Complex Lipids Research Articles

A conserved complex lipid signature marks human muscle aging and responds to short-term exercise.

Studies in preclinical models suggest that complex lipids, such as phospholipids, play a role in the regulation of longevity. However, identification of universally conserved complex lipid changes that occur during aging, and how these respond to interventions, is lacking. Here, to comprehensively map how complex lipids change during aging, we profiled ten tissues in young versus aged mice using a lipidomics platform. Strikingly, from >1,200 unique lipids, we found a tissue-wide accumulation of bis(monoacylglycero)phosphate (BMP) during mouse aging. To investigate translational value, we assessed muscle tissue of young and older people, and found a similar marked BMP accumulation in the human aging lipidome. Furthermore, we found that a healthy-aging intervention consisting of moderate-to-vigorous exercise was able to lower BMP levels in postmenopausal female research participants. Our work implicates complex lipid biology as central to aging, identifying a conserved aging lipid signature of BMP accumulation that is modifiable upon a short-term healthy-aging intervention.

Membrane specificity of the human cholesterol transfer protein STARD4

STARD4 regulates cholesterol homeostasis by transferring cholesterol between the plasma membrane and endoplasmic reticulum. The STARD4 structure features a helix-grip fold surrounding a large hydrophobic cavity holding the sterol. Its access is controlled by a gate formed by the Ω1 and Ω4 loops and the C-terminal α-helix. Little is known about the mechanisms by which STARD4 binds to membranes and extracts/releases cholesterol. All available structures of STARD4 are without a bound sterol and display the same closed conformation of the gate. The cholesterol transfer activity of the mouse STARD4 is enhanced in the presence of anionic lipids, and in particular of phosphatidylinositol biphosphates (PIP2) for which two binding sites were proposed on the mouse STARD4 surface. Yet only one of these sites is conserved in human STARD4. We here report the results of a liposome microarray-based assay and microseconds-long molecular dynamics simulations of human STARD4 with complex lipid bilayers mimicking the composition of the donor and acceptor membranes. We show that the binding of apo form of human STARD4 is sensitive to the presence of PIP2 through two specific binding sites, one of which was not identified on mouse STARD4. We report two novel conformations of the gate in holo-STARD4: a yet-unobserved close conformation and an open conformation of Ω4 shedding light on the opening/closure mechanism needed for cholesterol uptake/release. Overall, the modulation of human STARD4 membrane-binding by lipid composition, and by the presence of the cargo supports the capacity of human STARD4 to achieve directed transfer between specific organelle membranes.

A Study of Metabolites from Basidiomycota and Their Activities against Pseudomonas aeruginosa.

The World Health Organization (WHO) promotes research aimed at developing new drugs from natural compounds. Fungi are important producers of bioactive molecules, and they are often effective against other fungi and/or bacteria and are thus a potential source of new antibiotics. Basidiomycota crude extracts, which have previously been proven to be active against Pseudomonas aeruginosa ATCC27853, were subjected to liquid chromatographic separation by RP-18, leading to six macro-fractions for each fungal extract. The various fractions were tested for their bioactivities against P. aeruginosa ATCC27853, and ten of them were characterized by HPLC-HRMS and NMR. Further chromatographic separations were performed for a few selected macro-fractions, yielding seven pure compounds. Bioactivity was mainly found in the lipophilic fractions containing fatty acids and their derivatives, such as hydroxy or keto C-18 unsaturated acids, and in various complex lipids, such as glycolipids and related compounds. More hydrophilic molecules, such as GABA, phenethylamine, two chromogenic anthraquinoids and pistillarin, were also isolated, and their antibacterial activities were recorded. The novelties of this research are as follows: (i) the genera Cortinarius and Mycena have never been investigated before for the synthesis of antibiotic compounds; (ii) the molecules produced by these genera are known, but their production has never been reported in the investigated fungi; (iii) the determination of bacterial siderophore synthesis inhibition by certain compounds from Cortinarius and Mycena.

Clusters of adipose tissue dysfunction in adults with type 2 diabetes identify those with worse lipidomic profile despite similar glycaemic control.

To investigate clusters of adipose tissue dysfunction, that is, with adipose tissue insulin resistance (ADIPO-IR) and large waist circumference (WC), identify a worse lipidomic profile characterised by a high proportion of lipids rich in saturated fatty acids (SFA). Hierarchical clustering based on WC and ADIPO-IR (calculated as fasting plasma non-esterified fatty acids times fasting plasma insulin, FFA×INS), was performed in 192 adults with overweight/obesity and type 2 diabetes (T2D) treated with metformin (HbA1c=7.8%). Free fatty acid composition and lipidomic profile were measured by mass spectrometry (GC-MS and LC-MSQTOF). Indexes of fatty acid desaturation (stearoyl-coA desaturase-1 activity, SCD116=palmitoleic acid/palmitic acid and SCD118=oleic acid/stearic acid) and of insulin resistance (HOMA-IR) were also calculated. Three clusters were identified: CL1 (ADIPO-IR=4.9±2.4 and WC=96±7cm, mean±SD), CL2 (ADIPO-IR=6.5±2.5 and WC=114±7cm), and CL3 (ADIPO-IR=15.0±4.7 and WC=107±8cm). Insulin concentrations, ADIPO-IR, and HOMA-IR significantly increased from CL1 to CL3 (all p<0.001), while fasting glucose concentrations, HbA1c, dietary lipids and caloric intake were similar. Moreover, CL3 showed significantly higher concentrations of monounsaturated free fatty acids, oleic and palmitoleic acids, triglycerides (TAG) rich in saturated FA and associated with de novo lipogenesis (i.e., TAG 46-50), higher SCD116, SCD118, ceramide (d18:0/18:0), and phosphatidylcholine aa(36:5) compared with CL1/CL2 (all p<0.005). High ADIPO-IR and large WC identify a worse lipid profile in T2D characterised by complex lipids rich in SFA, likely due to de novo synthesis given higher plasma monounsaturated FFA and increased desaturase activity indexes. ID NCT00700856 https://clinicaltrials.gov.

The shift in the fatty acid composition of the circulating lipidome in Alzheimer's disease.

Fatty acids (FAs) are the building blocks of complex lipids and signaling compounds; the role of the lipidome fatty acid profile (LFA) in AD progression remains unclear. The LFA of plasma and cerebrospinal fluid (CSF) samples from 289 participants (103 AD patients, 92 MCI patients, and 94 controls) was determined by GC-FID. The MCI subjects were followed up for 58±12.5 months. In controls, CSF has a more neuroprotective LFA than plasma. In CSF, a higher content of docosahexaenoic acid was associated with a reduced risk of MCI-to-AD progression. In plasma, higher oleic acid content was associated with lower risk of AD, MCI, and MCI-to-AD progression, whereas higher levels of vaccenic acid and docosahexaenoic acid were associated with greater risk of AD and MCI, and higher rate of MCI-to-AD progression, respectively. The circulating LFA is involved in the pathogenesis and progression of AD. The lipidome fatty acid profile in CSF and plasma was markedly different. Higher levels of vaccenic acid and lower levels of oleic acid in plasma were associated with greater risk of Alzheimer's disease. In plasma, higher levels of oleic acid were associated with a reduced risk of MCI-to-AD progression. Higher levels of docosahexaenoic acid in CSF were associated with a lower risk of MCI-to-AD progression. Higher levels of docosahexaenoic acid in plasma were associated with a greater rate of MCI-to-AD progression.

Abstract 4765: Tumor, serum and plasma metabolome in patients with high-grade bladder cancer

Abstract Introduction: Bladder cancer (BlCa) ranks as the sixth most prevalent cancer in the world. A number of urine based BlCa detection tests have recently been approved for clinical use, including the NMP22 tests and the bladder tumor antigen (BTA) tests for both detection and monitoring of patients with known bladder cancer. The FDA have approved them both for surveillance in BlCa only in conjunction with cystoscopy. Unfortunately, the sensitivity and specificity of these tests are poor, and cannot distinguish between aggressive and non-aggressive tumors. This project aims to identify alternate markers that can overcome these drawbacks. Methods: We characterized the global metabolome of 47 bladder tumors retrieved by radical cystectomy (RC) or TURBT, including 14 specimens with matching plasma and serum samples. All specimens were subjected to untargeted metabolomic approaches with gas chromatography- time-of-flight mass spectrometry (GC-TOF MS) to profile primary metabolites, hydrophilic interaction liquid chromatography MS (HILIC-QTOF MS) to profile biogenic amines, and liquid chromatography charged surface hybrid MS (LC-CSH-QTOF MS) to characterize complex lipids. Differentially expressed metabolites were analyzed with respect to grade, stage, metastases, and the use of neoadjuvant chemotherapy. For selected metabolites, area under the ROC curve (AUC) is calculated as % sensitivity vs. 100% specificity. Expression of highly discriminatory metabolites were then validated in the plasma and serum samples to determine which forum would provide greater specificity. Results: We first assembled a discovery cohort of 33 patients who did not have matching blood specimen for primary metabolites, biogenic amines and complex lipids. Comparison of high vs low grade tumors yielded two lipids, a sphingomyelin (SM (d38) and a phosphatidylcholine PC(p-38), that were significantly different (adjusted p-value&amp;lt;0.05, log2fold change&amp;gt;2.5). These results were validated in the 14-sample cohort with matching plasma and serum of which the latter showed significant correlation between tumor and plasma (Spearman R=0.872, adj p=0.03). Conclusions: Our results indicate that certain sphingomyelins and phosphatidylcholines may be useful as plasma markers of high grade BlCa. Further analyses are required to evaluate whether these markers are more discriminatory compared to existing urinary markers. Citation Format: Maria-Malvina Tsamouri, Akshaya Karthikeyan, Christopher A. Lucchesi, Blythe P. Durbin-Johnson, Marc A. Dall'Era, Paramita M. Ghosh. Tumor, serum and plasma metabolome in patients with high-grade bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4765.

Disruption of polyunsaturated fatty acid biosynthesis drives STING-dependent acute myeloid leukemia cell maturation and death

The role of polyunsaturated fatty acid (PUFA) biosynthesis in acute myeloid leukemia (AML) remains largely undefined. A comparative expression analysis of 35 genes encoding fatty acid biosynthesis enzymes showed that fatty acid desaturase 1 (FADS1) was highly expressed across multiple AML subtypes relative to healthy controls and that elevated FADS1 expression correlates with worse overall AML patient survival. Functionally, shRNA-mediated inhibition of FADS1 reduced AML cell growth invitro and significantly delayed leukemia onset in an AML mouse model. AML cell lines depleted of FADS1 arrested in the G1/S-phase of the cell cycle, acquired characteristics of myeloid maturation and subsequently died. To understand the molecular consequences of FADS1 inhibition, a combination of mass spectrometry-based analysis of complex lipids and gene expression analysis (RNA-seq) was performed. FADS1 inhibition caused AML cells to exhibit significant lipidomic remodeling, including depletion of PUFAs from the phospholipids, phosphatidylserine, and phosphatidylethanolamine. These lipidomic alterations were accompanied by an increase induction of inflammatory and stimulator of interferon genes (STING)-mediated type-1 interferon signaling. Remarkably, genetic deletion of STING largely prevented the AML cell maturation and death phenotypes mediated by FADS1 inhibition. Highlighting the therapeutic implications of these findings, pharmacological blockade of PUFA biosynthesis reduced patient-derived AML cell numbers exvivo but not that of healthy donor cells. Similarly, STING agonism attenuated patient-derived-AML survival; however, STING activation also reduced healthy granulocyte numbers. Collectively, these data unveil a previously unrecognized importance of PUFA biosynthesis in leukemogenesis and that imbalances in PUFA metabolism can drive STING-mediated AML maturation and death.

Determination of fatty acid uptake and desaturase activity in mammalian cells by NMR-based stable isotope tracing

BackgroundMammalian cells both import exogenous fatty acids and synthesize them de novo. Palmitate, the end product of fatty acid synthase (FASN) is a substrate for stearoyl-CoA desaturases (Δ-9 desaturases) that introduce a single double bond into fatty acyl-CoA substrates such as palmitoyl-CoA and stearoyl-CoA. This process is particularly upregulated in lipogenic tissues and cancer cells. Tracer methodology is needed to determine uptake versus de novo synthesis of lipids and subsequent chain elongation and desaturation. Here we describe an NMR method to determine the uptake of 13C-palmitate from the medium into HCT116 human colorectal cancer cells, and the subsequent desaturation and incorporation into complex lipids. ResultsExogenous 13C16-palmitate was absorbed from the medium by HCT116 cells and incorporated primarily into complex glycerol lipids. Desaturase activity was determined from the quantification of double bonds in acyl chains, which was greatly reduced by ablation of the major desaturase SCD1. SignificanceThe NMR approach requires minimal sample preparation, is non-destructive, and provides direct information about the level of saturation and incorporation of fatty acids into complex lipids.

Unwrapping the (glyco-)lipidome in the microalgae Microchloropsis gaditana: Effects of eco-friendly extraction methods

Microalgae have recently captivated worldwide attention because of their extraordinary lipid composition, which includes essential omega-3 and omega-6 long-chain polyunsaturated fatty acids (LC-PUFA), neutral lipids, and more complex lipids (glycolipids and phospholipids), as well as eicosanoids. To explore this further, we present a novel (glyco-)lipidomics strategy, including a green chemistry extraction approach, liquid chromatography high-resolution mass spectrometry (LC-HRMS) lipid profiling, and a comprehensive data evaluation workflow. Using untargeted lipidomics based on LC-HRMS approaches, we studied the lipid composition up to the molecular species level of the microalgae Microchloropsis gaditana (formerly Nannochloropsis gaditana), focusing on the distribution of saturated, monounsaturated, and polyunsaturated fatty acids in different lipid classes. Our analysis successfully identified 520 lipid molecular species from 18 lipid classes. Regarding fatty acid composition, our primary focus was on elucidating the distribution of LC-PUFA across the different microalgal lipid classes. This particular strategy holds potential as it may unravel the use of microalgal lipids as innovative sources of food ingredients. Our findings show that LC-PUFA are relatively elevated in membrane lipids, in which cholesterol esters (ChE) have the highest PUFA share (94 %), followed by polar membrane lipids (glycolipids and phospholipids), with phosphatidylethanolamines (PE) up to 67 %, phosphatidylglycerols (PG) up to 47 %, and monogalactosyldiacylglycerols (MGDG) up to 47 % PUFA content. This highlights the high percentage of PUFA in the structural parts of microalgae and the importance of these lipid classes as a reservoir for PUFA. Finally, we also compared a classical Folch extraction method with environmentally friendly ultrasound-assisted extraction approaches and enzymatic pre-treatments to investigate the lipid composition and enrichment of valuable lipid classes. Thus, this extensive analysis of the (glyco-)lipidome of Microchloropsis gaditana provides valuable insights into the existing knowledge about this microalgae species, enhancing our understanding of the complex lipid composition and opening up new opportunities for potential applications in the biotechnology and food industry.

Abstract B053: Lipid metabolism and lipidomics of ovarian clear cell carcinoma: a comprehensive analysis

Abstract Ovarian clear cell carcinoma (OCCC) is a chemoresistant subtype of ovarian cancer accounting for 5-12% of all epithelial ovarian cancer cases. Lipid metabolism has been reported to be involved in platinum resistance and resistance to ferroptosis induction in various cancer types. However, the metabolomic and lipidomic landscape of OCCC has not been characterized. To explore the metabolic processes driving the aggressive nature of advanced OCCC, we carried out global metabolomic and lipidomic profiling on 64 early and advanced ovarian cancer samples, including tumors with corresponding metastases. Snap frozen tissue from 29 OCCC, 22 high-grade serous carcinoma (HGSOC), and 13 endometrioid ovarian carcinoma (EnOC) cases were included. Hydrophilic interaction liquid chromatography (HILIC)-mass spectrometry was performed to quantify the relative steady-state level of 318 unique metabolites. Principal component analysis (PCA) showed a distinct separation between OCCC and HGSOC/EnOC samples, mainly driven by short and medium chain acylcarnitine and hydroxylated acylcarnitine esters that were significantly more abundant in OCCC. Acylcarnitines are intermediaries of fatty acid beta-oxidation (FAO), and their enrichment in OCCC patient samples might indicate a difference in fatty acid utilization and lipid metabolism compared to OCCC and HGSOC/EnOC. FAO has been linked to redox homeostasis in cancer cells, acting as a protective factor against lipid peroxidation through selective depletion of certain lipid species. To explore whether deeper lipidomic changes in OCCC accompany increased acylcarnitine production, we performed global complex lipid detection on 7 OCCCs and 3 HGSOC tissue samples using reverse phase liquid chromatography and mass spectrometry. Six-hundred and fifty-seven unique lipid species were successfully identified. A comparison of lipid class abundances between OCCC and HGSOC uncovered increased levels of medium chain acylcarnitines in OCCC, corroborating the HILIC findings. Moreover, the levels of cholesterol esters and ceramides with polyunsaturated fatty acid chains were also more abundant in OCCC. Conversely, choline plasmalogens, a lipid class known to induce lipid peroxidation and ferroptosis cell death exhibited significantly lower abundance in OCCC. Additionally, saturated ethanolamine plasmalogens and ceramides containing saturated fatty acids were decreased in OCCC. In summary, our study reports significant differences in the lipid metabolism of OCCC compared to other major ovarian cancer subtypes. The observed decrease in peroxidation-prone lipid species might indicate the involvement of FAO in OCCC’s redox homeostasis and highlights FAO as a potential novel mechanism of platinum resistance in OCCC. Ongoing studies, presented at the meeting include the metabolomic and lipidomic profiling of a large panel of ovarian cancer cell lines to identify those that best model patient sample metabolism. These models will subsequently be used for in vitro and in vivo studies to evaluate FAO as a potential therapeutic target in OCCC. Citation Format: Agnes J. Bilecz, Roya AminiTabrizi, Hardik Shah, Ernst Lengyel. Lipid metabolism and lipidomics of ovarian clear cell carcinoma: a comprehensive analysis [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B053.

Hydrolysis of Phospholipids in Presence of Phospholipase D:Thrmodynamic and Kinetic Studies of Hydrolysis in Water and Alcoholic Environments

Phospholipase D(PLD)catalyzes the hydrolysis of phospholipids to produce phosphatidic acid and hydroxyl compounds. Phosphatidylcholine (PC) is the most abundant phospholipid in animals and plants, often constituting nearly 50% of total complex lipids in animals and plants. However, Phosphatidylserine (PS) makes up around 5–10% of all phospholipids. This work includes the monitoring thermodynamic and kinetic of the enzymatic hydrolysis of PC and PS in both water and ethanol. All experiments were performedusing the Schlink line technique inN2present as an inert gas to prevent the oxidative stress. UV/Vis. spectrophotometer was used to observe the kinetic of all enzymatic reactions. In addition, the Origin 2019 software was used to analyze and find the kinetic parameters of the enzymatic hydrolysis reactions. The results show that the enzymatic hydrolys is reactions of PC in water and in ethanol occurredat the same conditions, enzymatic activity 1.752 U/mg, temperature 37 ᵒC, and pH =7. However, the enzymatic hydrolysis reactions of PS in water occurred at different conditions than in ethanol. The enzymatic substitution reaction of PSinethanol was thermodynamic favorable reaction due to the value of the ∆G =_164.868 J, but in water was thermodynamically unfavorable ΔG = 65.048 J. However, the enzymatic hydrolysis reaction of PC in water was thermodynamically unfavorable ∆G = 345.319 Jaswellas in ethanol ∆G = 74.433 J. The study shows that there is clear impact of present nitrogen bases of PC and the environment of the hydrolysis on the activity of the enzymatic catalyzing.

Intraperitoneally injected d11-11(12)-epoxyeicosatrienoic acid is rapidly incorporated and esterified within rat plasma and peripheral tissues but not the brain

Epoxyeicosatrienoic acids (EpETrEs) are bioactive lipid mediators of arachidonic acid cytochrome P450 oxidation. In vivo, the free (unbound) form of EpETrEs regulate multiple processes including blood flow, angiogenesis and inflammation resolution. Free EpETrEs are thought to rapidly degrade via soluble epoxide hydrolase (sEH); yet, in many tissues, the majority of EpETrEs are esterified to complex lipids (e.g. phospholipids) suggesting that esterification may play a major role in regulating free, bioactive EpETrE levels. This hypothesis was tested by quantifying the metabolism of intraperitoneally injected free d11-11(12)-Epoxyeicosatrienoic acid (d11-11(12)-EpETrE) in male and female rats. Plasma and tissues (liver, adipose and brain) were obtained 3 to 4 min later and assayed for d11-11(12)-EpETrE and its sEH metabolite, d11-11,12-dihydroxyeicosatrienoic acid (d11-11,12-diHETrE) in both the free and esterified lipid fractions. In both males and females, the majority of injected tracer was recovered in liver followed by plasma and adipose. No tracer was detected in the brain, indicating that brain levels are maintained by endogenous synthesis from precursor fatty acids. In plasma, liver, and adipose, the majority (>54 %) of d11-11(12)-EpETrE was found esterified to phospholipids or neutral lipids (triglycerides and cholesteryl esters). sEH-derived d11-11,12-diHETrE was not detected in plasma or tissues, suggesting negligible conversion within the 3–4 min period post tracer injection. This study shows that esterification is the main pathway regulating free 11(12)-EpETrE levels in vivo.

GPCRs in the round: SMA-like copolymers and SMALPs as a platform for investigating GPCRs

G-protein-coupled receptors (GPCRs) are the largest family of membrane proteins, regulate a plethora of physiological responses and are the therapeutic target for 30–40% of clinically-prescribed drugs. They are integral membrane proteins deeply embedded in the plasma membrane where they activate intracellular signalling via coupling to G-proteins and β-arrestin. GPCRs are in intimate association with the bilayer lipids and that lipid environment regulates the signalling functions of GPCRs. This complex lipid ‘landscape’ is both heterogeneous and dynamic. GPCR function is modulated by bulk membrane properties including membrane fluidity, microdomains, curvature, thickness and asymmetry but GPCRs are also regulated by specific lipid:GPCR binding, including cholesterol and anionic lipids. Understanding the molecular mechanisms whereby GPCR signalling is regulated by lipids is a very active area of research currently. A major advance in membrane protein research in recent years was the application of poly(styrene-co-maleic acid) (SMA) copolymers. These spontaneously generate SMA lipid particles (SMALPs) encapsulating membrane protein in a nano-scale disc of cell membrane, thereby removing the historical need for detergent and preserving lipid:GPCR interaction. The focus of this review is how GPCR-SMALPs are increasing our understanding of GPCR structure and function at the molecular level. Furthermore, an increasing number of ‘second generation’ SMA-like copolymers have been reported recently. These are reviewed from the context of increasing our understanding of GPCR molecular mechanisms. Moreover, their potential as a novel platform for downstream biophysical and structural analyses is assessed and looking ahead, the translational application of SMA-like copolymers to GPCR drug discovery programmes in the future is considered.

A Metabolome-Wide Mendelian Randomization Study Identifies Dysregulated Arachidonic Acid Synthesis as a Potential Causal Risk Factor for Bipolar Disorder

BackgroundBipolar disorder (BPD) is a debilitating mood disorder with an unclear etiology. A better understanding of the underlying pathophysiological mechanisms will help to identify novel targets for improved treatment options and prevention strategies. In this metabolome-wide Mendelian randomization study, we screened for metabolites that may have a causal role in BPD. MethodsWe tested a total of 913 circulating metabolite exposures assessed in 14,296 Europeans using a mass spectrometry-based platform. For the BPD outcome, we used summary data from the largest and most recent genome-wide association study reported to date, including 41,917 BPD cases. ResultsWe identified 33 metabolites associated with BPD (padjusted < 5.48 × 10−5). Most of them were lipids, including arachidonic acid (β = −0.154, SE = 0.023, p = 3.30 × 10−11), a polyunsaturated omega-6 fatty acid, along with several complex lipids containing either an arachidonic or a linoleic fatty acid side chain. These associations did not extend to other closely related psychiatric disorders like schizophrenia or depression, although they may be involved in the regulation of lithium response. These lipid associations were driven by genetic variants within the FADS1/2/3 gene cluster, which is a robust BPD risk locus encoding a family of fatty acid desaturase enzymes that are responsible for catalyzing the conversion of linoleic acid into arachidonic acid. Statistical colocalization analyses indicated that 27 of the 33 metabolites shared the same genetic etiology with BPD at the FADS1/2/3 cluster, demonstrating that our findings are not confounded by linkage disequilibrium. ConclusionsOverall, our findings support the notion that arachidonic acid and other polyunsaturated fatty acids may represent potential targets for BPD.

Myomerger Induces Membrane Hemifusion and Regulates Fusion Pore Expansion.

Membrane fusion is a crucial mechanism in a wide variety of important events in cell biology from viral infection to exocytosis. However, despite many efforts and much progress, cell-cell fusion has remained elusive to our understanding. Along the life of the fusion pore, large conformational changes take place from the initial lipid bilayer bending, passing through the hemifusion intermediates, and ending with the formation of the first nascent fusion pore. In this sense, computer simulations are an ideal technique for describing such complex lipid remodeling at the molecular level. In this work, we studied the role played by the muscle-specific membrane protein Myomerger during the formation of the fusion pore. We have conducted μs length atomistic and coarse-grained molecular dynamics, together with free-energy calculations using ad hoc collective variables. Our results show that Myomerger favors the hemifusion diaphragm-stalk transition, reduces the nucleation-expansion energy difference, and promotes the formation of nonenlarging fusion pores.

Complex lipid oxidation induces two distinct membrane permeabilization mechanisms

Complex lipid oxidation induces two distinct membrane permeabilization mechanisms

Abstract A017: Lipidomic biomarkers of usual alcohol use and their association with pancreatic ductal adenocarcinoma risk

Abstract There is suggestive epidemiologic evidence for an association between alcohol use and pancreatic ductal adenocarcinoma (PDAC). Previous studies reported alcohol use to be associated with high-density lipoprotein and fatty acyl ethyl esters. Lipidomic metabolites may improve assessment of self-reported alcohol use and provide mechanistic insights for the alcohol-PDAC association. We aimed to identify lipidomic signatures of self-reported alcohol use and evaluate their associations with PDAC risk using comprehensive lipidomic measures from nested case-control studies within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study and the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial cohorts (716 case-control sets). Controls were matched to cases (1:1) by age, sex, race, date of blood draw and cohort. Serum samples and self-reported alcohol use were collected at baseline up to 24 years before PDAC diagnosis. Absolute concentrations of 611 lipid species across 15 classes were measured on the Complex Lipid Panel (Lipidizer Sciex SelexIon-5500 QTRAP platform) and grouped into 161 class-specific fatty acids (FA) and 28 total FAs. We conducted cross-sectional analyses using linear regression to identify associations between log-transformed alcohol and 1-standard deviation (SD) increase in log-lipid concentrations among controls within each cohort. Conditional logistic regression and fixed-effects meta-analysis were used to calculate PDAC odds ratios (OR) per 1-SD increase in the log-concentration identified alcohol-lipidomic metabolites in common in both cohorts. All models were adjusted for age, smoking, BMI, height, diabetes, education, and diet quality. Alcohol was associated with 36 species, 13 class-specific FAs and 3 total FAs at Bonferroni thresholds &amp;lt;8.18 × 10−5, &amp;lt;3.11 × 10−4, and &amp;lt;1.79 × 10−3, respectively. These belonged to ceramides, cholesterylesters, diacylglycerols, hexosylceramides, lactosylceramides, lysophosphatidylcholines, phosphatidylcholines, phospatidylethanolamines, phosphatidylinositols, and triacylglycerols lipid classes, and mostly holding FA15:0 (pentadecanoic acid), FA17:0 (margaric acid) and FA18:2 (linoleic acid). Nine triacylglycerols (TAG(49:3-FA18:2), TAG(51:3-FA18:2), TAG(49:2-FA18:2), TAG(49:3-FA15:0), TAG(51:3-FA15:0), TAG(51:4-FA18:3), TAG(51:2-FA18:2), TAG(51:5-FA18:3), TAG(51:2-FA15:0)) and one phosphatidylcholine (PC(15:0/18:2)) lipid species and one total FA (FA15:0) showed significant inverse associations with PDAC with ORs ranging from 0.82 to 0.86 (FDR&amp;lt;0.10). There was no association between class-specific FAs and PDAC. Our results support the hypothesis that alcohol use is associated with serum lipid species from 10 lipid classes, mostly with FA15:0, FA17:0 and FA18:2. Nine triacylglycerols, one phosphatidylcholine and total FA15:0 showed inverse prospective associations with PDAC, the latter having been shown to decrease inflammation and regulate glucose metabolism in previous studies. These findings may help understand the inconsistent association between alcohol use and PDAC. Citation Format: Sabine Naudin, Paloma Mitra, Ting Zhang, Demetrius Albanes, Steven C. Moore, Rachael Stolzenberg-Solomon. Lipidomic biomarkers of usual alcohol use and their association with pancreatic ductal adenocarcinoma risk [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A017.

Abstract A055: Context-dependent role of acid sphingomyelinase in pancreatic ductal adenocarcinoma

Abstract Sphingolipids, a class of lipids that includes the bioactive molecules ceramide and sphingosine-1-phosphate (S1P), play a contrasting role in the regulation of cancer cell death and survival. Acid sphingomyelinase (SMPD1) is the key enzyme that breaks down sphingomyelin into ceramide, accounting for more than 50% of the cellular ceramide supply and influencing the development of cancer. Here, we studied the role of SMPD1 in different cellular compartments during pancreatic ductal adenocarcinoma (PDAC) carcinogenesis. We performed a targeted quantitative plasma metabolite analysis of 637 patients (PDAC, 356; nonpancreatic disease, 281). We completed the IHC multiplex staining in the TMA of 124 patients with PDAC. Using CRISPR-Cas9 gene editing, SMPD1 was deleted in PDAC cell lines. The influence of SMPD1-KO on proliferation, colony formation, and migration was studied. Tumor progression and carcinogenesis was monitored in an orthotopic PDAC model and metastasis models using SMPD1 null cells, as well as in KC and KPC mice. Plasma metabolite analysis of 637 patients (PDAC, 356; nonpancreatic disease, 281) revealed the most pronounced changes in the group of complex lipids with the highest prevalence of sphingomyelins and ceramides. Overall survival was significantly correlated with high SMPD1 expression in tumor tissue in two independent cohorts. Interestingly, IHC multiplex analysis of SMPD1 'high expression' in cancer cells was associated with poorer survival, while SMPD1 'high expression' in immune cells was associated with longer overall survival. Expression level of Smpd1 differed from preneoplastic to neoplastic lesions in PDAC mouse tissue. Smpd1-KO in PDAC cell lines resulted in decreased proliferation, colony formation, and migration. In an orthotopic mouse model, Smpd1-KO clones resulted in significantly lower tumor weight and size compared to WT clones. Furthermore, the injection of WT clones into global Smpd1het-KO mice resulted in a significant increase in tumor weight compared to the injection of WT clones into WT mice. In two different metastasis models, injecting Smpd1-KO led to a significantly reduced formation of metastases in the lung and liver compared to WT clones. Interestingly, injecting WT clones into global Smpd1het-KO mice resulted in a significant decrease in metastasis formation in the lung compared to the injection of WT clones into WT mice. In general, we show a significant increase in survival in an orthotopic PDAC model using PDAC Smpd1-KO cells. In conclusion, SMPD1 has a context-dependent role in cancer cells and immune cells in PDAC. Inhibition of SMPD1 specifically in cancer cells is a viable therapeutic option, which should be explored in future studies. Citation Format: Ahmed Alnatsha, Qi Li, Klea Ricku, Prince Allawadhi, Jianying Xu, Didem Saka, Theresa Weltermann, Quan Zhaou, Nicole Schreiner, Kirsten Lauber, Ivonne Regel, Ujjwal Mukund Mahajan, Julia Mayerle. Context-dependent role of acid sphingomyelinase in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A055.

Human lipocalins bind and export fatty acids through the secretory pathway of yeast cells.

The activation of fatty acids to their acyl-CoA derivatives is a crucial step for their integration into more complex lipids or their degradation via beta-oxidation. Yeast cells employ five distinct acyl-CoA synthases to facilitate this ATP-dependent activation of acyl chains. Notably, mutant cells that are deficient in two of these fatty acid-activating (FAA) enzymes, namely, Faa1 and Faa4, do not take up free fatty acids but rather export them out of the cell. This unique fatty acid export pathway depends on small, secreted pathogenesis-related yeast proteins (Pry). In this study, we investigate whether the expression of human fatty acid-binding proteins, including Albumin, fatty acid-binding protein 4 (Fabp4), and three distinct lipocalins (ApoD, Lcn1, and Obp2a), could promote fatty acid secretion in yeast. To optimize the expression and secretion of these proteins, we systematically examined various signal sequences in both low-copy and high-copy number plasmids. Our findings reveal that directing these fatty-acid binding proteins into the secretory pathway effectively promotes fatty acid secretion from a sensitized quadruple mutant model strain (faa1∆ faa4∆ pry1∆ pry3∆). Furthermore, the level of fatty acid secretion exhibited a positive correlation with the efficiency of protein secretion. Importantly, the expression of all human lipid-binding proteins rescued Pry-dependent fatty acid secretion, resulting in the secretion of both long-chain saturated and unsaturated fatty acids. These results not only affirm the in vitro binding capabilities of lipocalins to fatty acids but also present a novel avenue for enhancing the secretion of valuable lipidic compounds. Given the growing interest in utilizing yeast as a cellular factory for producing poorly soluble compounds and the potential of lipocalins as platforms for engineering substrate-binding specificity, our model is considered as a powerful tool for promoting the secretion of high-value lipid-based molecules.

Fatty acid synthesis suppresses dietary polyunsaturated fatty acid use

Dietary polyunsaturated fatty acids (PUFA) are increasingly recognized for their health benefits, whereas a high production of endogenous fatty acids – a process called de novo lipogenesis (DNL) - is closely linked to metabolic diseases. Determinants of PUFA incorporation into complex lipids are insufficiently understood and may influence the onset and progression of metabolic diseases. Here we show that fatty acid synthase (FASN), the key enzyme of DNL, critically determines the use of dietary PUFA in mice and humans. Moreover, the combination of FASN inhibition and PUFA-supplementation decreases liver triacylglycerols (TAG) in mice fed with high-fat diet. Mechanistically, FASN inhibition causes higher PUFA uptake via the lysophosphatidylcholine transporter MFSD2A, and a diacylglycerol O-acyltransferase 2 (DGAT2)-dependent incorporation of PUFA into TAG. Overall, the outcome of PUFA supplementation may depend on the degree of endogenous DNL and combining PUFA supplementation and FASN inhibition might be a promising approach to target metabolic disease.