Enhancing fatty acid oxidation negatively regulates PPARs signaling in the heart

Abstract

High fatty acid oxidation (FAO) is associated with lipotoxicity, but whether it causes lipotoxic cardiomyopathy remains controversial. Molecular mechanisms that may be responsible for FAO-induced lipotoxic cardiomyopathy are also elusive. In this study, increasing FAO by genetic deletion of acetyl-CoA carboxylase 2 (ACC2) did not induce cardiac dysfunction after 16 weeks of high fat diet (HFD) feeding. This suggests that increasing FAO, per se, does not cause metabolic cardiomyopathy in obese mice. We compared transcriptomes of control and ACC2 deficient mouse hearts under chow- or HFD-fed conditions. ACC2 deletion had a significant impact on the global transcriptome including downregulation of the peroxisome proliferator-activated receptors (PPARs) signaling and fatty acid degradation pathways. Increasing fatty acids by HFD feeding normalized expression of fatty acid degradation genes in ACC2 deficient mouse hearts to the same level as the control mice. In contrast, cardiac transcriptome analysis of the lipotoxic mouse model (db/db) showed an upregulation of PPARs signaling and fatty acid degradation pathways. Our results suggest that enhancing FAO by genetic deletion of ACC2 negatively regulates PPARs signaling through depleting endogenous PPAR ligands, which can serve as a negative feedback mechanism to prevent excess activation of PPAR signaling under non-obese condition. In obesity, excessive lipid availability negates the feedback mechanism resulting in over activation of PPAR cascade, thus contributes to the development of cardiac lipotoxicity.

Keywords: Fatty acid oxidation; Lipotoxicity; PPAR; Transcriptomics.

Figure 1.. Four months’ HFD feeding did not alter cardiac function in ACC2 iKO mice.

(A) Schematic demonstration of the experimental procedure. (B-F) Con and ACC2 iKO mice were subjected to HFD feeding for 16 weeks. (B) Body weight was measured. (C-D) Hearts were perfused with a buffer containing ¹³C labeled fatty acids, ¹³C labeled glucose, lactate, and insulin (mixed substrates). (C) Contribution of ¹³C labeled substrates to tricarboxylic acid (TCA) cycle was determined by ¹³C NMR spectroscopy in heart extracts. Relative contribution of fatty acids, glucose, and other unlabeled substrates (lactate, endogenous) is reported as fold changes over Con-chow (dotted line) (*p<0.05 vs. Con/chow, #p<0.05 vs. Con/HFD, n=3–6). (D) Left ventricular pressure–volume relationship for end diastolic pressure is shown (n=3–6). (E) Left ventricular ejection fraction (LVEF %) was measured by echocardiography (n=4–6). (F) Evaluation of ROS generation by TBARS assay. Malondialdehyde (MDA) was quantified colorimetrically to monitor lipid peroxidation (*p<0.05 vs. Con/chow, #p<0.05 vs. Con/HFD, n=4).

Figure Figure .. Global transcriptome analysis in Con and ACC2 iKO mouse hearts under chow or HFD feeding.

(A) Principle component analysis (PCA) of RNA-seq data sets from Con and ACC2 iKO mouse hearts after 16-weeks chow and HFD feeding. PCA is based upon the abundance of all the transcripts detected in RNA-seq analysis. (B-C) Patterns of differential gene expression in ACC2 iKO mouse hearts compared with Con under chow-fed (B) and HFD-fed (C) conditions were analyzed by Enhanced Volcano Package. Red dots represent genes with p value < 0.001. (D) The number of shared DEGs between ACC2 iKO and Con mouse hearts under chow-fed and HFD-fed conditions was shown by Venn diagrams.

Figure 3.. Pathways enrichment and TF binding analysis of the downregulated DEGs in ACC2 iKO mouse heart under chow-fed condition.

(A) Pathway enrichment analysis of the downregulated genes in ACC2 iKO hearts under chow-fed condition was conducted using Cytoscape with ClueGo and CluePedia plugins. Top 10 clusters are shown. Adjusted p value was represented by dot color. Gene numbers were represented by dot size. Rich factor indicated the percentage of genes in the pathway. (B) Transfac Positional Weight Matrix for PPARα (upper panel) and PPARγ (lower panel). Motif name is attached. PPARs and RXR binds to direct repeat of a nuclear receptor-binding site (AGGTCA) spaced by one nucleotide. (C–D) The downregulated DEGs in ACC2 iKO hearts were subjected to transcriptional factor binding analysis. A network representing the selected targets (oval yellow nodes) regulated by the top potential transcriptional factor PPARα (C) and PPARγ (D) were shown. (E-F) Con and ACC2 iKO mice were subjected to HFD feeding for 16 weeks. ChIP-qPCR assays were performed with anti-PPARα (E) and anti-PPARγ (F) antibodies, respectively. The enrichment of individual PPARs’ targets was calculated as % of input DNA. (*p<0.05 vs. Con/chow, n=4–6).

Figure 4.. PPAR signaling and fatty acid degradation genes are upregulated in lipotoxic hearts but not in ACC2 iKO mouse hearts.

Pathway enrichment analysis of the downregulated DEGs in HFD-fed mice (A) and db/db mice (C) (GEO accession: GSE36875, p-value < 0.01) compared to their respective control. Pathway enrichment analysis of the upregulated DEGs in HFD-fed mice (B) and db/db mice (D) compared to their respective control.

Figure 5.. Differential transcriptional regulation of fatty acid degradation genes by FAO and fatty acid supply.

(A) Heatmap of FAO related gene transcription levels in ACC2 iKO/chow, Con/HFD, db/db and ACC2 iKO/HFD hearts. Color coding for each gene was assigned using a log2 fold change versus the mean value of Con/chow. (B) Heatmap of bioactive lipids in Con and ACC2 iKO hearts with chow or HFD feeding. Color coding for each gene was assigned using a log2 fold change versus the mean value of Con/chow. (C) Cardiac triglyceride (TG) content normalized to tissue weight (*p<0.05 vs. Con/chow, #p<0.05 vs. Con/HFD, n=5). (D) Schematic working hypothesis. Ligands mediated PPAR activation and enhanced FAO in HFD induced obesity or db/db mice may contribute to the development of lipotoxicity. On the other hand, enhancing FAO by ACC2 deletion may deplete intracellular fatty acids, which can serve as a negative feedback to prevent over activation of PPAR pathway.