Acyl Composition Research Articles

Dynamic Membrane Lipid Changes in Physcomitrium patens Reveal Developmental and Environmental Adaptations.

Membrane lipid composition is critical for an organism's growth, adaptation, and functionality. Mosses, as early non-vascular land colonizers, show significant adaptations and changes, but their dynamic membrane lipid alterations remain unexplored. Here, we investigated the temporal changes in membrane lipid composition of the moss Physcomitrium patens during five developmental stages and analyzed the acyl content and composition of the lipids. We observed a gradual decrease in total lipid content from the filamentous protonema stage to the reproductive sporophytes. Notably, we found significant levels of very long-chain polyunsaturated fatty acids, particularly arachidonic acid (C20:4), which are not reported in vascular plants and may aid mosses in cold and abiotic stress adaptation. During vegetative stages, we noted high levels of galactolipids, especially monogalactosyldiacylglycerol, associated with chloroplast biogenesis. In contrast, sporophytes displayed reduced galactolipids and elevated phosphatidylcholine and phosphatidic acid, which are linked to membrane integrity and environmental stress protection. Additionally, we observed a gradual decline in the average double bond index across all lipid classes from the protonema stage to the gametophyte stage. Overall, our findings highlight the dynamic nature of membrane lipid composition during moss development, which might contribute to its adaptation to diverse growth conditions, reproductive processes, and environmental challenges.

The role of lysophosphatidic acid acyltransferase 1 in reproductive growth of Arabidopsis thaliana.

Lysophosphatidic acid acyltransferase1 (LPAT1) catalyzes the second step of de novo glycerolipid biosynthesis in chloroplasts. However, the embryonic-lethal phenotype of the knockout mutant suggested an unknown role for LPAT1 in non-photosynthetic reproductive organs. Reciprocal genetic crossing of the lpat1-1 heterozygous line suggested a female gametophytic defect of the lpat1-1 knockout mutant. By suppressing LPAT1 specifically during seed development, we showed that LPAT1 suppression affected silique growth and seed production. Glycerolipid analysis of the LPAT1 knockdown lines revealed a pronounced decrease of phosphatidylcholine (PC) content in mature siliques along with an altered polyunsaturation level of the polar glycerolipids. In seeds, the acyl composition of triacylglycerol (TAG) was altered albeit not the content. These results indicate that plastidic LPAT1 plays an important role in reproductive growth and extraplastidic glycerolipid metabolism involving PC and TAG.

Novel theoretical database-assisted UHPLC-Q-TOF/MS strategy for profiling and identifying oxidized triglycerides in pharmaceutical excipient soybean oil

Pharmaceutical excipient soybean oil is widely used in injections. Its main components, triglycerides, are easily oxidized due to their unsaturated fatty acyls, raising safety concerns. However, it is hard to analyze those oxidized triglycerides due to their diverse compositions and low abundance. In this study, all theoretical oxidized triglycerides were predicted and a database consisting of 329 oxidized triglycerides was constructed. Then, a novel theoretical database-assisted ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) strategy was developed to finely profile and identify oxidized triglycerides in soybean oil. A total of 106 and 116 oxidized triglycerides were identified and relatively quantified in oxidized soybean oil and long-term stored soybean oil and preparations. It was found that oxidized triglycerides containing carbonyl groups were significantly more prevalent than other forms and oxidized triglycerides with two oxidized fatty acyl chains had the highest relative abundance. Fifteen markers indicating the oxidation of soybean oil were discovered. This strategy could rapidly and directly analyze the oxidized triglycerides and assign their fatty acyl compositions for the first time. This study will improve the quality control of soybean oil and its preparations.

ER-associated VAP27-1 and VAP27-3 proteins functionally link the lipid-binding ORP2A at the ER-chloroplast contact sites

The plant endoplasmic reticulum (ER) contacts heterotypic membranes at membrane contact sites (MCSs) through largely undefined mechanisms. For instance, despite the well-established and essential role of the plant ER-chloroplast interactions for lipid biosynthesis, and the reported existence of physical contacts between these organelles, almost nothing is known about the ER-chloroplast MCS identity. Here we show that the Arabidopsis ER membrane-associated VAP27 proteins and the lipid-binding protein ORP2A define a functional complex at the ER-chloroplast MCSs. Specifically, through in vivo and in vitro association assays, we found that VAP27 proteins interact with the outer envelope membrane (OEM) of chloroplasts, where they bind to ORP2A. Through lipidomic analyses, we established that VAP27 proteins and ORP2A directly interact with the chloroplast OEM monogalactosyldiacylglycerol (MGDG), and we demonstrated that the loss of the VAP27-ORP2A complex is accompanied by subtle changes in the acyl composition of MGDG and PG. We also found that ORP2A interacts with phytosterols and established that the loss of the VAP27-ORP2A complex alters sterol levels in chloroplasts. We propose that, by interacting directly with OEM lipids, the VAP27-ORP2A complex defines plant-unique MCSs that bridge ER and chloroplasts and are involved in chloroplast lipid homeostasis.

SS-31 treatment ameliorates cardiac mitochondrial morphology and defective mitophagy in a murine model of Barth syndrome

Barth syndrome (BTHS) is a lethal rare genetic disorder, which results in cardiac dysfunction, severe skeletal muscle weakness, immune issues and growth delay. Mutations in the TAFAZZIN gene, which is responsible for the remodeling of the phospholipid cardiolipin (CL), lead to abnormalities in mitochondrial membrane, including alteration of mature CL acyl composition and the presence of monolysocardiolipin (MLCL). The dramatic increase in the MLCL/CL ratio is the hallmark of patients with BTHS, which is associated with mitochondrial bioenergetics dysfunction and altered membrane ultrastructure. There are currently no specific therapies for BTHS. Here, we showed that cardiac mitochondria isolated from TAFAZZIN knockdown (TazKD) mice presented abnormal ultrastructural membrane morphology, accumulation of vacuoles, pro-fission conditions and defective mitophagy. Interestingly, we found that in vivo treatment of TazKD mice with a CL-targeted small peptide (named SS-31) was able to restore mitochondrial morphology in tafazzin-deficient heart by affecting specific proteins involved in dynamic process and mitophagy. This agrees with our previous data showing an improvement in mitochondrial respiratory efficiency associated with increased supercomplex organization in TazKD mice under the same pharmacological treatment. Taken together our findings confirm the beneficial effect of SS-31 in the amelioration of tafazzin-deficient dysfunctional mitochondria in a BTHS animal model.

Lipidomic Assessment of the Inhibitory Effect of Standardized Water Extract of Hydrangea serrata (Thunb.) Ser. Leaves during Adipogenesis.

Obesity is primarily exacerbated by excessive lipid accumulation during adipogenesis, with triacylglycerol (TG) as a major lipid marker. However, as the association between numerous lipid markers and various health conditions has recently been revealed, investigating the lipid metabolism in detail has become necessary. This study investigates the lipid metabolic effects of Hydrangea serrata (Thunb.) Ser. hot water leaf extract (WHS) on adipogenesis using LC-MS-based lipidomics analysis of undifferentiated, differentiated, and WHS-treated differentiated 3T3-L1 cells. WHS treatment effectively suppressed the elevation of glycerolipids, including TG and DG, and prevented a molecular shift in fatty acyl composition towards long-chain unsaturated fatty acids. This shift also impacted glycerophospholipid metabolism. Additionally, WHS stabilized significant lipid markers such as the PC/PE and LPC/PE ratios, SM, and Cer, which are associated with obesity and related comorbidities. This study suggests that WHS could reduce obesity-related risk factors by regulating lipid markers during adipogenesis. This study is the first to assess the underlying lipidomic mechanisms of the adipogenesis-inhibitory effect of WHS, highlighting its potential in developing natural products for treating obesity and related conditions. Our study provides a new strategy for the development of natural products for the treatment of obesity and related diseases.

Fatty acids of Helicobacter pylori lipoproteins CagT and Lpp20.

Bacterial lipoproteins are post-translationally modified by the addition of acyl chains that anchor the protein to bacterial membranes. This modification includes two ester-linked and one amide-linked acyl chain on lipoproteins from Gram-negative bacteria. Helicobacter pylori lipoproteins have important functions in pathogenesis (including delivering the CagA oncoprotein to mammalian cells) and are recognized by host innate and adaptive immune systems. The number and variety of acyl chains on lipoproteins impact the innate immune response through Toll-like receptor 2. The acyl chains added to lipoproteins are derived from membrane phospholipids. H. pylori membrane phospholipids have previously been shown to consist primarily of C14:0 and C19:0 cyclopropane-containing acyl chains. However, the acyl composition of H. pylori lipoproteins has not been determined. In this study, we characterized the acyl composition of two representative H. pylori lipoproteins, Lpp20 and CagT. Fatty acid methyl esters were prepared from both purified lipoproteins and analyzed by gas chromatography-mass spectrometry. For comparison, we also analyzed H. pylori phospholipids. Consistent with previous studies, we observed that the H. pylori phospholipids contain primarily C14:0 and C19:0 cyclopropane-containing fatty acids. In contrast, both the ester-linked and amide-linked fatty acids found in H. pylori lipoproteins were observed to be almost exclusively C16:0 and C18:0. A discrepancy between the acyl composition of membrane phospholipids and lipoproteins as reported here for H. pylori has been previously reported in other bacteria including Borrelia and Brucella. We discuss possible mechanisms.IMPORTANCEColonization of the stomach by Helicobacter pylori is an important risk factor in the development of gastric cancer, the third leading cause of cancer-related death worldwide. H. pylori persists in the stomach despite an immune response against the bacteria. Recognition of lipoproteins by TLR2 contributes to the innate immune response to H. pylori. However, the role of H. pylori lipoproteins in bacterial persistence is poorly understood. As the host response to lipoproteins depends on the acyl chain content, defining the acyl composition of H. pylori lipoproteins is an important step in characterizing how lipoproteins contribute to persistence.

Recent Analytical Methodologies in Lipid Analysis.

Lipids represent a large group of biomolecules that are responsible for various functions in organisms. Diseases such as diabetes, chronic inflammation, neurological disorders, or neurodegenerative and cardiovascular diseases can be caused by lipid imbalance. Due to the different stereochemical properties and composition of fatty acyl groups of molecules in most lipid classes, quantification of lipids and development of lipidomic analytical techniques are problematic. Identification of different lipid species from complex matrices is difficult, and therefore individual analytical steps, which include extraction, separation, and detection of lipids, must be chosen properly. This review critically documents recent strategies for lipid analysis from sample pretreatment to instrumental analysis and data interpretation published in the last five years (2019 to 2023). The advantages and disadvantages of various extraction methods are covered. The instrumental analysis step comprises methods for lipid identification and quantification. Mass spectrometry (MS) is the most used technique in lipid analysis, which can be performed by direct infusion MS approach or in combination with suitable separation techniques such as liquid chromatography or gas chromatography. Special attention is also given to the correct evaluation and interpretation of the data obtained from the lipid analyses. Only accurate, precise, robust and reliable analytical strategies are able to bring complex and useful lipidomic information, which may contribute to clarification of some diseases at the molecular level, and may be used as putative biomarkers and/or therapeutic targets.

P049 The lipidome of creeping fat in Crohn’s disease points towards a harmful microenvironment

Abstract Background A pathognomonic observation in Crohn’s disease (CD) is the existence of mesenteric adipose tissue (MAT) wrapping around the inflamed and fibrotic intestine, so-called creeping fat. The etiology of this phenomenon is unclear, and both a protective and harmful role in disease pathogenesis and progression have been proposed. Given this conflicting evidence, we aimed to further unravel the role of creeping fat by extensive profiling of its lipidome. Methods We prospectively included 22 CD patients who underwent an ileocecal resection with ileocolonic anastomosis (Table 1). Perioperatively paired adipose tissue samples were collected from 3 locations: (1) creeping fat, (2) MAT close to the proximal unaffected ileum, and (3) MAT far from the diseased ileum, defined as near the base of the ileocecal artery (Fig. 1A). The lipidome of all 66 samples was profiled by quantitative mass spectrometry (targeted C18 LC-MS/MS and HILIC LC-MS/MS; Lipometrix core KU Leuven). This approach enables the detection of mediator lipids, and membrane and storage lipids, of which the latter group represents more complex chemical structures and thus a higher diversity in classification and number of species. Data analysis and paired Wilcoxon rank- sum tests were performed with Python and R 4.3.2. Results Across all CD adipose tissue samples, we detected 38 mediator lipid species, and 456 membrane and storage lipid species . When paired-wise comparing creeping fat with MAT far from the diseased ileum, we observed a significant decrease in 16 lipid mediators (p<0.05), namely arachidonic acid (AA), docosahexaenoic acid (DHA), 4 epoxy and 10 hydroxy mediator lipids; with hydroxy mediator X being the top downregulated in creeping fat (fold change (FC)=-2.11, p=0.0003) (Fig. 1B). While AA and DHA are (semi)-essential fatty acids and function as precursors, the other 8 decreased lipids in creeping fat are known to be anti-inflammatory, and 3 (incl. mediator X) are also anti-fibrotic. The lipid mediator profile of MAT close to the proximal unaffected ileum was similar to that of creeping fat, except for a pro-inflammatory lipid being increased in creeping fat (FC=2.30, p=0.004) (Fig. 1B). Lastly, when evaluating the membrane and storage lipid abundances both at species level (sum notation; fatty acyl composition) and at class level, no paired-wise differences among the three adipose tissue locations were found (p>0.05). Conclusion In this pilot lipidomics study, we did not observe any changes in storage or membrane lipids in creeping fat as compared to paired unaffected MAT. In contrast, a decrease in anti-inflammatory and anti-fibrotic lipid species along with an increase in a pro-inflammatory species points towards a harmful microenvironment within creeping fat.

HILIC/MS quantitation of low-abundant phospholipids and sphingolipids in human plasma and serum: Dysregulation in pancreatic cancer

A new hydrophilic interaction liquid chromatography - mass spectrometry method is developed for low-abundant phospholipids and sphingolipids in human plasma and serum. The optimized method involves the Cogent Silica type C hydride column, the simple sample preparation by protein precipitation, and the removal of highly abundant lipid classes using the postcolumn valve directed to waste during two elution windows. The method allows a highly confident and sensitive identification of low-abundant lipid classes in human plasma (246 lipid species from 24 lipid subclasses) based on mass accuracy and retention dependencies in both polarity modes. The method is validated for quantitation using two internal standards (if available) for each lipid class and applied to human plasma and serum samples obtained from patients with pancreatic ductal adenocarcinoma (PDAC), healthy controls, and NIST SRM 1950. Multivariate data analysis followed by various statistical projection methods is used to determine the most dysregulated lipids. Significant downregulation is observed for lysophospholipids with fatty acyl composition 16:0, 18:0, 18:1, and 18:2. Distinct trends are observed for phosphatidylethanolamines (PE) in relation to the bonding type of fatty acyls, where most PE with acyl bonds are upregulated, while ether/plasmenyl PE are downregulated. For the sphingolipid category, sphingolipids with very long N-acyl chains are downregulated, while sphingolipids with shorter N-acyl chains were upregulated in PDAC. These changes are consistently observed for various classes of sphingolipids, ranging from ceramides to glycosphingolipids, indicating a possible metabolic disorder in ceramide biosynthesis caused by PDAC.

Dryland Endolithic Chroococcidiopsis andTemperate Fresh Water Synechocystis Have Distinct Membrane Lipid andPhotosynthesis Acclimation Strategies upon Desiccation andTemperature Increase.

An effect of climate change is the expansion of drylands in temperate regions, predicted to affect microbial biodiversity. Since photosynthetic organisms are at the base of ecosystem's trophic networks, we compared an endolithic desiccation-tolerant Chroococcidiopsis cyanobacteria isolated from gypsum rocks in the Atacama Desert with a freshwater desiccation-sensitive Synechocystis. We sought whether some acclimation traits in response to desiccation and temperature variations were shared, to evaluate the potential of temperate species to possibly become resilient to future arid conditions. When temperature varies, Synechocystis tunes the acyl composition of its lipids, via a homeoviscous acclimation mechanism known to adjust membrane fluidity, whereas no such change occurs in Chroococcidiopsis. Vice versa, a combined study of photosynthesis and pigment content shows that Chroococcidiopsis remodels its photosynthesis components and keeps an optimal photosynthetic capacity at all temperatures, whereas Synechocystis is unable to such adjustment. Upon desiccation on a gypsum surface, Synechocystis is rapidly unable to revive, whereas Chroococcidiopsis is capable to recover after three weeks. Using X-ray diffraction, we found no evidence that Chroococcidiopsis could use water extracted from gypsum crystals in such conditions as a surrogate for missing water. The sulfolipid sulfoquinovosyldiacylglycerol becomes the prominent membrane lipid in both dehydrated cyanobacteria, highlighting an overlooked function for this lipid. Chroococcidiopsis keeps a minimal level of monogalactosyldiacylglycerol, which may be essential for the recovery process. Results support that two independent adaptation strategies have evolved in these species to cope with temperature and desiccation increase and suggest some possible scenarios for microbial biodiversity change triggered by climate change.

LPGAT1 controls MEGDEL syndrome by coupling phosphatidylglycerol remodeling with mitochondrial transport

Phosphatidylglycerol (PG) is a mitochondrial phospholipid required for mitochondrial cristae structure and cardiolipin synthesis. PG must be remodeled to its mature form at the endoplasmic reticulum (ER) after mitochondrial biosynthesis to achieve its biological functions. Defective PG remodeling causes MEGDEL (non-alcohol fatty liver disease and 3-methylglutaconic aciduria with deafness, encephalopathy, and Leigh-like) syndrome through poorly defined mechanisms. Here, we identify LPGAT1, an acyltransferase that catalyzes PG remodeling, as a candidate gene for MEGDEL syndrome. We show that PG remodeling by LPGAT1 at the ER is closely coordinated with mitochondrial transport through interaction with the prohibitin/TIMM14 mitochondrial import motor. Accordingly, ablation of LPGAT1 or TIMM14 not only causes aberrant fatty acyl compositions but also ER retention of newly remodeled PG, leading to profound loss in mitochondrial crista structure and respiration. Consequently, genetic deletion of the LPGAT1 in mice leads to cardinal features of MEGDEL syndrome, including 3-methylglutaconic aciduria, deafness, dilated cardiomyopathy, and premature death, which are highly reminiscent of those caused by TIMM14 mutations in humans.

Comparative quantitative analysis of fruit oil from Hippophae rhamnoides (seabuckthorn) by qNMR, FTIR and GC–MS

ObjectiveTo develop a qNMR method for quantitative analysis of triacylglycerols in fruit oil of Hippophae rhamnoides (seabuckthorn, SBT) and analyze commercial samples of SBT oils using GC–MS and FTIR. MethodsSBT fruit oil (IPHRFH) was extracted with hexane and the triglyceride (TAG) was isolated by vacuum liquid chromatography. Six different branded SBT oils purchased from e-commerce suppliers (Amazon) and in-house prepared SBT oil was analyzed by qNMR and fatty acyl composition of TAGs determined by using NMR. In-house oil was also analysed by GC–MS and FTIR spectroscopy. ResultsThe qNMR results showed that the oil contained 80.3% of triacylglycerol (TAG). The SBT oil TAGs comprised of linolenate 6.6%, palmitoleate/oleate 65.4%, and total saturated fatty acyl chain including palmitate 28% as determined by qNMR. GC–MS analysis revealed that the major acyl functionalities present in the TAG were palmitoleic acid 36.5%, oleic acid 12.9%, palmitic acid 21.2%, and linoleic acid 18%. Of the six commercial samples analyzed, samples from only one supplier (SW) were fruit oil; All others were the seed oils or mix of fruit oil and seed oil. The labels for samples except for the SW did not indicate whether it was fruit oil or seed oil. ConclusionThe results suggest that SBT oil should be analyzed by combination of GC–MS, FTIR and qNMR for factual content of free fatty acid or TAGs, which are chemically different in nature and affect the quality of oil. GC–MS showed the content of omega free fatty acids after hydrolysis, while qNMR and FTIR showed the content of TAGs. The major acyl functionalities found in SBT fruit oil TAGs are palmitoleate/palmitate/oleate, while linoleate and linonelate make up a minor fraction. Furthermore, analysis of commercial samples showed discrepancies between label claims and actual content.

First insights into region-specific lipidome alterations of prefrontal cortex and hippocampus of mice exposed chronically to microcystins

Microcystins (MCs), a group of most widespread freshwater cyanotoxins that possess strong neurotoxicity, can adversely affect brain structures and functions and are linked to neurodegenerative diseases. Despite the essential role of lipids in brain structures and functions, the brain lipidome profile of mammals exposed to MCs remains unexplored, hindering a clear understanding of the neurotoxic effects of MCs and underlying mechanisms. In this study, we performed untargeted lipidomic profiling using ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) on the prefrontal cortex and hippocampus of mice orally exposed to 30 and 300 μg/kg body mass/day of microcystin-leucine arginine (MC-LR) for 180 days to evaluate the impacts of MC-LR on the brain lipidome profile and functions. Our results show that MC-LR resulted in a decline in cognitive parameters, as assessed by the Morris water maze test. Interestingly, apparent neurodegenerative changes were observed in the prefrontal cortex, but not in the hippocampus. Comprehensive lipidomic analyses uncovered profound, region-specific changes in the phospholipid and sphingolipid profile at the levels of lipid subclasses, lipid species, and fatty acyl composition. These changes showed overall decrease trends of lipid content in the prefrontal cortex yet increasing trends in the hippocampus. We identified distinct transcriptional regulations of lipid metabolism and apoptosis by MC-LR in the two regions, which appeared to underlie the neurodegenerative changes. Collectively, this study uncovers region-specific changes in the brain lipidome profile and functions induced by MCs, shedding light on the role of lipid dysfunction in neurotoxicity mechanism of MCs.

Effect of lipid saturation on the topology and oligomeric state of helical membrane polypeptides

Natural liquid crystalline membranes are made up of many different lipids carrying a mixture of saturated and unsaturated fatty acyl chains. Whereas in the past considerable attention has been paid to cholesterol content, the phospholipid head groups and the membrane surface charge the detailed fatty acyl composition was often considered less important. However, recent investigations indicate that the detailed fatty acyl chain composition has pronounced effects on the oligomerization of the transmembrane helical anchoring domains of the MHC II receptor or the membrane alignment of the cationic antimicrobial peptide PGLa. In contrast the antimicrobial peptides magainin 2 and alamethicin are less susceptible to lipid saturation. Using histidine-rich LAH4 designer peptides the high energetic contributions of lipid saturation in stabilizing transmembrane helical alignments are quantitatively evaluated. These observations can have important implications for the biological regulation of membrane proteins and should be taken into considerations during biophysical or structural experiments.

Pharmacological inhibition of ALCAT1 mitigates amyotrophic lateral sclerosis by attenuating SOD1 protein aggregation

ObjectiveMutations in the copper-zinc superoxide dismutase (SOD1) gene cause familial amyotrophic lateral sclerosis (ALS), a progressive fatal neuromuscular disease characterized by motor neurons death and severe skeletal muscle degeneration. However, there is no effective treatment for this debilitating disease, since the underlying cause for the pathogenesis remains poorly understood. Here, we investigated a role of acyl-CoA:lysocardiolipin acyltransferase 1 (ALCAT1), an acyltransferase that promotes mitochondrial dysfunction in age-related diseases by catalyzing pathological remodeling of cardiolipin, in promoting the development of ALS in the SOD1G93A transgenic mice. MethodsUsing SOD1G93A transgenic mice with targeted deletion of the ALCAT1 gene and treated with Dafaglitapin (Dafa), a very potent and highly selective ALCAT1 inhibitor, we determined whether ablation or pharmaceutical inhibition of ALCAT1 by Dafa would mitigate ALS and the underlying pathogenesis by preventing pathological remodeling of cardiolipin, oxidative stress, and mitochondrial dysfunction by multiple approaches, including lifespan analysis, behavioral tests, morphological and functional analysis of skeletal muscle, electron microscopic and Seahorse analysis of mitochondrial morphology and respiration, western blot analysis of the SOD1G93A protein aggregation, and lipidomic analysis of cardiolipin content and acyl composition in mice spinal cord. ResultsALCAT1 protein expression is potently upregulated in the skeletal muscle of the SOD1G93A mice. Consequently, ablation or pharmacological inhibition of ALCAT1 by Dafa attenuates motor neuron dysfunction, neuronal inflammation, and skeletal muscle atrophy in SOD1G93A mice by preventing SOD1G93A protein aggregation, mitochondrial dysfunction, and pathological CL remodeling, leading to moderate extension of lifespan in the SOD1G93A transgenic mice. ConclusionsALCAT1 promotes the development of ALS by linking SOD1G93A protein aggregation to mitochondrial dysfunction, implicating Dafa as a potential treatment for this debilitating disorder.

Changes in Phospholipid Composition of the Human Cerebellum and Motor Cortex during Normal Ageing.

(1) Background: Changes in phospholipid (phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, i.e., PC, PE and PS) composition with age in the mitochondrial and microsomal membranes of the human cerebellum and motor cortex were examined and compared to previous analyses of the prefrontal cortex, hippocampus and entorhinal cortex. (2) Methods: Nano-electrospray ionization on a hybrid triple quadrupole–linear ion trap mass spectrometer was used to analyse the brain regions of subjects aged 18–104 years. (3) Results: With age, the cerebellum showed many changes in the major phospholipids (>10% of the phospholipid class). In both membrane types, these included increases in PE 18:0_22:6 and PS 18:0_22:6, decreases in PE 18:0_20:4 and PS 18:0_18:1 and an increase in PC 16:0_16:0 (microsomal membrane only). In addition, twenty-one minor phospholipids also changed. In the motor cortex, only ten minor phospholipids changed with age. With age, the acyl composition of the membranes in the cerebellum increased in docosahexaenoic acid (22:6) and decreased in the arachidonic (20:4) and adrenic (22:4) acids. A comparison of phospholipid changes in the cerebellum, motor cortex and other brain areas is provided. (4) Conclusions: The cerebellum is exceptional in the large number of major phospholipids that undergo changes (with consequential changes in acyl composition) with age, whereas the motor cortex is highly resistant to change.

In Search of the Holy Grail: Toward a Unified Hypothesis on Mitochondrial Dysfunction in Age-Related Diseases.

Cardiolipin (CL) is a mitochondrial signature phospholipid that plays a pivotal role in mitochondrial dynamics, membrane structure, oxidative phosphorylation, mtDNA bioenergetics, and mitophagy. The depletion or abnormal acyl composition of CL causes mitochondrial dysfunction, which is implicated in the pathogenesis of aging and age-related disorders. However, the molecular mechanisms by which mitochondrial dysfunction causes age-related diseases remain poorly understood. Recent development in the field has identified acyl-CoA:lysocardiolipin acyltransferase 1 (ALCAT1), an acyltransferase upregulated by oxidative stress, as a key enzyme that promotes mitochondrial dysfunction in age-related diseases. ALCAT1 catalyzes CL remodeling with very-long-chain polyunsaturated fatty acids, such as docosahexaenoic acid (DHA). Enrichment of DHA renders CL highly sensitive to oxidative damage by reactive oxygen species (ROS). Oxidized CL becomes a new source of ROS in the form of lipid peroxides, leading to a vicious cycle of oxidative stress, CL depletion, and mitochondrial dysfunction. Consequently, ablation or the pharmacological inhibition of ALCAT1 have been shown to mitigate obesity, type 2 diabetes, heart failure, cardiomyopathy, fatty liver diseases, neurodegenerative diseases, and cancer. The findings suggest that age-related disorders are one disease (aging) manifested by different mitochondrion-sensitive tissues, and therefore should be treated as one disease. This review will discuss a unified hypothesis on CL remodeling by ALCAT1 as the common denominator of mitochondrial dysfunction, linking mitochondrial dysfunction to the development of age-related diseases.

222-LB: Switching Palmitoyl and Oleoyl Position in Sn-2 and Sn-3 of a Triacylglycerol Led to Differential Body Weight Gain Rates and Hepatic Gene Expressions in Mice Fed a High-Fat Diet

Alterations of fatty acid metabolism have been associated with diabetes. Triacylglycerol (TAG) can be obtained from dietary sources and synthesized de novo. The composition and location of the fatty acyl groups in a TAG can affect its physicochemical features such as melting temperature, and physiological characteristics such as digestion, absorption, and lipid metabolism. We hypothesize that the TAG fatty acyl position affects the body weight gain and lipid metabolism of mice in a high-fat diet (HFD) setting. Twenty-seven 6-week-old male C57/BL6J mice were randomly assigned to 3 dietary treatment groups, 1,2-dipalmitoyl-3-oleoylglycerol (PPO, 36 % w/w diet) , 1,3-dipalmitoyl-2-oleoylglycerol (POP, 36% w/w diet) , and the chow diet control groups. Mice in each group were fed their respective diet for 6 weeks, and food intake and body weight were measured daily. Mice fed the PPO diet had significantly higher food intake, calories intake and body wight gain rate, and lower liver to body weight ratio than those fed the POP diet. Mice fed the chow diet had significantly higher total grams of food intake, but lower calories intake, liver and epididymal fat weight, and body weight gain rate than those fed the PPO, but not the POP, diet. Mice in the PPO group had significantly higher serum glucose than that in other two groups, whereas mice fed the chow diet had significantly lower plasma cholesterol level than those HFD groups. The plasma TAG levels were not different among them. The hepatic levels of insulin receptor substrate 1, and fatty acid synthase, and phosphorylated acetyl CoA carboxylase in mice fed the chow diet are significantly higher than those fed the PPO or POP diet, whereas the hepatic expression level of stearoyl CoA desaturase 1 in mice fed the POP diet was lower than those fed the PPO or chow diet. We conclude that feeding a HFD affects the expression levels of hepatic proteins for lipid metabolism in mice. In addition, the position of fatty acyl group in a TAG affects the food intake, the body weight gain rate and lipid homeostasis in mice. Disclosure X. Hu: None. T. Wang: None. G. Chen: None.

De novo labeling and trafficking of individual lipid species in live cells

ObjectiveLipids exert dynamic biological functions which are determined both by their fatty acyl compositions and spatiotemporal distributions inside the cell. However, it remains a daunting task to investigate any of these features for each of the more than 1000 lipid species due to a lack of a universal labeling method for individual lipid moieties in live cells. Here we report a de novo lipid labeling method for individual lipid species with precise acyl compositions in live cells. The method is based on the principle of de novo lipid remodeling of exogenously added lysolipids with fluorescent acyl-CoA, leading to the re-synthesis of fluorescence-labeled lipids which can be imaged by confocal microscopy. MethodsThe cells were incubated with lysolipids and a nitro-benzoxadiazolyl (NBD) labeled acyl-CoA. The newly remodeled NBD-labeled lipids and their subcellular localization were analyzed by confocal imaging in live cells. Thin layer chromatography was carried out to verify the synthesis of NBD-labeled lipids. The mitochondrial trafficking of NBD-labeled lipids was validated in live cells with targeted deletion of phospholipids transporters, including TRIAP1/PRELI protein complex and StarD7. ResultsIncubation cells with lysolipids and NBD-acyl-CoA successfully labeled major lipid species with precise acyl compositions, including phospholipids, cholesterol esters, and neutral lipids, which can be analyzed by confocal imaging in live cells. In contrast to exogenously labeled lipids, the de novo labeled lipids retained full biological properties of their endogenous counterparts, including subcellular localization, trafficking, and recognition by lipid transporters. This method also uncovered some unexpected features of newly remodeled lipids and their transporters. ConclusionsThe de novo lipid labeling method not only provides a powerful tool for functional analysis of individual lipid species and lipid transporters, but also calls for re-evaluation of previously published results using exogenously labeled lipids.