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Discipline
Biological
Keywords
Liver X Receptors
Ceramide
Sphingolipids
Hepatocytes
Observation Type
Standalone
Nature
Standard Data
Submitted
Oct 28th, 2016
Published
Dec 19th, 2016
  • Abstract

    Sphingolipids are essential components of cellular membranes that also serve as signals to mediate important biological processes such as survival, proliferation, and differentiation under both physiological and pathological conditions. In this study, we provide evidence that the nuclear hormone receptor that regulates cholesterol metabolism, Liver X receptor (LXR), also regulates cellular sphingolipid content in mouse hepatic cells. Using a lipidomics approach, we showed that LXR activation leads to a significant increase in the levels of several sphingolipid species, including ceramides and sphingomyelin. These findings on the regulation of sphingolipids by LXRs may be useful for the development of novel drug therapies in diseases associated with sphingolipid alteration.

  • Figure
  • Introduction

    Sphingolipids are bioactive lipid molecules that have been implicated as key modulators of many diseases including cardiovascular, metabolic and autoimmune, and neurological disorders. Of the many classes of sphingolipids, ceramide and sphingosine-1-phosphate have been shown to be an important mediators in the cellular signal transduction involved in apoptosis, proliferation, stress responses, necrosis, inflammation, autophagy, senescence, and differentiation in multiple tissues. Understanding the complex regulation of sphingolipid synthesis would help to identify key ceramide, biosynthetic, and degradation pathways as potential pharmacological targets. LXRs are nuclear hormone receptors that serve as ligand-regulatable transcription factors to control the expression of genes involved in cholesterol metabolism, de novo lipogenesis, and anti-inflammatory pathways. Oxy-cholesterols are natural ligands of LXRs; however, the synthetic ligand, GW3965, is a highly specific LXR agonist for both in vitro and in vivo activation of LXRs. Given the importance of LXRs in cellular lipid metabolism, we have investigated their role(s) in sphingolipid biosynthesis in the AML-12 mouse hepatocyte cell line.

  • Objective

    The objective of this study was to determine the effect of LXR agonist GW3965 on cellular sphingolipid content.

  • Results & Discussion

    Our lipidomics analysis showed that several sphingolipid species were significantly upregulated by GW3965. Sphingolipids such as sphinganine, dihydroceramides, ceramides, glucosyl-ceramides, dihydrosphingomyelins, sphingomyelins, and sphingosine were significantly induced by GW3965 (Figure 1A-G). There are three major pathways for sphingolipid biosynthesis as shown in Figure 1H: (1) the sphingomyelinase pathway, which uses sphingomyelinase to break down sphingomyelin in the cell membrane and release ceramide; (2) the de novo pathway, which synthesizes ceramide from palmitate and serine; and (3) the salvage pathway, which can generate ceramide through breakdown of complex sphingolipids that are ultimately converted into sphingosine, which then undergoes reacylation to form ceramide. Therefore, we next looked if the expressions of the key genes (Figure 1H) regulating sphingolipid synthesis are affected by GW3965. Surprisingly, unlike its effect on the sphingolipid levels, GW3965 had only a modest effect on the expression of sphingolipid biosynthesis genes. Using fatty acid synthase (Fasn) expression as a positive control for LXR transcriptional activity, the relative expression levels of genes involved in the de novo and salvage pathways of ceramide synthesis (e.g., serine palmitoyl transferase [SPTLC1-3], ceramide synthase [CerS2-5], and dihydroceramide desaturase [DEGS1-2]) showed a tendency to increase with LXR activation but did not reach significance (Figure 1I). Similarly, the level of sphingomyelin synthase (SGMS1-2) also did not change. Additionally, GW3965 did not affect the gene expression of phosphatidylinositol-4-phosphate adaptor-2 (FAPP2), a protein needed for intracellular ceramide transport (Figure 1I). These results suggest that LXRs-mediated increase in sphingolipid content is possibly independent of the regulation of its biosynthetic genes by LXRs. Therefore, it is possible that LXR agonist may either increase the flux of substrate (i.e., palmitate) via the activity of endogenous Fasn activity or alter the activities of sphingolipid synthesis enzymes. The effect of GW3965 on Fasn was LXR-dependent as assessed by LXRalpha knockdown (Supplementary data). Further studies are needed to determine the specific mechanism(s).

  • Conclusions

    Our data conclusively show that LXR agonists are potent inducers of cellular sphingolipid synthesis. This is an important finding because it implies that LXR agonism or antagonism may be able to correct sphingolipid imbalances in specific disease contexts.

  • Limitations

    Further studies involving other cell types and in vivo experiments, involving disease models will be needed for further validation.

  • Methods

    Cell culture

    AML-12 (CRL-2254) cells were maintained at 37°C in DMEM-F12 1:1 containing 10% fetal bovine serum, 1× ITS, 10 nM dexamethasone, and 1× penicillin/streptomycin in a 5% CO2 atmosphere. Cells were treated with GW3965 (Sigma-Aldrich) at a dose of 10 μM.

    Lipidomic analysis
    Sphingolipid analysis was performed as described before. In brief, cells were harvested, washed with ice-cold phosphate-buffered saline (PBS), and spiked with an internal standard mixture (C17-sphingosine, C17-sphinganine, C17-sphingosine-1-phosphate, C17-sphinganine-1-phosphate, C12-ceramide, C12-ceramide-1-phosphate, C12-glucosylceramide, C12-lactosylceramide, C12-sphingomyelin, and 14:0 phosphocholine from Avanti Polar Lipids). Thereafter, the cells were resuspended in 900 μL ice-cold chloroform-methanol (1:2) and incubated in ice for 15 min with vortexing every 5 min. 300 μL ice-cold distilled water (dH2O) and 300 μL ice-cold chloroform were added to the samples, which were then vortexed and centrifuged at 8,000 g for 2 min at 4°C. The lower organic phase was transferred into a clean microcentrifuge tube. A second extraction was performed by adding 300 μL ice-cold chloroform, and the lower organic phase was pooled with that of the first extraction. The collected samples were dried under a stream of nitrogen and stored at -80°C until ready for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.

    RNA isolation and qRT-PCR
    Total RNA was isolated and qPCR performed using the QuantiTect SYBR Green PCR Kit (Qiagen, 204141) in accordance with the manufacturer’s instructions. Primers were purchased from Sigma-Aldrich (KiCqStart® SYBR® Green Predesigned Primers). Relative mRNA levels were assessed using 2-ddCt analysis.

    Calculations and statistics
    Results were expressed as mean ± SD. The statistical significance of differences (*p <0.05) was assessed by unpaired Student’s -t test when comparing different groups; t(DF) = 4.

  • Funding statement

    This work was supported by Singapore Ministry of Health NMRC/BNIG/2025/2014 (RAS).

  • Ethics statement

    Not applicable.

  • References
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    Matters Select25/30

    Activation of Liver X receptors (LXRs) increases sphingolipid biosynthesis in hepatic cells

    Abstractlink

    Sphingolipids are essential components of cellular membranes that also serve as signals to mediate important biological processes such as survival, proliferation, and differentiation under both physiological and pathological conditions. In this study, we provide evidence that the nuclear hormone receptor that regulates cholesterol metabolism, Liver X receptor (LXR), also regulates cellular sphingolipid content in mouse hepatic cells. Using a lipidomics approach, we showed that LXR activation leads to a significant increase in the levels of several sphingolipid species, including ceramides and sphingomyelin. These findings on the regulation of sphingolipids by LXRs may be useful for the development of novel drug therapies in diseases associated with sphingolipid alteration.

    Figurelink

    LXR-agonist GW3965 induces sphingolipid synthesis. Lipid levels of (A,B) Ceramides (1Cer), (C) Dihydroceramides (2Cer), (D) Glucosylceramides (GlcCer), (E) Dihydrosphingomyelin (DHSM), (F) Sphinganine & Sphingosine, (G) Sphingomyelin (SM) in control and GW3965-treated AML-12 cells. GW3965 was added at a dose of 10 µM for 24 h. Bars represent the mean of the respective individual ratios ±SD (n = 3, *p <0.05). (H) Sphingolipid synthesis pathway with important regulatory genes. (I) qRT-PCR analysis of genes involved in sphingolipid biosynthesis in AML-12 cells treated with GW3965 at a dose of 10 µM for 24 h. Bars represent the mean of the respective individual ratios ± SD (n = 3, *p <0.05).

    Introductionlink

    Sphingolipids are bioactive lipid molecules that have been implicated as key modulators of many diseases including cardiovascular, metabolic and autoimmune, and neurological disorders[1]. Of the many classes of sphingolipids, ceramide and sphingosine-1-phosphate have been shown to be an important mediators in the cellular signal transduction involved in apoptosis, proliferation, stress responses, necrosis, inflammation, autophagy, senescence, and differentiation in multiple tissues[2][3][4][5][6]. Understanding the complex regulation of sphingolipid synthesis would help to identify key ceramide, biosynthetic, and degradation pathways as potential pharmacological targets. LXRs are nuclear hormone receptors that serve as ligand-regulatable transcription factors to control the expression of genes involved in cholesterol metabolism, de novo lipogenesis, and anti-inflammatory pathways[7]. Oxy-cholesterols are natural ligands of LXRs; however, the synthetic ligand, GW3965, is a highly specific LXR agonist for both in vitro and in vivo activation of LXRs. Given the importance of LXRs in cellular lipid metabolism, we have investigated their role(s) in sphingolipid biosynthesis in the AML-12 mouse hepatocyte cell line.

    Objectivelink

    The objective of this study was to determine the effect of LXR agonist GW3965 on cellular sphingolipid content.

    Results & Discussionlink

    Our lipidomics analysis showed that several sphingolipid species were significantly upregulated by GW3965. Sphingolipids such as sphinganine, dihydroceramides, ceramides, glucosyl-ceramides, dihydrosphingomyelins, sphingomyelins, and sphingosine were significantly induced by GW3965 (Figure 1A-G). There are three major pathways for sphingolipid biosynthesis[8] as shown in Figure 1H: (1) the sphingomyelinase pathway, which uses sphingomyelinase to break down sphingomyelin in the cell membrane and release ceramide; (2) the de novo pathway, which synthesizes ceramide from palmitate and serine; and (3) the salvage pathway, which can generate ceramide through breakdown of complex sphingolipids that are ultimately converted into sphingosine, which then undergoes reacylation to form ceramide. Therefore, we next looked if the expressions of the key genes (Figure 1H) regulating sphingolipid synthesis are affected by GW3965. Surprisingly, unlike its effect on the sphingolipid levels, GW3965 had only a modest effect on the expression of sphingolipid biosynthesis genes. Using fatty acid synthase (Fasn) expression as a positive control for LXR transcriptional activity, the relative expression levels of genes involved in the de novo and salvage pathways of ceramide synthesis (e.g., serine palmitoyl transferase [SPTLC1-3], ceramide synthase [CerS2-5], and dihydroceramide desaturase [DEGS1-2]) showed a tendency to increase with LXR activation but did not reach significance (Figure 1I). Similarly, the level of sphingomyelin synthase (SGMS1-2) also did not change. Additionally, GW3965 did not affect the gene expression of phosphatidylinositol-4-phosphate adaptor-2 (FAPP2), a protein needed for intracellular ceramide transport (Figure 1I). These results suggest that LXRs-mediated increase in sphingolipid content is possibly independent of the regulation of its biosynthetic genes by LXRs. Therefore, it is possible that LXR agonist may either increase the flux of substrate (i.e., palmitate) via the activity of endogenous Fasn activity or alter the activities of sphingolipid synthesis enzymes. The effect of GW3965 on Fasn was LXR-dependent as assessed by LXRalpha knockdown (Supplementary data). Further studies are needed to determine the specific mechanism(s).

    Conclusionslink

    Our data conclusively show that LXR agonists are potent inducers of cellular sphingolipid synthesis. This is an important finding because it implies that LXR agonism or antagonism may be able to correct sphingolipid imbalances in specific disease contexts.

    Limitationslink

    Further studies involving other cell types and in vivo experiments, involving disease models will be needed for further validation.

    Methodslink

    Cell culture

    AML-12 (CRL-2254) cells were maintained at 37°C in DMEM-F12 1:1 containing 10% fetal bovine serum, 1× ITS, 10 nM dexamethasone, and 1× penicillin/streptomycin in a 5% CO2 atmosphere. Cells were treated with GW3965 (Sigma-Aldrich) at a dose of 10 μM.

    Lipidomic analysis
    Sphingolipid analysis was performed as described before[9][10]. In brief, cells were harvested, washed with ice-cold phosphate-buffered saline (PBS), and spiked with an internal standard mixture (C17-sphingosine, C17-sphinganine, C17-sphingosine-1-phosphate, C17-sphinganine-1-phosphate, C12-ceramide, C12-ceramide-1-phosphate, C12-glucosylceramide, C12-lactosylceramide, C12-sphingomyelin, and 14:0 phosphocholine from Avanti Polar Lipids). Thereafter, the cells were resuspended in 900 μL ice-cold chloroform-methanol (1:2) and incubated in ice for 15 min with vortexing every 5 min. 300 μL ice-cold distilled water (dH2O) and 300 μL ice-cold chloroform were added to the samples, which were then vortexed and centrifuged at 8,000 g for 2 min at 4°C. The lower organic phase was transferred into a clean microcentrifuge tube. A second extraction was performed by adding 300 μL ice-cold chloroform, and the lower organic phase was pooled with that of the first extraction. The collected samples were dried under a stream of nitrogen and stored at -80°C until ready for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.

    RNA isolation and qRT-PCR
    Total RNA was isolated and qPCR performed using the QuantiTect SYBR Green PCR Kit (Qiagen, 204141) in accordance with the manufacturer’s instructions. Primers were purchased from Sigma-Aldrich (KiCqStart® SYBR® Green Predesigned Primers). Relative mRNA levels were assessed using 2-ddCt analysis.

    Calculations and statistics
    Results were expressed as mean ± SD. The statistical significance of differences (*p <0.05) was assessed by unpaired Student’s -t test when comparing different groups; t(DF) = 4.

    Funding Statementlink

    This work was supported by Singapore Ministry of Health NMRC/BNIG/2025/2014 (RAS).

    Conflict of interestlink

    The authors declare no conflicts of interest.

    Ethics Statementlink

    Not applicable.

    No fraudulence is committed in performing these experiments or during processing of the data. We understand that in the case of fraudulence, the study can be retracted by Matters.

    Referenceslink
    1. Aburasayn Hanin, Al Batran Rami, Ussher John R.
      Targeting ceramide metabolism in obesity
      American Journal of Physiology - Endocrinology And Metabolism, 311/2016, pages E423-E435 DOI: 10.1152/ajpendo.00133.2016chrome_reader_mode
    2. Aguilera-Romero Auxiliadora, Gehin Charlotte, Riezman Howard
      Sphingolipid homeostasis in the web of metabolic routes
      Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1841/2014, pages 647-656 DOI: 10.1016/j.bbalip.2013.10.014chrome_reader_mode
    3. Chaurasia Bhagirath, Summers Scott A.
      Ceramides – Lipotoxic Inducers of Metabolic Disorders
      Trends in Endocrinology & Metabolism, 26/2015, pages 538-550 DOI: 10.1016/j.tem.2015.07.006chrome_reader_mode
    4. Jazvinšćak Jembrek Maja, Hof Patrick R., Šimić Goran
      Ceramides in Alzheimer’s Disease: Key Mediators of Neuronal Apoptosis Induced by Oxidative Stress and Aβ Accumulation
      Oxidative Medicine and Cellular Longevity, 2015/2015, pages 1-17 DOI: 10.1155/2015/346783chrome_reader_mode
    5. McDonald Georgia, Deepak Shantal, Miguel Laura,more_horiz, Jury Elizabeth C.
      Normalizing glycosphingolipids restores function in CD4+ T cells from lupus patients
      Journal of Clinical Investigation, 124/2014, pages 712-724 DOI: 10.1172/jci69571chrome_reader_mode
    6. Ong Wei-Yi, Herr Deron R, Farooqui Tahira,more_horiz, Farooqui Akhlaq A
      Role of sphingomyelinases in neurological disorders
      Expert Opinion on Therapeutic Targets, 19/2015, pages 1725-1742 DOI: 10.1517/14728222.2015.1071794chrome_reader_mode
    7. Hong Cynthia, Tontonoz Peter
      Liver X receptors in lipid metabolism: opportunities for drug discovery
      Nature Reviews Drug Discovery, 13/2014, pages 433-444 DOI: 10.1038/nrd4280chrome_reader_mode
    8. Sasset Linda, Zhang Yi, Dunn Teresa M., Di Lorenzo Annarita
      Sphingolipid De Novo Biosynthesis: A Rheostat of Cardiovascular Homeostasis
      Trends in Endocrinology & Metabolism, 27/2016, pages 807-819 DOI: 10.1016/j.tem.2016.07.005chrome_reader_mode
    9. Siddique M. M., Li Y., Wang L.,more_horiz, Summers S. A.
      Ablation of Dihydroceramide Desaturase 1, a Therapeutic Target for the Treatment of Metabolic Diseases, Simultaneously Stimulates Anabolic and Catabolic Signaling
      Molecular and Cellular Biology, 33/2013, pages 2353-2369 DOI: 10.1128/mcb.00226-13chrome_reader_mode
    10. Sinha Rohit A., Farah Benjamin L., Singh Brijesh K.,more_horiz, Yen Paul M.
      Caffeine stimulates hepatic lipid metabolism by the autophagy-lysosomal pathway in mice
      Hepatology, 59/2014, pages 1366-1380 DOI: 10.1002/hep.26667chrome_reader_mode
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