ACADL Promotes the Differentiation of Goat Intramuscular Adipocytes

Abstract

Intramuscular fat (IMF) deposits help improve meat quality such as marbling, juicy, flavor and tenderness. Long-chain acyl-CoA dehydrogenase (ACADL) is a key enzyme for catalyzing fatty acid oxidation, and studies have shown ACADL is involved in the deposition and differentiation of intramuscular adipocytes. However, the effect of ACADL on intramuscular adipocytes differentiation in goats needs further study. In this study, to explore the mechanism of ACADL on the development of goat intramuscular adipocytes, we constructed an over-expression plasmids and a SI-RNA of ACADL to explore the function of ACADL on the development of goat IMF. It was found that overexpression of ACADL promoted the differentiation of goat intramuscular adipocytes, and promoted the expression of fat cell differentiation marker genes lipoprotein lipase (LPL), peroxisome proliferator activated receptor gamma (PPARγ), APETALA-2-like transcription factor gene (AP2), CCAT enhancer binding protein (CEBPα), preadipocyte Factor 1 (Pref-1) and CCAT enhancer binding protein (CEBPβ), and the opposite trend occurred after interference. In addition, we screened of this related tumor necrosis factor (TNF) signaling pathway by RNA-Seq. So, we validate the signaling pathway with inhibitor of TNF signaling pathway. In summary, these results indicate that ACADL promotes intramuscular adipocytes differentiation through activation TNF signaling pathway. This study provides an important basis for the mechanism of IMF development.

Keywords: ACADL; RNA-seq; TNF signaling pathway; adipocyte; differentiation; mechanism.

Figure 1

Over-expression of goat ACADL promotes intramuscular adipocytes differentiation. (A) mRNA expression of ACADL was detected by qPCR after over-expressing ACADL for 48 h intramuscular adipocyte differentiation. (B) Photos of Bodipy staining and Oil red staining of the cells in the tests group (OE) and negative control group (NC) during intramuscular adipocyte differentiation. (C) The OD value of Oil red O staining at 492 nm during intramuscular adipocytes differentiation. (D) Effect of goat ACADL overexpression on the expression of adipocytes related genes. “*” significant difference and “**” very significant difference.

Figure 2

The effect of ACADL interference on goat adipocyte differentiation. (A) Expression efficiency detection after ACADL interference. (B) Morphology observation of Oil red O staining. (C) Oil red O staining showed the OD value at 492 nm during the differentiation of intramuscular adipocytes. (D) Effect of goat ACADL overexpression on adipocytes related gene’s expression. “*” significant difference and “**” very significant difference.

Figure 3

Adipocytes differentiated for 2 days. (A) Oil red staining, pre-differentiation (PD), after differentiation (AD). (B) OD value of preadipocytes and adipocytes, relative expression of ACADL. (C,D) Expression level of marker genes. “*” significant difference and “**” very significant difference.

Figure 4

Gene expression analysis. (A) Distribution of gene expression. (B) Heat map of sample-to-sample correlation. The horizontal ordinate coordinates in the plot are the squares of the correlation coefficients for each sample. (C) Co-expression of Venn diagram. The number of genes uniquely expressed in each group, overlapping regions showing the number of genes co-expressed in two groups. (D) Differential gene volcano. The abscissa is the log₂FoldChange value, the ordinate is log₁₀padjust or -log₁₀p-value, and the blue dotted line indicates the threshold line of the differential gene-screening criteria. (E) Heat map of differentially expressed gene clusters. Abscissa is the sample name, and the ordinate coordinate was the value of the FFPKM normalization of the differential gene, the redder the color, the higher the expression, the greener, and the lower the expression.

Figure 5

Enrichment analysis. (A) GO enrichment analysis histogram. The abscissa was GO term, the ordinate was the significance level of GO term enrichment, represented by −log₁₀ (p-value, and different colors indicate different functional classifications. (B) KEGG enrichment scatter plot. The abscissa coordinate is the ratio of the number of differential genes to the total number of differential genes annotated to the KEGG pathway, and the ordinate is the KEGG pathway, red circles indicate the selected pathway (C–F) Validation of representative by q-PCR (n = 5) Log₂p-value. TNFAIP3: tumor necrosis factor induced protein 3, CCL5: chemokine ligand 5, CCL20: chemokine ligand 20, SELE: selectin endothelial an adhesion molecule.

Figure 6

Overexpression of ACADL promotes intramuscular adipocytes differentiation through TNF-signaling pathway. (A) The mRNA level of ACADL in inhibitors of different concentrations. (B) MTT for the detection of cell activity over different time period. Screening of inhibitors for optimal concentrations (Unit nm). (C) The image of Bodipy and Oil red O staining of intramuscular adipocytes in VECTOR, OE, inhibitor, and OE combined with inhibitor. (D) OD value of VECTOR, OE, inhibitor, and OE combined with inhibitor. “*” significant difference and “**” very significant difference.

Figure 7

The recovery of gene expression is under the action of inhibitors. (a) Expression of TNF signaling pathway genes after the action of the inhibitor. (b) The expression of ACADL in different treatment control groups (OE, s1029, OE + S1029). (c–i) The expression level of differentiation marker genes in different treatment control groups. The mRNA level of ACADL (c), SREBP-1 (d), LPL (e), PPARγ (f), AP2 (g), CEBPα (h), PREF-1 (i), and CEBPβ in VECTOR, OE, and OE combined with inhibitors. n = 6, “*” p < 0.05, “**” p < 0.01.