Regular Research Articles
1-Deoxysphingolipid synthesis compromises anchorage-independent growth and plasma membrane endocytosis in cancer cells
Serine palmitoyltransferase (SPT) predominantly incorporates serine and fatty acyl-CoAs into diverse sphingolipids (SLs) that serve as structural components of membranes and signaling molecules within or amongst cells. However, SPT also uses alanine as a substrate in the contexts of low serine availability, alanine accumulation, or disease-causing mutations in hereditary sensory neuropathy type I, resulting in the synthesis and accumulation of 1-deoxysphingolipids (deoxySLs). These species promote cytotoxicity in neurons and impact diverse cellular phenotypes, including suppression of anchorage-independent cancer cell growth.Hepatocytes Deficient in Nuclear Envelope Protein Lamina-associated Polypeptide 1 are an Ideal Mammalian System to Study Intranuclear Lipid Droplets
Lipid droplets (LDs) are generally considered to be synthesized in the ER and utilized in the cytoplasm. However, LDs have been observed inside nuclei in some cells, although recent research on nuclear LDs has focused on cultured cell lines. To better understand nuclear LDs that occur in vivo, here we examined LDs in primary hepatocytes from mice following depletion of the nuclear envelope protein lamina-associated polypeptide 1 (LAP1). Microscopic image analysis showed that LAP1-depleted hepatocytes contain frequent nuclear LDs, which differ from cytoplasmic LDs in their associated proteins.ER Stress-Induced Sphingosine-1-Phosphate Lyase Phosphorylation Potentiates the Mitochondrial Unfolded Protein Response
The unfolded protein response (UPR) is an elaborate signaling network that evolved to maintain proteostasis in the endoplasmic reticulum (ER) and mitochondria (mt). These organelles are functionally and physically associated, and consequently, their stress responses are often intertwined. It is unclear how these two adaptive stress responses are coordinated during ER stress. The inositol-requiring enzyme-1 (IRE1), a central ER stress sensor and proximal regulator of the UPRER, harbors dual kinase and endoribonuclease (RNase) activities.Characterization of long-chain fatty acid-linked bile acids: a major conjugation form of 3β-hydroxy bile acids in feces
Although most bile acids (BAs) in feces are present in noncovalent forms that can be extracted with ethanol, non-negligible amounts of saponifiable BAs are also present. It is a major concern that such saponifiable BAs are routinely omitted from fecal BA measurements. We compared the BA profiles of healthy stools that were obtained with/without alkaline hydrolysis and found that as much as 29.7% (2.1–67.7%) of total BAs were saponifiable. Specifically, alkaline treatment led to significant elevations of isodeoxycholic acid (isoDCA) and isolithocholic acid (isoLCA) concentrations, suggesting that considerable proportions of isoDCA and isoLCA were esterified.RGMa promotes dedifferentiation of vascular smooth muscle cells into a macrophage-like phenotype in vivo and in vitro
Repulsive guidance molecule a (RGMa) is a glycosylphosphatidylinositol-anchored glycoprotein that has been demonstrated to influence inflammatory-related diseases in addition to regulating neuronal differentiation and survival during brain development. However, any function or mechanism of RGMa in dedifferentiation of contractile vascular smooth muscle cells (VSMCs) during inflammatory-related atherosclerosis is poorly understood. In the current study, we found that RGMa is expressed in VSMCs-derived macrophage-like cells from the fibrous cap of type V atherosclerotic plaques and the neointima of ligated carotid artery in ApoE−/− mice.Liposomes trigger bone marrow niche macrophage “foam” cell formation and affect hematopoiesis in mice
Liposomes are the most widely used nanocarrier platform for the delivery of therapeutic and diagnostic agents, and a number of liposomes have been approved for use in clinical practice. After systemic administration, most liposomes are cleared by macrophages in the mononuclear phagocyte system, such as the liver and bone marrow (BM). However, the majority of studies have focused on investigating the therapeutic results of liposomal drugs, and too few studies have evaluated the potential side effects of empty nanocarriers on the functions of macrophages in the mononuclear phagocyte system.PCSK9 is minimally associated with HDL but impairs the anti-atherosclerotic HDL effects on endothelial cell activation
Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) regulates the cell-surface localization of LDL receptors in hepatocytes and is associated with LDL and lipoprotein(a) [Lp(a)] uptake, reducing blood concentrations. However, the connection between PCSK9 and HDL is unclear. Here, we investigated the association of plasma PCSK9 with HDL subpopulations and examined the effects of PCSK9 on the atheroprotective function of HDL. We examined the association of PCSK9 with HDL in apoB-depleted plasma by ELISA, native PAGE, and immunoblotting.Identification and characterization of LPLAT7 as an sn-1-specific lysophospholipid acyltransferase
The main fatty acids at the sn-1 position of phospholipids (PLs) are saturated or monounsaturated fatty acids such as palmitic acid (C16:0), stearic acid (C18:0), and oleic acid (C18:1) and are constantly replaced, like unsaturated fatty acids at the sn-2 position. However, little is known about the molecular mechanism underlying the replacement of fatty acids at the sn-1 position, i.e., the sn-1 remodeling. Previously, we established a method to evaluate the incorporation of fatty acids into the sn-1 position of lysophospholipids (lyso-PLs).Associations between insulin-like growth factor binding protein-2 and lipoprotein kinetics in men
Low circulating concentrations of insulin-like growth factor binding protein-2 (IGFBP-2) have been associated with dyslipidemia, notably with high triglyceride (TG) levels. However, the determinants by which IGFBP-2 influences lipoprotein metabolism, especially that of TG-rich lipoproteins (TRLs), are poorly understood. Here, we aimed to assess the relationships between IGFBP-2 levels and lipoprotein production and catabolism in human subjects. Fasting IGFBP-2 concentrations were measured in the plasma of 219 men pooled from previous lipoprotein kinetics studies.Serum dihydroceramides correlate with insulin sensitivity in humans and decrease insulin sensitivity in vitro
Serum ceramides, especially C16:0 and C18:0 species, are linked to CVD risk and insulin resistance, but details of this association are not well understood. We performed this study to quantify a broad range of serum sphingolipids in individuals spanning the physiologic range of insulin sensitivity and to determine if dihydroceramides cause insulin resistance in vitro. As expected, we found that serum triglycerides were significantly greater in individuals with obesity and T2D compared with athletes and lean individuals.Acute retinol mobilization by retinol-binding protein 4 in mouse liver induces fibroblast growth factor 21 expression
Hepatocytes secrete retinol-binding protein 4 (RBP4) into circulation, thereby mobilizing vitamin A from the liver to provide retinol for extrahepatic tissues. Obesity and insulin resistance are associated with elevated RBP4 levels in the blood. However, in a previous study, we observed that chronically increased RBP4 by forced Rbp4 expression in the liver does not impair glucose homeostasis in mice. Here, we investigated the effects of an acute mobilization of hepatic vitamin A stores by hepatic overexpression of RBP4 in mice.Obesity reprograms the pulmonary polyunsaturated fatty acid-derived lipidome, transcriptome, and gene-oxylipin networks
Obesity exacerbates inflammation upon lung injury; however, the mechanisms by which obesity primes pulmonary dysregulation prior to external injury are not well studied. Herein, we tested the hypothesis that obesity dysregulates pulmonary PUFA metabolism that is central to inflammation initiation and resolution. We first show that a high-fat diet (HFD) administered to C57BL/6J mice increased the relative abundance of pulmonary PUFA-containing triglycerides and the concentration of PUFA-derived oxylipins (particularly prostaglandins and hydroxyeicosatetraenoic acids), independent of an increase in total pulmonary PUFAs, prior to onset of pulmonary inflammation.Individual and simultaneous treatment with antipsychotic aripiprazole and antidepressant trazodone inhibit sterol biosynthesis in the adult brain
Polypharmacy, or the simultaneous use of multiple drugs to treat a single patient, is a common practice in psychiatry. Unfortunately, data on the health effects of commonly used combinations of medications are very limited. In this study, we therefore investigated the effects and interactions between two commonly prescribed psychotropic medications with sterol inhibiting side effects, trazodone (TRZ), an antidepressant, and aripiprazole (ARI), an antipsychotic. In vitro cell culture experiments revealed that both medications alone disrupted neuronal and astroglial sterol biosynthesis in dose-dependent manners.Differential expression patterns of phospholipase D isoforms 1 and 2 in the mammalian brain and retina
Phosphatidic acid is a key signaling molecule heavily implicated in exocytosis due to its protein-binding partners and propensity to induce negative membrane curvature. One phosphatidic acid-producing enzyme, phospholipase D (PLD), has also been implicated in neurotransmission. Unfortunately, due to the unreliability of reagents, there has been confusion in the literature regarding the expression of PLD isoforms in the mammalian brain which has hampered our understanding of their functional roles in neurons.The small GTPase RAB10 regulates endosomal recycling of the LDL receptor and transferrin receptor in hepatocytes
The low-density lipoprotein receptor (LDLR) mediates the hepatic uptake of circulating low-density lipoproteins (LDLs), a process that modulates the development of atherosclerotic cardiovascular disease. We recently identified RAB10, encoding a small GTPase, as a positive regulator of LDL uptake in hepatocellular carcinoma cells (HuH7) in a genome-wide CRISPR screen, though the underlying molecular mechanism for this effect was unknown. We now report that RAB10 regulates hepatocyte LDL uptake by promoting the recycling of endocytosed LDLR from RAB11-positive endosomes to the plasma membrane.10,12-Conjugated linoleic acid supplementation improves HDL composition and function in mice
Obesity is associated with inflammation, insulin resistance, and type 2 diabetes, which are major risk factors for CVD. One dietary component of ruminant animal foods, 10,12-conjugated linoleic acid (10,12 CLA), has been shown to promote weight loss in humans. Previous work has shown that 10,12 CLA is atheroprotective in mice by a mechanism that may be distinct from its weight loss effects, but this exact mechanism is unclear. To investigate this, we evaluated HDL composition and function in obese LDL receptor (Ldlr−/−) mice that were losing weight because of 10,12 CLA supplementation or caloric restriction (CR; weight-matched control group) and in an obese control group consuming a high-fat high-sucrose diet.Inhibition of MAP4K4 signaling initiates metabolic reprogramming to protect hepatocytes from lipotoxic damage
The primary hepatic consequence of obesity is non-alcoholic fatty liver disease (NAFLD), affecting about 25% of the global adult population. Non-alcoholic steatohepatitis (NASH) is a severe form of NAFLD characterized by liver lipid accumulation, inflammation, and hepatocyte ballooning, with a different degree of hepatic fibrosis. In the light of rapidly increasing prevalence of NAFLD and NASH, there is an urgent need for improved understanding of the molecular pathogenesis of these diseases. The aim of this study was to decipher the possible role of STE20-type kinase MAP4K4 in the regulation of hepatocellular lipotoxicity and susceptibility to NAFLD.Identification and characterization of 3-ketosphinganine reductase activity encoded at the BT_0972 locus in Bacteroides thetaiotaomicron
Bacterial sphingolipid synthesis is important for the fitness of gut commensal bacteria with an implied potential for regulating mammalian host physiology. Multiple steps in bacterial sphingolipid synthesis pathways have been characterized previously, with the first step of de novo sphingolipid synthesis being well conserved between bacteria and eukaryotes. In mammals, the subsequent step of de novo sphingolipid synthesis is catalyzed by 3-ketosphinganine reductase, but the protein responsible for this activity in bacteria has remained elusive.ANGPTL4 silencing via antisense oligonucleotides reduces plasma triglycerides and glucose in mice without causing lymphadenopathy
Angiopoietin-like 4 (ANGPTL4) is an important regulator of plasma triglyceride (TG) levels and an attractive pharmacological target for lowering plasma lipids and reducing cardiovascular risk. Here, we aimed to study the efficacy and safety of silencing ANGPTL4 in the livers of mice using hepatocyte-targeting GalNAc-conjugated antisense oligonucleotides (ASOs). Compared with injections with negative control ASO, four injections of two different doses of ANGPTL4 ASO over 2 weeks markedly downregulated ANGPTL4 levels in liver and adipose tissue, which was associated with significantly higher adipose LPL activity and lower plasma TGs in fed and fasted mice, as well as lower plasma glucose levels in fed mice.Whole picture of human stratum corneum ceramides, including the chain-length diversity of long-chain bases
Ceramides are essential lipids for skin permeability barrier function, and a wide variety of ceramide species exist in the stratum corneum (SC). Although ceramides with long-chain bases (LCBs) of various lengths have been identified in the human SC, a quantitative analysis that distinguishes ceramide species with different LCB chain lengths has not been yet published. Therefore, the whole picture of human SC ceramides remains unclear. Here, we conducted LC/MS/MS analyses to detect individual ceramide species differing in both the LCB and FA chain lengths and quantified 1,327 unbound ceramides and 254 protein-bound ceramides: the largest number of ceramide species reported to date.Saroglitazar is noninferior to fenofibrate in reducing triglyceride levels in hypertriglyceridemic patients in a randomized clinical trial
Saroglitazar, being a dual PPAR-α/γ agonist, has shown beneficial effect in diabetic dyslipidemia and hypertriglyceridemia. Fibrates are commonly used to treat severe hypertriglyceridemia. However, the effect of saroglitazar in patients with moderate to severe hypertriglyceridemia was not evaluated. We conducted a study to compare the efficacy and safety of saroglitazar (4 mg) with fenofibrate (160 mg) in patients with moderate to severe hypertriglyceridemia. This was a multicenter, randomized, double-blinded, double-dummy, active-control, and noninferiority trial in adult patients with fasting triglyceride (TG) levels of 500–1,500 mg/dl.Plasma FA composition in familial LCAT deficiency indicates SOAT2-derived cholesteryl ester formation in humans
Mutations in the LCAT gene cause familial LCAT deficiency (Online Mendelian Inheritance in Man ID: #245900), a very rare metabolic disorder. LCAT is the only enzyme able to esterify cholesterol in plasma, whereas sterol O-acyltransferases 1 and 2 are the enzymes esterifying cellular cholesterol in cells. Despite the complete lack of LCAT activity, patients with familial LCAT deficiency exhibit circulating cholesteryl esters (CEs) in apoB-containing lipoproteins. To analyze the origin of these CEs, we investigated 24 carriers of LCAT deficiency in this observational study.Measurement of 7-dehydrocholesterol and cholesterol in hair can be used in the diagnosis of Smith-Lemli-Opitz syndrome
7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane.Direct anabolic metabolism of three-carbon propionate to a six-carbon metabolite occurs in vivo across tissues and species
Anabolic metabolism of carbon in mammals is mediated via the one- and two-carbon carriers S-adenosyl methionine and acetyl-coenzyme A. In contrast, anabolic metabolism of three-carbon units via propionate has not been shown to extensively occur. Mammals are primarily thought to oxidize the three-carbon short chain fatty acid propionate by shunting propionyl-CoA to succinyl-CoA for entry into the TCA cycle. Here, we found that this may not be absolute as, in mammals, one nonoxidative fate of propionyl-CoA is to condense to two three-carbon units into a six-carbon trans-2-methyl-2-pentenoyl-CoA (2M2PE-CoA).Identification of two lipid phosphatases that regulate sphingosine-1-phosphate cellular uptake and recycling
Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that serves as a potent extracellular signaling molecule. Metabolic regulation of extracellular S1P levels impacts key cellular activities through altered S1P receptor signaling. Although the pathway through which S1P is degraded within the cell and thereby eliminated from reuse has been previously described, the mechanism used for S1P cellular uptake and the subsequent recycling of its sphingoid base into the sphingolipid synthesis pathway is not completely understood.ω-O-Acylceramides but not ω-hydroxy ceramides are required for healthy lamellar phase architecture of skin barrier lipids
Epidermal omega-O-acylceramides (ω-O-acylCers) are essential components of a competent skin barrier. These unusual sphingolipids with ultralong N-acyl chains contain linoleic acid esterified to the terminal hydroxyl of the N-acyl, the formation of which requires the transacylase activity of patatin-like phospholipase domain containing 1 (PNPLA1). In ichthyosis with dysfunctional PNPLA1, ω-O-acylCer levels are significantly decreased, and ω-hydroxylated Cers (ω-OHCers) accumulate. Here, we explore the role of the linoleate moiety in ω-O-acylCers in the assembly of the skin lipid barrier.The effects of cardiolipin on the structural dynamics of the mitochondrial ADP/ATP carrier in its cytosol-open state
Cardiolipin (CL) has been shown to play a crucial role in regulating the function of proteins in the inner mitochondrial membrane. As the most abundant protein of the inner mitochondrial membrane, the ADP/ATP carrier (AAC) has long been the model of choice to study CL-protein interactions, and specifically bound CLs have been identified in a variety of crystal structures of AAC. However, how CL binding affects the structural dynamics of AAC in atomic detail remains largely elusive. Here we compared all-atom molecular dynamics simulations on bovine AAC1 in lipid bilayers with and without CLs.Adaptations of the 3T3-L1 adipocyte lipidome to defective ether lipid catabolism upon Agmo knockdown
Little is known about the physiological role of alkylglycerol monooxygenase (AGMO), the only enzyme capable of cleaving the 1-O-alkyl ether bond of ether lipids. Expression and enzymatic activity of this enzyme can be detected in a variety of tissues including adipose tissue. This labile lipolytic membrane-bound protein uses tetrahydrobiopterin as a cofactor, and mice with reduced tetrahydrobiopterin levels have alterations in body fat distribution and blood lipid concentrations. In addition, manipulation of AGMO in macrophages led to significant changes in the cellular lipidome, and alkylglycerolipids, the preferred substrates of AGMO, were shown to accumulate in mature adipocytes.Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer
The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs.Neuronal growth regulator 1 promotes adipocyte lipid trafficking via interaction with CD36
Neuronal growth regulator 1 (NEGR1) is a glycosylphosphatidylinositol-anchored membrane protein associated with several human pathologies, including obesity, depression, and autism. Recently, significantly enlarged white adipose tissue, hepatic lipid accumulation, and decreased muscle capacity were reported in Negr1-deficient mice. However, the mechanism behind these phenotypes was not clear. In the present study, we found NEGR1 to interact with cluster of differentiation 36 (CD36), the major fatty acid translocase in the plasma membrane.Sortilin enhances secretion of apolipoprotein(a) through effects on apolipoprotein B secretion and promotes uptake of lipoprotein(a)
Elevated plasma lipoprotein(a) (Lp(a)) is an independent, causal risk factor for atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Lp(a) is formed in or on hepatocytes from successive noncovalent and covalent interactions between apo(a) and apoB, although the subcellular location of these interactions and the nature of the apoB-containing particle involved remain unclear. Sortilin, encoded by the SORT1 gene, modulates apoB secretion and LDL clearance. We used a HepG2 cell model to study the secretion kinetics of apo(a) and apoB.Whole-exome sequencing reveals damaging gene variants associated with hypoalphalipoproteinemia
Low levels of high density lipoprotein-cholesterol (HDL-C) are associated with an elevated risk of arteriosclerotic coronary heart disease. Heritability of HDL-C levels is high. In this research discovery study, we used whole-exome sequencing to identify damaging gene variants that may play significant roles in determining HDL-C levels. We studied 204 individuals with a mean HDL-C level of 27.8 ± 6.4 mg/dl (range: 4–36 mg/dl). Data were analyzed by statistical gene burden testing and by filtering against candidate gene lists.Genetic dissection in mice reveals a dynamic crosstalk between the delivery pathways of vitamin A
Vitamin A is distributed within the body to support chromophore synthesis in the eyes and retinoid signaling in most other tissues. Two pathways exist for the delivery of vitamin A: the extrinsic pathway transports dietary vitamin A in lipoproteins from intestinal enterocytes to tissues, while the intrinsic pathway distributes vitamin A from hepatic stores bound to serum retinol binding protein (RBP). Previously, the intestine-specific homeodomain transcription factor (ISX) and the RBP receptor STRA6 were identified as gatekeepers of these pathways; however, it is not clear how mutations in the corresponding genes affect retinoid homeostasis.The SARS-CoV2 envelope differs from host cells, exposes procoagulant lipids, and is disrupted in vivo by oral rinses
The lipid envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an essential component of the virus; however, its molecular composition is undetermined. Addressing this knowledge gap could support the design of antiviral agents as well as further our understanding of viral-host protein interactions, infectivity, pathogenicity, and innate immune system clearance. Lipidomics revealed that the virus envelope comprised mainly phospholipids (PLs), with some cholesterol and sphingolipids, and with cholesterol/phospholipid ratio similar to lysosomes.Intravital lipid droplet labeling and imaging reveals the phenotypes and functions of individual macrophages in vivo
Macrophages play pivotal roles in the maintenance of tissue homeostasis. However, the reactivation of macrophages toward proinflammatory states correlates with a plethora of inflammatory diseases, including atherosclerosis, obesity, neurodegeneration, and bone marrow (BM) failure syndromes. The lack of methods to reveal macrophage phenotype and function in vivo impedes the translational research of these diseases. Here, we found that proinflammatory macrophages accumulate intracellular lipid droplets (LDs) relative to resting or noninflammatory macrophages both in vitro and in vivo, indicating that LD accumulation serves as a structural biomarker for macrophage phenotyping.Chronic cholesterol depletion increases F-actin levels and induces cytoskeletal reorganization via a dual mechanism
Previous work from us and others has suggested that cholesterol is an important lipid in the context of the organization of the actin cytoskeleton. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol is rarely addressed in the literature. In this work, we explored the signaling crosstalk between cholesterol and the actin cytoskeleton by using a high-resolution confocal microscopic approach to quantitatively measure changes in F-actin content upon cholesterol depletion.Maternal obesogenic diet enhances cholestatic liver disease in offspring
Human and animal model data show that maternal obesity promotes nonalcoholic fatty liver disease in offspring and alters bile acid (BA) homeostasis. Here we investigated whether offspring exposed to maternal obesogenic diets exhibited greater cholestatic injury. We fed female C57Bl6 mice conventional chow (CON) or high fat/high sucrose (HF/HS) diet and then bred them with lean males. Offspring were fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) for 2 weeks to induce cholestasis, and a subgroup was then fed CON for an additional 10 days.Consequences of excessive glucosylsphingosine in glucocerebrosidase-deficient zebrafish.
In Gaucher disease (GD), the deficiency of glucocerebrosidase causes lysosomal accumulation of glucosylceramide (GlcCer), which is partly converted by acid ceramidase to glucosylsphingosine (GlcSph) in the lysosome. Chronically elevated blood and tissue GlcSph is thought to contribute to symptoms in GD patients as well as to increased risk for Parkinson’s disease. On the other hand, formation of GlcSph may be beneficial since the water soluble sphingoid base is excreted via urine and bile. To study the role of excessive GlcSph formation during glucocerebrosidase deficiency, we studied zebrafish that have two orthologs of acid ceramidase, Asah1a and Asah1b.An anti-ANGPTL3/8 antibody decreases circulating triglycerides by binding to a LPL-inhibitory leucine zipper-like motif
Triglycerides (TG) are required for fatty acid transport and storage and are essential for human health. Angiopoietin-like-protein 8 (ANGPTL8) has previously been shown to form a complex with ANGPTL3 that increases circulating TG by potently inhibiting LPL. We also recently showed that the TG-lowering apolipoprotein A5 (ApoA5) decreases TG levels by suppressing ANGPTL3/8-mediated LPL inhibition. To understand how LPL binds ANGPTL3/8 and ApoA5 blocks this interaction, we used hydrogen-deuterium exchange mass-spectrometry and molecular modeling to map binding sites of LPL and ApoA5 on ANGPTL3/8.Hormone-sensitive lipase is localized at synapses and is necessary for normal memory functioning in mice
Hormone-sensitive lipase (HSL) is mainly present in adipose tissue where it hydrolyzes diacylglycerol. Although expression of HSL has also been reported in the brain, its presence in different cellular compartments is uncertain, and its role in regulating brain lipid metabolism remains hitherto unexplored. We hypothesized that HSL might play a role in regulating the availability of bioactive lipids necessary for neuronal function and therefore investigated whether dampening HSL activity could lead to brain dysfunction.Determination of tissue contributions to the circulating lipid pool in cold exposure via systematic assessment of lipid profiles
Plasma lipid levels are altered in chronic conditions such as type 2 diabetes and cardiovascular disease as well as during acute stresses such as fasting and cold exposure. Advances in MS-based lipidomics have uncovered a complex plasma lipidome of more than 500 lipids that serve functional roles, including as energy substrates and signaling molecules. This plasma lipid pool is maintained through regulation of tissue production, secretion, and uptake. A major challenge in understanding the lipidome complexity is establishing the tissues of origin and uptake for various plasma lipids, which is valuable for determining lipid functions.Pulmonary surfactant protein B carried by HDL predicts incident CVD in patients with type 1 diabetes
Atherosclerotic CVD is the major cause of death in patients with type 1 diabetes mellitus (T1DM). Alterations in the HDL proteome have been shown to associate with prevalent CVD in T1DM. We therefore sought to determine which proteins carried by HDL might predict incident CVD in patients with T1DM. Using targeted MS/MS, we quantified 50 proteins in HDL from 181 T1DM subjects enrolled in the prospective Coronary Artery Calcification in Type 1 Diabetes study. We used Cox proportional regression analysis and a case-cohort design to test associations of HDL proteins with incident CVD (myocardial infarction, coronary artery bypass grafting, angioplasty, or death from coronary heart disease).Hormone-sensitive lipase protects adipose triglyceride lipase-deficient mice from lethal lipotoxic cardiomyopathy
Lipid droplets (LDs) are multifunctional organelles that regulate energy storage and cellular homeostasis. The first step of triacylglycerol hydrolysis in LDs is catalyzed by adipose triglyceride lipase (ATGL), deficiency of which results in lethal cardiac steatosis. Although hormone-sensitive lipase (HSL) functions as a diacylglycerol lipase in the heart, we hypothesized that activation of HSL might compensate for ATGL deficiency. To test this hypothesis, we crossed ATGL-KO (AKO) mice and cardiac-specific HSL-overexpressing mice (cHSL) to establish homozygous AKO mice and AKO mice with cardiac-specific HSL overexpression (AKO+cHSL).Gut microbiome-derived glycine lipids are diet-dependent modulators of hepatic injury and atherosclerosis
Oral and gut Bacteroidetes produce unique classes of serine-glycine lipodipeptides and glycine aminolipids that signal through host Toll-like receptor 2. These glycine lipids have also been detected in human arteries, but their effects on atherosclerosis are unknown. Here, we sought to investigate the bioactivity of bacterial glycine lipids in mouse models of atherosclerosis. Lipid 654 (L654), a serine-glycine lipodipeptide species, was first tested in a high-fat diet (HFD)-fed Ldlr−/− model of atherosclerosis.Apolipoprotein A-V is a potential target for treating coronary artery disease: evidence from genetic and metabolomic analyses
Triglyceride (TG)-lowering LPL variants in combination with genetic LDL-C-lowering variants are associated with reduced risk of coronary artery disease (CAD). Genetic variation in the APOA5 gene encoding apolipoprotein A-V also strongly affects TG levels, but the potential clinical impact and underlying mechanisms are yet to be resolved. Here, we aimed to study the effects of APOA5 genetic variation on CAD risk and plasma lipoproteins through factorial genetic association analyses. Using data from 309,780 European-ancestry participants from the UK Biobank, we evaluated the effects of lower TG levels as a result of genetic variation in APOA5 and/or LPL on CAD risk with or without a background of reduced LDL-C.Ceramide kinase regulates acute wound healing by suppressing 5-oxo-ETE biosynthesis and signaling via its receptor OXER1
The sphingolipid, ceramide-1-phosphate (C1P), has been shown to promote the inflammatory phase and inhibit the proliferation and remodeling stages of wound repair via direct interaction with group IVA cytosolic phospholipase A2, a regulator of eicosanoid biosynthesis that fine-tunes the behaviors of various cell types during wound healing. However, the anabolic enzyme responsible for the production of C1P that suppresses wound healing as well as bioactive eicosanoids and target receptors that drive enhanced wound remodeling have not been characterized.LRP1 loss in airway epithelium exacerbates smoke-induced oxidative damage and airway remodeling
The LDL receptor-related protein 1 (LRP1) partakes in metabolic and signaling events regulated in a tissue-specific manner. The function of LRP1 in airways has not been studied. We aimed to study the function of LRP1 in smoke-induced disease. We found that bronchial epithelium of patients with chronic obstructive pulmonary disease and airway epithelium of mice exposed to smoke had increased LRP1 expression. We then knocked out LRP1 in human bronchial epithelial cells in vitro and in airway epithelial club cells in mice.Impact of pasteurization on the self-assembly of human milk lipids during digestion
Human milk is critical for the survival and development of infants. This source of nutrition contains components that protect against infections while stimulating immune maturation. In cases where the mother's own milk is unavailable, pasteurized donor milk is the preferred option. Although pasteurization has been shown to have minimal impact on the lipid and FA composition before digestion, no correlation has been made between the impact of pasteurization on the FFA composition and the self-assembly of lipids during digestion, which could act as delivery mechanisms for poorly water-soluble components.Neutral ceramidase deficiency protects against cisplatin-induced acute kidney injury
Cisplatin is a commonly used chemotherapeutic for the treatment of many solid organ cancers; however, its effectiveness is limited by the development of acute kidney injury (AKI) in 30% of patients. AKI is driven by proximal tubule cell death, leading to rapid decline in renal function. It has previously been shown that sphingolipid metabolism plays a role in regulating many of the biological processes involved in cisplatin-induced AKI. For example, neutral ceramidase (nCDase) is an enzyme responsible for converting ceramide into sphingosine, which is then phosphorylated to become sphingosine-1-phosphate, and our lab previously demonstrated that nCDase knockout (nCDase−/−) in mouse embryonic fibroblasts led to resistance to nutrient and energy deprivation–induced cell death via upregulation of autophagic flux.Cholesterol sulfate fluidizes the sterol fraction of the stratum corneum lipid phase and increases its permeability
Desulfation of cholesterol sulfate (CholS) to cholesterol (Chol) is an important event in epidermal homeostasis and necessary for stratum corneum (SC) barrier function. The CholS/Chol ratio decreases during SC maturation but remains high in pathological conditions, such as X-linked ichthyosis, characterized by dry and scaly skin. The aim of this study was to characterize the influence of the CholS/Chol molar ratio on the structure, dynamics, and permeability of SC lipid model mixtures. We synthesized deuterated CholS and investigated lipid models with specifically deuterated components using 2H solid-state NMR spectroscopy at temperatures from 25°C to 80°C.Damage response protein 1 (Dap1) functions in the synthesis of carotenoids and sterols in Xanthophyllomyces dendrorhous
Cytochrome P450s (P450s) are heme-containing proteins involved in several cellular functions, including biosynthesis of steroidal hormones, detoxification of xenobiotic compounds, among others. Damage response protein 1 (Dap1) has been described as a positive regulator of P450s through protein-protein interactions in organisms such as Schizosaccharomyces pombe. Three P450s in the carotenogenic yeast Xanthophyllomyces dendrorhous have thus far been characterized: Cyp51 and Cyp61, which are involved in ergosterol biosynthesis, and CrtS (astaxanthin synthase), which is involved in biosynthesis of the carotenoid astaxanthin.The SGLT2 inhibitor dapagliflozin promotes systemic FFA mobilization, enhances hepatic β-oxidation, and induces ketosis
Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to increase ketone bodies in patients with type 2 diabetes; however, the underlying mechanisms have not been fully elucidated. Here we examined the effect of the SGLT2 inhibitor dapagliflozin (1 mg/kg/day, formulated in a water, PEG400, ethanol, propylene glycol solution, 4 weeks) on lipid metabolism in obese Zucker rats. Fasting FFA metabolism was assessed in the anesthetized state using a [9,10-3H(N)]-palmitic acid tracer by estimating rates of plasma FFA appearance (Ra), whole-body FFA oxidation (Rox), and nonoxidative disposal (Rst).KIAA1363 affects retinyl ester turnover in cultured murine and human hepatic stellate cells
Large quantities of vitamin A are stored as retinyl esters (REs) in specialized liver cells, the hepatic stellate cells (HSCs). To date, the enzymes controlling RE degradation in HSCs are poorly understood. In this study, we identified KIAA1363 (also annotated as arylacetamide deacetylase 1 or neutral cholesterol ester hydrolase 1) as a novel RE hydrolase. We show that KIAA1363 is expressed in the liver, mainly in HSCs, and exhibits RE hydrolase activity at neutral pH. Accordingly, addition of the KIAA1363-specific inhibitor JW480 largely reduced RE hydrolase activity in lysates of cultured murine and human HSCs.Comparison between genetic and pharmaceutical disruption of Ldlr expression for the development of atherosclerosis
Antisense oligonucleotides (ASOs) against Ldl receptor (Ldlr-ASO) represent a promising strategy to promote hypercholesterolemic atherosclerosis in animal models without the need for complex breeding strategies. Here, we sought to characterize and contrast atherosclerosis in mice given Ldlr-ASO with those bearing genetic Ldlr deficiency. To promote atherosclerosis, male and female C57Bl6/J mice were either given weekly injections of Ldlr-ASO (5 mg/kg once per week) or genetically deficient in Ldlr (Ldlr−/−).Lipid droplet-mitochondria coupling via perilipin 5 augments respiratory capacity but is dispensable for FA oxidation
Disturbances in lipid homeostasis can cause mitochondrial dysfunction and lipotoxicity. Perilipin 5 (PLIN5) decorates intracellular lipid droplets (LDs) in oxidative tissues and controls triacylglycerol (TG) turnover via its interactions with adipose triglyceride lipase and the adipose triglyceride lipase coactivator, comparative gene identification-58. Furthermore, PLIN5 anchors mitochondria to the LD membrane via the outermost part of the carboxyl terminus. However, the role of this LD-mitochondria coupling (LDMC) in cellular energy catabolism is less established.Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux
Because of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three dimeric APOA1 molecules with their termini covalently joined in different combinations. The dimeric (d)-APOA1C-N mutant coupled the C-terminus of one APOA1 molecule to the N-terminus of a second with a short alanine linker, whereas the d-APOA1C-C and d-APOA1N-N mutants coupled the C-termini and the N-termini of two APOA1 molecules, respectively, using introduced cysteine residues to form disulfide linkages.Apolipoprotein F concentration, activity, and the properties of LDL controlling ApoF activation in hyperlipidemic plasma
Apolipoprotein F (ApoF) modulates lipoprotein metabolism by selectively inhibiting cholesteryl ester transfer protein activity on LDL. This ApoF activity requires that it is bound to LDL. How hyperlipidemia alters total plasma ApoF and its binding to LDL are poorly understood. In this study, total plasma ApoF and LDL-bound ApoF were quantified by ELISA (n = 200). Plasma ApoF was increased 31% in hypercholesterolemic plasma but decreased 20% in hypertriglyceridemia. However, in donors with combined hypercholesterolemia and hypertriglyceridemia, the elevated triglyceride ameliorated the rise in ApoF caused by hypercholesterolemia alone.Elevated granulocyte-colony stimulating factor and hematopoietic stem cell mobilization in Niemann-Pick type C1 disease
Niemann-Pick type C1 (NPC1) disease is a progressive lysosomal storage disorder caused by mutations of the NPC1 gene. While neurodegeneration is the most severe symptom, a large proportion of NPC1 patients also present with splenomegaly, which has been attributed to cholesterol and glycosphingolipid accumulation in late endosomes and lysosomes. However, recent data also reveal an increase in the inflammatory monocyte subset in the Npc1nih mouse model expressing an Npc1 null allele. We evaluated the contribution of hematopoietic cells to splenomegaly in NPC1 disease under conditions of hypercholesterolemia.Transgelin: a new gene involved in LDL endocytosis identified by a genome-wide CRISPR-Cas9 screen
A significant proportion of patients with elevated LDL and a clinical presentation of familial hypercholesterolemia do not carry known genetic mutations associated with hypercholesterolemia, such as defects in the LDL receptor. To identify new genes involved in the cellular uptake of LDL, we developed a novel whole-genome clustered regularly interspaced short palindromic repeat-Cas9 KO screen in HepG2 cells. We identified transgelin (TAGLN), an actin-binding protein, as a potentially new gene involved in LDL endocytosis.Apolipoprotein E content of VLDL limits LPL-mediated triglyceride hydrolysis
High levels of circulating triglycerides (TGs), or hypertriglyceridemia, are key components of metabolic diseases, such as type 2 diabetes, metabolic syndrome, and CVD. As TGs are carried by lipoproteins in plasma, hypertriglyceridemia can result from overproduction or lack of clearance of TG-rich lipoproteins (TRLs) such as VLDLs. The primary driver of TRL clearance is TG hydrolysis mediated by LPL. LPL is regulated by numerous TRL protein components, including the cofactor apolipoprotein C-II, but it is not clear how their effects combine to impact TRL hydrolysis across individuals.Monolayer autoxidation of arachidonic acid to epoxyeicosatrienoic acids as a model of their potential formation in cell membranes
In light of the importance of epoxyeicosatrienoic acids (EETs) in mammalian pathophysiology, a nonenzymatic route that might form these monoepoxides in cells is of significant interest. In the late 1970s, a simple system of arranging linoleic acid molecules on a monolayer on silica was devised and shown to yield monoepoxides as the main autoxidation products. Here, we investigated this system with arachidonic acid and characterized the primary products. By the early stages of autoxidation (∼10% conversion of arachidonic acid), the major products detected by LC-MS and HPLC-UV were the 14,15-, 11,12-, and 8,9-EETs, with the 5,6-EET mainly represented as the 5-δ-lactone-6-hydroxyeicosatrienoate as established by 1H-NMR.Loss of plasma membrane lipid asymmetry can induce ordered domain (raft) formation
In some cases, lipids in one leaflet of an asymmetric artificial lipid vesicle suppress the formation of ordered lipid domains (rafts) in the opposing leaflet. Whether this occurs in natural membranes is unknown. Here, we investigated this issue using plasma membrane vesicles (PMVs) from rat leukemia RBL-2H3 cells. Membrane domain formation and order was assessed by fluorescence resonance energy transfer and fluorescence anisotropy. We found that ordered domains in PMVs prepared from cells by N-ethyl maleimide (NEM) treatment formed up to ∼37°C, whereas ordered domains in symmetric vesicles formed from the extracted PMV lipids were stable up to 55°C, indicating the stability of ordered domains was substantially decreased in intact PMVs.1-Deoxysphinganine initiates adaptive responses to serine and glycine starvation in cancer cells via proteolysis of sphingosine kinase
Cancer cells may depend on exogenous serine, depletion of which results in slower growth and activation of adaptive metabolic changes. We previously demonstrated that serine and glycine (SG) deprivation causes loss of sphingosine kinase 1 (SK1) in cancer cells, thereby increasing the levels of its lipid substrate, sphingosine (Sph), which mediates several adaptive biological responses. However, the signaling molecules regulating SK1 and Sph levels in response to SG deprivation have yet to be defined.Ceramide-1-phosphate transfer protein promotes sphingolipid reorientation needed for binding during membrane interaction
Lipid transfer proteins acquire and release their lipid cargoes by interacting transiently with source and destination biomembranes. In the GlycoLipid Transfer Protein (GLTP) superfamily, the two-layer all-α-helical GLTP-fold defines proteins that specifically target sphingolipids (SLs) containing either sugar or phosphate headgroups via their conserved but evolutionarily-modified SL recognitions centers. Despite comprehensive structural insights provided by X-ray crystallography, the conformational dynamics associated with membrane interaction and SL uptake/release by GLTP superfamily members have remained unknown.Oxidized phospholipids cause changes in jejunum mucus that induce dysbiosis and systemic inflammation
We previously reported that adding a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to a Western diet (WD) ameliorated systemic inflammation. To determine the mechanism(s) responsible for these observations, Ldlr−/− mice were fed chow, a WD, or WD plus Tg6F. We found that a WD altered the taxonomic composition of bacteria in jejunum mucus. For example, Akkermansia muciniphila virtually disappeared, while overall bacteria numbers and lipopolysaccharide (LPS) levels increased.Loss of ABCA8B decreases myelination by reducing oligodendrocyte precursor cells in mice
The myelin sheath, which is wrapped around axons, is a lipid-enriched structure produced by mature oligodendrocytes. Disruption of the myelin sheath is observed in several neurological diseases, such as multiple sclerosis. A crucial component of myelin is sphingomyelin, levels of which can be increased by ABCA8, a member of the ATP-binding cassette transporter family. ABCA8 is highly expressed in the cerebellum, specifically in oligodendroglia. However, whether ABCA8 plays a role in myelination and mechanisms that would underlie this role remain unknown.Lipoprotein size is a main determinant for the rate of hydrolysis by exogenous LPL in human plasma
LPL is a key player in plasma triglyceride metabolism. Consequently, LPL is regulated by several proteins during synthesis, folding, secretion, and transport to its site of action at the luminal side of capillaries, as well as during the catalytic reaction. Some proteins are well known, whereas others have been identified but are still not fully understood. We set out to study the effects of the natural variations in the plasma levels of all known LPL regulators on the activity of purified LPL added to samples of fasted plasma taken from 117 individuals.Mass spectrometry imaging of phosphatidylcholine metabolism in lungs administered with therapeutic surfactants and isotopic tracers
Mass spectrometry imaging (MSI) visualizes molecular distributions throughout tissues but is blind to dynamic metabolic processes. Here, MSI with high mass resolution together with multiple stable isotope labeling provided spatial analyses of phosphatidylcholine (PC) metabolism in mouse lungs. Dysregulated surfactant metabolism is central to many respiratory diseases. Metabolism and turnover of therapeutic pulmonary surfactants were imaged from distributions of intact and metabolic products of an added tracer, universally 13C-labeled dipalmitoyl PC (U13C-DPPC).Membrane therapy using DHA suppresses epidermal growth factor receptor signaling by disrupting nanocluster formation
Summary Cellular membrane phospholipid enrichment of DHA suppresses EGFR-mediated phenotypes by reducing EGFR nanocluster formation across a variety of in vitro and in vivo models.The lipid substrate preference of CETP controls the biochemical properties of HDL in fat/cholesterol-fed hamsters
Cholesteryl ester transfer protein (CETP) modulates lipoprotein metabolism by transferring cholesteryl ester (CE) and triglyceride (TG) between lipoproteins. However, differences in the way CETP functions exist across species. Unlike human CETP, hamster CETP prefers TG over CE as a substrate, raising questions regarding how substrate preference may impact lipoprotein metabolism. To understand how altering the CE versus TG substrate specificity of CETP might impact lipoprotein metabolism in humans, we modified CETP expression in fat/cholesterol-fed hamsters, which have a human-like lipoprotein profile.Generation and validation of a conditional knockout mouse model for desmosterolosis
The enzyme 3β-hydroxysterol-Δ24 reductase (DHCR24, EC 1.3.1.72) catalyzes the conversion of desmosterol to cholesterol and is obligatory for post-squalene cholesterol synthesis. Genetic loss of this enzyme results in desmosterolosis (MIM # 602398 ), a rare disease that presents with multiple congenital anomalies, features of which overlap with subjects with the Smith-Lemli-Opitz syndrome (another post-squalene cholesterol disorder). Global knockout (KO) of Dhcr24 in mice recapitulates the biochemical phenotype, but pups die within 24 h from a lethal dermopathy, limiting its utility as a disease model.Human variant of scavenger receptor BI (R174C) exhibits impaired cholesterol transport functions
HDL and its primary receptor, scavenger receptor class B type I (SR-BI), work together to promote the clearance of excess plasma cholesterol, thereby protecting against atherosclerosis. Human variants of SR-BI have been identified in patients with high HDL-cholesterol levels, and at least one variant has been linked to cardiovascular disease. Therefore, while often regarded as beneficial, very high levels of HDL-cholesterol may result from impaired cholesterol clearance through SR-BI and contribute to cardiovascular risk.Impact of vitamin A transport and storage on intestinal retinoid homeostasis and functions
Lecithin:retinol acyltransferase and retinol-binding protein enable vitamin A (VA) storage and transport, respectively, maintaining tissue homeostasis of retinoids (VA derivatives). The precarious VA status of the lecithin:retinol acyltransferase–deficient (Lrat−/−) retinol-binding protein–deficient (Rbp−/−) mice rapidly deteriorates upon dietary VA restriction, leading to signs of severe vitamin A deficiency (VAD). As retinoids impact gut morphology and functions, VAD is often linked to intestinal pathological conditions and microbial dysbiosis.Plin2 deletion increases cholesteryl ester lipid droplet content and disturbs cholesterol balance in adrenal cortex
Cholesteryl esters (CEs) are the water-insoluble transport and storage form of cholesterol. Steroidogenic cells primarily store CEs in cytoplasmic lipid droplet (LD) organelles, as contrasted to the majority of mammalian cell types that predominantly store triacylglycerol (TAG) in LDs. The LD-binding Plin2 binds to both CE- and TAG-rich LDs, and although Plin2 is known to regulate degradation of TAG-rich LDs, its role for regulation of CE-rich LDs is unclear. To investigate the role of Plin2 in the regulation of CE-rich LDs, we performed histological and molecular characterization of adrenal glands from Plin2+/+ and Plin2−/− mice.Lipid droplet membrane proteome remodeling parallels ethanol-induced hepatic steatosis and its resolution
Lipid droplets (LDs) are composed of neutral lipids enclosed in a phospholipid monolayer, which harbors membrane-associated proteins that regulate LD functions. Despite the crucial role of LDs in lipid metabolism, remodeling of LD protein composition in disease contexts, such as steatosis, remains poorly understood. We hypothesized that chronic ethanol consumption, subsequent abstinence from ethanol, or fasting differentially affects the LD membrane proteome content and that these changes influence how LDs interact with other intracellular organelles.4β-Hydroxycholesterol is a prolipogenic factor that promotes SREBP1c expression and activity through the liver X receptor
Oxysterols are oxidized derivatives of cholesterol that play regulatory roles in lipid biosynthesis and homeostasis. How oxysterol signaling coordinates different lipid classes such as sterols and triglycerides remains incompletely understood. Here, we show that 4β-hydroxycholesterol (HC) (4β-HC), a liver and serum abundant oxysterol of poorly defined functions, is a potent and selective inducer of the master lipogenic transcription factor, SREBP1c, but not the related steroidogenic transcription factor SREBP2.High-fat diet activates liver iPLA2γ generating eicosanoids that mediate metabolic stress
High-fat (HF) diet–induced obesity precipitates multiple metabolic disorders including insulin resistance, glucose intolerance, oxidative stress, and inflammation, resulting in the initiation of cell death programs. Previously, we demonstrated murine germline knockout of calcium-independent phospholipase A2γ (iPLA2γ) prevented HF diet–induced weight gain, attenuated insulin resistance, and decreased mitochondrial permeability transition pore (mPTP) opening leading to alterations in bioenergetics.Molecular basis for the interaction of cellular retinol binding protein 2 (CRBP2) with nonretinoid ligands
Present in the small intestine, cellular retinol binding protein 2 (CRBP2) plays an important role in the uptake, transport, and metabolism of dietary retinoids. However, the recent discovery of the interactions of CRBP2 with 2-arachidonoylglycerol and other monoacylglycerols (MAGs) suggests the broader involvement of this protein in lipid metabolism and signaling. To better understand the physiological role of CRBP2, we determined its protein-lipid interactome using a fluorescence-based retinol replacement assay adapted for a high-throughput screening format.LRAT coordinates the negative-feedback regulation of intestinal retinoid biosynthesis from β-carotene
There is increasing recognition that dietary lipids can affect the expression of genes encoding their metabolizing enzymes, transporters, and binding proteins. This mechanism plays a pivotal role in controlling tissue homeostasis of these compounds and avoiding diseases. The regulation of retinoid biosynthesis from β-carotene (BC) is a classic example for such an interaction. The intestine-specific homeodomain transcription factor (ISX) controls the activity of the vitamin A-forming enzyme β-carotene oxygenase-1 in intestinal enterocytes in response to increasing concentration of the vitamin A metabolite retinoic acid.Lipid remodeling in response to methionine stress in MDA-MBA-468 triple-negative breast cancer cells
Methionine (Met) is an essential amino acid and critical precursor to the cellular methyl donor S-adenosylmethionine. Unlike nontransformed cells, cancer cells have a unique metabolic requirement for Met and are unable to proliferate in growth media where Met is replaced with its metabolic precursor, homocysteine. This metabolic vulnerability is common among cancer cells regardless of tissue origin and is known as “methionine dependence”, “methionine stress sensitivity”, or the Hoffman effect. The response of lipids to Met stress, however, is not well-understood.The ligand-bound state of a G protein-coupled receptor stabilizes the interaction of functional cholesterol molecules
Cholesterol is a major component of mammalian plasma membranes that not only affects the physical properties of the lipid bilayer but also is the function of many membrane proteins including G protein-coupled receptors. The oxytocin receptor (OXTR) is involved in parturition and lactation of mammals and in their emotional and social behaviors. Cholesterol acts on OXTR as an allosteric modulator inducing a high-affinity state for orthosteric ligands through a molecular mechanism that has yet to be determined.25-Hydroxycholesterol 3-sulfate is an endogenous ligand of DNA methyltransferases in hepatocytes
The oxysterol sulfate, 25-hydroxycholesterol 3-sulfate (25HC3S), has been shown to play an important role in lipid metabolism, inflammatory response, and cell survival. However, the mechanism(s) of its function in global regulation is unknown. The current study investigates the molecular mechanism by which 25HC3S functions as an endogenous epigenetic regulator. To study the effects of oxysterols/sterol sulfates on epigenetic modulators, 12 recombinant epigenetic enzymes were used to determine whether 25HC3S acts as their endogenous ligand.Vasculoprotective properties of plasma lipoproteins from brown bears (Ursus arctos)
Plasma cholesterol and triglyceride (TG) levels are twice as high in hibernating brown bears (Ursus arctos) than healthy humans. Yet, bears display no signs of early stage atherosclerosis development when adult. To explore this apparent paradox, we analyzed plasma lipoproteins from the same 10 bears in winter (hibernation) and summer using size exclusion chromatography, ultracentrifugation, and electrophoresis. LDL binding to arterial proteoglycans (PGs) and plasma cholesterol efflux capacity (CEC) were also evaluated.Simvastatin inhibits POVPC-mediated induction of endothelial-to-mesenchymal cell transition
Endothelial-to-mesenchymal transition (EndMT), the process by which an endothelial cell (EC) undergoes a series of molecular events that result in a mesenchymal cell phenotype, plays an important role in atherosclerosis. 1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), derived from the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine, is a proinflammatory lipid found in atherosclerotic lesions. Whether POVPC promotes EndMT and how simvastatin influences POVPC-mediated EndMT remains unclear.ApoA5 lowers triglyceride levels via suppression of ANGPTL3/8-mediated LPL inhibition
Triglyceride (TG) molecules represent the major storage form of fatty acids, and TG metabolism is essential to human health. However, the mechanistic details surrounding TG metabolism are complex and incompletely elucidated. Although it is known that angiopoietin-like protein 8 (ANGPTL8) increases TGs through an ANGPTL3/8 complex that inhibits LPL, the mechanism governing ApoA5, which lowers TGs, has remained elusive. Current hypotheses for how ApoA5 acts include direct stimulation of LPL, facilitation of TG-containing particle uptake, and regulation of hepatic TG secretion.Octanoate is differentially metabolized in liver and muscle and fails to rescue cardiomyopathy in CPT2 deficiency
Long-chain fatty acid oxidation is frequently impaired in primary and systemic metabolic diseases affecting the heart; thus, therapeutically increasing reliance on normally minor energetic substrates, such as ketones and medium-chain fatty acids, could benefit cardiac health. However, the molecular fundamentals of this therapy are not fully known. Here, we explored the ability of octanoate, an eight-carbon medium-chain fatty acid known as an unregulated mitochondrial energetic substrate, to ameliorate cardiac hypertrophy in long-chain fatty acid oxidation-deficient hearts because of carnitine palmitoyltransferase 2 deletion (Cpt2M−/−).Cannabinoid type 1 receptor inverse agonism attenuates dyslipidemia and atherosclerosis in APOE∗3-Leiden.CETP mice
Pharmacological blockade of the cannabinoid type 1 receptor, a G protein-coupled receptor expressed in the central nervous system and various peripheral tissues, reverses diet-induced obesity and dyslipidemia through the reduction of food intake and altered nutrient partitioning. This strategy is being explored for a number of therapeutic applications; however, its potency for the treatment of atherosclerotic cardiovascular disease via improvements in lipid metabolism remains unclear. Therefore, here, we aimed to investigate whether inhibition of the endocannabinoid system can attenuate atherosclerosis development through improvement of dyslipidemia.Structure of mouse cytosolic sulfotransferase SULT2A8 provides insight into sulfonation of 7α-hydroxyl bile acids
Cytosolic sulfotransferases (SULTs) catalyze the transfer of a sulfonate group from the cofactor 3’-phosphoadenosine 5’-phosphosulfate to a hydroxyl (OH) containing substrate and play a critical role in the homeostasis of endogenous compounds, including hormones, neurotransmitters, and bile acids. In human, SULT2A1 sulfonates the 3-OH of bile acids; however, bile acid metabolism in mouse is dependent on a 7α-OH sulfonating SULT2A8 via unknown molecular mechanisms. In this study, the crystal structure of SULT2A8 in complex with adenosine 3’,5’-diphosphate and cholic acid was resolved at a resolution of 2.5 Å.Carboxylesterase 2 proteins are efficient diglyceride and monoglyceride lipases possibly implicated in metabolic disease
Carboxylesterase 2 (CES2/Ces2) proteins exert established roles in (pro)drug metabolism. Recently, human and murine CES2/Ces2c have been discovered as triglyceride (TG) hydrolases implicated in the development of obesity and fatty liver disease. The murine Ces2 family consists of seven homologous genes as opposed to a single CES2 gene in humans. However, the mechanistic role of Ces2 protein family members is not completely understood. In this study, we examined activities of all Ces2 members toward TGs, diglycerides (DGs), and monoglycerides (MGs) as the substrate.Endothelial ether lipids link the vasculature to blood pressure, behavior, and neurodegeneration
Vascular disease contributes to neurodegeneration, which is associated with decreased blood pressure in older humans. Plasmalogens, ether phospholipids produced by peroxisomes, are decreased in Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders. However, the mechanistic links between ether phospholipids, blood pressure, and neurodegeneration are not fully understood. Here, we show that endothelium-derived ether phospholipids affect blood pressure, behavior, and neurodegeneration in mice.The acyl chains of phosphoinositide PIP3 alter the structure and function of nuclear receptor steroidogenic factor-1
Nuclear receptors are transcription factors that bind lipids, an event that induces a structural conformation of the receptor that favors interaction with transcriptional coactivators. The nuclear receptor steroidogenic factor-1 (SF-1, NR5A1) binds the signaling phosphoinositides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3), and our previous crystal structures showed how the phosphoinositide headgroups regulate SF-1 function. However, what role the acyl chains play in regulating SF-1 structure remains unaddressed.CRISPR/Cas9 deletion of ORMDLs reveals complexity in sphingolipid metabolism
The serine palmitoyltransferase (SPT) complex catalyzes the rate-limiting step in the de novo biosynthesis of ceramides, the precursors of sphingolipids. The mammalian ORMDL isoforms (ORMDL1-3) are negative regulators of SPT. However, the roles of individual ORMDL isoforms are unclear. Using siRNA against individual ORMDLs, only single siORMDL3 had modest effects on dihydroceramide and ceramide levels, whereas downregulation of all three ORMDLs induced more pronounced increases. With the CRISPR/Cas9-based genome-editing strategy, we established stable single ORMDL3 KO (ORMDL3-KO) and ORMDL1/2/3 triple-KO (ORMDL-TKO) cell lines to further understand the roles of ORMDL proteins in sphingolipid biosynthesis.Proteoglycan binding as proatherogenic function metric of apoB-containing lipoproteins and chronic kidney graft failure
Lipoprotein-proteoglycan binding is an early key event in atherosclerotic lesion formation and thus conceivably could play a major role in vasculopathy-driven chronic graft failure and cardiovascular mortality in renal transplant recipients. The present study investigated whether lipoprotein-proteoglycan binding susceptibility (LPBS) of apoB-containing lipoproteins and levels of the classical atherosclerosis biomarker LDL-C were associated with cardiovascular mortality (n = 130) and graft failure (n = 73) in 589 renal transplant recipients who were followed up from at least 1 year after transplantation for 9.5 years.Evaluation of the available cholesterol concentration in the inner leaflet of the plasma membrane of mammalian cells
Cholesterol is an essential component of the mammalian plasma membrane involved in diverse cellular processes. Our recent quantitative imaging analysis using ratiometric cholesterol sensors showed that the available cholesterol concentration in the inner leaflet of the plasma membrane (IPM) is low in unstimulated cells and increased in a stimulus-specific manner to trigger cell signaling events. However, the transbilayer distribution of cholesterol in the plasma membrane of mammalian cells remains controversial.Sar1b mutant mice recapitulate gastrointestinal abnormalities associated with chylomicron retention disease
Chylomicron retention disease (CRD) is an autosomal recessive disorder associated with biallelic Sar1b mutations leading to defects in intracellular chylomicron (CM) trafficking and secretion. To date, a direct cause-effect relationship between CRD and Sar1b mutation has not been established, but genetically modified animal models provide an opportunity to elucidate unrecognized aspects of these mutations. To examine the physiological role and molecular mechanisms of Sar1b function, we generated mice expressing either a targeted deletion or mutation of human Sar1b using the CRISPR-Cas9 system.The role of full-length apoE in clearance of Gram-negative bacteria and their endotoxins
ApoE is a well-known lipid-binding protein that plays a main role in the metabolism and transport of lipids. More recently, apoE-derived peptides have been shown to exert antimicrobial effects. Here, we investigated the antibacterial activity of apoE using in vitro assays, advanced imaging techniques, and in vivo mouse models. The formation of macromolecular complexes of apoE and endotoxins from Gram-negative bacteria was explored using gel shift assays, transmission electron microscopy, and CD spectroscopy followed by calculation of the α-helical content.Monomeric bile acids modulate the ATPase activity of detergent-solubilized ABCB4/MDR3
ABCB4, also called multidrug-resistant protein 3 (MDR3), is an ATP binding cassette transporter located in the canalicular membrane of hepatocytes that specifically translocates phosphatidylcholine (PC) lipids from the cytoplasmic to the extracellular leaflet. Due to the harsh detergent effect of bile acids, PC lipids provided by ABCB4 are extracted into the bile. While it is well known that bile acids are the major extractor of PC lipids from the membrane into bile, it is unknown whether only PC lipid extraction is improved or whether bile acids also have a direct effect on ABCB4.Analysis of 12/15-lipoxygenase metabolism of EPA and DHA with special attention to authentication of docosatrienes
A proposed beneficial impact of highly unsaturated “fish oil” fatty acids is their conversion by lipoxygenase (LOX) enzymes to specialized proresolving lipid mediators, including 12/15-LOX products from EPA and DHA. The transformations of DHA include formation of docosatrienes, named for the distinctive conjugated triene of the double bonds. To further the understanding of biosynthetic pathways and mechanisms, herein we meld together biosynthesis and NMR characterization of the unstable leukotriene A (LTA)-related epoxide intermediates formed by recombinant human 15-LOX-1, along with identification of the stable enzymatic products, and extend the findings into the 12/15-LOX metabolism in resident murine peritoneal macrophages.Inhibition of lysosomal phospholipase A2 predicts drug-induced phospholipidosis
Phospholipidosis, the excessive accumulation of phospholipids within lysosomes, is a pathological response observed following exposure to many drugs across multiple therapeutic groups. A clear mechanistic understanding of the causes and implications of this form of drug toxicity has remained elusive. We previously reported the discovery and characterization of a lysosome-specific phospholipase A2 (PLA2G15) and later reported that amiodarone, a known cause of drug-induced phospholipidosis, inhibits this enzyme.Atherosclerosis-associated hepatic secretion of VLDL but not PCSK9 is dependent on cargo receptor protein Surf4
Plasma LDL is produced from catabolism of VLDL and cleared from circulation mainly via the hepatic LDL receptor (LDLR). Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes LDLR degradation, increasing plasma LDL-C levels. Circulating PCSK9 is mainly secreted by the liver, whereas VLDL is exclusively secreted by hepatocytes. However, the mechanism regulating their secretion is not completely understood. Surfeit 4 (Surf4) is a cargo receptor localized in the ER membrane. It recruits cargos into coat protein complex II vesicles to facilitate their secretion.
