Irina Alecu

Lowering Plasma 1-Deoxysphingolipids Improves Neuropathy in Diabetic Rats

1-Deoxysphingolipids (1-deoxySLs) are atypical neurotoxic sphingolipids that are formed by the serine-palmitoyltransferase (SPT). Pathologically elevated 1-deoxySL concentrations cause hereditary sensory and autonomic neuropathy type 1 (HSAN1), an axonal neuropathy associated with several missense mutations in SPT. Oral L-serine supplementation suppressed the formation of 1-deoxySLs in patients with HSAN1 and preserved nerve function in an HSAN1 mouse model. Because 1-deoxySLs also are elevated in patients with type 2 diabetes mellitus, L-serine supplementation could also be a therapeutic option for diabetic neuropathy (DN). This was tested in diabetic STZ rats in a preventive and therapeutic treatment scheme. Diabetic rats showed significantly increased plasma 1-deoxySL concentrations, and L-serine supplementation lowered 1-deoxySL concentrations in both treatment schemes (P < 0.0001). L-serine had no significant effect on hyperglycemia, body weight, or food intake. Mechanical sensitivity was significantly improved in the preventive (P < 0.01) and therapeutic schemes (P < 0.001). Nerve conduction velocity (NCV) significantly improved in only the preventive group (P < 0.05). Overall NCV showed a highly significant (P = 5.2E-12) inverse correlation with plasma 1-deoxySL concentrations. In summary, our data support the hypothesis that 1-deoxySLs are involved in the pathology of DN and that an oral L-serine supplementation could be a novel therapeutic option for treating DN.

Fenofibrate lowers atypical sphingolipids in plasma of dyslipidemic patients: A novel approach for treating diabetic neuropathy?

BACKGROUND The condensation of palmitoyl-CoA and L-Serine is the first step in the de novo formation of sphingolipids and catalyzed by the serine-palmitoyltransferase (SPT). Besides other acyl-CoAs the SPT can also metabolize L-alanine and glycine, which forms an atypical category of neurotoxic 1-deoxy-sphingolipids (1-deoxySL). Several mutations in SPT are associated with pathologically increased 1-deoxySL levels, which cause the inherited sensory neuropathy HSAN1. 1-DeoxySL levels are also elevated in individuals with the metabolic syndrome and diabetes mellitus type II and seem to be involved in the pathology of the diabetic neuropathy. OBJECTIVE In previous studies, we observed a strong correlation between plasma 1-deoxySLs and triglycerides (TGs). We were therefore interested whether lowering plasma TG levels also affects plasma sphingolipid and in particular, 1-deoxySL levels. METHODS Sixty-six patients with dyslipidemia were treated for 6 wk with the TG-lowering drug fenofibrate (160 mg/d) or extended-release niacin (0.5 g/d for 3 wk, then 1 g/d) with 4 wk of washout between treatments. The sphingoid base profile was analyzed by liquid chromatography-mass spectrometry (LC-MS) before and after each treatment block. RESULTS Fenofibrate significantly lowered 1-deoxySLs and other atypical sphingoid bases (P < .001) but had no effect on the typical sphingolipids. In contrast, extended-release niacin had no effect on 1-deoxySL levels although both treatments lowered plasma TG levels. CONCLUSIONS The lowering of plasma 1-deoxySL levels by fenofibrate in dyslipidemic patients might be a novel therapeutic approach in the prevention and treatment of diabetic neuropathy.

Neurotoxic 1-deoxysphingolipids and paclitaxel-induced peripheral neuropathy

Peripheral neuropathy is a major dose‐limiting side effect of paclitaxel and cisplatin chemotherapy. In the current study, we tested the involvement of a novel class of neurotoxic sphingolipids, the 1‐deoxysphingolipids. 1‐Deoxysphingolipids are produced when the enzyme serine palmitoyltransferase uses l‐alanine instead of l‐serine as its amino acid substrate. We tested whether treatment of cells with paclitaxel (250 nM, 1 μM) and cisplatin (250 nM, 1 μM) would result in elevated cellular levels of 1‐deoxysphingolipids. Our results revealed that paclitaxel, but not cisplatin treatment, caused a dose‐dependent elevation of 1‐deoxysphingolipids levels and an increase in the message and activity of serine palmitoyltransferase (P < 0.05). We also tested whether there is an association between peripheral neuropathy symptoms [evaluated by the European Organization for Research and Treatment of Cancer (EORTC) QLQ‐chemotherapy‐induced peripheral neuropathy‐20 (CIPN20) instrument] and the 1‐deoxysphingolipid plasma levels (measured by mass spectrometry) in 27 patients with breast cancer who were treated with paclitaxel chemotherapy. Our results showed that there was an association between the incidence and severity of neuropathy and the levels of very‐long‐chain 1‐deoxyceramides such as C24 (P < 0.05), with the strongest association being with motor neuropathy (P < 0.001). Our data from cells and from patients with breast cancer suggest that 1‐deoxysphingolipids, the very‐long‐chain in particular, play a role as molecular intermediates of paclitaxel‐induced peripheral neuropathy.—Kramer, R., Bielawski, J., Kistner‐Griffin, E., Othman, A., Alecu, I., Ernst, D., Kornhauser, D., Hornemann, T., Spassieva, S. Neurotoxic 1‐deoxysphingolipids and paclitaxel‐induced peripheral neuropathy. FASEB J. 29, 4461‐4472 (2015). www.fasebj.org

HSAN1 mutations in serine palmitoyltransferase reveal a close structure–function–phenotype relationship

Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is a rare autosomal dominant inherited peripheral neuropathy caused by mutations in the SPTLC1 and SPTLC2 subunits of serine palmitoyltransferase (SPT). The mutations induce a permanent shift in the substrate preference from L-serine to L-alanine, which results in the pathological formation of atypical and neurotoxic 1-deoxy-sphingolipids (1-deoxySL). Here we compared the enzymatic properties of 11 SPTLC1 and six SPTLC2 mutants using a uniform isotope labelling approach. In total, eight SPT mutants (STPLC1p.C133W, p.C133Y, p.S331F, p.S331Y and SPTLC2p.A182P, p.G382V, p.S384F, p.I504F) were associated with increased 1-deoxySL synthesis. Despite earlier reports, canonical activity with l-serine was not reduced in any of the investigated SPT mutants. Three variants (SPTLC1p.S331F/Y and SPTLC2p.I505Y) showed an increased canonical activity and increased formation of C20 sphingoid bases. These three mutations are associated with an exceptionally severe HSAN1 phenotype, and increased C20 sphingosine levels were also confirmed in plasma of patients. A principal component analysis of the analysed sphingoid bases clustered the mutations into three separate entities. Each cluster was related to a distinct clinical outcome (no, mild and severe HSAN1 phenotype). A homology model based on the protein structure of the prokaryotic SPT recapitulated the same grouping on a structural level. Mutations associated with the mild form clustered around the active site, whereas mutations associated with the severe form were located on the surface of the protein. In conclusion, we showed that HSAN1 mutations in SPT have distinct biochemical properties, which allowed for the prediction of the clinical symptoms on the basis of the plasma sphingoid base profile.

Altered sphingoid base profiles in type 1 compared to type 2 diabetes

BackgroundSphingolipids are increasingly recognized to play a role in insulin resistance and diabetes. Recently we reported significant elevations of 1-deoxysphingolipids (1-deoxySL) - an atypical class of sphingolipids in patients with metabolic syndrome (MetS) and diabetes type 2 (T2DM). It is unknown whether 1-deoxySL in patients with diabetes type 1 (T1DM) are similarly elevated.FindingsWe analyzed the long chain base profile by LC-MS after hydrolyzing the N-acyl and O-linked headgroups in plasma from individuals with T1DM (N = 27), T2DM (N = 30) and healthy controls (N = 23). 1-deoxySLs were significantly higher in the groups with T2DM but not different between T1DM and controls. In contrast to patients with T2DM, 1-deoxSL levels are not elevated in T1DM.ConclusionsOur study indicates that the 1-deoxySL formation is not per-se caused by hyperglycemia but rather specifically associated with metabolic changes in T2DM, such as elevated triglyceride levels.

Cytotoxic 1-Deoxysphingolipids Are Metabolized by a Cytochrome P450-Dependent Pathway

The 1-deoxysphingolipids (1-deoxySLs) are atypical sphingolipids (SLs) that are formed when serine palmitoyltransferase condenses palmitoyl-CoA with alanine instead of serine during SL synthesis. The 1-deoxySLs are toxic to neurons and pancreatic β-cells. Pathologically elevated 1-deoxySLs cause the inherited neuropathy, hereditary sensory autonomic neuropathy type 1 (HSAN1), and are also found in T2D. Diabetic sensory polyneuropathy (DSN) and HSAN1 are clinically very similar, suggesting that 1-deoxySLs may be implicated in both pathologies. The 1-deoxySLs are considered to be dead-end metabolites, as they lack the C1-hydroxyl group, which is essential for the canonical degradation of SLs. Here, we report a previously unknown metabolic pathway, which is capable of degrading 1-deoxySLs. Using a variety of metabolic labeling approaches and high-resolution high-accuracy MS, we identified eight 1-deoxySL downstream metabolites, which appear to be formed by cytochrome P450 (CYP)4F enzymes. Comprehensive inhibition and induction of CYP4F enzymes blocked and stimulated, respectively, the formation of the downstream metabolites. Consequently, CYP4F enzymes might be novel therapeutic targets for the treatment of HSAN1 and DSN, as well as for the prevention of T2D.

ORMDL3 expression levels have no influence on the activity of serine palmitoyltransferase

ORMDL proteins are believed to be negative regulators of serine palmitoyl transferase (SPT), which catalyzes the first and rate limiting step in sphingolipid (SL) de novo synthesis. Several single‐nucleotide polymorphisms (SNPs) that are close to the ORMDL3 locus have been reported to increase ORMDL3 expression and to be associated with an elevated risk for early childhood asthma; however, the direct effect of ORMDL3 expression on SPT activity and its link to asthma remains elusive. In this study, we investigated whether ORMDL3 expression is associated with changes in SPT activity and to tal SL levels. Orm d l3‐knockout (Ormd l3‐/‐) and transgenic (Ormd l3Tg/wt) micewere generated to study the effect of ORMDL3 on to tal SL levels inplasma and tissues. Cellular SPT activity was measured in mouse embryonic fibroblasts from Ormd l3‐/‐ mice, as well as in HEK293 cells in which ORMDL3 was overexpressed and silenced. Further more, we analyzed the association of the reported ORMDL3 asthma SNPs with plasma sphingoid bases in a population‐based cohort of 971 individuals. Total C18‐long chain bases were not significantly altered in the plasma and tissues of Ormd l3‐/‐ mice, whereas C18‐sphinganine showed a small and significant increase inplasma, lung, andliver tissues. Mouseembryonic fibroblast cells from Ormd l3‐/‐ mice didnot show an altered SPT activity compared with Ormd l3+/2 and Ormd l3+/+ mice. Overexpression or knock down of ORMDL 3 in HEK 293 cells did not alter SPT activity; however, parallel knock down of all 3 ORMDL iso forms increased enzyme activity significantly. A significant association of the annotated ORMDL3 asthma SNPs with plasma long‐chain sphingoid base levels could not be confirmed. ORMDL3 expression levels seemnot to be directly associatedwith changes in SPT activity. ORMDL3 might influence de novo sphingolipid metabolism downstream of SPT.—Zhakupova, A., Debeuf, N., Krols, M., Toussaint, W., Vanhoutte, L., Alecu, I., Kutalik, Z., Vollenweider, P., Ernst, D., von Eckardstein, A., Lambrecht, B. N., Janssens, S., Hornemann, T. ORMDL3 expression levels have no influence on the activity of serine palmitoyltransferase. FASEB J. 30, 4289–4300 (2016). www.fasebj.org

Localization of 1-deoxysphingolipids to mitochondria induces mitochondrial dysfunction

1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids that are elevated in the plasma of patients with type 2 diabetes and hereditary sensory and autonomic neuropathy type 1 (HSAN1). Clinically, diabetic neuropathy and HSAN1 are very similar, suggesting the involvement of deoxySLs in the pathology of both diseases. However, very little is known about the biology of these lipids and the underlying pathomechanism. We synthesized an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, to trace the metabolism and localization of deoxySLs. Our results indicate that the metabolism of these lipids is restricted to only some lipid species and that they are not converted to canonical sphingolipids or fatty acids. Furthermore, exogenously added alkyne-doxSA [(2S,3R)-2-aminooctadec-17-yn-3-ol] localized to mitochondria, causing mitochondrial fragmentation and dysfunction. The induced mitochondrial toxicity was also shown for natural doxSA, but not for sphinganine, and was rescued by inhibition of ceramide synthase activity. Our findings therefore indicate that mitochondrial enrichment of an N-acylated doxSA metabolite may contribute to the neurotoxicity seen in diabetic neuropathy and HSAN1. Hence, we provide a potential explanation for the characteristic vulnerability of peripheral nerves to elevated levels of deoxySLs.

Disturbed sphingolipid metabolism with elevated 1-deoxysphingolipids in glycogen storage disease type I – A link to metabolic control

BACKGROUND 1-Deoxysphingolipids (1-deoxySLs) are atypical sphingolipids. They are formed during sphingolipid de novo synthesis by the enzyme serine palmitoyltransferase, due to the alternate use of alanine over its canonical substrate serine. Pathologically elevated 1-deoxySL are involved in several neurological and metabolic disorders. The objective of this study was to investigate the role of 1-deoxySL in glycogen storage disease type I (GSDI). METHODS In this prospective, longitudinal observational study (median follow-up 1.8y), the plasma 1-deoxySL profile was analyzed in 15 adult GSDI patients (12 GSDIa, 3 GSDIb), and 31 healthy controls, along with standard parameters for monitoring GSDI. RESULTS 1-Deoxysphinganine (1-deoxySA) concentrations were elevated in GSDI compared to controls (191 ± 129 vs 35 ± 14 nmol/l, p < 0.0001). Concordant with the mechanism of 1-deoxySL synthesis, plasma alanine was higher (625 ± 182 vs 398 ± 90 μmol/l, p < 0.0001), while serine was lower in GSDI than in controls (88 ± 22 vs 110 ± 18 μmol/l. p < 0.001). Accordingly, serine, alanine and triglycerides were determinants of 1-deoxySA in the longitudinal analysis of GSDIa. 1-deoxySA concentrations correlated with the occurrence of low blood glucose (area under the curve below 4 mmol/l) in continuous glucose monitoring. The 1-deoxySL profile in GSDIb was distinct from GSDIa, with a different ratio of saturated to unsaturated 1-deoxySL. CONCLUSION In addition to the known abnormalities of lipoproteins, GSDI patients also have a disturbed sphingolipid metabolism with elevated plasma 1-deoxySL concentrations. 1-DeoxySA relates to the occurrence of low blood glucose, and may constitute a potential new biomarker for assessing metabolic control. GSDIa and Ib have distinct 1-deoxySL profiles indicating that both GSD subtypes have diverse phenotypes regarding lipid metabolism.