Ceramide synthesis regulates biogenesis and packaging of exosomal MALAT1 from adipose derived stem cells, increases dermal fibroblast migration and mitochondrial function.

Background The function of exosomes, small extracellular vesicles (EVs) secreted from human adipose-derived stem cells (ADSC), is becoming increasingly recognized as a means of transferring the regenerative power of stem cells to injured cells in wound healing. Exosomes are rich in ceramides and long noncoding RNA (lncRNA) like metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). We identified putative ceramide responsive cis-elements (CRCE) in MALAT1. We hypothesized that CRCE respond to cellular ceramide levels to regulate EVs MALAT1 packaging. MALAT1 levels by many cells exceeds those of protein coding genes and it’s expression is equally high in exosomes. Ceramide also regulates exosome synthesis, however, the contents of exosome cargo via sphingomyelinase and ceramide synthase pathways has not been demonstrated. Methods ADSC were treated with an inhibitor of sphingomyelinase, GW4869, and stimulators of ceramide synthesis, C2- and C6-short chain ceramides, prior to collection of conditioned media (CM). EV were isolated from CM, and then used to treat human dermal fibroblast (HDF) cultures in cell migration scratch assays, and mitochondrial stress tests to evaluate oxygen consumption rates (OCR). Results Inhibition of sphingomyelinase by treatment of ADSC with GW4869 lowered levels of MALAT1 in small EVs. Stimulation of ceramide synthesis using C2- and C6- ceramides increased cellular, EVs levels of MALAT1. The functional role of EV MALAT1 was evaluated in HDF by applying EVs to HDF. Control EVs increased migration of HDF, and significantly increased ATP production, basal and maximal respiration OCR. EV from GW4869-treated ADSC inhibited cell migration and maximal respiration. However, EV from C2- and C6-treated cells, respectively, increased both functions but not significantly above control EV except for maximal respiration. EV were exosomes except when ADSC were treated with GW4869 and C6-ceramide, then they were larger and considered microvesicles. Conclusions Ceramide synthesis regulates MALAT1 EV content. Sphingomyelinase inhibition blocked MALAT1 from being secreted from ADSC EVs. Our report is consistent with those of MALAT1 increasing cell migration and mitochondrial MALAT1 altering maximal respiration in cells. Since MALAT1 is important for exosome function, it stands that increased exosomal MALAT1 should be beneficial for wound healing as shown with these assays.

Palmitic acid causes increased dihydroceramide levels when desaturase expression is directly silenced or indirectly lowered by silencing AdipoR2

Background AdipoR1 and AdipoR2 (AdipoRs) are plasma membrane proteins often considered to act as adiponectin receptors with a ceramidase activity. Additionally, the AdipoRs and their yeast and C. elegans orthologs are emerging as membrane homeostasis regulators that counter membrane rigidification by promoting fatty acid desaturation and incorporation of unsaturated fatty acids into phospholipids, thus restoring fluidity. Methods Using cultured cells, the effects of AdipoR silencing or over-expression on the levels and composition of several sphingolipid classes were examined. Results AdipoR2 silencing in the presence of exogenous palmitic acid potently causes increased levels of dihydroceramides, a ceramide precursor in the de novo ceramide synthesis pathway. Conversely, AdipoR2 over-expression caused a depletion of dihydroceramides. Conclusions The results are consistent with AdipoR2 silencing leading to increased intracellular supply of palmitic acid that in turn leads to increased dihydroceramide synthesis via the rate-limiting serine palmitoyl transferase step. In agreement with this model, inhibiting the desaturase SCD or SREBF1/2 (positive regulators of SCD) also causes a strong increase in dihydroceramide levels.

Long-Chain and Very Long-Chain Ceramides Mediate Doxorubicin-Induced Toxicity and Fibrosis

Doxorubicin (Dox) is a chemotherapeutic agent with cardiotoxicity associated with profibrotic effects. Dox increases ceramide levels with pro-inflammatory effects, cell death, and fibrosis. The purpose of our study was to identify the underlying ceramide signaling pathways. We aimed to characterize the downstream effects on cell survival, metabolism, and fibrosis. Human fibroblasts (hFSF) were treated with 0.7 µM of Dox or transgenically overexpressed ceramide synthase 2 (FLAG-CerS2). Furthermore, cells were pre-treated with MitoTempo (MT) (2 h, 20 µM) or Fumonisin B1 (FuB) (4 h, 100 µM). Protein expression was measured by Western blot or immunofluorescence (IF). Ceramide levels were determined with mass spectroscopy (MS). Visualizations were conducted using laser scanning microscopy (LSM) or electron microscopy. Mitochondrial activity was measured using seahorse analysis. Dox and CerS2 overexpression increased CerS2 protein expression. Coherently, ceramides were elevated with the highest peak for C24:0. Ceramide- induced mitochondrial ROS production was reduced with MT or FuB preincubation. Mitochondrial homeostasis was reduced and accompanied by reduced ATP production. Our data show that the increase in pro-inflammatory ceramides is an essential contributor to Dox side-effects. The accumulation of ceramides resulted in a lipotoxic shift and subsequently mitochondrial structural and functional damage, which was partially reversible following inhibition of ceramide synthesis.

Inhibition of ceramide synthesis improves the outcome in ischemia/reperfusion injury using human-induced pluripotent stem cell derived cardiomyocyte

Introduction and aim Ceramides are proven to be biologically active in apoptosis, inflammation, mitochondrial dysfunction, and as a second messenger in various signaling pathways1. However, the data linking the role of ceramides in ischemia/reperfusion injury (I/R injury) are lacking. We aimed to establish an I/R injury model using human-induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) and to evaluate ceramide levels, ceramide synthesis pathway, and outcome of CM with inhibition of ceramide synthesis during I/R injury. Methods HiPSC technology has been used to generate functional human CMs to elucidate the underlying mechanisms of the pathophysiology of the human heart. Results In our model, we observed an increase of mRNA levels of genes regulating ceramide synthesis after 6 h of ischemia followed by 16 h reperfusion, such as SPTLC1 (1.1±0.08 vs 1.0, p=0.2), CerS2 (1.6±0.3 vs 1.0, p<0.001), CerS4 (1.3±0.1 vs 1.0, p=0.02), CerS5 (1.3±0.1 vs 1.0, p=0.03), and SMPD (1.6±0.1 vs 1.0, p=0.008) compared to control. Also, both long- and very long-chain ceramide species levels measured with mass spectrometry were increased significantly after 6 h ischemia followed by 16 h reperfusion compared to control (C14:0: 1,1±0.3 pmol/million cells vs 0,3±0,2 pmol/ million cells, p=0.02 and C24:1: 26,3±7,1 pmol/ million cells vs 9,6±3,4 pmol/ million cells, p=0.02). Inhibition of ceramide synthesis with Fumonisin B1 (FB1) significantly increased the viability after 6h of ischemia followed by 16 h of reperfusion compared to CMs incubated without inhibitors (32.2%±1.5% vs 26.9%±2.6%, p=0.04). Interestingly, we identified two mechanisms with which the viability improves after incubation with ceramide inhibitor. The first mechanism observed could be the restoration of both intracellular calcium baseline (control 29±1.2, I/R 55±5.7 and I/R with FB1 35.6±2.5, p<0,001) and peak (control 45.1±5.6, I/R 94.3±5.7 and I/R with FB1 56.5±7.5, p<0,001) levels to nearly the same levels as observed in control samples. A possible cause of increased calcium oscillations after 6 h of ischemia followed by 3 h of reperfusion in the first place could be an upregulation of the RyR2 levels detected by qPCR (2.5±0.4 vs control 1.0, p=0.008). The second mechanism of improving viability in I/R injury could be a decrease of generation of reactive oxygen species (ROS) detected by MitoSOX dye after incubation with FB1 inhibitor to nearly the same levels as observed in control (control 22±5.1, I/R 33.8±5.8 and I/R with FB1 30.7±5.9, p=0,06). Conclusion We conclude that ceramides have important implications in either mediating or causing injury and their inhibition improves the outcome of I/R injury by decreasing ROS generation and improving calcium oscillations. FUNDunding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Jena University Hospital, Clinic for Internal Medicine 1Interdisciplinary Center for Clinical Research Jena

Antioxidants Supplementation Reduces Ceramide Synthesis Improving the Cardiac Insulin Transduction Pathway in a Rodent Model of Obesity

Obesity-related disruption in lipid metabolism contributes to cardiovascular dysfunction. Despite numerous studies on lipid metabolism in the left ventricle, there is no data describing the influence of n-acetylcysteine (NAC) and α-lipoic acid (ALA), as glutathione precursors, on sphingolipid metabolism, and insulin resistance (IR) occurrence. The aim of our experiment was to evaluate the influence of chronic antioxidants administration on myocardial sphingolipid state and intracellular insulin signaling as a potential therapeutic strategy for obesity-related cardiovascular IR. The experiment was conducted on male Wistar rats fed a standard rodent chow or a high-fat diet with intragastric administration of NAC or ALA for eight weeks. Cardiac and plasma sphingolipid species were assessed by high-performance liquid chromatography (HPLC). The proteins expressed from sphingolipid and insulin signaling pathways were determined by Western blot. Antioxidant supplementation markedly reduced ceramide accumulation by lowering the expression of selected proteins from the sphingolipid pathway and simultaneously increased the myocardial sphingosine-1-phosphate level. Moreover, NAC and ALA augmented the expression of GLUT4 and the phosphorylation state of Akt (Ser473) and GSK3β (Ser9), which improved the intracellular insulin transduction pathway. Based on our results, we may postulate that NAC and ALA have a beneficial influence on the cardiac ceramidose under IR conditions.

Lycoperoside H, a Tomato Seed Saponin, Improves Epidermal Dehydration by Increasing Ceramide in the Stratum Corneum and Steroidal Anti-Inflammatory Effect

Tomatoes are widely consumed, however, studies on tomato seeds are limited. In this study, we isolated 11 compounds including saponins and flavonol glycosides from tomato seeds and evaluated their effects on epidermal hydration. Among the isolated compounds, tomato seed saponins (10 µM) significantly increased the mRNA expression of proteins related to epidermal hydration, including filaggrin, involucrin, and enzymes for ceramide synthesis, by 1.32- to 1.91-fold compared with the control in HaCaT cells. Tomato seed saponins (10 µM) also decreased transepidermal water loss by 7 to 13 g/m2·h in the reconstructed human epidermal keratinization (RHEK) models. Quantitative analysis of the ceramide content in the stratum corneum (SC) revealed that lycoperoside H (1–10 µM) is a promising candidate to stimulate ceramide synthesis via the upregulation of ceramide synthase-3, glucosylceramide synthase, and β-glucocerebrosidase, which led to an increase in the total SC ceramides (approximately 1.5-fold) in concert with ceramide (NP) (approximately 2-fold) in the RHEK models. Evaluation of the anti-inflammatory and anti-allergic effects of lycoperoside H demonstrated that lycoperoside H is suggested to act as a partial agonist of the glucocorticoid receptor and exhibits anti-inflammatory effects (10 mg/kg in animal test). These findings indicate that lycoperoside H can improve epidermal dehydration and suppress inflammation by increasing SC ceramide and steroidal anti-inflammatory activity.

M6A methylation of DEGS2, a key ceramide-synthesizing enzyme, is involved in colorectal cancer progression through ceramide synthesis

N6-methyladenosine (m6A) is the most prevalent RNA epigenetic regulator in cancer. However, the understanding of m6A modification on lipid metabolism regulation in colorectal cancer (CRC) is very limited. Here, we observed that human CRCs exhibited increased m6A mRNA methylation mediated by dysregulation of m6A erasers and readers. By performing methylated RNA-immunoprecipitation sequencing (MeRIP-seq) and transcriptomic sequencing (RNA-seq), we identified DEGS2 as a downstream target of m6A dysregulation. Overexpression or knockdown of DEGS2 confirmed the role of DEGS2 in proliferation, invasion and metastasis of CRC both in vitro and in vivo. Mechanistic studies identified the specific m6A modification site within DEGS2 mRNA, and mutation of this target site was found to drastically enhance the proliferative and invasive ability of CRC cells in vitro and promote tumorigenicity in vivo. Lipidome analysis showed that lipid metabolism was dysregulated in CRC. Moreover, ceramide synthesis was suppressed due to DEGS2 upregulation mediated by m6A modification in CRC tissues. Our findings highlight that the function of DEGS2 m6A methylation in CRC and extend the understanding of the importance of RNA epigenetics in cancer biology.

Effects of serine palmitoyltransferase inhibition by myriocin in ad libitum-fed and nutrient-restricted ewes

The fungal isolate myriocin inhibits serine palmitoyltransferase and de novo ceramide synthesis in rodents; however, the effects of myriocin on ceramide concentrations and metabolism have not been previously investigated in ruminants. In our study, twelve non-lactating crossbred ewes received an intravenous bolus of myriocin (0, 0.1, 0.3, or 1.0 mg/kg/body weight [BW]; CON, LOW, MOD, or HIGH) every 48 h for 17 d. Ewes consumed a high-energy diet from d 1-14, and were nutrient-restricted (straw only) from d 15-17. Blood was collected preprandial and at 1, 6, and 12 h relative to bolus and nutrient restriction. Tissues were collected following euthanasia on d 17. Plasma was analyzed for free fatty acids (FFA), glucose, and insulin. Plasma and tissue ceramides were quantified using mass spectrometry. HIGH selectively decreased metabolizable energy intake, BW, and plasma insulin, and increased plasma FFA (Dose, P < 0.05). Myriocin linearly decreased plasma very-long-chain (VLC) ceramide and dihydroceramide (DHCer) by d 13 (Linear, P < 0.05). During nutrient restriction, fold-change in FFA was lower with increasing dose (P < 0.05). Nutrient restriction increased plasma C16:0-Cer, an effect suppressed by MOD and HIGH (Dose × Time, P < 0.05). Myriocin linearly decreased most ceramide and DHCer species in liver and omental and mesenteric adipose, VLC ceramide and DHCer in pancreas, and C18:0-Cer in skeletal muscle and subcutaneous adipose tissue (Linear, P ≤ 0.05). We conclude that the intravenous delivery of 0.3 mg of myriocin/kg of BW/48 h decreases circulating and tissue ceramide without modifying energy intake in ruminants.