Cholesterol is regarded as a signaling molecule in regulating the metabolism and function of fat cells, in which 7-Dehydrocholesterol reductase (DHCR7) is a key enzyme that catalyzes the conversion of 7-dehydrocholesterol to cholesterol, however, the exact function of DHCR7 in goat adipocytes remains unknown. Here, the effect of DHCR7 on the formation of subcutaneous and intramuscular fat in goats was investigated in vitro, and the result indicated that the mRNA level of DHCR7 showed a gradual downward trend in subcutaneous adipogenesis, but an opposite trend in intramuscular adipogenesis. In the process of subcutaneous preadipocytes differentiation, overexpression of DHCR7 inhibited the expression of adipocytes differentiation marker genes (CEBP/α, CEBP/β, SREBP1 and AP2), lipid metabolism-related genes (AGPAT6, FASN, SCD1 and LPL), and the lipid accumulation. However, in intramuscular preadipocyte differentiation, DHCR7 overexpression showed a promoting effect on adipocyte differentiation marker genes (CEBP/α, CEBP/β, PPARγ and SREBP1) and lipid metabolism-related genes (GPAM, AGPAT6, DGAT1 and SCD1) expression, and on lipid accumulation. In summary, our work demonstrated that DHCR7 played an important role in regulating adipogenic differentiation and lipid metabolism in preadipocytes in goats, which is of great significance for uncovering the underlying molecular mechanism of adipocyte differentiation and improving goat meat quality.

Goat intramuscular fat (IMF) deposition is precisely regulated by many key genes as well as transcription factors. Nevertheless, the potential of the regulators of goat IMF deposition remains undefined. In this work, we reported that the transcription factor FOS is expressed at a low level at the early differentiation stage and at a high level in late differentiation. The overexpression of FOS inhibited intramuscular adipocyte lipid accumulation and significantly downregulated the expressions of PPARγ, C/EBPβ, C/EBPα, AP2, SREBP1, FASN, ACC, HSL, and ATGL. Consistently, the knockdown of FOS, facilitated by two distinct siRNAs, significantly promoted intramuscular adipocyte lipid accumulation. Moreover, our analysis revealed multiple potential binding sites for FOS on the promoters of PPARγ, C/EBPβ, and C/EBPα. The expression changes in PPARγ, C/EBPβ, and C/EBPα during intramuscular adipogenesis were opposite to that of FOS. In summary, FOS inhibits intramuscular lipogenesis in goats and potentially negatively regulates the expressions of PPARγ, C/EBPβ, and C/EBPα genes. Our research will provide valuable data for the underlying molecular mechanism of the FOS regulation network of intramuscular lipogenesis.

The aim of this study was to elucidate the effect of FAM13A on the differentiation of goat intramuscular precursor adipocytes and its mechanism of action. Here, we cloned the CDS region 2094 bp of the goat FAM13A gene, encoding a total of 697 amino acid residues. Functionally, overexpression of FAM13A inhibited the differentiation of goat intramuscular adipocytes with a concomitant reduction in lipid droplets, whereas interference with FAM13A expression promoted the differentiation of goat intramuscular adipocytes. To further investigate the mechanism of FAM13A inhibiting adipocyte differentiation, 104 differentially expressed genes were screened by RNA-seq, including 95 up-regulated genes and 9 down-regulated genes. KEGG analysis found that the RIG-I receptor signaling pathway, NOD receptor signaling pathway and toll-like receptor signaling pathway may affect adipogenesis. We selected the RIG-I receptor signaling pathway enriched with more differential genes as a potential adipocyte differentiation signaling pathway for verification. Convincingly, the RIG-I like receptor signaling pathway inhibitor (HY-P1934A) blocked this pathway to save the phenotype observed in intramuscular adipocyte with FAM13A overexpression. Finally, the upstream miRNA of FAM13A was predicted, and the targeted inhibition of miR-21-5p on the expression of FAM13A gene was confirmed. In this study, it was found that FAM13A inhibited the differentiation of goat intramuscular adipocytes through the RIG-I receptor signaling pathway, and the upstream miRNA of FAM13A (miR-21-5p) promoted the differentiation of goat intramuscular adipocytes. This work extends the genetic regulatory network of IMF deposits and provides theoretical support for improving human health and meat quality from the perspective of IMF deposits.

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.

Intramuscular fat (IMF) is a crucial indicator of meat quality. Recent studies increasingly suggest that methionine (Met) plays a role in fat accumulation. However, the mechanism by which Met affects the proliferation and differentiation of intramuscular adipocytes remains unclear. In this study, goat intramuscular adipocytes were cultured in basal complete medium supplemented with Met at 0, 50, 100, 200, 400, and 800 μM. This study demonstrates a quadratic relationship between the level of Met supplementation and cellular proliferation and differentiation, with an optimal effect observed at 100 μM Met. The results of RNA-seq analysis demonstrated that supplementation with Met led to the downregulation of genes involved in lipid metabolism, including the genes PER2, PRKG1, and SLC5A5. Furthermore, we found that overexpression of SLC5A5 inhibits adipogenic differentiation in goat intramuscular adipocytes, while Met supplementation reversed this inhibitory effect. In addition, silencing SLC5A5 significantly promoted adipogenic differentiation, whilst Met supplementation further enhanced this promotion effect. In conclusion, this study indicates that Met supplementation of basal complete medium benefits adipogenic differentiation of goat intramuscular adipocytes, likely by inhibiting the expression of SLC5A5. This research provides a foundation for improving goat meat quality.

The differential fat deposition in intramuscular (IMF) and subcutaneous (SCF) adipose tissues significantly influences goat meat quality. While microRNAs (miRNAs) are key post-transcriptional regulators, the shared key miRNAs governing adipogenesis across different adipose depots remain largely unexplored in goats. In this study, intramuscular and subcutaneous preadipocytes were isolated from the same healthy 7-day-old male Jianzhou Da’er goat, and each experimental group comprised five independently cultured cell replicates derived from this donor. Integrated miRNA and mRNA sequencing was performed on undifferentiated intramuscular (JNE) and subcutaneous (PE) preadipocytes and their corresponding adipocytes after 72 h of differentiation (JNC and PC, respectively), allowing the identification of depot-specific and shared differentially expressed miRNAs (DEmiRNAs) and mRNAs (DEmRNAs). By constructing miRNA–mRNA regulatory networks, chi-miR-1 was identified as the highest-degree miRNA node in the shared network and was therefore prioritized for functional evaluation. RT-qPCR, chi-miR-1 mimic and inhibitor transfection, Oil Red O staining, and BODIPY staining showed that chi-miR-1 overexpression increased lipid droplet accumulation, whereas its inhibition produced the opposite effect in both intramuscular and subcutaneous adipocytes. Furthermore, bioinformatic target prediction and network analysis identified chi-miR-1–PPARG and chi-miR-206–IGFBP3 as predicted potential regulatory relationships; these interactions were not directly validated experimentally. Our findings, obtained using cells derived from a single donor, suggest that chi-miR-1 may serve as a candidate shared pro-adipogenic regulator under the present in vitro conditions and provide insights into miRNA-mediated regulation of adipogenesis in different goat adipose depots.