Comparative gene expression profiles induced by PPARγ and PPARα/γ agonists in human hepatocytes

Abstract

Background: Several glitazones (PPARγ agonists) and glitazars (dual PPARα/γ agonists) have been developed to treat hyperglycemia and, simultaneously, hyperglycemia and dyslipidemia, respectively. However, most have caused idiosyncratic hepatic or extrahepatic toxicities through mechanisms that remain largely unknown. Since the liver plays a key role in lipid metabolism, we analyzed changes in gene expression profiles induced by these two types of PPAR agonists in human hepatocytes.

Methodology/principal findings: Primary human hepatocytes and the well-differentiated human hepatoma HepaRG cells were exposed to different concentrations of two PPARγ (troglitazone and rosiglitazone) and two PPARα/γ (muraglitazar and tesaglitazar) agonists for 24 h and their transcriptomes were analyzed using human pangenomic Agilent microarrays. Principal Component Analysis, hierarchical clustering and Ingenuity Pathway Analysis® revealed large inter-individual variability in the response of the human hepatocyte populations to the different compounds. Many genes involved in lipid, carbohydrate, xenobiotic and cholesterol metabolism, as well as inflammation and immunity, were regulated by both PPARγ and PPARα/γ agonists in at least a number of human hepatocyte populations and/or HepaRG cells. Only a few genes were selectively deregulated by glitazars when compared to glitazones, indicating that PPARγ and PPARα/γ agonists share most of their target genes. Moreover, some target genes thought to be regulated only in mouse or to be expressed in Kupffer cells were also found to be responsive in human hepatocytes and HepaRG cells.

Conclusions/significance: This first comprehensive analysis of gene regulation by PPARγ and PPARα/γ agonists favor the conclusion that glitazones and glitazars share most of their target genes and induce large differential changes in gene profiles in human hepatocytes depending on hepatocyte donor, the compound class and/or individual compound, thereby supporting the occurrence of idiosyncratic toxicity in some patients.

Figure 1. Intracellular ATP content in primary human hepatocytes and HepaRG cells treated with PPARγ or PPARα/γ agonists.

Intracellular ATP content was measured in cells treated with TRO, ROSI, MURA or TESA for 24 h. Results are normalized to control cells and expressed as means ± S.D. of three independent experiments.* p<0.05, N.T.: non tested.

Figure 2. Caspase 3 activity in HepaRG cells treated with PPARγ or PPARα/γ agonists.

Caspase 3 levels were determined in HepaRG cells after a 24 h treatment with TRO, ROSI, MURA or TESA. Staurosporine (2 µM) was used as a positive control. Results are normalized to control cells and expressed as means ± S.D. of three independent experiments.* p<0.05.

Figure 3. ROS levels in HepaRG cells treated by PPARγ or PPARα/γ agonists.

ROS levels were estimated by measurement of intracellular DCFDA in HepaRG cells after a 24 h treatment by TRO, ROSI, MURA or TESA. Menadione (2 mM) was used as a positive control. Results are normalized to control cells and expressed as means ± S.D. of three independent experiments.* p<0.05.

Figure 4. PPARα and PPARγ1 transcript levels in freshly isolated hepatocytes, primary human hepatocytes and HepaRG cells.

Comparative expression of PPARα and PPARγ1 in freshly isolated hepatocytes (FIH), primary human hepatocytes (PHH) and differentiated HepaRG cells incubated in a medium containing 0.01% DMSO. The results are expressed relative to 18S and are the mean ± S.D.of at least three independent experiments.

Figure 5. PPARγ protein level in primary human hepatocytes and HepaRG cells.

Primary human hepatocytes and differentiated HepaRG cells were incubated in a medium containing 0.01% DMSO. The results are expressed as optical density per 100 µg total protein and are the mean ± S.D. of at least three independent experiments. ns, not statistically significant.

Figure 6. Two-dimensional hierarchical clustering of gene expression profiles in hepatocytes treated with PPAR agonists.

The clustering was generated by using Resolver system software with an agglomerative algorithm Ward's min variance link heuristic criteria and Euclidean distance metric (FC≥1.5 and p≤0.01). Two-dimensional clustering was performed on gene expression profiles in hepatocytes treated with glitazones (a) and glitazars (b).

Figure 7. Venn diagram representation of differentially expressed genes in glitazone- and glitazar-treated hepatocytes.

Venn diagrams showed overlap of gene signatures (FC≥1.5 and p≤0.01) in at least two concentrations of each glitazone and glitazar in PHH (a) and HepaRG cells (b).

Figure 8. NTCP activity in primary human hepatocytes and HepaRG cells after 24 h treatment with PPAR agonists.

NTCP activity was determined at two concentrations of TRO, ROSI, MURA or one concentration of TESA. Each bar chart colour represents a cell condition treatment. Results are normalized to control cells and expressed as means ± S.D.of three independent experiments.* p<0.05.