Search

Genetic determinants of interindividual differences in energy expenditure (EE) are largely unknown. Sphingolipids, such as ceramides, have been implicated in the regulation of human EE via mitochondrial uncoupling. In this study, we investigated whether genetic variants within enzymes involved in sphingolipid synthesis and degradation affect EE and insulin-related traits in a cohort of American Indians informative for 24-h EE and glucose disposal rates during a hyperinsulinemic-euglycemic clamp. Association analysis of 10,084 genetic variants within 28 genes involved in sphingolipid pathways identified a missense variant (rs267738, A>C, E115A) in exon 4 of CERS2 that was associated with higher sleeping EE (116 kcal/day) and increased rates of endogenous glucose production during basal (5%) and insulin-stimulated (43%) conditions, both indicators of hepatic insulin resistance. The rs267738 variant did not affect ceramide synthesis in HepG2 cells but resulted in a 30% decrease in basal mitochondrial respiration. In conclusion, we provide evidence that the CERS2 rs267738 missense variant may influence hepatic glucose production and postabsorptive sleeping metabolic rate., (© 2024 by the American Diabetes Association.)

The mechanisms underlying lipid metabolic disorders in Parkinson's diseases (PD) remain unclear. Weighted Gene Co-Expression Network Analysis (WGCNA) was conducted to identify PD-related modular genes and differentially expressed genes (DEGs). Lipid metabolism-related genes (LMRGs) were extracted from Molecular Signatures Database. Candidate genes were assessed with overlapping modular genes, DEGs, and LMRGs for the purpose of building protein-protein interaction (PPI) networks. Then, biomarkers were generated by machine learning and Backpropagation Neural Network development according to candidate genes. Biomarker-based enrichment and network modulation analyses were executed to investigate related signaling pathways. Following dimensionality reduction clustering and annotation, scRNA-seq was submitted to cellular interactions and trajectory analysis to analyze regulatory mechanisms of critical cells. Finally, qRT-PCR was conducted to confirm the expression of biomarkers in PD patients. Four biomarkers (MSMO1, ELOVL6, AACS, and CERS2) were obtained and highly predictive after analysis mentioned above. Then, OPC, Oli, and Neu cells were the primary expression sites for biomarkers according to scRNA-seq studies. Finally, we confirmed mRNA of MSMO1, ELOVL6 and AACS were downregulated in PD patients comparing with control, while CERS2 was upregulated. In conclusion, MSMO1, ELOVL6, AACS, and CERS2 related to LMRGs could be new biomarkers for diagnosing and treating PD., (© 2024. The Author(s).)

Background: Stress is implicated in various pathological conditions leading to liver injury. Existing evidence suggests that excessive stress can induce mitochondrial damage in hepatocytes, yet the underlying mechanism remains unclear. Ceramide synthase 6 (CerS6)-derived C16:0 ceramide is recognised as a lipotoxic substance capable of causing mitochondrial damage. However, the role of CerS6 in stress has received insufficient attention. This study aimed to explore the involvement of CerS6 in stress-induced hepatic damage and its associated mechanisms., Methods: The rat restraint stress model and a corticosterone (CORT)-induced hepatocyte stress model were employed for in vivo and in vitro experimental analyses, respectively. Changes in mitochondrial damage and ceramide metabolism in hepatocytes induced by stress were evaluated. The impact of CORT on mitochondrial damage and ceramide metabolism in hepatocytes was assessed following CerS6 knockdown. Mitochondria were isolated using a commercial kit, and ceramides in liver tissue and hepatocytes were detected by LC-MS/MS., Results: In comparison to the control group, rats subjected to one week of restraint exhibited elevated serum CORT levels. The liver displayed significant signs of mitochondrial damage, accompanied by increased CerS6 and mitochondrial C16:0 ceramide, along with activation of the AMPK/p38 MAPK pathway. In vitro studies demonstrated that CORT treatment of hepatocytes resulted in mitochondrial damage, concomitant with elevated CerS6 and mitochondrial C16:0 ceramide. Furthermore, CORT induced sequential phosphorylation of AMPK and p38 MAPK proteins, and inhibition of the p38 MAPK pathway using SB203580 mitigated the CORT-induced elevation in CerS6 protein. Knocking down CerS6 in hepatocytes inhibited both the increase in C16:0 ceramide and the release of mitochondrial cytochrome c induced by CORT., Conclusions: CerS6-associated C16:0 ceramide plays a mediating role in stress-induced mitochondrial damage in hepatocytes. The molecular mechanism is linked to CORT-induced activation of the AMPK/p38 MAPK pathway, leading to upregulated CerS6., (© 2024. The Author(s).)

Background: The benefits of first-line, cisplatin-based chemotherapy for muscle-invasive bladder cancer are limited due to intrinsic or acquired resistance to cisplatin. Increasing evidence has revealed the implication of cancer stem cells in the development of chemoresistance. However, the underlying molecular mechanisms remain to be elucidated. This study investigates the role of LASS2, a ceramide synthase, in regulating Wnt/β-catenin signaling in a subset of stem-like bladder cancer cells and explores strategies to sensitize bladder cancer to cisplatin treatment., Methods: Data from cohorts of our center and published datasets were used to evaluate the clinical characteristics of LASS2. Flow cytometry was used to sort and analyze bladder cancer stem cells (BCSCs). Tumor sphere formation, soft agar colony formation assay, EdU assay, apoptosis analysis, cell viability, and cisplatin sensitivity assay were used to investigate the functional roles of LASS2. Immunofluorescence, immunoblotting, coimmunoprecipitation, LC-MS, PCR array, luciferase reporter assays, pathway reporter array, chromatin immunoprecipitation, gain-of-function, and loss-of-function approaches were used to investigate the underlying mechanisms. Cell- and patient-derived xenograft models were used to investigate the effect of LASS2 overexpression and a combination of XAV939 on cisplatin sensitization and tumor growth., Results: Patients with low expression of LASS2 have a poorer response to cisplatin-based chemotherapy. Loss of LASS2 confers a stem-like phenotype and contributes to cisplatin resistance. Overexpression of LASS2 results in inhibition of self-renewal ability of BCSCs and increased their sensitivity to cisplatin. Mechanistically, LASS2 inhibits PP2A activity and dissociates PP2A from β-catenin, preventing the dephosphorylation of β-catenin and leading to the accumulation of cytosolic phospho-β-catenin, which decreases the transcription of the downstream genes ABCC2 and CD44 in BCSCs. Overexpression of LASS2 combined with a tankyrase inhibitor (XAV939) synergistically inhibits tumor growth and restores cisplatin sensitivity., Conclusions: Targeting the LASS2 and β-catenin pathways may be an effective strategy to overcome cisplatin resistance and inhibit tumor growth in bladder cancer patients., (© 2024. The Author(s).)

Sphingomyelin plays a key role in cellular cholesterol homeostasis by binding to and sequestering cholesterol in the plasma membrane. We discovered that synthesis of very long chain (VLC) sphingomyelins is inversely regulated by cellular cholesterol levels; acute cholesterol depletion elicited a rapid induction of VLC-sphingolipid synthesis, increased trafficking to the Golgi apparatus and plasma membrane, while cholesterol loading reduced VLC-sphingolipid synthesis. This sphingolipid-cholesterol metabolic axis is distinct from the sterol responsive element binding protein pathway as it requires ceramide synthase 2 (CerS2) activity, epidermal growth factor receptor signaling, and was unaffected by inhibition of protein translation. Depletion of VLC-ceramides reduced plasma membrane cholesterol content, reduced plasma membrane lipid packing, and unexpectedly resulted in the accumulation of cholesterol in the cytoplasmic leaflet of the lysosome membrane. This study establishes the existence of a cholesterol-sphingolipid regulatory axis that maintains plasma membrane lipid homeostasis via regulation of sphingomyelin synthesis and trafficking., (© 2023 Kim et al.)

The human CERS2 gene encodes a ceramide synthase enzyme, known as CERS2 (ceramide synthase 2). This protein is also known as LASS2 (LAG1 longevity assurance homolog 2) and TMSG1 (tumor metastasis-suppressor gene 1). Although previously described as a tumor suppressor for different types of cancer, such as prostate or liver cancer, it has also been observed to promote tumor growth in adenocarcinoma. In this review, we focus on the influence of CERS2 in bladder cancer (BC), approaching the existing literature about its structure and activity, as well as the miRNAs regulating its expression. From a mechanistic point of view, different explanations for the role of CERS2 as an antitumor protein have been proposed, including the production of long-chain ceramides, interaction with vacuolar ATPase, and its function as inhibitor of mitochondrial fission. In addition, we reviewed the literature specifically studying the expression of this gene in both BC and biopsy-derived tumor cell lines, complementing this with an analysis of public gene expression data and its association with disease progression. We also discuss the importance of CERS2 as a biomarker and the presence of CERS2 mRNA in extracellular vesicles isolated from urine.

Lipid metabolism disorders and mitochondrial dysfunction contribute to the progression of diabetes and chronic liver disease (CLD). Ferroptosis, as a form of cell death centered on reactive oxygen species (ROS) accumulation and lipid peroxidation, is closely related to mitochondrial dysfunction. However, whether there exists mechanistic links between these processes remains unknown. Here, to explore the molecular mechanism of diabetes complicated with CLD, we showed that high glucose could restrain the activity of antioxidant enzymes, promote mitochondrial ROS (mtROS) production, and induce a state of oxidative stress in the mitochondria of human normal liver (LO2) cells. We demonstrated that high glucose induced ferroptosis and promoted the development of CLD, which was reversed by the ferroptosis inhibitor Ferrostatin-1 (Fer-1). In addition, the mitochondria-targeting antioxidant Mito-TEMPO was used to intervene LO2 cells in high-glucose culture, and ferroptosis was found to be inhibited, whereas markers of liver injury and fibrosis improved. Furthermore, high glucose could promote ceramide synthetase 6 (CerS6) synthesis through the TLR4/IKKβ pathway. The knockout of CerS6 in LO2 cells showed that mitochondrial oxidative stress was attenuated, ferroptosis was inhibited, and markers of liver injury and fibrosis were ameliorated. In contrast, the overexpression of CerS6 in LO2 cells showed the opposite changes and these changes were inhibited by Mito-TEMPO. In short, we positioned the study of lipid metabolism to a specific enzyme CerS6, with a high degree of specificity. Our findings revealed the mechanism by which the mitochondria act as a bridge linking CerS6 and ferroptosis, confirming that under high glucose conditions, CerS6 promotes ferroptosis through mitochondrial oxidative stress, eventually leading to CLD., Competing Interests: Declaration of competing interest None., (Copyright © 2023. Published by Elsevier B.V.)

Cervical cancer (CC) is a common disease in women characterized by high recurrence rate. LncRNA ceramide synthase 6 antisense RNA 1 (CERS6-AS1) has been found to play a crucial role in the progression of breast cancer and pancreatic cancer. Nevertheless, the regulatory role of CERS6-AS1 in CC remains largely unclear. Here, we found that the expression of CERS6-AS1 was upregulated in CC tissues and cell lines compared with adjacent tissues and normal human cervical epithelial cells. CERS6-AS1 overexpression promoted proliferation and invasion, and inhibited apoptosis in CC cells, while silencing of CERS6-AS1 led to the opposite results. CERS6-AS1 was verified as a sponge of miR-6838-5p by RNA pull-down and luciferase reporter gene assays. Functional investigations revealed that CERS6-AS1 knockdown inhibited proliferation and invasion, and promoted apoptosis in CC cells, which was reversed by miR-6838-5p inhibitor. Furthermore, forkhead box P2 (FOXP2) was identified as a target for miR-6838-5p, and overexpression of miR-6838-5p decreased the expression level of FOXP2. Besides, CERS6-AS1 was able to sponge miR-6838-5p to accelerate CC cell proliferation and invasion and inhibited cell apoptosis through upregulating FOXP2 expression. In general, CERS6-AS1 was able to regulate CC cell proliferation, invasion and apoptosis by the miR-6838-5p/FOXP2 axis, which suggested that CERS6-AS1 may be a potential target for the treatment of CC., (©The Author(s) 2023. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

Background: Colon cancer (CC) belongs to a common cancer of digestive system. Long non-coding RNAs (lncRNAs) are dysregulated in numerous cancers and affect their development. The function of lncRNA CERS6 antisense RNA 1 (CERS6-AS1) in CC remains unclear., Materials and Methods: CERS6-AS1 expression in colon adenocarcinoma tissues and CC cell lines was assessed by The Cancer Genome Atlas database and quantitative real-time polymerase chain reaction analysis. The function of CERS6-AS1 in CC was analysed by 5-ethynyl-2'-deoxyuridine, colony formation, flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labelling, wound healing, Transwell and immunofluorescence assays. Mechanistic analyses including RNA pull down, RNA-binding protein immunoprecipitation and luciferase reporter assay revealed the interaction between RNAs., Results: CERS6-AS1 expression was aberrantly upregulated in colon adenocarcinoma tissues and CC cell lines. CERS6-AS1 knockdown inhibited CC cell malignant phenotypes and in vivo tumour growth. CERS6-AS1 served as the competing endogenous RNA of microRNA-16-5p in CC, and microRNA-16-5p inhibition partly rescued the effects of CERS6-AS1 depletion on CC development. Mitochondrial calcium uniporter was targeted by microRNA-16-5p. Mitochondrial calcium uniporter upregulation completely remedied the influence of CERS6-AS1 silencing in CC progression. Moreover, CERS6-AS1 enhanced the stability of mitochondrial calcium uniporter messenger RNA via recruiting RNA-binding protein embryonic lethal abnormal vision like 1., Conclusion: CERS6-AS1 promotes the development of CC via upregulating mitochondrial calcium uniporter expression., (© 2023 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.)

Objective: CERS6 antisense RNA 1 (CERS6-AS1), a long non-coding RNA (lncRNA), plays a role in the malignant progression of a variety of cancers. However, it is unclear whether it affects the malignant behavior of cervical cancer (CC) cells., Methods: CERS6-AS1 and miR-195-5p expression was estimated in CC via qRT-PCR. CCK-8, caspase-3 activity, scratch, and Transwell assays were performed to detect CC cell viability, caspase-3 activity, migration, and invasion in vitro . A tumor xenograft experiment was designed to study the growth of CC tumors in vivo . RIP and luciferase reporter experiments verified the relationship between CERS6-AS1 and miR-195-5p., Results: CERS6-AS1 overexpression and poor miR-195-5p levels were observed in CC. Inhibition of CERS6-AS1 impaired the viability, invasion, and migration of CC cells, promoted apoptosis, and suppressed tumor growth. In terms of the underlying mechanism, CERS6-AS1, as a competitive endogenous RNA (ceRNA), participated in the regulation of miR-195-5p levels in CC cells. Functionally, miR-195-5p interference attenuated the inhibitory effect of CERS6-AS1 on the malignant behaviors of CC cells., Conclusion: CERS6-AS1 acts as an oncogene in CC, in vivo and in vitro , by negatively regulating miR-195-5p., (© 2023 by the Association of Clinical Scientists, Inc.)

In a previous study by the authors, the longevity assurance homolog 2 ( LASS2 ) gene was determined to inhibit activity of vacuolar H + ‑ATPase (V‑ATPase) by combining with the C subunit (ATP6L) of V‑ATPase. However, the influence of LASS2 overexpression and silencing on apoptosis of human lung cancer cells 95D or 95C remains unclear. Thus, the effect of LASS2 on apoptosis and its potential mechanisms were investigated in 95D and 95C cells. Using the lentiviral transfection method, lentiviral vectors of LASS2 overexpression and silencing were transfected into 95D and 95C cells, respectively. The apoptotic ability of tumor cells was observed by flow cytometry. The expression levels of LASS2 , Bcl‑2, Bax, cytochrome c , caspase‑9, and caspase‑3 were detected by western blotting. CCK‑8 assay was used to detect the growth ability of tumor cells in vitro . Flow cytometric analysis revealed that LASS2 overexpression could promote the early apoptosis of lung cancer cells 95D. CCK‑8 assay demonstrated that LASS2 overexpression inhibited the proliferation of 95D cells. Additionally, LASS2 overexpression decreased the expression of Bcl‑2, induced the release of cytochrome c from mitochondria, and promoted the activation of caspase‑9 and caspase‑3. There was a significant difference in the expression of Bcl‑2, cytochrome c , caspase‑9 and caspase‑3 in the LASS2 ‑overexpression group compared with the normal and negative control groups. Alternatively, the aforementioned experiments in lung cancer cells 95C following LASS2 silencing produced the opposite effects. LASS2 may induce early apoptosis of lung cancer cells by influencing the caspase‑dependent mitochondrial pathway.

Ethnopharmacological Relevance: Danhe granule (DHG) is used by Chinese doctors to treat blood stasis, phlegm and dampness. Its lipid-lowering ability has been investigated in our previous research. However, the anti-liver inflammatory and fibrotic effects and mechanism of action of DHG in non-alcoholic steatohepatitis (NASH) have not been explored., Aim of the Study: To evaluate the ameliorative effects of DHG on liver inflammation and fibrosis in a methionine/choline-deficient (MCD) diet-induced NASH rat model, and its underlying mechanism., Materials and Methods: Sprague-Dawley rats were fed an MCD diet for two weeks and then treated with or without DHG by oral gavage for eight weeks. Their body weight and liver index were measured. The serum alanine aminotransferase (ALT) and aspartate transaminase (AST) activities as well as the liver triglyceride (TG) and free fatty acid (FFA) levels were tested using reagent kits. Inflammatory cytokines, including Tnf-α, Il-β and Il-6, and fibrosis genes, including Acta2, Col1a1, Col1a2 and Tgf-β were examined by real-time quantitative PCR (RT-qPCR). Hematoxylin-eosin (H&E), Oil Red O, Masson's and Sirius Red staining were used to observe liver changes. The plasma and liver ceramide levels were analyzed using HPLC-QQQ-MS/MS. The expression of serine palmitoyl-CoA transferase (Spt), ceramide synthase 6 (Cers6), dihydroceramide desaturase 1 (Des1), glucosylceramide synthase (Gcs), and ceramide kinase (Cerk) mRNA was assayed by RT-qPCR, while the protein expression of CerS6, DES1, GCS, CerK, and casein kinase 2α (CK2α) was tested by western blotting (WB). CerS6 degradation was evaluated using a cycloheximide (CHX) assay in vitro., Results: The liver index decreased by 20% in DHG groups and the serum ALT and AST decreased by approximately 50% and 30%, respectively in the DHG-H group. The liver Oil Red O staining, TG, and FFA changes showed that DHG reduced hepatic lipid accumulation by approximately 30% in NASH rats. H&E, Masson's and Sirius Red staining and the mRNA levels of Tnf-α, Il-β, Il-6, Acta2, Col1a1, Col1a2 and Tgf-β revealed that DHG alleviated liver inflammation and fibrosis in NASH rats. The ceramide (Cer 16:0), and hexosylceramide (HexCer 16:0, HexCer 18:0, HexCer 22:0, HexCer 24:0 and HexCer 24:1) levels decreased by approximately 17-56% in the plasma of the DHG-M and H rats. The Cer 16:0 content in the liver decreased by 20%, 50%, and 70% with the DHG-L, M, and H treatments; additionally, the dhCer 16:0, Cer 18:0, HexCer 18:0, HexCer 20:0 Cer 22:0-1P, Cer 24:0-1p, Cer 24:1-1p, and Cer 26:1-1p levels decreased in the DHG groups. The mRNA and protein expression levels of DES1, GCS, Cerk, CerS6, and CHX assay indicated that DHG decreased the mRNA and protein expression levels of CerK and reduced CerS6 protein expression by promoting its degradation. Additionally, DHG attenuated the protein expression of CK2α which could increase CerS6 enzymatic activity by phosphorylating its C-terminal region., Conclusion: DHG ameliorated the levels of liver FFA and TG and inflammation and fibrosis in MCD-induced rats, which were associated with decreasing ceramide species in the plasma and liver by reducing the expression levels of CerS6 and CerK., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The important membrane lipid, ceramide, is generated by a family of homologous enzymes, the ceramide synthases (CerSs), multi-spanning membrane proteins located in the endoplasmic reticulum. Six CerS isoforms exist in mammals with each using a subset of acyl-CoAs for (dihydro)ceramide synthesis. A number of mice have been generated in which one or other CerS has been genetically manipulated, including complete knock-outs, with each displaying phenotypes concomitant with the expression levels of the CerS in question and the presumed biological function of the ceramide species that it generates. We recently described a short C-terminal motif in the CerS which is involved in CerS dimer formation; deleting this motif had no effect on the ability of the CerS to synthesize ceramide in vitro. In the current study, we generated a CerS6 mouse using CRISPR-Cas9, in which the DDRSDIE motif was replaced by ADAAAIA. While levels of CerS6ADAAAIA expression were unaffected in the CerS6ADAAAIA mouse, and CerS6ADAAAIA was able to generate C16-ceramide in vitro, ceramide levels were significantly reduced in the CerS6ADAAAIA mouse, suggesting that replacing this motif affects an as-yet unknown mechanism of regulation of ceramide synthesis via the DDRSDIE motif in vivo. Crossing CerS6ADAAAIA mice with CerS5 null mice led to generation of viable mice in which C16-ceramide levels were reduced by up to 90%, suggesting that depletion of C16-ceramide levels is compensated for by other ceramide species with different acyl chain lengths., Competing Interests: The authors declare that no competing interests exist.

Pancreatic cancer (PC) is a lethal malignancy that threatens human health. Long noncoding RNAs (lncRNAs) act as important mediators in PC development. Our study aimed to investigate the function and mechanism of lncRNA ceramide synthase 6 antisense RNA 1 (CERS6-AS1) in PC. As shown by RT-qPCR, CERS6-AS1 was significantly upregulated in PC cells and tissues. Silencing CERS6-AS1 suppressed PC cell viability and proliferation while enhancing cell apoptosis according to colony formation assays, EdU assays, and flow cytometry analyses. Mechanistically, CERS6-AS1 interacted with miR-195-5p to elevate the expression level of the WD repeat domain phosphoinositide interacting 2 (WIPI2), which is a downstream target gene of miR-195-5p in PC. Moreover, miR-195-5p expression was negatively associated with CERS6-AS1 expression (or WIPI2 expression) in PC tissues. Rescue assays revealed that WIPI2 overexpression rescued the effects of CERS6-AS1 deficiency on cell viability, proliferation, and apoptosis. In summary, CERS6-AS1 facilitates PC cell proliferation while inhibiting PC cell apoptosis by upregulating WIPI2 via miR-195-5p. This study might provide promising insight into the role of CERS6-AS1 in PC development., (© 2022 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia, Ltd on behalf of Kaohsiung Medical University.)

Objectives: Patients with hepatocellular carcinoma (HCC) have poor prognosis due to lack of early diagnosis and effective treatment. Therefore, there is an urgent need to better understand the molecular mechanisms associated with HCC and to identify effective targets for early diagnosis and treatment. This study is to explore the expression and biological role of ceramide synthase 3 (CerS3) in HCC., Methods: A total of 159 pairs of HCC tissues and adjacent non-tumor tissues were obtained from the patients underwent radical resection in Shenzhen People's Hospital, and the total RNA and proteins from HCC tissues and adjacent non-tumor tissues were obtained. The expression of CerS3 protein and mRNA in HCC was detected by immunohistochemistry, Western blotting and real-time PCR. In vitro experiments, Hep3B cells were divided into a control vector group and a CerS3 vector group, and the cells were transfected with retroviral vector containing control cDNA or CerS3 cDNA, respectively. HCCLM3 cells were divided into a normal control shRNA group and a CerS3 shRNA group, and the cells were transfected with lentiviral vectors containing normal control shRNA or CerS3 shRNA, respectively. MTT, EdU, Transwell and scratch method were used to detect cell proliferation, migration and invasion. RNA sequencing was performed to determine the downstream signal of CerS3., Results: Compared with the corresponding adjacent tissues,the mRNA and protein levels of CerS3 were elevated in the HCC tissues, with significant difference (both P <0.05). The Univariate and multivariate analysis showed that the overall survival rate was significantly correlated with the presence of venous invasion (95% CI 1.8-9.2, P <0.01), TNM stage (95% CI 2.3-5.2, P <0.05), poor histological grade (95% CI 1.4-6.8, P <0.05), and CerS3 (95% CI 1.5-3.9, P <0.05). Furthermore, the high CerS3 expression levels in tumor tissues were significantly associated with shorter overall survival rates compared with the low CerS3 expression ( P <0.05). Compared with the vector control group, the Hep3B cell viability, EdU positive cells, and migration and invasion cell numbers in the CerS3 vector group were significantly increased (all P <0.05). Compared with the shRNA normal control group, the HCCLM3 cell viability, EdU positive cells, and numbers of migrating and invasive cells in the CerS3 shRNA group were significantly lower (all P <0.05). The RNA sequencing confirmed that the small mothers against decapentaplegic family member 6 ( SMAD6 ) gene as an oncogenic gene could promote the HCC metastasis., Conclusions: Clinically, the overexpression of CerS3 is closely related to poor clinical features and poor prognosis. Functionally, CerS3 participates in the proliferation, invasion and metastasis of liver cancer cells via activating SMAD6 gene.

Accumulating evidence indicates that long noncoding RNAs (lncRNAs) act as tumor promoters or suppressors in various types of cancer. Previous investigations suggest that ceramide synthase 6 (CERS6) antisense RNA 1 (CERS6-AS1) acts as an oncogene in breast cancer; however, its role in colorectal cancer is unknown. This study aimed to explore the molecular mechanism of CERS6-AS1 in colorectal cancer. Gene expression in colorectal cancer was examined using reverse transcription-quantitative polymerase chain reaction and western blot analyses. The viability and proliferation of colorectal cancer cells were measured by Cell Counting Kit-8 assays and colony formation assays. The migratory and invasive capacities of the colorectal cancer cells were assessed by Transwell assay. Cell stemness was examined by sphere-formation assay. Mechanistically, RNA pull-down assays, RNA immunoprecipitation assays, and luciferase reporter assays were performed to explore the relationship among CERS6-AS1, miR-15b-5p and spectrin beta, non-erythrocytic 2 (SPTBN2). Moreover, a xenograft tumor model was established to investigate the role of CERS6-AS1 in vivo. We found that CERS6-AS1 and SPTBN2 were highly expressed in colorectal cancer tissues and cells. CERS6-AS1 depletion inhibited cell viability, proliferation, migration, and invasion; the epithelial-mesenchymal transition process and stemness. It suppressed xenograft tumor growth in colorectal cancer. Moreover, SPTBN2 levels were positively regulated by CERS6-AS1 and negatively regulated by miR-15b-5p in colorectal cancer cells. Rescue assays revealed that SPTBN2 reversed the inhibitory effect of CERS6-AS1 deficiency on the malignant behaviors of colorectal cancer cells. Overall, the lncRNA CERS6-AS1 facilitates malignant phenotypes of colorectal cancer cells by targeting miR-15b-5p to upregulate SPTBN2., (© 2022 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia, Ltd on behalf of Kaohsiung Medical University.)

Dihydroceramide is a lipid molecule generated via the action of (dihydro)ceramide synthases (CerSs), which use two substrates, namely sphinganine and fatty acyl-CoAs. Sphinganine is generated via the sequential activity of two integral membrane proteins located in the endoplasmic reticulum. Less is known about the source of the fatty acyl-CoAs, although a number of cytosolic proteins in the pathways of acyl-CoA generation modulate ceramide synthesis via direct or indirect interaction with the CerSs. In this study, we demonstrate, by proteomic analysis of immunoprecipitated proteins, that fatty acid transporter protein 2 (FATP2) (also known as very long-chain acyl-CoA synthetase) directly interacts with CerS2 in mouse liver. Studies in cultured cells demonstrated that other members of the FATP family can also interact with CerS2, with the interaction dependent on both proteins being catalytically active. In addition, transfection of cells with FATP1, FATP2, or FATP4 increased ceramide levels although only FATP2 and 4 increased dihydroceramide levels, consistent with their known intracellular locations. Finally, we show that lipofermata, an FATP2 inhibitor which is believed to directly impact tumor cell growth via modulation of FATP2, decreased de novo dihydroceramide synthesis, suggesting that some of the proposed therapeutic effects of lipofermata may be mediated via (dihydro)ceramide rather than directly via acyl-CoA generation. In summary, our study reinforces the idea that manipulating the pathway of fatty acyl-CoA generation will impact a wide variety of down-stream lipids, not least the sphingolipids, which utilize two acyl-CoA moieties in the initial steps of their synthesis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Ceramide is a lipid moiety synthesized via the enzymatic activity of ceramide synthases (CerSs), six of which have been identified in mammalian cells, and each of which uses a unique subset of acyl-CoAs for ceramide synthesis. The CerSs are part of a larger gene family, the Tram-Lag-CLN8 domain family. Here, we identify a unique, C-terminal motif, the DxRSDxE motif, which is only found in CerSs and not in other Tram-Lag-CLN8 family members. Deletion of this motif in either CerS2 or in CerS6 did not affect the ability of either enzyme to generate ceramide using both an in vitro assay and metabolic labeling, but deletion of this motif did affect the activity of CerS2 when coexpressed with CerS6. Surprisingly, transfection of cells with either CerS2 or CerS6 lacking the motif did not result in changes in cellular ceramide levels. We found that CerS2 and CerS6 interact with each other, as shown by immunoprecipitation, but deletion of the DxRSDxE motif impeded this interaction. Moreover, proteomics analysis of cells transfected with CerS6 Δ338-344 indicated that deletion of the C-terminal motif impacted cellular protein expression, and in particular, the levels of ORMDL1, a negative regulator of sphingolipid synthesis. We suggest that this novel C-terminal motif regulates CerS dimer formation and thereby impacts ceramide synthesis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Oncogenic multidrug resistance is commonly intrinsic to renal cancer based on the physiological expression of detoxification transporters, particularly ABCB1, thus hampering chemotherapy. ABCB1 activity is directly dependent on its lipid microenvironment, localizing to cholesterol- and sphingomyelin (SM)-rich domains. As ceramides are the sole source for SMs, we hypothesized that ceramide synthase (CerS)-derived ceramides regulate ABCB1 activity. Using data from RNA-Seq databases, we found that patient kidney tumors exhibited increased CerS2 mRNA, which was inversely correlated with CerS6 mRNA in ABCB1 + clear cell carcinomas. Endogenous elevated CerS2 and lower CerS5/6 mRNA and protein resulted in disproportionately higher CerS2 to CerS5/6 activities (approximately twofold) in chemoresistant ABCB1 high (A498, Caki-1) compared with chemosensitive ABCB1 low (ACHN, normal human proximal convoluted tubule cell) cells. In addition, lipidomics analyses by HPLC-MS/MS showed bias toward CerS2-associated C20:0/C20:1-ceramides compared with CerS5/6-associated C14:0/C16:0-ceramides (2:1). SMs were similarly altered. We demonstrated that chemoresistance to doxorubicin in ABCB1 high cells was partially reversed by inhibitors of de novo ceramide synthesis (l-cycloserine) and CerS (fumonisin B 1 ) in cell viability assays. Downregulation of CerS2/6, but not CerS5, attenuated ABCB1 mRNA, protein, plasma membrane localization, rhodamine 123 + efflux transport activity, and doxorubicin resistance. Similar findings were observed with catalytically inactive CerS6-H212A. Furthermore, CerS6-targeting siRNA shifted ceramide and SM composition to ultra long-chain species (C22-C26). Inhibitors of endoplasmic reticulum-associated degradation (eeyarestatin I) and the proteasome (MG132, bortezomib) prevented ABCB1 loss induced by CerS2/6 downregulation. We conclude that a critical balance in ceramide/SM species is prerequisite to ABCB1 expression and functionalization, which could be targeted to reverse multidrug resistance in renal cancers., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Skeletal muscle is perceived as a major tissue in glucose and lipid metabolism. High fat diet (HFD) lead to the accumulation of intramuscular lipids, including: long chain acyl-CoA, diacylglycerols, and ceramides. Ceramides are considered to be one of the most important lipid groups in the generation of skeletal muscle insulin resistance. So far, it has not been clearly established whether all ceramides adversely affect the functioning of the insulin pathway, or whether there are certain ceramide species that play a pivotal role in the induction of insulin resistance. Therefore, we designed a study in which the expression of CerS1 and CerS5 genes responsible for the synthesis of C18:0-Cer and C16:0-Cer, respectively, was locally silenced in the gastrocnemius muscle of HFD-fed mice through in vivo electroporation-mediated shRNA plasmids. Our study indicates that HFD feeding induced both, the systemic and skeletal muscle insulin resistance, which was accompanied by an increase in the intramuscular lipid levels, decreased activation of the insulin pathway and, consequently, a decrease in the skeletal muscle glucose uptake. CerS1 silencing leads to a reduction in C18:0-Cer content, with a subsequent increase in the activity of the insulin pathway, and an improvement in skeletal muscle glucose uptake. Such effects were not visible in case of CerS5 silencing, which indicates that the accumulation of C18:0-Cer plays a decisive role in the induction of skeletal muscle insulin resistance.

Cell division produces two viable cells of a defined size. Thus, all cells require mechanisms to measure growth and trigger cell division when sufficient growth has occurred. Previous data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals in budding yeast. However, the conservation of mechanisms that govern growth control is poorly understood. In fission yeast, ceramide synthase is encoded by two genes, Lac1 and Lag1. Here, we characterize them by using a combination of genetics, microscopy, and lipid analysis. We showed that Lac1 and Lag1 co-immunoprecipitate and co-localize at the endoplasmic reticulum. However, each protein generates different species of ceramides and complex sphingolipids. We further discovered that Lac1, but not Lag1, is specifically required for proper control of cell growth and size in Schizosaccharomyces pombe . We propose that specific ceramide and sphingolipid species produced by Lac1 are required for normal control of cell growth and size in fission yeast.

Multiple noncoding natural antisense transcripts (ncNAT) are known to modulate key biological events such as cell growth or differentiation. However, the actual impact of ncNATs on cancer progression remains largely unknown. In this study, we identified a complete list of differentially expressed ncNATs in hepatocellular carcinoma. Among them, a previously undescribed ncNAT HNF4A-AS1L suppressed cancer cell growth by regulating its sense gene HNF4A , a well-known cancer driver, through a promoter-specific mechanism. HNF4A-AS1L selectively activated the HNF4A P1 promoter via HNF1A, which upregulated expression of tumor suppressor P1-driven isoforms, while having no effect on the oncogenic P2 promoter. RNA-seq data from 23 tissue and cancer types identified approximately 100 ncNATs whose expression correlated specifically with the activity of one promoter of their associated sense gene. Silencing of two of these ncNATs ENSG00000259357 and ENSG00000255031 (antisense to CERS2 and CHKA , respectively) altered the promoter usage of CERS2 and CHKA . Altogether, these results demonstrate that promoter-specific regulation is a mechanism used by ncNATs for context-specific control of alternative isoform expression of their counterpart sense genes. SIGNIFICANCE: This study characterizes a previously unexplored role of ncNATs in regulation of isoform expression of associated sense genes, highlighting a mechanism of alternative promoter usage in cancer., (©2021 The Authors; Published by the American Association for Cancer Research.)

Folic acid-fortified foods and multi-vitamin supplements containing folic acid (FA) are widely used around the world, but the exact mechanisms/metabolic effects of FA are not precisely identified. We have demonstrated that Ceramide Synthase 6 (CerS6) and C 16:0 -ceramide mediate response to folate stress in cultured cells. Here we investigated the dietary FA effects on mouse liver metabolome, with a specific focus on sphingolipids, CerS6 and C 16:0 -ceramide. Wild-type and CerS6 -/- mice were fed FA-deficient, control, or FA over-supplemented diets for 4 weeks. After dietary treatment, liver concentrations of ceramides, sphingomyelins and hexosylceramides were measured by LC-MS/MS and complemented by untargeted metabolomic characterization of mouse livers. Our study shows that alterations in dietary FA elicit multiple sphingolipid responses mediated by CerS6 in mouse livers. Folic acid-deficient diet elevated C 14:0 -, C 18:0 - and C 20:0 - but not C 16:0 -ceramide in WT male and female mice. Additionally, FA over-supplementation increased multiple sphingomyelin species, including total sphingomyelins, in both sexes. Of note, concentrations of C 14:0 - and C 16:0 -ceramides and hexosylceramides were significantly higher in female livers than in male. The latter were increased by FD diet, with no difference between sexes in total pools of these sphingolipid classes. Untargeted liver metabolomic analysis concurred with the targeted measurements and showed broad effects of dietary FA and CerS6 status on multiple lipid classes including sex-specific effects on phosphatidylethanolamines and diacylglycerols. Our study demonstrates that both dietary FA and CerS6 status exhibit pleiotropic and sex-dependent effects on liver metabolism, including hepatic sphingolipids, diacylglycerols, long chain fatty acids, and phospholipids., Competing Interests: Declaration of competing interests The authors declare that there are no conflicts of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

Background: Ceramide plays an important role in the occurrence and development of tumor. The synthesis of ceramide needs the participation of LASS. Current studies have shown that different LASS family members play different functions in tumors, especially LASS6, has been proved to play a key role in breast cancer, gastric cancer, melanoma and so on, but the research on ovarian cancer is very limited., Methods: Bioinformatics web resources, including Oncomine, UALCAN, Kaplan-Meier Plotter and TIMER were used to analyze the expression profile, prognostic value and immune infiltration of LASS6. The related genes of LASS6 in ovarian cancer were mined by Regulome Explorer and LinkedOmics database, and cluster analysis was done by DAVID. The PPI network involving LASS6 was constructed by STRING database. Finally, the correlation between 10 genes and LASS6 was analyzed by GEPIA database, and their prognostic value in ovarian cancer was analyzed by Kaplan-Meier plotter., Results: The expression of LASS6 was up-regulated in ovarian cancer, which was related to the progression and poor prognosis of ovarian cancer. Through GO/KEGG cluster analysis, we also found that LASS6 may affect calcium ion channel and its transport pathways. The analysis of regulatory network involved in LASS6 showed that the high mRNAs of 7 key genes were associated with poor prognosis of OS in patients with ovarian cancer, among which DEGS1 was the most significant., Conclusions: LASS6 may play an important role in the regulation of calcium pathway and become a new therapeutic target and potential prognostic marker in ovarian cancer., (© 2021. The Author(s).)

Dietary sphingolipids such as glucosylceramide and sphingomyelin are known to improve the skin barrier function of damaged skin. In this study, we focused on free-ceramide prepared from soy sauce lees, which is a byproduct of soy sauce production. The effects of dietary soy sauce lees ceramide on the skin of normal mice were evaluated and compared with those of dietary maize glucosylceramide. We found that transepidermal water loss value was significantly suppressed by dietary supplementation with soy sauce lees ceramide as effectively as or more effectively than maize glucosylceramide. Although the content of total and each subclass of ceramide in the epidermis was not significantly altered by dietary sphingolipids, that of 12 types of ceramide molecules, which were not present in dietary sources, was significantly increased upon ingestion of maize glucosylceramide and showed a tendency to increase with soy sauce lees ceramide intake. In addition, the mRNA expression of ceramide synthase 4 and involucrin in the skin was downregulated by sphingolipids. This study, for the first time, demonstrated that dietary soy sauce lees ceramide enhances skin barrier function in normal hairless mice, although further studies are needed to clarify the molecular mechanism.

Background: Abnormal expression of miR 20a is reported in various types of malignancy neoplasms. However, its function is not consistent in different tumors. This study aims to explore the potential functions of miR 20a and its underlying mechanisms in bladder cancer., Methods: Ninety-six patients diagnosed with bladder cancer were recruited into the study. The expression levels of miR-20a in bladder cancer samples and adjacent non-tumor samples were investigated by qRT-PCR. Wound healing, CCK8, and transwell migration assays were carried out for determining the functions of miR20a. Bioinformatics analysis was used for predicting the downstream gene of miR-20a. Western blot, qRT-PCR, and fluorescent reporter assays were used to verify the target gene., Results: MiR-20a was significantly increased in bladder cancer tissues, and its rising level was closely correlated with histological grade, clinical stage, recurrence and metastasis in bladder cancer. Exogenous upregulation of miR-20a expression obviously enhanced the aggressive biological functions of bladder cancer in vitro. LASS2 was verified to be a target gene of miR-20a. Moreover, miR-20a can negatively regulate LASS2 at protein and mRNA levels., Conclusions: Increasing miR-20a is closely related to aggressive clinicopathological features. MiR 20a plays an oncogenic role in bladder cancer, which contributes to target LASS2 directly.

Ceramide synthase 6 (CERS6) promotes lung cancer metastasis by stimulating cancer cell migration. To examine the underlying mechanisms, we performed luciferase analysis of the CERS6 promoter region and identified the Y-box as a cis-acting element. As a parallel analysis of database records for 149 non-small-cell lung cancer (NSCLC) cancer patients, we screened for trans-acting factors with an expression level showing a correlation with CERS6 expression. Among the candidates noted, silencing of either CCAAT enhancer-binding protein γ (CEBPγ) or Y-box binding protein 1 (YBX1) reduced the CERS6 expression level. Following knockdown, CEBPγ and YBX1 were found to be independently associated with reductions in ceramide-dependent lamellipodia formation as well as migration activity, while only CEBPγ may have induced CERS6 expression through specific binding to the Y-box. The mRNA expression levels of CERS6, CEBPγ, and YBX1 were positively correlated with adenocarcinoma invasiveness. YBX1 expression was observed in all 20 examined clinical lung cancer specimens, while 6 of those showed a staining pattern similar to that of CERS6. The present findings suggest promotion of lung cancer migration by possible involvement of the transcription factors CEBPγ and YBX1., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Sphingolipids are a family of lipids that are critical to cell function and survival. Much of the recent work done on sphingolipids has been performed by a closely-knit family of sphingolipid researchers, which including our colleague, Dr. Lina Obeid, who recently passed away. We now briefly review where the sphingolipid field stands today, focusing in particular on areas of sphingolipid research to which Dr. Obeid made valued contributions. These include the 'many-worlds' view of ceramides and the role of a key enzyme in the sphingolipid biosynthetic pathway, namely the ceramide synthases (CerS). The CerS contain a number of functional domains and also interact with a number of other proteins in lipid metabolic pathways, fulfilling Dr. Obeid's prophecy that ceramides, and the enzymes that generate ceramides, form the critical hub of the sphingolipid metabolic pathway., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Global dysregulation of RNA splicing and imbalanced sphingolipid metabolism has emerged as promoters of cancer cell transformation. Here, we present specific signature of alternative splicing (AS) events of sphingolipid genes for each breast cancer subtype from the TCGA-BRCA dataset. We show that ceramide synthase 2 (CERS2) undergoes a unique cassette exon event specifically in Luminal B subtype tumors. We validated this exon 8 skipping event in Luminal B cancer cells compared to normal epithelial cells, and in patient-derived tumor tissues compared to matched normal tissues. Differential AS-based survival analysis shows that this AS event of CERS2 is a poor prognostic factor for Luminal B patients. As Exon 8 corresponds to catalytic Lag1p domain, overexpression of AS transcript of CERS2 in Luminal B cancer cells leads to a reduction in the level of very-long-chain ceramides compared to overexpression of protein-coding (PC) transcript of CERS2. We further demonstrate that this AS event-mediated decrease of very-long-chain ceramides leads to enhanced cancer cell proliferation and migration. Therefore, our results show subtype-specific AS of sphingolipid genes as a regulatory mechanism that deregulates sphingolipids like ceramides in breast tumors, and can be explored further as a suitable therapeutic target.

Regulation of sphingolipid metabolism plays a role in cellular homeostasis, and dysregulation of these pathways is involved in cancer progression. Previously, our reports identified ceramide as an anti-metastatic lipid. In the present study, we investigated the biochemical alterations in ceramide-centered metabolism of sphingolipids that were associated with metastatic potential. We established metastasis-prone sublines of SKOV3 ovarian cancer cells using an in vivo selection method. These cells showed decreases in ceramide levels and ceramide synthase (CerS) 2 expression. Moreover, CerS2 downregulation in ovarian cancer cells promoted metastasis in vivo and potentiated cell motility and invasiveness. Moreover, CerS2 knock-in suppressed the formation of lamellipodia required for cell motility in this cell line. In order to define specific roles of ceramide species in cell motility controlled by CerS2, the effect of exogenous long- and very long-chain ceramide species on the formation of lamellipodia was evaluated. Treatment with distinct ceramides increased cellular ceramides and had inhibitory effects on the formation of lamellipodia. Interestingly, blocking the recycling pathway of ceramides by a CerS inhibitor was ineffective in the suppression of exogenous C 24:1 -ceramide for the formation of lamellipodia. These results suggested that C 24:1 -ceramide, a CerS2 metabolite, predominantly suppresses the formation of lamellipodia without the requirement for deacylation/reacylation. Moreover, knockdown of neutral ceramidase suppressed the formation of lamellipodia concomitant with upregulation of C 24:1 -ceramide. Collectively, the CerS2-C 24:1 -ceramide axis, which may be countered by neutral ceramidase, is suggested to limit cell motility and metastatic potential. These findings may provide insights that lead to further development of ceramide-based therapy and biomarkers for metastatic ovarian cancer., (© 2021 Federation of American Societies for Experimental Biology.)

Background and Aims: The wide prevalence of chemoresistance and compromised early diagnosis of gallbladder cancer (GBC) has led to poor patient prognosis, requiring sustained efforts for the identification of effective biomarkers and therapeutic intervention. Ceramides have emerged as intracellular signaling molecules linked to tumorigenesis and therapeutic response in cancers. However, the clinical relevance of ceramides with GBC has not been investigated., Approach and Results: In the present study, we revealed aberrant gene expressions (e.g., serine palmitoyltransferase 1 [SPTLC1] and ceramide synthase 2 [CERS2]) of de novo ceramide biosynthesis and length-specific ceramide production in GBC tissues. Analyses of serum ceramide pattern in healthy controls, gallbladder stone, and GBC patients identified C24-Ceramide as a potential diagnostic biomarker for patients with GBC. Importantly, elevation of SPTLC1, CERS2, and its product, C24-Ceramide, was associated with tumor staging, distal metastasis, and worse prognosis. In line with this, C 24 -Ceramide promoted GBC cell proliferation and migration in vitro and in vivo. Mechanistically, C24-Ceramide directly bound to phosphatidylinositol 5-phosphate 4-kinase type-2 gamma (PIP4K2C), a regulator of mammalian target of rapamycin (mTOR), to facilitate mTOR complex formation and activation. C6-Ceramide, an analogue of natural ceramide, competed with C24-Ceramide for PIP4K2C binding, thereby abrogating C24-Ceramide-mediated mTOR signaling activation and oncogenic activity. Furthermore, stimulation with C6-Ceramide significantly suppressed the proliferative and metastatic capacity of GBC cells in vitro and in vivo, which was dependent on PIP4K2C., Conclusions: Our findings highlight the clinical relevance of ceramide metabolism with GBC progression and identify C24-Ceramide as a diagnostic biomarker for GBC. We propose that PIP4K2C is indispensable for C6-Ceramide as a potential therapeutic intervention for GBC through a direct competition with C24-Ceramide., (© 2020 by the American Association for the Study of Liver Diseases.)

Senescence is an antiproliferative mechanism that can suppress tumor development and can be induced by oncogenes such as genes of the Ras family. Although studies have implicated bioactive sphingolipids (SL) in senescence, the specific mechanisms remain unclear. Here, using MCF10A mammary epithelial cells, we demonstrate that oncogenic K-Ras (Kirsten rat sarcoma viral oncogene homolog) is sufficient to induce cell transformation as well as cell senescence-as revealed by increases in the percentage of cells in the G1 phase of the cell cycle, p21 WAF1/Cip1/CDKN1A (p21) expression, and senescence-associated β-galactosidase activity (SA-β-gal). Furthermore, oncogenic K-Ras altered SL metabolism, with an increase of long-chain (LC) C18, C20 ceramides (Cer), and very-long-chain (VLC) C22:1, C24 Cer, and an increase of sphingosine kinase 1 (SK1) expression. Since Cer and sphingosine-1-phosphate have been shown to exert opposite effects on cellular senescence, we hypothesized that targeting SK1 could enhance oncogenic K-Ras-induced senescence. Indeed, SK1 downregulation or inhibition enhanced p21 expression and SA-β-gal in cells expressing oncogenic K-Ras and impeded cell growth. Moreover, SK1 knockdown further increased LC and VLC Cer species (C18, C20, C22:1, C24, C24:1, C26:1), especially the ones increased by oncogenic K-Ras. Fumonisin B1 (FB1), an inhibitor of ceramide synthases (CerS), reduced p21 expression induced by oncogenic K-Ras both with and without SK1 knockdown. Functionally, FB1 reversed the growth defect induced by oncogenic K-Ras, confirming the importance of Cer generation in the senescent phenotype. More specifically, downregulation of CerS2 by siRNA blocked the increase of VLC Cer (C24, C24:1, and C26:1) induced by SK1 knockdown and phenocopied the effects of FB1 on p21 expression. Taken together, these data show that targeting SK1 is a potential therapeutic strategy in cancer, enhancing oncogene-induced senescence through an increase of VLC Cer downstream of CerS2.

The lipid composition of HIV-1 virions is enriched in sphingomyelin (SM), but the roles that SM or other sphingolipids (SLs) might play in the HIV-1 replication pathway have not been elucidated. In human cells, SL levels are regulated by ceramide synthase (CerS) enzymes that produce ceramides, which can be converted to SMs, hexosylceramides, and other SLs. In many cell types, CerS2, which catalyzes the synthesis of very long chain ceramides, is the major CerS. We have examined how CerS2 deficiency affects the assembly and infectivity of HIV-1. As expected, we observed that very long chain ceramide, hexosylceramide, and SM were reduced in CerS2 knockout cells. CerS2 deficiency did not affect HIV-1 assembly or the incorporation of the HIV-1 envelope (Env) protein into virus particles, but it reduced the infectivites of viruses produced in the CerS2-deficient cells. The reduced viral infection levels were dependent on HIV-1 Env, since HIV-1 particles that were pseudotyped with the vesicular stomatitis virus glycoprotein did not exhibit reductions in infectivity. Moreover, cell-cell fusion assays demonstrated that the functional defect of HIV-1 Env in CerS2-deficient cells was independent of other viral proteins. Overall, our results indicate that the altered lipid composition of CerS2-deficient cells specifically inhibit the HIV-1 Env receptor binding and/or fusion processes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Sphingolipids constitute a class of bio-reactive molecules that transmit signals and exhibit a variety of physical properties in various cell types, though their functions in cancer pathogenesis have yet to be elucidated. Analyses of gene expression profiles of clinical specimens and a panel of cell lines revealed that the ceramide synthase gene CERS6 was overexpressed in non-small-cell lung cancer (NSCLC) tissues, while elevated expression was shown to be associated with poor prognosis and lymph node metastasis. NSCLC profile and in vitro luciferase analysis results suggested that CERS6 overexpression is promoted, at least in part, by reduced miR-101 expression. Under a reduced CERS6 expression condition, the ceramide profile became altered, which was determined to be associated with decreased cell migration and invasion activities in vitro. Furthermore, CERS6 knockdown suppressed RAC1-positive lamellipodia/ruffling formation and attenuated lung metastasis efficiency in mice, while forced expression of CERS6 resulted in an opposite phenotype in examined cell lines. Based on these findings, we consider that ceramide synthesis by CERS6 has important roles in lung cancer migration and metastasis., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The evolution of eukaryotic genomes has been propelled by a series of gene duplication events, leading to an expansion in new functions and pathways. While duplicate genes may retain some functional redundancy, it is clear that to survive selection they cannot simply serve as a backup but rather must acquire distinct functions required for cellular processes to work accurately and efficiently. Understanding these differences and characterizing gene-specific functions is complex. Here we explore different gene pairs and families within the context of the endoplasmic reticulum (ER), the main cellular hub of lipid biosynthesis and the entry site for the secretory pathway. Focusing on each of the ER functions, we highlight specificities of related proteins and the capabilities conferred to cells through their conservation. More generally, these examples suggest why related genes have been maintained by evolutionary forces and provide a conceptual framework to experimentally determine why they have survived selection.

LAG1 longevity assurance homolog 2 (LASS2), a highly conserved transmembrane protein, has been reported to be associated with nonalcoholic fatty liver disease (NAFLD). However, the effect of LASS2 on energy homeostasis and its mechanism remains unknown. In this study, we found lower expression levels of LASS2 in the livers of mice with liver steatosis induced by a high-fat diet (HFD) and free fatty acids (FFAs)-treated hepatocytes. In FFAs-treated Hepa1-6 cells and mouse primary hepatocytes (MPHs), LASS2 overexpression significantly decreased intracellular lipid content compared with the control cells. LASS2 overexpression also significantly upregulated lipolysis-related proteins, such as ATGL and HSL, and inhibited lipogenesis-related proteins, such as SREBP1 and FAS. In addition, the phosphorylation levels of AMPK and ACC increased significantly. On the contrary, LASS2 knockdown in FFAs-treated hepatocytes aggravated lipid accumulation via facilitating lipogenesis and inhibiting lipolysis. Further, co-IP and LC-MS analysis found that LASS2 might interacted with NDUFS2 to inhibit lipogenesis. The production of mitochondrial reactive oxygen species (mtROS) may be related to the interaction between LASS2 and NDUFS2. Collectively, we are the first time to showed that LASS2 might promote the phosphorylation of AMPK via mtROS produced by interaction with NDUFS2/OXPHOS, thus inhibiting lipogenesis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Weaned piglets (n = 3 × 6) were fed 0, 15 and 30 mg/kg diet fumonisin (FB 1 , FB 2 and FB 3 , i.e., FBs, a sphinganine analogue mycotoxin), from the age of 35 days for 21 days, to assess mycotoxin induced, dose-dependent changes in the red cells' membrane. Ouabain sensitive Na + /K + ATPase activity was determined from lysed red cell membranes, membrane fatty acid (FA) profile was analysed, as well as antioxidant and lipid peroxidation endpoints. Final body weight was higher in the 30 mg/kg group (vs. control), even besides identical cumulative feed intake. After 3 weeks, there was a difference between control and the 30 mg/kg group in red cell membrane sodium pump activity; this change was dose-dependent (sig.: 0.036; R 2 = 0.58). Membrane FA profile was strongly saturated with non-systematic inter-group differences; pooled data provided negative correlation with sodium pump activity (all individual membrane n6 FAs). Intracellular antioxidants (reduced glutathione and glutathione peroxidase) and lipid peroxidation indicators (conj. dienes, trienes and malondialdehyde) were non-responsive. We suppose a ceramide synthesis inhibitor (FB 1 ) effect exerted onto the cell membrane, proven to be toxin dose-dependent and increasing sodium pump activity, with only indirect FA compositional correlations and lack of lipid peroxidation.

Background and Aims: Dysfunctional hepatic lipid metabolism is a cause of nonalcoholic fatty liver disease (NAFLD), the most common chronic liver disorder worldwide, and is closely associated with insulin resistance and type 2 diabetes. ELOVL fatty acid elongase 6 (Elovl6) is responsible for converting C16 saturated and monounsaturated fatty acids (FAs) into C18 species. We have previously shown that Elovl6 contributes to obesity-induced insulin resistance by modifying hepatic C16/C18-related FA composition., Approach and Results: To define the precise molecular mechanism by which hepatic Elovl6 affects energy homeostasis and metabolic disease, we generated liver-specific Elovl6 knockout (LKO) mice. Unexpectedly, LKO mice were not protected from high-fat diet-induced insulin resistance. Instead, LKO mice exhibited higher insulin sensitivity than controls when consuming a high-sucrose diet (HSD), which induces lipogenesis. Hepatic patatin-like phospholipase domain-containing protein 3 (Pnpla3) expression was down-regulated in LKO mice, and adenoviral Pnpla3 restoration reversed the enhancement in insulin sensitivity in HSD-fed LKO mice. Lipidomic analyses showed that the hepatic ceramide(d18:1/18:0) content was lower in LKO mice, which may explain the effect on insulin sensitivity. Ceramide(d18:1/18:0) enhances protein phosphatase 2A (PP2A) activity by interfering with the binding of PP2A to inhibitor 2 of PP2A, leading to Akt dephosphorylation. Its production involves the formation of an Elovl6-ceramide synthase 4 (CerS4) complex in the endoplasmic reticulum and a Pnpla3-CerS4 complex on lipid droplets. Consistent with this, liver-specific Elovl6 deletion in ob/ob mice reduced both hepatic ceramide(d18:1/18:0) and PP2A activity and ameliorated insulin resistance., Conclusions: Our study demonstrates the key role of hepatic Elovl6 in the regulation of the acyl-chain composition of ceramide and that C18:0-ceramide is a potent regulator of hepatic insulin signaling linked to Pnpla3-mediated NAFLD., (© 2019 by the American Association for the Study of Liver Diseases.)

Gentiana lutea is a bitter herb that is traditionally used to improve gastric disorders. Recently, we have shown that Gentiana lutea extract (GE) also modulates the lipid metabolism of human keratinocytes in vitro and in vivo. In the present study, we investigated the role of GE on ceramide synthesis in human primary keratinocytes (HPKs) and psoriasis-like keratinocytes. We could demonstrate that GE increased the concentrations of glucosylceramides and the ceramide AS/AdS subclass without affecting the overall ceramide content in HPKs. The expression of ceramide synthase 3 ( CERS3 ) and elongases ( ELOVL1 and 4 ) was reduced in psoriasis lesions compared to healthy skin. Psoriasis-like HPKs, generated by stimulating HPKs with cytokines that are involved in the pathogenesis of psoriasis (IL-17, TNF-α, IL-22 and IFN-γ) showed increased levels of IL-6, IL-8 and increased expression of DEFB4A , as well as decreased expression of ELOVL4 . The treatment with GE partly rescued the reduced expression of ELOVL4 in psoriasis-like HPKs and augmented CERS3 expression. This study has shown that GE modulates ceramide synthesis in keratinocytes. Therefore, GE might be a novel topical treatment for skin diseases with an altered lipid composition such as psoriasis.

Sphingolipids (SLs) synthesis involves a complex metabolic pathway occurring between the endoplasmic reticulum (ER) and Golgi apparatus, generating ceramide synthesis and complex lipids, respectively. Here we show that E. histolytica, apparently lacking cellular organelles (ER and Golgi apparatus), synthesizes a wide variety of sphingolipid subspecies, being particularly abundant those of long-chain fatty acids. In silico analysis showed five putative genes coding for ceramide synthases (CerS), all of them coding for proteins containing the TLC domain, a region conserved in CerS of multiple organisms. These genes are abundantly expressed in different growth phases. Silencing and overexpression of CerS C4M4U4 (the closest homolog of human CerS 2 and 3) demonstrated its involvement in the synthesis of ceramide. Additionally, we identify C4M4U4, SMS2 and PKC (α, βII) proteins and their subcellular localization of E. histolytica, suggesting that these subcellular compartments might be involved in the biosynthesis and signaling pathway of sphingolipids, and evidencing different sphingolipid synthesis pathways in Entamoeba., (Copyright © 2019 Elsevier Inc. All rights reserved.)

To explore the role and mechanism of CERS1 in hypophysoma and investigate whether CERS1 overexpression can change the autophagy process of hypophysoma, and then to explore whether CERS1's effect was regulated by the PI3K/AKT signaling pathway. Western blot and RT-PCR were used to analyze the expression or mRNA level of CERS1 at different tissues or cell lines. Afterwards, the occurrence and development of hypophysoma in vivo and in vitro, respectively, was observed by using CERS1 overexpression by lentivirus. Finally, MK-2206 and LY294002 were applied to discuss whether the role of CERS1 was regulated by the PI3K/AKT signaling pathway. Results show that the CERS1 expression and mRNA level in tumor or AtT-20 cells were decreased. CERS1 over-expressed by lentivirus could inhibit hypophysoma development in vivo and in vitro by reducing tumor volume and weight, weakening tumor proliferation and invasion, and enhancing apoptosis. In addition, shCERS1 could reverse the process. The above results indicate that CERS1 is possibly able to enhance autophagy in hypophysoma through the PI3K/AKT signaling pathway.

Sphingolipids (SLs) are an important class of membrane lipids containing a long chain sphingoid base backbone. SL synthesis is compartmentalized between two major cell organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. The initial steps of sphingolipid synthesis take place in the ER, where the simplest SL, ceramide, is synthesized. Although ceramide is a critical membrane component, an imbalance of ceramide levels can have significant deleterious effects on cell properties leading to events such as apoptosis. For this reason and others, ER ceramide levels must be tightly regulated. Here, we describe the biological and biophysical properties of ceramide and discuss how this might impact the ER membrane. This article is part of a special issue entitled: ER Platforms for Membrane Lipid Dynamics., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The development and progression of colorectal cancer (CRC), a major cause of cancer-related death in the western world, is accompanied with alterations of sphingolipid (SL) composition in colon tumors. A number of enzymes involved in the SL metabolism have been found to be deregulated in human colon tumors, in experimental rodent studies, and in human colon cancer cells in vitro. Therefore, the enzymatic pathways that modulate SL levels have received a significant attention, due to their possible contribution to CRC development, or as potential therapeutic targets. Many of these enzymes are associated with an increased sphingosine-1-phosphate/ceramide ratio, which is in turn linked with increased colon cancer cell survival, proliferation and cancer progression. Nevertheless, more attention should also be paid to the more complex SLs, including specific glycosphingolipids, such as lactosylceramides, which can be also deregulated during CRC development. In this review, we focus on the potential roles of individual SLs/SL metabolism enzymes in colon cancer, as well as on the pros and cons of employing the current in vitro models of colon cancer cells for lipidomic studies investigating the SL metabolism in CRC.

Ataxin-2 (human gene symbol ATXN2 ) acts during stress responses, modulating mRNA translation and nutrient metabolism. Ataxin-2 knockout mice exhibit progressive obesity, dyslipidemia, and insulin resistance. Conversely, the progressive ATXN2 gain of function due to the fact of polyglutamine (polyQ) expansions leads to a dominantly inherited neurodegenerative process named spinocerebellar ataxia type 2 (SCA2) with early adipose tissue loss and late muscle atrophy. We tried to understand lipid dysregulation in a SCA2 patient brain and in an authentic mouse model. Thin layer chromatography of a patient cerebellum was compared to the lipid metabolome of Atxn2 -CAG100-Knockin (KIN) mouse spinocerebellar tissue. The human pathology caused deficits of sulfatide, galactosylceramide, cholesterol, C22/24-sphingomyelin, and gangliosides GM1a/GD1b despite quite normal levels of C18-sphingomyelin. Cerebellum and spinal cord from the KIN mouse showed a consistent decrease of various ceramides with a significant elevation of sphingosine in the more severely affected spinal cord. Deficiency of C24/26-sphingomyelins contrasted with excess C18/20-sphingomyelin. Spinocerebellar expression profiling revealed consistent reductions of CERS protein isoforms, Sptlc2 and Smpd3 , but upregulation of Cers2 mRNA, as prominent anomalies in the ceramide-sphingosine metabolism. Reduction of Asah2 mRNA correlated to deficient S1P levels. In addition, downregulations for the elongase Elovl1 , Elovl4 , Elovl5 mRNAs and ELOVL4 protein explain the deficit of very long-chain sphingomyelin. Reduced ASMase protein levels correlated to the accumulation of long-chain sphingomyelin. Overall, a deficit of myelin lipids was prominent in SCA2 nervous tissue at prefinal stage and not compensated by transcriptional adaptation of several metabolic enzymes. Myelination is controlled by mTORC1 signals; thus, our human and murine observations are in agreement with the known role of ATXN2 yeast, nematode, and mouse orthologs as mTORC1 inhibitors and autophagy promoters., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The endoplasmic reticulum (ER) is not only important for protein synthesis and folding but is also crucial for lipid synthesis and metabolism. In the current study, we demonstrate an important role of ceramide synthases (CerS) in ER stress and NAFLD progression. Ceramide is important in sphingolipid metabolism, and its acyl chain length is determined by a family of six CerS in mammals. CerS2 generates C22-C24 ceramides, and CerS5 or CerS6 produces C16 ceramide. To gain insight into the role of CerS in NAFLD, we used a high-fat diet (HFD)-induced NAFLD mouse model. Decreased levels of CerS2 and increased levels of CerS6 were observed in the steatotic livers of mice fed a HFD. In vitro experiments with Hep3B cells indicated the protective role of CerS2 and the detrimental role of CerS6 in the ER stress response induced by palmitate treatment. In particular, CerS6 overexpression increased sterol regulatory element-binding protein-1 (SREBP-1) cleavage with decreased levels of INSIG-1, leading to increased lipogenesis. Blocking ER stress abrogated the detrimental effects of CerS6 on palmitate-induced SREBP-1 cleavage. In accordance with the protective role of CerS2 in the palmitate-induced ER stress response, CerS2 knockdown enhanced ER stress and SREBP-1 cleavage, and CerS2 heterozygote livers exhibited a stronger ER stress response and higher triglyceride levels following HFD. Finally, treatment with a low dose of bortezomib increased hepatic CerS2 expression and protected the development of NAFLD following HFD. These results indicate that CerS and its derivatives impact hepatic ER stress and lipogenesis differently and might be therapeutic targets for NAFLD.

The ceramide synthase 2 ( CERS2 ) gene has been linked to tumour recurrence and invasion in many different types of cancers including bladder cancer. In this study, the expression levels of CERS2 in bladder cancer cell lines were analysed using qRT-PCR and the protein expression in clinical bladder cancer histopathological specimens were examined via immunohistochemistry. The potential utility of CERS2 as a predictive biomarker of response to oncolytic virotherapy was assessed by correlating the CERS2 mRNA expression to IC 50 values of cells treated with the Newcastle disease virus (NDV), AF2240 strain. This study demonstrates that CERS2 is differentially expressed in different types of bladder cancer cell lines and that the siRNA-mediated downregulation of the expression of CERS2 reduces the migratory potential of UMUC1 bladder cancer cells. However, there were no significant correlations between the expression levels of the CERS2 protein with bladder cancer grade/stage or between the IC50 values of cells treated with NDV and CERS2 expression. Although the utility of CERS2 expression may be limited, its potential as an antimigration cancer therapeutic should be further examined., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 Ahmed Faris Aldoghachi et al.)

Homo sapiens longevity assurance homolog 2 of yeast LAG1 (LASS2), is a gene isolated from a human liver complementary DNA library. In this study, we found that LASS2 protein level was positively related to International Federation of Gynecology and Obstetrics (FIGO) stage and LASS2-negative tumors showed significant association with longer disease-free survival (DFS) and overall survival (OS) in ovarian cancer patients. The heterogeneous expression of LASS2 had been exhibited in diverse ovarian cancer cells. A significantly lower messenger RNA (mRNA) and protein level of LASS2 was seen in 3AO cell compared with those in other types of ovarian cancer cells. Meanwhile, the mRNA and protein levels of LASS2 in ES-2 and NIH:OVCAR-3 cells were obviously higher. LASS2 overexpression in 3AO cell could promote migration, invasion, and metastasis abilities in vitro and in vivo, while LASS2 knockdown in ES-2 and NIH:OVCAR-3 cells had the opposite effects. The oncogenic capacity of LASS2 in ovarian cancer may be mediated by increased expression of YAP/TAZ. It is indicated that lowering the expression of LASS2 is likely to serve as an unprecedented approach for the treatment of ovarian cancer., (© 2018 Wiley Periodicals, Inc.)