C2 Ceramide Research Articles

Cerebrospinal Fluid C18 Ceramide Associates with Markers of Alzheimer's Disease and Inflammation at the Pre- and Early Stages of Dementia.

Background:Understanding how dysregulation in lipid metabolism relates to the severity of Alzheimer‘s disease (AD) pathology might be critical in developing effective treatments.Objective:To identify lipid species in cerebrospinal fluid (CSF) associated with signature AD pathology and to explore their relationships with measures reflecting AD-related processes (neurodegeneration, inflammation, deficits in verbal episodic memory) among subjects at the pre- and early symptomatic stages of dementia.Methods: A total of 60 subjects that had been referred to an Icelandic memory clinic cohort were classified as having CSF AD (n = 34) or non-AD (n = 26) pathology profiles. Untargeted CSF lipidomic analysis was performed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) for the detection of mass-to-charge ratio (m/z) features. CSF proteins reflecting neurodegeneration (neurofilament light [NFL]) and inflammation (chitinase-3-like protein 1 [YKL-40], S100 calcium-binding protein B [S100B], glial fibrillary acidic protein [GFAP]) were also measured. Rey Auditory Verbal Learning (RAVLT) and Story tests were used for the assessment of verbal episodic memory.Results: Eight out of 1008 features were identified as best distinguishing between the CSF profile groups. Of those, only the annotation of the m/z feature assigned to lipid species C18 ceramide was confirmed with a high confidence. Multiple regression analyses, adjusted for age, gender, and education, demonstrated significant associations of CSF core AD markers (Aβ42: st.β= –0.36, p = 0.007; T-tau: st.β= 0.41, p = 0.005) and inflammatory marker S100B (st.β= 0.51, p = 0.001) with C18 ceramide levels.Conclusion: Higher levels of C18 ceramide associated with increased AD pathology and inflammation, suggesting its potential value as a therapeutic target.

Downregulation of ceramide synthase 1 promotes oral cancer through endoplasmic reticulum stress

C18 ceramide plays an important role in the occurrence and development of oral squamous cell carcinoma. However, the function of ceramide synthase 1, a key enzyme in C18 ceramide synthesis, in oral squamous cell carcinoma is still unclear. The aim of our study was to investigate the relationship between ceramide synthase 1 and oral cancer. In this study, we found that the expression of ceramide synthase 1 was downregulated in oral cancer tissues and cell lines. In a mouse oral squamous cell carcinoma model induced by 4-nitroquinolin-1-oxide, ceramide synthase 1 knockout was associated with the severity of oral malignant transformation. Immunohistochemical studies showed significant upregulation of PCNA, MMP2, MMP9, and BCL2 expression and downregulation of BAX expression in the pathological hyperplastic area. In addition, ceramide synthase 1 knockdown promoted cell proliferation, migration, and invasion in vitro. Overexpression of CERS1 obtained the opposite effect. Ceramide synthase 1 knockdown caused endoplasmic reticulum stress and induced the VEGFA upregulation. Activating transcription factor 4 is responsible for ceramide synthase 1 knockdown caused VEGFA transcriptional upregulation. In addition, mild endoplasmic reticulum stress caused by ceramide synthase 1 knockdown could induce cisplatin resistance. Taken together, our study suggests that ceramide synthase 1 is downregulated in oral cancer and promotes the aggressiveness of oral squamous cell carcinoma and chemotherapeutic drug resistance.

Ceramide Synthase 2 Null Mice Are Protected from Ovalbumin-Induced Asthma with Higher T Cell Receptor Signal Strength in CD4+ T Cells.

(1) Background: six mammalian ceramide synthases (CerS1–6) determine the acyl chain length of sphingolipids (SLs). Although ceramide levels are increased in murine allergic asthma models and in asthmatic patients, the precise role of SLs with specific chain lengths is still unclear. The role of CerS2, which mainly synthesizes C22–C24 ceramides, was investigated in immune responses elicited by airway inflammation using CerS2 null mice. (2) Methods: asthma was induced in wild type (WT) and CerS2 null mice with ovalbumin (OVA), and inflammatory cytokines and CD4 (cluster of differentiation 4)+ T helper (Th) cell profiles were analyzed. We also compared the functional capacity of CD4+ T cells isolated from WT and CerS2 null mice. (3) Results: CerS2 null mice exhibited milder symptoms and lower Th2 responses than WT mice after OVA exposure. CerS2 null CD4+ T cells showed impaired Th2 and increased Th17 responses with concomitant higher T cell receptor (TCR) signal strength after TCR stimulation. Notably, increased Th17 responses of CerS2 null CD4+ T cells appeared only in TCR-mediated, but not in TCR-independent, treatment. (4) Conclusions: altered Th2/Th17 immune response with higher TCR signal strength was observed in CerS2 null CD4+ T cells upon TCR stimulation. CerS2 and very-long chain SLs may be therapeutic targets for Th2-related diseases such as asthma.

CERTL reduces C16 ceramide, amyloid-\u03b2 levels, and inflammation in a model of Alzheimer\u2019s disease

BackgroundDysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer’s disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain.MethodsA plasmid expressing CERTL, the long isoform of CERTs, was used to study the interaction of CERTL with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTL protein was employed to study interaction of CERTL with amyloid-β (Aβ), Aβ aggregation process in presence of CERTL, and the resulting changes in Aβ toxicity in neuroblastoma cells. CERTL was overexpressed in neurons by adeno-associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety, and locomotion. At week 12, brains were investigated for sphingolipid levels by mass spectrometry, plaques, and neuroinflammation by immunohistochemistry, gene expression, and/or immunoassay.ResultsHere, we report that CERTL binds to APP, modifies Aβ aggregation, and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERTL in vivo over-expression has a mild effect on animal locomotion, decreases Aβ formation, and modulates microglia by decreasing their pro-inflammatory phenotype.ConclusionOur results demonstrate a crucial role of CERTL in regulating ceramide levels in the brain, in amyloid plaque formation and neuroinflammation, thereby opening research avenues for therapeutic targets of AD and other neurodegenerative diseases.

Serum Ceramides as Prognostic Biomarkers of Large Thrombus Burden in Patients with STEMI: A Micro-Computed Tomography Study.

ST-elevation myocardial infarction (STEMI) remains one of the leading causes of mortality worldwide. The identification of novel metabolic and imaging biomarkers could unveil key pathophysiological mechanisms at the molecular level and promote personalized care in patients with acute coronary syndromes. We studied 38 patients with STEMI who underwent primary percutaneous coronary intervention and thrombus aspiration. We sought to correlate serum ceramide levels with micro-CT quantified aspirated thrombus volume and relevant angiographic outcomes, including modified TIMI thrombus grade and pre- or post-procedural TIMI flow. Higher ceramide C16:0 levels were significantly but weakly correlated with larger aspirated thrombus volume (Spearman r = 0.326, p = 0.046), larger intracoronary thrombus burden (TB; p = 0.030) and worse pre- and post-procedural TIMI flow (p = 0.049 and p = 0.039, respectively). Ceramides C24:0 and C24:1 were also significantly associated with larger intracoronary TB (p = 0.008 and p = 0.001, respectively). Receiver operating characteristic analysis demonstrated that ceramides C24:0 and C24:1 could significantly predict higher intracoronary TB (area under the curve: 0.788, 95% CI: 0.629–0.946 and 0.846, 95% CI: 0.706–0.985, respectively). In conclusion, serum ceramide levels were higher among patients with larger intracoronary and aspirated TB. This suggests that quantification of serum ceramides might improve risk-stratification of patients with STEMI and facilitate an individualized approach in clinical practice.

C6 Ceramide (d18:1/6:0) as a Novel Treatment of Cutaneous T Cell Lymphoma.

Simple SummaryThere is no curative treatment for mycosis fungoides and Sézary syndrome, which are the most frequent forms of cutaneous T cell lymphoma (CTCL). Short-chain ceramides like C6 Ceramide are known to induce cell death by both apoptosis and necrosis. Here, we demonstrate that C6 Ceramide strongly reduced cell viability and induced cell death in CTCL cell lines but not in HaCaT keratinocytes and primary human keratinocytes. C6 Ceramide was rapidly metabolized by both keratinocyte cell types but not by CTCL cells. These results provide the basis for further clinical trials with topical applicated C6 Ceramide against mycosis fungoides and Sézary syndrome.Cutaneous T cell lymphomas (CTCLs) represent a heterogeneous group of T cell lymphomas that primarily affect the skin. The most frequent forms of CTCL are mycosis fungoides and Sézary syndrome. Both are characterized by frequent recurrence, developing chronic conditions and high mortality with a lack of a curative treatment. In this study, we evaluated the effect of short-chain, cell-permeable C6 Ceramide (C6Cer) on CTCL cell lines and keratinocytes. C6Cer significantly reduced cell viability of CTCL cell lines and induced cell death via apoptosis and necrosis. In contrast, primary human keratinocytes and HaCaT keratinocytes were less affected by C6Cer. Both keratinocyte cell lines showed higher expressions of ceramide catabolizing enzymes and HaCaT keratinocytes were able to metabolize C6Cer faster and more efficiently than CTCL cell lines, which might explain the observed protective effects. Along with other existing skin-directed therapies, C6Cer could be a novel well-tolerated drug for the topical treatment of CTCL.

A homologous-targeting "nanoconverter" with variable size for deep tumor penetration and immunotherapy.

Tumor-associated immunosuppression, as a key barrier, prevents immunotherapy-resistant tumors. In this study, an ingenious "nanoconverter" was designed to convert immunosuppression into immunoactivation, which was a C6-ceramide (C6)-modified tumor cytomembrane-coated polydopamine-paclitaxel system (PTX/PDA@M-C6). The co-administration of C6-ceramide and tumor cytomembrane changed an adaptive immune state to an activation state, which induced a robust antigen presentation ability of tumor-infiltrating dendritic cells to activate T1 helper cells and cytotoxic T lymphocytes. Meanwhile, C6-ceramide regulated the phenotype of macrophages via the reactive oxygen species pathway, which resulted in the conversion of M2-like macrophages by infiltration within tumors into M2-like macrophages, and therefore, M2-like macrophage-mediated immunosuppression was weakened distinctly. The "nanoconverter"-mediated conversion process upregulated the expression of related immune factors including interleukin-12, interleukin-6, tumor necrosis factor-α and interferon-γ and executed positive anti-tumor effects. In addition, under the protection of tumor-homologous cytomembrane, the "nanoconverter" exhibited excellent delivery efficiency (23.22%), and subsequently, accumulated special structural "nanoconverter" could break down into smaller nanoparticles for deep penetration into the tumor tissue under a NIR laser. Ultimately, chemo/thermal therapy-assisted immunotherapy completely eliminated the tumors of tumor-bearing mice, and a potent memory response relying on effector memory T cells still persisted to protect against tumor relapse after the end of treatment. The "nanoconverter" serves as a promising nanodrug delivery system for the conversion of immunosuppression and enhanced chemo/thermal therapy. Therefore, the highly cumulative "nanoconverter" has great potential for promoting the effect and clinical application of immunotherapy.

Inhibition of acid sphingomyelinase by ambroxol prevents SARS-CoV-2 entry into epithelial cells

The acid sphingomyelinase/ceramide system has been shown to be important for cellular infection with at least some viruses, for instance, rhinovirus or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Functional inhibition of the acid sphingomyelinase using tricyclic antidepressants prevented infection of epithelial cells, for instance with SARS-CoV-2. The structure of ambroxol, that is, trans-4-[(2,4-dibromanilin-6-yl)-methyamino]-cyclohexanol, a mucolytic drug applied by inhalation, suggests that the drug might inhibit the acid sphingomyelinase and thereby infection with SARS-CoV-2. To test this, we used vesicular stomatitis virus pseudoviral particles presenting SARS-CoV-2 spike protein on their surface (pp-VSV-SARS-CoV-2 spike), a bona fide system for mimicking SARS-CoV-2 entry into cells. Viral uptake and formation of ceramide localization were determined by fluorescence microscopy, activity of the acid sphingomyelinase by consumption of [14C]sphingomyelin and ceramide was quantified by a kinase method. We found that entry of pp-VSV-SARS-CoV-2 spike required activation of acid sphingomyelinase and release of ceramide, events that were all prevented by pretreatment with ambroxol. We also obtained nasal epithelial cells from human volunteers prior to and after inhalation of ambroxol. Inhalation of ambroxol reduced acid sphingomyelinase activity in nasal epithelial cells and prevented pp-VSV-SARS-CoV-2 spike-induced acid sphingomyelinase activation, ceramide release, and entry of pp-VSV-SARS-CoV-2 spike ex vivo. The addition of purified acid sphingomyelinase or C16 ceramide restored entry of pp-VSV-SARS-CoV-2 spike into ambroxol-treated epithelial cells. We propose that ambroxol might be suitable for clinical studies to prevent coronavirus disease 2019.

Evaluation of Serum C24 Ceramide as a Predictor of Hepatic Decompensation in Patients with Liver Cirrhosis

Background: It was found that there is a significant progressive decrease in the serum level of C24 ceramide with increasing severity of cirrhosis. Our aim was to evaluate the efficacy of C24 Ceramide (C24Cer) as a predictor of decompensation in cirrhotic patients at Suez Canal University hospital, Ismailia, Egypt. Subjects and Methods: Patients with viral related liver cirrhosis were consecutively confronted in the outpatient and inpatient sections. They were classified according to Child-Pugh into A, B, C groups. A control group (13 individuals) was compared with each group. Clinical assessment and Liver profile were performed. The serum level of C24Cer was measured using 20μL extracted serum with methanol: chloroform: HCl (15:83:2), using liquid chromatography coupled to tandem mass spectrometry. Results: Patients with Child-A cirrhosis showed a significantly higher mean C24Cer level compared to Child-C patients (p <0.001). Pairwise comparisons showed a statistically significant stepwise decrease in the mean C24Cer level from control group to Child-A, B then C cirrhotic patients respectively (p-value: 0.029, <0.001, and <0.001). There was a highly significant negative correlation between C24Cer and each of total bilirubin, PT, ascites grade, hepatic encephalopathy grade, and Child score (p < 0.001 for each) as well as a highly significant positive correlation with albumin level (p < 0.001). Conclusion: a low serum C24Cer is associated with hepatic decompensation and is good with a sensitivity of 100% and a specificity of 73%. Further studies are needed to elucidate the real-life significance of C24Cer as a non-invasive mortality predictor in cirrhosis.

Plasma Ceramides and Risk of Impaired Lower-Extremity Function in Older Adults: A Nested Case-Control Study.

Higher levels of ceramides have been linked to several chronic diseases; also there is emerging cross-sectional evidence that ceramides are associated with lower physical functioning. This research assessed for the first time the prospective relationship between ceramide species and impaired lower-extremity function (ILEF) in older adults. Case-control study with 43 cases of ILEF and 86 age- and sex-matched controls, which was nested in the Seniors-ENRICA cohort of community-dwelling older adults. Incident ILEF from 2015 to 2017 was ascertained with the Short Physical Performance Battery. In 2015, 27 ceramide species were measured in plasma by liquid chromatography-tandem mass spectrometry. Conditional logistic regression models were used to assess the longitudinal relationship between ceramides concentration and incidence of ILEF. After adjusting for education level, body mass index, alcohol and total energy intake, physical activity, and presence of chronic conditions, some ceramide species were related to 2-year incidence of ILEF. Specifically, the odds ratios of ILEF per 1-SD increase in ceramide concentration were: 1.66 [95% CI = (1.03, 2.68)] for ceramide C14:0, 1.61 (1.00, 2.59) for ceramide C16:0, and 1.64 (1.03, 2.60) for ceramide C16:1 (n-7). In the case of ceramides C16:0 and C16:1 (n-7), a stronger relationship was found in those with a higher body mass index; systolic blood pressure could also mediate the relationship between ceramide C16:1 (n-7) and ILEF (p for interaction = .03). Higher plasma levels of ceramides C14:0, C16:0, and C16:1 (n-7) are associated with higher risk of ILEF, and might serve as risk markers for functional decline in older adults.

Increased Level of Serum Ceramides Correlate with Liver Steatosis, Hba1c and Cholesterol in Obese Patients

Objective: Sphingolipids including ceramides are biological active components of all cell membranes. They play a great role in cell interconnections in the process of proliferation, maturation, cell apoptosis and any fluctuations of their level can lead to development of metabolic diseases such as type 2 diabetes (T2D) and nonalcoholic fatty liver disease. Nevertheless, there is lack of information about what type of ceramides play a role in aforementioned diseases. Here we investigated the relationship between the serum level of some type of ceramides and parameters of metabolic syndrome that is commonly present in obese patients. Design: We performed cross-sectional study in two groups. One of them was control group – lean healthy people (n=10, body mass index, BMI 21, 2±1, 89) and the second group included patients with the obesity (n=24, BMI 33, 9±3, 1). We quantified the levels of serum ceramide by liquid chromatography-mass spectrometry, analyzed the parameters for insulin resistance, liver function and lipid metabolism by biochemical blood test. Results: The subjects with obesity had elevated level of ceramide C16:0, C18:0, C24:0 comparing with control group (p&lt;0,001). As results of our study, we found correlation of the level ceramide C16:0, C18:0, C24:0 with the results of steatometry and some metabolic parameters (glycosylated hemoglobin (Hb A1C), cholesterol). Conclusion: These results demonstrate that obese subjects had increased level of ceramide C16:0, C18:0, C24:0 that correlated with some metabolic parameters supposedly recognizing them as new biomarkers of metabolic syndrome.

Caveolin-1 regulates the ASMase/ceramide-mediated radiation response of endothelial cells in the context of tumor\u2013stroma interactions

The integral membrane protein caveolin-1 (CAV1) plays a central role in radioresistance-mediating tumor–stroma interactions of advanced prostate cancer (PCa). Among the tumor–stroma, endothelial cells (EC) evolved as critical determinants of the radiation response. CAV1 deficiency in angiogenic EC was already shown to account for increased apoptosis rates of irradiated EC. This study explores the potential impact of differential CAV1 levels in EC on the acid sphingomyelinase (ASMase)/ceramide pathway as a key player in the regulation of EC apoptosis upon irradiation and cancer cell radioresistance. Enhanced apoptosis sensitivity of CAV1-deficient EC was associated with increased ASMase activity, ceramide generation, formation of large lipid platforms, and finally an altered p38 mitogen-activated protein kinase (MAPK)/heat-shock protein 27 (HSP27)/AKT (protein kinase B, PKB) signaling. CAV1-deficient EC increased the growth delay of LNCaP and PC3 PCa cells upon radiation treatment in direct 3D spheroid co-cultures. Exogenous C6 and C16 ceramide treatment in parallel increased the growth delay of PCa spheroids and induced PCa cell apoptosis. Analysis of the respective ceramide species in PCa cells with increased CAV1 levels like those typically found in radio-resistant advanced prostate tumors further revealed an upregulation of unsaturated C24:1 ceramide that might scavenge the effects of EC-derived apoptosis-inducing C16 ceramide. Higher ASMase as well as ceramide levels could be confirmed by immunohistochemistry in human advanced prostate cancer specimen bearing characteristic CAV1 tumor–stroma alterations. Conclusively, CAV1 critically regulates the generation of ceramide-dependent (re-)organization of the plasma membrane that in turn affects the radiation response of EC and adjacent PCa cells. Understanding the CAV1-dependent crosstalk between tumor cells and the host-derived tumor microvasculature and its impact on radiosensitivity may allow to define a rational strategy for overcoming tumor radiation resistance improving clinical outcomes by targeting CAV1.

Sex‐dependent Effects of High‐fat Diet and Stress on CNS Inflammation and Ceramides Production

The intake of a high‐fat diet (HFD) and exposition to stress are associated with development of a neuroinflammatory state in the central nervous system (CNS). Previously we found high levels of long‐chain (C18:0 and C20:0) ceramides in blood of rats fed an HFD that correlated with increased immobility in the forced swim test, suggesting that ceramides could contribute to the development of depressive‐like behavior. In the current study, we aimed to determine whether adding short‐term unpredictable stress (STUS) would exacerbate the effects of HFD on neuroinflammation and ceramide metabolism in the ventral hippocampus (VH) and the development of depressive‐like behavior. We hypothesized that exposure to HFD and STUS would cause a neuroinflammatory state and increased levels of long‐chained ceramides in the VH leading to depressive‐like behavior. To test this, male and female Sprague Dawley rats were administered either a low‐fat or high‐fat diet for eight weeks, while other groups of female and male rats were administered a low‐fat or high‐fat diet for 10 weeks with the inclusion of 4 days of random unpredictable stressors. After sacrifice, we assessed the expression of microglial (Iba‐1), neuroinflammatory state (NLRP3, HMGB1 and TNF‐α) and ceramide production markers (CerS5, CerS6, SMPD1 and SMPD3) in the VH. Female HFD rats showed increased expression of TNF‐α and HMGB‐1 independent of STUS, while male rats increased expression of Iba‐1, TNF‐α and NLRP3 after exposition to STUS, suggesting a sex‐dependent activation of inflammatory mediators. The assessment of enzymes that produce long‐chained ceramides revealed increased CerS5, CerS6 and SMPD1 mRNA only in the VH of female HFD rats exposed to STUS, suggesting sex differences in the production of long‐chained ceramides due to combined HFD and STUS. VH tissue and blood were collected to quantify ceramides by gas chromatography‐mass spectrometry (GC‐MS). Based on the expression of the enzymes involved in the production of ceramides, we expect that the GC‐MS assay will show increased C16 ceramides in female rats exposed to HFD and STUS. After diet completion, rats were subjected to sucrose grooming test and forced swim test to assess anhedonia and behavioral despair. Female HFD with STUS showed trending increases in grooming activity, reduced immobility and increased swimming time while male rats did not present any behavioral changes, suggesting less depressive‐like behavior in female rats. In conclusion, an HFD in combination with STUS causes sex‐dependent changes inflammatory state and long‐chained ceramides production without increasing depressive‐like behavior.Support or Funding InformationNIH‐NIGMS R25GM082406 (PHSU RISE Graduate Training Program), U54 MD007579, NIGMS/INBRE P20 GM103475, and NIH‐NIGMS R25GM096955 (UPR‐Ponce PRISE Program).

Increased Level of Serum Ceramides Correlate with Liver Steatosis, Hba1c and Cholesterol in Obese Patients

Objective: Sphingolipids including ceramides are biological active components of all cell membranes. They play a great role in cell interconnections in the process of proliferation, maturation, cell apoptosis and any fluctuations of their level can lead to development of metabolic diseases such as type 2 diabetes (T2D) and nonalcoholic fatty liver disease. Nevertheless, there is lack of information about what type of ceramides play a role in aforementioned diseases. Here we investigated the relationship between the serum level of some type of ceramides and parameters of metabolic syndrome that is commonly present in obese patients. Design: We performed cross-sectional study in two groups. One of them was control group – lean healthy people (n=10, body mass index, BMI 21, 2±1, 89) and the second group included patients with the obesity (n=24, BMI 33, 9±3, 1). We quantified the levels of serum ceramide by liquid chromatography-mass spectrometry, analyzed the parameters for insulin resistance, liver function and lipid metabolism by biochemical blood test. Results: The subjects with obesity had elevated level of ceramide C16:0, C18:0, C24:0 comparing with control group (p&lt;0,001). As results of our study, we found correlation of the level ceramide C16:0, C18:0, C24:0 with the results of steatometry and some metabolic parameters (glycosylated hemoglobin (Hb A1C), cholesterol). Conclusion: These results demonstrate that obese subjects had increased level of ceramide C16:0, C18:0, C24:0 that correlated with some metabolic parameters supposedly recognizing them as new biomarkers of metabolic syndrome.

Ceramide-Induced Cell Death Depends on Calcium and Caspase-Like Activity in Rice.

Ceramide sphingolipids are major components of membranes. C2 and C6 ceramides induce programmed cell death (PCD) in animals and plants, and we previously showed that C2 and C6 ceramides induce PCD in rice (Oryza sativa) protoplasts. However, the mechanistic link between sphingolipids and PCD in rice remains unclear. Here, we observed that calcium levels increased rapidly after ceramide treatment. Moreover, the calcium channel inhibitor LaCl3 and the intracellular calcium chelator acetoxymethyl-1, 2-bis (2-aminophenoxy) ethic acid (BAPTA-AM) inhibited this calcium increase and prevented ceramide-induced PCD. Moreover, caspase-3-like protease activity increased significantly in C6 ceramide-treated protoplasts, and a caspase-specific inhibitor prevented C6 ceramide-induced cell death. We also detected the other typical PCD events including ATP loss. DIDS (4, 49-diisothiocyanatostilbene- 2, 29-disulfonic acid), an inhibitor of voltage-dependent anion channels (VDACs), decreased C6 ceramide-induced cell death. Together, this evidence suggests that mitochondria played an important role in C6 ceramide-induced PCD.

Transcriptomics Reveal Altered Metabolic and Signaling Pathways in Podocytes Exposed to C16 Ceramide-Enriched Lipoproteins.

Sphingolipids are bioactive lipids associated with cellular membranes and plasma lipoproteins, and their synthesis and degradation are tightly regulated. We have previously determined that low plasma concentrations of certain ceramide species predict the development of nephropathy in diabetes patients with normal albumin excretion rates at baseline. Herein, we tested the hypothesis that altering the sphingolipid content of circulating lipoproteins can alter the metabolic and signaling pathways in podocytes, whose dysfunction leads to an impairment of glomerular filtration. Cultured human podocytes were treated with lipoproteins from healthy subjects enriched in vitro with C16 ceramide, or D-erythro 2-hydroxy C16 ceramide, a ceramide naturally found in skin. The RNA-Seq data demonstrated differential expression of genes regulating sphingolipid metabolism, sphingolipid signaling, and mTOR signaling pathways. A multiplex analysis of mTOR signaling pathway intermediates showed that the majority (eight) of the pathway phosphorylated proteins measured (eleven) were significantly downregulated in response to C16 ceramide-enriched HDL2 compared to HDL2 alone and hydroxy ceramide-enriched HDL2. In contrast, C16 ceramide-enriched HDL3 upregulated the phosphorylation of four intermediates in the mTOR pathway. These findings highlight a possible role for lipoprotein-associated sphingolipids in regulating metabolic and signaling pathways in podocytes and could lead to novel therapeutic targets in glomerular kidney diseases.

PKCζ mediated anti-proliferative effect of C2 ceramide on neutralization of the tumor microenvironment and melanoma regression.

Immunotherapy, which has advantages over chemotherapy due to lesser toxicity and higher specificity, is on the rise to treat cancer. Recently, pro-apoptotic glycolipid, ceramide has emerged as a key regulator in cancer immunotherapy. The present study elucidated the potential anti-melanoma efficacy of cell-permeable, exogenous C2 ceramide on cell death and amelioration of tumor microenvironment (TME). We, for the first time, demonstrated that C2 ceramide triggered apoptosis of melanoma cells by augmenting PKCζ along with pro-inflammatory cytokines and signaling factors. C2 ceramide showed a PKCζ-mediated tumor-suppressive role in melanoma without exhibiting hepatotoxicity and nephrotoxicity. Moreover, PKCζ was revealed as one of the key regulators of Akt and ceramide during C2 ceramide-mediated apoptosis. C2 ceramide was effective in repolarization of M2 macrophage phenotype and reduction of angiogenic factors such as VEGF, VEGFR1, VEGFR2, HIF1α. Interestingly, PKCζ knockdown attenuated C2 ceramide-mediated inhibition of melanoma progression. Restoration of the Th1 type TME by C2 ceramide enhanced cytotoxic T cell-mediated killing of melanoma cells. Altogether, the study unraveled that C2 ceramide-induced PKCζ was associated with favorable immune cell functioning in TME leading to melanoma regression. Thus, our findings explored a novel mechanistic insight into C2 ceramide as a promising immunotherapeutic agent in melanoma treatment.

Effect of ER stress on sphingolipid levels and apoptotic pathways in retinal pigment epithelial cells

BackgroundWe aimed to determine sphingolipid levels and examine apoptotic pathways in human retinal pigment epithelial cells (ARPE-19) undergoing endoplasmic reticulum (ER) stress. MethodsCells were treated with tunicamycin (TM) to induce ER stress and tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, was administered to decrease cytotoxicity. Cell viability was measured by MTT assay. Levels of C16–C24 sphingomyelins (SM) and C16–C24 ceramides (CERs) were determined by LC-MS/MS. Glucose-regulated protein 78-kd (GRP78) and nuclear factor kappa-b subunit 1 (NFκB1) gene expressions were evaluated by quantitative PCR analysis, while GRP 78, NF-κB p65, cleaved caspase-3 and caspase-12 protein levels were assesed by immunofluorescence. Ceramide-1-phosphate (C1P) levels were determined by immunoassay, while caspase −3 and −12 activity in cell lysates were measured via a fluorometric method. ResultsInduction of ER stress in TM treated groups were confirmed by significantly increased mRNA and protein levels of GRP78. TM significantly decreased cell viability compared to controls. Treatment with TUDCA along with TM significantly increased cell viability compared to the TM group. A significant increase was observed in C22–C24 CERs, C1P, caspase-3, caspase-12, NFκB1 mRNA and NF-κB p65 protein levels in cells treated with TM compared to controls. Administration of TUDCA lead to a partial decrease in GRP78 expression, NFκB1 mRNA, NF-κB p65 protein, C22–C24 CERs and C1P levels along with a decrease in caspase-3 and -12 activity. ConclusionsThe results of this study reveal the presence of increased long chain CERs, C1P and apoptotic markers in retinal cells undergoing ER stress.

Effect of acute and short-term dietary fat ingestion on postprandial skeletal muscle protein synthesis rates in middle-aged, overweight, and obese men.

Muscle anabolic resistance to dietary protein is associated with obesity and insulin resistance. However, the contribution of excess consumption of fat to anabolic resistance is not well studied. The aim of these studies was to test the hypothesis that acute and short-term dietary fat overload will impair the skeletal muscle protein synthetic response to dietary protein ingestion. Eight overweight/obese men [46.4 ± 1.4 yr, body mass index (BMI) 32.3 ± 5.4 kg/m2] participated in the acute feeding study, which consisted of two randomized crossover trials. On each occasion, subjects ingested an oral meal (with and without fat emulsion), 4 h before the coingestion of milk protein, intrinsically labeled with [1-13C]phenylalanine, and dextrose. Nine overweight/obese men (44.0 ± 1.7 yr, BMI 30.1 ± 1.1 kg/m2) participated in the chronic study, which consisted of a baseline, 1-wk isocaloric diet, followed by a 2-wk high-fat diet (+25% energy excess). Acutely, incorporation of dietary amino acids into the skeletal muscle was twofold higher (P < 0.05) in the lipid trial compared with control. There was no effect of prior lipid ingestion on indices of insulin sensitivity (muscle glucose uptake, pyruvate dehydrogenase complex activity, and Akt phosphorylation) in response to the protein/dextrose drink. Fat overfeeding had no effect on muscle protein synthesis or glucose disposal in response to whey protein ingestion, despite increased muscle diacylglycerol C16:0 (P = 0.06) and ceramide C16:0 (P < 0.01) levels. Neither acute nor short-term dietary fat overload has a detrimental effect on the skeletal muscle protein synthetic response to dietary protein ingestion in overweight/obese men, suggesting that dietary-induced accumulation of intramuscular lipids per se is not associated with anabolic resistance.

CCR1 Activation Promotes Neuroinflammation Through CCR1/TPR1/ERK1/2 Signaling Pathway After Intracerebral Hemorrhage in Mice.

The activation of C-C chemokine receptor type 1 (CCR1) has been shown to be pro-inflammatory in several animal models of neurological diseases. The objective of this study was to investigate the activation of CCR1 on neuroinflammation in a mouse model of intracerebral hemorrhage (ICH) and the mechanism of CCR1/tetratricopeptide repeat 1 (TPR1)/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway in CCR1-mediated neuroinflammation. Adult male CD1 mice (n= 210) were used in the study. The selective CCR1 antagonist Met-RANTES was administered intranasally at 1h after autologous blood injection. To elucidate potential mechanism, a specific ERK1/2 activator (ceramide C6) was administered prior to Met-RANTES treatment; CCR1 activator (recombinant CCL5, rCCL5) and TPR1 CRISPR were administered in naïve mouse. Neurobehavioral assessments, brain water content, immunofluorescence staining, and western blot were performed. The endogenous expressions of CCR1, CCL5, TPR1, and p-ERK1/2 were increased in the brain after ICH. CCR1 were expressed on microglia, neurons, and astrocytes. The inhibition of CCR1 with Met-RANTES improved neurologic function, decreased brain edema, and suppressed microglia/macrophage activations and neutrophil infiltration after ICH. Met-RANTES treatment decreased expressions of CCR1, TPR1, p-ERK, TNF-α, and IL-1β, which was reversed by ceramide C6. The brain CCR1 activation by rCCL5 injection in naïve mouse resulted in neurological deficits and increased expressions of CCR1, TPR1, p-ERK, TNF-α, and IL-1β. These detrimental effects of rCCL5 were reversed by TPR1 knockdown using TPR1 CRISPR. Our study demonstrated that CCR1 activation promoted neuroinflammation through CCR1/TPR1/ERK1/2 signaling pathway after ICH in mice. CCR1 inhibition with Met-RANTES attenuated neuroinflammation, thereby reducing brain edema and improving neurobehavioral functions. Targeting CCR1 activation may provide a promising therapeutic approach in the management of ICH patients.