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. 2020 Jun 29;11(7):720.
doi: 10.3390/genes11070720.

Cardiac Transcriptome Analysis Reveals Nr4a1 Mediated Glucose Metabolism Dysregulation in Response to High-Fat Diet

Lihui Men  1 , Wenting Hui  1 , Xin Guan  1 , Tongtong Song  1 , Xuan Wang  1 , Siwei Zhang  1 , Xia Chen  1
Affiliations

Cardiac Transcriptome Analysis Reveals Nr4a1 Mediated Glucose Metabolism Dysregulation in Response to High-Fat Diet

Lihui Men et al. Genes (Basel). .

Abstract

Obesity is associated with an increased risk of developing cardiovascular disease (CVD), with limited alterations in cardiac genomic characteristics known. Cardiac transcriptome analysis was conducted to profile gene signatures in high-fat diet (HFD)-induced obese mice. A total of 184 differentially expressed genes (DEGs) were identified between groups. Based on the gene ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs, the critical role of closely interlocked glucose metabolism was determined in HFD-induced cardiac remodeling DEGs, including Nr4a1, Fgf21, Slc2a3, Pck1, Gck, Hmgcs2, and Bpgm. Subsequently, the expression levels of these DEGs were evaluated in both the myocardium and palmitic acid (PA)-stimulated H9c2 cardiomyocytes using qPCR. Nr4a1 was highlighted according to its overexpression resulting from the HFD. Additionally, inhibition of Nr4a1 by siRNA reversed the PA-induced altered expression of glucose metabolism-related DEGs and hexokinase 2 (HK2), the rate-limiting enzyme in glycolysis, thus indicating that Nr4a1 could modulate glucose metabolism homeostasis by regulating the expression of key enzymes in glycolysis, which may subsequently influence cardiac function in obesity. Overall, we provide a comprehensive understanding of the myocardium transcript molecular framework influenced by HFD and propose Nr4a1 as a key glucose metabolism target in obesity-induced CVD.

Keywords: Nr4a1; cardiac transcriptome analysis; glucose metabolism; high-fat diet.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
HFD-induced biological characteristics and histopathological alterations. (A) Changes in body weight after eight weeks of a normal diet (ND) or high-fat diet (HFD), n = 12. (B–C) Serum levels of TC and TG in mice in the ND and HFD groups. (D-E) Alterations of serum LDH and CK activities between groups, n = 5. (F) H&E staining of myocardium tissues (×400) from ND and HFD groups. (G) Quantitative analysis of cardiomyocyte cross-sectional area between groups, n = 4. (H) Sirius Red staining of the myocardium tissues (original magnification × 400). (I) Quantitative analysis of collagen deposition volume between groups, n = 4. Data represent means ± SD; * p <0.05, ** p <0.01, HFD vs. ND group.
Figure 2
Figure 2
HFD-induced differentially expressed genes (DEGs) in the myocardium. (A) FPKM box plot shows the expression levels of transcripts between the ND group and the HFD group. (B) Volcano plot showing differential expression with increased DEGs colored in red and decreased DEGs colored in green. (C) Altered number of cardiac DEGs induced by HFD. (D) Heatmap with hierarchical clustering analyses of DEGs in different samples. The color from blue to red represents the transcript level from low to high.
Figure 3
Figure 3
Top 30 GO terms for (A) upregulated DEGs and (B) downregulated DEGs.
Figure 4
Figure 4
Bubble plot of the significantly enriched KEGG pathways in the HFD group vs. ND group. Colors represent minus logarithms of p-values, from purple to red, indicating higher significance. The lengths of the columns represent the numbers of genes enriched in a pathway.
Figure 5
Figure 5
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway classification and network analysis of enriched genes. (A) Enriched KEGG pathway classified into four major levels distinguished by colors. The length of the bar represents the percentage of DEGs involved in secondary KEGG classification. (B) The connection of enriched KEGG pathways increased in size, indicating a high degree of certain pathways.
Figure 6
Figure 6
Effect of Nr4a1 on glucose metabolism homeostasis (A) Expression of glucose metabolism related DEGS and key genes in glycolysis metabolism in the myocardium, in response to a normal diet (ND) or high-fat diet (HFD) using qPCR. (B,C) Protein expression of NR4A1 and HK2 between groups. Data represent means ± SD; n = 3, * p < 0.05, ** p < 0.01, HFD vs. ND group.
Figure 7
Figure 7
(A) PA induction altered expression of DEGs and hexokinase 2 (Hk2) in H9c2 cells, and the effect of knockdown Nr4a1 by siRNA on the expression of glucose homeostasis-related DEGs under PA treatment conditions. * p < 0.05, ** p < 0.01, vs. the CON group; # p < 0.05, ## p < 0.01, vs. the PA-treated group. (B,C) Nr4a1 knockdown significantly decreased the protein level of NR4A1 and increased the protein level of HK2 in H9c2 cells under both regular and PA-treated conditions. n = 3, * p < 0.05, ** p < 0.01, vs. the CON group; # p < 0.05, ## p < 0.01, vs. the PA-treated group.
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