Destabilization of ß Cell FIT2 by saturated fatty acids alter lipid droplet numbers and contribute to ER stress and diabetes.

SignificanceWith obesity on the rise, there is a growing appreciation for intracellular lipid droplet (LD) regulation. Here, we show how saturated fatty acids (SFAs) reduce fat storage-inducing transmembrane protein 2 (FIT2)-facilitated, pancreatic ß cell LD biogenesis, which in turn induces ß cell dysfunction and death, leading to diabetes. This mechanism involves direct acylation of FIT2 cysteine residues, which then marks the FIT2 protein for endoplasmic reticulum (ER)-associated degradation. Loss of ß cell FIT2 and LDs reduces insulin secretion, increases intracellular ceramides, stimulates ER stress, and exacerbates diet-induced diabetes in mice. While palmitate and stearate degrade FIT2, unsaturated fatty acids such as palmitoleate and oleate do not, results of which extend to nutrition and diabetes.

Erythrocytes efficiently utilize exogenous sphingosines for S1P synthesis and export via Mfsd2b.

Sphingosine-1-phosphate (S1P) is a potent lipid mediator that exerts its activity via activation of five different G protein-coupled receptors, designated as S1P1-5. This potent lipid mediator is synthesized from the sphingosine precursor by two sphingosine kinases (SphK1 and 2) and must be exported to exert extracellular signaling functions. We recently identified Mfsd2b as the S1P transporter in the hematopoietic system. However, the sources of sphingosine for S1P synthesis and the transport mechanism of Mfsd2b in erythrocytes remain to be determined. Here, we show that erythrocytes efficiently take up exogenous sphingosine and that a de novo synthesis pathway in part provides sphingosines to erythrocytes. The uptake of sphingosine in erythrocytes is facilitated by the activity of SphK1. By converting sphingosine into S1P, SphK1 indirectly increases the influx of sphingosine, a process that is irreversible in erythrocytes. Our results explain for the abnormally high amount of sphingosine accumulation in Mfsd2b knockout erythrocytes. Furthermore, we show that Mfsd2b utilizes a proton gradient to facilitate the release of S1P. The negatively charged residues D95 and T157 are essential for Mfsd2b transport activity. Of interest, we also discovered an S1P analog that inhibits S1P export from erythrocytes, providing evidence that sphingosine analogs can be used to inhibit S1P export by Mfsd2b. Collectively, our results highlight that erythrocytes are efficient in sphingosine uptake for S1P production and the release of S1P is dependent on Mfsd2b functions.

Mapping the distribution of double bond location isomers in lipids across mouse tissues.

Lipids are highly diverse and essential biomolecules in all living systems. As lipid homeostasis is often perturbed in metabolic diseases, these molecules can serve as both biomarkers and drug targets. The development of modern mass spectrometry (MS) provided the platform for large-scale lipidomic studies at the level of molecular species. Traditionally, more detailed structural information, such as the C[double bond, length as m-dash]C location, was mostly assumed instead of properly measured, though the specific isomers were indicated as potential biomarkers of cancers or cardiovascular diseases. Recent C[double bond, length as m-dash]C localization methods, including the Paternò-Büchi (PB) reaction, have shown the prevalent and heterogeneous distribution of C[double bond, length as m-dash]C location in lipids across tissues. Mapping the lipidome of model animals at the level of C[double bond, length as m-dash]C position would increase the understanding of the metabolism and function of lipid isomers, facilitating clinical research. In this study, we employed an online PB reaction on a liquid chromatography-high resolution MS platform to map C[double bond, length as m-dash]C location isomers in five different murine tissues. We analyzed phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins; we relatively quantified and mapped the distribution of ∼30 groups of co-existing isomers, characterized by different chain lengths and degrees of unsaturation. More specifically, we performed relative quantitation of four isomers of the C16:1 fatty acyl, which included rarely reported n-10 and n-5 species besides n-9 and n-7 isomers. We showed a small variation of the isomers' relative composition among individual animals (<20%) but significant differences across different lipid species and mouse tissues. Our results provided an initial database to map alternative lipid metabolic pathways at the tissue level.

Immunolipidomics Reveals a Globoside Network During the Resolution of Pro-Inflammatory Response in Human Macrophages.

Toll-like receptor 4 (TLR4)-mediated changes in macrophages reshape intracellular lipid pools to coordinate an effective innate immune response. Although this has been previously well-studied in different model systems, it remains incompletely understood in primary human macrophages. Here we report time-dependent lipidomic and transcriptomic responses to lipopolysaccharide (LPS) in primary human macrophages from healthy donors. We grouped the variation of ~200 individual lipid species measured by LC-MS/MS into eight temporal clusters. Among all other lipids, glycosphingolipids (glycoSP) and cholesteryl esters (CE) showed a sharp increase during the resolution phase (between 8h or 16h post LPS). GlycoSP, belonging to the globoside family (Gb3 and Gb4), showed the greatest inter-individual variability among all lipids quantified. Integrative network analysis between GlycoSP/CE levels and genome-wide transcripts, identified Gb4 d18:1/16:0 and CE 20:4 association with subnetworks enriched for T cell receptor signaling (PDCD1, CD86, PTPRC, CD247, IFNG) and DC-SIGN signaling (RAF1, CD209), respectively. Our findings reveal Gb3 and Gb4 globosides as sphingolipids associated with the resolution phase of inflammatory response in human macrophages.

Sphingolipid metabolism during Toll-like receptor 4 (TLR4)-mediated macrophage activation.

Macrophage activation in response to stimulation of Toll-like receptor 4 (TLR4) provides a paradigm for investigating energy metabolism that regulates the inflammatory response. TLR4-mediated pro-inflammatory macrophage activation is characterized by increased glycolysis and altered mitochondrial metabolism, supported by selective amino acid uptake and/or usage. Fatty acid metabolism remains as a highly complex rewiring that accompanies classical macrophage activation. TLR4 activation leads to de novo synthesis of fatty acids, which flux into sphingolipids, complex lipids that form the building blocks of eukaryotic cell membranes and regulate cell function. Here, we review the importance of TLR4-mediated de novo synthesis of membrane sphingolipids in macrophages. We first highlight fatty acid metabolism during TLR4-driven macrophage immunometabolism. We then focus on the temporal dynamics of sphingolipid biosynthesis and emphasize the modulatory role of some sphingolipid species (i.e. sphingomyelins, ceramides and glycosphingolipids) on the pro-inflammatory and pro-resolution phases of LPS/TLR4 activation in macrophages.

A reference map of sphingolipids in murine tissues.

Sphingolipids (SPs) have both a structural role in the cell membranes and a signaling function that regulates many cellular processes. The enormous structural diversity and low abundance of many SPs pose a challenge for their identification and quantification. Recent advances in lipidomics, in particular liquid chromatography (LC) coupled with mass spectrometry (MS), provide methods to detect and quantify many low-abundant SP species reliably. Here we use LC-MS to compile a "murine sphingolipid atlas," containing the qualitative and quantitative distribution of 114 SPs in 21 tissues of a widely utilized wild-type laboratory mouse strain (C57BL/6). We report tissue-specific SP fingerprints, as well as sex-specific differences in the same tissue. This is a comprehensive, quantitative sphingolipidomic map of mammalian tissues collected in a systematic fashion. It will complement other tissue compendia for interrogation into the role of SP in mammalian health and disease.

Deletion of Mfsd2b impairs thrombotic functions of platelets.

We recently discovered that Mfsd2b, which is the S1P exporter found in blood cells. Here, we report that Mfsd2b is critical for the release of all S1P species in both resting and activated platelets. We show that resting platelets store S1P in the cytoplasm. After activation, this S1P pool is delivered to the plasma membrane, where Mfsd2b is predominantly localized for export. Employing knockout mice of Mfsd2b, we reveal that platelets contribute a minor amount of plasma S1P. Nevertheless, Mfsd2b deletion in whole body or platelets impairs platelet morphology and functions. In particular, Mfsd2b knockout mice show significantly reduced thrombus formation. We show that loss of Mfsd2b affects intrinsic platelet functions as part of remarkable sphingolipid accumulation. These findings indicate that accumulation of sphingolipids including S1P by deletion of Mfsd2b strongly impairs platelet functions, which suggests that the transporter may be a target for the prevention of thrombotic disorders.

TDP-43 mediates SREBF2-regulated gene expression required for oligodendrocyte myelination.

Cholesterol metabolism operates autonomously within the central nervous system (CNS), where the majority of cholesterol resides in myelin. We demonstrate that TDP-43, the pathological signature protein for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), influences cholesterol metabolism in oligodendrocytes. TDP-43 binds directly to mRNA of SREBF2, the master transcription regulator for cholesterol metabolism, and multiple mRNAs encoding proteins responsible for cholesterol biosynthesis and uptake, including HMGCR, HMGCS1, and LDLR. TDP-43 depletion leads to reduced SREBF2 and LDLR expression, and cholesterol levels in vitro and in vivo. TDP-43-mediated changes in cholesterol levels can be restored by reintroducing SREBF2 or LDLR. Additionally, cholesterol supplementation rescues demyelination caused by TDP-43 deletion. Furthermore, oligodendrocytes harboring TDP-43 pathology from FTD patients show reduced HMGCR and HMGCS1, and coaggregation of LDLR and TDP-43. Collectively, our results indicate that TDP-43 plays a role in cholesterol homeostasis in oligodendrocytes, and cholesterol dysmetabolism may be implicated in TDP-43 proteinopathies-related diseases.

Long acyl chain ceramides govern cholesterol and cytoskeleton dependence of membrane outer leaflet dynamics.

The spatiotemporal dynamics of the plasma membrane is a consequence of fine-tuned interactions between membrane components. However, the precise identity of molecular factors that maintain this delicate balance, which is lost even in cell membrane derived mimics, remains elusive. Here, we use two cell lines, CHO-K1 and RBL-2H3, which show differences in outer membrane organization, dynamics, and cytoskeleton coupling, to investigate the underlying factors. To our surprise, knock-down of the cytoskeleton-interacting Immunoglobulin E receptor, which is abundant in RBL-2H3 but not in CHO-K1 cells, is not responsible for lipid confinement or cytoskeleton coupling. A subsequent lipidomic analysis of the two cell membranes revealed differences in total membrane ceramide content (C16 to C24). Analysis of the dynamics and organization of ceramide treated live cell membranes by imaging fluorescence correlation spectroscopy demonstrates that C24 and C16 saturated ceramides uniquely alter membrane dynamics by promoting the formation of cholesterol-independent domains and by elevating the inter-leaflet coupling.

Sphingolipid Analysis in Clinical Research.

Sphingolipids are the most diverse class of lipids due to the numerous variations in their structural components. This diversity is also reflected in their extremely different functions. Sphingolipids are not only constituents of cell membranes but have also emerged as key signaling molecules involved in a variety of cellular functions, such as cell growth and differentiation, proliferation, and apoptotic cell death. Lipidomic analyses in clinical research have identified pathways and products of sphingolipid metabolism that are altered in several human pathologies. In this article, we describe how to properly design a lipidomic experiment in clinical research, how to handle plasma and serum samples for this purpose, and how to measure sphingolipids using liquid chromatography-mass spectrometry.

Enriched Expression of Neutral Sphingomyelinase 2 in the Striatum is Essential for Regulation of Lipid Raft Content and Motor Coordination.

Sphingomyelinases are a family of enzymes that hydrolyze sphingomyelin to generate phosphocholine and ceramide. The brain distribution and function of neutral sphingomyelinase 2 (nSMase2) were elucidated in this study. nSMase2 mRNA expression was greatest in the striatum, followed by the prefrontal cortex, hippocampus, cerebellum, thalamus, brainstem, and olfactory bulb. The striatum had the highest level of nSMase2 protein expression, followed by the prefrontal cortex, thalamus, hippocampus, brainstem, and cerebellum. Dense immunolabeling was observed in the striatum, including the caudate-putamen, while moderately dense staining was found in the olfactory bulb and cerebral neocortex. Electron microscopy of the caudate-putamen showed nSMase2 immunoreaction product was present in small diameter dendrites or dendritic spines, that formed asymmetrical synapses with unlabeled axon terminals containing small round vesicles; and characteristics of glutamatergic axons. Lipidomic analysis of the striatum showed increase in long chain sphingomyelins, SM36:1 and SM38:1 after inhibition of nSMase activity. Quantitative proteomic analysis of striatal lipid raft fraction showed many proteins were downregulated by more than 2-fold after inhibition or antisense knockdown of nSMase; consistent with the notion that nSMase2 activity is important for aggregation or clustering of proteins in lipid rafts. Inhibition or antisense knockdown of nSMase2 in the caudate-putamen resulted in motor deficits in the rotarod and narrow beam tests; as well as decreased acoustic startle and improved prepulse inhibition of the startle reflex. Together, results indicate an important function of nSMase2 in the striatum.