BACKGROUND. Sphingolipids are important components of cellular membranes and functionally associated with fundamental processes such as cell differentiation, neuronal signaling, and myelin sheath formation. Defects in the synthesis or degradation of sphingolipids leads to various neurological pathologies; however, the entire spectrum of sphingolipid metabolism disorders remains elusive. METHODS. A combined approach of genomics and lipidomics was applied to identify and characterize a human sphingolipid metabolism disorder. RESULTS. By whole-exome sequencing in a patient with a multisystem neurological disorder of both the central and peripheral nervous systems, we identified a homozygous p.Ala280Val variant in DEGS1, which catalyzes the last step in the ceramide synthesis pathway. The blood sphingolipid profile in the patient showed a significant increase in dihydro sphingolipid species that was further recapitulated in patient-derived fibroblasts, in CRISPR/Cas9–derived DEGS1-knockout cells, and by pharmacological inhibition of DEGS1. The enzymatic activity in patient fibroblasts was reduced by 80% compared with wild-type cells, which was in line with a reduced expression of mutant DEGS1 protein. Moreover, an atypical and potentially neurotoxic sphingosine isomer was identified in patient plasma and in cells expressing mutant DEGS1. CONCLUSION. We report DEGS1 dysfunction as the cause of a sphingolipid disorder with hypomyelination and degeneration of both the central and peripheral nervous systems. TRIAL REGISTRATION. Not applicable. FUNDING. Seventh Framework Program of the European Commission, Swiss National Foundation, Rare Disease Initiative Zurich.

High-density lipoproteins (HDL) contain hundreds of lipid species and proteins and exert many potentially vasoprotective and antidiabetogenic activities on cells. To resolve structure-function-disease relationships of HDL, we characterized HDL of 51 healthy subjects and 98 patients with diabetes (T2DM), coronary heart disease (CHD), or both for protein and lipid composition, as well as functionality in 5 cell types. The integration of 40 clinical characteristics, 34 nuclear magnetic resonance (NMR) features, 182 proteins, 227 lipid species, and 12 functional read-outs by high-dimensional statistical modeling revealed, first, that CHD and T2DM are associated with different changes of HDL in size distribution, protein and lipid composition, and function. Second, different cellular functions of HDL are weakly correlated with each other and determined by different structural components. Cholesterol efflux capacity (CEC) was no proxy of other functions. Third, 3 potentially novel determinants of HDL function were identified and validated by the use of artificially reconstituted HDL, namely the sphingadienine-based sphingomyelin SM 42:3 and glycosylphosphatidylinositol-phospholipase D1 for the ability of HDL to inhibit starvation-induced apoptosis of human aortic endothelial cells and apolipoprotein F for the ability of HDL to promote maximal respiration of brown adipocytes.

Sphingolipids (SLs) are structurally diverse lipids that are defined by the presence of a long-chain base (LCB) backbone. Typically, LCBs contain a single Δ4E double bond (DB) (mostly d18:1), whereas the dienic LCB sphingadienine (d18:2) contains a second DB at the Δ14Z position. The enzyme introducing the Δ14Z DB is unknown. We analyzed the LCB plasma profile in a gender-, age-, and BMI-matched subgroup of the CoLaus cohort (n = 658). Sphingadienine levels showed a significant association with gender, being on average ∼30% higher in females. A genome-wide association study (GWAS) revealed variants in the fatty acid desaturase 3 (FADS3) gene to be significantly associated with the plasma d18:2/d18:1 ratio (p = −log 7.9). Metabolic labeling assays, FADS3 overexpression and knockdown approaches, and plasma LCB profiling in FADS3-deficient mice confirmed that FADS3 is a bona fide LCB desaturase and required for the introduction of the Δ14Z double bond. Moreover, we showed that FADS3 is required for the conversion of the atypical cytotoxic 1-deoxysphinganine (1-deoxySA, m18:0) to 1-deoxysphingosine (1-deoxySO, m18:1). HEK293 cells overexpressing FADS3 were more resistant to m18:0 toxicity than WT cells. In summary, using a combination of metabolic profiling and GWAS, we identified FADS3 to be essential for forming Δ14Z DB containing LCBs, such as d18:2 and m18:1. Our results unravel FADS3 as a Δ14Z LCB desaturase, thereby disclosing the last missing enzyme of the SL de novo synthesis pathway.

Sphingolipids (SLs) are chemically diverse lipids that have important structural and signaling functions within mammalian cells. SLs are commonly defined by the presence of a long-chain base (LCB) that is normally formed by the conjugation of l-serine and palmitoyl-CoA. This pyridoxal 5-phosphate (PLP)-dependent reaction is mediated by the enzyme serine-palmitoyltransferase (SPT). However, SPT can also metabolize other acyl-CoAs, in the range of C₁₄ to C₁₈, forming a variety of LCBs that differ by structure and function. Mammalian SPT consists of three core subunits: SPTLC1, SPTLC2, and SPTLC3. Whereas SPTLC1 and SPTLC2 are ubiquitously expressed, SPTLC3 expression is restricted to certain tissues only. The influence of the individual subunits on enzyme activity is not clear. Using cell models deficient in SPTLC1, SPTLC2, and SPTLC3, we investigated the role of each subunit on enzyme activity and the LCB product spectrum. We showed that SPTLC1 is essential for activity, whereas SPTLC2 and SPTLC3 are partly redundant but differ in their enzymatic properties. SPTLC1 in combination with SPTLC2 specifically formed C18, C19, and C20 LCBs while the combination of SPTLC1 and SPTLC3 yielded a broader product spectrum. We identified anteiso-branched-C18 SO (meC18SO) as the primary product of the SPTLC3 reaction. The meC18SO was synthesized from anteiso-methyl-palmitate, in turn synthesized from a precursor metabolite generated in the isoleucine catabolic pathway. The meC18SO is metabolized to ceramides and complex SLs and is a constituent of human low- and high-density lipoproteins. Significance Sphingolipids (SLs) are complex lipids that constitute hundreds of subspecies. All SLs share a long-chain base (LCB) as a defining structural component. LCBs are formed by serine-palmitoyltransferase (SPT) in the first and rate-limiting step of SL de novo synthesis. SPT consists of three subunits that show tissue-specific expression. In presence of the SPTLC3 subunit, the enzyme forms a spectrum of straight and branched LCBs with distinct biochemical and biophysical properties. This alters the composition of cellular membranes and might influence the dynamics of membrane-related transport and signaling events. SPTLC3 is particularly abundant in skin, and changes in SPT activity are related to dermal pathologies. Genetic variants of SPTLC3 are associated with metabolic conditions such as dyslipidemia and atherosclerosis.

Endothelial cell senescence is a key driver of cardiovascular aging, yet little is known about the mechanisms by which it is induced in vivo. Here we show that the gut bacterial metabolite phenylacetic acid (PAA) and its byproduct, phenylacetylglutamine (PAGln), are elevated in aged humans and mice. Metagenomic analyses reveal an age-related increase in PAA-producing microbial pathways, positively linked to the bacterium Clostridium sp. ASF356 (Clos). We demonstrate that colonization of young mice with Clos increases blood PAA levels and induces endothelial senescence and angiogenic incompetence. Mechanistically, we find that PAA triggers senescence through mitochondrial H₂O₂ production, exacerbating the senescence-associated secretory phenotype. By contrast, we demonstrate that fecal acetate levels are reduced with age, compromising its function as a Sirt1-dependent senomorphic, regulating proinflammatory secretion and redox homeostasis. These findings define PAA as a mediator of gut–vascular crosstalk in aging and identify sodium acetate as a potential microbiome-based senotherapy to promote healthy aging.

Impaired proinsulin-to-insulin processing in pancreatic β-cells is a key defective step in both type 1 diabetes and type 2 diabetes (T2D) (refs. ^1,2), but the mechanisms involved remain to be defined. Altered metabolism of sphingolipids (SLs) has been linked to development of obesity, type 1 diabetes and T2D (refs. ^3–8); nonetheless, the role of specific SL species in β-cell function and demise is unclear. Here we define the lipid signature of T2D-associated β-cell failure, including an imbalance of specific very-long-chain SLs and long-chain SLs. β-cell-specific ablation of CerS2, the enzyme necessary for generation of very-long-chain SLs, selectively reduces insulin content, impairs insulin secretion and disturbs systemic glucose tolerance in multiple complementary models. In contrast, ablation of long-chain-SL-synthesizing enzymes has no effect on insulin content. By quantitatively defining the SL–protein interactome, we reveal that CerS2 ablation affects SL binding to several endoplasmic reticulum–Golgi transport proteins, including Tmed2, which we define as an endogenous regulator of the essential proinsulin processing enzyme Pcsk1. Our study uncovers roles for specific SL subtypes and SL-binding proteins in β-cell function and T2D-associated β-cell failure.