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. 2023 Apr 6:14:1128033.
doi: 10.3389/fgene.2023.1128033. eCollection 2023.

Integrative metabolomic and transcriptomic analysis reveals difference in glucose and lipid metabolism in the longissimus muscle of Luchuan and Duroc pigs

Liyan Deng  1   2   3   4 , Wangchang Li  1   2   3   5   6 , Weiwei Liu  1   2   3   5 , Yanwen Liu  1   2   3 , Bingkun Xie  7 , Martien A M Groenen  4 , Ole Madsen  4 , Xiaogan Yang  5 , Zhonglin Tang  1   2   3   5   6
Affiliations

Integrative metabolomic and transcriptomic analysis reveals difference in glucose and lipid metabolism in the longissimus muscle of Luchuan and Duroc pigs

Liyan Deng et al. Front Genet. .

Abstract

Luchuan pig, an obese indigenous Chinese porcine breed, has a desirable meat quality and reproductive capacity. Duroc, a traditional western breed, shows a faster growth rate, high feed efficiency and high lean meat rate. Given the unique features these two porcine breeds have, it is of interest to investigate the underlying molecular mechanisms behind their distinctive nature. In this study, the metabolic and transcriptomic profiles of longissimus dorsi muscle from Duroc and Luchuan pigs were compared. A total of 609 metabolites were identified, 77 of which were significantly decreased in Luchuan compared to Duroc, and 71 of which were significantly elevated. Most differentially accumulated metabolites (DAMs) upregulated in Luchuan were glycerophospholipids, fatty acids, oxidized lipids, alcohols, and amines, while metabolites downregulated in Luchuan were mostly amino acids, organic acids and nucleic acids, bile acids and hormones. From our RNA-sequencing (RNA-seq) data we identified a total of 3638 differentially expressed genes (DEGs), 1802 upregulated and 1836 downregulated in Luchuan skeletal muscle compared to Duroc. Combined multivariate and pathway enrichment analyses of metabolome and transcriptome results revealed that many of the DEGs and DAMs are associated with critical energy metabolic pathways, especially those related to glucose and lipid metabolism. We examined the expression of important DEGs in two pathways, AMP-activated protein kinase (AMPK) signaling pathway and fructose and mannose metabolism, using Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). Genes related to glucose uptake, glycolysis, glycogen synthesis, fatty acid synthesis (PFKFB1, PFKFB4, MPI, TPI1, GYS1, SLC2A4, FASN, IRS1, ULK1) are more activated in Luchuan, while genes related to fatty acid oxidation, cholesterol synthesis (CPT1A, HMGCR, FOXO3) are more suppressed. Energy utilization can be a decisive factor to the distinctive metabolic, physiological and nutritional characteristics in skeletal muscle of the two breeds we studied. Our research may facilitate future porcine breeding projects and can be used to reveal the potential molecular basis of differences in complex traits between various breeds.

Keywords: AMPK signaling pathway; Duroc; Luchuan; RNA-seq; fructose and mannose metabolism; glycolysis; lipid metabolism; metabolic profiling.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Multivariate and cluster analyses results of Duroc and Luchuan skeletal muscle metabolites. (A) PCA analysis of metabolites detected in Duroc and Luchuan samples. Duroc is highlighted in green and Luchuan in orange. (B) OPLS-DA score plot demonstrates separation of the Duroc and Luchuan groups. (C) Hierarchical cluster analysis of metabolites from Luchuan and Duroc samples. The color represents accumulation of metabolites, from low (green) to high (red). The Z score scale marks the deviation from the mean by standard deviation units. (D) Heatmap of all DAMs. The metabolites were classified into 16 classes, and the colors display the abundance of metabolites.
FIGURE 2
FIGURE 2
Significant DAMs in Luchuan and Duroc. (A) Volcano plot of all 609 metabolites detected. Upregulated metabolites were defined with fold change ≥2 (red) while downregulated metabolites were with fold change ≤0.5 (green). In addition, a threshold of VIP>1 was applied to distinguish DAMs from the unchanged ones. (B) Metabolites with the highest VIP score. (C) Metabolites with the highest log2-transformed fold change. (D) The top 20 pathways with the lowest p-values. Rich factor is the ratio of the number of DAMs to all metabolites that were annotated to a pathway. The color of the dots represents level of enrichment, varying from red (p = 0) to purple (p = 1). The size of the dots indicates the number of DAMs annotated to a pathway.
FIGURE 3
FIGURE 3
Analysis of DEGs between Luchuan and Duroc. (A) Correlation of gene expression of DEGs based on TPM. (B) Number of upregulated and downregulated DEGs. (C) Top 10 most enriched pathways of the DEGs based on p-value. (D) Top 10 GO enrichment terms based on p-value; the size of dots indicates number of genes related to the term.
FIGURE 4
FIGURE 4
Integrative analysis of metabolome and transcriptome. (A) Heatmap of correlation between all upregulated and downregulated DEGs and DAMs. (B) Correlation between genes and metabolites, and the expression level of several key genes in AMPK signaling pathway in different individuals. (C) Correlation between genes and metabolites, and the expression level of several key genes in fructose and mannose metabolism in different individuals. (D)The Log2 Fold Change (FC) of several metabolites involved in these two pathways. (E) Relative mRNA expression level (qPCR) of several genes that are essential in these two pathways. (F) Linear regression between Log2 FC of gene expression (RNA-seq) and Log2 FC of relative mRNA expression (qPCR).
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