Phosphatidylethanolamine Ratio Research Articles

Abstract 2057: Serine Hydroxymethyltransferase 2 In Hepatocytes Regulates One-carbon Metabolism, Circulating VLDL And Atherosclerosis

While the role of lipids in atherosclerotic cardiovascular disease (ASCVD) is well studied, the contribution of amino acids is becoming increasingly recognized. We and others reported that the ratio of glycine to serine is reduced in association with ASCVD severity. Primarily expressed in the liver, serine hydroxymethyltransferase 2 (SHMT2) is thought to be responsible for the bulk of glycine biosynthesis from serine. We recently reported that reversed SHMT2 activity in fatty livers drives glycine depletion ( Ghrayeb et al. Cell Metab 2024 ). Nevertheless, whether the regulation of glycine/serine metabolism by hepatic SHMT2 affects atherosclerosis remains unknown. Utilizing novel hepatocyte-specific Shmt2 knockout mice ( Shmt2 HKO ) crossed with apolipoprotein E-deficient ( Apoe -/- ) mice, metabolomics, and transcriptomics, we studied the role of hepatocyte SHMT2 in atherosclerosis. We found a significant increase in glycine and the glycine:serine ratio in plasma from Shmt2 HKO / Apoe -/- mice compered to Shmt2 flox / Apoe -/- mice fed a Western diet. In livers from Shmt2 HKO / Apoe -/- mice, decreased S-adenosylmethionine (SAM) and increased adenosylhomocysteine (SAH) resulted in reduced SAM:SAH ratio. While hepatic steatosis was unaltered, Shmt2 HKO / Apoe -/- mice had lower phosphatidylcholine (PC) and PC:phosphatidylethanolamine (PE) ratio, key regulators of hepatic VLDL assembly and secretion. Accordingly, FPLC demonstrated reduced triglyceride and cholesterol contents in VLDL from Shmt2 HKO / Apoe -/- mice. Moreover, RNA-seq and qPCR analyses revealed a downregulation of proinflammatory mediators ( Tnf, Ir1b, Ccr5 ) and an upregulation of fatty acid β-oxidation regulators ( Acadm , Acadl ) in livers from Shmt2 HKO / Apoe -/- mice. Finally, histological analysis of aortic sinuses revealed lower plaque size in female Shmt2 HKO / Apoe -/- mice. These studies uncover hepatic SHMT2 as a regulator of one-carbon and VLDL metabolism in atherosclerosis.

Multi-molecular hyperspectral PRM-SRS microscopy

Lipids play crucial roles in many biological processes. Mapping spatial distributions and examining the metabolic dynamics of different lipid subtypes in cells and tissues are critical to better understanding their roles in aging and diseases. Commonly used imaging methods (such as mass spectrometry-based, fluorescence labeling, conventional optical imaging) can disrupt the native environment of cells/tissues, have limited spatial or spectral resolution, or cannot distinguish different lipid subtypes. Here we present a hyperspectral imaging platform that integrates a Penalized Reference Matching algorithm with Stimulated Raman Scattering (PRM-SRS) microscopy. Using this platform, we visualize and identify high density lipoprotein particles in human kidney, a high cholesterol to phosphatidylethanolamine ratio inside granule cells of mouse hippocampus, and subcellular distributions of sphingosine and cardiolipin in human brain. Our PRM-SRS displays unique advantages of enhanced chemical specificity, subcellular resolution, and fast data processing in distinguishing lipid subtypes in different organs and species.

Lipid profiling reveals Leymus Chinensis root insensitivity to Ca limitation

BackgroundLeymus chinensis (L. chinensis) is a perennial native forage grass widely distributed in the steppe of Inner Mongolia as the dominant species. Calcium (Ca) is an essential mineral element important for plant adaptation to the growth environment. Ca limitation was previously shown to strongly inhibit Arabidopsis (Arabidopsis thaliana) seedling growth and disrupt plasma membrane stability and selectivity, increasing fluid-phase-based endocytosis and contents of all major membrane lipids.ResultsIn this study, we investigated the significance of Ca for L. chinensis growth and membrane stability relative to Arabidopsis. Our results showed that Ca limitation did not affect L. chinensis seedling growth and endocytosis in roots. Moreover, the plasma membrane maintained high selectivity. The lipid phosphatidylcholine (PC): phosphatidylethanolamine (PE) ratio, an indicator of the membrane stability, was five times higher in L. chinensis than in Arabidopsis. Furthermore, in L. chinensis, Ca limitation did not affect the content of any major lipid types, decreased malondialdehyde (MDA) content, and increased superoxide dismutase (SOD) activity, showing an opposite pattern to that in Arabidopsis. L. chinensis roots accumulated higher contents of PC, phosphatidylinositol (PI), monogalactosyldiacylglycerol (MGDG), phosphatidylglycerol (PG), cardiolipin (CL), digalactosyldiacylglycerol (DGDG), and lysophosphatidylcholine (LPC) but less phosphatidylethanolamine (PE), diacylglycerol (DAG), triacylglycerolv (TAG), phosphatidylserine (PS), lysobisphosphatidic acids (LPAs), lysophosphatidylethanolamine (LPE), and lysophosphatidylserine (LPS) than Arabidopsis roots. Moreover, we detected 31 and 66 unique lipids in L. chinensis and Arabidopsis, respectively.ConclusionsThis study revealed that L. chinensis roots have unique membrane lipid composition that was not sensitive to Ca limitation, which might contribute to the wider natural distribution of this species.

Skeletal Muscle Consequences of Phosphatidylethanolamine Synthesis Deficiency.

The maintenance of phospholipid homeostasis is increasingly being implicated in metabolic health. Phosphatidylethanolamine (PE) is the most abundant phospholipid on the inner leaflet of cellular membranes, and we have previously shown that mice with a heterozygous ablation of the PE synthesizing enzyme, Pcyt2 (Pcyt2+/-), develop obesity, insulin resistance, and NASH. Skeletal muscle is a major determinant of systemic energy metabolism, making it a key player in metabolic disease development. Both the total PE levels and the ratio of PE to other membrane lipids in skeletal muscle are implicated in insulin resistance; however, the underlying mechanisms and the role of Pcyt2 regulation in this association remain unclear. Here, we show how reduced phospholipid synthesis due to Pcyt2 deficiency causes Pcyt2+/- skeletal muscle dysfunction and metabolic abnormalities. Pcyt2+/- skeletal muscle exhibits damage and degeneration, with skeletal muscle cell vacuolization, disordered sarcomeres, mitochondria ultrastructure irregularities and paucity, inflammation, and fibrosis. There is intramuscular adipose tissue accumulation, and major disturbances in lipid metabolism with impaired FA mobilization and oxidation, elevated lipogenesis, and long-chain fatty acyl-CoA, diacylglycerol, and triacylglycerol accumulation. Pcyt2+/- skeletal muscle exhibits perturbed glucose metabolism with elevated glycogen content, impaired insulin signaling, and reduced glucose uptake. Together, this study lends insight into the critical role of PE homeostasis in skeletal muscle metabolism and health with broad implications on metabolic disease development.

A Trifolium repens flavodoxin-like quinone reductase 1 (TrFQR1) improves plant adaptability to high temperature associated with oxidative homeostasis and lipids remodeling.

Maintenance of stable mitochondrial respiratory chains could enhance adaptability to high temperature, but the potential mechanism was not elucidated clearly in plants. In this study, we identified and isolated a TrFQR1 gene encoding the flavodoxin-like quinone reductase 1 (TrFQR1) located in mitochondria of leguminous white clover (Trifolium repens). Phylogenetic analysis indicated that amino acid sequences of FQR1 in various plant species showed a high degree of similarities. Ectopic expression of TrFQR1 protected yeast (Saccharomyces cerevisiae) from heat damage and toxic levels of benzoquinone, phenanthraquinone and hydroquinone. Transgenic Arabidopsis thaliana and white clover overexpressing TrFQR1 exhibited significantly lower oxidative damage and better photosynthetic capacity and growth than wild-type in response to high-temperature stress, whereas AtFQR1-RNAi A. thaliana showed more severe oxidative damage and growth retardation under heat stress. TrFQR1-transgenic white clover also maintained better respiratory electron transport chain than wild-type plants, as manifested by significantly higher mitochondrial complex II and III activities, alternative oxidase activity, NAD(P)H content, and coenzyme Q10 content in response to heat stress. In addition, overexpression of TrFQR1 enhanced the accumulation of lipids including phosphatidylglycerol, monogalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol and cardiolipin as important compositions of bilayers involved in dynamic membrane assembly in mitochondria or chloroplasts positively associated with heat tolerance. TrFQR1-transgenic white clover also exhibited higher lipids saturation level and phosphatidylcholine:phosphatidylethanolamine ratio, which could be beneficial to membrane stability and integrity during a prolonged period of heat stress. The current study proves that TrFQR1 is essential for heat tolerance associated with mitochondrial respiratory chain, cellular reactive oxygen species homeostasis, and lipids remodeling in plants. TrFQR1 could be selected as a key candidate marker gene to screen heat-tolerant genotypes or develop heat-tolerant crops via molecular-based breeding.

Discovery of Myeloid-Derived Suppressor Cell-Specific Metabolism by Metabolomic and Lipidomic Profiling.

The endogenous factors that control the differentiation of myeloid-derived suppressor cells (MDSCs) are not yet fully understood. The purpose of this study was to find MDSC-specific biomolecules through comprehensive metabolomic and lipidomic profiling of MDSCs from tumor-bearing mice and to discover potential therapeutic targets for MDSCs. Partial least squares discriminant analysis was performed on the metabolomic and lipidomic profiles. The results showed that inputs for the serine, glycine, and one-carbon pathway and putrescine are increased in bone marrow (BM) MDSC compared to normal BM cells. Splenic MDSC showed an increased phosphatidylcholine to phosphatidylethanolamine ratio and less de novo lipogenesis products, despite increased glucose concentration. Furthermore, tryptophan was found to be at the lowest concentration in splenic MDSC. In particular, it was found that the concentration of glucose in splenic MDSC was significantly increased, while that of glucose 6-phosphate was not changed. Among the proteins involved in glucose metabolism, GLUT1 was overexpressed during MDSC differentiation but decreased through the normal maturation process. In conclusion, high glucose concentration was found to be an MDSC-specific feature, and it was attributed to GLUT1 overexpression. These results will help to develop new therapeutic targets for MDSCs.

Changing the phospholipid composition of lipid droplets alters localization of select lipid droplet proteins.

Our experiments aim to determine if decreasing the amount of phosphatidylcholine (PC) relative to phosphatidylethanolamine (PE) at the lipid droplet surface changes the localization of specific lipid droplet proteins. We manipulate lipid droplet phospholipids in both a cultured mouse hepatocyte (AML12) cell line and on synthetic lipid droplets. Decreasing the PC:PE ratio increases perilipin 2, decreases DGAT2, and does not change rab18 or lanosterol synthase levels on lipid droplets. These differences may be explained by the distinct structural motifs that mediate the protein-lipid droplet interactions.

Oxidative stress is inhibited by plant-based supplements: A quantitative lipidomic analysis of antioxidant activity and lipid compositional change

Dietary supplements containing plant extracts are often marketed as having antioxidant properties. Using complex lipid mixtures derived from cellular tissue, we perform a targeted lipidomic assessment of the antioxidant activity of five plant extract supplements. Formulations derived from grape seed, milk thistle, pine bark, turmeric and hawthorn extract were evaluated. Overall, we find evidence to suggest that all supplements suppressed in vitro lipid oxidation to some degree. Suppression of oxidation was most effective in the dominant lipid classes, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which contain significant numbers of polyunsaturated lipids. Under the conditions of study, grape seed extract supplement was the most effective antioxidant showing statistically significant suppression of lipid oxidation in 11 of the 15 lipid classes quantified. Lipid compositional changes and metrics such as the PC: PE ratio and the double bond index were determined to provide an insight into the effect of oxidative stress and antioxidants on collective membrane properties. Finally, the likely stress that in vivo lipid oxidation could cause to cells, and the mitigating effects of supplements in this context, is also discussed.

Assessment of a combination of plasma anti-histone autoantibodies and PLA2/PE ratio as potential biomarkers to clinically predict autism spectrum disorders

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficiencies in social interaction and repetitive behaviors. Multiple studies have reported abnormal cell membrane composition and autoimmunity as known mechanisms associated with the etiopathogenesis of ASD. In this study, multiple regression and combined receiver operating characteristic (ROC) curve as statistic tools were done to clarify the relationship between phospholipase A2 and phosphatidylethanolamine (PE) ratio (PLA2/PE) as marker of lipid metabolism and membrane fluidity, and antihistone-autoantibodies as marker of autoimmunity in the etiopathology of ASD. Furthermore, the study intended to define the linear combination that maximizes the partial area under an ROC curve for a panel of markers. Forty five children with ASD and forty age- and sex-matched controls were enrolled in the study. Using ELISA, the levels of antihistone-autoantibodies, and PLA2 were measured in the plasma of both groups. PE was measured using HPLC. Statistical analyses using ROC curves and multiple and logistic regression models were performed. A notable rise in the area under the curve was detected using combined ROC curve models. Additionally, higher specificity and sensitivity of the combined markers were documented. The present study indicates that the measurement of the predictive value of selected biomarkers related to autoimmunity and lipid metabolism in children with ASD using a ROC curve analysis should lead to a better understanding of the pathophysiological mechanism of ASD and its link with metabolism. This information may enable the early diagnosis and intervention.

Decreasing Phosphatidylcholine on the Lipid Droplet Surface Selectively Recruits Proteins with Amphipathic Alpha Helices

Lipid droplets are ubiquitous, intracellular fat‐storage organelles. Each lipid droplet is surrounded by a phospholipid monolayer primarily composed of two phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE). We have previously shown that lipid droplets accumulating in a rat model of alcoholic fatty liver disease have a decreased PC:PE ratio compared to healthy control animals. Moreover, altering the phospholipid composition of the lipid droplet surface changes the amount of specific lipid droplet proteins, suggesting a mechanism for fat accumulation in alcoholic fatty liver disease. Using subcellular fractionation and immunoblotting, we investigated the abundance and lipid droplet association of multiple proteins in AML12 cells cultured under conditions that alter the PC:PE ratio and mimic the lipid droplet structure in alcoholic fatty liver disease. With this approach, we measured the relative binding of proteins that interact with lipid droplets through a variety of structural motifs, including amphipathic alpha helices, lipid anchors, and hairpin loops. Our results indicate that the amphipathic alpha helix‐containing proteins perilipin 1, perilipin 2, and CIDE‐C are significantly more likely to associate with lipid droplets with decreased PC:PE ratios. This same change in PC:PE levels did not change association of rab 18, a protein that interacts with lipid droplets through a lipid anchor, and decreased lipid droplet association of a protein (DGAT2) that trafficks to lipid droplets as they bud from the ER. Our results support a model where increased binding of the amphipathic alpha helical domains of some lipid droplet proteins contributes to lipid droplet overaccumulation in alcoholic fatty liver disease. This work builds on our understanding of alcoholic fatty liver disease and the mechanisms that facilitate protein binding to lipid droplets.

LPGAT1 controls the stearate/palmitate ratio of phosphatidylethanolamine and phosphatidylcholine in sn-1 specific remodeling

Most mammalian phospholipids contain a saturated fatty acid at the sn-1 carbon atom and an unsaturated fatty acid at the sn-2 carbon atom of the glycerol backbone group. While the sn-2 linked chains undergo extensive remodeling by deacylation and reacylation (Lands cycle), it is not known how the composition of saturated fatty acids is controlled at the sn-1 position. Here, we demonstrate that lysophosphatidylglycerol acyltransferase 1 (LPGAT1) is an sn-1 specific acyltransferase that controls the stearate/palmitate ratio of phosphatidylethanolamine (PE) and phosphatidylcholine. Bacterially expressed murine LPGAT1 transferred saturated acyl-CoAs specifically into the sn-1 position of lysophosphatidylethanolamine (LPE) rather than lysophosphatidylglycerol and preferred stearoyl-CoA over palmitoyl-CoA as the substrate. In addition, genetic ablation of LPGAT1 in mice abolished 1-LPE:stearoyl-CoA acyltransferase activity and caused a shift from stearate to palmitate species in PE, dimethyl-PE, and phosphatidylcholine. Lysophosphatidylglycerol acyltransferase 1 KO mice were leaner and had a shorter life span than their littermate controls. Finally, we show that total lipid synthesis was reduced in isolated hepatocytes of LPGAT1 knockout mice. Thus, we conclude that LPGAT1 is an sn-1 specific LPE acyltransferase that controls the stearate/palmitate homeostasis of PE and the metabolites of the PE methylation pathway and that LPGAT1 plays a central role in the regulation of lipid biosynthesis with implications for body fat content and longevity.

PH-Responsive Liposomes of Dioleoyl Phosphatidylethanolamine and Cholesteryl Hemisuccinate for the Enhanced Anticancer Efficacy of Cisplatin.

The current study aimed to develop pH-responsive cisplatin-loaded liposomes (CDDP@PLs) via the thin film hydration method. Formulations with varied ratios of dioleoyl phosphatidylethanolamine (DOPE) to cholesteryl hemisuccinate (CHEMS) were investigated to obtain the optimal particle size, zeta potential, entrapment efficiency, in vitro release profile, and stability. The particle size of the CDDP@PLs was in the range of 153.2 ± 3.08–206.4 ± 2.26 nm, zeta potential was −17.8 ± 1.26 to −24.6 ± 1.72, and PDI displayed an acceptable size distribution. Transmission electron microscopy revealed a spherical shape with ~200 nm size. Fourier transform infrared spectroscopic analysis showed the physicochemical stability of CDDP@PLs, and differential scanning calorimetry analysis showed the loss of the crystalline nature of cisplatin in liposomes. In vitro release study of CDDP@PLs at pH 7.4 depicted the lower release rate of cisplatin (less than 40%), and at a pH of 6.5, an almost 65% release rate was achieved compared to the release rate at pH 5.5 (more than 80%) showing the tumor-specific drug release. The cytotoxicity study showed the improved cytotoxicity of CDDP@PLs compared to cisplatin solution in MDA-MB-231 and SK-OV-3 cell lines, and fluorescence microscopy also showed enhanced cellular internalization. The acute toxicity study showed the safety and biocompatibility of the developed carrier system for the potential delivery of chemotherapeutic agents. These studies suggest that CDDP@PLs could be utilized as an efficient delivery system for the enhancement of therapeutic efficacy and to minimize the side effects of chemotherapy by releasing cisplatin at the tumor site.

Application of Lipid Class Ratios for Sample Stability Monitoring-Evaluation of Murine Tissue Homogenates and SDS as a Stabilizer.

Lipids are a ubiquitous class of structurally complex molecules involved in various biological processes. In the fast-growing field of lipidomics, preanalytical issues are frequently neglected. Here, we investigated the stability of lipid profiles of murine liver, brain, lung, heart, and spleen homogenates by quantitative flow injection analysis using tandem mass spectrometry and high-resolution mass spectrometry. Storage of tissue homogenates at room temperature showed substantial alterations of the lipid profiles reflecting lipolytic action. Therefore, ratios of ceramide to sphingomyelin, lysophosphatidylethanolamine to phosphatidylethanolamine, lysophosphatidylcholine to phosphatidylcholine, and diglyceride to triglyceride were applied to monitor sample stability and the effect of sodium dodecyl sulfate (SDS) as a potential stabilizing agent. The addition of SDS led to a concentration-dependent stabilization of lipid profiles in liver, brain, and heart homogenates, while in lung and spleen homogenates, in particular, the lysophosphatidylethanolamine to phosphatidylethanolamine ratio increased upon addition of SDS. In conclusion, we demonstrated that lipid class ratios reflecting lipolytic activity could be applied to evaluate both the stability of samples and the influence of stabilizers.

Exercise training improves mitochondrial respiration and is associated with an altered intramuscular phospholipid signature in women with obesity

Aims/hypothesisWe sought to determine putative relationships among improved mitochondrial respiration, insulin sensitivity and altered skeletal muscle lipids and metabolite signature in response to combined aerobic and resistance training in women with obesity.MethodsThis study reports a secondary analysis of a randomised controlled trial including additional measures of mitochondrial respiration, skeletal muscle lipidomics, metabolomics and protein content. Women with obesity were randomised into 12 weeks of combined aerobic and resistance exercise training (n = 20) or control (n = 15) groups. Pre- and post-intervention testing included peak oxygen consumption, whole-body insulin sensitivity (intravenous glucose tolerance test), skeletal muscle mitochondrial respiration (high-resolution respirometry), lipidomics and metabolomics (mass spectrometry) and lipid content (magnetic resonance imaging and spectroscopy). Proteins involved in glucose transport (i.e. GLUT4) and lipid turnover (i.e. sphingomyelin synthase 1 and 2) were assessed by western blotting.ResultsThe original randomised controlled trial showed that exercise training increased insulin sensitivity (median [IQR]; 3.4 [2.0–4.6] to 3.6 [2.4–6.2] x10−5 pmol l−1 min−1), peak oxygen consumption (mean ± SD; 24.9 ± 2.4 to 27.6 ± 3.4 ml kg−1 min−1), and decreased body weight (84.1 ± 8.7 to 83.3 ± 9.7 kg), with an increase in weight (pre intervention, 87.8± 10.9 to post intervention 88.8 ± 11.0 kg) in the control group (interaction p < 0.05). The current study shows an increase in mitochondrial respiration and content in response to exercise training (interaction p < 0.05). The metabolite and lipid signature at baseline were significantly associated with mitochondrial respiratory capacity (p < 0.05) but were not associated with whole-body insulin sensitivity or GLUT4 protein content. Exercise training significantly altered the skeletal muscle lipid profile, increasing specific diacylglycerol(32:2) and ceramide(d18:1/24:0) levels, without changes in other intermediates or total content of diacylglycerol and ceramide. The total content of cardiolipin, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) increased with exercise training with a decrease in the PC:PE ratios containing 22:5 and 20:4 fatty acids. These changes were associated with content-driven increases in mitochondrial respiration (p < 0.05), but not with the increase in whole-body insulin sensitivity or GLUT4 protein content. Exercise training increased sphingomyelin synthase 1 (p < 0.05), with no change in plasma-membrane-located sphingomyelin synthase 2.Conclusions/interpretationThe major findings of our study were that exercise training altered specific intramuscular lipid intermediates, associated with content-driven increases in mitochondrial respiration but not whole-body insulin sensitivity. This highlights the benefits of exercise training and presents putative target pathways for preventing lipotoxicity in skeletal muscle, which is typically associated with the development of type 2 diabetes.Graphical abstract

Stable Isotopic Tracer Phospholipidomics Reveals Contributions of Key Phospholipid Biosynthetic Pathways to Low Hepatocyte Phosphatidylcholine to Phosphatidylethanolamine Ratio Induced by Free Fatty Acids.

There is a strong association between hepatocyte phospholipid homeostasis and non-alcoholic fatty liver disease (NAFLD). The phosphatidylcholine to phosphatidylethanolamine ratio (PC/PE) often draws special attention as genetic and dietary disruptions to this ratio can provoke steatohepatitis and other signs of NAFLD. Here we demonstrated that excessive free fatty acid (1:2 mixture of palmitic and oleic acid) alone was able to significantly lower the phosphatidylcholine to phosphatidylethanolamine ratio, along with substantial alterations to phospholipid composition in rat hepatocytes. This involved both a decrease in hepatocyte phosphatidylcholine (less prominent) and an increase in phosphatidylethanolamine, with the latter contributing more to the lowered ratio. Stable isotopic tracer phospholipidomic analysis revealed several previously unidentified changes that were triggered by excessive free fatty acid. Importantly, the enhanced cytidine diphosphate (CDP)-ethanolamine pathway activity appeared to be driven by the increased supply of preferred fatty acid substrates. By contrast, the phosphatidylethanolamine N-methyl transferase (PEMT) pathway was restricted by low endogenous methionine and consequently low S-adenosylmethionine, which resulted in a concomitant decrease in phosphatidylcholine and accumulation of phosphatidylethanolamine. Overall, our study identified several previously unreported links in the relationship between hepatocyte free fatty acid overload, phospholipid homeostasis, and the development of NAFLD.

Aging and β3-adrenergic stimulation alter mitochondrial lipidome of adipose tissue

Mitochondrial abundance and thermogenic capacity are two imperative components that distinguish brown, beige and white adipose tissues. Most importantly, the lipid composition is vital for maintaining the quantity, quality and function of mitochondria. Therefore, we employed quantitative lipidomics to probe the mitochondrial lipidome of adipose tissues. The mitochondrial lipidome reveals β3-adrenergic stimulation and aging drastically altered the levels of phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio and acyl chain desaturation. Precisely, PC36:2 and PE38:4 levels correlate with the increased brown and beige fat activity in young mice. While aging increased lysoPC species in white adipose tissue (WAT) mitochondria, CL-316,243 administration reduced lysoPC species and increased lyso-PE18:1 and 18:2 content during WAT browning. Also, non-thermogenic mitochondria accumulate sphingomyelin (SM), phosphatidylserine (PS), phosphatidic acid (PA) and ether-linked PC (ePC). Similarly, enrichment of phosphatidylglycerol (PG) and cardiolipin (CL) levels are associated with thermogenic mitochondria. Also, our in vitro experiment supports that blocking the de novo sphingolipid synthesis pathway by myriocin, SPT1 inhibitor increased the thermogenic capacity and oxygen consumption rate in mature adipocytes. Overall, our study suggests mitochondria of brown, beige and white adipose tissues own a unique pattern of lipid molecular species and their levels are altered by aging and CL-316,243 administration.

Bridging the Antimicrobial Activity of Two Lactoferricin Derivatives in E. coli and Lipid-Only Membranes.

We coupled the antimicrobial activity of two well-studied lactoferricin derivatives, LF11-215 and LF11-324, in Escherichia coli and different lipid-only mimics of its cytoplasmic membrane using a common thermodynamic framework for peptide partitioning. In particular, we combined an improved analysis of microdilution assays with ζ-potential measurements, which allowed us to discriminate between the maximum number of surface-adsorbed peptides and peptides fully partitioned into the bacteria. At the same time, we measured the partitioning of the peptides into vesicles composed of phosphatidylethanolamine (PE), phosphatidylgylcerol (PG), and cardiolipin (CL) mixtures using tryptophan fluorescence and determined their membrane activity using a dye leakage assay and small-angle X-ray scattering. We found that the vast majority of LF11-215 and LF11-324 readily enter inner bacterial compartments, whereas only 1−5% remain surface bound. We observed comparable membrane binding of both peptides in membrane mimics containing PE and different molar ratios of PG and CL. The peptides' activity caused a concentration-dependent dye leakage in all studied membrane mimics; however, it also led to the formation of large aggregates, part of which contained collapsed multibilayers with sandwiched peptides in the interstitial space between membranes. This effect was least pronounced in pure PG vesicles, requiring also the highest peptide concentration to induce membrane permeabilization. In PE-containing systems, we additionally observed an effective shielding of the fluorescent dyes from leakage even at highest peptide concentrations, suggesting a coupling of the peptide activity to vesicle fusion, being mediated by the intrinsic lipid curvatures of PE and CL. Our results thus show that LF11-215 and LF11-324 effectively target inner bacterial components, while the stored elastic stress makes membranes more vulnerable to peptide translocation.

Alterations in cellular and organellar phospholipid compositions of HepG2 cells during cell growth

The human hepatoblastoma cell line, HepG2, has been used for investigating a wide variety of physiological and pathophysiological processes. However, less information is available about the phospholipid metabolism in HepG2 cells. In the present report, to clarify the relationship between cell growth and phospholipid metabolism in HepG2 cells, we examined the phospholipid class compositions of the cells and their intracellular organelles by using enzymatic fluorometric methods. In HepG2 cells, the ratios of all phospholipid classes, but not the ratio of cholesterol, markedly changed with cell growth. Of note, depending on cell growth, the phosphatidic acid (PA) ratio increased and phosphatidylcholine (PC) ratio decreased in the nuclear membranes, the sphingomyelin (SM) ratio increased in the microsomal membranes, and the phosphatidylethanolamine (PE) ratio increased and the phosphatidylserine (PS) ratio decreased in the mitochondrial membranes. Moreover, the mRNA expression levels of enzymes related to PC, PE, PS, PA, SM and cardiolipin syntheses changed during cell growth. We suggest that the phospholipid class compositions of organellar membranes are tightly regulated by cell growth. These findings provide a basis for future investigations of cancer cell growth and lipid metabolism.

Milk fat globule size development in the mammary epithelial cell: a potential role for ether phosphatidylethanolamine

Milk fat globule (MFG) size is a milk production trait characteristic to the individual animal and has important effects on the functional and nutritional properties of milk. Although the regulation of MFG size in the mammary epithelial cell is not fully understood, lipid droplet (LD) fusion prior to secretion is believed to play a role. We selected cows that consistently produced milk with predominantly small or large MFGs to compare their lipidomic profiles, with focus on the polar lipid fraction. The polar lipid composition of the monolayer surrounding the LD is believed to either promote or prevent LD fusion. Using a targeted LC–MS/MS approach we studied the relative abundance of 301 detected species and found significant differences between the studied groups. Here we show that the lipidomic profile of milk from small MFG cows is characterised by higher phosphatidylcholine to phosphatidylethanolamine ratios. In contrast, the milk from large MFG cows contained more ether-phosphatidylethanolamine species. This is the first time that a potential role for ether-phosphatidylethanolamine in MFG size development has been suggested.

Lipids, proteins and extracellular metabolites of Trichoderma harzianum modifications caused by 2,4-dichlorophenoxyacetic acid as a plant growth stimulator

Strains of Trichoderma harzianum are well-known producers of bioactive secondary metabolites and have a beneficial effect on plants. However, to the best of our knowledge, the effect of the commonly used pesticides on the activity of this fungus is not yet investigated. Therefore, in the present study, the effect of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on the lipidome and selected extracellular compounds synthesized by T. harzianum IM 0961 was examined. It was observed that the herbicide 2,4-D caused changes in the lipid composition of the mycelium and that the herbicide exhibited lipophilic properties. In addition, the herbicide disturbed the phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio and increased membrane permeability. The higher amount of cardiolipin CL 72:7 and the lower amount of CL 72:8 could have been associated with a decreased ratio of 18:2 and 18:1 fatty acids in the herbicide-treated samples. Moreover, in the presence of 2,4-D, an increased lipid peroxidation (twofold), as well as a higher content of oxylipin (9-HODE and 13-HODE) and phosphatidic acid (PA), was noted, confirming that 2,4-D induced lipid peroxidation in the mycelium. The herbicide also exerted its toxic effect on the production of 14-aminoacid peptaibols and two compounds, harzianic acid and t22-azaphilone, exhibiting antibiotic and plant growth-promoting activity. During proteomic analysis, the synthesis of some proteins, such as calcineurin-like phosphoesterase metallophosphatases (MPPs), which modulate the properties of cell walls, was found to be inhibited by the herbicide. These presented findings may be of significant value in understanding the effect of 2,4-D on the activity of T. harzianum.