Transcriptional profiling of PPARα−/− and CREB3L3−/− livers reveals disparate regulation of hepatoproliferative and metabolic functions of PPARα
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Peroxisome Proliferator-Activated receptor α (PPARα) and cAMP-Responsive Element Binding Protein 3-Like 3 (CREB3L3) are transcription factors involved in the regulation of lipid metabolism in the liver. The aim of the present study was to characterize the interrelationship between PPARα and CREB3L3 in regulating hepatic gene expression. Male wild-type, PPARα−/−, CREB3L3−/− and combined PPARα/CREB3L3−/− mice were subjected to a 16-h fast or 4 days of ketogenic diet. Whole genome expression analysis was performed on liver samples.
Under conditions of overnight fasting, the effects of PPARα ablation and CREB3L3 ablation on plasma triglyceride, plasma β-hydroxybutyrate, and hepatic gene expression were largely disparate, and showed only limited interdependence. Gene and pathway analysis underscored the importance of CREB3L3 in regulating (apo)lipoprotein metabolism, and of PPARα as master regulator of intracellular lipid metabolism. A small number of genes, including Fgf21 and Mfsd2a, were under dual control of PPARα and CREB3L3. By contrast, a strong interaction between PPARα and CREB3L3 ablation was observed during ketogenic diet feeding. Specifically, the pronounced effects of CREB3L3 ablation on liver damage and hepatic gene expression during ketogenic diet were almost completely abolished by the simultaneous ablation of PPARα. Loss of CREB3L3 influenced PPARα signalling in two major ways. Firstly, it reduced expression of PPARα and its target genes involved in fatty acid oxidation and ketogenesis. In stark contrast, the hepatoproliferative function of PPARα was markedly activated by loss of CREB3L3.
These data indicate that CREB3L3 ablation uncouples the hepatoproliferative and lipid metabolic effects of PPARα. Overall, except for the shared regulation of a very limited number of genes, the roles of PPARα and CREB3L3 in hepatic lipid metabolism are clearly distinct and are highly dependent on dietary status.
KeywordsLiver CREB3L3 PPARα Fasting Ketogenic diet Transcriptomics
cAMP-Responsive Element Binding Protein 3-Like 3
Gene Set Enrichment Analysis
non-esterified fatty acids
Peroxisome Proliferator-Activated Receptor
Selective PPAR Modulator
The liver plays a critical role in the metabolic response to changes in the diet. An important regulatory mechanism in the control of metabolism is via changes in the expression of relevant genes. Indeed, changes in nutrient composition and nutrient availability trigger profound changes in the hepatic expression of numerous genes involved in glucose and lipid metabolism. An important transcription factor that is involved in the adaptive response to changes in nutrient supply is PPARα [1, 2]. PPARα is a member of the family of nuclear receptors and part of the subfamily of Peroxisome Proliferators Activated Receptors, which also includes PPARβ/δ and PPARγ . The PPARs share a common mode of action that involves heterodimerization with the nuclear receptor RXR (Retinoid X Receptor), followed by binding of the PPAR-RXR complex to specific DNA sequences in the regulatory regions of target genes [4, 5, 6]. Activation of transcription is triggered by binding of a ligand, which include fatty acids and fatty acid derivatives such as eicosanoids and oxidized fatty acids, as well as a variety of synthetic compounds collectively referred to as peroxisome proliferators .
Evidence abounds indicating that PPARα is crucial for the transcriptional regulation of hepatic lipid metabolism during fasting. Indeed, studies employing expression profiling of whole body or liver-specific PPARα−/− mice have demonstrated that PPARα induces the expression of hundreds of genes involved in nearly every branch of hepatic lipid metabolism [8, 9, 10, 11, 12]. Hence, PPARα can be aptly described as the master regulator of hepatic lipid metabolism, especially under conditions of elevated hepatic lipid load, as occurs during fasting, high fat feeding, and a ketogenic diet. In line with this notion, the absence of PPARα during fasting leads to a host of metabolic disturbances, including a fatty liver, elevated plasma non-esterified fatty acids, hypoglycemia and hypoketonemia [8, 9, 10, 11, 12].
Cyclic AMP-responsive element-binding protein 3 Like 3 (CREB3L3, encoded by Creb3l3) is a bZiP transcription factor that is highly expressed in the liver . CREB3L3 is produced as an ER precursor form and is proteolytically activated in the Golgi to liberate the N-terminal portion that functions as a transcriptional activator . Growing evidence implicates CREB3L3 in the regulation of glucose and lipid metabolism in the liver . Specifically, CREB3L3 has been shown to stimulate gluconeogenesis  and glycogenolysis , plasma triglyceride clearance , and lipid droplet formation .
Both PPARα and CREB3L3 are activated in the liver by fasting and play important roles in the utilization of fatty acids for energy in the fasted state [8, 9, 10, 19]. Several lines of evidence point to an interaction between PPARα- and CREB3L3-mediated gene regulation. First, PPARα has been shown to regulate CREB3L3 expression in human and mouse hepatocytes , likely via a PPRE located upstream of exon 3 , indicating that Creb3l3 is a direct PPARα target gene. Second, there is strong evidence that PPARα and CREB3L3 cooperate in the regulation of certain genes such as Fgf21, encoding Fibroblast Growth Factor 21. Specifically, PPARα and CREB3L3 physically interact to form a complex that binds to an integrated CRE-PPAR-responsive element-binding motif in the Fgf21 gene promoter . The physical interaction between PPARα and CREB3L3 is enhanced by fasting and dependent on CREB3L3 acetylation at K294 . More recently, it was shown that during fasting, PPARα and CREB3L3 also cooperate in the stimulation of hepatic gluconeogenesis by targeting genes such as Pck1, encoding Phosphoenolpyruvate Carboxykinase 1 . Other genes that are under dual control of PPARα and CREB3L3 in liver include Cidec, encoding Cell Death Inducing DFFA Like Effector C [18, 24]. The data presented above suggest that part of the effects of PPARα may be mediated by CREB3L3 and point towards cooperativity in gene regulation by PPARα and CREB3L3. Based on the analysis of the phenotype of single and combined PPARα−/− and CREB3L3−/− mice, it was proposed that CREB3L3 co-operates with PPARα by directly and indirectly regulating the expression of genes involved in fatty acid oxidation and ketogenesis .
To further characterize the cooperativity between PPARα and CREB3L3 in hepatic gene regulation, we studied the effect of PPARα and CREB3L3 ablation, either individually or combined, on overall hepatic gene regulation using whole genome expression profiling, in mice after a 16-h fast and after 4 days of ketogenic diet.
Effect of PPARα and/or CREB3L3 ablation on fasting plasma metabolites
Effects of PPARα and CREB3L3 ablation on hepatic gene expression in the fasted state are largely independent
To study the potential similarity between the effect of PPARα and CREB3L3 deficiency on liver gene expression, we performed principle component analysis (Fig. 2c) and hierarchical clustering (Fig. 2d). Both analyses indicated the separate clustering of the four experimental groups, with limited variation between the individual mice in each group, and the more pronounced effect of PPARα deficiency compared to CREB3L3 deficiency. In addition, both analyses showed that the whole genome effects of combined PPARα/CREB3L3 deficiency are largely taken up by PPARα deficiency (Fig. 2c, d).
Hierarchical biclustering of samples and genes visualized in a heatmap further supported the conclusions reached above, showing the much more pronounced effect of PPARα deficiency on hepatic gene expression and the more significant contribution of PPARα towards the effect of combined PPARα/CREB3L3 deficiency (Fig. 2e). The heat map also shows that for certain genes, the effects of PPARα and CREB3L3 deficiency are additive and seemingly independent, whereas for other genes the effect of PPARα and CREB3L3 deficiency appears to be synergistic and thus dependent.
A limited number of genes is commonly downregulated by PPARα and CREB3L3 deficiency in liver during fasting
CREB3L3 deficiency leads to downregulation of genesets related to lipoprotein and lipid transport
Consistent with the notion that the effects of combined PPARα/CREB3L3 deficiency are largely taken up by PPARα deficiency, the far majority of genesets downregulated in the combined PPARα/CREB3L3−/− mice were also downregulated in the PPARα−/− mice (Fig. 6d). Indeed, the enrichment scores of the most highly downregulated genesets in the combined PPARα/CREB3L3−/− mice were very similar in the single PPARα−/− mice, suggesting that the functional impact of combined PPARα/CREB3L3−/− deficiency is mostly accounted for by deficiency of PPARα. The exception were two genesets related to lipoprotein and lipid transport, which had similar enrichment scores in the combined PPARα/CREB3L3−/− mice and single CREB3L3−/− mice (Fig. 6d), suggesting that the regulation of these two genesets is driven by CREB3L3 deficiency.
Deficiency of PPARα led to the upregulation of genesets related to the unfolded protein response and inflammatory signalling (Additional file 1: Figure S1A). By contrast, deficiency of CREB3L3 led to upregulation of genesets related to cholesterol synthesis and protein translation (Additional file 1: Figure S1B). Consistent with this result, genes involved in cholesterol metabolism feature prominently among the top 40 most highly upregulated genes in CREB3L3−/− mice (Additional file 1: Figure S1C).
Overall, the above analyses indicate that the effects of PPARα and CREB3L3 deficiency on hepatic gene expression during fasting are very distinct. Only a limited number of genes is under regulation of both PPARα and CREB3L3. The PPARα/CREB3L3−/− mice reflect the combined effect of especially PPARα and to a lesser extent CREB3L3 deficiency, showing a minor degree of synergism.
Effect of PPARα and/or CREB3L3 deficiency on plasma metabolites during ketogenic diet
Effects of CREB3L3 deficiency on hepatic gene expression during ketogenic diet are dependent on PPARα
To study the similarity between the three different genetic models in liver gene expression, we performed principle component analysis (Fig. 8c) and hierarchical clustering (Fig. 8d). Principle component analysis and hierarchical clustering of samples showed that the CREB3L3−/− mice formed a distinct cluster, underscoring the profound effect of CREB3L3 deficiency on hepatic gene expression during ketogenic diet. Surprisingly, the PPARα−/− mice and combined PPARα/CREB3L3−/− mice clustered together and were very distinct from the CREB3L3−/− mice. Hierarchical biclustering of samples and genes visualized in a heatmap further confirmed that at the level of hepatic gene expression, the PPARα−/− mice and combined PPARα/CREB3L3−/− mice were nearly indistinguishable, whereas the CREB3L3−/− mice showed a very different gene expression profile (Fig. 8e). These data thus show that deficiency of CREB3L3 has no effect on hepatic gene expression in the absence of PPARα, indicating that the major liver phenotype triggered by CREB3L3 deficiency during ketogenic diet is dependent on PPARα.
Scatter plot analysis confirmed that the effects of PPARα and CREB3L3 deficiency on hepatic gene expression are very dissimilar, whereas the effect of PPARα deficiency and combined PPARα/CREB3L3 deficiency are similar (Additional file 1: Figure S2A). Venn diagram of significantly changed genes confirmed that deficiency of CREB3L3 leads to the up- and downregulation of a large set of genes that are not affected in the PPARα−/− or PPARα/CREB3L3−/− mice (Additional file 1: Figure S2B).
Induction of mitogenic genes in CREB3L3−/− mice during ketogenic diet is mediated by PPARα
In line with the known mitogenic effect of PPARα activation on hepatocyte proliferation, pharmacological activation of PPARα in vivo has been shown to cause the induction of numerous genes and proteins involved in cell cycle control , which is specifically mediated by mouse PPARα and not human PPARα . Previously, we found that treating mice with the specific PPARα agonist Wy-14,643 markedly induced numerous genesets related to cell cycle . A heatmap of the most highly enriched genes in the geneset Mitotic.M.M.G1 phase underscores the marked induction of cell cycle-related genes by Wy-14,643, which is entirely PPARα dependent (Fig. 10d). Strikingly, most of these genes are also highly upregulated in the CREB3L3−/− mice on ketogenic diet, which again is entirely PPARα dependent (Fig. 10d), indicating that the pronounced upregulation of the cell cycle in CREB3L3−/− mice is mediated by PPARα. Taken together, these data indicate that CREB3L3 deficiency uncouples the hepatoproliferative and lipid metabolic effects of PPARα.
In this paper we studied the effect of individual and combined PPARα and CREB3L3 deficiency on hepatic gene expression after a 16-h fast and a 4-day ketogenic diet. Under conditions of overnight fasting, the effect of PPARα deficiency and CREB3L3 deficiency on hepatic gene expression are largely independent, and only show a very limited degree of synergism. A small number of genes is under dual control of PPARα and CREB3L3, including Fgf21 and Mfsd2a. Our data do not support a strong co-dependence of PPARα and CREB3L3 in hepatic gene regulation during fasting. By contrast, a strong interaction between PPARα and CREB3L3 exists during ketogenic diet feeding. Previously, it was shown that CREB3L3−/− mice on a ketogenic diet exhibit a strong phenotype characterized by hepatomegaly and steatohepatitis, and elevated expression of inflammatory marker genes [19, 25]. Here, using whole genome expression profiling, we corroborate these findings. In addition, we show that deficiency of CREB3L3 has virtually no effect on hepatic gene expression in the absence of PPARα, indicating that the major liver phenotype triggered by CREB3L3 deficiency during ketogenic diet is dependent on PPARα. Furthermore, we find that CREB3L3 has a dual impact on PPARα signalling during ketogenic diet. On the one hand, CREB3L3 deficiency leads to reduced expression of PPARα and PPARα target genes involved in fatty acid oxidation and ketogenesis. On the other hand, CREB3L3 deficiency leads to the marked activation of the hepatoproliferative effect of PPARα. Overall, our data suggest that CREB3L3 deficiency during ketogenic diet uncouples the mitogenic and lipid metabolic effects of PPARα in the liver.
It is unclear how CREB3L3 deficiency promotes liver damage and hepatoproliferation during ketogenic diet and how this effect is dependent on PPARα. It could be envisioned that deficiency of CREB3L3 disrupts a certain metabolic pathway, such as fatty acid oxidation or fatty acid elongation and desaturation, leading to accumulation of intermediate lipid species that ligand-activate PPARα and specifically stimulate the mitogenic action of PPARα. In addition, these lipid species may promote liver damage. Additionally, it is possible that CREB3L3 deficiency alters a specific metabolic pathway, possibly involving accumulation of damaging intermediates, and that these effects are dependent on an enzyme/factor whose expression is maintained by PPARα. Insofar as CREB3L3 and PPARα regulate the expression of many genes, it is not possible to pinpoint the exact causal gene(s) downstream of CREB3L3 and PPARα.
Other examples exist of the uncoupling of the mitogenic and metabolic actions of PPARα. For example, human PPARα upregulates genes involved in fatty acid oxidation but not the cell cycle, as shown by studies in mice carrying human PPARα [29, 30]. Another example is the activation of mouse PPARα by dietary n-3 poly-unsaturated fatty acids, which leads to upregulation of PPARα targets involved in lipid metabolism but does not trigger hepatocyte proliferation . These findings strongly indicate that the mechanisms by which PPARα affects lipid metabolism and hepatocyte proliferation are distinct [29, 30]. Mechanistically, how ligand-activated PPARα could selectively activate mitogenic and not metabolic pathways is unclear but could be related to the SPPARM concept [32, 33, 34]. According to this concept, different PPAR agonists have only partially overlapping effects on gene expression based on selective receptor-coregulator interactions. Borrowing from this notion, it can be hypothesized that the epigenetic mechanisms that drive the PPARα-dependent activation of genes involved in fatty acid oxidation and ketogenesis are different from the epigenetic mechanisms that support the induction of mitogenic pathways by PPARα, and additionally that these mechanisms are differentially affected by CREB3L3 deficiency.
An intriguing question is why CREB3L3 deficiency leads to a pronounced phenotype in mice fed a ketogenic but has much more limited effects in fasted mice. Direct comparison of hepatic gene expression in wild-type mice after fasting and ketogenic diet showed that expression of SREBP1 and its target genes involved in lipogenesis and cholesterogenesis was much higher after the ketogenic diet than after fasting (not shown). Previously, it was shown that CREB3L3 is a negative regulator of SREBP-1c production and hepatic lipogenesis , which is in line with our observation that genes involved in lipogenesis/cholesterogenesis are highly elevated in CREB3L3−/− mice under regular fasting conditions. Hence, placing CREB3L3−/− mice on a ketogenic diet is expected to lead to markedly increased lipogenesis/cholesterogenesis, which in turn may lead to the generation of a specific (set of) lipids that could trigger the mitogenic effect of PPARα (Fig. 11b). The upregulation of cholesterogenesis upon CREB3L3 deficiency in the fasted state is seemingly at odds with a previous study that suggested that CREB3L3 stimulates lipogenesis and cholesterogenesis . However, closer inspection at the individual gene levels shows substantial correspondence and indicates that CREB3L3 downregulates SREBP-dependent genes.
Despite lower expression of Cidec, which promotes lipid droplet formation [41, 42], PPARα−/−, CREB3L3−/−, and PPARα/CREB3L3−/− mice have elevated hepatic triglyceride levels. Similarly, expression of Plin5, which also promotes hepatic fat storage , is lower in PPARα−/− mice, despite these mice showing more pronounced steatosis. Accordingly, these data suggest that the elevated hepatic triglycerides in the PPARα−/−, CREB3L3−/−, and PPARα/CREB3L3−/− mice are not mediated by changes in Cidec and Plin5 expression. It should be noted that an increase in liver triglycerides does not necessarily have to be accompanied by elevated hepatic expression of Cidec and/or Plin5.
One limitation of our study is that we used whole body PPARα−/− and CREB3L3−/− mice. Ideally, it would have been better to use liver-specific PPARα and CREB3L3 deficient mice. Nevertheless, due to the high expression of PPARα and CREB3L3 in liver, we believe the results presented here reflect the hepatic function of the two transcription factors [2, 13]. An additional limitation is that we did not unveil the molecular details of the interaction between PPARα and CREB3L3 during ketogenic diet. These aspects should be further addressed in future studies.
We find that PPARα and CREB3L3 regulate distinct genes in the liver during fasting, with the exception of a limited number of common targets such as Fgf21. Strikingly, deficiency of CREB3L3 in mice during ketogenic diet uncouples the hepatoproliferative and metabolic effects of PPARα. Our data underscore the distinct functions of PPARα and CREB3L3 in the regulation of hepatic gene expression.
CREB3L3−/− mice were backcrossed onto a C57BL/6 background at least 10 times . PPARα−/− mice that had been backcrossed on a pure C57Bl/6J background for more than 10 generations were acquired from Jackson Laboratories (no. 008154, B6;129S4-Pparatm1Gonz/J) . The two lines were interbred to generate combined PPARα/CREB3L3−/− mice. Mice were housed in a specific pathogen free facility at the Weill Cornell Medical College on a 12 h light/dark cycles and fed ad libitum standard chow diet (PicoLab Rodent diet 20, #5058, Lab diet). The four different mouse lines (wild-type, PPARα−/−, CREB3L3−/−, and PPARα/CREB3L3−/−) were either fasted for 16 h or fed a ketogenic diet for 4 days (# F3666, Bio-Serv). The mice used for experiments were all male and approximately 8 weeks old. The euthanasia was carried out at around 10 a.m., with the ketogenic diet group being non-fasted (ad libitum fed). Blood was taken by orbital puncture under isoflurane anesthesia, followed by euthanasia of the mice by cervical dislocation. Tissues were excised and immediately frozen in liquid nitrogen followed by storage at − 80 °C.
For the adenoviral-mediated CREB3L3 overexpression, two-month-old male mice were injected intravenously via the tail vein at a dose of 3 × 10^9 particles of the adenoviruses per g body weight in 0.15 ml of saline. Mice injected with GFP-expressing adenovirus were used as control. Mice were euthanized four days after adenovirus injection and livers of three mice per group were used for whole genome expression profiling as detailed below. All animal experiments were approved by the Institutional Animal Care and Use Committee at Weill Cornell Medical College (Protocol #2012–0048) and performed in accordance with the approved guidelines.
Plasma triglycerides, non-esterified fatty acids, ketone bodies, alanine aminotransferase, and FGF21 concentrations were determined using assay kits (Serum Triglyceride Determination Kit, Sigma; NEFA-HR (2), Wako Chemicals; Autokit Total Ketone Bodies, Wako Chemicals; ALT Kit, Bio-Quant; Mouse/Rat FGF-21 Quantikine ELISA Kit, R&D Systems; Human FGF-21 Quantikine ELISA Kit, R&D Systems). Lipids were extracted from liver tissues with chloroform/methanol mixture (2:1 v/v), as described previously .
Microarray analysis was performed on liver samples using 3–4 biological replicates per group. Total RNA was extracted from cells using TRIzol reagent (Life Technologies, Bleiswijk, The Netherlands) and subsequently purified using the RNeasy Micro kit (Qiagen, Venlo, The Netherlands). RNA integrity was verified with RNA 6000 Nano chips on an Agilent 2100 bioanalyzer (Agilent Technologies, Amsterdam, The Netherlands). Purified RNA (100 ng) was labelled with the Ambion WT expression kit (Carlsbad, CA) and hybridized to an Affymetrix Mouse Gene 1.1 ST array plate (Affymetrix, Santa Clara, CA). Hybridization, washing, and scanning were carried out on an Affymetrix GeneTitan platform according to the manufacturer’s instructions. Normalized expression estimates were obtained from the raw intensity values applying the robust multi-array analysis preprocessing algorithm available in the Bioconductor library AffyPLM with default settings [46, 47]. Probe sets were defined according to Dai et al. . In this method probes are assigned to Entrez IDs as a unique gene identifier. In this study, probes were reorganized based on the Entrez Gene database, build 37, version 1 (remapped CDF v22). The P values were calculated using an Intensity-Based Moderated T-statistic (IBMT) . Genes were defined as significantly changed when P < 0.001.
Geneset enrichment analysis (GSEA) was used to identify gene sets that were enriched among the upregulated or downregulated genes . Genes were ranked based on the IBMT-statistic and subsequently analyzed for over- or underrepresentation in predefined genesets derived from Gene Ontology, KEGG, National Cancer Institute, PFAM, Biocarta, Reactome and WikiPathways pathway databases. Only genesets consisting of more than 15 and fewer than 500 genes were taken into account. Statistical significance of GSEA results was determined using 1000 permutations.
Statistical analysis of the transcriptomics data was performed as described in the previous paragraph. Statistical analysis of the other parameters was performed by two-way ANOVA and Student’s t-test. Data are presented as mean ± SEM. P < 0.05 was considered statistically significant.
This research was supported by CVON ENERGISE grant CVON2014–02. This funding body had no role in the design of the study, collection, analysis, and interpretation of data, or in writing the manuscript.
Availability of data and materials
The datasets generated and/or analyzed during the current study are available in the Gene Expression Omnibus repository: GSE121096.
SK and AHL conceived and designed the study. PMMR, JGP, XX and KYH acquired and analyzed the data. SK and PMMR drafted the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All animal experiments were approved by the Institutional Animal Care and Use Committee at Weill Cornell Medical College (Protocol #2012–0048) and performed in accordance with the approved guidelines.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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