- The whole transcriptome effects of the PPAR α agonist fenofibrate on livers of hepatocyte humanized mice. - The aim of the present study was to better characterize the impact of PPAR α activation on gene regulation in human liver. - To that end, chimeric mice containing hepatocyte humanized livers were given an oral dose of 300 mg/kg fenofibrate daily for 4 days. - Results: Of the different human liver models, the gene expression profile of hepatocyte humanized livers most closely resembled actual human liver. - In the hepatocyte humanized mouse livers, the human hepatocytes exhibited excessive lipid accumulation. - Fenofibrate increased the size of the mouse but not human hepatocytes, and tended to reduce steatosis in the human hepatocytes. - According to gene set enrichment analysis, fenofibrate upregulated interferon/cytokine signaling-related pathways in hepatocyte humanized liver, but downregulated these pathways in normal mouse liver. - downregulated pathways related to DNA synthesis in hepatocyte humanized liver but not in normal mouse liver.. - Keywords: Hepatocyte humanized mice, Fenofibrate, PPAR α , Transcriptomics, Lipid metabolism, DNA synthesis. - to replacement of the host hepatocytes at a repopulation rate exceeding . - An important advantage of the hepatocyte humanized livers is that the hepatocytes still replicate, in contrast to cultured human primary he- patocytes or liver slices. - First, we compared the whole genome expression profile of human liver biopsies with the whole genome expression profile of hepatocyte humanized mouse livers, human pri- mary hepatocytes, and human precision-cut liver slices.. - Interestingly, for highly expressed genes, hepatocyte humanized livers more closely resembled actual human liver tissue as compared to human primary hepatocytes and human precision-cut liver slices, as reflected by the smaller scatter at the high expression range.. - Besides the scatter analysis, we utilized principle compo- nent analysis to compare the transcriptome of human liver of different types of subjects (normal, healthy obese, steatosis, and NASH) with hepatocyte humanized mouse liver and other liver models, including human primary he- patocytes and human liver slices (Fig. - As a final validation of the model, mRNA expression levels of PPAR α in hepatocyte humanized livers were similar to the levels measured in human liver biopsies (Fig. - These data support the notion that hepatocyte humanized livers are a suitable model for human liver, with some restrictions.. - To study the effect of PPARα activation on gene ex- pression in hepatocyte humanized livers, chimeric mice with hepatocyte humanized livers were given fenofibrate or vehicle at a daily oral dose of 300 mg/kg for 4 days.. - Histological examination of the H&E-stained livers showed clearly distinctive clusters of human and mouse hepatocytes.. - A ten- dency toward reduced steatosis by fenofibrate was ob- served in the sections of the liver populated by human hepatocytes (Fig. - Transcriptomics was carried out on human liver biopsies (GSE hepatocyte humanized livers, primary human hepatocytes (GSE and human precision-cut liver slices (GSE . - a Scatter plot analysis of normalized expression values comparing the whole genome expression profile of the human liver biopsies (x-axis) with the whole genome expression profile of the hepatocyte humanized livers (left panel), human precision-cut liver slices (middle panel), and human primary hepatocytes (right panel) (all y-axis). - b Genes that were expressed at much higher levels in human liver biopsies than in hepatocyte humanized livers ( Δ 2 log[normalized mean expression value] >. - c Principle component analysis was performed to compare the transcriptome of human liver of different types of subjects (normal, healthy obese, steatosis, and NASH) with hepatocyte humanized mouse liver and other liver models, including human primary hepatocytes and human liver slices. - Principle component analysis of the transcriptomics data showed that the livers of the fenofibrate-treated hepatocyte humanized mice clearly separated from the livers of the control-treated hepatocyte humanized mice (Fig. - A dendogram confirmed the separate clustering of the two sets of samples (Fig. - 2 Fenofibrate does not cause any changes in basic parameters in hepatocyte humanized mice. - Chimeric mice containing hepatocyte humanized livers were treated with 300 mg/kg fenofibrate daily for 4 days. - a mRNA expression of PPAR α in 15 human liver biopsies collected during bariatric surgery and in liver samples from 3 chimeric mice containing hepatocyte humanized livers.. - e Histological examination of livers of chimeric mice containing hepatocyte humanized livers that received control or fenofibrate treatment. - To compare the effect of fenofibrate in normal mouse liver and hepatocyte humanized liver, we performed a comparative analysis of the three tran- scriptomics datasets. - 3 Parallel induction of mouse and human PPAR α target genes by fenofibrate in hepatocyte humanized livers. - a qPCR was performed on cDNA generated from livers of control-treated and fenofibrate-treated mice containing hepatocyte humanized livers, using human primers (upper panel) or mouse primers (lower panel). - b Concentration of ANGPTL4 in plasma of control-treated and fenofibrate-treated mice containing hepatocyte humanized livers, as determined by ELISA. - These data suggest that in vivo, human liver cells are less sensitive to the effect of fenofibrate as compared to mouse liver cells, confirming the results of the qPCR on the hep- atocyte humanized livers. - Unfortunately, no transcripto- mics dataset was available from normal mice treated with fenofibrate at the same dose and for the same duration as the hepatocyte humanized mice.. - To further compare the effects of fenofibrate between normal mouse liver and hepatocyte humanized liver, we performed gene set enrichment analysis (GSEA). - expected, pathways covering PPARα signaling and fatty acid oxidation featured prominently among the most significantly induced pathways in both normal mouse liver and hepatocyte humanized liver (Fig. - 8, showing a consistent pattern of upregu- lation in hepatocyte humanized liver and normal mouse liver. - Surprisingly, certain immune-related pathways such as interferon signaling were strongly upregulated in hepatocyte humanized liver, but were markedly down- regulated in normal mouse liver (Fig. - The differential regulation of the geneset INTERFERO- N.ALPHA.BETA.SIGNALING by fenofibrate in hepato- cyte humanized liver and normal mouse liver is visualized in Fig. - 4 Distinct clustering of livers of control-treated and fenofibrate-treated hepatocyte humanized mice. - Transcriptomics was performed on livers of chimeric mice containing hepatocyte humanized livers. - The dendogram reveals the distinct clustering and separation of the fenofibrate and control groups. - The changes in gene expression are expressed relative to the mean of the control group as a signal log ratio. - pathways by fenofibrate in hepatocyte humanized liver were related to DNA synthesis (Fig. - The differential regulation of the geneset DNA.STRAN- D.ELONGATION by fenofibrate between hepatocyte humanized liver and normal mouse liver is visualized in Fig. - Activation of the pre-replicative complex_Homo sapiens_R-HSA-68962 G2/M Checkpoints_Homo sapiens_R-HSA-69481. - 5 Pathway analysis of differential gene expression between control-treated and fenofibrate-treated hepatocyte humanized mice.. - Transcriptomics was performed on livers of hepatocyte humanized mice treated with 300 mg/kg fenofibrate daily for 4 days (n = 3) or vehicle (control, n = 3). - in hepatocyte humanized liver, which also was not seen in normal mouse liver (Fig. - Overall, GSEA shows that the effects of fenofibrate in normal mouse liver and hepatocyte humanized liver are quite distinct, especially in relation to DNA synthesis pathways and interferon signaling pathways.. - The studies were not carried out with fenofi- brate but with Wy14,643, another PPARα agonist, pre- cluding a whole genome comparison with the study in chimeric mice carrying hepatocyte humanized livers.. - Nevertheless, we took the top 40 most highly induced genes by fenofibrate in hepatocyte humanized livers and compared the fenofibrate-induced expression changes with the Wy-14,643-induced expression changes in hu- man primary hepatocytes and human precision cut liver slices (Fig. - The most apparent difference was the regulation of several interferon-sensitive genes, including IFI6, IFITM1, PSMB9 and ISG15, which were upregu- lated by fenofibrate in the hepatocyte humanized mouse livers but downregulated by Wy-14,643 in human pri- mary hepatocytes and human precision cut liver slices.. - By harbouring clusters of mouse and hu- man hepatocytes, the hepatocyte humanized livers are. - The main findings of the present study are: 1) The hepatocyte humanized livers recapitulate the principal effects of PPARα activation on lipid metabolism revealed by other model systems of human liver. - cytokine signalling were upregulated by fenofibrate in mice with hepatocyte humanized livers, yet are downregu- lated by fenofibrate in normal mouse liver. - 5) Chimeric mice with hepatocyte humanized livers can be used to study the effect of activation of PPARα and other nuclear receptors on secretion of hepatokines into plasma.. - The position of the fenofibrate-induced genes in cellular lipid metabolism is illustrated in Fig. - Hepatocyte humanized mouse livers, 4 days. - 6 Comparative analysis of the effect of fenofibrate in normal mouse liver and hepatocyte humanized liver. - study, we found that PPARα activation in chimeric mice with hepatocyte humanized livers causes the downregu- lation of genes and pathways connected to DNA synthe- sis, further strengthening the notion that the effects of PPARα activation on DNA synthesis, cell proliferation and hepato-carcinogenesis are distinct between mouse liver and human liver. - The differential regulation is viv- idly illustrated by MCM4, which was markedly downreg- ulated by fenofibrate in hepatocyte humanized liver but strongly upregulated by fenofibrate in normal mouse liver. - One of the major discrepancies between the effect of PPARα activation in hepatocyte humanized liver and. - 7 Comparative pathway analysis of the effect of fenofibrate in normal mouse liver and hepatocyte humanized liver. - Gene set enrichment analysis was performed on the effect of treatment of chimeric mice containing hepatocyte humanized livers with 300 mg/kg fenofibrate daily for 4 days (n = 3 per group), and on the effect of treatment of wildtype mice with fenofibrate for 14 days via the feed (0.03 wt/wt, equivalent to approximately 1 mg/mouse/day, n = 8 per group). - a The top 20 most highly upregulated genesets in hepatocyte humanized livers (left panel) and normal mouse livers (right panel). - b The top 20 most highly downregulated genesets in hepatocyte humanized livers (left panel) and normal mouse livers (right panel). - Whereas PPARα activation causes the downregulation of inter- feron signaling in mouse liver, it led to upregulation of interferon signaling in hepatocyte humanized liver. - Whether the upregulation of interferon signaling by PPARα activation in hepatocyte humanized liver is an arte- fact of the interaction between human hepatocytes and mouse Kupffer cells, is related to the immune-deficiency in the SCID host mice, or in fact most accurately reflects the response to PPARα activation in human liver remains to be established.. - Another pathway that appears to be differentially regu- lated by PPARα in hepatocyte humanized liver and nor- mal mouse liver is complement and coagulation. - With respect to the complement pathway, previously we demonstrated that mannose-binding lectin (MBL2)—the primary component of the lectin branch of the complement system — is a target of PPAR α and is markedly induced by PPAR α activation in primary hepato- cytes and human liver slices [21, 39]. - However, mRNA levels of MBL2 and other complement-related genes were not altered by fenofibrate in hepatocyte humanized liver. - 8 Effect of fenofibrate on specific genesets in normal mouse liver and hepatocyte humanized liver. - Gene set enrichment analysis was performed on the effect of treatment of chimeric mice containing hepatocyte humanized livers with 300 mg/kg fenofibrate daily for 4 days (n = 3 per group). - Control Fenofibrate Wy-14,643 Wy-14,643 Hepatocyte humanized. - A) The top 40 most significantly induced genes by fenofibrate in hepatocyte humanized livers were ranked according to IBMT P-value. - Interestingly, in hepatocyte humanized mouse liver, fenofibrate treatment did not sig- nificantly change the expression of APOA1 and APOC3 mRNA, while it increased mRNA levels of APOA4 (fold-- change = 3.9, P <. - The residual presence of mouse hepatocytes in the hepatocyte humanized mouse livers allowed for direct comparison of the effect of fenofibrate on human and mouse hepatocytes. - The use of chimeric mice with hepatocyte humanized livers in principle allows for study of the secretion of hu- man liver proteins into the blood. - Indeed, we were able to detect human ANGPTL4 in blood plasma of mice with hepatocyte humanized livers. - Furthermore, follow- ing the induction of ANGPTL4 mRNA by PPAR α , treat- ment of the mice with fenofibrate significantly increased plasma ANGPTL4 levels. - Overall, these data sug- gest that chimeric mice with hepatocyte humanized livers are a suitable model to study the secretion of hu- man liver proteins into the blood.. - In our study, the livers of the hepatocyte humanized mice were very fatty, which was observed specifically in the liver sections populated by human hepatocytes. - In conclusion, using transcriptomics, we show that chimeric mice containing hepatocyte humanized livers are a highly valuable tool to study the in vivo function of PPARα in human liver. - Sequences of the primers used are listed in Table 1. - YI performed the animal study and contributed to the writing of the manuscript. - CT designed the animal study and contributed to the writing of the manuscript. - SK is responsible for the integrity of the work as a whole. - Chimeric mice with hepatocyte-humanized liver as an appropriate model to study human peroxisome proliferator- activated receptor-alpha. - Interaction of the peroxisome-proliferator-activated receptor and retinoid X receptor. - The retinoid X receptor enhances the function of the peroxisome proliferator activated receptor.. - Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. - Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. - evaluation of the risk of hypolipidemic drugs and industrial plasticizers to humans. - The role and regulation of the peroxisome proliferator activated receptor alpha in human liver. - Genomewide comparison of the inducible transcriptomes of nuclear receptors CAR, PXR and PPARalpha in primary human hepatocytes.. - Mechanism of action of the. - nongenotoxic peroxisome proliferators: role of the peroxisome proliferator- activator receptor alpha. - A map of the PPARalpha transcription regulatory network for primary human hepatocytes.
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