Dataset Information

Comprehensive evaluation of the metabolic effects of porcine CRTC3 overexpression on subcutaneous adipocytes with metabolomic and transcriptomic analyses.

ABSTRACT:

Background

Meat quality is largely driven by fat deposition, which is regulated by several genes and signaling pathways. The cyclic adenosine monophosphate (cAMP) -regulated transcriptional coactivator 3 (CRTC3) is a coactivator of cAMP response element binding protein (CREB) that mediates the function of protein kinase A (PKA) signaling pathway and is involved in various biological processes including lipid and energy metabolism. However, the effects of CRTC3 on the metabolome and transcriptome of porcine subcutaneous adipocytes have not been studied yet. Here, we tested whether porcine CRTC3 expression would be related to fat deposition in Heigai pigs (a local fatty breed in China) and Duroc×Landrace×Yorkshire (DLY, a lean breed) pigs in vivo. The effects of adenovirus-induced CRTC3 overexpression on the metabolomic and transcriptomic profiles of subcutaneous adipocytes were also determined in vitro by performing mass spectrometry-based metabolomics combined with RNA sequencing (RNA-seq).

Results

Porcine CRTC3 expression is associated with fat deposition in vivo. In addition, CRTC3 overexpression increased lipid accumulation and the expression of mature adipocyte-related genes in cultured porcine subcutaneous adipocytes. According to the metabolomic analysis, CRTC3 overexpression induced significant changes in adipocyte lipid, amino acid and nucleotide metabolites in vitro. The RNA-seq analysis suggested that CRTC3 overexpression alters the expression of genes and pathways involved in adipogenesis, fatty acid metabolism and glycerophospholipid metabolism in vitro.

Conclusions

We identified significant alterations in the metabolite composition and the expression of genes and pathways involved in lipid metabolism in CRTC3-overexpressing adipocytes. Our results suggest that CRTC3 might play an important regulatory role in lipid metabolism and thus affects lipid accumulation in porcine subcutaneous adipocytes.

SUBMITTER: Liu J

PROVIDER: S-EPMC7927250 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Publications

Comprehensive evaluation of the metabolic effects of porcine CRTC3 overexpression on subcutaneous adipocytes with metabolomic and transcriptomic analyses.

Liu Jiaqi J Li Jie J Chen Wentao W Xie Xintao X Chu Xingang X Valencak Teresa G TG Wang Yizhen Y Shan Tizhong T

Journal of animal science and biotechnology 20210303 1

<h4>Background</h4>Meat quality is largely driven by fat deposition, which is regulated by several genes and signaling pathways. The cyclic adenosine monophosphate (cAMP) -regulated transcriptional coactivator 3 (CRTC3) is a coactivator of cAMP response element binding protein (CREB) that mediates the function of protein kinase A (PKA) signaling pathway and is involved in various biological processes including lipid and energy metabolism. However, the effects of CRTC3 on the metabolome and trans ...[more]

PMID: 33653408

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Project description:BackgroundLivestock production aims to provide meats of high and consistent eating quality. Insufficient intramuscular (IM) fat and excessive subcutaneous (SC) fat are paramount pork quality challenges. IM fat and SC fat, which are modulated by the adipogenesis of IM and SC adipocytes, play key roles in pork quality. Galectin-12 (LGALS12) was proven to be an important regulator of fat deposition in porcine. However, the current knowledge of the transcriptome-wide role of LGALS12 in adipocytes is still limited. This study was aimed to discover the different regulatory mechanisms of LGALS12 in porcine IM and SC adipocyte.ResultsThe siRNA-mediated knockdown of the expression of LGALS12 identified 1075 and 3016 differentially expressed genes (DEGs) in IM and SC adipocytes, respectively. Among these, 585 were up- and 490 were downregulated in the IM adipocytes, while 2186 were up- and 830 were downregulated in the SC adipocytes. Moreover, 418 DGEs were observed only in the IM adipocytes, 2359 DGEs only in the SC adipocytes, and 657 DGEs in both types of adipocytes. According to Gene Ontology (GO) analysis, DEGs in both IM and SC adipocytes were mainly enriched in categories related to lipids or fat cell differentiation. Pathway analysis of the DEGs revealed 88 changed signaling pathways in the IM adipocytes and 86 in the SC adipocytes. The signaling pathways present in only one type of adipocyte were identified from among the top 50 signaling pathways in each type of adipocyte. Four signaling pathways, encompassing PI3K-AKT, cardiac muscle contraction, fatty acid metabolism and Ras, were significantly enriched in the IM adipocytes. On the other hand, four different signaling pathways, encompassing TNF, WNT, cGMP-PKG and NF-kappa B, were greatly enriched in the SC ones. The pathway changes were confirmed by chemical inhibition assays.ConclusionsOur data reveals that LGALS12 knockdown alters the expression of numerous genes involved in key biological processes in the development of adipocytes. These observations provide a global view of the role of LGALS12 in porcine IM and SC adipocytes; thus, improving our understanding of the regulatory mechanisms by which this gene acts in fat development.

Project description:Abstract CRTC3 is a member of cAMP-responsive element-binding protein (CREB)-regulated transcription coactivators (CRTCs) that plays important roles in regulating CREB-target gene expression and energy metabolism. However, there were nearly no reports about the porcine CRTC3 (pCRTC3). In the current study, we cloned the full length cDNA of pCRTC3 gene and examine its expression pattern and function in intestinal epithelial cells. The full length cDNA sequence of pCRTC3 was 2173 bp (GenBank accession no. MF964215), with a 1,860-bp open reading frame encoding a 620-AA protein. Comparison of the deduced AA sequence with different species including human, mouse, rat, papio, cattle and rabbit showed 89 to 91.9% similarity. The pCRTC3 was highly expressed in small intestine and spleen, to a lesser degree in lung, liver and adipose tissue, and was expressed at a low but detectable level in skeletal muscle, kidney and heart. In addition, high protein levels of pCRTC3 were found in IPEC-J2 cells, in which pCRTC3 was mainly localized in cytoplasm. Furthermore, we constructed pCRTC3-sgRNA1-4 expression plasmids to knockdown pCRTC3 in IPEC-J2 cells. Compared with the Lipofectamine 2000 or pX330-Cas9-empty vector control group, the expression of pCRTC3 was obviously decreased by pCRTC3-sgRNA1-4. Notably, knockdown of pCRTC3 significantly decreased the expression of the porcine tight junction related genes including ZO-1, ZO-2, Occludin, and Claudin-1 by 57.88% (P < 0.01), 40.19% (P < 0.01), 51.59% (P < 0.01) and 35.70% (P < 0.05), respectively. Meanwhile, we found oxidative stress treatment affects the expression of CRTC3 in IPEC-J2 cells.Taken together, we first cloned the full-length sequence of pCRTC3 and revealed the tissue-specific expression pattern, localization and function of pCRTC3 in regulating the expression of intestinal tight junction related genes. This study could provide some useful information for understanding the function of CRTC3 in pigs.

Dataset Information

Comprehensive evaluation of the metabolic effects of porcine CRTC3 overexpression on subcutaneous adipocytes with metabolomic and transcriptomic analyses.

Background

Results

Conclusions

Publications

Comprehensive evaluation of the metabolic effects of porcine CRTC3 overexpression on subcutaneous adipocytes with metabolomic and transcriptomic analyses.

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