拟南芥TAG1基因对脂类合成调控作用的研究进展

三酰甘油（TAG）是真核生物中能量贮存的最主要形式。植物中贮存的三酰甘油是食用油类和工业用油的主要来源。TAG1基因的表达产物甘油二酯酰基转移酶（DGAT）能够调控三酰甘油的合成。as11是TAG1基因突变获得的脂类代谢相关突变体。该文概述了拟南芥（Arabidopsis thaliana）突变体as11的生物学特征及TAG1基因对脂类合成调控的最新进展。     

拟南芥TAG1 基因对脂类合成调控作用的研究进展

三酰甘油(TAG)是真核生物中能量贮存的最主要形式。植物中贮存的三酰甘油是食用油类和工业用油的主要来源。TAG1基因的表达产物甘油二酯酰基转移酶(DGAT)能够调控三酰甘油的合成。as11是TAG1基因突变获得的脂类代谢相关突变体。该文概述了拟南芥(Arabidopsis thaliana)突变体as11的生物学特征及TAG1基因对脂类合成调控的最新进展。     

The role of triacylglycerol in cardiac energy provision

Triacylglycerols (TAGs) constitute the main energy storage resource in mammals, by virtue of their high energy density. This in turn is a function of their highly reduced state and hydrophobicity. Limited water solubility, however, imposes specific requirements for delivery and uptake mechanisms on TAG-utilising tissues, including the heart, as well as intracellular disposition. TAGs constitute potentially the major energy supply for working myocardium, both through blood-borne provision and as intracellular TAG within lipid droplets, but also provide the heart with fatty acids (FAs) which the myocardium cannot itself synthesise but are required for glycerolipid derivatives with (non-energetic) functions, including membrane phospholipids and lipid signalling molecules. Furthermore they serve to buffer potentially toxic amphipathic fatty acid derivatives. Intracellular handling and disposition of TAGs and their FA and glycerolipid derivatives similarly requires dedicated mechanisms in view of their hydrophobic character. Dysregulation of utilisation can result in inadequate energy provision, accumulation of TAG and/or esterified species, and these may be responsible for significant cardiac dysfunction in a variety of disease states. This review will focus on the role of TAG in myocardial energy provision, by providing FAs from exogenous and endogenous TAG sources for mitochondrial oxidation and ATP production, and how this can change in disease and impact on cardiac function. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.     

Quantitative profiling and pattern analysis of triacylglycerol species in Arabidopsis seeds by electrospray ionization mass spectrometry

Plant triacylglycerols (TAGs), or vegetable oils, provide approximately 25% of dietary calories to humans and are becoming an increasingly important source of renewable bioenergy and industrial feedstocks. TAGs are assembled by multiple enzymes in the endoplasmic reticulum from building blocks that include an invariable glycerol backbone and variable fatty acyl chains. It remains a challenge to elucidate the mechanism of synthesis of hundreds of different TAG species in planta. One reason is the lack of an efficient analytical approach quantifying individual molecular species. Here we report a rapid and quantitative TAG profiling approach for Arabidopsis seeds based on electrospray ionization tandem mass spectrometry with direct infusion and multiple neutral loss scans. The levels of 93 TAG molecular species, identified by their acyl components, were determined in Arabidopsis seeds. Quantitative TAG pattern analyses revealed that the TAG assembly machinery preferentially produces TAGs with one elongated fatty acid. The importance of the selectivity in oil synthesis was consistent with an observation that an Arabidopsis mutant overexpressing a patatin‐like phospholipase had enhanced seed oil content with elongated fatty acids. This quantitative TAG profiling approach should facilitate investigations aimed at understanding the biochemical mechanisms of TAG metabolism in plants.     

A roadmap of tandemly arrayed genes in the genomes of human, mouse, and rat

Tandemly arrayed genes (TAGs) play an important functional and physiological role in the genome. Most previous studies have focused on individual TAG families in a few species, yet a broad characterization of TAGs is not available. Here we identified all TAGs in the genomes of humans, mouse, and rat and performed a comprehensive analysis of TAG distribution, TAG sizes, TAG orientations and intergenic distances, and TAG functions. TAGs account for about 14-17% of all genes in the genome and nearly one-third of all duplicated genes, highlighting the predominant role that tandem duplication plays in gene duplication. For all species, TAG distribution is highly heterogeneous along chromosomes and some chromosomes are enriched with TAG forests, whereas others are enriched with TAG deserts. The majority of TAGs are of size 2 for all genomes, similar to the previous findings in Caenorhabditis elegans, Arabidopsis thaliana, and Oryza sativa, suggesting that it is a rather general phenomenon in eukaryotes. The comparison with the genome patterns shows that TAG members have a significantly higher proportion of parallel gene orientation in all species, corroborating Graham's claim that parallel orientation is the preferred form of orientation in TAGs. Moreover, TAG members with parallel orientation tend to be closer to each other than all neighboring genes in the genome with parallel orientation. The analyses of Gene Ontology function indicate that genes with receptor or binding activities are significantly overrepresented by TAGs. Computer simulation reveals that random gene rearrangements have little effect on the statistics of TAGs for all genomes. Finally, the average proportion of TAGs shows a trend of increase with the increase of family sizes, although the correlation between TAG proportions in individual families and family sizes is not significant.     

Chemistry and liquid chromatography methods for the analyses of primary oxidation products of triacylglycerols

Abstract

Triacylglycerols (TAGs) are one of the major components of the cells in higher biological systems, which can act as an energy reservoir in the living cells. The unsaturated fatty acid moiety is the key site of oxidation and formation of oxidation compounds. The TAG free radical generates several primary oxidation compounds. These include hydroperoxides, hydroxides, epidioxides, hydroperoxy epidioxides, hydroxyl epidioxides, and epoxides. The presence of these oxidized TAGs in the cell increases the chances of several detrimental processes. For this purpose, several liquid chromatography (LC) methods were reported in their analyses. This review is therefore focused on the chemistry, oxidation, extraction, and the LC methods reported in the analyses of oxidized TAGs. The studies on thin-layer chromatography were mostly focused on the total oxidized TAGs separation and employ hexane as major solvent. High-performance LC (HPLC) methods were discussed in details along with their merits and demerits. It was found that most of the HPLC methods employed isocratic elution with methanol and acetonitrile as major solvents with an ultraviolet detector. The coupling of HPLC with mass spectrometry (MS) highly increases the efficiency of analysis as well as enables reliable structural elucidation. The use of MS was found to be helpful in studying the oxidation chemistry of TAGs and needs to be extended to the complex biological systems.     

A homolog of Arabidopsis SDP1 lipase in Nannochloropsis is involved in degradation of de novo-synthesized triacylglycerols in the endoplasmic reticulum

Organisms of the microalgal genus Nannochloropsis produce high levels of triacylglycerols (TAGs), an efficient raw material for biofuels. A complete understanding of the TAG-breakdown pathway is critical for improving the productivity of TAGs to meet future needs. Among a number of lipases annotated as TAG lipase in the genomes of every organism, Arabidopsis SUGAR-DEPENDENT 1 (AtSDP1) lipases are characterized as a type of crucial TAG lipase in plants, similar to ScTgl3–5 in Saccharomyces cerevisiae. Homologs of the AtSDP1 TAG lipases are universally found in the genomes of plants, fungi, and algae. Here we identified two homologs of AtSDP1 TAG lipases in the oleaginous microalga species Nannochloropsis oceanica, NoTGL1 and NoTGL2. We generated single- and double-knockout strains for these lipases by homologous recombination. Whereas overall TAG content in the NoTGL2 single-knockout mutant was identical to that of wild type, the NoTGL1 knockout showed a two-fold increase in TAG content per cell in early log phase under nutrient-sufficient conditions without affecting growth. Homologs of AtSDP1 in S. cerevisiae are localized to the surface of lipid droplets, and AtSDP1 is transported from peroxisomes to the surface of lipid droplets. In contrast, NoTGL1 localized to the endoplasmic reticulum in both Nannochloropsis and yeast. We suggest that homologs of AtSDP1 lipases in Nannochloropsis modulate de novo TAG biosynthesis in the endoplasmic reticulum, unlike the roles of these lipases in other organisms. These results provide important insights into the mechanisms of TAG metabolism catalyzed by homologs of AtSDP1 lipase, which are highly conserved across species.     

Triacylglycerol accumulation and profiling in the model diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum (Baccilariophyceae) during starvation

Although substantial economic barriers exist, marine diatoms such as Thalassiosira pseudonana and Phaeodactylum tricornutum hold promise as feedstock for biodiesel because of their ability to manufacture and store triacylglycerols (TAGs). The recent sequencing of these two marine diatom genomes by the United States Department of Energy Joint Genome Institute and the development of improved systems for genetic manipulation should allow a more systematic approach to understanding and maximizing TAG production. However, in order to best utilize these genomes and genetic tools, we must first gain a deeper understanding of the nutrient-mediated regulation of TAG anabolism. By determining both the yield and molecular species distribution of TAGs we will, in the future, be able to fully characterize the effects of genetic manipulation. Here, we lay the groundwork for understanding TAG production in T. pseudonana and P. tricornutum, as a function of nitrate and silicate depletion. Diatoms were starved of either nitrate or silicate, and TAGs were extracted with hexane from lyophilized samples taken at various time intervals following starvation. The timing of TAG production and the relative abundance of TAGs were estimated by fluorescence spectroscopy using Nile red and the total yield per biomass determined by gravimetric assay. TAGs were analyzed using thin layer chromatography, gas chromatography–mass spectrometry, and electrospray ionization mass spectrometry to identify the major TAG species produced during the growth curve. Under our conditions, the TAG yield from T. pseudonana is about 14–18% of total dry weight. The TAG yield from P. tricornutum is about 14% of total dry weight. Silicate-starved T. pseudonana accumulated an average of 24% more TAGs than those starved for nitrate; however, the chemotypes of the TAGs produced were generally similar regardless of the starvation condition employed.     

Analysis of changes in triacylglycerol ratios in mouse liver and plasma in response to a liver X receptor agonist

An LC-MS method was developed for the analysis of triacylglycerols (TAG) in mouse liver extracts and plasma samples. C57 Mice were treated with two LXR agonists that have been shown to upregulate TAGs, T0901317 (T1317) or Org 264693 and compared to vehicle dosed animals. The dose used was 30 mg kg⁻¹, once daily, with three different dose regimes; 24 H, 48 H and 5 day. The TAG ratios measured were C52:2/C54:3 and C52:3/C54:4, which corresponded to a decrease in the palmitate and an increase in oleate composition of the TAGs. A significant change in the C52:2/C54:3 ratio was observed with all dose regimes and a good correlation was obtained between liver and plasma samples. In a separate study, the same compounds were dosed to LXR α and LXR β knock-out (KO) mice at 30 mg kg⁻¹, once daily, for 5 days. The LXR β KO mice showed similar TAG ratio changes to the C57 mice, whereas the LXR α KO mice showed no change in TAG ratios versus vehicle dosed animals. Measurements of lipid liability in response to an LXR agonist are typically made by measuring total liver TAG levels, which here, only showed a significant effect after the 48 H and 5 day dose regimes. By using a ratio measurement analysis could be performed on plasma samples, greatly simplifying the sample preparation procedure, without the requirement for either calibration curves or an internal standard.     

微藻三酰甘油合成途径及其最新研究进展

三酰甘油(triacylglycerols,TAGs)是动物、植物、微生物和微藻细胞主要的储藏性脂类,它可应用于食品、轻工业和生物燃料等方面,是一种新型可再生能源——生物柴油生产的重要原料。与高等油料作物相比,微藻具有光合作用效率高、生长速度快、油脂产量高、不占用农业耕地和适应多种生长环境等优势,是一种潜在的新型生物柴油生产原料。然而,目前人们对有机体,尤其是微藻细胞内TAG合成与积累的分子机制及细胞的代谢调控机制还知之甚少。对TAG合成的一系列重要过程,包括脂肪酸的合成,TAG生物合成的主要途径和旁路途径,以及与TAG合成相关的关键酶和重要基因等进行了综述,特别对微藻细胞中与TAG合成相关的关键基因的最新研究进展进行了总结,旨在更好地了解油脂代谢的调控途径,为最大限度地供应生物柴油的生产原料提供理论基础。     

Sequential one-step extraction and analysis of triacylglycerols and fatty acids in plant tissues

A method for plant tissue digestion and triacylglycerol (TAG) extraction followed by transmethylation of TAGs to produce the fatty acid methyl esters (FAMEs) from small storage tissue samples is presented. The method allows the analysis of both TAGs and FAMEs from the same sample. Several reagent mixtures and different experimental conditions were tested on sliced sunflower seeds. The best results were obtained using a mixture that was 33.3% a solution of NaCl (0.17 M) in methanol and 66.6% heptane by volume. The TAGs in the heptane solution were transmethylated with a mixture containing methanol:toluene:dimethoxypropane:H(4)SO(2) (39:20:5:2, by vol). The method was also tested on other oil seed storage tissue (soybean) and fruit tissues from olive and acorn. In all cases, sunflower, soybean, olive, and acorn, the TAGs and FAMEs composition data obtained by this method were quite similar to data from a standard analysis method. In samples with high protein content, such as soybean and sunflower seeds, the TAG extraction was incomplete. The water content of fruit samples did not interfere with TAG extraction obtained by this method.     

Increased glucose oxidation in H-FABP null soleus muscle is associated with defective triacylglycerol accumulation and mobilization, but not with the defect of exogenous fatty acid oxidation

Heart-type fatty acid-binding protein (H-FABP) is a major fatty acid-binding factor in skeletal muscles. Genetic lack of H-FABP severely impairs the esterification and oxidation of exogenous fatty acids in soleus muscles isolated from chow-fed mice (CHOW-solei) and high fat diet-fed mice (HFD-solei), and prevents the HFD-induced accumulation of muscle triacylglycerols (TAGs). Here, we examined the impact of H-FABP deficiency on the relationship between fatty acid utilization and glucose oxidation. Glucose oxidation was measured in isolated soleus muscles in the presence or absence of 1 mM palmitate (simple protocol) or in the absence of fatty acid after preincubation with 1 mM palmitate (complex protocol). With the simple protocol, the mutation slightly reduced glucose oxidation in CHOW-muscles, but markedly increased it in HFD-muscles; unexpectedly, this pattern was not altered by the addition of palmitate, which reduced glucose oxidation in both CHOW- and HFD-solei irrespective of the mutation. In the complex protocol, the mutation first inhibited the synthesis and accumulation of TAGs and then their mobilization; with this protocol, the mutation increased glucose oxidation in both CHOW- and HFD-solei. We conclude: (i) H-FABP mediates a non-acute inhibition of muscle glucose oxidation by fatty acids, likely by enabling both the accumulation and mobilization of a critical mass of muscle TAGs; (ii) H-FABP does not mediate the acute inhibitory effect of extracellular fatty acids on muscle glucose oxidation; (iii) H-FABP affects muscle glucose oxidation in opposing ways, with inhibition prevailing at high muscle TAG contents.     

Metabolic engineering of oilseed crops to produce high levels of novel acetyl glyceride oils with reduced viscosity,freezing point and calorific value

Seed oils have proved recalcitrant to modification for the production of industrially useful lipids. Here, we demonstrate the successful metabolic engineering and subsequent field production of an oilseed crop with the highest accumulation of unusual oil achieved so far in transgenic plants. Previously, expression of the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene in wild‐type Arabidopsis seeds resulted in the accumulation of 45 mol% of unusual 3‐acetyl‐1,2‐diacyl‐sn‐glycerols (acetyl‐TAGs) in the seed oil (Durrett et al., 2010 PNAS 107:9464). Expression of EaDAcT in dgat1 mutants compromised in their ability to synthesize regular triacylglycerols increased acetyl‐TAGs to 65 mol%. Camelina and soybean transformed with the EaDAcT gene accumulate acetyl‐triacylglycerols (acetyl‐TAGs) at up to 70 mol% of seed oil. A similar strategy of coexpression of EaDAcT together with RNAi suppression of DGAT1 increased acetyl‐TAG levels to up to 85 mol% in field‐grown transgenic Camelina. Additionally, total moles of triacylglycerol (TAG) per seed increased 20%. Analysis of the acetyl‐TAG fraction revealed a twofold reduction in very long chain fatty acids (VLCFA), consistent with their displacement from the sn‐3 position by acetate. Seed germination remained high, and seedlings were able to metabolize the stored acetyl‐TAGs as rapidly as regular triacylglycerols. Viscosity, freezing point and caloric content of the Camelina acetyl‐TAG oils were reduced, enabling use of this oil in several nonfood and food applications.     

Triacylglycerol accumulation of Phaeodactylum tricornutum with different supply of inorganic carbon

The diatom Phaeodactylum tricornutum produces large quantities of lipids, especially triacylglycerols (TAGs) under nitrogen or phosphorus limitation. In this study, production of lipids and TAGs during this process was compared under conditions with different inputs of inorganic carbon. With an abundant supply of inorganic carbon, considerable accumulation of biomass, lipids, and TAGs was identified after a nitrogen/phosphorus-limiting “induction incubation.” TAGs were still synthesized and accumulated even under inorganic carbon limitation with a cessation in the production of biomass and cellular lipids. This part of accumulated TAGs could be synthesized through recycling and transformation of other lipids such as glycolipids and phospholipids. Additionally, some alterations in the fatty acid profile following TAG accumulation were found. The content of the C16:0 fatty acid increased with decreases in C16:3 and C20:5, which could have been caused by enzymatic selectivity for these fatty acids during the process of TAG synthesis. It was concluded that nitrogen and phosphorus metabolism regulates the synthesis of TAG, while carbon metabolism promotes it by providing sufficient substrates.     

Cytosolic triacylglycerol biosynthetic pathway in oilseeds. Molecular cloning and expression of peanut cytosolic diacylglycerol acyltransferase

Triacylglycerols (TAGs) are the most important storage form of energy for eukaryotic cells. TAG biosynthetic activity was identified in the cytosolic fraction of developing peanut (Arachis hypogaea) cotyledons. This activity was NaF insensitive and acyl-coenzyme A (CoA) dependent. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step in TAG biosynthesis that acylates diacylglycerol to TAG. Soluble DGAT was identified from immature peanuts and purified by conventional column chromatographic procedures. The enzyme has a molecular mass of 41 +/- 1.0 kD. Based on the partial peptide sequence, a degenerate probe was used to obtain the full-length cDNA. The isolated gene shared less than 10% identity with the previously identified DGAT1 and 2 families, but has 13% identity with the bacterial bifunctional wax ester/DGAT. To differentiate the unrelated families, we designate the peanut gene as AhDGAT. Expression of peanut cDNA in Escherichia coli resulted in the formation of labeled TAG and wax ester from [14C]acetate. The recombinant E. coli showed high levels of DGAT activity but no wax ester synthase activity. TAGs were localized in transformed cells with Nile blue A and oil red O staining. The recombinant and native DGAT was specific for 1,2-diacylglycerol and did not utilize hexadecanol, glycerol-3-phosphate, monoacylglycerol, lysophosphatidic acid, and lysophosphatidylcholine. Oleoyl-CoA was the preferred acyl donor as compared to palmitoyl- and stearoyl-CoAs. These data suggest that the cytosol is one of the sites for TAG biosynthesis in oilseeds. The identified pathway may present opportunities of bioengineering oil-yielding plants for increased oil production.     

Effects of palm oil and transesterified palm oil on chylomicron and VLDL triacylglycerol structures and postprandial lipid response

The effects of positional distribution of triacylglycerol (TAG) fatty acids to TAG structures in chylomicrons and VLDL, and to postprandial lipemia, were studied in 10 healthy premenopausal women using a 6-h oral fat load test and a randomized, double-blind cross-over design. Molecular level information of TAG regioisomerism was obtained with a tandem mass spectrometric method. The positional distribution of fatty acids in chylomicron TAGs was similar to the respective dietary fat; 79% of the analyzed regioisomers in palm oil and 84% of the analyzed regioisomers in transesterified oil were found in chylomicron TAGs 3 h after the oral fat loads. VLDL TAGs were equal after the two fat loads in all but one regioisomer. Similarities in the fatty acid compositions of chylomicron TAGs suggest that palmitic acid was absorbed equally from both test fats. The proportion of palmitoleic acid in the chylomicrons was increased. Fat with palmitic acid predominantly in the sn-1 and sn-3 positions caused a larger incremental area of total TAGs in plasma and reduced plasma insulin values at the beginning of the postprandial response (0-90 min) compared with fat with palmitic acid randomly distributed. The relationship between TAG molecular structures in dietary fats and in lipoproteins provides new means for understanding the effects of fatty acid positional distribution on human lipid metabolism.