Browsing by Author "Moura, Teresa F."
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- Conjugated linoleic acid reduces permeability and fluidity of adipose plasma membranes from obese Zucker ratsPublication . Martins, Ana Paula; Lopes, Paula A.; Martins, Susana V.; Madeira, Ana; Santos, Nuno C.; Prates, José A. M.; Moura, Teresa F.; Soveral, GraçaConjugated linoleic acid (CLA) is a dietary fatty acid frequently used as a body fat reducing agent whose effects upon cell membranes and cellular function remain unknown. Obese Zucker rats were fed atherogenic diets containing saturated fats of vegetable or animal origin with or without 1% CLA, as a mixture of cis(c)9,trans(t)11 and t10,c12 isomers. Plasma membrane vesicles obtained from visceral adi- pose tissue were used to assess the effectiveness of dietary fat and CLA membrane incorporation and its outcome on fluidity and permeability to water and glycerol. A significant decrease in adipose membrane fluidity was correlated with the changes observed in permeability, which seem to be caused by the incor- poration of the t10,c12 CLA isomer into membrane phospholipids. These results indicate that CLA supple- mentation in obese Zucker rats fed saturated and cholesterol rich diets reduces the fluidity and permeability of adipose membranes, therefore not supporting CLA as a body fat reducing agent through membrane fluidification in obese fat consumers.
- Conjugated linoleic acid reduces permeability and fluidity of adipose plasma membranes from obese Zucker ratsPublication . Martins, Ana P.; Lopes de Almeida, Paula A.; Martins, Susana V.; Madeira, Ana; Santos, Nuno C.; Prates, José A. M.; Moura, Teresa F.; Soveral, GraçaConjugated linoleic acid (CLA) is a dietary fatty acid frequently used as a body fat reducing agent whose effects upon cell membranes and cellular function remain unknown. Obese Zucker rats were fed atherogenic diets containing saturated fats of vegetable or animal origin with or without 1% CLA, as a mixture of cis(c)9,trans(t)11 and t10,c12 isomers. Plasma membrane vesicles obtained from visceral adipose tissue were used to assess the effectiveness of dietary fat and CLA membrane incorporation and its outcome on fluidity and permeability to water and glycerol. A significant decrease in adipose membrane fluidity was correlated with the changes observed in permeability, which seem to be caused by the incorporation of the t10,c12 CLA isomer into membrane phospholipids. These results indicate that CLA supplementation in obese Zucker rats fed saturated and cholesterol rich diets reduces the fluidity and permeability of adipose membranes, therefore not supporting CLA as a body fat reducing agent through membrane fluidification in obese fat consumers.
- Differences in lipid deposition and adipose membrane biophysical properties from lean and obese pigs under dietary protein restrictionPublication . Martins, Ana P.; Lopes, Paula A.; Madeira, Marta S.; Martins, Susana V.; Santos, Nuno C.; Moura, Teresa F.; Prates, José A. M.; Soveral, GraçaObesity consists in fat accumulation leading to increase in adipose cells number and size. Adipocyte membrane biophysical properties are critical to maintain cellular viability in metabolically healthy obesity. This study investigated the effect of the genetic background and dietary protein restriction on fat tissue lipid composition, adipocyte membrane fluidity and water permeability using the pig as experimental model. Twenty-four male pigs from distinct genotypes, lean and obese, were fed on normal and reduced protein diets within a 2 x 2 factorial arrangement (two genotypes and two diets). Backfat thickness was twofold higher in obese than in lean pigs but unrelated to dietary protein level. In contrast, total fatty acids in the subcutaneous adipose tissue were dependent on both breed and diet, with increased lipid content promoted by the fatty genotype and by the restriction of dietary protein. Adipose membranes isolated from obese pig’s subcutaneous fat tissue showed higher permeability to water, in line with an increased fluidity. Moreover, the reduced content of dietary protein influenced positively the fluidity of adipose membranes. Neither genotype nor diet affected total cholesterol concentration in the adipose membranes. Membrane-saturated fatty acids’ content was influenced by genotype, while membrane-polyunsaturated fatty acids, particularly from the n-6 family, was influenced by diet. The ratio of oleic (18:1c9)/linoleic (18:2n-6) acids was positively correlated with membrane fluidity. All together, these findings reinforce the genetic background as a determinant player on adipose membrane biophysical properties and point to the dietary protein level as an important factor for subcutaneous lipid deposition as well as for regulation of membrane function, factors that may have impact on human obesity and metabolic syndrome.
- Differential mesenteric fat deposition in bovines fed on silage or concentrate is independent of glycerol membrane permeabilityPublication . Martins, Ana Paula; Lopes, Paula A.; Costa, Ana Sofia H.; Martins, Susana V.; Santos, Nuno C.; Prates, José A. M.; Moura, Teresa F.; Soveral, GraçaIn the meat industry, the manipulation of fat deposition in cattle is of pivotal importance to improve production efficiency, carcass composition and ultimately meat quality. There is an increasing interest in the identification of key factors and molecular mechanisms responsible for the development of specific fat depots. This study aimed at elucidating the influence of breed and diet on adipose tissue membrane permeability and fluidity and their interplay on fat deposition in bovines. Two Portuguese autochthonous breeds, Alentejana and Barrosã, recognized as late and early maturing breeds, respectively, were chosen to examine the effects of breed and diet on fat deposition and on adipose membrane composition and permeability. Twenty-four male bovines from these breeds were fed on silage- or concentrate-based diets for 11 months. Animals were slaughtered to determine their live slaughter and hot carcass weights, as well as weights of subcutaneous and visceral adipose depots. Mesenteric fat depots were excised and used to isolate adipocyte membrane vesicles where cholesterol content, fatty acid profile as well as permeability and fluidity were determined. Total accumulation of neither subcutaneous nor visceral fat was influenced by breed. In contrast, mesenteric and omental fat depots weights were higher in concentrate-fed bulls relative to silage-fed animals. Membrane fluidity and permeability to water and glycerol in mesenteric adipose tissue were found to be independent of breed and diet. Moreover, the deposition of cholesterol and unsaturated fatty acids, which may influence membrane properties, were unchanged among experimental groups. Adipose membrane lipids from the mesenteric fat depot of ruminants were rich in saturated fatty acids, and unaffected by polyunsaturated fatty acids (PUFA) dietary levels. Our results provide evidence against the involvement of cellular membrane permeability to glycerol on fat accumulation in mesenteric fat tissue of concentrate-fed bovines, which is consistent with the unchanged membrane lipid profile found among experimental groups.
- Effect of dietary conjugated linoleic acid isomers on water and glycerol permeability of kidney membranesPublication . Soveral, Graca; Martins, Ana P.; Martins, Susana V.; Lopes, Paula A.; Alfaia, Cristina M.; Prates, Jose A. M.; Moura, Teresa F.Conjugated linoleic acid (CLA) refers to a group of positional and geometrical isomers of linoleic acid in which the double bonds are conjugated. Dietary CLA has been associated with various health benefits although details of its molecular mode of action remain elusive. The effect of CLA supplemented to palm oil-based diets in Wistar Fats, as a mixture of both or isolated c9,t11 and t11,c12 isomers, was examined on water and glycerol membrane permeability of kidney proximal tubule. Although water permeability was unaltered, an increase in glycerol permeability was obtained for the group supplemented with CLA mixture, even though the activation energy for glycerol permeation remained high. This effect was correlated with an increased CLA isomeric membrane incorporation for the same dietary group. These results suggest that diet supplementation with CLA mixture, in contrast to its individual isomers, may enhance membrane fluidity subsequently raising kidney glycerol reabsorption. (C) 2009 Elsevier Inc. All rights reserved.. - FCT [POCT1/CVT/2002/44750, PTDC/CVT/2006/66114, SFRH/BD/2005/22566, SFRH/BPD/2005/23931]. - This study was supported by FCT Grants POCT1/CVT/2002/44750 and PTDC/CVT/2006/66114 and individual FCT fellowships to Susana Martins (SFRH/BD/2005/22566) and Paula Alexandra Lopes (SFRH/BPD/2005/23931). The authors acknowledge Ana Madeira for technical support. We also thank Susana Alves and Rui Bessa (Unidade de Proclucao Animal, L-INIA-REQUIMTE) for providing fatty acid profile analyses.
- Effect of dietary conjugated linoleic acid isomers on water and glycerol permeability of kidney membranesPublication . Soveral, Graça; Martins, Ana Paula; Martins, Susana V.; Lopes, Paula A.; Alfaia, Cristina M.; Prates, José A. M.; Moura, Teresa F.Conjugated linoleic acid (CLA) refers to a group of positional and geometrical isomers of linoleic acid in which the double bonds are conjugated. Dietary CLA has been associated with various health benefits although details of its molecular mode of action remain elusive. The effect of CLA supplemented to palm oil-based diets in Wistar rats, as a mixture of both or isolated c9,t11 and t10,c12 isomers, was examined on water and glycerol membrane permeability of kidney proximal tubule. Although water permeability was unaltered, an increase in glycerol permeability was obtained for the group supplemented with CLA mixture, even though the activation energy for glycerol permeation remained high. This effect was correlated with an increased CLA isomeric membrane incorporation for the same dietary group. These results suggest that diet supplementation with CLA mixture, in contrast to its individual isomers, may enhance membrane fluidity subsequently raising kidney glycerol reabsorption.
- Exploring the three PIPs and three TIPs of grapevine for transport of water and atypical substrates through heterologous expression in aqy-null yeastPublication . Sabir, Farzana; Leandro, Maria José; Martins, Ana Paula; Loureiro-Dias, Maria; Moura, Teresa F.; Soveral, Graça; Prista, CatarinaAquaporins are membrane channels that facilitate the transport of water and other small molecules across the cellular membranes. We examined the role of six aquaporins of Vitis vinifera (cv. Touriga nacional) in the transport of water and atypical substrates (other than water) in an aqy-null strain of Saccharomyces cerevisiae. Their functional characterization for water transport was performed by stopped-flow fluorescence spectroscopy. The evaluation of permeability coefficients (Pf) and activation energies (Ea) revealed that three aquaporins (VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2) are functional for water transport, while the other three (VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1) are non-functional. TIPs (VvTnTIP1;1 and VvTnTIP2;2) exhibited higher water permeability than VvTnPIP2;1. All functional aquaporins were found to be sensitive to HgCl2, since their water conductivity was reduced (24–38%) by the addition of 0.5 mM HgCl2. Expression of Vitis aquaporins caused different sensitive phenotypes to yeast strains when grown under hyperosmotic stress generated by KCl or sorbitol. Our results also indicate that Vitis aquaporins are putative transporters of other small molecules of physiological importance. Their sequence analyses revealed the presence of signature sequences for transport of ammonia, boron, CO2, H2O2 and urea. The phenotypic growth variations of yeast cells showed that heterologous expression of Vitis aquaporins increased susceptibility to externally applied boron and H2O2, suggesting the contribution of Vitis aquaporins in the transport of these species
- Grapevine aquaporins: gating of a tonoplast intrinsic protein (TIP2; 1) by cytosolic pHPublication . Leitão, Luís; Prista, Catarina; Moura, Teresa F.; Loureiro-Dias, Maria; Soveral, GraçaGrapevine (Vitis vinifera L.) is one of the oldest and most important perennial crops being considered as a fruit ligneous tree model system in which the water status appears crucial for high fruit and wine quality, controlling productivity and alcohol level. V. vinifera genome contains 28 genes coding for aquaporins, which acting in a concerted and regulated manner appear relevant for plant withstanding extremely unfavorable drought conditions essential for the quality of berries and wine. Several Vv aquaporins have been reported to be expressed in roots, shoots, berries and leaves with clear cultivar differences in their expression level, making their in vivo biochemical characterization a difficult task. In this work V. vinifera cv. Touriga nacional VvTnPIP1;1, VvTnPIP2;2 and VvTnTIP2;1 were expressed in yeast and water transport activity was characterized in intact cells of the transformants. The three aquaporins were localized in the yeast plasma membrane but only VvTnTIP2;1 expression enhanced the water permeability with a concomitant decrease of the activation energy of water transport. Acidification of yeast cytosol resulted in loss of VvTnTIP2;1 activity. Sequence analysis revealed the presence of a His131 residue, unusual in TIPs. By site directed mutagenesis, replacement of this residue by aspartic acid or alanine resulted in loss of pHin dependence while replacement by lysine resulted in total loss of activity. In addition to characterization of VvTn aquaporins, these results shed light on the gating of a specific tonoplast aquaporin by cytosolic pH
- Lack of Aquaporin 3 in bovine erythrocyte membranes correlates with low glycerol permeationPublication . Campos, Elisa; Moura, Teresa F.; Oliva, Abel; Leandro, Paula; Soveral, GraçaIn general, erythrocytes are highly permeable to water, urea and glycerol. However, expression of aquaporin isoforms in erythrocytes appears to be species characteristic. In the present study, human (hRBC) and bovine (bRBC) erythrocytes were chosen for comparative studies due to their significant difference in membrane glycerol permeability. Osmotic water permeability (Pf) at 23 ºC was (2.89 ± 0.37) × 10-2 and (5.12 ± 0.61) × 10-2 cm s-1 for human and bovine cells respectively, with similar activation energies for water transport. Glycerol permeability (Pgly) for human ((1.37 ± 0.26) × 10-5 cm s-1) differed in three orders of magnitude from bovine erythrocytes ((5.82 ± 0.37) ×10-8 cm s-1) that also showed higher activation energy for glycerol transport. When compared to human, bovine erythrocytes showed a similar expression pattern of AQP1 glycosylated forms on immunoblot analysis, though in slight higher levels, which could be correlated with the 1.5-fold larger Pf found. However, AQP3 expression was not detectable. Immunofluorescence analysis confirmed the absence of AQP3 expression in bovine erythrocyte membranes. In conclusion, lack of AQP3 in bovine erythrocytes points to the lipid pathway as responsible for glycerol permeation and explains the low glycerol permeability and high Ea for transport observed in ruminants.
- Membrane tension regulates water transport in yeastPublication . Soveral, Graça; Madeira, Ana; Loureiro-Dias, Maria C.; Moura, Teresa F.Evidence that membrane surface tension regulates water fluxes in intact cells of a Saccharomyces cerevisiae strain overexpressing aquaporin AQY1 was obtained by assessing the osmotic water transport parameters in cells equilibrated in different osmolarities. The osmotic water permeability coefficients (Pf) obtained for yeast cells overexpressing AQY1 incubated in low osmolarity buffers were similar to those obtained for a double mutant aqy1aqy2 and approximately three times lower (with higher activation energy, Ea) than values obtained for cells incubated in higher osmolarities (with lower Ea). Moreover, the initial inner volumes attained a maximum value for cells equilibrated in lower osmolarities (below 0.75 M) suggesting a preswollen state with the membrane under tension, independent of aquaporin expression. In this situation, the impairment of water channel activity suggested by lower Pf and higher Ea could probably be the first available volume regulatory tool that, in cooperation with other osmosensitive solute transporters, aims to maintain cell volume. The results presented point to the regulation of yeast water channels by membrane tension, as previously described in other cell systems