Nuclear Receptor Subfamily 4 Group A Member 1 (NR4A1) Promotes the Adipogenesis of Intramuscular Preadipocytes through PI3K/AKT Pathway in Goats

Abstract

As a transcription factor, Nuclear Receptor Subfamily 4 Group A Member 1 (NR4A1) binds to downstream target genes to participate in cell proliferation and cell differentiation. We found that the NR4A1 reached the highest expression at 60 h after the differentiation of goat intramuscular preadipocytes. Overexpression of goat NR4A1 increased the number of intracellular lipid droplets and up-regulated the expression of adipocyte-differentiation-related marker genes including AP2, SREBP1, ACC, GPAM, and DGAT2, while the relative expression levels of Pref-1 and HSL were significantly decreased. On the contrary, after NR4A1 was knocked down by siRNA, the number of intracellular lipid droplets and the relative expression levels of LPL, CEBPα, CEBPβ, ACC, and DGAT2 were significantly decreased, and the relative expression levels of Pref-1 and HSL were significantly up-regulated. These results suggest that NR4A1 promotes the differentiation of goat intramuscular preadipocytes. Transcriptome sequencing was carried out after overexpression of goat NR4A1, and the KEGG enrichment analysis result showed that the most differentially expressed genes were related to adipocyte differentiation and were enriched in the PI3K-Akt signaling pathway. LY249002, an inhibitor of the PI3K-Akt signaling pathway, was introduced and decreased the number of intracellular lipid droplets, and the relative expression levels of C/EBPα, SREBP1, AP2, C/EBPβ, GPAM, ACC, DGAT1, DGAT2, and ATGL were decreased accordingly. The above results indicate that overexpression of goat NR4A1 may promote the differentiation of intramuscular preadipocytes through the PI3K-Akt signaling pathway.

Keywords: NR4A1; adipogenesis; goat; intramuscular preadipocyte.

Figure 1

Relative expression level of NR4A1 during intramuscular adipocyte differentiation. The expression level of NR4A1 mRNA was detected by qPCR, and the UXT gene was used as the reference gene. Data are expressed as “Means ± SD”, n = 6, * p < 0.05.

Figure 2

Overexpression of NR4A1 promotes the differentiation of intramuscular adipocytes in goats. (A) NR4A1 overexpression efficiency detection. (B) The result of oil red O staining images of 2-day induced differentiation between NC and NR4A1 overexpression groups. (C) The result of OD value at 490 nm. (D) The relative expression levels of goat intramuscular adipocyte differentiation marker genes and lipid-metabolism-related genes after overexpression of NR4A1. Data are expressed as “Means ± SD”, n = 3. ** p < 0.01, * p < 0.05.

Figure 3

Knockdown of NR4A1 expression blocks the differentiation of intramuscular adipocytes in goats. (A) The knockdown efficiency of NR4A1. (B) The result of oil red O staining images of 2-day induced differentiation between NC and NR4A1 overexpression groups. (C) The result of OD value at 490 nm. (D) The relative expression levels of goat intramuscular adipocyte differentiation marker genes and lipid-metabolism-related genes after knockdown of NR4A1. Data are expressed as “Means ± SD”, n = 3. ** p < 0.01, * p < 0.05.

Figure 4

KEGG enrichment analysis of differentially expressed mRNAs.

Figure 5

Differentially expressed mRNAs were enriched into adipocyte differentiation and lipid metabolism pathways.

Figure 6

Verification of differentially expressed mRNAs.

Figure 7

LY249002 blocks NR4A1 overexpression and promotes the differentiation of intramuscular preadipocytes in goats. (A) The resulting oil red O staining images of 2-day induced differentiation among NC, NR4A1 overexpression, and NR4A1 overexpression with LY294002 added groups. (B) The result of OD value at 490 nm. (C) Overexpression of NR4A1 followed by addition of LY249002 to the relative expression levels of goat intramuscular adipocyte differentiation marker genes and lipid-metabolism-related genes. Data are expressed as “Means ± SD”, n = 3. ** p < 0.01, * p < 0.05.