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Adaptation to hypothermic stress is important for skeletal muscle cells, but a comprehensive knowledge of molecular mediators is lacking. We show that adaptation to mild hypothermia (320C) improves the ability of skeletal muscle myoblasts to differentiate into myotubesin vitro. We performed proteomic analysis of mouse myoblasts exposed to mild hypothermia for various time points and identified dynamic changes in mitochondrial metabolism and proteostasis. This revealed that RBM3, an RNA-binding protein, increases progressively with acute and chronic exposure to hypothermic stress, and is necessary for the enhanced differentiation upon hypothermic adaptation. We also demonstrate that overexpression of RBM3 at physiological temperatures is sufficient to (i) enhance mitochondrial metabolism as judged by a decrease in the AMPK energy-sensing pathway, (ii) increase levels of proteins associated with translation and increase levels of 4E-BP1 phosphorylation, (iii) increase stem cell markers (MyoD1, PAX7), and improve differentiation of myoblasts from both young and aged mice.

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.

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., The mechanisms by which platelets release sphingosine-1-phosphate (S1P) is not well characterized. Here the authors show that Mfsd2b is required for S1P release from both resting and activated platelets and that deletion of Mfsd2b impairs thrombotic functions of platelets.

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 β cell LD biogenesis, which in turn induces β 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 β 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. Ministry of Education (MOE) Nanyang Technological University National Research Foundation (NRF) Published version This work was supported by the Singapore Ministry of Education grants under its Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2015-T2-2-087 and MOE2018-T2-1-085 to Y.A.) and Tier 1 (2017-T1-001-220 and 2019-T1-001-059 to Y.A.); the LKCMedicine, Nanyang Technological University Singapore Start-up Grant (P.-O.B.); and the LKCMedicine Healthcare Research Fund (Diabetes Research), established through the generous support of alumni of Nanyang Technological University, Singapore (Y.A.). Q.W.C.H. is supported by the Nanyang President’s Graduate Scholarship award, Nanyang Technological University, Singapore. J.N.F. is supported by Singapore National Research Foundation Fellowship award (NRF-NRFF2016-03). Work in the M.R.W. and F.T. laboratories is supported by grants from the National University of Singapore via the Life Sciences Institute, the National Research Foundation (NRF; NRFI2015-05 and NRFSBP-P4) and the NRF and Agency for Science, Technology and Research (A*STAR) Industry Alignment Fund–Industry Collaborative Project I1901E0040. N.T. is supported by the 1.3.5 Project for Disciplines of Excellence grant, West China Hospital, Sichuan University (ZYGD18017). P.-O.B. is supported by the grants from the Swedish Research Council, the Family Erling-Persson Foundation, the Novo Nordisk Foundation, the Stichting af Jochnick Foundation, the Swedish Diabetes Association, the Scandia Insurance Company Ltd., the Diabetes Research and Wellness Foundation, Berth von Kantzow’s Foundation, the Strategic Research Program in Diabetes at Karolinska Institutet, the ERC-2018-AdG 834860 EYELETS, and the Center of Excellence–International Collaboration Initiative Grant (139180012, China). X.Z. is currently sup-ported by the National Natural Science Foundation of China grant (82070846). G.A.R. was supported by the Wellcome Trust Investigator (WT212625/Z/18/Z) Award, the Medical Research Council Programme grant (MR/R022259/1), and the European Union Horizon2020 research and innovation programme via the Innovative Medicines Initiative 2 Joint Undertaking under Grant Agreement No. 115881 (RHAPSODY).

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 Paterno-Buchi (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 (

Western type diets are linked to obesity and diabetes partly because of their high saturated fatty acid (SFA) content. We found that SFAs, but not unsaturated fatty acids (USFAs), reduced the number of lipid droplets (LDs) within pancreatic β-cells. Mechanistically, SFAs but not USFAs disabled LD biogenesis by inducing palmitoylation and subsequent ERAD-C mediated degradation of LD formation protein, Fat storage-Inducing Transmembrane protein 2 (FIT2). Targeted ablation of FIT2 reduced β-cell LD numbers, lowered β-cell ATP levels, reduced Ca2+ signaling, downregulated β-cell transcription factors (RNA sequencing analysis), and exacerbated diet-induced diabetes in mice. Subsequent mass spectrometry studies revealed increased C16:0 ceramide accumulation in islets of mice lacking β-cell FIT2 under lipotoxic conditions. Inhibition of ceramide synthases ameliorated the enhanced ER stress. Overexpression of FIT2 increased number of intracellular LDs and rescued SFA-induced ER-stress and apoptosis thereby highlighting the protective role of FIT2 and LDs against β-cell lipotoxicity and diet-induced diabetes.

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.

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.

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.

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.

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.

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. NMRC (Natl Medical Research Council, S’pore) Published version