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. 2015 Oct 8:16:760.
doi: 10.1186/s12864-015-1969-3.

The impact of PPARα activation on whole genome gene expression in human precision cut liver slices

Aafke W F Janssen  1 , Bark Betzel  2 , Geert Stoopen  3 , Frits J Berends  4 , Ignace M Janssen  5 , Ad A Peijnenburg  6 , Sander Kersten  7
Affiliations

The impact of PPARα activation on whole genome gene expression in human precision cut liver slices

Aafke W F Janssen et al. BMC Genomics. .

Abstract

Background: Studies in mice have shown that PPARα is an important regulator of lipid metabolism in liver and key transcription factor involved in the adaptive response to fasting. However, much less is known about the role of PPARα in human liver.

Methods: Here we set out to study the function of PPARα in human liver via analysis of whole genome gene regulation in human liver slices treated with the PPARα agonist Wy14643.

Results: Quantitative PCR indicated that PPARα is well expressed in human liver and human liver slices and that the classical PPARα targets PLIN2, VLDLR, ANGPTL4, CPT1A and PDK4 are robustly induced by PPARα activation. Transcriptomics analysis indicated that 617 genes were upregulated and 665 genes were downregulated by PPARα activation (q value < 0.05). Many genes induced by PPARα activation were involved in lipid metabolism (ACSL5, AGPAT9, FADS1, SLC27A4), xenobiotic metabolism (POR, ABCC2, CYP3A5) or the unfolded protein response, whereas most of the downregulated genes were involved in immune-related pathways. Among the most highly repressed genes upon PPARα activation were several chemokines (e.g. CXCL9-11, CCL8, CX3CL1, CXCL6), interferon γ-induced genes (e.g. IFITM1, IFIT1, IFIT2, IFIT3) and numerous other immune-related genes (e.g. TLR3, NOS2, and LCN2). Comparative analysis of gene regulation by Wy14643 between human liver slices and primary human hepatocytes showed that down-regulation of gene expression by PPARα is much better captured by liver slices as compared to primary hepatocytes. In particular, PPARα activation markedly suppressed immunity/inflammation-related genes in human liver slices but not in primary hepatocytes. Finally, several putative new target genes of PPARα were identified that were commonly induced by PPARα activation in the two human liver model systems, including TSKU, RHOF, CA12 and VSIG10L.

Conclusion: Our paper demonstrates the suitability and superiority of human liver slices over primary hepatocytes for studying the functional role of PPARα in human liver. Our data underscore the major role of PPARα in regulation of hepatic lipid and xenobiotic metabolism in human liver and reveal a marked immuno-suppressive/anti-inflammatory effect of PPARα in human liver slices that may be therapeutically relevant for non-alcoholic fatty liver disease.

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Figures

Fig. 1
Fig. 1
Classical PPARα targets genes are robustly induced by PPARα activation in human PCLS. a Expression level of PPARα in human liver biopsies (n = 15) and human PCLS (n = 5). b Gene expression changes of selected PPARα target genes in human PCLS in response to 24 h Wy14643 treatment as determined by quantitative real-time PCR. c Gene expression changes of the same genes according to microarray. Error bars represent SEM. Asterisk indicates statistically significant (P < 0.05, Student's t-test)
Fig. 2
Fig. 2
Top 25 genes induced or repressed by Wy14643 in human PCLS. Heatmap showing gene expression changes of the top 25 most significantly induced (a) and repressed (b) genes in human PCLS treated with Wy14643 for 24 h, as determined by microarray analysis. Genes were ranked based on statistical significance in the form of q-value (IBMT regularised paired t-test). P1 to P4 represent the four human subjects that donated a liver specimen for preparation of PCLS
Fig. 3
Fig. 3
Top 20 gene sets induced or repressed by Wy14643 in human PCLS. The top 20 most significantly induced or repressed pathways in human PCLS in response to 24 h Wy14643 treatment were determined by gene set enrichment analysis. Gene sets were ranked according to normalized enrichment score (NES)
Fig. 4
Fig. 4
Comparative analysis of effect Wy14643 on gene expression in human PCLS and primary hepatocytes. a The number of differentially expressed genes in human PCLS and human primary hepatocytes in response to 24 h Wy14643 treatment in a common geneset of 17,351 genes was calculated based on a statistical significance cut-off of P < 0.001 (IBMT regularised paired t-test) and fold-change >1.20. Genes were separated according to up- or down-regulation. b Correlation plot showing changes in gene expression in response to Wy14643 (expressed as signal log ratio) in human PCLS (x-axis) and primary human hepatocytes (y-axis). Selected PPARα target genes commonly induced by Wy14643 in PCLS and hepatocytes are highlighted in red. Selected inflammation-related genes specifically repressed by Wy14643 in PCLS are highlighted in blue. Selected lipid metabolism-related genes specifically induced by Wy14643 in primary hepatocytes are highlighted in green. c Volcano plot showing relative changes in gene expression in response to Wy14643 (expressed as signal log ratio, x-axis) plotted against statistical significance (expressed as IBMT regularised paired t-test P-value, y-axis) for the PCLS and primary hepatocytes
Fig. 5
Fig. 5
Comparative heatmap analysis of effect Wy14643 on gene expression in human PCLS and primary hepatocytes. Genes that were statistically significantly regulated by Wy14643 in human PCLS (P < 0.001, IBMT regularised paired t-test) were ranked according to fold-change in expression. The top 40 genes with highest fold-induction (a) or fold-repression (b) are shown. Expression changes of the same gene set in primary hepatocytes is shown in the right panel. P1 to P4 represent the four human subjects that donated a liver specimen for preparation of PCLS. P5 to P10 represent the six human subjects that donated a liver specimen for preparation of human hepatocytes
Fig. 6
Fig. 6
Regulation of selected gene sets by Wy14643 in human PCLS and primary hepatocytes. Heatmap showing gene expression changes of enriched genes that are part of the gene sets “metabolism of xenobiotics by cytochrome P450” (a), “IRE1a activated chaperones” (b), and “Interferon alpha beta signaling” (c) in human PCLS and primary hepatocytes. P1 to P4 represent the four human subjects that donated a liver specimen for preparation of PCLS. P5 to P10 represent the six human subjects that donated a liver specimen for preparation of human hepatocytes
Fig. 7
Fig. 7
Overview of regulation of lipid metabolism by PPARα in human liver. A detailed overview map was created of metabolic genes upregulated by PPARα in human liver based on transcriptomics analysis of human PCLS and primary hepatocytes treated with Wy14643. Genes indicated in red are significantly induced by Wy14643 in human PCLS and primary hepatocytes. Genes indicated in green are significantly induced by Wy14643 in human PCLS but not primary hepatocytes. Genes indicated in blue are significantly induced by Wy14643 in human primary hepatocytes but not PCLS. Statistical significance was determined by IBMT regularised paired t-test (P < 0.01)
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