Diabetic retinopathy is a ceramidopathy reversible by anti-ceramide immunotherapy.

Diabetic retinopathy is a microvascular disease that causes blindness. Using acid sphingomyelinase knockout mice, we reported that ceramide generation is critical for diabetic retinopathy development. Here, in patients with proliferative diabetic retinopathy, we identify vitreous ceramide imbalance with pathologic long-chain C16-ceramides increasing and protective very long-chain C26-ceramides decreasing. C16-ceramides generate pro-inflammatory/pro-apoptotic ceramide-rich platforms on endothelial surfaces. To geo-localize ceramide-rich platforms, we invented a three-dimensional confocal assay and showed that retinopathy-producing cytokines TNFα and IL-1ß induce ceramide-rich platform formation on retinal endothelial cells within seconds, with volumes increasing 2-logs, yielding apoptotic death. Anti-ceramide antibodies abolish these events. Furthermore, intravitreal and systemic anti-ceramide antibodies protect from diabetic retinopathy in standardized rodent ischemia reperfusion and streptozotocin models. These data support (1) retinal endothelial ceramide as a diabetic retinopathy treatment target, (2) early-stage therapy of non-proliferative diabetic retinopathy to prevent progression, and (3) systemic diabetic retinopathy treatment; and they characterize diabetic retinopathy as a "ceramidopathy" reversible by anti-ceramide immunotherapy.

Untargeted and temporal analysis of retinal lipidome in bacterial endophthalmitis.

Bacterial endophthalmitis is a blinding infectious disease typically acquired during ocular surgery. We previously reported significant alterations in retinal metabolism during Staphylococcus (S) aureus endophthalmitis. However, the changes in retinal lipid composition during endophthalmitis are unknown. Here, using a mouse model of S. aureus endophthalmitis and an untargeted lipidomic approach, we comprehensively analyzed temporal alterations in total lipids and oxylipin in retina. Our data showed a time-dependent increase in the levels of lipid classes, sphingolipids, glycerolipids, sterols, and non-esterified fatty acids, whereas levels of phospholipids decreased. Among lipid subclasses, phosphatidylcholine decreased over time. The oxylipin analysis revealed increased prostaglandin-E2, hydroxyeicosatetraenoic acids, docosahexaenoic acid, eicosapentaenoic acid, and α-linolenic acid. In-vitro studies using mouse bone marrow-derived macrophages showed increased lipid droplets and lipid-peroxide formation in response to S. aureus infection. Collectively, these findings suggest that S. aureus-infection alters the retinal lipid profile, which may contribute to the pathogenesis of bacterial endophthalmitis.

Dietary fish oil enriched in very-long-chain polyunsaturated fatty acid reduces cardiometabolic risk factors and improves retinal function.

Very-long-chain polyunsaturated fatty acids (VLCPUFAs; C24-38) constitute a unique class of PUFA that have important biological roles, but the lack of a suitable dietary source has limited research in this field. We produced an n-3 C24-28-rich VLCPUFA-oil concentrated from fish oil to study its bioavailability and physiological functions in C57BL/6J mice. The serum and retinal C24:5 levels increased significantly compared to control after a single-dose gavage, and VLCPUFAs were incorporated into the liver, brain, and eyes after 8-week supplementation. Dietary VLCPUFAs resulted in favorable cardiometabolic changes, and improved electroretinography responses and visual performance. VLCPUFA supplementation changed the expression of genes involved in PPAR signaling pathways. Further in vitro studies demonstrated that the VLCPUFA-oil and chemically synthesized C24:5 are potent agonists for PPARs. The multiple potential beneficial effects of fish oil-derived VLCPUFAs on cardiometabolic risk and eye health in mice support future efforts to develop VLCPUFA-oil into a supplemental therapy.

Loss of carnitine palmitoyltransferase 1a reduces docosahexaenoic acid-containing phospholipids and drives sexually dimorphic liver disease in mice.

BACKGROUND AND AIMS: Genome and epigenome wide association studies identified variants in carnitine palmitoyltransferase 1a (CPT1a) that associate with lipid traits. The goal of this study was to determine the role of liver-specific CPT1a on hepatic lipid metabolism. APPROACH AND RESULTS: Male and female liver-specific knockout (LKO) and littermate controls were placed on a low-fat or high-fat diet (60% kcal fat) for 15 weeks. Mice were necropsied after a 16 h fast, and tissues were collected for lipidomics, matrix-assisted laser desorption ionization mass spectrometry imaging, kinome analysis, RNA-sequencing, and protein expression by immunoblotting. Female LKO mice had increased serum alanine aminotransferase levels which were associated with greater deposition of hepatic lipids, while male mice were not affected by CPT1a deletion relative to male control mice. Mice with CPT1a deletion had reductions in DHA-containing phospholipids at the expense of monounsaturated fatty acids (MUFA)-containing phospholipids in whole liver and at the level of the lipid droplet (LD). Male and female LKO mice increased RNA levels of genes involved in LD lipolysis (Plin2, Cidec, G0S2) and in polyunsaturated fatty acid metabolism (Elovl5, Fads1, Elovl2), while only female LKO mice increased genes involved in inflammation (Ly6d, Mmp12, Cxcl2). Kinase profiling showed decreased protein kinase A activity, which coincided with increased PLIN2, PLIN5, and G0S2 protein levels and decreased triglyceride hydrolysis in LKO mice. CONCLUSIONS: Liver-specific deletion of CPT1a promotes sexually dimorphic steatotic liver disease (SLD) in mice, and here we have identified new mechanisms by which females are protected from HFD-induced liver injury.

Loss of Carnitine Palmitoyltransferase 1a Reduces Docosahexaenoic Acid-Containing Phospholipids and Drives Sexually Dimorphic Liver Disease in Mice.

Background and Aims: Genome and epigenome wide association studies identified variants in carnitine palmitoyltransferase 1a (CPT1a) that associate with lipid traits. The goal of this study was to determine the impact by which liver-specific CPT1a deletion impacts hepatic lipid metabolism. Approach and Results: Six-to-eight-week old male and female liver-specific knockout (LKO) and littermate controls were placed on a low-fat or high-fat diet (HFD; 60% kcal fat) for 15 weeks. Mice were necropsied after a 16 hour fast, and tissues were collected for lipidomics, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), kinome analysis, RNA-sequencing, and protein expression by immunoblotting. Female LKO mice had increased serum alanine aminotransferase (ALT) levels which were associated with greater deposition of hepatic lipids, while male mice were not affected by CPT1a deletion relative to male control mice. Mice with CPT1a deletion had reductions in DHA-containing phospholipids at the expense of monounsaturated fatty acids (MUFA)-containing phospholipids in both whole liver and at the level of the lipid droplet (LD). Male and female LKO mice increased RNA levels of genes involved in LD lipolysis ( Plin2 , Cidec , G0S2 ) and in polyunsaturated fatty acid (PUFA) metabolism ( Elovl5, Fads1, Elovl2 ), while only female LKO mice increased genes involved in inflammation ( Ly6d, Mmp12, Cxcl2 ). Kinase profiling showed decreased protein kinase A (PKA) activity, which coincided with increased PLIN2, PLIN5, and G0S2 protein levels and decreased triglyceride hydrolysis in LKO mice. Conclusions: Liver-specific deletion of CPT1a promotes sexually dimorphic steatotic liver disease (SLD) in mice, and here we have identified new mechanisms by which females are protected from HFD-induced liver injury.

Untargeted Analysis of Lipids Containing Very Long Chain Fatty Acids in Retina and Retinal Tight Junctions.

Several recent studies suggest that C24-C38 very long chain fatty acids (VLCFA) play an important role in vision, and decreased levels of retina VLCFA have been associated with vision disorders including the onset and progression of diabetic retinopathy in animal models. Traditional methods for VLCFA analysis lack the sensitivity and specificity needed to enable detailed characterization of VLCFA incorporation into complex lipids in tissues and subcellular components. To assess whether decreased VLCFA in diabetic retina are directly implicated in diabetes-induced breakdown of the blood-retinal barrier, we demonstrated the utility of performing untargeted lipid analysis via Orbitrap high resolution/accurate mass MS and MS/MS-based shotgun lipidomics to identify structural lipids containing VLCFA substituents. This comprehensive and highly sensitive approach to untargeted lipid identification enabled us to characterize low-abundance sphingolipids containing very long chain fatty acids from isolated retinal tight junction complexes, as well as VLCFA-containing phospholipids in retinal tissues. To facilitate future biochemical and physiological studies of the roles of VLCFA in blood-retina barrier integrity and maintenance of vision, this chapter describes steps to isolate tight junction complexes from a cell culture model of the outer blood-retinal barrier and perform untargeted Orbitrap high resolution/accurate mass-based lipid analysis to identify VLCFA in tight junctions and retina tissue.

Diabetes Retinopathy: New Ways to Detect and Treat.

Recent clinical trials demonstrated strong association between lipid abnormalities and progression of diabetic retinopathy (DR); however, whether circulating lipid levels or retinal lipid metabolism, or both, contributes to the pathogenesis of DR is not well understood. Limited amounts of retinal tissue available from animal models, such as mouse models of DR, have proved. Limited amount of retinal tissue was especially challenging for cholesterol and oxysterol detection as it precluded identification of individual isomers of each nonesterified sterol class. To measure cholesterol and oxysterols from limited retinal tissue samples, we developed extremely sensitive electrospray ionization liquid chromatography high-resolution/accurate mass measurements on an LTQ Orbitrap Velos mass spectrometer that are able to resolve sterols and oxysterols separated by reverse-phase HPLC using a gradient of 85-100% methanol containing 0.1% formic acid, with subsequent detection in positive ionization mode. This methodology will aid in our understanding of diabetes-induced changes in retinal cholesterol and oxysterol metabolism.

Androgen-mediated Perturbation of the Hepatic Circadian System Through Epigenetic Modulation Promotes NAFLD in PCOS Mice.

In women, excess androgen causes polycystic ovary syndrome (PCOS), a common fertility disorder with comorbid metabolic dysfunctions including diabetes, obesity, and nonalcoholic fatty liver disease. Using a PCOS mouse model, this study shows that chronic high androgen levels cause hepatic steatosis while hepatocyte-specific androgen receptor (AR)-knockout rescues this phenotype. Moreover, through RNA-sequencing and metabolomic studies, we have identified key metabolic genes and pathways affected by hyperandrogenism. Our studies reveal that a large number of metabolic genes are directly regulated by androgens through AR binding to androgen response element sequences on the promoter region of these genes. Interestingly, a number of circadian genes are also differentially regulated by androgens. In vivo and in vitro studies using a circadian reporter [Period2::Luciferase (Per2::LUC)] mouse model demonstrate that androgens can directly disrupt the hepatic timing system, which is a key regulator of liver metabolism. Consequently, studies show that androgens decrease H3K27me3, a gene silencing mark on the promoter of core clock genes, by inhibiting the expression of histone methyltransferase, Ezh2, while inducing the expression of the histone demethylase, JMJD3, which is responsible for adding and removing the H3K27me3 mark, respectively. Finally, we report that under hyperandrogenic conditions, some of the same circadian/metabolic genes that are upregulated in the mouse liver are also elevated in nonhuman primate livers. In summary, these studies not only provide an overall understanding of how hyperandrogenism associated with PCOS affects liver gene expression and metabolism but also offer insight into the underlying mechanisms leading to hepatic steatosis in PCOS.

BSCL2/Seipin deficiency in hearts causes cardiac energy deficit and dysfunction via inducing excessive lipid catabolism.

BACKGROUND: Heart failure (HF) is one of the leading causes of death worldwide and is associated with cardiac metabolic perturbations. Human Type 2 Berardinelli-Seip Congenital Lipodystrophy (BSCL2) disease is caused by mutations in the BSCL2 gene. Global lipodystrophic Bscl2-/- mice exhibit hypertrophic cardiomyopathy with reduced cardiac steatosis. Whether BSCL2 plays a direct role in regulating cardiac substrate metabolism and/or contractile function remains unknown. METHODS: We generated mice with cardiomyocyte-specific deletion of Bscl2 (Bscl2cKO ) and studied their cardiac substrate utilisation, bioenergetics, lipidomics and contractile function under baseline or after either a treatment regimen using fatty acid oxidation (FAO) inhibitor trimetazidine (TMZ) or a prevention regimen with high-fat diet (HFD) feeding. Mice with partial ATGL deletion and cardiac-specific deletion of Bscl2 were also generated followed by cardiac phenotyping. RESULTS: Different from hypertrophic cardiomyopathy in Bscl2-/- mice, mice with cardiac-specific deletion of Bscl2 developed systolic dysfunction with dilation. Myocardial BSCL2 deletion led to elevated ATGL expression and FAO along with reduced cardiac lipid contents. Cardiac dysfunction in Bscl2cKO mice was independent of mitochondrial dysfunction and oxidative stress, but associated with decreased metabolic reserve and ATP levels. Importantly, cardiac dysfunction in Bscl2cKO mice could be partially reversed by FAO inhibitor TMZ, or prevented by genetic abolishment of one ATGL allele or HFD feeding. Lipidomic analysis further identified markedly reduced glycerolipids, glycerophospholipids, NEFA and acylcarnitines in Bscl2cKO hearts, which were partially normalised by TMZ or HFD. CONCLUSIONS: We identified a new form of cardiac dysfunction with excessive lipid utilisation which ultimately causes cardiac substrate depletion and bioenergetics failure. Our findings also uncover a crucial role of BSCL2 in controlling cardiac lipid catabolism and contractile function and provide novel insights into metabolically treating energy-starved HF using FAO inhibitor or HFD.

Hexosylceramides and Glycerophosphatidylcholine GPC(36:1) Increase in Multi-Organ Dysfunction Syndrome Patients with Pediatric Intensive Care Unit Admission over 8-Day Hospitalization.

Glycero- and sphingo-lipids are important in plasma membrane structure, caloric storage and signaling. An un-targeted lipidomics approach for a cohort of critically ill pediatric intensive care unit (PICU) patients undergoing multi-organ dysfunction syndrome (MODS) was compared to sedation controls. After IRB approval, patients meeting the criteria for MODS were screened, consented (n = 24), and blood samples were collected from the PICU at HDVCH, Michigan; eight patients needed veno-arterial extracorporeal membrane oxygenation (VA ECMO). Sedation controls were presenting for routine sedation (n = 4). Plasma lipid profiles were determined by nano-electrospray (nESI) direct infusion high resolution/accurate mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Biostatistics analysis was performed using R v 3.6.0. Sixty-one patient samples over three time points revealed a ceramide metabolite, hexosylceramide (Hex-Cer) was high across all time points (mean 1.63-3.19%; vs. controls 0.22%). Fourteen species statistically differentiated from sedation controls (p-value ≤ 0.05); sphingomyelin (SM) [SM(d18:1/23:0), SM(d18:1/22:0), SM(d18:1/23:1), SM(d18:1/21:0), SM(d18:1/24:0)]; and glycerophosphotidylcholine (GPC) [GPC(36:01), GPC(18:00), GPC(O:34:02), GPC(18:02), GPC(38:05), GPC(O:34:03), GPC(16:00), GPC(40:05), GPC(O:36:03)]. Hex-Cer has been shown to be involved in viral infection and may be at play during acute illness. GPC(36:01) was elevated in all MODS patients at all time points and is associated with inflammation and brain injury.

Fasting and fasting-mimicking treatment activate SIRT1/LXRα and alleviate diabetes-induced systemic and microvascular dysfunction.

AIMS/HYPOTHESIS: Homo sapiens evolved under conditions of intermittent food availability and prolonged fasting between meals. Periods of fasting are important for recovery from meal-induced oxidative and metabolic stress, and tissue repair. Constant high energy-density food availability in present-day society contributes to the pathogenesis of chronic diseases, including diabetes and its complications, with intermittent fasting (IF) and energy restriction shown to improve metabolic health. We have previously demonstrated that IF prevents the development of diabetic retinopathy in a mouse model of type 2 diabetes (db/db); however the mechanisms of fasting-induced health benefits and fasting-induced risks for individuals with diabetes remain largely unknown. Sirtuin 1 (SIRT1), a nutrient-sensing deacetylase, is downregulated in diabetes. In this study, the effect of SIRT1 stimulation by IF, fasting-mimicking cell culture conditions (FMC) or pharmacological treatment using SRT1720 was evaluated on systemic and retinal metabolism, systemic and retinal inflammation and vascular and bone marrow damage. METHODS: The effects of IF were modelled in vivo using db/db mice and in vitro using bovine retinal endothelial cells or rat retinal neuroglial/precursor R28 cell line serum starved for 24 h. mRNA expression was analysed by quantitative PCR (qPCR). SIRT1 activity was measured via histone deacetylase activity assay. NR1H3 (also known as liver X receptor alpha [LXRα]) acetylation was measured via western blot analysis. RESULTS: IF increased Sirt1 mRNA expression in mouse liver and retina when compared with non-fasted animals. IF also increased SIRT1 activity eightfold in mouse retina while FMC increased SIRT1 activity and expression in retinal endothelial cells when compared with control. Sirt1 expression was also increased twofold in neuronal retina progenitor cells (R28) after FMC treatment. Moreover, FMC led to SIRT1-mediated LXRα deacetylation and subsequent 2.4-fold increase in activity, as measured by increased mRNA expression of the genes encoding ATP-binding cassette transporter (Abca1 and Abcg1). These changes were reduced when retinal endothelial cells expressing a constitutively acetylated LXRα mutant were tested. Increased SIRT1/LXR/ABC-mediated cholesterol export resulted in decreased retinal endothelial cell cholesterol levels. Direct activation of SIRT1 by SRT1720 in db/db mice led to a twofold reduction of diabetes-induced inflammation in the retina and improved diabetes-induced visual function impairment, as measured by electroretinogram and optokinetic response. In the bone marrow, there was prevention of diabetes-induced myeloidosis and decreased inflammatory cytokine expression. CONCLUSIONS/INTERPRETATION: Taken together, activation of SIRT1 signalling by IF or through pharmacological activation represents an effective therapeutic strategy that provides a mechanistic link between the advantageous effects associated with fasting regimens and prevention of microvascular and bone marrow dysfunction in diabetes.

Pediatric Multi-Organ Dysfunction Syndrome: Analysis by an Untargeted "Shotgun" Lipidomic Approach Reveals Low-Abundance Plasma Phospholipids and Dynamic Recovery over 8-Day Period, a Single-Center Observational Study.

Lipids are molecules involved in metabolism and inflammation. This study investigates the plasma lipidome for markers of severity and nutritional status in critically ill children. Children with multi-organ dysfunction syndrome (MODS) (n = 24) are analyzed at three time-points and cross-referenced to sedation controls (n = 4) for a total of N = 28. Eight of the patients with MODS, needed veno-arterial extracorporeal membrane oxygenation (VA ECMO) support to survive. Blood plasma lipid profiles are quantified by nano-electrospray (nESI), direct infusion high resolution/accurate mass spectrometry (MS), and tandem mass spectrometry (MS/MS), and compared to nutritional profiles and pediatric logistic organ dysfunction (PELOD) scores. Our results show that PELOD scores were not significantly different between MODS and ECMO cases across time-points (p = 0.66). Lipid profiling provides stratification between sedation controls and all MODS patients for total lysophosphatidylserine (lysoPS) (p-value = 0.004), total phosphatidylserine (PS) (p-value = 0.015), and total ether-linked phosphatidylethanolamine (ether-PE) (p-value = 0.03) after adjusting for sex and age. Nutrition intake over time did not correlate with changes in lipid profiles, as measured by caloric and protein intake. Lipid measurement in the intensive care environment shows dynamic changes over an 8-day pediatric intensive care unit (PICU) course, suggesting novel metabolic indicators for defining critically ill children.

The Feasibility of Studying Metabolites in PICU Multi-Organ Dysfunction Syndrome Patients over an 8-Day Course Using an Untargeted Approach.

Metabolites are generated from critical biological functions and metabolism. This pediatric study reviewed plasma metabolites in patients suffering from multi-organ dysfunction syndrome (MODS) in the pediatric intensive care unit (PICU) using an untargeted metabolomics approach. Patients meeting the criteria for MODS were screened for eligibility and consented (n = 24), and blood samples were collected at baseline, 72 h, and 8 days; control patients (n = 4) presented for routine sedation in an outpatient setting. A subset of MODS patients (n = 8) required additional support with veno-atrial extracorporeal membrane oxygenation (VA-ECMO) therapy. Metabolites from thawed blood plasma were determined from ion pairing reversed-phase liquid chromatography-mass spectrometry (LC-MS) analysis. Chromatographic peak alignment, identification, relative quantitation, and statistical and bioinformatics evaluation were performed using MAVEN and MetaboAnalyst 4.0. Metabolite analysis revealed 115 peaks per sample. From the partial least squares-discriminant analysis (PLS-DA) with variance of importance (VIP) scores above ≥2.0, 7 dynamic metabolites emerged over the three time points: tauro-chenodeoxycholic acid (TCDCA), hexose, p-hydroxybenzoate, hydroxyphenylacetic acid (HPLA), 2_3-dihydroxybenzoic acid, 2-keto-isovalerate, and deoxyribose phosphate. After Bonferroni adjustment for repeated measures, hexose and p-hydroxybenzoate were significant at one time point or more. Kendall's tau-b test was used for internal validation of creatinine. Metabolites may be benign or significant in describing a patient's pathophysiology and require operator interpretation.

Mitochondrial Ceramide Effects on the Retinal Pigment Epithelium in Diabetes.

Mitochondrial damage in the cells comprising inner (retinal endothelial cells) and outer (retinal pigment epithelium (RPE)) blood-retinal barriers (BRB) is known to precede the initial BRB breakdown and further histopathological abnormalities in diabetic retinopathy (DR). We previously demonstrated that activation of acid sphingomyelinase (ASM) is an important early event in the pathogenesis of DR, and recent studies have demonstrated that there is an intricate connection between ceramide and mitochondrial function. This study aimed to determine the role of ASM-dependent mitochondrial ceramide accumulation in diabetes-induced RPE cell damage. Mitochondria isolated from streptozotocin (STZ)-induced diabetic rat retinas (7 weeks duration) showed a 1.64 ± 0.29-fold increase in the ceramide-to-sphingomyelin ratio compared to controls. Conversely, the ceramide-to-sphingomyelin ratio was decreased in the mitochondria isolated from ASM-knockout mouse retinas compared to wild-type littermates, confirming the role of ASM in mitochondrial ceramide production. Cellular ceramide was elevated 2.67 ± 1.07-fold in RPE cells derived from diabetic donors compared to control donors, and these changes correlated with increased gene expression of IL-1ß, IL-6, and ASM. Treatment of RPE cells derived from control donors with high glucose resulted in elevated ASM, vascular endothelial growth factor (VEGF), and intercellular adhesion molecule 1 (ICAM-1) mRNA. RPE from diabetic donors showed fragmented mitochondria and a 2.68 ± 0.66-fold decreased respiratory control ratio (RCR). Treatment of immortalized cell in vision research (ARPE-19) cells with high glucose resulted in a 25% ± 1.6% decrease in citrate synthase activity at 72 h. Inhibition of ASM with desipramine (15 µM, 1 h daily) abolished the decreases in metabolic functional parameters. Our results are consistent with diabetes-induced increase in mitochondrial ceramide through an ASM-dependent pathway leading to impaired mitochondrial function in the RPE cells of the retina.

Regulation of lipid metabolism in pancreatic beta cells by interferon gamma: A link to anti-viral function.

Interferons (IFN) have been shown to alter lipid metabolism in immune and some non-hematopoietic cells and this affects host cell response to pathogens. In type 1 diabetes, IFNγ acts as a proinflammatory cytokine that, along with other cytokines, is released during pancreatic beta cell autoinflammation and contributes to immune response and beta cell dysfunction. The hypothesis tested herein is that IFN modifies beta cell lipid metabolism and this is associated with enhanced anti-viral response and beta cell stress. Treatment of INS-1 cells with IFNγ for 6 to 24 h led to a dynamic change in TAG and lipid droplet (LD) levels, with a decrease at 6 h and an increase at 24 h. The later accumulation of TAG was associated with increased de novo lipogenesis (DNL), and impaired mitochondrial fatty acid oxidation (FAO). Gene expression results suggested that IFNγ regulates lipolytic, lipogenic, LD and FAO genes in a temporal manner. The changes in lipid gene expression are dependent on the classical Janus kinase (JAK) pathway. Pretreatment with IFNγ robustly enhanced anti-viral gene expression induced by the viral mimetic polyinosinic: polycytidylic acid (PIC), and this potentiating effect of IFNγ was markedly attenuated by inhibitors of DNL. The IFNγ-induced accumulation of lipid, however, was insufficient to cause endoplasmic reticulum (ER) stress. These studies demonstrated a non-canonical effect of IFNγ in regulation of pancreatic beta cell lipid metabolism that is intimately linked with host cell defense and might alter cellular function early in the progression to type 1 diabetes.

Lipidome Profiles Are Related to Depressive Symptoms and Preterm Birth Among African American Women.

African American women have the highest rate of preterm birth (PTB; <37 completed weeks' gestation) of any racial and ethnic group in the United States (14.1%). Depressive symptoms (DS) have been linked to PTB risk of African American women. We hypothesized that maternal lipidomic profiles are related to prenatal DS and gestational age at birth among African American women. Women were enrolled at 9-25 weeks' gestation, completed questionnaires, and provided plasma samples. Lipidomic profiles were determined by "shotgun" Orbitrap high-resolution/accurate mass spectrometry. Data were analyzed using SIMCA P+ software. There was a clear separation in the orthogonal projections to latent structures discriminant analysis score plot between women with Center for Epidemiologic Studies Depression Scale (CES-D) scores ≥23 and women with CES-D scores ≤22. Similarly, a clear separation was observed in the model between PTB and full-term birth. Corresponding S-plot, loading plot, and variable importance in projection plot/list were used to identify the lipids responsible for the groupings. Higher levels of specific triglyceride (TG) species and lower levels of specific phosphatidylcholines (PCs) PC(37:1), PC(41:6), and PC(39:3) were associated with PTB. PC PC(37:1) levels were also lower among women with CES-D scores ≥23, pointing toward a possible connection between DS and PTB. Although overweight pregnant women showed higher levels of TGs, the PTB model showed specific TGs unique to PTB. Lipidomic profiles in pregnant African American women are related to DS, and our data suggest a role for specific TGs and PCs in PTB.

Cholesterol Acceptors Regulate the Lipidome of Macrophage Foam Cells.

Arterial foam cells are central players of atherogenesis. Cholesterol acceptors, apolipoprotein A-I (apoA-I) and high-density lipoprotein (HDL), take up cholesterol and phospholipids effluxed from foam cells into the circulation. Due to the high abundance of cholesterol in foam cells, most previous studies focused on apoA-I/HDL-mediated free cholesterol (FC) transport. However, recent lipidomics of human atherosclerotic plaques also identified that oxidized sterols (oxysterols) and non-sterol lipid species accumulate as atherogenesis progresses. While it is known that these lipids regulate expression of pro-inflammatory genes linked to plaque instability, how cholesterol acceptors impact the foam cell lipidome, particularly oxysterols and non-sterol lipids, remains unexplored. Using lipidomics analyses, we found cholesterol acceptors remodel foam cell lipidomes. Lipid subclass analyses revealed various oxysterols, sphingomyelins, and ceramides, species uniquely enriched in human plaques were significantly reduced by cholesterol acceptors, especially by apoA-I. These results indicate that the function of lipid-poor apoA-I is not limited to the efflux of cholesterol and phospholipids but suggest that apoA-I serves as a major regulator of the foam cell lipidome and might play an important role in reducing multiple lipid species involved in the pathogenesis of atherosclerosis.

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids.

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

Differential composition of DHA and very-long-chain PUFAs in rod and cone photoreceptors.

Long-chain PUFAs (LC-PUFAs; C20-C22; e.g., DHA and arachidonic acid) are highly enriched in vertebrate retina, where they are elongated to very-long-chain PUFAs (VLC-PUFAs; C 28) by the elongation of very-long-chain fatty acids-4 (ELOVL4) enzyme. These fatty acids play essential roles in modulating neuronal function and health. The relevance of different lipid requirements in rods and cones to disease processes, such as age-related macular degeneration, however, remains unclear. To better understand the role of LC-PUFAs and VLC-PUFAs in the retina, we investigated the lipid compositions of whole retinas or photoreceptor outer segment (OS) membranes in rodents with rod- or cone-dominant retinas. We analyzed fatty acid methyl esters and the molecular species of glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine) by GC-MS/GC-flame ionization detection and ESI-MS/MS, respectively. We found that whole retinas and OS membranes in rod-dominant animals compared with cone-dominant animals had higher amounts of LC-PUFAs and VLC-PUFAs. Compared with those of rod-dominant animals, retinas and OS membranes from cone-dominant animals also had about 2-fold lower levels of di-DHA (22:6/22:6) molecular species of glycerophospholipids. Because PUFAs are necessary for optimal G protein-coupled receptor signaling in rods, these findings suggest that cones may not have the same lipid requirements as rods.

Staphylococcus aureus Utilizes Host-Derived Lipoprotein Particles as Sources of Fatty Acids.

Methicillin-resistant Staphylococcus aureus (MRSA) is a threat to global health. Consequently, much effort has focused on the development of new antimicrobials that target novel aspects of S. aureus physiology. Fatty acids are required to maintain cell viability, and bacteria synthesize fatty acids using the type II fatty acid synthesis (FASII) pathway. FASII is significantly different from human fatty acid synthesis, underscoring the therapeutic potential of inhibiting this pathway. However, many Gram-positive pathogens incorporate exogenous fatty acids, bypassing FASII inhibition and leaving the clinical potential of FASII inhibitors uncertain. Importantly, the source(s) of fatty acids available to pathogens within the host environment remains unclear. Fatty acids are transported throughout the body by lipoprotein particles in the form of triglycerides and esterified cholesterol. Thus, lipoproteins, such as low-density lipoprotein (LDL), represent a potentially rich source of exogenous fatty acids for S. aureus during infection. We sought to test the ability of LDLs to serve as a fatty acid source for S. aureus and show that cells cultured in the presence of human LDLs demonstrate increased tolerance to the FASII inhibitor triclosan. Using mass spectrometry, we observed that host-derived fatty acids present in the LDLs are incorporated into the staphylococcal membrane and that tolerance to triclosan is facilitated by the fatty acid kinase A, FakA, and Geh, a triacylglycerol lipase. Finally, we demonstrate that human LDLs support the growth of S. aureus fatty acid auxotrophs. Together, these results suggest that human lipoprotein particles are a viable source of exogenous fatty acids for S. aureus during infection.IMPORTANCE Inhibition of bacterial fatty acid synthesis is a promising approach to combating infections caused by S. aureus and other human pathogens. However, S. aureus incorporates exogenous fatty acids into its phospholipid bilayer. Therefore, the clinical utility of targeting bacterial fatty acid synthesis is debated. Moreover, the fatty acid reservoir(s) exploited by S. aureus is not well understood. Human low-density lipoprotein particles represent a particularly abundant in vivo source of fatty acids and are present in tissues that S. aureus colonizes. Herein, we establish that S. aureus is capable of utilizing the fatty acids present in low-density lipoproteins to bypass both chemical and genetic inhibition of fatty acid synthesis. These findings imply that S. aureus targets LDLs as a source of fatty acids during pathogenesis.