Intramuscular fat (IMF) deposition is one of the most important factors affecting meat quality and is closely associated with the expression of carnitine palmitoyl transferase 1A (CPT1A) which facilitates the transfer of long-chain fatty acids (LCFAs) into the mitochondria. However, the role of how CPT1A regulates the IMF formation remains unclear. Herein, we established the temporal expression profile of CPT1A during the differentiation of goat intramuscular precursor adipocytes. Functionally, the knockdown of CPT1A by siRNA treatment significantly increased the mRNA expression of adipogenic genes and promoted lipid deposition in goat intramuscular precursor adipocytes. Meanwhile, a CPT1A deficiency inhibited cell proliferation and promoted cell apoptosis significantly. CPT1A was then supported by the overexpression of CPT1A which significantly suppressed the cellular triglyceride deposition and promoted cell proliferation although the cell apoptosis also was increased. For RNA sequencing, a total of 167 differential expression genes (DEGs), including 125 upregulated DEGs and 42 downregulated DEGs, were observed after the RNA silencing of CPT1A compared to the control, and were predicted to enrich in the focal adhesion pathway, cell cycle, apoptosis and the MAPK signaling pathway by KEGG analysis. Specifically, blocking the MAPK signaling pathway by a specific inhibitor (PD169316) rescued the promotion of cell proliferation in CPT1A overexpression adipocytes. In conclusion, the expression variation of CPT1A may reconstruct the lipid distribution between cellular triglyceride deposition and cell proliferation in goat intramuscular precursor adipocyte. Furthermore, we demonstrate that CPT1A promotes the proliferation of goat adipocytes through the MAPK signaling pathway. This work widened the genetic regulator networks of IMF formation and delivered theoretical support for improving meat quality from the aspect of IMF deposition.

Meat quality in goats is partly determined by the intramuscular fat (IMF) content, which is associated with the proliferation and differentiation of intramuscular preadipocytes. Emerging studies have suggested that miRNA plays a crucial role in adipocyte proliferation and differentiation. In our recent study, we observed the expression variations in miR-196a in the longissimus dorsi muscle of Jianzhou goats at different ages. However, the specific function and underlying mechanism of miR-196a in IMF deposition are still unclear. This study demonstrated that miR-196a significantly enhanced adipogenesis and apoptosis and reduced the proliferation of preadipocytes. Subsequently, RNA-seq was employed to determine genes regulated by miR-196a, and 677 differentially expressed genes were detected after miR-196a overexpression. The PI3K-Akt pathway was identified as activated in miR-196a regulating intramuscular adipogenesis via Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and further verified via Western blot and rescue assays. Lastly, using RT-qPCR, Western blot, dual-luciferase, and rescue assays, we found that miR-196a promoted adipogenesis and suppressed the proliferation of intramuscular preadipocytes by the downregulation of MAP3K1. In summary, these results suggest that miR-196a regulates IMF deposition by targeting MAP3K1 and activating the PI3K-Akt pathway and provide a theoretical foundation for improving goat meat quality through molecular breeding.

Silent Information Regulator 5 (SIRT5) has been established as a crucial regulator of cellular alanylation modification. Furthermore, accumulating evidence suggests that SIRT5 plays a significant regulatory role in key metabolic pathways, including glycolysis, the tricarboxylic acid (TCA) cycle, and fatty acid oxidation, all of which are closely associated with cellular lipid metabolism. Despite these advancements, the specific role of SIRT5 in regulating intramuscular fat (IMF) deposition in goats, as well as the underlying molecular mechanisms, remains largely unexplored. In this study, we cloned the complete coding sequence of the goat SIRT5 gene and, through amino acid sequence alignment, demonstrated its closest phylogenetic relationship with sheep. Additionally, we characterized the higher expression of SIRT5 during the differentiation of goat intramuscular precursor adipocytes. The silencing of SIRT5 by siRNA-mediated knockdown significantly upregulated the expression of lipogenesis-related genes and enhanced lipid deposition in goat intramuscular preadipocytes. Concurrently, SIRT5 deficiency led to the inhibition of cell proliferation and a marked reduction in apoptosis. Interestingly, although overexpression of SIRT5 promoted cell proliferation, it did not significantly alter lipid deposition in goat intramuscular precursor adipocytes. RNA sequencing (RNA-seq) analysis identified a total of 106 differentially expressed genes (DEGs) following SIRT5 silencing in goat preadipocytes, predominantly involved in the Focal adhesion, HIF-1, PI3K-Akt, and MAPK signaling pathways by KEGG pathway enrichment analysis. Notably, we successfully reversed the phenotypic effects observed in SIRT5 knockdown goat precursor adipocytes by inhibiting the PI3K-Akt and MAPK signaling pathways using the AKT inhibitor LY294002 and the p38 MAPK pathway inhibitor PD169316, respectively. In conclusion, our findings demonstrated that SIRT5 may modulate intramuscular fat deposition in goats through PI3k-Akt and MAPK signaling pathways. These results expand the gene regulatory network associated with IMF formation and provide a theoretical foundation for improving meat quality by targeting IMF deposition.

The role of fatty acid transporter 4 (FATP4) in regulating lipid metabolism has been well studied. However, how it affects IMF deposition, especially in goats, remains poorly understood. Here, we cloned the whole coding sequence of the goat FATP4 gene and revealed its closest affinity to sheep by amino acid sequence blast analysis. In addition, we found that the FATP4 reached its highest expression level at day 6 of goat preadipocyte differentiation in vitro. Functionally, in cultured goat intramuscular preadipocytes, siRNA-induced FATP4 knockdown dramatically raised the mRNA expression of lipogenesis-related genes and encouraged lipid deposition. At the same time, FATP4 deficiency inhibited cell proliferation and significantly decreased apoptosis. Unexpectedly, although the overexpression of FATP4 promoted cell proliferation and suppressed apoptosis, it only slightly decreased cellular lipid deposition in goat intramuscular preadipocytes. For RNA-seq (performed on pooled cell samples with three technical replicates), a total of 467 differential genes (DEGs) were identified after silencing of FATP4 in goat preadipocytes, including 47 upregulated genes and 420 downregulated genes. These DEGs were mainly enriched in the signaling pathways of Focal adhesion, HIF-1, and PI3K-Akt by KEGG analysis. To validate these findings, knockdown of FATP4 increased the expression of phosphatidylinositol 3-kinase (PI3k) and vice versa. Convincingly, we rescued the phenotype observed in FATP4 knockout goat preadipocytes by blocking the PI3k-Akt signaling pathway with an AKT inhibitor (LY294002). In summary, in our in vitro model, FATP4 plays a crucial role in directing fatty acids toward cell proliferation (prioritized over cellular lipid deposition) via the PI3K-Akt signaling pathway in goat intramuscular preadipocytes. These findings provide preliminary mechanistic insights into the regulatory network of IMF formation at the cellular level, and offer theoretical clues for future research aimed at enhancing meat quality from the standpoint of IMF deposition.