Misexpression of FATTY ACID ELONGATION1 in the Arabidopsis Epidermis Induces Cell Death and Suggests a Critical Role for Phospholipase A2 in This Process

Very-long-chain fatty acids (VLCFAs) are important functional components of various lipid classes, including cuticular lipids in the higher plant epidermis and lipid-derived second messengers. Here, we report the characterization of transgenic Arabidopsis thaliana plants that epidermally express FATTY ACID ELONGATION1 (FAE1), the seed-specific β-ketoacyl-CoA synthase (KCS) catalyzing the first rate-limiting step in VLCFA biosynthesis. Misexpression of FAE1 changes the VLCFAs in different classes of lipids but surprisingly does not complement the KCS fiddlehead mutant. FAE1 misexpression plants are similar to the wild type but display an essentially glabrous phenotype, owing to the selective death of trichome cells. This cell death is accompanied by membrane damage, generation of reactive oxygen species, and callose deposition. We found that nuclei of arrested trichome cells in FAE1 misexpression plants cell-autonomously accumulate high levels of DNA damage, including double-strand breaks characteristic of lipoapoptosis. A chemical genetic screen revealed that inhibitors of KCS and phospholipase A2 (PLA2), but not inhibitors of de novo ceramide biosynthesis, rescue trichome cells from death. These results support the functional role of acyl chain length of fatty acids and PLA2 as determinants for programmed cell death, likely involving the exchange of VLCFAs between phospholipids and the acyl-CoA pool.     

Lipoapoptosis: its mechanism and its diseases

The balance between cell division and cell death determines the cell population of an organ. When cell death exceeds cell replacement in an organ, a functional deficit is created. A metabolic cause of programmed cell death, lipoapoptosis, has recently been identified to occur in obesity and aging. If nonadipose tissues are exposed to an excess of long-chain fatty acids, unless leptin action increases their oxidation sufficiently, unoxidized fatty acids enter nonoxidative pathways. While initially they are sequestered as harmless neutral fat, ultimately some will enter more toxic pathways. One of these, the de novo ceramide pathway, has been implicated in the lipoapoptosis of beta-cells and myocardiocytes of congenitally obese rats in which leptin action is defective. Here we review the mechanisms of lipoapoptosis and the diseases that result from this cause of a diminishing cell population of these organs. We suggest that some of the components of the metabolic syndrome of obese humans and the sarcopenia of aging may be result of failure of leptin liporegulation to prevent lipid overload of lean body mass and lipoapoptosis in certain organ systems.     

Long-chain fatty acid transport in bacteria andyeast. Paradigms for defining the mechanism underlying this protein-mediated process

Protein-mediated transport of exogenous long-chain fatty acids across the membrane has been defined in a number of different systems. Central to understanding the mechanism underlying this process is the development of the appropriate experimental systems which can be manipulated using the tools of molecular genetics. Escherichia coli and Saccharomyces cerevisiae are ideally suited as model systems to study this process in that both [1] exhibit saturable long-chain fatty acid transport at low ligand concentration; [2] have specific membrane-bound and membrane-associated proteins that are components of the transport apparatus; and [3] can be easily manipulated using the tools of molecular genetics. In E. coli, this process requires the outer membrane-bound fatty acid transport protein FadL and the inner membrane associated fatty acyl CoA synthetase (FACS). FadL appears to represent a substrate specific channel for long-chain fatty acids while FACS activates these compounds to CoA thioesters thereby rendering this process unidirectional. This process requires both ATP generated from either substrate-level or oxidative phosphorylation and the proton electrochemical gradient across the inner membrane. In S. cerevisiae, the process of long-chain fatty acid transport requires at least the membrane-bound protein Fat1p. Exogenously supplied fatty acids are activated by the fatty acyl CoA synthetases Faa1p and Faa4p but unlike the case in E. coli, there is not a tight linkage between transport and activation. Studies evaluating the growth parameters in the presence of long-chain fatty acids and long-chain fatty acid transport profiles of a fat1 strain support the hypothesis that Fat1p is required for optimal levels of long-chain fatty acid transport.     

Fish Oil Supplementation Alters the Plasma Lipidomic Profile and Increases Long-Chain PUFAs of Phospholipids and Triglycerides in Healthy Subjects

Background

While beneficial health effects of fish and fish oil consumption are well documented, the incorporation of n-3 polyunsaturated fatty acids in plasma lipid classes is not completely understood. The aim of this study was to investigate the effect of fish oil supplementation on the plasma lipidomic profile in healthy subjects.

Methodology/Principal Findings

In a double-blinded randomized controlled parallel-group study, healthy subjects received capsules containing either 8 g/d of fish oil (FO) (1.6 g/d EPA+DHA) (n = 16) or 8 g/d of high oleic sunflower oil (HOSO) (n = 17) for seven weeks. During the first three weeks of intervention, the subjects completed a fully controlled diet period. BMI and total serum triglycerides, total-, LDL- and HDL-cholesterol were unchanged during the intervention period. Lipidomic analyses were performed using Ultra Performance Liquid Chromatography (UPLC) coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (QTOFMS), where 568 lipids were detected and 260 identified. Both t-tests and Multi-Block Partial Least Square Regression (MBPLSR) analysis were performed for analysing differences between the intervention groups. The intervention groups were well separated by the lipidomic data after three weeks of intervention. Several lipid classes such as phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, sphingomyelin, phosphatidylserine, phosphatidylglycerol, and triglycerides contributed strongly to this separation. Twenty-three lipids were significantly decreased (FDR<0.05) in the FO group after three weeks compared with the HOSO group, whereas fifty-one were increased including selected phospholipids and triglycerides of long-chain polyunsaturated fatty acids. After seven weeks of intervention the two intervention groups showed similar grouping.

Conclusions/Significance

In healthy subjects, fish oil supplementation alters lipid metabolism and increases the proportion of phospholipids and triglycerides containing long-chain polyunsaturated fatty acids. Whether the beneficial effects of fish oil supplementation may be explained by a remodeling of the plasma lipids into phospholipids and triglycerides of long-chain polyunsaturated fatty acids needs to be further investigated.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01034423","term_id":"NCT01034423"}}NCT01034423     

Patterns of variation in the lipid class and fatty acid composition of Nannochloropsis oculata (Eustigmatophyceae) during batch culture

Changes in the lipid and fatty acyl compositions of the marine microalga Nannochloropsis oculata Droop were examined during a batch culture growth cycle. During the early phase of batch culture the cellular proportion of triacylglycerols (TAG) increased. This was in addition to the increases in TAG observed in many microalgal species in the stationary-phase. Concomitant increases in the relative proportions of both saturated and monounsaturated fatty acids and decreases in the proportion of polyunsaturated fatty acids in total lipid were also associated with this phase. The separated individual lipid classes were found to have characteristic fatty acyl compositions. The relative proportion of lipid per cell, the relative proportions of the individual lipid classes and the fatty acyl compositions of the individual classes were all subject to variability during the growth cycle. The changing total lipid fatty acyl composition of N. oculata was found to be determined by the proportion of the total lipid present as TAG. The data suggest that the changes observed in the fatty acyl composition of N. oculata are a result of the partitioning of photosynthetically fixed carbon between polar and neutral lipid class biosynthesis and fatty acyl desaturation and elongation pathways. The effect of such a partitioning of carbon is discussed in relation to the effects of environmental variables and growth phase upon the balance of lipid class and polyunsaturated fatty acids (PUFA) synthesis in marine microalgae.     

Delay of Membrane Lipid Degradation by Calcium Treatment during Cabbage Leaf Senescence

Cabbage leaf discs (Brassica oleracea L., Capitata group) were floated adaxial side up in 0, 0.05, or 0.25 m CaCl₂ solutions at 15°C for 14 d in the dark. To assess whether the delay of senescence by calcium treatment involved protection of membrane lipids, chlorophyll and protein content and the lipid composition of the membranes were determined during incubation. Chlorophyll and protein content decreased with time, in correlation with a reduction in the amount of phospholipids. The degree of unsaturation of phospholipids and free fatty acids decreased, whereas the ratio of sterol to phospholipid increased. The proportions of phospholipid classes did not change during senescence. The catabolism of phospholipids was delayed by 0.05 m calcium, but accelerated by 0.25 m, as compared to the untreated control. Based on the levels of the lipid intermediates, phospholipase D, phosphatidic acid phosphatase, lipolytic acyl hydrolase, and lipoxygenase appeared to be involved in the breakdown of phospholipids during senescence. Phospholipase D and phosphatidic acid phosphatase may be directly influenced by calcium. The calcium treatment apparently did not affect the activity of acyl hydrolase. Lipoxygenase, responsible for the peroxidation of the polyunsaturated fatty acids, was probably indirectly influenced by calcium. We conclude that the delay of senescence of cabbage leaf discs by calcium treatment involved protection of membrane lipids from degradation.     

Stable Cell Surface Expression of GPI‐Anchored Proteins,but not Intracellular Transport,Depends on their Fatty Acid Structure

Glycosylphosphatidylinositol‐anchored proteins (GPI‐APs) are a class of lipid anchored proteins expressed on the cell surface of eukaryotes. The potential interaction of GPI‐APs with ordered lipid domains enriched in cholesterol and sphingolipids has been proposed to function in the intracellular transport of these lipid anchored proteins. Here, we examined the biological importance of two saturated fatty acids present in the phosphatidylinositol moiety of GPI‐APs. These fatty acids are introduced by the action of lipid remodeling enzymes and required for the GPI‐AP association within ordered lipid domains. We found that the fatty acid remodeling is not required for either efficient Golgi‐to‐plasma membrane transport or selective endocytosis via GPI‐enriched early endosomal compartment (GEEC)/ clathrin‐independent carrier (CLIC) pathway, whereas cholesterol depletion significantly affects both pathways independent of their fatty acid structure. Therefore, the mechanism of cholesterol dependence does not appear to be related to the interaction with ordered lipid domains mediated by two saturated fatty acids. Furthermore, cholesterol extraction drastically releases the unremodeled GPI‐APs carrying an unsaturated fatty acid from the cell surface, but not remodeled GPI‐APs carrying two saturated fatty acids. This underscores the essential role of lipid remodeling to ensure a stable membrane association of GPI‐APs particularly under potential membrane lipid perturbation.      

Lipid-induced up-regulation of human acyl-CoA synthetase 5 promotes hepatocellular apoptosis

In the pathogenesis of nonalcoholic fatty liver disease, accumulation of lipids in hepatocytes and hepatocyte apoptosis are strongly implicated in disease progression from the potentially reversible condition of steatosis to severe acute and chronic liver injury. Acyl-CoA synthetase 5, a member of the ACSL gene family that catalyzes the activation of long-chain fatty acids for lipid biosynthesis, is the only ACSL isoform that is both, located on mitochondria and functionally involved in enterocyte apoptosis. In this study, the regulation of human ACSL5 in hepatocellular fatty acid degeneration and its involvement in hepatocyte apoptosis was investigated using models of in vitro and in vivo steatosis as well as plasmid-mediated stable gene transfer and RNAi-mediated gene silencing. ACSL5 mRNA and protein were strongly increased by uptake of dietary derived fatty acids in primary human hepatocytes, HepG2 cells and human steatotic liver. Over-expression of ACSL5 decreased HepG2 cell viability and increased susceptibility to TRAIL- and TNFα-, but not FAS- induced apoptosis, whereas knock down of ACSL5 reduced apoptosis susceptibility. High ACSL5 activity resulted in enhanced caspase-3/7 activity, but was not accompanied by up-regulation of death receptors, DR4, DR5 or TNF-R1. This study gives evidence that hepatocyte steatosis is associated with ACSL5 up-regulation resulting in increased susceptibility to hepatic cell death. We propose that ACSL5 could play a role in promoting fatty acid-induced lipoapoptosis in hepatocytes as important mechanism in fatty liver-related disorders.     

Phospholipid turnover and acyl chain remodeling in the yeast ER

The turnover of phospholipids plays an essential role in membrane lipid homeostasis by impacting both lipid head group and acyl chain composition. This review focusses on the degradation and acyl chain remodeling of the major phospholipid classes present in the ER membrane of the reference eukaryote Saccharomyces cerevisiae, i.e. phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Phospholipid turnover reactions are introduced, and the occurrence and important functions of phospholipid remodeling in higher eukaryotes are briefly summarized. After presenting an inventory of established mechanisms of phospholipid acyl chain exchange, current knowledge of phospholipid degradation and remodeling by phospholipases and acyltransferases localized to the yeast ER is summarized. PC is subject to the PC deacylation-reacylation remodeling pathway (PC-DRP) involving a phospholipase B, the recently identified glycerophosphocholine acyltransferase Gpc1p, and the broad specificity acyltransferase Ale1p. PI is post-synthetically enriched in C18:0 acyl chains by remodeling reactions involving Cst26p. PE may undergo turnover by the phospholipid: diacylglycerol acyltransferase Lro1p as first step in acyl chain remodeling. Clues as to the functions of phospholipid acyl chain remodeling are discussed.     

Diabetes and apoptosis: lipotoxicity

Obesity is an established risk factor in the pathogenesis of insulin resistance, type 2 diabetes mellitus and cardiovascular disease; all components that are part of the metabolic syndrome. Traditionally, insulin resistance has been defined in a glucocentric perspective. However, elevated systemic levels of fatty acids are now considered significant contributors towards the pathophysiological aspects associated with the syndrome. An overaccumulation of unoxidized long-chain fatty acids can saturate the storage capacity of adipose tissue, resulting in a lipid ‘spill over’ to non-adipose tissues, such as the liver, muscle, heart, and pancreatic-islets. Under these circumstances, such ectopic lipid deposition can have deleterious effects. The excess lipids are driven into alternative non-oxidative pathways, which result in the formation of reactive lipid moieties that promote metabolically relevant cellular dysfunction (lipotoxicity) and programmed cell-death (lipoapoptosis). Here, we focus on how both of these processes affect metabolically significant cell-types and highlight how lipotoxicity and sequential lipoapoptosis are as major mediators of insulin resistance, diabetes and cardiovascular disease.     

Lipids of fish parasites and their hosts: fatty acids of phospholipids of Paratenuisentis ambiguus and its host eel (Anguilla anguilla)

The fatty acid composition of various phospholipid classes, e.g. phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines and sphingomyelins, of the fish endoparasite Paratenuisentis ambiguus and of intestinal tissue of its host, eel (Anguilla anguilla), were investigated. Phospholipids from parasite and infected host intestine show considerable differences in the fatty acid compositions. High proportions of saturated long-chain and very long-chain acyl moieties were found in phosphatidylcholines, ethanolamines and serines of the parasite. Membrane phospholipids of P. ambiguus contain remarkably high proportions of polyunsaturated acyl moieties, in particular eicosapentaenoyl moieties (20:5 n-3), which undoubtedly originate from aquatic feed or host intestine. It is suggested that the parasite's membranes are stabilized by incorporation of high proportions of saturated long-chain and very long-chain fatty acids to counterbalance the destabilizing effects of the polyunsaturated fatty acids.     

Lipid composition of the three co-existing <Emphasis Type="Italic">Calanus</Emphasis> species in the Arctic: impact of season,location and environment

Arctic species of Calanus are critical to energy transfer between higher and lower trophic levels and their relative abundance, and lipid content is influenced by the alternation of cold and warm years. All three species of Calanus were collected during different periods in Kongsfjorden (Svalbard, 79°N) and adjacent shelf during the abnormally warm year of 2006. Lipid composition and fatty acid structure of individual lipid classes were examined in relation with population structure. Wax esters dominated the neutral lipid fraction. Phosphatidylcholine (PC) dominated the structural lipids followed by phosphatidylethanolamine (PE). PC/PE ratios of 3–6 suggested an increase in PC proportions compared to earlier studies. Depending on the time scale, fatty acids of wax esters illustrated either trophic differences between fjord and offshore conditions for C. hyperboreus and C. finmarchicus or trophic differences related to seasonality for C. glacialis. Similarly, seasonality and trophic conditions controlled the changes in fatty acids of triglycerides, but de novo synthesis of long-chain monoenes suggested energy optimization to cope with immediate metabolic needs. Polar lipids fatty acid composition was species specific and on the long-term (comparison with data from the past decade) composition appears related to changes in trophic environment. Fatty acid composition of PC and PE indicated relative dominance of 20:5n-3 in PC and 22:6n-3 in PE for all three species. The combination of PE and PC acyl chain and phospholipid head group restructuring indicates an inter-annual variability and suggests that membrane lipids are the most likely candidate to evaluate adaptive changes in Arctic copepods to hydrothermal regime.     

Significance of cytoplasmic fatty acid-binding protein for the ischemic heart

Ischemia of the heart is accompanied by the tissue accumulation of long-chain fatty acids and their metabolic derivatives such as -hydroxy fatty acids and fatty acyl-CoA and acyl-L-carnitine esters. These substances might be detrimental for proper myocardial function. Previously, it has been suggested that intracellular lipid binding proteins like cytoplasmic fatty acid-binding protein (FABP) and acyl-CoA binding protein (ACBP) may bind these accumulating fatty acyl moieties to prevent their elevated levels from potentially harmful actions. In addition, the suggestion has been made that the abundantly present FABP may scavenge free radicals which are generated during reperfusion of the ischemic heart. However, these protective actions are challenged by the continuous physico-chemical partition of fatty acyl moieties between FABP and membrane structures and by the rapid release of FABP from ischemic and reperfused cardiac muscle. Careful evaluation of the available literature data reveals that at present no definite conclusion can be drawn about the potential protective effect of FABP on the ischemic and reperfused heart. Biochem123: 167–173, 1993)Abbreviations FABP Fatty Acid-Binding Protein - ACBP Acyl-CoA Binding Protein - MDGI Mammary-Derived Growth Inhibitor - CK Creatine Kinase - LDH Lactate Dehydrogenase     

Methylmalonic and propionic acidemias: lipid profiles of normal and affected human skin fibroblasts incubated with [1-14C]propionate

Normal human skin fibroblasts and those from methylmalonic acidemia and propionic acidemia patients were grown in culture. Following incubation with [1-14C]propionate, the major lipid classes in the cells were separated by thin layer chromatography and isolated fractions analyzed by radio gas chromatography for the presence of odd-numbered long-chain fatty acids; the pattern of even-numbered long-chain fatty acids was obtained also. Normal fibroblasts incorporated a small percentage of propionate into odd-numbered fatty acids which were present in all lipids studied. The abnormal cells incorporated a larger amount while maintaining the characteristic ratios of odd-numbered fatty acids found in the normal line. Most of the radioactivity was associated with phospholipids which are the predominant constituents of cell membranes. A characteristic C15/C17 ratio was found for different phospholipids and the triglyceride fraction; pentadecanoic acid was the principal odd-numbered fatty acid utilized in the assembly of complex lipids. Compared to even-numbered long-chain fatty acids the absolute amount of odd-numbered fatty acids was low (1-2%), even in affected cells. An unusual polar lipid fraction was isolated in the course of the study. In the normal cell it contained several unlabeled eicosanoids which were missing from the same fraction of both affected cell lines.     

Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis

Elevated serum free fatty acids (FFAs) and hepatocyte lipoapoptosis are features of non-alcoholic fatty liver disease. However, the mechanism by which FFAs mediate lipoapoptosis is unclear. Because JNK activation is pivotal in both the metabolic syndrome accompanying non-alcoholic fatty liver disease and cellular apoptosis, we examined the role of JNK activation in FFA-induced lipoapoptosis. Multiple hepatocyte cell lines and primary mouse hepatocytes were treated in culture with monounsaturated fatty acids and saturated fatty acids. Despite equal cellular steatosis, apoptosis and JNK activation were greater during exposure to saturated versus monounsaturated FFAs. Inhibition of JNK, pharmacologically as well as genetically, reduced saturated FFA-mediated hepatocyte lipoapoptosis. Cell death was caspase-dependent and associated with mitochondrial membrane depolarization and cytochrome c release indicating activation of the mitochondrial pathway of apoptosis. JNK-dependent lipoapoptosis was associated with activation of Bax, a known mediator of mitochondrial dysfunction. As JNK can activate Bim, a BH3 domain-only protein capable of binding to and activating Bax, its role in lipoapoptosis was also examined. Small interfering RNA-targeted knock-down of Bim attenuated both Bax activation and cell death. Collectively the data indicate that saturated FFAs induce JNK-dependent hepatocyte lipoapoptosis by activating the proapoptotic Bcl-2 proteins Bim and Bax, which trigger the mitochondrial apoptotic pathway.     

Importance of lipidic acyl chains in the process of biochemical thermal adaptation of <Emphasis Type="Italic">Cunninghamella japonica</Emphasis> mucoraceous fungus

The temperature of C. japonica cultivation influences the lipid content and composition of acyl chains, especially the content of such polyunsaturated acids as linoleic and linolenic. Thermal adaptation is accompanied by the modulation of fatty acid isomeric composition and acyl chain length and, at low temperatures, promotes the appearance of fatty acids uncommon to the fungus, in particular, arachidonic acid. The changes occur on a background of significant alterations in the fungus metabolism (in glucose uptake, ATP content, economic coefficient value, etc.). In experiments on the inhibition of translation with cycloheximide, abrupt temperature change (supraoptimal to cold) did not lead to desaturase de novo synthesis, but rather stimulated the activity of the named enzymes, except for palmitoleoyl-CoA desaturase. In the process of temperature adaptation, polar lipid microviscosity modulating compounds influenced fatty acid acyl chain composition. Microviscosity differences between polar and neutral lipids and correlation to the degree of fatty acid unsaturation during temperature fluctuation were established.     

Cytosolic fatty acid binding proteins catalyze two distinct steps in intracellular transport of their ligands

Cytosolic long-chain fatty acid binding proteins (FABPs) are found in tissues that metabolize fatty acids. Like most lipid binding proteins, their specific functions remain unclear. Two classes have been described. Membrane-active FABPs interact directly with membranes during exchange of fatty acids between the protein binding site and the membrane, while membrane-inactive FABPs bind only to fatty acids that are already in aqueous solution. Despite these binding proteins, most fatty acids in cell cytoplasm appear to be bound to membranes. This paper reviews data suggesting that FABPs catalyze transfer of fatty acids between intracellular membranes, often across considerable intracellular distances. This process occurs in three distinct steps: dissociation of the fatty acid from a donor membrane, diffusion of the fatty acid across the intervening water layer, and binding to an acceptor membrane. Membrane-active FABPs catalyze dissociation of the fatty acid from the donor membrane and binding to the acceptor membrane, while membrane-inactive FABPs catalyze diffusion of fatty acids across the aqueous cytosol. Thus, FABPs catalyze all three steps in intracellular transport. A simple quantitative model has been developed that predicts the rate of intracellular transport as a function of the concentration, affinity and diffusional mobility of the binding protein. Different FABPs may have evolved to match the specific transport requirements of the cell type within which they are found.     

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (ω-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.