Involvement of low molecular mass soluble acyl-CoA-binding protein in seed oil biosynthesis

Acyl-CoA-binding protein (ACBP), a low molecular mass (m) (～ 10 kDa) soluble protein ubiquitous in eukaryotes, plays an important housekeeping role in lipid metabolism by maintaining the intracellular acyl-CoA pool. ACBP is involved in lipid biosynthesis and transport, gene expression, and membrane biogenesis. In plants, low m ACBP and high m ACBPs participate in response mechanisms to biotic and abiotic factors, acyl-CoA transport in phloem, and biosynthesis of structural and storage lipids. In light of current research on the modification of seed oil, insight into mechanisms of substrate trafficking within lipid biosynthetic pathways is crucial for developing rational strategies for the production of specialty oils with the desired alterations in fatty acid composition. In this review, we summarize our knowledge of plant ACBPs with emphasis on the role of low m ACBP in seed oil biosynthesis, based on in vitro studies and analyses of transgenic plants. Future prospects and possible applications of low m ACBP in seed oil modification are discussed.     

Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii: with focus on proteins involved in lipid metabolism

Oil bodies are sites of energy and carbon storage in many organisms including microalgae. As a step toward deciphering oil accumulation mechanisms in algae, we used proteomics to analyze purified oil bodies from the model microalga Chlamydomonas reinhardtii grown under nitrogen deprivation. Among the 248 proteins (≥ 2 peptides) identified by LC-MS/MS, 33 were putatively involved in the metabolism of lipids (mostly acyl-lipids and sterols). Compared with a recently reported Chlamydomonas oil body proteome, 19 new proteins of lipid metabolism were identified, spanning the key steps of the triacylglycerol synthesis pathway and including a glycerol-3-phosphate acyltransferase (GPAT), a lysophosphatidic acid acyltransferase (LPAT) and a putative phospholipid:diacylglycerol acyltransferase (PDAT). In addition, proteins putatively involved in deacylation/reacylation, sterol synthesis, lipid signaling and lipid trafficking were found to be associated with the oil body fraction. This data set thus provides evidence that Chlamydomonas oil bodies are not only storage compartments but also are dynamic structures likely to be involved in processes such as oil synthesis, degradation and lipid homeostasis. The proteins identified here should provide useful targets for genetic studies aiming at increasing our understanding of triacyglycerol synthesis and the role of oil bodies in microalgal cell functions.     

Expression, purification and functional characterization of recombinant human acyl-CoA-binding protein (ACBP) from erythroid cells

Fatty acyl-CoA esters are extremely important in cellular homeostasis. They are intermediates in both lipid metabolism and post-translational protein modifications. Among these modification events, protein palmitoylation seems to be unique by its reversibility which allows dynamic regulation of the protein hydrophobicity. The recent discovery of an enzyme family that catalyze protein palmitoylation has increased the understanding of the enzymology of the covalent attachment of fatty acids to proteins. Despite that, the molecular mechanism of supplying acyl-CoA esters to this reaction is yet to be established. Acyl-coenzyme A-binding proteins are known to bind long-chain acyl-CoA esters with very high affinity. Therefore, they play a significant role in intracellular acyl-CoA transport and pool formation. The purpose of this work is to explore the potential of one of the acyl-CoA-binding proteins to participate in the protein palmitoylation. In this study, a recombinant form of ACBP derived from human erythroid cells was expressed in E. coli, purified, and functionally characterized. We demonstrate that recombinant hACBP effectively binds palmitoyl-CoA in vitro, undergoing a shift from a monomeric to a dimeric state, and that this ligand-binding ability is involved in erythrocytic membrane phosphatidylcholine (PC) remodeling but not in protein acylation.     

Phosphatidylcholine synthesis and its catabolism by yeast neuropathy target esterase 1

Phosphatidylcholine (PtdCho) is the major phospholipid component of eukaryotic membranes and deciphering the molecular mechanisms regulating PtdCho homeostasis is necessary to fully understand many pathophysiological situations where PtdCho metabolism is altered. This concept is illustrated in this review by summarizing recent evidence on Nte1p, a yeast endoplasmic reticulum resident phospholipase B that deacylates PtdCho producing intracellular glycerophosphocholine. The mammalian and Drosophila homologues, neuropathy target esterase and swiss cheese, respectively, have been implicated in normal brain development with increased intracytoplasmic vesicularization and multilayered membrane stacks as cytological signatures of their absence. Consistent with a role in lipid and membrane homeostasis, Nte1p-mediated PtdCho deacylation is strongly affected by Sec14p, a component of the yeast secretory machinery characterized by its ability to interface between lipid metabolism and vesicular trafficking. The preference of Nte1p toward PtdCho produced through the CDP-choline pathway and the downstream production of choline by the Gde1p glycerophosphodiesterase for resynthesis of PtdCho by the CDP-choline pathway are also highlighted.     

Metabolism of apical versus basolateral sn-2-monoacylglycerol and fatty acids in rodent small intestine

The metabolic fates of radiolabeled sn-2-monoacylglycerol (MG) and oleate (FA) in rat and mouse intestine, added in vivo to the apical (AP) surface in bile salt micelles, or to the basolateral (BL) surface via albumin-bound solution, were examined. Mucosal lipid products were quantified, and the results demonstrate a dramatic difference in the esterification patterns for both MG and FA, depending upon their site of entry into the enterocyte. For both lipids, the ratio of triacylglycerol to phospholipid (TG:PL) formed was approximately 10-fold higher for delivery at the AP relative to the BL surface. Further, a 3-fold higher level of FA oxidation was found for BL compared with AP substrate delivery. Incorporation of FA into individual PL species was also significantly different, with >2-fold greater incorporation into phosphatidylethanolamine (PE) and a 3-fold decrease in the phosphatidylcholine:PE ratio for AP- compared with BL-added lipid. Overnight fasting increased the TG:PL incorporation ratio for both AP and BL lipid addition, suggesting that metabolic compartmentation is a physiologically regulated phenomenon. These results support the existence of separate pools of TG and glycerolipid intermediates in the intestinal epithelial cell, and underscore the importance of substrate trafficking in the regulation of enterocyte lipid metabolism.     

Electrospray ionization mass spectrometry and exogenous heavy isotope-labeled lipid species provide detailed information on aminophospholipid acyl chain remodeling

Mammalian cells maintain the phospholipid compositions of their different membranes remarkably constant. Beside de novo synthesis, degradation, and intracellular trafficking, acyl chain remodeling plays an important role in phospholipid homeostasis. However, many key details of this process remain unresolved, largely because of limitations of existing methodologies. Here we describe a novel approach that allows one to study metabolism of individual phospholipid species in unprecedented detail. Forty different phosphatidylethanolamine (PE) or -serine (PS) species with a deuterium-labeled head group were synthesized and introduced to BHK21 or HeLa cells using cyclodextrin-mediated transfer. Their metabolism was then monitored in detail by electrospray ionization mass spectrometry. Atypical PE and PS species were rapidly remodeled at both sn1 and sn2 position, yielding a molecular species profile similar to that the endogenous PE and PS. In contrast, remodeling of exogenous species identical or similar to major endogenous ones was more limited and much slower. Major differences in remodeling pathways and kinetics were observed between species within a class, as well as between corresponding PE and PS species. These data along with those obtained with pharmacological inhibitors strongly suggest that multiple lipid class-specific A-type phospholipases and acyl transferases are involved in aminophospholipid remodeling. In conclusion, the approach described here provides highly detailed information on remodeling of exogenously added (amino)glycerophospholipids and should thus be very helpful when elucidating the proteins and processes maintaining molecular species homeostasis.     

Effect of slaughter age and feeding system on the neutral and polar lipid composition of horse meat

This study was undertaken to provide a thorough analysis of the neutral lipid (NL) and polar lipid (PL) fractions of horse meat that included the content and distribution of acyl and alkenyl moieties in foals under different rearing conditions. Two groups of crossbred horses were studied; the first group was selected from suckling foals produced under grazing conditions and slaughtered at 4 months of age (n=8), and the second group was selected from concentrate-finished foals and slaughtered at 12 months of age (n=7). There were significant differences related to the age and feeding practices of foals which affected the intramuscular (IM) fat content and the fatty acid (FA) composition of NL and PL fractions. Samples from suckling foals were leaner and provided the highest content of methylation products from the plasmalogenic lipids, and total and n-3 polyunsaturated fatty acid (PUFA). By contrast, the meat from concentrate-finished foals had a higher IM fat level resulting in a greater accumulation of 16:0 and total monounsaturated FAs in the NL fraction, whereas the muscle PL fraction retained a similar FA composition between both groups. Linolenic acid was preferentially deposited in the NL fraction, but linoleic acid and the long-chain n-3 and n-6 PUFAs were incorporated into the PL fraction where they served as cell membrane constituents and in eicosanoid formation.     

Good Fat, Essential Cellular Requirements for Triacylglycerol Synthesis to Maintain Membrane Homeostasis in Yeast

Storage triacylglycerols (TAG) and membrane phospholipids share common precursors, i.e. phosphatidic acid and diacylglycerol, in the endoplasmic reticulum. In addition to providing a biophysically rather inert storage pool for fatty acids, TAG synthesis plays an important role to buffer excess fatty acids (FA). The inability to incorporate exogenous oleic acid into TAG in a yeast mutant lacking the acyltransferases Lro1p, Dga1p, Are1p, and Are2p contributing to TAG synthesis results in dysregulation of lipid synthesis, massive proliferation of intracellular membranes, and ultimately cell death. Carboxypeptidase Y trafficking from the endoplasmic reticulum to the vacuole is severely impaired, but the unfolded protein response is only moderately up-regulated, and dispensable for membrane proliferation, upon exposure to oleic acid. FA-induced toxicity is specific to oleic acid and much less pronounced with palmitoleic acid and is not detectable with the saturated fatty acids, palmitic and stearic acid. Palmitic acid supplementation partially suppresses oleic acid-induced lipotoxicity and restores carboxypeptidase Y trafficking to the vacuole. These data show the following: (i) FA uptake is not regulated by the cellular lipid requirements; (ii) TAG synthesis functions as a crucial intracellular buffer for detoxifying excess unsaturated fatty acids; (iii) membrane lipid synthesis and proliferation are responsive to and controlled by a balanced fatty acid composition.     

Mechanisms and functions of membrane lipid remodeling in plants

Lipid remodeling, defined herein as post-synthetic structural modifications of membrane lipids, play crucial roles in regulating the physicochemical properties of cellular membranes and hence their many functions. Processes affected by lipid remodeling include lipid metabolism, membrane repair, cellular homeostasis, fatty acid trafficking, cellular signaling and stress tolerance. Glycerolipids are the major structural components of cellular membranes and their composition can be adjusted by modifying their head groups, their acyl chain lengths and the number and position of double bonds. This review summarizes recent advances in our understanding of mechanisms of membrane lipid remodeling with emphasis on the lipases and acyltransferases involved in the modification of phosphatidylcholine and monogalactosyldiacylglycerol, the major membrane lipids of extraplastidic and photosynthetic membranes, respectively. We also discuss the role of triacylglycerol metabolism in membrane acyl chain remodeling. Finally, we discuss emerging data concerning the functional roles of glycerolipid remodeling in plant stress responses. Illustrating the molecular basis of lipid remodeling may lead to novel strategies for crop improvement and other biotechnological applications such as bioenergy production.     

Fatty acid transport by vectorial acylation in mammals: roles played by different isoforms of rat long-chain acyl-CoA synthetases

Mammals express multiple isoforms of acyl-CoA synthetase (ACSL1 and ACSL3-6) in various tissues. These enzymes are essential for fatty acid metabolism providing activated intermediates for complex lipid synthesis, protein modification, and beta-oxidation. Yeast in contrast express four major ACSLs, which have well-defined functions. Two, Faa1p and Faa4p, are specifically required for fatty acid transport by vectorial acylation. Four ACSLs from the rat were expressed in a yeast faa1delta faa4delta strain and their roles in fatty acid transport and trafficking characterized. All four restored ACS activity yet varied in substrate preference. ACSL1, 4, and 6 were able to rescue fatty acid transport activity and triglyceride synthesis. ACSL5, however, was unable to facilitate fatty acid transport despite conferring robust oleoyl-CoA synthetase activity. This is the first study evaluating the role of the mammalian ACSLs in fatty acid transport and supports a role for ACSL1, 4, and 6 in transport by vectorial acylation.     

The influence of dietary essential fatty acids on uterine C20 and C22 fatty acid composition.

The effect of dietary fatty acids on uterine fatty acid composition was studied in rats fed control diet or semi-synthetic diet supplemented with 1.5 microliter/g/day evening primrose oil (EPO) or fish oil (FO). Diet-related changes in uterine lipid were detected within 21 days. Changes of 2- to 20-fold were detected in the uterine n-6 and n-3 essential fatty acids (EFA) and in certain saturated and monounsaturated fatty acids. The FO diet was associated with higher uterine C20 and C22 n-3, and the EPO diet, with higher uterine n-6 fatty acid. High uterine C18:2 n-6 was detected in neutral lipid (NL) of rats fed high concentrations of this fatty acid, but there was little evidence of selective incorporation or retention of C18:2 n-6 by uterine NL. The incorporation of EFA into uterine phospholipids (PL) was greater than NL EFA incorporation, and uterine PL n-3/n-6 ratios showed greater diet dependence. Tissue/diet fatty acid ratios in NL and PL also indicated preferential incorporation/synthesis of C16:1 n-9, and C16:0, and there was greater incorporation of C12:0 and C14:0 into uteri of rats fed EPO and FO. Replacement of 50-60% of arachidonate with n-3 EFA in uterine PL may inhibit n-6 EFA metabolism necessary for uterine function at parturition.     

PPARA/RXRA signalling regulates the fate of hepatic non-esterified fatty acids in a sheep model of maternal undernutrition

Maternal undernutrition during late gestation accelerates body fat mobilization to provide more energy for foetal growth and development, which unbalances metabolic homeostasis and results in serious lipid metabolism disorder. However, detailed regulatory mechanisms are poorly understood. Here, a sheep model was used to explore the regulatory role of PPARA/RXRA signalling in hepatic lipid metabolism in undernutrition based on RNA sequencing and cell experiments. KOG function classification showed that lipid transport and metabolism was markedly altered in an undernourished model. In detail, when compared with the controls, fatty acid transport and oxidation and triglyceride metabolism were up-regulated in an undernourished model, while fatty acid synthesis, steroid synthesis, and phospholipid metabolism were down-regulated. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis demonstrated that PPARA/RXRA signalling pathway was altered. Moreover, PPARA signalling associated genes were positively correlated with hepatic non-esterified fatty acid (NEFA) levels, while retinol metabolism associated genes were negatively correlated with blood beta-hydroxybutyric acid (BHBA) levels. Results of primary hepatocytes showed that NEFAs could activate PPARA signalling and facilitate fatty acid oxidation (FAO) and ketogenesis, while BHBA could inhibit RXRA signalling and repress FAO and ketogenesis. Excessively accumulated NEFAs in hepatocytes promoted triglyceride synthesis. Furthermore, activation of PPARA/RXRA signalling by WY14643 and 9-cis-retinoic acid could enhance FAO and ketogenesis and reduce NEFAs accumulation and esterification. Our findings elucidate the regulatory mechanisms of NEFAs and BHBA on lipid metabolism as well as the potential role of the PPARA/RXRA signalling pathway in hepatic lipid metabolism, which may contribute to exploring new strategies to maintain lipid metabolic homeostasis in human beings.     

Regulation of adiponectin on lipid metabolism in large yellow croaker (Larimichthys crocea)

Adiponectin (APN), an adipose tissue-derived hormone, plays a key role in regulating energy metabolism in mammals. However, its physiological roles in teleosts remain poorly understood. In the present study, the apn gene was cloned from large yellow croaker, which was mainly expressed in the adipose, muscle and liver. Further studies showed that adaptor protein phosphotyrosine interaction PH domain and leucine zipper 1 (APPL1) was localized in the cytoplasm near the cell membrane and was directly bounded to adiponectin receptors (AdipoRs). Meanwhile, APN played a crucial role in lipid metabolism of primary muscle cells by promoting the synthesis, oxidation and transport of fatty acids, and the promoting effects were blocked by knockdown of appl1 and AdipoRs. Furthermore, the activation/inhibition of peroxisome proliferators activated receptor γ (PPARγ) enhanced/suppressed the APN-mediated lipid metabolism. Overall, results showed that APN mediated lipid metabolism through AdipoRs-APPL1 activated PPARγ and further regulated the synthesis, oxidation and transport of FA. This study will facilitate the investigation of APN functions in lipid metabolism and energy homeostasis and reveal the evolution of lipids utilization and energy homeostasis in vertebrates.     

Fatty acid synthesis is critical for stem cell pluripotency via promoting mitochondrial fission

Pluripotent stem cells are known to display distinct metabolic phenotypes than their somatic counterparts. While accumulating studies are focused on the roles of glucose and amino acid metabolism in facilitating pluripotency, little is known regarding the role of lipid metabolism in regulation of stem cell activities. Here, we show that fatty acid (FA) synthesis activation is critical for stem cell pluripotency. Our initial observations demonstrated enhanced lipogenesis in pluripotent cells and during cellular reprogramming. Further analysis indicated that de novo FA synthesis controls cellular reprogramming and embryonic stem cell pluripotency through mitochondrial fission. Mechanistically, we found that de novo FA synthesis regulated by the lipogenic enzyme ACC1 leads to the enhanced mitochondrial fission via (i) consumption of AcCoA which affects acetylation‐mediated FIS1 ubiquitin–proteasome degradation and (ii) generation of lipid products that drive the mitochondrial dynamic equilibrium toward fission. Moreover, we demonstrated that the effect of Acc1 on cellular reprogramming via mitochondrial fission also exists in human iPSC induction. In summary, our study reveals a critical involvement of the FA synthesis pathway in promoting ESC pluripotency and iPSC formation via regulating mitochondrial fission.     

Identification and characterization of a major liver lysophosphatidylcholine acyltransferase

Phosphatidylcholine (PC) is synthesized through the Kennedy pathway, but more than 50% of PC is remodeled through the Lands cycle, i.e. the deacylation and reacylation of PC to attain the final and proper fatty acids within PC. The reacylation step is catalyzed by lysophosphatidylcholine acyltransferase (LPCAT), and we report here the identification of a novel LPCAT, which we named LPCAT3. LPCAT3 belongs to the membrane-bound O-acyltransferase (MBOAT) family and encodes a protein of 487 amino acids with a calculated molecular mass of 56 kDa. Membranes from HEK293 cells overexpressing LPCAT3 showed significantly increased LPCAT activity as assessed by thin layer chromatography analysis with substrate preference toward unsaturated fatty acids. LPCAT3 is localized within the endoplasmic reticulum and is primarily expressed in metabolic tissues including liver, adipose, and pancreas. In a human hepatoma Huh7 cells, RNA interference-mediated knockdown of LPCAT3 resulted in virtually complete loss of membrane LPCAT activity, suggesting that LPCAT3 is primarily responsible for hepatic LPCAT activity. Furthermore, peroxisome proliferator-activated receptor alpha agonists dose-dependently regulated LPCAT3 in liver in a peroxisome proliferator-activated receptor alpha-dependent fashion, implicating a role of LPCAT3 in lipid homeostasis. Our studies identify a long-sought enzyme that plays a critical role in PC remodeling in metabolic tissues and provide an invaluable tool for future investigations on how PC remodeling may potentially impact glucose and lipid homeostasis.     

Overexpression of rat long chain acyl-coa synthetase 1 alters fatty acid metabolism in rat primary hepatocytes

Long chain acyl-CoA synthetases (ACSL) activate fatty acids (FA) and provide substrates for both anabolic and catabolic pathways. We have hypothesized that each of the five ACSL isoforms partitions FA toward specific downstream pathways. Acsl1 mRNA is increased in cells under both lipogenic and oxidative conditions. To elucidate the role of ACSL1 in hepatic lipid metabolism, we overexpressed an Acsl1 adenovirus construct (Ad-Acsl1) in rat primary hepatocytes. Ad-ACSL1, located on the endoplasmic reticulum but not on mitochondria or plasma membrane, increased ACS specific activity 3.7-fold. With 100 or 750 mum [1-(14)C]oleate, Ad-Acsl1 increased oleate incorporation into diacylglycerol and phospholipids, particularly phosphatidylethanolamine and phosphatidylinositol, and decreased incorporation into cholesterol esters and secreted triacylglycerol. Ad-Acsl1 did not alter oleate incorporation into triacylglycerol, beta-oxidation products, or total amount of FA metabolized. In pulse-chase experiments to examine the effects of Ad-Acsl1 on lipid turnover, more labeled triacylglycerol and phospholipid, but less labeled diacylglycerol, remained in Ad-Acsl1 cells, suggesting that ACSL1 increased reacylation of hydrolyzed oleate derived from triacylglycerol and diacylglycerol. In addition, less hydrolyzed oleate was used for cholesterol ester synthesis and beta-oxidation. The increase in [1,2,3-(3)H]glycerol incorporation into diacylglycerol and phospholipid was similar to the increase with [(14)C]oleate labeling suggesting that ACSL1 increased de novo synthesis. Labeling Ad-Acsl1 cells with [(14)C]acetate increased triacylglycerol synthesis but did not channel endogenous FA away from cholesterol ester synthesis. Thus, consistent with the hypothesis that individual ACSLs partition FA, Ad-Acsl1 increased FA reacylation and channeled FA toward diacylglycerol and phospholipid synthesis and away from cholesterol ester synthesis.     

Effect of weak constant magnetic field on the composition and content of lipids in radish seedlings at various temperatures

We studied the effects of weak permanent homogenous hirizontal magnetic field (PMF) (400 A/m) on the composition and content of lipids and composition of their fatty acids (FAs) in radish (Raphanus sativus L. var. radicula D.C., cv. Rosovo-krashyi s belym konchikom) seedlings at temperatures of 20 and 10°C. We compared lipid composition and content in seedlings at the phase of developed cotyledons (20°C, 5-day-old, and 10°C, 8-day-old seedlings) under low light and in darkness with the lipid composition and content in dry seeds. The seedlings grown in geomagnetic field (GMF) served as a control. In dry seeds, about 99% of total lipids comprised neutral lipids (NL) and only 1% were polar lipids (PL). Triacylglycerols predominated among NL comprising 93% of total seed lipids. During seed germination, NLs were consumed and PL were produced: the amount of glycolipids increased in control by 3.5–5 times and the amount of phospholipis, by 1.5–2 times.In the light at 20°C, PMF suppressed the formation of PL (by 18%), whereas in darkness, it stimulated it approximately by 80% as compared with control. In the light at 10°C, PMF slightly stimulated PL formation; in darkness, it did not almost affect their synthesis. In all treatments, PMF increased the ratio of phospholipids to sterols by 30–100%. Among FA, PMF exerted the strongest effect on the content of erucic acid: it increased in the light and in darkness at 20°C approximately by 25% and decreased at 10°C in the light by 13%. PMF behaved as a correction factor affecting lipid metabolism on the background of light and temperature action.