Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Jun 30.
Published in final edited form as: Toxicology. 2021 Jun 9;458:152831. doi: 10.1016/j.tox.2021.152831

The Aryl Hydrocarbon Receptor Activates Ceramide Biosynthesis in Mice Contributing to Hepatic Lipogenesis

Qing Liu 1,#, Limin Zhang 3,#, Erik L Allman 1, Troy D Hubbard 1, Iain A Murray 1, Fuhua Hao 1, Yuan Tian 1, Wei Gui 1, Robert G Nichols 1, Philip B Smith 2, Mallappa Anitha 1, Gary H Perdew 1, Andrew D Patterson 1,*
PMCID: PMC8327397  NIHMSID: NIHMS1714100  PMID: 34097992

Abstract

Aryl hydrocarbon receptor (AHR) activation via 2,3,7,8-tetrachlorodibenzofuran (TCDF) induces the accumulation of hepatic lipids. Here we report that AHR activation by TCDF (24 μg/kg body weight given orally for five days) induced significant elevation of hepatic lipids including ceramides in mice, was associated with increased expression of key ceramide biosynthetic genes, and increased activity of their respective enzymes. Results from chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA) and cell-based reporter luciferase assays indicated that AHR directly activated the serine palmitoyltransferase long chain base subunit 2 (Sptlc2, encodes serine palmitoyltransferase 2 (SPT2)) gene whose product catalyzes the initial rate-limiting step in de novo sphingolipid biosynthesis. Hepatic ceramide accumulation was further confirmed by mass spectrometry-based lipidomics. Taken together, our results revealed that AHR activation results in the up-regulation of Sptlc2, leading to ceramide accumulation, thus promoting lipogenesis, which can induce hepatic lipid accumulation.

Keywords: aryl hydrocarbon receptor (AHR), lipogenesis, ceramide, Sptlc2

1. Introduction

The aryl hydrocarbon receptor (AHR) mediates toxicologic and carcinogenic reactions to polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons. AHR binds environmental persistent organic pollutants (POPs), such as 2,3,7,8-tetrachlorodibenzofuran (TCDF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Upon activation, AHR translocates to the nucleus and dimerizes with the AHR nuclear translocator (ARNT). The AHR-ARNT heterodimer then binds to dioxin response elements (DREs) of target genes (such as Cyp1a1, Cyp1a2, and Cyp1b1), thus promoting changes in gene expression. AHR mediates differential expression in genes associated with lipid transport, processing, and metabolism in mice (Boverhof et al. 2005; Duval et al. 2017; Kim et al. 2021; Lee et al. 2010; Tanos et al. 2012).

Sphingolipids are a class of diverse lipids that serve multiple biological roles (Poss and Summers 2020). Ceramides, essential precursors for complex sphingolipids, are linked to obesity-associated metabolic dysfunction (Holland et al. 2007). There are three main pathways for ceramide production (Plotegher et al. 2019): the de novo synthesis pathway, the sphingomyelin phosphodiesterase (SMase) pathway, and the salvage pathway. De novo synthesis starts in the endoplasmic reticulum (ER) with the condensation of fatty acids and amino acids to generate a sphingoid base by serine palmitoyltransferase (SPT), and then produces ceramide by the action of ceramide synthase (CerS) and dihydroceramide desaturases (Des). Once ceramides or dihydroceramides are synthesized in ER, they translocate to the Golgi apparatus and are converted into complex sphingolipids by spermine synthase (Sms) or ceramide kinase (CerK). Ceramides can be reformed by the salvage pathway that involves many enzymes, such as acid sphingomyelin phosphodiesterase (A-SMase), acid ceramidases (A-CDase), and glycosyl synthase (GCS) within the endo-lysosomal route (Plotegher et al. 2019).

Previous studies have shown that elevated ceramide levels in rodent liver are associated with the development of abnormal liver metabolism, hepatic insulin resistance, and steatosis (Xia et al. 2014). It has been reported that AHR activation by high dose TCDD induced elevation of ceramide levels (in liver and skin) and increased the activity of related rate-limiting enzymes, such as SPT and SMase (Kennedy et al. 2013; Muenyi et al. 2014; Nault et al. 2017). A recent study showed that AHR bound and activated the gene promoter of serine palmitoyltransferase small subunit A (SPTSSA), thus acting as a positive regulator of sphingolipid levels in the biosynthetic pathway (Majumder et al. 2020).

In this current study, a combination of LC-MS-based lipidomics, targeted ceramide metabolite profiling, ChIP-qPCR, cell-based reporter luciferase assays, and EMSA were used to investigate how the AHR controls ceramide biosynthesis and results in liver lipid accumulation. In particular, we focused on sphingolipid metabolism along with the regulation and expression of genes involved in the ceramide pathway. Our findings provide new evidence that AHR contributes to hepatic lipogenesis via direct activation of Sptlc2 gene expression, leading to an elevation in the rate limiting enzyme of the de novo pathway of ceramide synthesis.

2. Materials and methods

2.1. Animal Experiments.

Animal procedures were performed according to protocols approved by the Pennsylvania State University Institutional Animal Care and Use Committee. Wild type male C57BL/6J mice (4 weeks of age) were purchased from the Jackson Laboratory. Ahr-null (Ahr−/−) congenic male mice on a C57BL/6J background were bred in house and utilized at 6 weeks of age. C57BL/6J mice were acclimated for one week after arriving in the mouse facility. Wild-type and Ahr−/− mice underwent one week of transgenic dough pill (Bio-Serve) training. Mice were fed with untreated dough pills (acetone solution as vehicle) or dough pills containing TCDF (Cambridge Isotope Laboratories, Inc.) to achieve a final dose of 24 μg/kg body weight daily for five days (one pill per mouse per day). TCDF was provided through the diet as it represents a common exposure route. Tissues were collected immediately following sacrifice by CO2 asphyxiation on day 7. All samples were stored at −80°C until analysis.

2.2. RNA Isolation and Quantitative Real-Time PCR.

RNA was extracted from 50 mg of frozen liver using TRIzol reagent (Invitrogen). cDNA was synthesized from 1 μg of total RNA using qScript cDNA SuperMix (Quanta Biosciences) as described in the Supporting Information. Quantitative real-time PCR assays were performed using PowerUp™ SYBR Green qPCR Master Mix (Applied Biosystems) on QuantStudio™ 3 Real-Time PCR System (Applied Biosystems).

2.3. Quantification of Hepatic Triglycerides.

Liver triglycerides were extracted using a 2:1 chloroform/methanol solution and measured with a triglyceride colorimetric assay kit, according to the manufacturer’s instructions (Cayman).

2.4. Determination of sphingomyelinase activity.

10 mg frozen liver samples were homogenized in 100 μL of PBS (containing 0.1% BSA), followed by centrifugation at 18,000 x g for 5 min. Supernatants were collected and analyzed with a colorimetric sphingomyelinase assay kit (Sigma-Aldrich).

2.5. Electrophoretic Mobility Shift Assay.

Gel retardation assays were performed using in vitro translated murine AHRb-1 and human ARNT as described previously(Chiaro et al. 2008). Briefly, in vitro translated murine AHRb-1, ARNT were incubated with 32P-radiolabeled oligonucleotide probes for Cyp1a1 (5’-gatctggctcttctcacgcaactgcg-3’, 5’-gatccggagtggcgtgagaagagcca-3’), Sptlc2-DRE1 (5’-agtgctgagattaaaggcgtgcgccaccacacccgg-3’, 5’-ccgggtgtggtggcgcacgcctttaatctcagcact-3’) or Sptlc2-DRE2 (5’-gggtggccccgccctcgcgtgacggccggaccggga-3’, 5’-tcccggtccggccgtcacgcgagggcggggccaccc-3’), as appropriate. Complexes were incubated with vehicle or 10 nM TCDD, as indicated. Protein-oligonucleotide complexes were resolved on 8% non-denaturing polyacrylamide gels, dried and visualized by autoradiography.

2.6. Transient Transfection and Luciferase Assays.

Hepa1 cells were cultured at 37°C in a humidified atmosphere composed of 95% air and 5% CO2 in α-modified essential media (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan) and 100 lU/ml penicillin/100 μg/ml streptomycin (Sigma-Aldrich). Cells were plated to approximately 90% confluency in 12-well cell culture plates and transfected using the Lipofectamine® 3000 transfection reagent (Life Technologies) with a total 1.5 μg of total plasmid per well. Transient transfections used 600 ng pcDNA3 expression plasmid, 400 ng of reporter plasmid pGL3-basic, Sptlc2 wild type or Sptlc2 DRE mutant, and 500 ng of pSV-βgal internal control. Sptlc2 DRE is a Sptlc2 promoter driven reporter construct or its mutated DRE form, and empty vector controls. The empty vector pGL4 and pGudLuc 6.1 (Cyp1a1 DRE-driven luciferase reporter) are served as the negative and positive controls, respectively. Upon completion of the transfection at 4 h in 0.5 mL OptiMEM media, an additional 0.5 mL of OptiMEM contain FBS (to a final concentration of 2%) and Pen/Strep was added for another 4 hrs to allow the cells to recover. Finally, the cells were treated for 4 hrs with the vehicle control (VEH, Dimethyl sulfoxide) or 20 nM TCDF. Following treatment, cells were lysed in lysis buffer (2 mM trans-1,2-diaminocyclohexane-N,N,N′ ,N′ -tetraacetic acid, 2 mM dithiothreitol, 10% glycerol, and 1% Triton X-100). Lysates were assayed for luciferase activity using the Promega luciferase assay system (Promega) as specified by the manufacturer. Light production was measured at room temperature using a TD-20e luminometer (Turner Designs). For normalization and transfection efficiency, β-galactosidase activity was measure at 410 nM using a colorimetric assay on the Tecan Infinite m200Pro plate reader. Luciferase activity was normalized against β-galactosidase activity and expressed as normalized luciferase activity.

2.7. UHPLC-MS quantitation of Ceramides.

Quantitation of hepatic ceramides was performed by multiple reaction monitoring (MRM) and/or parent ion scanning using a Waters UHPLC Acquity system coupled to a Waters Xevo TQMS with a Waters Acquity C18 BEH column (2.1 × 100 mm). The internal standard C17:0 ceramide was obtained from Sigma-Aldrich. Sample preparation and ceramide measurements were carried out as described previously (Jiang et al. 2015a).

2.8. Orbitrap-MS Analysis of Lipids.

Liver (25 mg) was extracted twice with 0.5 mL of pre-cooled isopropanol/water/ethyl acetate (30:10:60, v:v:v) containing 1:1000 Equisplash (Avanti) and 0.1 μM d7-C17-sphingosine. After homogenization (Precellys, Bertin Technologies, Rockville, MD) and centrifugation (Eppendorf, Hamburg, Germany), the supernatant was collected, evaporated to dryness (Thermo Scientific, Waltham, MA) and dissolved in 100 μL of isopropanol/acetonitrile/H2O (45:35:20, v:v:v). After centrifugation, supernatants were transferred to autosampler vials for LC-MS analysis.

Total lipids were analyzed by Vanquish UHPLC system coupled to an Orbitrap Fusion Lumos Tribrid™ mass spectrometer using a H-ESI™ ion source (all Thermo Fisher Scientific) with a Waters (Milford, MA) CSH C18 column (1.0 × 150 mm × 1.7 um particle size) as previously described(Tian et al. 2020). LC-MS data were analyzed by the open-source software MS-DIAL(Tsugawa et al. 2015), identifications were performed using the in-silico lipid library and the spectra was manually checked to confirm assignments. The experiment was processed in technical duplicate and the averages were used for statistical purposes.

2.9. ChIP-PCR/qPCR assay.

ChIP-PCR/qPCR assay was conducted by SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) according to the manufacturer’s instructions (Cell Signaling Technology, #9005). Briefly, liver tissue was collected, proteins and DNA were cross-linked with 1% formaldehyde in PBS at room temperature for 10 min. Chromatin was extracted and immunoprecipitated with 2 μg of antibodies to AHR (Enzo, BML-SA210-0100) at 4°C overnight. The immune complexes were collected by incubation with protein G magnetic beads and the beads were washed with low salt, high salt and ChIP buffer. After the elution of bound chromatin, the genomic DNA was purified using a spin column and subjected to PCR or qPCR using primers specific for the Cyp1a1, Sptlc2 and Asah1 promoter regions which are known to contain DREs or the non-binding region as a negative control.

2.10. Clinical Biochemistry.

Serum ALT and ALP were measured with VetScan VS2 using the Mammalian Liver Profile disk (Abaxis).

2.11. Statistical Data Analysis.

All the experimental values are presented as mean ± S.D.. Graphical illustrations and statistical analysis were performed with GraphPad Prism version 7.0 (GraphPad). Two-tailed Student’s t-test was used for multiple-comparison. P-values less than 0.05 were considered as significant difference.

3. Results

3.1. TCDF modulated liver enzymes and AHR signaling.

24 μg/kg body weight TCDF exposure led to significant elevation in the expression of the AHR target gene Cyp1a1 in mouse liver (Fig. S1A). Exposure to TCDF at 24 μg/kg body weight daily for five days resulted in slight but significant elevation of serum ALT and ALP levels in TCDF treated mice (Fig. S1B). In contrast, after 24 μg/kg body weight TCDF exposure, Ahr−/− mice exhibited no significant changes in liver enzymes ALT and ALP, or increased expression of Cyp1a1 mRNA in the liver.

3.2. TCDF regulates ceramide synthesis metabolism.

To investigate how TCDF affects the ceramide synthesis pathway, we analyzed sphingolipid metabolism of liver and serum from vehicle- and TCDF-treated mice via lipidomics and visualized via heatmap (Fig. 1 and Fig. S2). Significant differences were found in the levels of various lipid species. SMs (sphingomyelins) and PE-Cers (ceramide phosphoethanolamines) were depleted in TCDF-treated mouse liver while ceramides including Cer-NDS, Cer-AS, Cer-NS, and Cer-AP were elevated in TCDF-treated mouse liver (Fig. 1). Cer-NS and Cer-NDS were also found to increase in mouse serum (Fig. S2). In addition, ceramides in liver samples were quantified via triple quadrupole mass spectrometry. C16:0 (FC = 1.75, p < 0.01), C18:0 (FC = 1.85, p < 0.01), C18:1 (FC = 1.89, p < 0.05) and C24:0 (FC = 1.96, p < 0.05) ceramide levels were significantly elevated in TCDF-treated mice compared to levels in vehicle mice (Fig. 2C). However, no significant changes in ceramide levels were observed after treatment of Ahr−/− mice with TCDF.

Figure 1. AHR activation contributes to hepatic sphingolipid changes.

Figure 1.

Open in a new tab

Heatmap of log2 mean centered normalized sphingolipid data from vehicle and TCDF-treated mouse liver. The red and blue color of the tiles indicates high abundance and low abundance, respectively. log2 mean centered data were imported into R, and the ComplexHeatmap package was used to create the heatmap. Metabolites were sorted and separated into groups based on the metabolite category. Cer, Ceramide; PE-Cer, Ceramide phosphoethanolamines; SM: Sphingomyelin; HexCer: Hexosylceramide; SHerCer: sulfatide; NS, non-hydroxyfatty acid-sphingosine; NDS, non-hydroxyfatty acid-dihydrosphingosine; AS: alpha-hydroxy fatty acid-sphingosine; AP: alpha-hydroxy fatty acid-phytospingosine.

Figure 2. TCDF induced ceramide production and regulated gene expression in the ceramide biosynthesis pathway.

Figure 2.

Open in a new tab

(A) qPCR analysis of mRNA levels of ceramide biosynthesis-related genes (Smpd1, Smpd2, Smpd3 and Smpd4, Sptlc1, Sptlc2, Cers2, Cers4, Cser5, Cers6, Degs1, Degs2, Cerk, Sgms1, Sgms2, Ugcg, Asah1, Asah2) expression in vehicle and TCDF-treated mice, n = 5. (B) mRNA expression for genes important in the ceramide biosynthesis pathway in vehicle and TCDF-treated mouse liver. (C) Quantification of ceramides in vehicle and TCDF-treated WT and Ahr−/− mouse liver by UPLC-TQS-MS, n = 5. (D) N-SMase activity of vehicle and TCDF-treated mouse liver, n = 5. All data are presented as the mean ± S.D., *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed Student’s t-test.

Based on the changes of sphingolipid concentrations, we set out to examine how AHR influences the transcriptional regulation of genes encoding key enzymes involved in the ceramide synthesis pathway. Ceramide synthesis-related mRNAs such as serine palmitoyltransferse long-chain base subunits (Sptlc1, FC = 1.67; Sptlc2, FC = 1.35), ceramide synthases (Cers2, FC = 1.33; Cers6, FC = 1.53), acid ceramidase (Asah1, FC = 1.43), and sphingomyelin phosphodiesterase (Smpd1, FC = 1.56; Smpd2, FC = 1.36; Smpd3, FC = 2.13; Smpd4, FC = 1.38), were significantly up-regulated (p < 0.05) by TCDF in liver (Fig. 2A and 2B). These ceramide synthesis-related mRNAs were not significantly affected by TCDF exposure in Ahr−/− mice (Fig. S3). In addition, N-Smase activity (FC = 1.28, p < 0.001) was increased in the liver after TCDF treatment (Fig. 2D).

3.3. Ceramide synthesis gene Sptlc2 is targeted and regulated by AHR.

An examination of the Sptlc2 promoter (−3kb/+80) revealed the presence of two putative dioxin response elements (DREs) located at −2457 bp (DRE1) and −40 bp (DRE2) suggesting that the observed in vivo TCDF-mediated induction of Sptlc2 mRNA may be a consequence of direct AHR-dependent transcriptional activation of Sptlc2. EMSA performed using oligonucleotide probes spanning these putative DREs revealed minimal AHR association with DRE1. However robust TCDD-dependent AHR binding to DRE2 was observed, comparable to that observed with DREs located within the enhancer region of the prototypical AHR target Cyp1a1 (Fig. 3A).

Figure 3. Ceramide synthesis gene Sptlc2 is targeted by AHR.

Figure 3.

Open in a new tab

(A) In vitro translated murine AHRb-1/ARNT gel shift assay displaying the capacity of ligand-transformed murine AHR/ARNT heterodimer to bind 32P-labeled oligonucleotides containing DRE sequences from the promoter regions of Cyp1a1 and Sptlc2 (indicated by arrow). (B) Transient transfection of Hepa1 cells with a combination of murine AHR (Ahrb-1), Sptlc2 promoter driven luciferase constructs or its mutated DRE form to assess transcriptional regulation at the Sptlc2 promoter by the Ah receptor in the context of 20 nM TCDF treatments. (C) ChIP-PCR results for Cyp1a1, Sptlc2, and Asah1gene promoters in the vehicle and TCDF-treated mice, n = 3. (D) ChIP-qPCR results for Cyp1a1, Sptlc2 and Asah1 gene promoters that were quantified by normalization with the corresponding input signal, n = 3, data are presented as the mean ± S.D., *p < 0.05, **p < 0.01, ****p < 0.0001 by two-tailed Student’s t-test.

To determine if ligand-mediated binding of AHR to DRE2 within the promoter of Sptlc2 is functionally significant with regard to Sptlc2 induction, we generated a heterologous reporter construct comprising the −335/+80 bp Sptlc2 promoter cloned upstream of luciferase. Transient co-transfection of this reporter constructs together with AHR into Hepa1 cells and subsequent exposure to 20 nM TCDF for 4 h revealed a statistically significant 50% increase in Sptlc2 reporter activity while no significant change was found for Sptlc2 DRE mutant reporter after TCDF exposure (Fig. 3B). Thus, confirmation that TCDF-mediated AHR binding to DRE2 is permissive for Sptlc2 induction was established using a mutant reporter in which the minimal core consensus sequence of DRE2 was mutated to ablate AHR binding. It is important to note that some baseline activity of AHR in Hepa1 cells is expected given the ubiquitous presence of AHR ligands in the cell culture media and/or contamination from plasticizers (Roblin et al. 2004). These results support the observed in vivo induction of hepatic Sptlc2 following exposure to TCDF and indicate that elevated hepatic ceramide concentrations may be a consequence of direct AHR transcriptional activity upon rate-limiting components of the de novo ceramide synthesis.

To further investigate the potential ceramide gene targets of AHR, genomic DNA fragments bound by AHR were analyzed by ChIP assay (Fig. 3C–D). Immunoprecipitated DNA was amplified and quantified by qPCR with primers for Cyp1a1 (Cyp1a1 served as positive control), Sptlc2, and Asah1 gene promoters containing known and suspected DREs (Table S2). The PCR products for Cyp1a1 and Sptlc2 show a significant increase when viewed on an agarose gel. Consistently, the percentage of Cyp1a1 and Sptlc2 in AHR recognized genomic DNA fragments increased significantly after TCDF treatment; however, the Asah1 gene did not show significant change after TCDF treatment in both PCR and qPCR results. These results suggest that AHR binds directly to the Sptlc2 promoter.

The promoter from position +90 to −548 bp of the Sptlc2 sequence from mouse was aligned with the Sptlc2 promoter of human using CLUSTALW (Fig. 4). We found that the promoter of the mouse Sptlc2 gene is 72.4% identical to the corresponding human sequence, which is consistent with previous reports (Linn et al. 2006). Moreover, the position and sequence of DREs were also conserved between the two species. The apparent conservation highlights the DRE as an important functional element in Sptlc2/SPTLC2 promoters.

Figure 4. DRE of Sptlc2 conserved in mouse and human.

Figure 4.

Open in a new tab

The sequence of the putative dioxin responsive element (DRE) sites in the proximal promoter region (sequences spanning positions +90 through −548 bp) of mouse Sptlc2, and the alignments with the corresponding human SPTLC2 promoter sequences are shown. The asterisk “*” indicates positions which have a single, fully conserved residue. The gray color indicates the core DRE 5’-TNGCGTG-3’. The blue color indicates the primer region used for the ChIP assay.

3.4. AHR activation contributes to hepatic lipid accumulation.

It was reported that ceram ides are positively correlated with total cholesterol (TC) and triglycerides (TG) in human plasma (Ichi et al. 2006). Quantification of hepatic TG showed that hepatic TG content was significantly increased in mice after 24 μg/kg body weight TCDF treatment (Fig. 5A), which was confirmed by significant up-regulation of the triglyceride biosynthetic genes, diacylglycerol O-acyltransferase 1 (Dgat1) and diacylglycerol O-acyltransferase 2 (Dgat2) (Fig. 5B) . DGAT1 and DGAT2 are key enzymes catalyzing the final step in mammalian triglyceride synthesis (Millar et al. 2006). Sterol response element-binding protein 1c (Srebp1c), the hepatic lipogenesis-related gene, mRNA was also increased in mouse liver after TCDF treatment. TCDF did not alter the mRNA expression levels of Dgat1, Dgat2, and Srebp1c in Ahr−/− mice compared to WT mice. In addition, differences in levels of hepatic triglyceride (TG) were not observed in the presence or absence of TCDF in Ahr−/− mice. These observations suggest the TCDF contributes to hepatic lipogenesis which is associated with ceramide accumulation upon AHR activation.

Figure 5. AHR Activation contributes to TCDF-induced Hepatic Lipogenesis.

Figure 5.

Open in a new tab

(A) Quantification of liver triglycerides, n = 5 mice per group. (B) RT-qPCR analyses of Srebp1c, Dgat1 and Dgat2 mRNA levels in the liver of vehicle and TCDF-treated WT and Ahr−/− mice, n = 5 mice per group. Data are presented as mean ± S.D., *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed Student’s t-test.

4. Discussion and conclusion

Ceramides are key molecules in sphingolipid metabolism, which is a key cellular process that constitutes a highly complex network of interconnected pathways (Ségui et al. 2006). In this study, we discovered that TCDF activated the ceramide biosynthesis pathway by directly targeting the Sptlc2 gene through transcriptional regulation in mouse liver (Fig. 6). Exposure to TCDF resulted in significant elevation of ceramides, depletion of sphingomyelins, and glycosphingolipid disorder in mouse liver. Consistent with these findings, the levels of mRNAs and enzyme activity involved in ceramide biosynthetic pathways were increased in liver from TCDF-treated mice.

Figure 6. Schematic of sphingolipid metabolism and the enzymes directly participating in the ceramide synthesis pathway.

Figure 6.

Open in a new tab

Metabolic pathways for ceramide synthesis are composed of the de novo pathway, the sMase pathway, and the salvage pathway. Red and blue colors represent the genes or the metabolites upregulated and downregulated following TCDF treatment compared with the vehicle group. Black indicates no significant change or was not measured in this study. Cer, Ceramide; PE-Cer, Ceramide phosphoethanolamines; SPT, Serine Palmitoyltransferase; CerS, ceramide synthases; Des, dihydroceramide desaturases; CerK, ceramide kinase; N-SMase, Neutral Sphingomyelin phosphodiesterase; A-SMase, acid Sphingomyelin phosphodiesterase; Sms, Spermine synthase; CDase, neutral ceramidase; A-CDase, acid ceramidase; GCS, glucosylceramide synthase. Created with BioRender.com.

SPT catalyzes the first, rate-limiting step in the de novo ceramide synthesis pathway and is encoded by the genes Sptlc1 and Sptlc2. The active form of SPT is likely a heterodimer formed with SPT1 and SPT2 (Gault et al. 2010). Sptlc1 and Sptlc2 were significantly up-regulated following TCDF exposure in our study. Consistently, it has been reported that the ceramide content in plasma is positively correlated with the activity of hepatic SPT and serum SMase in humans (Pagadala et al. 2012). The function of SPT1 is thought to stabilize SPT2 in the ER rather than contributing to catalytic activity (Gault et al. 2010). A previous study reported that although the adenoviral expression of Sptlc1 or Sptlc2 increased the protein level of each SPT subunit, only the increase in Sptlc2 expression promoted the production of ceramide in HepG2 cells (Kim et al. 2020). This evidence suggests that Sptlc2 (rather than Sptlc1) expression is positively correlated with ceramide production. CerS1-6 have been identified in mammals and they are encoded by six distinct genes (Lahiri et al. 2007; Pewzner-Jung et al. 2006), which differ in tissue distribution and substrate specificity. CerS2 utilizes C20-C26 acyl CoA species and preferentially produces ultra-long chain ceramides species. CerS6 prefers palmitoyl CoA as substrates, mainly producing C16-ceramide (Laviad et al. 2008; Mullen et al. 2012). In our study, the expression of Cers2 and Cers6 was significantly increased in TCDF-exposed mice, and these findings are consistent with the elevation of C16:0 and C24:0 ceramide levels.

Ceramide is also produced by the hydrolysis of sphingomyelin catalyzed by N-SMase in the cell membranes. N-SMase activity increased significantly in TCDF-treated mouse liver which was supported by the up-regulation of Smpd2, Smpd3 and Smpd4, and an increase in ceramide levels and a decrease in SM levels. Changes in the enzymatic activity of N-SMase resulted in significant changes in ceramide levels. Others have reported similar changes in N-SMase activity and found them to be associated with disorders such as fatty liver disease (Jiang et al. 2015b).

Ceramide could be recycled by the ceramide synthase catalyzed reacylation of sphingosine which is generated from catabolism of complex sphingolipids in the salvage pathway. Our findings showed the upregulation of Asah1 in endo-lysosomes, which is associated with the intracellular glycosphingolipid disorder.

Previous RNA-Seq data published by the Zacharewski group (Fader et al. 2017) (Accession: GSE87519) reported increased expression of hepatic Cyp1a1 (FC = 2271.44 for 10 μg/kg TCDD and FC = 1770.99 for 30 μg/kg TCDD), Sptlc2 (FC = 1.22 for 10 μg/kg TCDD and FC= 1.81 for 30 μg/kg TCDD) and Asah1 (FC = 1.00 for 10 μg/kg TCDD and FC= 1.19 for 30 μg/kg TCDD) after 28 days of continuous treatment. Consistently, we also found significant increase for Cyp1a1 (FC = 599.30, p < 0.0001), Sptlc2 (FC = 1.35, p < 0.05) and Asah1 (FC = 1.43, p < 0.05) in liver qPCR data from mice treated with the dose of 24 μg/kg TCDF for 5 days. There may be several reasons for the modest fold change of Sptlc2 including high baseline transcriptional activity as well as the lower dose of TCDF used. Subtle changes with Sptlc2 gene expression have been noted in previous studies (Jiang et al. 2015b). However, the change in enzyme activity as well as the change in ceramide levels supports these modest changes in gene expression have clear physiologic impact. Moreover, it was found that AHR might associate with the promoter region of Sptlc2 on AHR ChIP-Seq dataset in the GEO database (Accession: GSE97634) (Fader et al. 2017). The max log2 (fold-change) of Cyp1a1, Sptlc2, and Asah1 peaks enriched by AHR was 4.49, 1.72, and 0.93, respectively, in liver samples from male C57BL/6 mice 2 h following a single oral dose of 30 μg/kg TCDD in the published ChlP-Seq dataset (Fader et al. 2017). Our findings confirm this change by showing a similar significant increase for the enrichment of Cyp1a1 (FC = 5.55, p < 0.05), Sptlc2 (FC = 2.27, p < 0.05) and Asah1 (FC = 1.32, p > 0.05) promoters which contain DREs in liver from TCDF treated mice (24 μg/kg, 5 days), although the fold change for Cyp1a1 by TCDF (24 μg/kg) treatment is about 1/3 of that by TCDD (30 μg/kg) treatment (Fader et al. 2017). Transcriptomic approaches for TCDD toxicity testing have been widely applied in vivo (Fader et al. 2019; Li et al. 2013), but relatively few studies have used TCDF, particularly at low doses (usually 0.3~3000 μg/kg) (Burgoon et al. 2009; Fader et al. 2017). Notably, our results show a significant increase of AHR target genes involved in the de novo ceramide synthesis pathway, which directly supports that TCDF induced de novo ceram ide biosynthesis at doses not associated with overt toxicity. Moreover, the high sequence conservation between the promoter of mouse and human along with the presence of conserved DREs in the regulatory region upstream of Sptlc2/SPTLC2 suggests that AHR activity is an important component of the signaling pathway in rodents and humans (Batzoglou et al. 2000).

TCDF exposure caused the accumulation of triglycerides in mouse liver through AHR activation at low dose, which was closely related to the significant increase in ceramide and related ceramide synthesis mRNA levels. Here the TCDF-associated increase in hepatic ceramides is a consequence of direct AHR-mediated transcription of Sptlc2, which encodes the key rate-limiting enzyme for de novo ceramide synthesis. This study revealed that TCDF-induced hepatic lipogenesis by activation of AHR is characterized by the increase of ceramides in mouse liver.

Our results establish the direct role of the AHR in the hepatic regulation of the key rate-limiting ceramide synthesis gene, Sptlc2. The evidence that AHR regulates the biosynthesis pathway of ceramide may be important for understanding the role AHR plays in liver lipid disorders including non-alcoholic fatty liver disease (NAFLD).

Supplementary Material

1

Acknowledgment

This work was supported by National Institute of Environmental Health Sciences [Grants ES004869 (GHP), ES019964 (GHP), ES028244 (GHP), and ES028288 (ADP), ES026684 (ADP), ES022186 (ADP), S10 OD021750-01A1 (ADP)] and the National Natural Science Foundation of China (Z.L.M., 21577169 and 21635006).

Footnotes

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Batzoglou S, Pachter L, Mesirov JP, Berger B and Lander ES 2000. Human and mouse gene structure: comparative analysis and application to exon prediction. Genome Res 10, 950–958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boverhof DR, Burgoon LD, Tashiro C, Chittim B, Harkema JR, Jump DB and Zacharewski TRJTS 2005. Temporal and dose-dependent hepatic gene expression patterns in mice provide new insights into TCDD-Mediated hepatotoxicity. 85, 1048–1063. [DOI] [PubMed] [Google Scholar]
  3. Burgoon LD, Ding Q, N’Jai A, Dere E, Burg AR, Rowlands JC, Budinsky RA, Stebbins KE and Zacharewski TR 2009. Automated dose-response analysis of the relative hepatic gene expression potency of TCDF in C57BL/6 mice. Toxicol Sci 112, 221–228. [DOI] [PubMed] [Google Scholar]
  4. Chiaro CR, Patel RD and Perdew GH 2008. 12(R)-Hydroxy-5(Z),8(Z), 10(E), 14(Z)-eicosatetraenoic acid [12(R)-HETE], an arachidonic acid derivative, is an activator of the aryl hydrocarbon receptor. Molecular pharmacology 74, 1649–1656. [DOI] [PubMed] [Google Scholar]
  5. Duval C, Teixeira-Clerc F, Leblanc AF, Touch S, Emond C, Guerre-Millo M, Lotersztajn S, Barouki R, Aggerbeck M and Coumoul X.J.E.h.p. 2017. Chronic exposure to low doses of dioxin promotes liver fibrosis development in the C57BL/6J diet-induced obesity mouse model. 125, 428–436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fader KA, Nault R, Doskey CM, Fling RR and Zacharewski TR 2019. 2,3,7,8-Tetrachlorodibenzo-p-dioxin abolishes circadian regulation of hepatic metabolic activity in mice. Sci Rep-Uk 9, 6514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fader KA, Nault R, Kirby MP, Markous G, Matthews J and Zacharewski TR 2017. Convergence of hepcidin deficiency, systemic iron overloading, heme accumulation, and REV-ERBα/β activation in aryl hydrocarbon receptor-elicited hepatotoxicity. Toxicology and applied pharmacology 321, 1–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gault CR, Obeid LM and Hannun YA 2010. An overview of sphingolipid metabolism: from synthesis to breakdown. Advances in experimental medicine and biology 688, 1–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Holland WL, Brozinick JT, Wang LP, Hawkins ED, Sargent KM, Liu Y, Narra K, Hoehn KL, Knotts TA, Siesky A, Nelson DH, Karathanasis SK, Fontenot GK, Birnbaum MJ and Summers SA 2007. Inhibition of ceramide synthesis ameliorates glucocorticoid-, saturated-fat-, and obesity-induced insulin resistance. Cell metabolism 5, 167–179. [DOI] [PubMed] [Google Scholar]
  10. Ichi I, Nakahara K, Miyashita Y, Hidaka A, Kutsukake S, Inoue K, Maruyama T, Miwa Y, Harada-Shiba M, Tsushima M, Kojo S and Kisei Cohort Study G 2006. Association of ceramides in human plasma with risk factors of atherosclerosis. Lipids 41, 859–863. [DOI] [PubMed] [Google Scholar]
  11. Jiang C, Xie C, Li F, Zhang L, Nichols RG, Krausz KW, Cai J, Qi Y, Fang ZZ, Takahashi S, Tanaka N, Desai D, Amin SG, Albert I, Patterson AD and Gonzalez FJ 2015a. Intestinal farnesoid X receptor signaling promotes nonalcoholic fatty liver disease. The Journal of clinical investigation 125, 386–402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jiang C, Xie C, Lv Y, Li J, Krausz KW, Shi J, Brocker CN, Desai D, Amin SG, Bisson WH, Liu Y, Gavrilova O, Patterson AD and Gonzalez FJ 2015b. Intestine-selective farnesoid X receptor inhibition improves obesity-related metabolic dysfunction. Nature Communications 6, 10166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kennedy LH, Sutter CH, Leon Carrion S, Tran QT, Bodreddigari S, Kensicki E, Mohney RP and Sutter TR 2013. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated production of reactive oxygen species is an essential step in the mechanism of action to accelerate human keratinocyte differentiation. Toxicol Sci 132, 235–249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kim GT, Kim SJ, Park SH, Lee D and Park TS 2020. Hepatic Expression of the Serine Palmitoyltransferase Subunit Sptlc2 Reduces Lipid Droplets in the Liver by Activating VLDL Secretion. Journal of lipid and atherosclerosis 9, 291–303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kim JH, Matsubara T, Lee J, Fenollar-Ferrer C, Han K, Kim D, Jia S, Chang CJ, Yang H, Nagano T, Krausz KW, Yim SH and Gonzalez FJ 2021. Lysosomal SLC46A3 modulates hepatic cytosolic copper homeostasis. Nat Commun 12, 290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lahiri S, Lee H, Mesicek J, Fuks Z, Haimovitz-Friedman A, Kolesnick RN and Futerman AH 2007. Kinetic characterization of mammalian ceramide synthases: determination of K(m) values towards sphinganine. FEBS letters 581, 5289–5294. [DOI] [PubMed] [Google Scholar]
  17. Laviad EL, Albee L, Pankova-Kholmyansky I, Epstein S, Park H, Merrill AH Jr. and Futerman AH 2008. Characterization of ceramide synthase 2: tissue distribution, substrate specificity, and inhibition by sphingosine 1-phosphate. J Biol Chem 283, 5677–5684. [DOI] [PubMed] [Google Scholar]
  18. Lee JH, Wada T, Febbraio M, He J, Matsubara T, Lee MJ, Gonzalez FJ and Xie WJG 2010. A novel role for the dioxin receptor in fatty acid metabolism and hepatic steatosis. 139, 653–663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Li ZH, Xu H, Zheng W, Lam SH and Gong Z 2013. RNA-sequencing analysis of TCDD-induced responses in zebrafish liver reveals high relatedness to in vivo mammalian models and conserved biological pathways. PLoS One 8, e77292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Linn SC, Andras LM, Kim H-S, Wei J, Nagiec MM, Dickson RC and Merrill AH 2006. Functional characterization of the promoter for the mouse SPTLC2 gene, which encodes subunit 2 of serine palmitoyltransferase. FEBS letters 580, 6217–6223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Majumder S, Kono M, Lee YT, Byrnes C, Li C, Tuymetova G and Proia RL 2020. A genome-wide CRISPR/Cas9 screen reveals that the aryl hydrocarbon receptor stimulates sphingolipid levels. J Biol Chem 295, 4341–4349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Millar JS, Stone SJ, Tietge UJ, Tow B, Billheimer JT, Wong JS, Hamilton RL, Farese RV Jr. and Rader DJ 2006. Short-term overexpression of DGAT1 or DGAT2 increases hepatic triglyceride but not VLDL triglyceride or apoB production. Journal of lipid research 47, 2297–2305. [DOI] [PubMed] [Google Scholar]
  23. Muenyi CS, Carrion SL, Jones LA, Kennedy LH, Slominski AT, Sutter CH and Sutter TR 2014. Effects of in utero exposure of C57BL/6J mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin on epidermal permeability barrier development and function. Environ Health Perspect 122, 1052–1058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mullen TD, Hannun YA and Obeid LM 2012. Ceramide synthases at the centre of sphingolipid metabolism and biology. Biochem J 441, 789–802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nault R, Fader KA, Lydic TA and Zacharewski TR 2017. Lipidomic Evaluation of Aryl Hydrocarbon Receptor-Mediated Hepatic Steatosis in Male and Female Mice Elicited by 2,3,7,8-Tetrachlorodibenzo-p-dioxin. Chemical research in toxicology 30, 1060–1075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pagadala M, Kasumov T, McCullough AJ, Zein NN and Kirwan JP 2012. Role of ceramides in nonalcoholic fatty liver disease. Trends in endocrinology and metabolism: TEM 23, 365–371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pewzner-Jung Y, Ben-Dor S and Futerman AH 2006. When do Lasses (longevity assurance genes) become CerS (ceramide synthases)?: Insights into the regulation of ceramide synthesis. J Biol Chem 281, 25001–25005. [DOI] [PubMed] [Google Scholar]
  28. Plotegher N, Bubacco L, Greggio E and Civiero L 2019. Ceramides in Parkinson’s Disease: From Recent Evidence to New Hypotheses. 13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Poss AM and Summers SA 2020. Too Much of a Good Thing? An Evolutionary Theory to Explain the Role of Ceramides in NAFLD. Front Endocrinol (Lausanne) 11, 505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Roblin S, Okey AB and Harper PA 2004. AH receptor antagonist inhibits constitutive CYP1A1 and CYP1B1 expression in rat BP8 cells. Biochemical and biophysical research communications 317, 142–148. [DOI] [PubMed] [Google Scholar]
  31. Ségui B, Andrieu-Abadie N, Jaffrézou J-P, Benoist H and Levade T.J.B.e.B.A.-B. 2006. Sphingolipids as modulators of cancer cell death: potential therapeutic targets. 1758, 2104–2120. [DOI] [PubMed] [Google Scholar]
  32. Tanos R, Patel RD, Murray IA, Smith PB, Patterson AD and Perdew GHJH 2012. Aryl hydrocarbon receptor regulates the cholesterol biosynthetic pathway in a dioxin response element - independent manner. 55, 1994–2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tian Y, Gui W, Rimal B, Koo I, Smith PB, Nichols RG, Cai J, Liu Q and Patterson AD 2020. Metabolic impact of persistent organic pollutants on gut microbiota. Gut Microbes 12, 1848209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tsugawa H, Cajka T, Kind T, Ma Y, Higgins B, Ikeda K, Kanazawa M, VanderGheynst J, Fiehn O and Arita M 2015. MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis. Nature methods 12, 523–526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Xia JY, Morley TS and Scherer PE 2014. The adipokine/ceramide axis: key aspects of insulin sensitization. Biochimie 96, 130–139. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1714100-supplement-1.docx (9MB, docx)

RESOURCES