers: control (CLA free) group, t10, c12 CLA group, c9, t11 CLA group and Mixture (50% c9, t11 CLA + 50% t10, c12) group. In Experiment 3, zygotes were allocated in 2 groups: CLA group, containing 100 μM of t10, c12 CLA isomer (determined in Experiment 2) and control (CLA free) group. Blastocysts were separated according to their stage (Bx or Bl) and subjected to vitrification or freezing. As viability criteria, cleavage and blastocyst rates were assessed after IVC and reexpanding and hatching rates were assessed after re-warming. Cell counting and mRNA expression of the ACCa, SCD1 and FASN enzymes were also assessed. The highest concentration of t10, c12 CLA that did not impair blastocyst rates was 100μM.In Experiment 2, the highest hatching rate after re-warming was obtained in c9, t11 group, vitrified at Bx stage (68.6%), not differing from the other treatments vitrified at this stage. The lowest hatching rate was obtained in Mixture group, vitrified at Bl stage (8.0%), not differing from the groups t10, c12 and c9, t11, vitrified at the same stage. In all treatments, Bx stage embryos showed higher viability than Bl stage embryos, except for the Control group, in whose the viability was similar. In Experiment 3, the highest hatching rate was obtained in Bx stage Control vitrified group (67.4%), which was similar to the Bx stage vitrified CLA group. The xv lowest hatching rate was observed in Bl stage frozen CLA group (10.3%), not differing from the other cryopreserved treatments at the same stage. In all frozen groups, despite CLA addition, hatching rates were all similar. There was also no difference in cell counting or mRNA expression of the enzymes ACCa, SCD1 and FASN, among embryos.Under the conditions of this study, the addition of CLA isomers t10, c12 and c9, t11 to culture media does not improve cryotolerance of IVP bovine embryos, and the isomer t10, c12 CLA does not influences the cell number and mRNA expression of enzymes ACCa, SCD1 and FASN / A criopreservação de embriões é uma importante ferramenta que permite o armazenamento de material genético por período prolongado, sem causar perda de atividade funcional ou alterações genéticas nessas estruturas. A criopreservação de embriões gerados in vivo está bem estabelecida e proporciona resultados muito próximos aos obtidos após a transferência de embriões frescos. Já os embriões bovinos produzidos in vitro (PIV) são extremamente sensíveis aos métodos convencionais de criopreservação, apresentando reduzidas taxas de sobrevivência após o reaquecimento. Essa sensibilidade parece estar relacionada ao acúmulo excessivo de lipídeos no citoplasma dos embriões durante o desenvolvimento in vitro, principalmente quando o cultivo é realizado em meios adicionados de soro. Estudos realizados com embriões em estágios iniciais de desenvolvimento evidenciaram que a remoção física das gotas lipídicas é capaz de aumentar a tolerância destes embriões à criopreservação. Contudo, o método de delipidação é demasiadamente trabalhoso e demorado, além de alterar o potencial de desenvolvimento dos embriões após a transferência para as receptoras. Dessa forma, os estudos têm buscado métodos não invasivos e menos trabalhosos, que reduzam a quantidade de lipídeos no citoplasma dos embriões bovinos PIV, melhorando assim a sua criotolerância. O trans-10, cis-12 CLA, um isômero conjugado do ácido linoleico, é capaz de inibir a síntese de ácidos graxos, diminuindo o teor lipídico em diversos tecidos de animais e humanos. Sabe-se que um dos mecanismos pelos quais este isômero exerce esse efeito é através da diminuição da expressão de RNAm de enzimas responsáveis pela síntese de lipídeos. Estudos mostraram que a adição do t10, c12 CLA ao meio de cultivo pode reduzir o acúmulo lipídico de embriões bovinos PIV, aumentando a sua criotolerância. Assim, este estudo teve como objetivo avaliar o efeito dos dois principais isômeros do CLA na criotolerância de embriões bovinos PIV e adicionalmente a expressão de RNAm das enzimas acetil-CoA carboxilase (ACCa), estearoil-CoA dessaturase (SCD1) e ácido graxo sintase (FASN) desses embriões. No Experimento 1, embriões foram cultivados em meio com concentrações crescentes do isômero t10, c12 CLA: grupo controle (0 μM), grupo 50CLA (50 μM), grupo 100CLA (100 μM) e grupo 200CLA (200 μM), buscando a maior concentração que não afete o posterior desenvolvimento embrionário. No Experimento 2, os zigotos foram cultivados em meio com 100 μM, (concentração apontada no Experimento 1) de diferentes isômeros de CLA, sendo: grupo controle, sem adição de CLA, grupo t10, c12 CLA, grupo c9, t11 CLA e grupo Mistura, com 50% de cada isômero. No Experimento 3, os zigotos foram distribuídos em 2 grupos: grupo CLA, contendo 100 μM do isômero t10, c12 CLA (determinado no Experimento 2) e grupo controle, sem CLA. Os blastocistos obtidos foram separados em função do estágio (Bx ou Bl) e submetidos ao processo de vitrificação ou congelamento. Como critérios de viabilidade foram avaliados as taxas de clivagem e de blastocistos após o cultivo, e de re-expansão e eclosão após o reaquecimento. Ainda, foi determinada a densidade celular e a expressão de RNAm das enzimas ACCa, SCD1 e FASN destes embriões. A maior concentração de t10, c12 CLA que não reduziu a taxa de blastocistos foi a de 100μM. No Experimento 2, a maior taxa de eclosão após o reaquecimento foi a do grupo c9, t11 vitrificado no estágio de Bx xiii (68,6%), que não diferiu dos demais tratamentos vitrificados neste estágio. A menor taxa de eclosão foi a do grupo Mistura (c9, t11 + t10, c12, no estágio de Bl (8,0%), que não diferiu dos grupos t10, c12 e c9, t11, vitrificados no mesmo estágio. Em todos os tratamentos, embriões em estágio de Bx apresentaram taxas superiores aos Bl, com exceção do grupo Controle, cujas taxas foram similares. No Experimento 3, a maior taxa de eclosão foi observada no grupo Controle com Bx vitrificados (67,4%), que não diferiu do grupo CLA, com Bx vitrificados. A menor taxa de eclosão foi a do grupo CLA com Bl congelados (10,3%), que não diferiu dos demais tratamentos criopreservados neste estágio. Nos grupos submetidos ao congelamento, as taxas de desenvolvimento foram semelhantes, independente da exposição ao CLA. Não houve diferença na densidade celular entre os embriões expostos ou não ao t10, c12 CLA, e nem na expressão de RNAm das enzimas ACCa, SCD1 e FASN. Conclui-se que nas condições deste estudo a adição dos isômeros do CLA t10, c12 e c9, t11 ao meio de cultivo não melhora a criotolerância de embriões bovinos PIV, bem como que o isômero t10, c12 CLA, não interfere na densidade celular e na expressão de RNAm das enzimas ACCa, SCD1 e FASN destes embriões