The aryl hydrocarbon receptor activates ceramide biosynthesis in mice contributing to hepatic lipogenesis

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); Ceramide; Lipogenesis; Sptlc2.

Figure 1.. AHR activation contributes to hepatic sphingolipid changes

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

(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.

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

(A) In vitro translated murine AHR^b-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.

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

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.

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

(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.

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

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.