Sn-2-monoacylglycerol, not glycerol, is preferentially utilised for triacylglycerol and phosphatidylcholine biosynthesis in Atlantic salmon (Salmo salar L.) intestine

Pathways of lipid resynthesis in the intestine of fish are relatively unknown. Various reports have suggested the existence of both sn-1,3-specific (pancreatic) and non-specific (bile salt-activated) lipase activity operating on dietary triacylglycerol (TAG) in the intestinal lumen of fish during digestion. Thus, sn-2-monoacylglycerol (2-MAG) and glycerol, respective hydrolytic products of each lipase, are absorbed and utilised for glycerolipid synthesis in enterocytes via two alternative routes: monoacylglycerol (MAG) and glycerol-3-phosphate (G3P) pathways. Despite different precursors, both pathways converge at the production of sn-1,2-diacylglycerol (1,2-DAG) where TAG or phosphatidylcholine (PC) synthesis can occur. To elucidate the relative activities of MAG and G3P pathways in Atlantic salmon enterocytes, intestinal segments were mounted in Ussing chambers where equimolar mixtures of sn-2-oleoyl-[1,2,3-(3)H]glycerol (2-MAG) and [(14)C(U)]glycerol, plus unlabelled 16:0 and 18:2n-6 as exogenous fatty acid sources, were delivered in bile salt-containing Ringer solution to the mucosa. The MAG pathway predominated, over the G3P pathway, synthesizing ca. 95% of total TAG and ca. 80% of total PC after a 3 h incubation period at 10 degrees C. Further, the 1,2-DAG branch point into TAG or PC was polarised towards TAG synthesis (6:1) via the MAG pathway but more evenly distributed between TAG and PC (1:1) via the G3P pathway. Effect of long-chain saturated, monounsaturated and polyunsaturated fatty acids on the synthesized TAG/PC ratio was assessed by individually exchanging 16:0, 18:1n-9 or 18:2n-6, for 16:0+18:2n-6, in mucosal solutions. TAG synthesis was influenced considerably more than PC synthesis, via either pathway, by exogenous fatty acids utilised. 18:1n-9 significantly stimulated TAG synthesis via the MAG pathway yielding a TAG/PC ratio of 12:1. Alternatively, 18:2n-6 stimulated TAG synthesis the most via the G3P pathway (TAG/PC=4:1). 16:0 significantly attenuated TAG synthesis via either pathway. Micellar fatty acid species also significantly affected intestinal active transport mechanisms as shown by decreasing transepithelial potential (TEP) and short-circuit current (SSC) with increasing fatty acid unsaturation. The epithelial integrity was, however, not compromised after 3 h of exposure to any of the fatty acids. The implications of these findings on dietary fatty acid composition and enterocytic lipid droplet accumulation are discussed.     

Comparative evaluation of rumen‐protected fat,coconut oil and various oilseeds supplemented to fattening bulls

The effects of five different dietary fat supplements on fatty acid composition and oxidative stability of subcutaneous and kidney fat were evaluated in 36 Brown Swiss bulls and compared to a low fat diet in a monofactorial design. The following fat supplements were provided as additional fat at 30 g per kg feed dry matter: crystalline rumen‐protected fat, coconut oil, and three types of crushed whole oilseeds (rapeseed, sunflower seed and linseed). Adipose tissues reflected differences (P < 0.05) in dietary fatty acid composition although to a lower extent. Using protected fat, which contained elevated levels of trans fatty acids, and sunflower seed, containing a high proportion of linoleic acid, significantly increased C18:1 trans fatty acid proportion in the adipose tissues. The use of sunflower seed increased conjugated linoleic acid. The oilseeds resulted in lower amounts of C16:0 in favour of C18:0. Except for linseed, all fat supplemented groups improved oxidative stability of adipose tissues as compared with control. This was explained by lower proportions of unsaturated fatty acids in adipose tissue (protected fat), by elevated α‐tocopherol contents (rapeseed, sunflower seed) or by a combination of both (coconut oil). Fat colour remained unaffected by treatments. Compared to other fat supplements oilseeds, especially sunflower seed and rapeseed, can therefore be recommended to be fed to bulls in order to increase the proportions of C18 unsaturated fatty acids in adipose tissues and to maintain or improve oxidative stability.     

Lipids,Proteins, and Structure of Seed Oil Bodies from Diverse Species

Oil bodies isolated from the mature seeds of rape (Brassica napus L.), mustard (Brassica juncea L.), cotton (Gossypium hirsutum L.), flax (Linus usitatis simum), maize (Zea mays L.), peanut (Arachis hypogaea L.), and sesame (Sesamum indicum L.) had average diameters that were different but within a narrow range (0.6-2.0 [mu]m), as measured from electron micrographs of serial sections. Their contents of triacylglycerols (TAG), phospholipids, and proteins (oleosins) were correlated with their sizes. The correlation fits a formula that describes a spherical particle surrounded by a shell of a monolayer of phospholipids embedded with oleosins. Oil bodies from the various species contained substantial amounts of the uncommon negatively charged phosphatidylserine and phosphatidylinositol, as well as small amounts of free fatty acids. These acidic lipids are assumed to interact with the basic amino acid residues of the oleosins on the surface of the phospholipid layer. Isoelectrofocusing revealed that the oil bodies from the various species had an isoelectric point of 5.7 to 6.6 and thus possessed a negatively charged surface at neutral pH. We conclude that seed oil bodies from diverse species are very similar in structure. In rapeseed during maturation, TAG and oleosins accumulated concomitantly. TAG-synthesizing acyltransferase activities appeared at an earlier stage and peaked during the active period of TAG accumulation. The concomitant accumulation of TAG and oleosins is similar to that reported earlier for maize and soybean, and the finding has an implication for the mode of oil body synthesis during seed maturation.     

Characterization of seed oils from fresh Bokbunja (Rubus coreanus Miq.) and wine processing waste

The physicochemical characteristics, fatty acid (FA) profile, and triacylglyceride (TAG) composition of seed oils from fresh Bokbunja (Rubus coreanus Miq.) fruits and traditional Bokbunja wine processing waste were determined in this study. Oil contents of the fresh seeds and the seeds from wine processing waste were similar, accounting for about 18% of dry weight. The free fatty acid (FFA) content between the two seed oils was significantly different (0.50% for fresh seed oil and 73.14% for wine seed oil). Iodine, conjugated diene, saponification values, and unsaponifiable matter were very similar in the oil samples, but the specific extinction coefficients at 232 and 270 nm of wine seed oil were higher than those of fresh seed oil. Linoleic (C18:2, 50.45-53.18%, L) and linolenic (C18:3, 29.36-33.25%, Ln) acids were the dominant FAs in the two seed oils, whereas oleic (C18:1, 7.32-8.04%, O), palmitic (C16:0, 1.55-1.65%, P), and stearic (C18:0, 0.65-0.68%, S) acids were the minor FAs. LLL, OLL, LLLn, OOL, LLnLn, and OOO were the abundant TAGs, representing >90% of the oils.     

Organ- and development-specific acyl coenzyme a lysophosphatidate acyltransferases in palm and meadowfoam

Of the three acyl coenzyme A acyltransferases (AT) directly involved in the assembly of fatty acids into triacylglycerols (TAG) in maturing seed, lysophosphatidate (LPA) AT has the highest substrate stringence and dictates which fatty acids can be used. We studied LPA-AT in the microsomes from various organs of palm (Syragrus cocoides) and found that only the microsomes from maturing seed could act on 1-lauroyl-LPA and lauroyl-coenzyme A to produce dilauroyl-phosphatidate. Similarly, of the microsomes from various organs of meadowfoam (Limnanthes alba), only those from maturing seed were active with 1-erucoyl-LPA and erucoyl-coenzyme A to generate dierucoyl-phosphatidate. During maturation of the seeds of both species, the pattern of appearance of LPA-AT that produced dioleoyl phosphatidate was different from that of LPA-AT that generated dilauroyl or dierucoyl phosphatidate. The results show that in seeds, at least those that contain unusual fatty acids in the storage TAG, LPA-AT for the synthesis of TAG is different from the enzyme(s) for the synthesis of membrane lipids. They also suggest that there may be distinct pathways and/or compartments for the synthesis of TAG and membrane phospholipids.     

Alteration of seed fatty acid composition by an ethyl methanesulfonate-induced mutation in Arabidopsis thaliana affecting diacylglycerol acyltransferase activity.

In characterizing the enzymes involved in the formation of very long-chain fatty acids (VLCFAs) in the Brassicaceae, we have generated a series of mutants of Arabidopsis thaliana that have reduced VLCFA content. Here we report the characterization of a seed lipid mutant, AS11, which, in comparison to wild type (WT), has reduced levels of 20:1 and 18:1 and accumulates 18:3 as the major fatty acid in triacylglycerols. Proportions of 18:2 remain similar to WT. Genetic analyses indicate that the fatty acid phenotype is caused by a semidominant mutation in a single nuclear gene, designated TAG1, located on chromosome 2. Biochemical analyses have shown that the AS11 phenotype is not due to a deficiency in the capacity to elongate 18:1 or to an increase in the relative delta 15 or delta 12 desaturase activities. Indeed, the ratio of desaturase/elongase activities measured in vitro is virtually identical in developing WT and AS11 seed homogenates. Rather, the fatty acid phenotype of AS11 is the result of reduced diacylglycerol acyltransferase activity throughout development, such that triacylglycerol biosynthesis is reduced. This leads to a reduction in 20:1 biosynthesis during seed development, leaving more 18:1 available for desaturation. Thus, we have demonstrated that changes to triacylglycerol biosynthesis can result in dramatic changes in fatty acid composition and, in particular, in the accumulation of VLCFAs in seed storage lipids.     

Genetic enhancement of palmitic acid accumulation in cotton seed oil through RNAi down‐regulation of ghKAS2 encoding β‐ketoacyl‐ACP synthase II (KASII)

Palmitic acid (C16:0) already makes up approximately 25% of the total fatty acids in the conventional cotton seed oil. However, further enhancements in palmitic acid content at the expense of the predominant unsaturated fatty acids would provide increased oxidative stability of cotton seed oil and also impart the high melting point required for making margarine, shortening and confectionary products free of trans fatty acids. Seed‐specific RNAi‐mediated down‐regulation of β‐ketoacyl‐ACP synthase II (KASII) catalysing the elongation of palmitoyl‐ACP to stearoyl‐ACP has succeeded in dramatically increasing the C16 fatty acid content of cotton seed oil to well beyond its natural limits, reaching up to 65% of total fatty acids. The elevated C16 levels were comprised of predominantly palmitic acid (C16:0, 51%) and to a lesser extent palmitoleic acid (C16:1, 11%) and hexadecadienoic acid (C16:2, 3%), and were stably inherited. Despite of the dramatic alteration of fatty acid composition and a slight yet significant reduction in oil content in these high‐palmitic (HP) lines, seed germination remained unaffected. Regiochemical analysis of triacylglycerols (TAG) showed that the increased levels of palmitic acid mainly occurred at the outer positions, while C16:1 and C16:2 were predominantly found in the sn‐2 position in both TAG and phosphatidylcholine. Crossing the HP line with previously created high‐oleic (HO) and high‐stearic (HS) genotypes demonstrated that HP and HO traits could be achieved simultaneously; however, elevation of stearic acid was hindered in the presence of high level of palmitic acid.     

Regiospecific distribution of fatty acids in triacylglycerols of Artemia franciscana nauplii enriched with fatty acid ethyl esters

This paper reports the positional distribution of fatty acids in triacylglycerols (TAG) of Artemia franciscana nauplii enriched with each of palmitic (16:0), oleic (18:1n-9), linoleic (18:2n-6), linolenic (18:3n-3), eicosapentaenoic (20:5n-3), and docosahexaenoic (22:6n-3) acid ethyl esters. TAG extracted from the enriched and unenriched nauplii were subjected to regiospecific analysis to determine the fatty acid compositions of the sn-1(3) and sn-2 positions of TAG. In the unenriched nauplii, 16:0, 18:1n-9, and 18:2n-6 were preferentially located in the sn-1(3) position followed by the sn-2 position [i.e. sn-1(3)>sn-2], whereas 18:3n-3 was concentrated in the sn-2 position [i.e. sn-2>sn-1(3)]. Contents of 20:5n-3 and 22:6n-3 were low. After the nauplii were enriched with each of the ethyl esters for 18 h, fatty acid fed to the nauplii showed higher content in the sn-1(3) position than in the sn-2 position [i.e. sn-1(3)>sn-2]. Distribution pattern of 18:3n-3 changed from sn-2>sn-1(3) to sn-1(3)>sn-2 during the enrichment with 18:3n-3 ethyl ester. Increases in all of the fatty acids in TAG were attributed to that in the sn-1(3) position much more than that in the sn-2 position. Artemia nauplii appear to be characterized by preferential incorporation of exogenous fatty acids into the sn-1(3) position of TAG, even though endogenous fatty acids are esterified in the opposite position.     

Competing carboxylases: circadian and metabolic regulation of Rubisco in C3 and CAM Mesembryanthemum crystallinum L

The temporal co-ordination of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPc) activities by Mesembryanthemum crystallinum L. in C(3) and crassulacean acid metabolism (CAM) modes was investigated under conventional light-dark (LD) and continuous light (LL) conditions. When C(3) , net CO(2) assimilation rate increased during each subjective night under LL with maximum carboxylation unrelated to Rubisco activation state. The CAM circadian rhythm of CO(2) uptake was more pronounced, with CO(2) assimilation rate maximal towards the end of each subjective night. In vivo and in vitro techniques were integrated to map carboxylase enzyme regulation to the framework provided by CAM LL gas exchange activity. Rubisco was activated in vitro throughout each subjective dark period and consistently deactivated at each subjective dawn, similar to that observed at true dawn in constitutive CAM species. Instantaneous carbon isotope discrimination showed in vivo carboxylase co-dominance during the CAM subjective night, initially by Rubisco and latterly C(4) (PEPc), despite both enzymes seemingly activated in vitro. The circadian rhythm in titratable acidity accumulation was progressively damped over successive subjective nights, but maintenance of PEPc carboxylation capacity ensures that CAM plants do not become progressively more 'C(3) -like' with time under LL.     

Multifunctional acetyl-CoA carboxylase 1 is essential for very long chain fatty acid elongation and embryo development in Arabidopsis

Acetyl-CoA carboxylase (ACCase) catalyses the carboxylation of acetyl-CoA, forming malonyl-CoA, which is used in the plastid for fatty acid synthesis and in the cytosol in various biosynthetic pathways including fatty acid elongation. In Arabidopsis thaliana, ACC1 and ACC2, two genes located in a tandem repeat within a 25-kbp genomic region near the centromere of chromosome 1, encode two multifunctional ACCase isoforms. Both genes, ACC1 and ACC2, appear to be ubiquitously expressed, but little is known about their respective function and importance. Here, we report the isolation and characterisation of two allelic mutants disrupted in the ACC1 gene. Both acc1-1 and acc1-2 mutations are recessive and embryo lethal. Embryo morphogenesis is impaired and both alleles lead to cucumber-like structures lacking in cotyledons, while the shortened hypocotyl and root exhibit a normal radial pattern organisation of the body axis. In this abnormal embryo, the maturation process still occurs. Storage proteins accumulate normally, while triacylglycerides (TAG) are synthesised at a lower concentration than in the wild-type seed. However, these TAG are totally devoid of very long chain fatty acids (VLCFA) and consequently enriched in C18:1, like all lipid fractions analysed in the mutant seed. These data demonstrate, in planta, the role of ACCase 1 in VLCFA elongation. Furthermore, this multifunctional enzyme also plays an unexpected and central function in embryo morphogenesis, especially in apical meristem development.     

高油酸油菜遗传育种研究进展

油酸（C18∶1）是双低菜籽油的主要脂肪酸之一,合理的高油酸菜籽油脂肪酸组分更有利于人体健康,因此提高油酸含量是双低油菜品质育种的一个重要方向。当前我国高油酸油菜品种选育相关研究进展缓慢,高油酸菜籽油产业化进程急需提速。围绕高油酸油菜主要从四个方面开展论述：国内外利用理化方法成功创建的油菜高油酸种质资源及其高油酸性状遗传模式;油菜高油酸性状的控制基因及其突变位点;世界上高油酸品种的培育以及我国高油酸品种的发展趋势;当前高油酸品种的不足及今后高油酸油菜品种的改良途径。为油菜育种工作者较全面地展示了国际上高油酸油菜在遗传育种方面的研究成果,也为今后我国高油酸油菜的进一步发展提供了参考。     

13C NMR, GC and HPLC characterization of lipid components of the salted and dried mullet (Mugil cephalus) roe "bottarga"

13C NMR spectroscopy, in conjunction with HPLC and GC techniques, has been used to study the molecular composition of lipids extracted from commercial products of bottarga. To this goal, both the saponifiable and unsaponifiable fractions of lipid extracts were also examined by 13C NMR. Among the major lipid classes wax esters (WE) showed a concentration of more than 50mol%, triacylglycerols (TAG) and phospholipids (PL) represented a minor fraction. Concentrations up to 29mol% of free fatty acids (FFA) were found. The most represented fatty alcohol was 16:0 that accounted for more than 50%, among fatty acids the most represented were 16:1 n-7, 22:6 n-3, 18:1 n-9, 16:0, and 20:5 n-3, in particular the n-3 polyunsaturated fatty acids (PUFA) averaged 40mg/g of the edible portion. 13C NMR spectroscopy put in evidence that cholesterol was present in its free and esterified forms and its total content was measured as ca. 10mg/g of the edible portion.     

Phospholipid and triacylglycerol profiles modified by PLD suppression in soybean seed

Phospholipase D (PLD) is capable of hydrolyzing membrane phospholipids, producing phosphatidic acid. To alter phospholipid profiles in soybean seed, we attenuated PLD enzyme activity by an RNA interference construct using the partial sequence from a soybean PLDα gene. Two transgenic soybean lines were established by particle inflow gun (PIG) bombardment by co‐bombarding with pSPLDi and pHG1 vectors. The lines were evaluated for the presence and expression of transgenes thoroughly through the T₄ generation. PLD‐suppressed soybean lines were characterized by decreased PLDα enzyme activity and decreased PLDα protein both during seed development and in mature seeds. There was no change in total phospholipid amount; however, the PLD‐attenuated transgenic soybean seed had higher levels of di18 : 2 (dilinoleoyl)‐phosphatidylcholine (PC) and ‐phosphatidylethanolamine (PE) in seeds than the non‐transgenic lines. The increased polyunsaturation was at the expense of PC and PE species containing monounsaturated or saturated fatty acids. In addition to increased unsaturation in the phospholipids, there was a decrease in unsaturation of the triacylglycerol (TAG) fraction of the soybean seeds. Considering recent evidence for the notion that desaturation of fatty acids occurs in the PC fraction and that the PC → DAG (diacylglycerol) → TAG pathway is the major route of TAG biosynthesis in developing soybean seed, the current data suggest that PLDα suppression slows the conversion of PC to TAG. This would be consistent with PLD playing a positive role in that conversion. The data indicate that soybean PLD attenuation is a potentially useful approach to altering properties of edible and industrial soybean lecithin.     

Triacylglycerol catabolism in the prawn Macrobrachium borellii (Crustacea: Palaemoniade)

While invertebrates store neutral lipids as their major energy source, little is known about triacylglycerol (TAG) class composition and their differential catabolism in aquatic arthropods. This study focuses on the composition of the main energy source and its catabolism by lipase from the midgut gland (hepatopancreas) of the crustacean Macrobrachium borellii. Silver-ion thin-layer chromatography of prawn large TAG deposit (80% of total lipids) and its subsequent fatty acid analysis by gas chromatography allowed the identification of 4 major fractions. These are composed of fatty acids of decreasing unsaturation and carbon chain length, the predominant being 18:1n-9. Fraction I, the most unsaturated one, contained mainly 20:5n-3; fraction II 18:2n-6; fraction III 18:1n-9 while the most saturated fraction contained mostly 16:0. Hepatopancreas main lipase (Mr 72 kDa) cross-reacted with polyclonal antibodies against insect lipase, was not dependent on the presence of Ca²⁺ and had an optimum activity at 40 °C and pH 8.0. Kinetic analysis showed a Michaelis–Menten behavior. A substrate competition assay evidenced lipase specificity following the order: 18:1n-9-TAG > PUFA-enriched-TAG > 16:0-TAG different from that in vertebrates. These data indicate there is a reasonable correspondence between the fatty acid composition of TAG and the substrate specificity of lipase, which may be an important factor in determining which fatty acids are mobilized during lipolysis for oxidation in crustaceans.     

A jojoba beta-Ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants.

beta-Ketoacyl-coenzyme A (CoA) synthase (KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoA. This reaction is the initial step of the microsomal fatty acyl-CoA elongation pathway responsible for formation of very long chain fatty acids (VLCFAs, or fatty acids with chain lengths > 18 carbons). Manipulation of this pathway is significant for agriculture, because it is the basis of conversion of high erucic acid rapeseed into canola. High erucic acid rapeseed oil, used as an industrial feedstock, is rich in VLCFAs, whereas the edible oil extracted from canola is essentially devoid of VLCFAs. Here, we report the cloning of a cDNA from developing jojoba embryos involved in microsomal fatty acid elongation. The jojoba cDNA is homologous to the recently cloned Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene that has been suggested to encode KCS. We characterize the jojoba enzyme and present biochemical data indicating that the jojoba cDNA does indeed encode KCS. Transformation of low erucic acid rapeseed with the jojoba cDNA restored KCS activity to developing embryos and altered the transgenic seed oil composition to contain high levels of VLCFAs. The data reveal the key role KCS plays in determining the chain lengths of fatty acids found in seed oils.     

Myristic acid increases delta6-desaturase activity in cultured rat hepatocytes

In order to study the effects of saturated fatty acids on delta6-desaturase activity, rat hepatocytes in primary culture were incubated with lauric (C12:0), myristic (C14:0) or palmitic (C16:0) acids. After optimization, the standard in vitro conditions for the measurement of delta6-desaturase activity were as follows: 60 micromol x L(-1) alpha-linolenic acid (C18:3n-3), reaction time of 20 min and protein content of 0.4 mg. Data showed that cell treatment with 0.5 mmol x L(-1) myristic acid during 43 h specifically increased delta6-desaturase activity. This improvement, reproducible for three substrates of delta6-desaturase, i.e. oleic acid (C18:1n-9), linoleic acid (C18:2n-6) and alpha-linoleic acid (C18:3n-3) was dose-dependent in the range 0.1-0.5 mmol x L(-1) myristic acid concentration.