Alterations In Sphingolipid Metabolism Research Articles

The relationship between sphingomyelin and ceramide levels and soft neurological signs in ADHD.

Attention deficit hyperactivity disorder (ADHD), characterized by attention deficit, hyperactivity, and impulsivity, has recently been associated with lipid metabolism. In particular, the roles of sphingomyelin, ceramide, andgalactosylceramidase in the pathophysiology of ADHD are being investigated. This study aims to explore the relationship between sphingolipid metabolism markers and soft neurological signs (SNS) in children diagnosed with ADHD who are not undergoing medication treatment. A cross-sectional analysis was conducted on 41 children and adolescents aged 7-12 years diagnosed with ADHD and 39 neurotypically developing controls. Plasma levels of ceramide, sphingomyelin, and galactosylceramidase were measuredusing Enzyme-Linked Immunosorbent Assay (ELISA). SNS were assessed using the Physical and Neurological Examination for Soft Signs (PANESS). Statistical analyses included Student's t-tests, Mann-Whitney U tests, and Multivariate Analysis ofCovariance (MANCOVA), along with logistic regression analysis. Plasma levels of ceramide and sphingomyelin in children with ADHD showed significant differences compared to the neurotypically developing control group; however, there were no significant differences in galactosylceramidase levels between the two groups. Positive correlations were found between plasma levels of ceramide and sphingomyelin and the PANESS subscales F1 (Total Gait and Station) and F3 (Total Dysrhythmia). Additionally, logistic regression analysis indicated that high ceramide levels were positively associated with ADHD. This study underscores a significant association between alterations in sphingolipid metabolism (specifically increased levels of ceramide and sphingomyelin) and the presence of SNS in children with ADHD. These findings elucidate the potential role of sphingolipid metabolism in the pathophysiology of ADHD and provide suggestions for future therapeutic research targeting sphingolipid metabolism in the treatment of ADHD.

Normal and Dysregulated Sphingolipid Metabolism: Contributions to Podocyte Injury and Beyond.

Podocyte health is vital for maintaining proper glomerular filtration in the kidney. Interdigitating foot processes from podocytes form slit diaphragms which regulate the filtration of molecules through size and charge selectivity. The abundance of lipid rafts, which are ordered membrane domains rich in cholesterol and sphingolipids, near the slit diaphragm highlights the importance of lipid metabolism in podocyte health. Emerging research shows the importance of sphingolipid metabolism to podocyte health through structural and signaling roles. Dysregulation in sphingolipid metabolism has been shown to cause podocyte injury and drive glomerular disease progression. In this review, we discuss the structure and metabolism of sphingolipids, as well as their role in proper podocyte function and how alterations in sphingolipid metabolism contributes to podocyte injury and drives glomerular disease progression.

Recurrent de novo SPTLC2 variant causes childhood-onset amyotrophic lateral sclerosis (ALS) by excess sphingolipid synthesis

BackgroundAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the upper and lower motor neurons with varying ages of onset, progression and pathomechanisms. Monogenic childhood-onset ALS, although rare, forms an...

LRRK2 exonic variants are associated with lysosomal hydrolase activities and lysosphingolipid alterations in Parkinson's disease

Last data demonstrated that exonic variants of LRRK2 (p.G2019S, p.M1646T) may affect the catalytic activity of lysosomal enzyme glucocerebrosidase (GCase) probably through the phosphorylation of Rab10 protein. We aimed to evaluate an association of LRRK2 exonic variants previously associated with alteration of phosphorylation levels for Rab10Thr73 with PD risk in Russian population and analyze an impact of p.G2019S mutation and selected LRRK2 variants on lysosomal hydrolase activities. LRRK2 variants were determined by full sequencing of LRRK2 in 508 PD patients and 470 controls from Russian population. Activity of lysosomal enzymes (glucocerebrosidase (GCase), alpha-galactosidase A (GLA), acid sphingomyelinase (ASMase) and concentrations of their corresponded substrates (hexosylsphingosine (HexSph), globotriaosylsphingosine (LysoGb3), lysosphingomyelin (LysoSM), respectively) were estimated in 211 PD patients and 179 controls by liquid chromatography with tandem mass spectrometry (LC-MS-MS) in dry blood spots. p.M1646T and p.N2081D were associated with PD (OR = 2.33, CI 95%: 1.1215 to 4.8253, p = 0.023; OR = 1.89, 95%CI: 1.0727 to 3.3313, p = 0.028, respectively) in Russian population. An increased LysoGb3 concentration was found in p.G2019S and p.N2081D LRRK2 carriers among PD patients compared to both PD patients and controls (p.G2019S: p = 0.00086, p = 0.0004, respectively; p.N2081D: p = 0.012, p = 0.0076, respectively). A decreased ASMase activity in p.G2019S LRRK2 carriers among PD patients (p = 0.014) was demonstrated as well. Our study supported possible involvement of LRRK2 dysfunction in an alteration of sphingolipid metabolism in PD.

Lipid signatures of West Nile virus infection unveil alterations of sphingolipid metabolism providing novel biomarkers

ABSTRACT West Nile virus (WNV) is a neurotropic flavivirus transmitted by the bites of infected mosquitoes. Severe forms of West Nile disease (WND) can curse with meningitis, encephalitis or acute flaccid paralysis. A better understanding of the physiopathology associated with disease progression is mandatory to find biomarkers and effective therapies. In this scenario, blood derivatives (plasma and serum) constitute the more commonly used biofluids due to its ease of collection and high value for diagnostic purposes. Therefore, the potential impact of this virus in the circulating lipidome was addressed combining the analysis of samples from experimentally infected mice and naturally WND patients. Our results unveil dynamic alterations in the lipidome that define specific metabolic fingerprints of different infection stages. Concomitant with neuroinvasion in mice, the lipid landscape was dominated by a metabolic reprograming that resulted in significant elevations of circulating sphingolipids (ceramides, dihydroceramides, and dihydrosphingomyelins), phosphatidylethanolamines and triacylglycerols. Remarkably, patients suffering from WND also displayed an elevation of ceramides, dihydroceramides, lactosylceramides, and monoacylglycerols in their sera. The dysregulation of sphingolipid metabolism by WNV may provide new therapeutic opportunities and supports the potential of certain lipids as novel peripheral biomarkers of WND progression.

The Presence of Periodontitis Exacerbates Non-Alcoholic Fatty Liver Disease via Sphingolipid Metabolism-Associated Insulin Resistance and Hepatic Inflammation in Mice with Metabolic Syndrome.

Clinical studies have shown that periodontitis is associated with non-alcoholic fatty liver disease (NAFLD). However, it remains unclear if periodontitis contributes to the progression of NAFLD. In this study, we generated a mouse model with high-fat diet (HFD)-induced metabolic syndrome (MetS) and NAFLD and oral P. gingivalis inoculation-induced periodontitis. Results showed that the presence of periodontitis increased insulin resistance and hepatic inflammation and exacerbated the progression of NAFLD. To determine the role of sphingolipid metabolism in the association between NAFLD and periodontitis, we also treated mice with imipramine, an inhibitor of acid sphingomyelinase (ASMase), and demonstrated that imipramine treatment significantly alleviated insulin resistance and hepatic inflammation, and improved NAFLD. Studies performed in vitro showed that lipopolysaccharide (LPS) and palmitic acid (PA), a major saturated fatty acid associated with MetS and NAFLD, synergistically increased the production of ceramide, a bioactive sphingolipid involved in NAFLD progression in macrophages but imipramine effectively reversed the ceramide production stimulated by LPS and PA. Taken together, this study showed for the first time that the presence of periodontitis contributed to the progression of NAFLD, likely due to alterations in sphingolipid metabolism that led to exacerbated insulin resistance and hepatic inflammation. This study also showed that targeting ASMase with imipramine improves NAFLD by reducing insulin resistance and hepatic inflammation.

Molecular profiling of the stroke-induced alterations in the cerebral microvasculature reveals promising therapeutic candidates

Stroke-induced cerebral microvascular dysfunction contributes to aggravation of neuronal injury and compromises the efficacy of current reperfusion therapies. Understanding the molecular alterations in cerebral microvessels in stroke will provide original opportunities for scientific investigation of novel therapeutic strategies. Toward this goal, using a recently optimized method which minimizes cell activation and preserves endothelial cell interactions and RNA integrity, we conducted a genome-wide transcriptomic analysis of cerebral microvessels in a mouse model of stroke and compared these transcriptomic alterations with the ones observed in human, nonfatal, brain stroke lesions. Results from these unbiased comparative analyses have revealed the common alterations in mouse stroke microvessels and human stroke lesions and identified shared molecular features associated with vascular disease (e.g., Serpine1/Plasminogen Activator Inhibitor-1, Hemoxygenase-1), endothelial activation (e.g., Angiopoietin-2), and alterations in sphingolipid metabolism and signaling (e.g., Sphigosine-1-Phosphate Receptor 2). Sphingolipid profiling of mouse cerebral microvessels validated the transcript data and revealed the enrichment of sphingomyelin and sphingoid species in the cerebral microvasculature compared to brain and the stroke-induced increase in ceramide species. In summary, our study has identified novel molecular alterations in several microvessel-enriched, translationally relevant, and druggable targets, which are potent modulators of endothelial function. Our comparative analyses have revealed the presence of molecular features associated with cerebral microvascular dysfunction in human chronic stroke lesions. The results shared here provide a detailed resource for therapeutic discovery of candidates for neurovascular protection in stroke and potentially, other pathologies exhibiting cerebral microvascular dysfunction.

Nanosecond pulsed electric field ablation-induced modulation of sphingolipid metabolism is associated with Ly6c2+ mononuclear phagocyte differentiation in liver cancer.

Preclinical studies have proven that nanosecond pulsed electric field (nsPEF) ablation can be a safe and effective treatment for humans with unresectable liver cancer that are ineligible for thermal ablation. The concomitant activation of antitumor immunity by nsPEF can also potentially prevent tumor recurrence. However, whether nsPEF exhibits similar efficacy in a clinical setting remains to be investigated. A prospective clinical trial (clinicaltrials.gov identifier: NCT04039747) was conducted to evaluate the safety and efficacy of ultrasound (US)-guided nsPEF ablation in 15 patients with unresectable liver cancer that were ineligible for thermal ablation. We found that nsPEF ablation was safe and produced a 12-month recurrence-free survival (RFS) and local RFS of 60% (9/15) and 86.7% (13/15), respectively, in the enrolled patients. Integrative proteomic and metabolomic analysis showed that sphingolipid metabolism was the most significantly enriched pathway in patient sera after nsPEF without recurrence within 8 months. A similar upregulation of sphingolipid metabolism was observed in the intratumoral mononuclear phagocytes (MNPs), rather than other immune and nonimmune cells, of an nsPEF-treated mouse model. We then demonstrated that lymphocyte antigen 6 complex, locus C2-positive (Ly6c2+ ) monocytes first differentiated into Ly6c2+ monocyte-derived macrophages with an increase in sphingolipid metabolic activity, and subsequently into Ly6c2+ dendritic cells (DCs). Ly6c2+ DCs communicated with CD8+ T cells and increased the proportions of IFN-γ+ CD8+ memory T cells after nsPEF, and this finding was subsequently confirmed by depletion of liver Ly6c2+ MNPs. In conclusion, nsPEF was a safe and effective treatment for liver cancer. The alteration of sphingolipid metabolism induced by nsPEF was associated with the differentiation of Ly6c2+ MNPs, and subsequently induced the formation of memory CD8+ T cells with potent antitumor effect.

Acid sphingomyelinase inhibition improves motor behavioral deficits and neuronal loss in an amyotrophic lateral sclerosis mouse model

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by the degeneration of motor neurons in the spinal cord. Main symptoms are manifested as weakness, muscle loss, and muscle atrophy. Some studies have reported that alterations in sphingolipid metabolism may be intimately related to neurodegenerative diseases, including ALS. Acid sphingomyelinase (ASM), a sphingolipid-metabolizing enzyme, is considered an important mediator of neurodegenerative diseases. Herein, we show that ASM activity increases in samples from patients with ALS and in a mouse model. Moreover, genetic inhibition of ASM improves motor function impairment and spinal neuronal loss in an ALS mouse model. Therefore, these results suggest the role of ASM as a potentially effective target and ASM inhibition may be a possible therapeutic approach for ALS. [BMB Reports 2022; 55(12): 621-626].

Lipid metabolism characterization in gastric cancer identifies signatures to predict prognostic and therapeutic responses.

Purpose: Increasing evidence has elucidated the significance of lipid metabolism in predicting therapeutic efficacy. Obviously, a systematic analysis of lipid metabolism characterizations of gastric cancer (GC) needs to be reported. Experimental design: Based on two proposed computational algorithms (TCGA-STAD and GSE84437), the lipid metabolism characterization of 367 GC patients and its systematic relationship with genomic characteristics, clinicopathologic features, and clinical outcomes of GC were analyzed in our study. Differentially expressed genes (DEGs) were identified based on the lipid metabolism cluster. At the same time, we applied single-factor Cox regression and random forest to screen signature genes to construct a prognostic model, namely, the lipid metabolism score (LMscore). Next, we deeply explored the predictive value of the LMscore for GC. To verify the specific changes in lipid metabolism, a total of 90 serum, 30 tumor, and non-tumor adjacent tissues from GC patients, were included for pseudotargeted metabolomics analysis via SCIEX triple quad 5500 LC-MS/MS system. Results: Five lipid metabolism signature genes were identified from a total of 3,104 DEGs. The LMscore could be a prognosticator for survival in different clinicopathological GC cohorts. As well, the LMscore was identified as a predictive biomarker for responses to immunotherapy and chemotherapeutic drugs. Additionally, significant changes in sphingolipid metabolism and sphingolipid molecules were discovered in cancer tissue from GC patients by pseudotargeted metabolomics. Conclusion: In conclusion, multivariate analysis revealed that the LMscore was an independent prognostic biomarker of patient survival and therapeutic responses in GC. Depicting a comprehensive landscape of the characteristics of lipid metabolism may help to provide insights into the pathogenesis of GC, interpret the responses of gastric tumors to therapies, and achieve a better outcome in the treatment of GC. In addition, significant alterations of sphingolipid metabolism and increased levels of sphingolipids, in particular, sphingosine (d16:1) and ceramide, were discovered in GC tissue by lipidome pseudotargeted metabolomics, and most of the sphingolipid molecules have the potential to be diagnostic biomarkers for GC.

Decreased Prosaposin and Progranulin in the Cingulate Cortex Are Associated with Schizophrenia Pathophysiology.

Prosaposin (PSAP) and progranulin (PGRN) are two lysosomal proteins that interact and modulate the metabolism of lipids, particularly sphingolipids. Alterations in sphingolipid metabolism have been found in schizophrenia. Genetic associations of PSAP and PGRN with schizophrenia have been reported. To further clarify the role of PSAP and PGRN in schizophrenia, we examined PSAP and PGRN levels in postmortem cingulate cortex tissue from healthy controls along with patients who had suffered from schizophrenia, bipolar disorder, or major depressive disorder. We found that PSAP and PGRN levels are reduced specifically in schizophrenia patients. To understand the role of PSAP in the cingulate cortex, we used an AAV strategy to knock down PSAP in neurons located in this region. Neuronal PSAP knockdown led to the downregulation of neuronal PGRN levels and behavioral abnormalities. Cingulate-PSAP-deficient mice exhibited increased anxiety-like behavior and impaired prepulse inhibition, as well as intact locomotion, working memory, and a depression-like state. The behavioral changes were accompanied by increased early growth response protein 1 (EGR-1) and activity-dependent cytoskeleton-associated protein (ARC) levels in the sensorimotor cortex and hippocampus, regions implicated in circuitry dysfunction in schizophrenia. In conclusion, PSAP and PGRN downregulation in the cingulate cortex is associated with schizophrenia pathophysiology.

Resveratrol Affects Sphingolipid Metabolism in A549 Lung Adenocarcinoma Cells.

Resveratrol is a naturally occurring polyphenol which has various beneficial effects, such as anti-inflammatory, anti-tumor, anti-aging, antioxidant, and neuroprotective effects, among others. The anti-cancer activity of resveratrol has been related to alterations in sphingolipid metabolism. We analyzed the effect of resveratrol on the enzymes responsible for accumulation of the two sphingolipids with highest functional activity—apoptosis promoting ceramide (CER) and proliferation-stimulating sphingosine-1-phosphate (S1P)—in human lung adenocarcinoma A549 cells. Resveratrol treatment induced an increase in CER and sphingosine (SPH) and a decrease in sphingomyelin (SM) and S1P. Our results showed that the most common mode of CER accumulation, through sphingomyelinase-induced hydrolysis of SM, was not responsible for a CER increase despite the reduction in SM in A549 plasma membranes. However, both the activity and the expression of CER synthase 6 were upregulated in resveratrol-treated cells, implying that CER was accumulated as a result of stimulated de novo synthesis. Furthermore, the enzyme responsible for CER hydrolysis, alkaline ceramidase, was not altered, suggesting that it was not related to changes in the CER level. The enzyme maintaining the balance between apoptosis and proliferation, sphingosine kinase 1 (SK1), was downregulated, and its expression was reduced, resulting in a decrease in S1P levels in resveratrol-treated lung adenocarcinoma cells. In addition, incubation of resveratrol-treated A549 cells with the SK1 inhibitors DMS and fingolimod additionally downregulated SK1 without affecting its expression. The present studies provide information concerning the biochemical processes underlying the influence of resveratrol on sphingolipid metabolism in A549 lung cancer cells and reveal possibilities for combined use of polyphenols with specific anti-proliferative agents that could serve as the basis for the development of complex therapeutic strategies.

Treatment with THI, an inhibitor of sphingosine-1-phosphate lyase, modulates glycosphingolipid metabolism and results therapeutically effective in experimental models of Huntington’s disease

Huntington's disease (HD) is a fatal neurodegenerative disorder with no effective cure currently available. Over the past few years our research has shown that alterations in sphingolipid metabolism represent a critical determinant in HD pathogenesis. In particular, aberrant metabolism of sphingosine-1-phosphate (S1P) has been reported in multiple disease settings, including human postmortem brains from HD patients. In this study, we investigate the potential therapeutic effect of the inhibition of S1P degradative enzyme SGPL1, by the chronic administration of the 2-acetyl-5-tetrahydroxybutyl imidazole (THI) inhibitor. We show that THI mitigated motor dysfunctions in both mouse and fly models of HD. The compound evoked the activation of pro-survival pathways, normalized levels of brain-derived neurotrophic factor, preserved white matter integrity, and stimulated synaptic functions in HD mice. Metabolically, THI restored normal levels of hexosylceramides and stimulated the autophagic and lysosomal machinery, facilitating the reduction of nuclear inclusions of both wild-type and mutant huntingtin proteins.

Temporal Alterations of Sphingolipids in Optic Nerves After Indirect Traumatic Optic Neuropathy

PurposeTo identify optic nerve (ON) lipid alterations associated with sonication-induced traumatic optic neuropathy (TON).DesignExperimental study.SubjectsA mouse model of indirect TON was generated using sound energy concentrated focally at the entrance of the optic canal using a laboratory sonifier with a microtip probe.MethodsAnalyses of datasets generated from high-performance liquid chromatography-electrospray tandem mass spectrometry of ONs dissected from the head of the ON to the optic chiasm at 1 day, 7 days, and 14 days postsonication compared with that in nonsonicated controls.Main Outcome MeasuresLipid abundance alterations in postsonicated ONs were evaluated using 1-way analysis of variance (false discovery rate-adjusted significant P value < 0.01), lipid-related gene sets, biochemical properties, and receiver operating characteristic to identify lipids associated with optic neuropathy.ResultsThere were 28 lipid species with significantly different abundances across the control and postsonication groups. The 2 most significantly upregulated lipids included a sphingomyelin (SM) species, SM(d40:7), and a hexosylceramide (CerG1) species, CerG1(d18:1/24:2). Hexosylceramide (d18:1/24:2) was noted to have a stepwise increasing trend from day 1 to day 14 after sonication-induced optic neuropathy. Investigation of biophysical properties showed notable enrichment of lipids with high and above-average transition temperatures at day 14 after sonication. Lipid-related gene set analysis revealed enrichment in sphingolipid and glycosphingolipid metabolic processes. The best classifier to differentiate day 14 postsonication from controls, based on area under the receiver operating characteristic curve, was CerG1(d18:1/24:2) (area under the receiver operating characteristic curve: 1).ConclusionsTemporal alterations in sphingolipid metabolism and biochemical properties were observed in the ON of mice after sonication-induced optic neuropathy, with notable elevations in sphingomyelin and hexosylceramide species. Hexosylceramide (d18:1/24:2) may be associated with damage after indirect trauma, indicating that lipid membrane abnormalities may be a mediator of pathology due to trauma.

Screening of sphingolipid metabolism-related genes associated with immune cells in myocardial infarction: a bioinformatics analysis.

BackgroundInflammation and immune cell infiltration in infarcted myocardial tissue are critical to myocardial infarction (MI) prognosis, and alterations in sphingolipid metabolism (SM) have been shown to potentially influence the inflammatory response and induce cardioprotection, but the underlying mechanisms are unclear. We therefore performed bioinformatics analysis to screen for key genes of SM in MI immune cells.MethodsThree matrix files including GSE61145, GSE23294, and GSE71906 were downloaded from the Gene Expression Omnibus (GEO) database. GSE61145 was a human peripheral blood database, and GSE23294 and GSE71906 were 2 mouse myocardial tissue databases. R and annotation packages were used to screen for differentially-expressed genes (DEGs). Datasets of human and mouse cardiac tissues were downloaded from the GEO database for subsequent validation. The downloaded platform and matrix files were processed using R language and annotation packages. Key targets and enrichment pathways were identified using Gene Ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The Wilcoxon test was performed on the genes involved in SM pathways in neutrophils.ResultsA total of 261 DEGs were obtained from human peripheral blood datasets, among which 101 were immune-related. GO analysis revealed that neutrophil activation, T cell activation, and T cell differentiation were significantly enriched in the immune-related DEGs. Three types of immune cells were identified in infarcted myocardial tissues. In addition, 194 DEGs were obtained from mouse myocardial tissue data, among which 6 SM-related genes (Asah1, Degs1, Neu1, Sptlc2, Sphk1, and Gba2) were significantly associated with MI. Evaluation of the relationships between these DEGs and neutrophils showed that the expression of the Sptlc2 gene was significantly upregulated in neutrophils of the MI group, while the expression levels of the Asah1 and Degs1 genes were downregulated.ConclusionsWe identified 3 SM-related genes that were highly associated with neutrophils in MI, which may advance our understanding of SM in immune cells after MI.

Ceramide/Sphingosine 1-Phosphate Axis as a Key Target for Diagnosis and Treatment in Alzheimer's Disease and Other Neurodegenerative Diseases.

Alzheimer’s disease (AD) is considered the most prevalent neurodegenerative disease and the leading cause of dementia worldwide. Sphingolipids, such as ceramide or sphingosine 1-phosphate, are bioactive molecules implicated in structural and signaling functions. Metabolic dysfunction in the highly conserved pathways to produce sphingolipids may lead to or be a consequence of an underlying disease. Recent studies on transcriptomics and sphingolipidomics have observed alterations in sphingolipid metabolism of both enzymes and metabolites involved in their synthesis in several neurodegenerative diseases, including AD. In this review, we highlight the most relevant findings related to ceramide and neurodegeneration, with a special focus on AD.

Hydroxychloroquine Causes Early Inner Retinal Toxicity and Affects Autophagosome-Lysosomal Pathway and Sphingolipid Metabolism in the Retina.

Hydroxychloroquine (HCQ) is an anti-malarial drug but also widely used to treat autoimmune diseases like arthritis and lupus. Although there have been multiple reports of the adverse effect of prolonged HCQ usage on the outer retina, leading to bull's-eye maculopathy, the effect of HCQ toxicity on the inner retina as well as on overall visual functions has not been explored in detail. Furthermore, lack of an established animal model of HCQ toxicity hinders our understanding of the underlying molecular mechanisms. Here, using a small clinical study, we confirmed the effect of HCQ toxicity on the inner retina, in particular the reduction in central inner retinal thickness, and established a mouse model of chronic HCQ toxicity that recapitulates the effects observed in human retina. Using the mouse model, we demonstrated that chronic HCQ toxicity results in loss of inner retinal neurons and retinal ganglion cells (RGC) and compromises visual functions. We further established that HCQ treatment prevents autophagosome-lysosome fusion and alters the sphingolipid homeostasis in mouse retina. Our results affirm the notion that HCQ treatment causes early damage to the inner retina and affects visual functions before leading to characteristic toxicity in the macular region of the outer retina, 'bull's-eye maculopathy.' We also provide insights into the underlying molecular mechanisms of HCQ retinal toxicity that may involve autophagy-lysosomal defects and alterations in sphingolipid metabolism.

Grade-dependent changes in sphingolipid metabolism in clear cell renal cell carcinoma.

There is a host of evidence for the role of bioactive sphingolipids in cancer biology, and dysregulated sphingolipid metabolism was observed in many malignant tumors. The aim of the present study was to provide more detailed data on sphingolipid metabolism in different stages of clear cell renal cell carcinoma (ccRCC). Samples of the tumor and noncancerous fragments of the same kidney were collected from patients who underwent a radical nephrectomy. The subjects were stratified according to the degree of malignancy of the tumor (n = 14 for G2, 12 for G3, and 9 for G4). The content of bioactive sphingolipids/glycosphingolipids was measured with an HPLC and HPTLC method, and the mRNA and protein expression of sphingolipid transporters and metabolizing enzymes was evaluated using real-time polymerase chain reaction (PCR) and Western blot, respectively. Compared to healthy kidney tissue, ccRCC was characterized by accumulation of sphingosine, sphingosine-1-phosphate (S1P), ceramide, dihydrosphingosine, and dihydroceramide. However, in the case of the latter two, the accumulation was limited to higher malignancy grades. In addition, compared to the healthy tissue, the content of gangliosides in the tumor was increased at the expense of globosides. We also found profound grade-dependent changes in the mRNA level of S1P-metabolizing enzymes, and spinster homolog 2. In general, their expression was much higher in G2 tumors compared to higher malignancy grades. We conclude that ccRCC is characterized by profound and multilevel alterations in sphingolipid metabolism, which to a large extent are grade-dependent. We hypothesize that dysregulation of sphingolipid metabolism contributes to the progression of ccRCC.

The sphingosine 1-phosphate receptor 2/4 antagonist JTE-013 elicits off-target effects on sphingolipid metabolism

Sphingosine 1-phosphate (S1P) is a signaling lipid that has broad roles, working either intracellularly through various protein targets, or extracellularly via a family of five G-protein coupled receptors. Agents that selectively and specifically target each of the S1P receptors have been sought as both biological tools and potential therapeutics. JTE-013, a small molecule antagonist of S1P receptors 2 and 4 (S1P2 and S1P4) has been widely used in defining the roles of these receptors in various biological processes. Indeed, our previous studies showed that JTE-013 had anti-acute myeloid leukaemia (AML) activity, supporting a role for S1P2 in the biology and therapeutic targeting of AML. Here we examined this further and describe lipidomic analysis of AML cells that revealed JTE-013 caused alterations in sphingolipid metabolism, increasing cellular ceramides, dihydroceramides, sphingosine and dihydrosphingosine. Further examination of the mechanisms behind these observations showed that JTE-013, at concentrations frequently used in the literature to target S1P2/4, inhibits several sphingolipid metabolic enzymes, including dihydroceramide desaturase 1 and both sphingosine kinases. Collectively, these findings demonstrate that JTE-013 can have broad off-target effects on sphingolipid metabolism and highlight that caution must be employed in interpreting the use of this reagent in defining the roles of S1P2/4.

Altered hepatic sphingolipid metabolism in insulin resistant mice: Role of advanced glycation endproducts

High plasma levels of the sphingolipid intermediates ceramide (Cer) and sphingosine-1-phosphate (S1P) are suggested to be involved in the development of insulin resistance (IR). Recent evidence indicates that advanced glycation endproducts (AGEs) can alter the sphingolipids metabolism equilibrium. Since enzymes responsible for sphingolipid rheostat maintenance are highly expressed in liver, we thus investigated whether AGEs accumulation can affect hepatic sphingolipids metabolism in insulin resistant mice.Two different models of IR were examined: genetically diabetic LeptrDb-/- (DbDb) and diet-induced insulin resistant C57Bl/6J mice fed a 60% trans-fat diet (HFD). In addition, a group of HFD mice was supplemented with the anti-AGEs compound pyridoxamine. AGEs were evaluated in the liver by western blotting. Cer and S1P were measured by UHPLC-MS/MS. The expression of RAGE and of enzymes involved in sphingolipid metabolism were assessed by RT-PCR and western blotting. HepG2 cells were used to study the effect of the major AGE Nε-(carboxymethyl)lysine (CML)-albumin on sphingolipid metabolism and the role of the receptor of AGEs (RAGE).High levels of AGEs and RAGE were detected in the liver of both DbDb and HFD mice in comparison to controls. The expression of enzymes of sphingolipid metabolism was altered in both models, accompanied by increased levels of Cer and S1P. Specifically, ceramide synthase 5 and sphingosine kinase 1 were increased, while neutral ceramidase was reduced. Pyridoxamine supplementation to HFD mice diminished hepatic AGEs and prevented alterations of sphingolipid metabolism and the development of IR. CML administration to HepG2 cells evoked alterations similar to those observed in vivo, that were in part mediated by the binding to RAGE.The present study shows a direct involvement of AGEs in alterations of sphingolipid metabolism associated to the development of IR. The modulation of sphingolipids metabolism through the prevention of AGEs accumulation by pyridoxamine may reduce the development of IR.