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L-Serine Deficiency Elicits Intracellular Accumulation of Cytotoxic Deoxysphingolipids and Lipid Body Formation.

ABSTRACT: L-serine is required to synthesize membrane lipids such as phosphatidylserine and sphingolipids. Nevertheless, it remains largely unknown how a diminished capacity to synthesize L-serine affects lipid homeostasis in cells and tissues. Here, we show that deprivation of external L-serine leads to the generation of 1-deoxysphingolipids (doxSLs), including 1-deoxysphinganine, in mouse embryonic fibroblasts (KO-MEFs) lacking D-3-phosphoglycerate dehydrogenase (Phgdh), which catalyzes the first step in the de novo synthesis of L-serine. A novel mass spectrometry-based lipidomic approach demonstrated that 1-deoxydihydroceramide was the most abundant species of doxSLs accumulated in L-serine-deprived KO-MEFs. Among normal sphingolipid species in KO-MEFs, levels of sphinganine, dihydroceramide, ceramide, and hexosylceramide were significantly reduced after deprivation of external L-serine, whereas those of sphingomyelin, sphingosine, and sphingosine 1-phosphate were retained. The synthesis of doxSLs was suppressed by supplementing the culture medium with L-serine but was potentiated by increasing the ratio of L-alanine to L-serine in the medium. Unlike with L-serine, depriving cells of external L-leucine did not promote the occurrence of doxSLs. Consistent with results obtained from KO-MEFs, brain-specific deletion of Phgdh in mice also resulted in accumulation of doxSLs in the brain. Furthermore, L-serine-deprived KO-MEFs exhibited increased formation of cytosolic lipid bodies containing doxSLs and other sphingolipids. These in vitro and in vivo studies indicate that doxSLs are generated in the presence of a high ratio of L-alanine to L-serine in cells and tissues lacking Phgdh, and de novo synthesis of L-serine is necessary to maintain normal sphingolipid homeostasis when the external supply of this amino acid is limited.

SUBMITTER: Esaki K

PROVIDER: S-EPMC4505526 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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L-Serine Deficiency Elicits Intracellular Accumulation of Cytotoxic Deoxysphingolipids and Lipid Body Formation.

Esaki Kayoko K Sayano Tomoko T Sonoda Chiaki C Akagi Takumi T Suzuki Takeshi T Ogawa Takuya T Okamoto Masahiro M Yoshikawa Takeo T Hirabayashi Yoshio Y Furuya Shigeki S

The Journal of biological chemistry 20150422 23

L-serine is required to synthesize membrane lipids such as phosphatidylserine and sphingolipids. Nevertheless, it remains largely unknown how a diminished capacity to synthesize L-serine affects lipid homeostasis in cells and tissues. Here, we show that deprivation of external L-serine leads to the generation of 1-deoxysphingolipids (doxSLs), including 1-deoxysphinganine, in mouse embryonic fibroblasts (KO-MEFs) lacking D-3-phosphoglycerate dehydrogenase (Phgdh), which catalyzes the first step i ...[more]

PMID: 25903138

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Project description:1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids of clinical relevance as they are elevated in plasma of patients suffering from hereditary sensory and autonomic neuropathy (HSAN1) or type 2 diabetes. Their neurotoxicity is described best but they inflict damage to various cell types by an uncertain pathomechanism. Using mouse embryonic fibroblasts and an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, we here study the impact of deoxySLs on macroautophagy/autophagy, the regulated degradation of dysfunctional or expendable cellular components. We find that deoxySLs induce autophagosome and lysosome accumulation indicative of an increase in autophagic flux. The autophagosomal machinery targets damaged mitochondria that have accumulated N-acylated doxSA metabolites, presumably deoxyceramide and deoxydihydroceramide, and show aberrant swelling and tubule formation. Autophagosomes and lysosomes also interact with cellular lipid aggregates and crystals that occur upon cellular uptake and N-acylation of monomeric doxSA. As crystals entering the lysophagosomal apparatus in phagocytes are known to trigger the NLRP3 inflammasome, we also treated macrophages with doxSA. We demonstrate the activation of the NLRP3 inflammasome by doxSLs, prompting the release of IL1B from primary macrophages. Taken together, our data establish an impact of doxSLs on autophagy and link doxSL pathophysiology to inflammation and the innate immune system.Abbreviations: alkyne-doxSA: (2S,3R)-2-aminooctadec-17yn-3-ol; alkyne-SA: (2S,3R)-2- aminooctadec-17yn-1,3-diol; aSA: alkyne-sphinganine; ASTM-BODIPY: azido-sulfo-tetramethyl-BODIPY; CerS: ceramide synthase; CMR: clonal macrophage reporter; deoxySLs: 1-deoxysphingolipids; dox(DH)Cer: 1-deoxydihydroceramide; doxCer: 1-deoxyceramide; doxSA: 1-deoxysphinganine; FB1: fumonisin B1; HSAN1: hereditary sensory and autonomic neuropathy type 1; LC3: MAP1LC3A and MAP1LC3B; LPS: lipopolysaccharide; MEF: mouse embryonal fibroblasts; MS: mass spectrometry; N3635P: azido-STAR635P; N3Cy3: azido-cyanine 3; N3picCy3: azido-picolylcyanine 3; NLRP3: NOD-like receptor pyrin domain containing protein 3; P4HB: prolyl 4-hydroxylase subunit beta; PINK1: PTEN induced putative kinase 1; PYCARD/ASC: PYD and CARD domain containing; SPTLC1: serine palmitoyltransferase long chain base subunit 1; SQSTM1: sequestosome 1; TLC: thin layer chromatography.

Project description:Imbalanced dietary habits are closely associated with poor micronutrients status and the development of obesity. Previous studies have shown that serum folate level is decreased in obese individuals. However, whether folate deficiency could result in adiposity is still unclear. The aim of this study was to investigate the effects of dietary folate on lipid accumulation and leptin production using both in vivo and in vitro studies. Male C57BL/6 mice were fed with a diet with (f1) or without (f0) folate in a high-fat (HF) diet containing high-sucrose (HFS-f1, HFS-f0) for 4.5-5 months in Experiment 1, or an HF diet (HF-f1, HF-f0) for 12 months in Experiment 2, or an HF diet containing high-fructose (HFF-f1, HFF-f0) for 12 months in Experiment 3, compared with the normal-fat (NF-f1, NF-f0) diet, respectively. The serum levels of folate and leptin, white adipose tissue (WAT), size of adipocytes, hepatic contents of triglyceride (TG), and cholesterol were measured. In vitro study, TG contents, proinflammatory cytokines, leptin, and expressions of hypoxia-inducible factor (HIF)-1α and lipogenesis-related genes of 3T3-L1 adipocytes cultured with (f1) or without (f0) folate were assayed. The results showed that folate deficiency together with a high-fat diet (HFS-f0, HF-f0, HFF-f0) had higher WAT mass, adipocyte size, serum leptin level, and hepatic TG compared to those of the folate-sufficient groups (HFS-f1, HF-f1, and HFF-f1). Folate deficiency with a high-fat high -sucrose or -fructose diet (HFS-f0, HFF-f0) significantly increased the body weight of the mice. Increased intracellular TG, leptin, monocyte chemotactic protein (MCP)-1 and interleukin (IL)-6 levels, and the expression of Hif1α and lipogenesis-related genes Cebpα, Cebpβ, Acc1, Fasn, and Fabp4 were also detected in folate-deficient 3T3-L1 adipocytes. Our results suggested that folate deficiency increased lipid accumulation and leptin production of adipocytes, and thus, inadequate folate status might be one of the risk factors for adiposity.

Project description:We recently discovered that exposure of enterohemorrhagic Escherichia coli (EHEC) to d-serine resulted in accumulation of this unusual amino acid, induction of the SOS regulon, and downregulation of the type III secretion system that is essential for efficient colonization of the host. Here, we have investigated the physiological relevance of this elevated SOS response, which is of particular interest given the presence of Stx toxin-carrying lysogenic prophages on the EHEC chromosome that are activated during the SOS response. We found that RecA elevation in response to d-serine, while being significant, was heterogeneous and not capable of activating stx expression or stx phage transduction to a nonlysogenic recipient. This "SOS-like response" was, however, capable of increasing the mutation frequency associated with low-level RecA activity, thus promoting genetic diversity. Furthermore, this response was entirely dependent on RecA and enhanced in the presence of a DNA-damaging agent, indicating a functional SOS response, but did not result in observable cleavage of the LexA repressor alone, indicating a controlled mechanism of induction. This work demonstrates that environmental factors not usually associated with DNA damage are capable of promoting an SOS-like response. We propose that this modulated induction of RecA allows EHEC to adapt to environmental insults such as d-serine while avoiding unwanted phage-induced lysis.ImportanceThe SOS response is a global stress network that is triggered by the presence of DNA damage due to breakage or stalled replication forks. Activation of the SOS response can trigger the replication of lytic bacteriophages and promote genetic diversification through error-prone polymerases. We have demonstrated that the host-associated metabolite d-serine contributes to Escherichia coli niche specification and accumulates inside cells that cannot catabolize it. This results in a modulated activation of the SOS antirepressor RecA that is insufficient to trigger lytic bacteriophage but capable of increasing the SOS-associated mutation frequency. These findings describe how relevant signals not normally associated with DNA damage can hijack the SOS response, promoting diversity as E. coli strains adapt while avoiding unwanted phage lysis.

Dataset Information

L-Serine Deficiency Elicits Intracellular Accumulation of Cytotoxic Deoxysphingolipids and Lipid Body Formation.

Publications

L-Serine Deficiency Elicits Intracellular Accumulation of Cytotoxic Deoxysphingolipids and Lipid Body Formation.

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