Aquaporins in adipocytes:insights into the pathophysiology of human obesity

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2014

Biological membranes : biophysical properties and aquaporin function

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2014

Effect of fatty acids on hyphal growth in the pathogenic yeast Candida albicans

Candida albicans est une levure pathogène qui, à l’état commensal, colonise les muqueuses de la cavité orale et du tractus gastro-intestinal. De nature opportuniste, C. albicans cause de nombreuses infections, allant des candidoses superficielles (muguet buccal, vulvo-vaginite) aux candidoses systémiques sévères. C. albicans a la capacité de se développer sous diverses morphologies, telles que les formes levures, pseudohyphes et hyphes. Des stimuli environnementaux mimant les conditions retrouvées chez l’hôte (température de 37°C, pH neutre, présence de sérum) induisent la transition levure-à-hyphe (i.e. morphogenèse ou filamentation). Cette transition morphologique contribue à la pathogénicité de C. albicans, du fait que des souches présentant un défaut de filamentation sont avirulentes. Non seulement la morphogenèse est un facteur de virulence, mais elle constituerait aussi une cible pour le développement d’antifongiques. En effet, il a déjà été démontré que l’inhibition de la transition levure-à-hyphe atténuait la virulence de C. albicans lors d’infections systémiques. Par ailleurs, des études ont démontré que de nombreuses molécules pouvaient moduler la morphogenèse. Parmi ces molécules, certains acides gras, dont l’acide linoléique conjugué (CLA), inhibent la formation d’hyphes. Ainsi, le CLA posséderait des propriétés thérapeutiques, du fait qu’il interfère avec un déterminant de pathogénicité de C. albicans. Par contre, avant d’évaluer son potentiel thérapeutique dans un contexte clinique, il est essentiel d’étudier son mode d’action. Ce projet vise à caractériser l’activité anti-filamentation des acides gras et du CLA et à déterminer le mécanisme par lequel ces molécules inhibent la morphogenèse chez C. albicans. Des analyses transcriptomiques globales ont été effectuées afin d’obtenir le profil transcriptionnel de la réponse de C. albicans au CLA. L’acide gras a entraîné une baisse des niveaux d’expression de gènes encodant des protéines hyphes-spécifiques et des régulateurs de morphogenèse, dont RAS1. Ce gène code pour la GTPase Ras1p, une protéine membranaire de signalisation qui joue un rôle important dans la transition levure-à-hyphe. Des analyses de PCR quantitatif ont confirmé que le CLA inhibait l’induction de RAS1. De plus, le CLA a non seulement causé une baisse des niveaux cellulaires de Ras1p, mais a aussi entraîné sa délocalisation de la membrane plasmique. En affectant les niveaux et la localisation cellulaire de Ras1p, le CLA nuit à l’activation de la voie de signalisation Ras1p-dépendante, inhibant ainsi la morphogenèse. Il est possible que le CLA altère la structure de la membrane plasmique et affecte indirectement la localisation membranaire de Ras1p. Ces travaux ont permis de mettre en évidence le mode d’action du CLA. Le potentiel thérapeutique du CLA pourrait maintenant être évalué dans un contexte d’infection, permettant ainsi de vérifier qu’une telle approche constitue véritablement une stratégie pour le traitement des candidoses.

Quantification of ruminal Clostridium proteoclasticum by real-time PCR using a molecular beacon approach

Aims: All members of the ruminal Butyrivibrio group convert linoleic acid (cis-9,cis-12-18 : 2) via conjugated 18 : 2 metabolites (mainly cis-9,trans-11-18 : 2, conjugated linoleic acid) to vaccenic acid (trans-11-18 : 1), but only members of a small branch, which includes Clostridium proteoclasticum, of this heterogeneous group further reduce vaccenic acid to stearic acid (18 : 0, SA). The aims of this study were to develop a real-time polymerase chain reaction (PCR) assay that would detect and quantify these key SA producers and to use this method to detect diet-associated changes in their populations in ruminal digesta of lactating cows. Materials and Results: The use of primers targeting the 16S rRNA gene of Cl. proteoclasticum was not sufficiently specific when only binding dyes were used for detection in real-time PCR. Their sequences were too similar to some nonproducing strains. A molecular beacon probe was designed specifically to detect and quantify the 16S rRNA genes of the Cl. proteoclasticum subgroup. The probe was characterized by its melting curve and validated using five SA-producing and ten nonproducing Butyrivibrio-like strains and 13 other common ruminal bacteria. Analysis of ruminal digesta collected from dairy cows fed different proportions of starch and fibre indicated a Cl. proteoclasticum population of 2-9% of the eubacterial community. The influence of diet on numbers of these bacteria was less than variations between individual cows. Conclusion: A molecular beacon approach in qPCR enables the detection of Cl. proteoclasticum in ruminal digesta. Their numbers are highly variable between individual animals. Signifance and Impact of the Study: SA producers are fundamental to the flow of polyunsaturated fatty acid and vaccenic acid from the rumen. The method described here enabled preliminary information to be obtained about the size of this population. Further application of the method to digesta samples from cows fed diets of more variable composition should enable us to understand how to control these bacteria in order to enhance the nutritional characteristics of ruminant-derived foods, including milk and beef.

Improving the nutritional quality of milk

Epidemiological evidence suggests high milk consumption protects against certain chronic diseases, and yet the high saturated fatty acid (SFA) concentration of milk fat has lead to research into reducing SFA and trans- fatty acid content, and enhancing conjugated linoleic acid (CLA) content. The most successful method of reducing SFA content is by feeding dairy cows supplemental oilseeds, although amount, type and form of oilseed influence the degree of effect. Also, the high unsaturated fatty acid content of oilseeds leads to increases in milk fat trans- fatty acids unless the oilseed is protected from rumen metabolism.

Dietary fatty acids and human health

A considerable amount of evidence has accumulated to support the view that the very long chain omega 3 fatty acids (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) have beneficial cardiovascular and anti-inflammatory properties and that levels of their consumption are insufficient in most Western diets. More recently, attention has been given to the possibility that the precursor omega-3 PUFA, alpha linolenic acid (ALNA), may share some of the beneficial actions of EPA/DHA on human health. Further research into the metabolism and physiological actions of ALNA, and comparisons with EPA/DHA, is needed before conclusions regarding the optimal amounts and types of omega-3 PUFA for human health can be defined. Conjugated linoleic acid (CLA), which arises as a metabolic by-product of rumen hydrogenation and which is found in foods of animal origin, has been proposed to possess potent health promoting properties, but much of this research has been conducted in experimental animals. There is an urgent need for complementary studies in human volunteers, to confirm the putative anti-carcinogenic, anti-atherogenic, anti-lipogenic and immuno-suppressive properties of CLA.

Meat quality and health implications of organic and conventional beef production

Recommendation to reduce fat consumption from ruminant meat does not consider the contribution of nutritionally beneficial fatty acids in lean beef. Here we report effects of production system (organic vs conventional) and finishing season on meat and fat quality of sirloin steaks from retail outlets and simulated fatty acid intakes by consumers. There was little difference in meat quality (pH, shear force and colour), but the fat profiles varied considerably between production systems and season. Meat fat from organic and summer finished cattle contained higher concentrations of conjugated linoleic acid, its precursor vaccenic acid and individual omega-3 fatty acids and had a lower ratio of omega-6 to omega-3 fatty acids compared with non-organic and winter finished cattle respectively. The fat profile from summer finished organic beef aligns better to recommended dietary guideline including those for long chain omega-3 fatty acids compared with that from winter finished, non-organic steak.

Effect of feeding intensity and milking system on nutritionally relevant milk components in dairy farming systems in the North East of England

There is increasing concern that the intensification of dairy production reduces the concentrations of nutritionally desirable compounds in milk. This study therefore compared important quality parameters (protein and fatty acid profiles; α-tocopherol and carotenoid concentrations) in milk from four dairy systems with contrasting production intensities (in terms of feeding regimens and milking systems). The concentrations of several nutritionally desirable compounds (β-lactoglobulin, omega-3 fatty acids, omega-3/omega-6 ratio, conjugated linoleic acid c9t11, and/or carotenoids) decreased with increasing feeding intensity (organic outdoor ≥ conventional outdoor ≥ conventional indoors). Milking system intensification (use of robotic milking parlors) had a more limited effect on milk composition, but increased mastitis incidence. Multivariate analyses indicated that differences in milk quality were mainly linked to contrasting feeding regimens and that milking system and breed choice also contributed to differences in milk composition between production systems.

Fat composition of organic and conventional retail milk in northeast England

This study of UK retail milk identified highly significant variations in fat composition. The survey, conducted over 2 yr replicating summer and winter, sampled 22 brands, 10 of which indicated organic production systems. Results corroborate earlier farm-based findings considering fat composition of milk produced under conventional and organic management. Organic milk had higher concentrations of beneficial fatty acids (FA) than conventional milk, including total polyunsaturated fatty acids (PUFA; 39.4 vs. 31.8 g/kg of total FA), conjugated linoleic acid cis-9,trans-11 (CLA9; 7.4 v 5.6 g/kg of FA), and α-linolenic acid (α-LN; 6.9 vs. 4.4 g/kg of FA). As expected, purchase season had a strong effect on fat composition: compared with milk purchased in winter, summer milk had a lower concentration of saturated fatty acids (682 vs. 725 g/kg of FA) and higher concentrations of PUFA (37.6 vs. 32.8 g/kg of FA), CLA9 (8.1 vs. 4.7 g/kg of FA), and α-LN (6.5 vs. 4.6 g/kg of FA). Differences identified between sampling years were more surprising: compared with that in yr 2, milk purchased in year 1 had higher concentrations of PUFA (37.5 vs. 32.9 g/kg of FA), α-LN (6.0 vs. 5.1 g/kg of FA), and linoleic acid (19.9 vs. 17.5 g/kg of FA) and lower concentrations of C16:0 and C14:0 (332 vs. 357 and 110 vs. 118 g/kg of FA, respectively). Strong interactions were identified between management and season as well as between season and year of the study. As in the earlier farm studies, differences in fat composition between systems were greater for summer compared with winter milk. Large between-year differences may be due to changes in weather influencing milk composition through forage availability, quality, and intake. If climate change predictions materialize, both forage and dairy management may have to adapt to maintain current milk quality. Considerable variation existed in milk fat composition between brands.

The effects of dairy management and processing on quality characteristics of milk and dairy products

Studies within the QLIF project reviewed in this article suggest that organic or low-input management is more likely to result in milk with fatty acid profiles that are higher in α-linolenic acid and/or beneficial isomers of conjugated linoleic acid and antioxidants with up to a 2.5-fold increase in some cases, relative to milk from conventional production. These advantages are preserved during processing, resulting in elevated contents or concentrations of these constituents in processed dairy products of organic or low input origin. Much of the literature suggests that these benefits are very likely to be a result of a greater reliance on forages in the dairy diets (especially grazed grass). Since the adoption of alternative breeds or crosses is often an integral part sustaining these low-input systems, it is not possible to rule out an interaction with genotype in these monitored herds. The results suggest that milk fat composition with respect to human health can be optimized by exploiting grazing in the diet of dairy cows. However, in many European regions this may not be possible due to extremes in temperature, soil moisture levels or both. In such cases milk quality can be maintained by the inclusion of oil seeds in the dairy diets.

Higher PUFA and omega-3 PUFA, CLA, α-tocopherol and iron, but lower iodine and selenium concentrations in organic milk: a systematic literature review and meta- and redundancy analyses

Demand for organic milk is partially driven by consumer perceptions that it is more nutritious. However, there is still considerable uncertainty over whether the use of organic production standards affects milk quality. Here we report results of meta-analyses based on 170 published studies comparing the nutrient content of organic and conventional bovine milk. There were no significant differences in total SFA and MUFA concentrations between organic and conventional milk. However, concentrations of total PUFA and n-3 PUFA were significantly higher in organic milk, by an estimated 7 (95 % CI −1, 15) % and 56 (95 % CI 38, 74) %, respectively. Concentrations of α-linolenic acid (ALA), very long-chain n-3 fatty acids (EPA+DPA+DHA) and conjugated linoleic acid were also significantly higher in organic milk, by an 69 (95 % CI 53, 84) %, 57 (95 % CI 27, 87) % and 41 (95 % CI 14, 68) %, respectively. As there were no significant differences in total n-6 PUFA and linoleic acid (LA) concentrations, the n-6:n-3 and LA:ALA ratios were lower in organic milk, by an estimated 71 (95 % CI −122, −20) % and 93 (95 % CI −116, −70) %. It is concluded that organic bovine milk has a more desirable fatty acid composition than conventional milk. Meta-analyses also showed that organic milk has significantly higher α-tocopherol and Fe, but lower I and Se concentrations. Redundancy analysis of data from a large cross-European milk quality survey indicates that the higher grazing/conserved forage intakes in organic systems were the main reason for milk composition differences.

Composição química e perfil de ácidos graxos da carne de bovinos de diferentes condições sexuais recebendo silagem de milho e concentrado ou cana-de-açúcar e concentrado contendo grãos de girassol

O objetivo neste trabalho foi avaliar as características químicas e a composição em ácidos graxos do contrafilé (músculo Longissimus) de tourinhos, novilhos e novilhas da raça Canchim. Os animais foram terminados em confinamento com duas dietas experimentais, uma com silagem de milho e concentrado e outra com cana-de-açúcar e concentrado contendo grãos de girassol. Os teores de umidade, proteína e minerais no músculo não diferiram entre as dietas e as condições sexuais dos animais. A carne de novilhos terminados com a dieta com grãos de girassol apresentou maior teor de lipídios (3,31%) em comparação à dos animais de outras categorias. Os animais que receberam a dieta com grãos de girassol apresentaram maiores concentrações de ácido linoléico conjugado (18:2 cis9, trans-11) (0,73%) e ácidos graxos poliinsaturados (8,12%) no músculo, e também relações mais elevadas de ácidos graxos insaturados:saturados (0,93) e ácidos graxos poliinsaturados:saturados (0,16) em comparação àqueles que receberam a dieta convencional, à base de silagem de milho (0,34%; 6,31%; 0,86; e 0,11, respectivamente). A composição em ácidos graxos da carne de bovinos pode ser melhorada com a utilização de cana-de-açúcar e grãos de girassol na dieta dos animais terminados em confinamento.

Composição em ácidos graxos e qualidade da carne de tourinhos Nelore e Canchim alimentados com dietas à base de cana-de-açúcar e dois níveis de concentrado

O objetivo neste trabalho foi avaliar a composição de ácidos graxos e a qualidade do contrafilé (músculo Longissimus lumborum) de tourinhos das raças Nelore e Canchim. Os animais foram terminados em confinamento e alimentados com dietas contendo cana-de-açúcar e dois níveis de concentrado (40 e 60% na matéria seca). Os concentrados foram compostos de grãos de girassol, milho, farelo de soja, levedura seca de cana-de-açúcar, uréia e núcleo mineral. O delineamento experimental foi o inteiramente ao acaso, em esquema fatorial 2 × 2 (grupo genético × nível de concentrado). Não foram observadas diferenças nos teores de umidade, proteína e extrato etéreo da carne. Os animais da raça Nelore apresentaram maiores concentrações de ácido linoléico conjugado (0,52%), ácidos graxos insaturados (46,82%) e também relações mais elevadas de ácidos graxos insaturados:saturados (1,02) e monoinsaturados:saturados (0,86) em comparação aos tourinhos da raça Canchim. Os tourinhos da raça Canchim apresentaram maior intensidade das cores vermelha e amarela no contrafilé e maior luminosidade da gordura de cobertura. Houve interação para força de cisalhamento, que foi menor nos tourinhos Nelore alimentados com 40% de concentrado. Tourinhos da raça Nelore apresentam carne com melhor composição de ácidos graxos na gordura intramuscular do ponto de vista da saúde humana.

Efeito da suplementação com ácido linoléico conjugado e do treinamento em natação sobre a composição corporal e os parâmetros bioquímicos de ratos Wistar em crescimento

OBJETIVO: Avaliar os efeitos da suplementação com ácido linoléico conjugado, associada ao treinamento moderado em natação, sobre a composição corporal, o consumo e a eficiência alimentar, a glicemia, o perfil lipídico e o glicogênio muscular e hepático de ratos Wistar. MÉTODOS: Ratos Wistar (30 dias) foram divididos em: sedentário, sedentário suplementado, treinado e treinado suplementado. Permaneceram em gaiolas individuais com comida e água ad libitum, temperatura de 23ºC (com variação de1ºC) e ciclo claro-escuro de 12 horas, durante 8 semanas. A sessão de natação durou 1 hora e foi realizada três vezes/semana, bem como a suplementação com ácido linoléico conjugado a 2%. Após sacrifício, o plasma, os tecidos adiposos brancos e o marrom, o músculo gastrocnêmio e o fígado foram coletados e pesados. RESULTADOS: A suplementação per se não promoveu modificação na ingestão alimentar e na massa corporal dos animais. Houve aumento na glicemia de jejum (p<0,05), nas lipoproteínas de alta densidade (p<0,05), no colesterol total (p<0,05) e redução dos triacilgliceróis. A suplementação associada ao treinamento reduziu a massa corporal (p<0,05) e aumentou o peso relativo do tecido adiposo, do fígado e a glicemia de jejum. CONCLUSÃO: A suplementação com ácido linoléico conjugado associada à prática de exercício físico parece ter influência no balanço energético, mas, por outro lado, o aumento no peso do fígado indica que a ingestão deste ácido graxo pode ter efeitos indesejáveis, aumentando as chances de desenvolvimento do fígado gorduroso. Estes achados apontam perspectivas para novos estudos envolvendo análises histológicas do fígado, expressão gênica de enzimas chaves do metabolismo lipídico e de carboidratos, associados ou não a diferentes protocolos de treinamento físico.

Meat characteristics of Nellore steers fed whole cottonseed

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)