Lipid content and fatty acid profile of Senegalese sole (Solea senegalensis Kaup, 1858) juveniles as affected by feed containing different amounts of plant protein sources

A growth trial with Senegalese Sole (Solea senegalensis Kaup, 1858) juveniles fed with diets containing increasing replacement levels of fishmeal by mixtures of plant protein sources was conducted over 12 weeks. Total fat contents of muscle, liver, viscera, skin, fins and head tissues were determined, as well as fatty acid profiles of muscle and liver (GC-FID analysis). Liver was the preferential local for fat deposition (5.5–10.8% of fat) followed by fins (3.4–6.7% fat). Increasing levels of plant protein in the diets seems to be related to increased levels of total lipids in the liver. Sole muscle is lean (2.4–4.0% fat), with total lipids being similar among treatments. Liver fatty acid profile varied significantly among treatments. Plant protein diets induced increased levels of C16:1 and C18:2 n -6 and a decrease in ARA and EPA levels. Muscle fatty acid profile also evidenced increasing levels of C18:2 n 6, while ARA and DHA remained similar among treatments. Substitution of fishmeal by plant protein is hence possible without major differences on the lipid content and fatty acid profile of the main edible portion of the fish – the muscle.

Engineering of fatty acid production and secretion in Saccharomyces cerevisiae

Tese de Doutoramento em Ciências - Especialidade em Biologia

Synthesis of medium-chain-length polyhydroxyalkanoates in arabidopsis thaliana using intermediates of peroxisomal fatty acid beta-oxidation.

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCL-PHAs) are synthesized in bacteria by using intermediates of the beta-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants, Arabidopsis thaliana was transformed with the PhaC1 synthase from Pseudomonas aeruginosa modified for peroxisome targeting by addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the beta-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.

Fatty acid signaling in Arabidopsis.

Many organisms use fatty acid derivatives as biological regulators. In plants, for example, fatty acid-derived signals have established roles in the regulation of developmental and defense gene expression. Growing numbers of these compounds, mostly derived from fatty acid hydroperoxides, are being characterized. The model plant Arabidopsis thaliana is serving a vital role in the discovery of fatty acid-derived signal molecules and the genetic analysis of their synthesis and action. The Arabidopsis genome sequencing project, the availability of large numbers of mutants in fatty acid biosynthesis and signal transduction, as well as excellent pathosystems, make this plant a tremendously useful model for research in fatty acid signaling. This review summarizes recent progress in understanding fatty acid signaling in A. thaliana and highlights areas of research where progress is rapid. Particular attention is paid to the growing literature on the jasmonate family of regulators and their role in defense against insects and microbial pathogens.

Differential involvement of peroxisome-proliferator-activated receptors alpha and delta in fibrate and fatty-acid-mediated inductions of the gene encoding liver fatty-acid-binding protein in the liver and the small intestine.

Liver fatty-acid-binding protein (L-FABP) is a cytoplasmic polypeptide that binds with strong affinity especially to long-chain fatty acids (LCFAs). It is highly expressed in both the liver and small intestine, where it is thought to have an essential role in the control of the cellular fatty acid (FA) flux. Because expression of the gene encoding L-FABP is increased by both fibrate hypolipidaemic drugs and LCFAs, it seems to be under the control of transcription factors, termed peroxisome-proliferator-activated receptors (PPARs), activated by fibrate or FAs. However, the precise molecular mechanism by which these regulations take place remain to be fully substantiated. Using transfection assays, we found that the different PPAR subtypes (alpha, gamma and delta) are able to mediate the up-regulation by FAs of the gene encoding L-FABP in vitro. Through analysis of LCFA- and fibrate-mediated effects on L-FABP mRNA levels in wild-type and PPARalpha-null mice, we have found that PPARalpha in the intestine does not constitute a dominant regulator of L-FABP gene expression, in contrast with what is known in the liver. Only the PPARdelta/alpha agonist GW2433 is able to up-regulate the gene encoding L-FABP in the intestine of PPARalpha-null mice. These findings demonstrate that PPARdelta can act as a fibrate/FA-activated receptor in tissues in which it is highly expressed and that L-FABP is a PPARdelta target gene in the small intestine. We propose that PPARdelta contributes to metabolic adaptation of the small intestine to changes in the lipid content of the diet.

Postinfarction heart failure in rats is associated with upregulation of GLUT-1 and downregulation of genes of fatty acid metabolism.

OBJECTIVES: Increasing evidence suggests that left ventricular remodeling is associated with a shift from fatty acid to glucose metabolism for energy production. The aim of this study was to determine whether left ventricular remodeling with and without late-onset heart failure after myocardial infarction is associated with regional changes in the expression of regulatory proteins of glucose or fatty acid metabolism. METHODS: Myocardial infarction was induced in rats by ligation of the left anterior descending coronary artery (LAD). In infarcted and sham-operated hearts the peri-infarction region (5-mm zone surrounding the region at risk), the interventricular septum and the right ventricular free wall were separated for analysis. RESULTS: At 8 and 20 weeks after LAD ligation, the peri-infarction region and the septum exhibited marked re-expression of atrial natriuretic factor [+252+/-37 and +1093+/-279%, respectively, in the septum (P<0.05)] and of alpha-smooth muscle actin [+34+/-10 and +43+/-14%, respectively, in the septum (P<0.05)]. At 8 weeks, when left ventricular hypertrophy was present without signs of heart failure, myocardial mRNA expression of glucose transporters (GLUT-1 and GLUT-4) was not altered, whereas mRNA expression of medium-chain acyl-CoA dehydrogenase (MCAD) was significantly reduced in the peri-infarction region (-25+/-7%; P<0.05). In hearts exhibiting heart failure 20 weeks after infarct-induction there was a change in all three ventricular regions of both mRNA and protein content of GLUT-1 [+72+/-28 and +121+/-15%, respectively, in the peri-infarction region (P<0.05)] and MCAD [-29+/-9 and -56+/-4%, respectively, in the peri-infarction region (P<0.05)]. CONCLUSION: In rats with large myocardial infarction, progression from compensated remodeling to overt heart failure is associated with upregulation of GLUT-1 and downregulation of MCAD in both the peri-infarction region and the septum.

Fatty Acid and Sterol Composition of Three Phytomonas Species

Fatty acid and sterol analysis were performed on Phytomonas serpens and Phytomonas sp. grown in chemically defined and complex medium, and P. françai cultivated in complex medium. The three species of the genus Phytomonas had qualitatively identical fatty acid patterns. Oleic, linoleic, and linolenic were the major unsaturated fatty acids. Miristic and stearic were the major saturated fatty acids. Ergosterol was the only sterol isolated from Phytmonas sp. and P. serpens grown in a sterol-free medium, indicating that it was synthesized de novo. When P. françai that does not grow in defined medium was cultivated in a complex medium, cholesterol was the only sterol detected. The fatty acids and sterol isolated from Phytomonas sp. and P. serpens grown in a chemically defined lipid-free medium indicated that they were able to biosynthesize fatty acids and ergosterol from acetate or from acetate precursors such as glucose or threonine.

Decreased expression of peroxisome proliferator-activated receptor alpha and liver fatty acid binding protein after partial hepatectomy of rats and mice.

BACKGROUND AIMS: Marked changes in metabolism, including liver steatosis and hypoglycemia, occur after partial hepatectomy. Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a nuclear hormone receptor that is activated by fatty acids and involved in hepatic fatty acid metabolism and regeneration. Liver fatty acid binding protein (LFABP) is an abundant protein in liver cytosol whose expression is regulated by PPAR alpha. It is involved in fatty acid uptake and diffusion and in PPAR alpha signaling. The aim of this study was to investigate the expression of PPAR alpha and LFABP during liver regeneration. METHODS: Male Sprague-Dawley rats and male C57 Bl/6 mice were subjected to 2/3 hepatectomy and LFABP and PPAR alpha mRNA and protein levels were measured at different time points after surgery. The effect of partial hepatectomy was followed during 48 h in rats and 72 h in mice. RESULTS: PPAR alpha mRNA and protein levels were decreased 26 h after hepatectomy of rats. The LFABP mRNA and protein levels paralleled those of PPAR alpha and were also decreased 26 h after hepatectomy. In mice, the mRNA level was decreased after 36 and 72 h after hepatectomy. In this case, LFABP mRNA levels decreased more slowly after partial hepatectomy than in rats. CONCLUSIONS: A marked decrease in PPAR alpha expression may be important for changed gene expression, e.g. LFABP, and metabolic changes, such as hypoglycemia, during liver regeneration.

Characterization of Aspergillus species based on fatty acid profiles

Cellular fatty acid (FA) composition was utilized as a taxonomic tool to discriminate between different Aspergillus species. Several of the tested species had the same FA composition and different relative FA concentrations. The most important FAs were palmitic acid (C16:0), estearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2), which represented 95% of Aspergillus FAs. Multivariate data analysis demonstrated that FA analysis is a useful tool for differentiating species belonging to genus Aspergillus. All the species analyzed showed significantly FA acid profiles (p < 0.001). Furthermore, it will be possible to distinguish among Aspergillus spp. in the Flavi Section. FA composition can serve as a useful tool for the identification of filamentous fungi.

Common variants of the liver fatty acid binding protein gene influence the risk of type 2 diabetes and insulin resistance in Spanish population

SUMMARY The main objective was to evaluate the association between SNPs and haplotypes of the FABP1-4 genes and type 2 diabetes, as well as its interaction with fat intake, in one general Spanish population. The association was replicated in a second population in which HOMA index was also evaluated. METHODS 1217 unrelated individuals were selected from a population-based study [Hortega study: 605 women; mean age 54 y; 7.8% with type 2 diabetes]. The replication population included 805 subjects from Segovia, a neighboring region of Spain (446 females; mean age 52 y; 10.3% with type 2 diabetes). DM2 mellitus was defined in a similar way in both studies. Fifteen SNPs previously associated with metabolic traits or with potential influence in the gene expression within the FABP1-4 genes were genotyped with SNPlex and tested. Age, sex and BMI were used as covariates in the logistic regression model. RESULTS One polymorphism (rs2197076) and two haplotypes of the FABP-1 showed a strong association with the risk of DM2 in the original population. This association was further confirmed in the second population as well as in the pooled sample. None of the other analyzed variants in FABP2, FABP3 and FABP4 genes were associated. There was not a formal interaction between rs2197076 and fat intake. A significant association between the rs2197076 and the haplotypes of the FABP1 and HOMA-IR was also present in the replication population. CONCLUSIONS The study supports the role of common variants of the FABP-1 gene in the development of type 2 diabetes in Caucasians.

Total lipid and fatty acid accumulation during basidiospore formation in the ectomycorrhizal fungus Pisolithus sp.

The basidiospores of Pisolithus sp. contain large amounts of lipids, indicating provision for future germination in the host rhizosphere. However, the accumulation, composition, and mobilization of lipids during formation and germination of these spores are largely unknown. In this study, lipid storage and fatty acid composition during basidiosporogenesis were analyzed in fresh basidiocarps using bright-field microscopy and gas chromatography. Abundant lipid bodies are found in the hyphae, basidia, and basidiospores of fungal basidiocarps. This evidences a considerable C transport in the basidiocarp to meet the C demand during basidiospore formation. Fatty acid composition analysis revealed the presence of 24 compounds with chains of 9 to 18 C atoms, either saturated or insaturated, with one or two insaturations. The fatty acid composition and content varied according to the developmental stage of the peridioles. In free basidiospores, the predominant compounds were 16:0, 16:1w5c, 18:1w9c, and 18:2w6,9c/18:0ante, at concentrations of 76, 46, 192, and 51 µg g-1 dry matter, respectively. Our results indicate that oleic acid is the major constituent of lipid reserves in Pisolithus sp. basidiospores. Further studies are being conducted to determine the factors that induce lipid mobilization during spore germination.

Total fatty acid composition in the characterization and identification of orchid mycorrhizal fungi Epulorhiza spp.

Rhizoctonia-like fungi are the main mycorrhizal fungi in orchid roots. Morphological characterization and analysis of conserved sequences of genomic DNA are frequently employed in the identification and study of fungi diversity. However, phytopathogenic Rhizoctonia-like fungi have been reliably and accurately characterized and identified through the examination of the fatty acid composition. To evaluate the efficacy of fatty acid composition in characterizing and identifying Rhizoctonia-like mycorrhizal fungi in orchids, three Epulorhiza spp. mycorrhizal fungi from Epidendrum secundum, two unidentified fungi isolated from Epidendrum denticulatum, and a phytopathogenic fungus, Ceratorhiza sp. AGC, were grouped based on the profile of their fatty acids, which was assessed by the Euclidian and Mahalanobis distances and the UPGMA method. Dendrograms distinguished the phytopathogenical isolate of Ceratorhiza sp. AGC from the mycorrhizal fungi studied. The symbionts of E. secundum were grouped into two clades, one containing Epulorhiza sp.1 isolates and the other the Epulorhiza sp.2 isolate. The similarity between the symbionts of E. denticulatum and Epulorhiza spp. fungi suggests that symbionts found in E. denticulatum may be identified as Epulorhiza. These results were corroborated by the analysis of the rDNA ITS region. The dendrogram constructed based on the Mahalanobis distance differentiated the clades most clearly. Fatty acid composition analysis proved to be a useful tool for characterizing and identifying Rhizoctonia-like mycorrhizal fungi.

Beta-oxidation in fatty acid degradation and beyond.

The degradation of fatty acids in plants occurs primarily in the peroxisomes through the beta-oxidation cycle. Enzymes that are involved in various aspects of beta-oxidation have been identified recently and shown to act biochemically on a diversity of fatty acids and derivatives. Analysis of several mutants has revealed essential roles for beta-oxidation in the breakdown of reserve triacylglycerols, seed development, seed germination and post-germinative growth before the establishment of photosynthesis. Beta-oxidation has also a considerable importance during the vegetative and reproductive growth phases, and plays a role in plant responses to stress, particularly in the synthesis of jasmonic acid.

Characterization of rapeseed (Brassica napus) oils by bulk C, O, H, and fatty acid C stable isotope analyses

Rapeseed (Brassica napus) oils differing in cultivar, sites of growth, and harvest year were characterized by fatty acid concentrations and carbon, hydrogen, and oxygen stable isotope analyses of bulk oils (delta(13)C(bulk), delta(2)H(bulk), delta(18)O(bulk) values) and individual fatty acids (delta(13)C(FA)). The delta(13)C(bulk), delta(2)H(bulk), and delta(18)O(bulk) values were determined by continuous flow combustion and high-temperature conversion elemental analyzer isotope ratio mass spectrometry (EA/IRMS, TC-EA/IRMS). The delta(13)C(FA) values were determined using gas chromatography-combustion isotope ratio mass spectrometry (GC/C/IRMS). For comparison, other C(3) vegetable oils rich in linolenic acid (flax and false flax oils) and rich in linoleic acid (poppy, sunflower, and safflower oils) were submitted to the same chemical and isotopic analyses. The bulk and molecular delta(13)C values were typical for C(3) plants. The delta(13)C value of palmitic acid (delta(13)C(16:0)) and n-3 alpha-linolenic acid (delta(13)C(18:3n-3)) differed (p < 0.001) between rape, flax, and poppy oils. Also within species, significant differences of delta(13)C(FA) were observed (p < 0.01). The hydrogen and oxygen isotope compositions of rape oil differed between cultivars (p < 0.05). Major differences in the individual delta(13)C(FA) values were found. A plant-specific carbon isotope fractionation occurs during the biosynthesis of the fatty acids and particularly during desaturation of C(18) acids in rape and flax. Bulk oil and specific fatty acid stable isotope analysis might be useful in tracing dietary lipids differing in their origin.

Impact of unusual fatty acid synthesis on futile cycling through beta-oxidation and on gene expression in transgenic plants.

Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12-epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal beta-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward beta-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through beta-oxidation than the expression profile of genes involved in lipid metabolism.