Sphingolipid Precursor Research Articles

Ceramides-Emerging Biomarkers of Lipotoxicity in Obesity, Diabetes, Cardiovascular Diseases, and Inflammation.

Abnormalities in lipid homeostasis have been associated with many human diseases, and the interrelation between lipotoxicity and cellular dysfunction has received significant attention in the past two decades. Ceramides (Cers) are bioactive lipid molecules that serve as precursors of all complex sphingolipids. Besides their function as structural components in cell and mitochondrial membranes, Cers play a significant role as key mediators in cell metabolism and are involved in numerous cellular processes, such as proliferation, differentiation, inflammation, and induction of apoptosis. The accumulation of various ceramides in tissues causes metabolic and cellular disturbances. Recent studies suggest that Cer lipotoxicity has an important role in obesity, metabolic syndrome, type 2 diabetes, atherosclerosis, and cardiovascular diseases (CVDs). In humans, elevated plasma ceramide levels are associated with insulin resistance and impaired cardiovascular and metabolic health. In this review, we summarize the role of ceramides as key mediators of lipotoxicity in obesity, diabetes, cardiovascular diseases, and inflammation and their potential as a promising diagnostic tool.

Loss of dihydroceramide desaturase drives neurodegeneration by disrupting endoplasmic reticulum and lipid droplet homeostasis in glial cells.

Dihydroceramide desaturases convert dihydroceramides to ceramides, the precursors of all complex sphingolipids. Reduction of DEGS1 dihydroceramide desaturase function causes pediatric neurodegenerative disorder hypomyelinating leukodystrophy-18 (HLD-18). We discovered that infertile crescent (ifc), the Drosophila DEGS1 homolog, is expressed primarily in glial cells to promote CNS development by guarding against neurodegeneration. Loss of ifc causes massive dihydroceramide accumulation and severe morphological defects in cortex glia, including endoplasmic reticulum (ER) expansion, failure of neuronal ensheathment, and lipid droplet depletion. RNAi knockdown of the upstream ceramide synthase schlank in glia of ifc mutants rescues ER expansion, suggesting dihydroceramide accumulation in the ER drives this phenotype. RNAi knockdown of ifc in glia but not neurons drives neuronal cell death, suggesting that ifc function in glia promotes neuronal survival. Our work identifies glia as the primary site of disease progression in HLD-18 and may inform on juvenile forms of ALS, which also feature elevated dihydroceramide levels.

Membrane contact sites regulate vacuolar fission via sphingolipid metabolism

Membrane contact sites (MCSs) are junctures that perform important roles including coordinating lipid metabolism. Previous studies have indicated that vacuolar fission/fusion processes are coupled with modifications in the membrane lipid composition. However, it has been still unclear whether MCS-mediated lipid metabolism controls the vacuolar morphology. Here, we report that deletion of tricalbins (Tcb1, Tcb2, and Tcb3), tethering proteins at endoplasmic reticulum (ER)–plasma membrane (PM) and ER–Golgi contact sites, alters fusion/fission dynamics and causes vacuolar fragmentation in the yeast Saccharomyces cerevisiae. In addition, we show that the sphingolipid precursor phytosphingosine (PHS) accumulates in tricalbin-deleted cells, triggering the vacuolar division. Detachment of the nucleus–vacuole junction (NVJ), an important contact site between the vacuole and the perinuclear ER, restored vacuolar morphology in both cells subjected to high exogenous PHS and Tcb3-deleted cells, supporting that PHS transport across the NVJ induces vacuole division. Thus, our results suggest that vacuolar morphology is maintained by MCSs through the metabolism of sphingolipids.

Membrane contact sites regulate vacuolar fission via sphingolipid metabolism.

Membrane contact sites (MCSs) are junctures that perform important roles including coordinating lipid metabolism. Previous studies have indicated that vacuolar fission/fusion processes are coupled with modifications in the membrane lipid composition. However, it has been still unclear whether MCS-mediated lipid metabolism controls the vacuolar morphology. Here, we report that deletion of tricalbins (Tcb1, Tcb2, and Tcb3), tethering proteins at endoplasmic reticulum (ER)-plasma membrane (PM) and ER-Golgi contact sites, alters fusion/fission dynamics and causes vacuolar fragmentation in the yeast Saccharomyces cerevisiae. In addition, we show that the sphingolipid precursor phytosphingosine (PHS) accumulates in tricalbin-deleted cells, triggering the vacuolar division. Detachment of the nucleus-vacuole junction (NVJ), an important contact site between the vacuole and the perinuclear ER, restored vacuolar morphology in both cells subjected to high exogenous PHS and Tcb3-deleted cells, supporting that PHS transport across the NVJ induces vacuole division. Thus, our results suggest that vacuolar morphology is maintained by MCSs through the metabolism of sphingolipids.

Front Cover: Characterization of an Unusual α‐Oxoamine Synthase Off‐Loading Domain from a Cyanobacterial Type I Fatty Acid Synthase (ChemBioChem 18/2023)

A new fatty acid synthase off-loading domain. Fatty acid synthase (FAS) usually releases its products by hydrolysis or transesterification. Here, an α-oxoamine synthase off-loading domain is identified in a cyanobacterial type I FAS. This domain releases the fatty acyl chain through a condensation with L-serine, offering a novel pathway to α-amino ketones that share similarities with sphingolipid precursors. More information can be found in the Research Article by A. E. Fraley, S. Sasso et al.

Quantification of α-hydroxy ceramides in mice serum by LC-MS/MS: Application to sepsis study

Alpha-hydroxy ceramides are not only the precursors of many complex sphingolipids, also play a major role in membrane homeostasis and cellular signal transduction. However, current research rarely involved quantitative methods for α-hydroxy ceramides, which greatly restricts the study of its biological function. This work aimed to develop a reliable assay for the accurate quantification of α-hydroxy ceramides in vivo study. LC-MS/MS method was developed for the accurate quantification of six α-hydroxy ceramides of Cer(d18:1/16:0(2OH)), Cer(d18:1/18:0(2OH)), Cer(d18:1/18:1(2OH)), Cer(d18:1/20:0(2OH)), Cer(d18:1/22:0(2OH)) and Cer(d18:1/24:1(2OH)) in mice serum. This assay was validated with low limit of quantitation of 3.125 ng/mL, a dynamic range of 3.125–400 ng/mL (R2 > 0.99), precision (<15 %), and accuracy (88 % to 115 %). Applying the method to the determination of α-hydroxy ceramides in the serum of sepsis mice, the levels of Cer(d18:1/16:0(2OH)), Cer(d18:1/20:0(2OH)), Cer(d18:1/24:1(2OH)) were significantly elevated in LPS-induced septic as compared to the normal control. In conclusion, this LC-MS method was qualified in α-hydroxy ceramides quantification in vivo and a significant association was found between α-hydroxy ceramides and sepsis.

Yeast Svf1 binds ceramides and contributes to sphingolipid metabolism at the ER cis-Golgi interface.

Ceramides are essential precursors of complex sphingolipids and act as potent signaling molecules. Ceramides are synthesized in the endoplasmic reticulum (ER) and receive their head-groups in the Golgi apparatus, yielding complex sphingolipids (SPs). Transport of ceramides between the ER and the Golgi is executed by the essential ceramide transport protein (CERT) in mammalian cells. However, yeast cells lack a CERT homolog, and the mechanism of ER to Golgi ceramide transport remains largely elusive. Here, we identified a role for yeast Svf1 in ceramide transport between the ER and the Golgi. Svf1 is dynamically targeted to membranes via an N-terminal amphipathic helix (AH). Svf1 binds ceramide via a hydrophobic binding pocket that is located in between two lipocalin domains. We showed that Svf1 membrane-targeting is important to maintain flux of ceramides into complex SPs. Together, our results show that Svf1 is a ceramide binding protein that contributes to sphingolipid metabolism at Golgi compartments.

Mechanisms of Nonvesicular Ceramide Transport.

Ceramides, as key components of cellular membranes, play essential roles in various cellular processes, including apoptosis, cell proliferation, and cell signaling. Ceramides are the precursors of all complex sphingolipids in eukaryotic cells. They are synthesized in the endoplasmic reticulum and are further processed at the Golgi apparatus. Therefore, ceramides have to be transported between these two organelles. In mammalian cells, the ceramide transfer protein forms a contact site between the ER and the trans-Golgi region and transports ceramide utilizing its steroidogenic acute regulatory protein-related lipid transfer domain. In yeast, multiple mechanisms of nonvesicular ceramide transport have been described. This involves the nuclear-vacuolar junction protein Nvj2, the yeast tricalbin proteins, and the lipocalin-like protein Svf1. This review aims to provide a comprehensive overview of nonvesicular ceramide transport mechanisms and their relevance in cellular physiology. We will highlight the physiological and pathological consequences of perturbations in nonvesicular ceramide transport and discuss future challenges in identifying and analyzing ceramide transfer proteins.

Inimitable Impacts of Ceramides on Lipid Rafts Formed in Artificial and Natural Cell Membranes

Ceramide is the simplest precursor of sphingolipids and is involved in a variety of biological functions ranging from apoptosis to the immune responses. Although ceramide is a minor constituent of plasma membranes, it drastically increases upon cellular stimulation. However, the mechanistic link between ceramide generation and signal transduction remains unknown. To address this issue, the effect of ceramide on phospholipid membranes has been examined in numerous studies. One of the most remarkable findings of these studies is that ceramide induces the coalescence of membrane domains termed lipid rafts. Thus, it has been hypothesised that ceramide exerts its biological activity through the structural alteration of lipid rafts. In the present article, we first discuss the characteristic hydrogen bond functionality of ceramides. Then, we showed the impact of ceramide on the structures of artificial and cell membranes, including the coalescence of the pre-existing lipid raft into a large patch called a signal platform. Moreover, we proposed a possible structure of the signal platform, in which sphingomyelin/cholesterol-rich and sphingomyelin/ceramide-rich domains coexist. This structure is considered to be beneficial because membrane proteins and their inhibitors are separately compartmentalised in those domains. Considering the fact that ceramide/cholesterol content regulates the miscibility of those two domains in model membranes, the association and dissociation of membrane proteins and their inhibitors might be controlled by the contents of ceramide and cholesterol in the signal platform.

Microdomain Protein Nce102 Is a Local Sensor of Plasma Membrane Sphingolipid Balance.

ABSTRACTSphingolipids are essential building blocks of eukaryotic membranes and important signaling molecules that are regulated tightly in response to environmental and physiological inputs. While their biosynthetic pathway has been well-described, the mechanisms that facilitate the perception of sphingolipid levels at the plasma membrane remain to be uncovered. In Saccharomyces cerevisiae, the Nce102 protein has been proposed to function as a sphingolipid sensor as it changes its plasma membrane distribution in response to sphingolipid biosynthesis inhibition. We show that Nce102 redistributes specifically in regions of increased sphingolipid demand, e.g., membranes of nascent buds. Furthermore, we report that the production of Nce102 increases following sphingolipid biosynthesis inhibition and that Nce102 is internalized when excess sphingolipid precursors are supplied. This finding suggests that the total amount of Nce102 in the plasma membrane is a measure of the current need for sphingolipids, whereas its local distribution marks sites of high sphingolipid demand. The physiological role of Nce102 in the regulation of sphingolipid synthesis is demonstrated by mass spectrometry analysis showing reduced levels of hydroxylated complex sphingolipids in response to heat stress in the nce102Δ deletion mutant. We also demonstrate that Nce102 behaves analogously in the widespread human fungal pathogen Candida albicans, suggesting a conserved principle of local sphingolipid control across species.IMPORTANCE Microorganisms are challenged constantly by their rapidly changing environment. To survive, they have developed diverse mechanisms to quickly perceive stressful situations and adapt to them appropriately. The primary site of both stress sensing and adaptation is the plasma membrane. We identified the yeast protein Nce102 as a marker of local sphingolipid levels and fluidity in the plasma membrane. Nce102 is an important structural and functional component of the membrane compartment Can1 (MCC), a plasma membrane microdomain stabilized by a large cytosolic hemitubular protein scaffold, the eisosome. The MCC/eisosomes are widely conserved among fungi and unicellular algae. To determine if Nce102 carries out similar functions in other organisms, we analyzed the human fungal pathogen Candida albicans and found that Nce102 responds to sphingolipid levels also in this organism, which has potential applications for the development of novel therapeutic approaches. The presented study represents a valuable model for how organisms regulate plasma membrane sphingolipids.

Membrane Contact Sites in Yeast: Control Hubs of Sphingolipid Homeostasis.

Sphingolipids are the most diverse class of membrane lipids, in terms of their structure and function. Structurally simple sphingolipid precursors, such as ceramides, act as intracellular signaling molecules in various processes, including apoptosis, whereas mature and complex forms of sphingolipids are important structural components of the plasma membrane. Supplying complex sphingolipids to the plasma membrane, according to need, while keeping pro-apoptotic ceramides in check is an intricate task for the cell and requires mechanisms that tightly control sphingolipid synthesis, breakdown, and storage. As each of these processes takes place in different organelles, recent studies, using the budding yeast Saccharomyces cerevisiae, have investigated the role of membrane contact sites as hubs that integrate inter-organellar sphingolipid transport and regulation. In this review, we provide a detailed overview of the findings of these studies and put them into the context of established regulatory mechanisms of sphingolipid homeostasis. We have focused on the role of membrane contact sites in sphingolipid metabolism and ceramide transport, as well as the mechanisms that prevent toxic ceramide accumulation.

Ceramide Metabolism Enzymes-Therapeutic Targets against Cancer.

Sphingolipids are both structural molecules that are essential for cell architecture and second messengers that are involved in numerous cell functions. Ceramide is the central hub of sphingolipid metabolism. In addition to being the precursor of complex sphingolipids, ceramides induce cell cycle arrest and promote cell death and inflammation. At least some of the enzymes involved in the regulation of sphingolipid metabolism are altered in carcinogenesis, and some are targets for anticancer drugs. A number of scientific reports have shown how alterations in sphingolipid pools can affect cell proliferation, survival and migration. Determination of sphingolipid levels and the regulation of the enzymes that are implicated in their metabolism is a key factor for developing novel therapeutic strategies or improving conventional therapies. The present review highlights the importance of bioactive sphingolipids and their regulatory enzymes as targets for therapeutic interventions with especial emphasis in carcinogenesis and cancer dissemination.

Ceramide Metabolism and Parkinson's Disease-Therapeutic Targets.

Ceramide is a bioactive sphingolipid involved in numerous cellular processes. In addition to being the precursor of complex sphingolipids, ceramides can act as second messengers, especially when they are generated at the plasma membrane of cells. Its metabolic dysfunction may lead to or be a consequence of an underlying disease. Recent reports on transcriptomics and electrospray ionization mass spectrometry analysis have demonstrated the variation of specific levels of sphingolipids and enzymes involved in their metabolism in different neurodegenerative diseases. In the present review, we highlight the most relevant discoveries related to ceramide and neurodegeneration, with a special focus on Parkinson’s disease.

The structural basis of fatty acid elongation by the ELOVL elongases.

Very long chain fatty acids (VLCFAs) are essential building blocks for the synthesis of ceramides and sphingolipids. The first step in the fatty acid elongation cycle is catalyzed by the 3-keto acyl-coenzyme A (CoA) synthases (in mammals, ELOVL elongases). Although ELOVLs are implicated in common diseases, including insulin resistance, hepatic steatosis and Parkinson's, their underlying molecular mechanisms are unknown. Here we report the structure of the human ELOVL7 elongase, which comprises an inverted transmembrane barrel surrounding a 35-Å long tunnel containing a covalently attached product analogue. The structure reveals the substrate-binding sites in the narrow tunnel and an active site deep in the membrane. We demonstrate that chain elongation proceeds via an acyl-enzyme intermediate involving the second histidine in the canonical HxxHH motif. The unusual substrate-binding arrangement and chemistry suggest mechanisms for selective ELOVL inhibition, relevant for diseases where VLCFAs accumulate, such as X-linked adrenoleukodystrophy.

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. While ORMDL3-KO modestly increased dihydroceramide and ceramide levels, ORMDL-TKO cells had dramatic increases in the accumulation of these sphingolipid precursors. SPT activity was increased only in ORMDL-TKO cells. In addition, ORMDL-TKO but not ORMDL3-KO dramatically increased levels of galactosylceramides, glucosylceramides, and lactosylceramides, the elevated N-acyl chain distributions of which broadly correlated with the increases in ceramide species. Surprisingly, although C16:0 is the major sphingomyelin species, it was only increased in ORMDL3-KO, whereas all other N-acyl chain sphingomyelin species were significantly increased in ORMDL-TKO cells. Analysis of sphingoid bases revealed that although sphingosine was only increased 2-fold in ORMDL-TKO cells, levels of dihydrosphingosine, dihydrosphingosine-1-phosphate, and sphingosine-1-phosphate were hugely increased in ORMDL-TKO cells and not in ORMDL3-KO cells. Thus, ORMDL proteins may have a complex, multifaceted role in the biosynthesis and regulation of cellular sphingolipids.

Simplified Mass Spectrometric Analysis of Ceramides using a Common Collision Energy.

Ceramides (CER) are biologically active sphingolipid precursors that are mechanistically linked to several pathogenic states including cancer, insulin resistance, and neurodegeneration. CER are commonly quantified through mass spectrometry-based methods founded upon a product ion scan (PIS) in positive mode to produce a characteristic m/z 264 ion. The ionization efficiency (IE) of CER species decreases with an increase in CER mass, thus quantitation of CER typically involves application of mass-dependent response factors (RF) for each CER species. In this work, we observed that the RF were systematically dependent on the number of fatty acid acyl carbons and the collision energy (CE) used to generate the m/z 264 ion. Using these complimentary trends, we determined an "isosbestic" CE where the RF for all CER species were equivalent, allowing for CER quantitation without postcollection correction factors. A comparison of this common CE/common RF method to the multiple RF method demonstrated good agreement between the two methods. Use of the common CE/common RF method will reduce data processing and reduce the use of multiple CER species standards.

Astrocytic ceramide as possible indicator of neuroinflammation

BackgroundNeurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease dementia (PDD), and frontotemporal lobar dementia (FTLD) are characterized by progressive neuronal loss but differ in their underlying pathological mechanisms. However, neuroinflammation is commonly observed within these different forms of dementia. Recently, it has been suggested that an altered sphingolipid metabolism may contribute to the pathogenesis of a variety of neurodegenerative conditions. Especially ceramide, the precursor of all complex sphingolipids, is thought to be associated with pro-apoptotic cellular processes, thereby propagating neurodegeneration and neuroinflammation, although it remains unclear to what extent. The current pathological study therefore investigates whether increased levels of ceramide are associated with the degree of neuroinflammation in various neurodegenerative disorders.MethodsImmunohistochemistry was performed on human post-mortem tissue of PDD and FTLD Pick’s disease cases, which are well-characterized cases of dementia subtypes differing in their neuroinflammatory status, to assess the expression and localization of ceramide, acid sphingomyelinase, and ceramide synthase 2 and 5. In addition, we determined the concentration of sphingosine, sphingosine-1-phosphate (S1P), and ceramide species differing in their chain-length in brain homogenates of the post-mortem tissue using HPLC-MS/MS.ResultsOur immunohistochemical analysis reveals that neuroinflammation is associated with increased ceramide levels in astrocytes in FTLD Pick’s disease. Moreover, the observed increase in ceramide in astrocytes correlates with the expression of ceramide synthase 5. In addition, HPLC-MS/MS analysis shows a shift in ceramide species under neuroinflammatory conditions, favoring pro-apoptotic ceramide.ConclusionsTogether, these findings suggest that detected increased levels of pro-apoptotic ceramide might be a common denominator of neuroinflammation in different neurodegenerative diseases.

Lipidomic profiling reveals early-stage metabolic dysfunction in overweight or obese humans

BackgroundAdvances in mass spectrometry and lipidomics techniques are providing new insights into the role of lipid metabolism in obesity-related diseases. However, human lipidomic studies have been inconsistent, owing to the use of indirect proxy measures of metabolic outcomes and relatively limited coverage of the lipidome. Here, we employed comprehensive lipid profiling and gold-standard metabolic measures to test the hypothesis that distinct lipid signatures in obesity may signify early stages of pathogenesis toward type 2 diabetes. MethodsUsing high-performance liquid chromatography-electrospray tandem mass spectrometry, we profiled >450 lipid species across 26 classes in 65 overweight or obese non-diabetic individuals. Intensive metabolic testing was conducted using direct gold-standard measures of adiposity (% body fat by dual X-ray absorptiometry), insulin sensitivity (hyperinsulinaemic-euglycaemic clamps), and insulin secretion (intravenous glucose tolerance tests), as well as measurement of serum inflammatory cytokines and adipokines (multiplex assays; flow cytometry). Univariable and multivariable linear regression models were computed using Matlab R2011a, and all analyses were corrected for multiple testing using the Benjamini-Hochberg method. ResultsWe present new evidence showing a strong and independent positive correlation between the lysophosphatidylinositol (LPI) lipid class and insulin secretion in vivo in humans (β [95% CI] = 781.9 [353.3, 1210.4], p = 0.01), supporting the insulinotropic effects of LPI demonstrated in mouse islets. Dihydroceramide, a sphingolipid precursor, was independently and negatively correlated with insulin sensitivity (β [95% CI] = −1.9 [−2.9, −0.9], p = 0.01), indicating a possible upregulation in sphingolipid synthesis in obese individuals. These associations remained significant in multivariable models adjusted for age, sex, and % body fat. The dihexosylceramide class correlated positively with interleukin-10 before and after adjustment for age, sex, and % body fat (p = 0.02), while the phosphatidylethanolamine class and its vinyl ether-linked (plasmalogen) derivatives correlated negatively with % body fat in both univariable and age- and sex-adjusted models (all p < 0.04). ConclusionsOur data suggest that these lipid classes may signify early pathogenesis toward type 2 diabetes and could serve as novel therapeutic targets or biomarkers for diabetes prevention.

Intra- and intercellular trafficking in sphingolipid metabolism in myelination.

The myelin sheath, produced by oligodendrocytes in the central nervous system, provides essential electrical insulation to neurons, but also is critical for viability of neurons. Both the protein and lipid composition of this fascinating membrane is unique. Here the focus is on the sphingolipids that are highly abundant in myelin and, in particular, how they are produced. This review discusses how sphingolipid metabolism is regulated. In particular the subcellular localization of lipid metabolic enzymes is discussed and how inter-organelle transport can affect the metabolic routes that sphingolipid precursors take. Understanding the regulation of sphingolipid metabolism in formation of the myelin membrane will have a significant impact on strategies to treat demyelinating diseases.

Candida albicans gains azole resistance by altering sphingolipid composition

Fungal infections by drug-resistant Candida albicans pose a global public health threat. However, the pathogen’s diploid genome greatly hinders genome-wide investigations of resistance mechanisms. Here, we develop an efficient piggyBac transposon-mediated mutagenesis system using stable haploid C. albicans to conduct genome-wide genetic screens. We find that null mutants in either gene FEN1 or FEN12 (encoding enzymes for the synthesis of very-long-chain fatty acids as precursors of sphingolipids) exhibit resistance to fluconazole, a first-line antifungal drug. Mass-spectrometry analyses demonstrate changes in cellular sphingolipid composition in both mutants, including substantially increased levels of several mannosylinositolphosphoceramides with shorter fatty-acid chains. Treatment with fluconazole induces similar changes in wild-type cells, suggesting a natural response mechanism. Furthermore, the resistance relies on a robust upregulation of sphingolipid biosynthesis genes. Our results shed light into the mechanisms underlying azole resistance, and the new transposon-mediated mutagenesis system should facilitate future genome-wide studies of C. albicans.