Alaa Othman

Bile Acid Metabolites in Serum: Intraindividual Variation and Associations with Coronary Heart Disease, Metabolic Syndrome and Diabetes Mellitus

Bile acids (BAs) regulate glucose and lipid metabolism. In longitudinal and case-control-studies, we investigated the diurnal variation of serum concentrations of the 15 major BAs as well as the biosynthetic precursor 7α-hydroxy-4-cholesten-3-one (C4) and their associations, respectively, with coronary artery disease (CAD), diabetes mellitus type 2 (T2DM), and non-diabetic metabolic syndrome (MetS). In hourly taken blood samples of four healthy probands, the intraindividual 24 h variation of C4, conjugated and unconjugated BAs ranged from 42% to 72%, from 23% to 91%, and from 49% to 90%, respectively. Conjugated BA concentrations mainly increased following food intake. Serum levels of C4 and unconjugated BAs changed with daytime with maxima varying interindividually between 20h00 and 1h00 and between 3h00 and 8h00, respectively. Comparisons of data from 75 CAD patients with 75 CAD-free controls revealed no statistically significant association of CAD with BAs or C4. Comparisons of data from 50 controls free of T2DM or MetS, 50 MetS patients, and 50 T2DM patients revealed significantly increased fasting serum levels of C4 in patients with MetS and T2DM. Multiple regression analysis revealed body mass index (BMI) and plasma levels of triglycerides (TG) as independent determinants of C4 levels. Upon multivariate and principle component analyses the association of C4 with T2DM and/or MetS was not independent of or superior to the canonical MetS components. In conclusion, despite large intra- and interindividual variation, serum levels of C4,are significantly increased in patients with MetS and T2DM but confounded with BMI and TG.

Deoxysphingoid bases as plasma markers in Diabetes mellitus

BackgroundSphingoid bases are formed from the precursors L-serine and palmitoyl-CoA-a reaction which is catalyzed by the serine-palmitoyltransferase (SPT). SPT metabolizes, besides palmitoyl-CoA also other acyl-CoAs but shows also variability towards the use of other amino acid substrates. The enzyme is also able to metabolize alanine, which results in the formation of an atypical deoxy-sphingoid base (DSB). This promiscuous activity is greatly increased in the case of the sensory neuropathy HSAN1, and pathologically elevated DSB levels have been identified as the cause of this disease. Clinically, HSAN1 shows a pronounced similarity to the diabetic sensory neuropathy (DSN), which is the most common chronic complication of diabetes mellitus. Since serine and alanine metabolism is functionally linked to carbohydrate metabolism by their precursors 3-phosphoglycerate and pyruvate, we were interested to see whether the levels of certain sphingoid base metabolites are altered in patients with diabetes.ResultsIn a case-control study we compared plasma sphingoid base levels between healthy and diabetic individuals. DSB levels were higher in the diabetic group whereas C16 and C18 sphingoid bases were not significantly different. Plasma serine, but not alanine levels were lower in the diabetic group. A subsequent lipoprotein fractionation showed that the DSBs are primarily present in the LDL and VLDL fraction.ConclusionOur results suggest that DSBs are a novel category of plasma biomarkers in diabetes which reflect functional impairments of carbohydrate metabolism. Furthermore, elevated DSB levels as we see them in diabetic patients might also contribute to the progression of the diabetic sensory neuropathy, the most frequent complication of diabetes.

Deoxysphingolipids, Novel Biomarkers for Type 2 Diabetes, Are Cytotoxic for Insulin- Producing Cells

Irreversible failure of pancreatic β-cells is the main culprit in the pathophysiology of diabetes, a disease that is now a global epidemic. Recently, elevated plasma levels of deoxysphingolipids, including 1-deoxysphinganine, have been identified as a novel biomarker for the disease. In this study, we analyzed whether deoxysphingolipids directly compromise the functionality of insulin-producing Ins-1 cells and primary islets. Treatment with 1-deoxysphinganine induced dose-dependent cytotoxicity with senescent, necrotic, and apoptotic characteristics and compromised glucose-stimulated insulin secretion. In addition, 1-deoxysphinganine altered cytoskeleton dynamics, resulting in intracellular accumulation of filamentous actin and activation of the Rho family GTPase Rac1. Moreover, 1-deoxysphinganine selectively upregulated ceramide synthase 5 expression and was converted to 1-deoxy-dihydroceramides without altering normal ceramide levels. Inhibition of intracellular 1-deoxysphinganine trafficking and ceramide synthesis improved the viability of the cells, indicating that the intracellular metabolites of 1-deoxysphinganine contribute to its cytotoxicity. Analyses of signaling pathways identified Jun N-terminal kinase and p38 mitogen-activated protein kinase as antagonistic effectors of cellular senescence. The results revealed that 1-deoxysphinganine is a cytotoxic lipid for insulin-producing cells, suggesting that the increased levels of this sphingolipid observed in diabetic patients may contribute to the reduced functionality of pancreatic β-cells. Thus, targeting deoxysphingolipid synthesis may complement the currently available therapies for diabetes.

Plasmalogens of high-density lipoproteins (HDL) are associated with coronary artery disease and anti-apoptotic activity of HDL

OBJECTIVE Low high-density lipoprotein (HDL) cholesterol and loss of atheroprotective functions of HDL are associated with coronary artery disease (CAD). Here, we investigated the associations of HDL phospholipids with acute and stable CAD as well as with the anti-apoptotic activity of HDL. METHODS 49 species of phosphatidylcholines (PCs), lysophosphatidylcholines and sphingomyelins (SMs) as well as three species of sphingosine-1-phosphate (S1P) were quantified by liquid chromatography - mass spectrometry in HDL isolated from 22 healthy subjects as well as 23 and 22 patients with stable CAD and acute coronary syndrome (ACS), respectively. Native HDL and artificially reconstituted HDL (rHDL) were tested for their capacity to inhibit apoptosis of endothelial cells (ECs) induced by serum deprivation. RESULTS HDL of CAD or ACS patients differed from HDL of healthy controls by the content in nine of the 52 quantified phospholipid species as well as reduced anti-apoptotic activity. The capacity of HDL to inhibit EC apoptosis correlated significantly with five of eleven odd-chain PCs (= plasmalogens), two S1Ps, SM42:2, PC34:2, and PC32:0. An orthogonal partial least square - discriminant analysis revealed independent associations of stable CAD with HDL-associated PC34:2, PC33:3 and PC35:2 as well as anti-apoptotic activity of HDL and of ACS with HDL-associated PC33:3, PC35:2, SM42:1, PC34:2 and PC36:2. rHDL reconstituted with apoA-I, PC34:1, and PC35:2 inhibited apoptosis of ECs more effectively than rHDL containing only apoA-I and PC34:1. CONCLUSIONS The inverse association of HDL-plasmalogen levels with both stable and acute CAD may reflect direct anti-apoptotic effects of plasmologens on ECs.

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.

Viral serine palmitoyltransferase induces metabolic switch in sphingolipid biosynthesis and is required for infection of a marine alga.

Significance This work investigates the metabolic basis of the interactions between the cosmopolitan bloom-forming alga Emiliania huxleyi and its specific large DNA virus. We demonstrate some of the basic metabolic principles used by the EhV virus to “engineer” its host sphingolipid metabolism, to produce a unique suite of virus-specific lipids. These sphingolipids were essential for the virus assembly and infectivity. These results present novel insight into the chemical “arm race” at sea that coevolved around a unique metabolic pathway. Furthermore, it may provide important evolutionary and biochemical insights into sphingolipid biosynthesis and its functional role in other host–pathogen interactions (e.g. HIV, hepatitis C virus, dengue virus). Marine viruses are the most abundant biological entities in the oceans shaping community structure and nutrient cycling. The interaction between the bloom-forming alga Emiliania huxleyi and its specific large dsDNA virus (EhV) is a major factor determining the fate of carbon in the ocean, thus serving as a key host-pathogen model system. The EhV genome encodes for a set of genes involved in the de novo sphingolipid biosynthesis, not reported in any viral genome to date. We combined detailed lipidomic and biochemical analyses to characterize the functional role of this virus-encoded pathway during lytic viral infection. We identified a major metabolic shift, mediated by differential substrate specificity of virus-encoded serine palmitoyltransferase, a key enzyme of sphingolipid biosynthesis. Consequently, unique viral glycosphingolipids, composed of unusual hydroxylated C17 sphingoid bases (t17:0) were highly enriched in the infected cells, and their synthesis was found to be essential for viral assembly. These findings uncover the biochemical bases of the virus-induced metabolic rewiring of the host sphingolipid biosynthesis during the chemical “arms race” in the ocean.

Apolipoprotein M modulates erythrocyte efflux and tubular reabsorption of sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) mediates several cytoprotective functions of HDL. apoM acts as a S1P binding protein in HDL. Erythrocytes are the major source of S1P in plasma. After glomerular filtration, apoM is endocytosed in the proximal renal tubules. Human or murine HDL elicited time- and dose-dependent S1P efflux from erythrocytes. Compared with HDL of wild-type (wt) mice, S1P efflux was enhanced in the presence of HDL from apoM transgenic mice, but not diminished in the presence of HDL from apoM knockout (Apom−/−) mice. Artificially reconstituted and apoM-free HDL also effectively induced S1P efflux from erythrocytes. S1P and apoM were not measurable in the urine of wt mice. Apom−/− mice excreted significant amounts of S1P. apoM was detected in the urine of mice with defective tubular endocytosis because of knockout of the LDL receptor-related protein, chloride-proton exchanger ClC-5 (Clcn5−/−), or the cysteine transporter cystinosin. Urinary levels of S1P were significantly elevated in Clcn5−/− mice. In contrast to Apom−/− mice, these mice showed normal plasma concentrations for apoM and S1P. In conclusion, HDL facilitates S1P efflux from erythrocytes by both apoM-dependent and apoM-independent mechanisms. Moreover, apoM facilitates tubular reabsorption of S1P from the urine, however, with no impact on S1P plasma concentrations.

Plasma 1-deoxysphingolipids are predictive biomarkers for type 2 diabetes mellitus

Objective Serine palmitoyltransferase (SPT) catalyzes the condensation of serine and palmitoyl coenzyme A, the first step in the de novo sphingolipid synthesis. Apart from these canonical substrates, SPT can also metabolize alanine and other acyl coenzyme As. This forms a spectrum of atypical sphingoid bases which are altered in the context of the metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM). We investigated whether atypical sphingolipids can be used as prospective markers to predict the incidence of T2DM. Research design and methods Using liquid chromatography/mass spectrometry, we analyzed the sphingoid base profile in a prospective cohort with 339 individuals. All individuals were followed up for a period of 8 years. Results Confirming earlier results, we found 1-deoxysphingolipids (1-deoxySLs) to be significantly elevated in patients with MetS, impaired fasting glucose, and T2DM. Patients who developed T2DM during the follow-up period (n=32) showed significantly higher 1-deoxySL levels at baseline compared with those who did not develop T2DM until the end of the study (n=70). 1-Deoxysphingosine levels were independent predictors for T2DM even after adjusting for glycated hemoglobin (standardized adjusted OR=2.1, CI 95% (1.19 to 3.71); p=0.010), MetS (standardized adjusted OR=1.97, CI 95% (1.13 to 3.43); p=0.017), and other risk factors such as age, sex, BMI, and lipid-lowering drugs. Similar results were observed for the 1-deoxysphinganine levels. Conclusions Our results support a novel role for 1-deoxySL as predictive biomarkers for the development of T2DM in risk patients and warrants further larger prospective trials in lower risk cohorts.

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