Comparative Transcriptional Network Modeling of Three PPAR-α/γ Co-Agonists Reveals Distinct Metabolic Gene Signatures in Primary Human Hepatocytes

Aims: To compare the molecular and biologic signatures of a balanced dual peroxisome proliferator-activated receptor (PPAR)-α/γ agonist, aleglitazar, with tesaglitazar (a dual PPAR-α/γ agonist) or a combination of pioglitazone (Pio; PPAR-γ agonist) and fenofibrate (Feno; PPAR-α agonist) in human hepatocytes. Methods and Results: Gene expression microarray profiles were obtained from primary human hepatocytes treated with EC50-aligned low, medium and high concentrations of the three treatments. A systems biology approach, Causal Network Modeling, was used to model the data to infer upstream molecular mechanisms that may explain the observed changes in gene expression. Aleglitazar, tesaglitazar and Pio/Feno each induced unique transcriptional signatures, despite comparable core PPAR signaling. Although all treatments inferred qualitatively similar PPAR-α signaling, aleglitazar was inferred to have greater effects on high- and low-density lipoprotein cholesterol levels than tesaglitazar and Pio/Feno, due to a greater number of gene expression changes in pathways related to high-density and low-density lipoprotein metabolism. Distinct transcriptional and biologic signatures were also inferred for stress responses, which appeared to be less affected by aleglitazar than the comparators. In particular, Pio/Feno was inferred to increase NFE2L2 activity, a key component of the stress response pathway, while aleglitazar had no significant effect. All treatments were inferred to decrease proliferative signaling. Conclusions: Aleglitazar induces transcriptional signatures related to lipid parameters and stress responses that are unique from other dual PPAR-α/γ treatments. This may underlie observed favorable changes in lipid profiles in animal and clinical studies with aleglitazar and suggests a differentiated gene profile compared with other dual PPAR-α/γ agonist treatments

Clustering of the hypotheses into broader biologic processes/pathways.

<p>Numbers (in the boxes) indicate the number of genes/hypotheses in molecular pathway clusters.</p

Treatment-induced RNA and gene expression changes.

<p>(<b>A</b>) Total number of RNA state changes (up- or downregulation) in human hepatocytes treated with aleglitazar, tesaglitazar or Pio/Feno. (<b>B</b>) Unique and shared gene expression following treatment with aleglitazar, tesaglitazar or Pio/Feno.</p

Cellular stress response-associated RNA changes and inferred signaling pathways.

<p>(<b>A</b>) Biologic processes associated with the cellular stress response inferred to increase or decrease upon treatment with aleglitazar, tesaglitazar or Pio/Feno. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035012#pone-0035012-g002" target="_blank"><i>Figure 2A</i></a> for treatment dosages. As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035012#pone-0035012-g005" target="_blank"><i>Figure 5A</i></a>, proxy hypotheses are shown. (<b>B</b>) Molecular signaling pathway, predicted by RNA state changes shown in panel A, that could lead to changes in the stress response. Numbers in the blue or yellow boxes indicate the number of gene expression state changes that support the inference: negative numbers indicate an inferred decrease and positive numbers indicate an inferred increase. Numbers in the green or red boxes indicate the log fold change. * Denotes genes/pathways responsive to the agent cited. ER, endoplasmic reticulum; Med, medium; ROS, reactive oxygen species.</p

Molecular pathways modulated in response to treatment with aleglitazar, tesaglitazar or Pio/Feno.

<p>Number of RNA expression changes observed following treatment with aleglitazar, tesaglitazar or Pio/Feno. Of the 2,000 unique mechanisms represented in the knowledgebase, 280 were considered statistically significant in at least one treatment condition and are designated as “hypotheses”.</p

PPAR-α signaling, lipid metabolism-associated RNA changes and inferred signaling pathways.

<p>(<b>A</b>) Observed RNA state changes and Causal Network Modeling–based predictions of PPAR-α signaling and lipid metabolism upon treatment with aleglitazar, tesaglitazar or Pio/Feno. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035012#pone-0035012-g002" target="_blank"><i>Figure 2A</i></a> for treatment dosages. “Bile duct ligation” is known as a “proxy” hypothesis and describes the finding that changes in gene expression observed in the current experiments are consistent with gene expression modulations in other experiments where bile duct ligation was the perturbation. Similarly “Response to osmotic stress”, “Reponse to shear stress”, “Corticotropin”, “Response to DNA damage stimulus”, “shear stress” and “Response to stress-inducing agents” (e.g. nelfinavir) are also proxy hypotheses. (<b>B</b>) Molecular signaling pathways, predicted by RNA state changes shown in panel A, that could lead to changes in lipid parameters. Depiction is based on data derived from treatment with the medium concentration of aleglitazar. Numbers in the blue or yellow boxes indicate the number of gene expression state changes that support the inference: negative numbers indicate an inferred decrease and positive numbers indicate an inferred increase. Numbers in the green or red boxes indicate the log fold change. HDL, high-density lipoprotein; LDL, low-density lipoprotein; Med, medium; TG, triglycerides.</p

Insulin signaling and biologic response-associated RNA changes and inferred signaling pathways.

<p>(<b>A</b>) Observed RNA state changes related to insulin signaling and biologic processes inferred to increase or decrease upon treatment with aleglitazar, tesaglitazar or Pio/Feno. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035012#pone-0035012-g002" target="_blank"><i>Figure 2A</i></a> for treatment dosages. (<b>B</b>) Molecular signaling pathway that could lead to changes in insulin signaling to decrease cellular proliferation. Depiction is based on data derived from treatment with the medium concentration of aleglitazar. Numbers in the blue or yellow boxes indicate the number of gene expression state changes that support the inference: negative numbers indicate an inferred decrease and positive numbers indicate an inferred increase. Numbers in the green or red boxes indicate the log fold change. AMP, adenosine monophosphate; CDK, cyclin-dependent kinase; Med, medium.</p