FOS Inhibits the Differentiation of Intramuscular Adipocytes in Goats.

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/EBPß, 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/EBPß, and C/EBPα. The expression changes in PPARγ, C/EBPß, 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/EBPß, and C/EBPα genes. Our research will provide valuable data for the underlying molecular mechanism of the FOS regulation network of intramuscular lipogenesis.

[Gene cloning and sequence analysis of the RPL29 gene and its effect on lipogenesis in goat intramuscular adipocytes].

The aim of this study was to clone the goat RPL29 gene and analyze its effect on lipogenesis in intramuscular adipocytes. Using Jianzhou big-eared goats as the object, the goat RPL29 gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR), the gene structure and expressed protein sequence were analyzed by bioinformatics, and the mRNA expression levels of RPL29 in various tissues and different differentiation stages of intramuscular adipocytes of goats were detected by quantitative real-time PCR (qRT-PCR). The RPL29 overexpression vector pEGFP-N1-RPL29 constructed by gene recombination was used to transfect into goat intramuscular preadipocytes and induce differentiation. Subsequently, the effect of overexpression of RPL29 on fat droplet accumulation was revealed morphologically by oil red O and Bodipy staining, and changes in the expression levels of genes related to lipid metabolism were detected by qRT-PCR. The results showed that the length of the goat RPL29 was 507 bp, including a coding sequence (CDS) region of 471 bp which encodes 156 amino acid residues. It is a positively charged and stable hydrophilic protein mainly distributed in the nucleus of cells. Tissue expression profiling showed that the expression level of this gene was much higher in subcutaneous adipose tissue and inter-abdominal adipose tissue of goats than in other tissues (P < 0.05). The temporal expression profile showed that the gene was expressed at the highest level at 84 h of differentiation in goat intramuscular adipocytes, which was highly significantly higher than that in the undifferentiated period (P < 0.01). Overexpression of RPL29 promoted lipid accumulation in intramuscular adipocytes, and the optical density values of oil red O staining were significantly increased (P < 0.05). In addition, overexpression of RPL29 was followed by a highly significant increase in ATGL and ACC gene expression (P < 0.01) and a significant increase in FASN gene expression (P < 0.05). In conclusion, the goat RPL29 may promote intra-muscular adipocyte deposition in goats by up-regulating FASN, ACC and ATGL.

[Cloning, identification and functional analysis of the goat transcription factor c-fos].

C-fos is a transcription factor that plays an important role in cell proliferation, differentiation and tumor formation. The aim of this study was to clone the goat c-fos gene, clarify its biological characteristics, and further reveal its regulatory role in the differentiation of goat subcutaneous adipocytes. We cloned the c-fos gene from subcutaneous adipose tissue of Jianzhou big-eared goats by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed its biological characteristics. Using real-time quantitative PCR (qPCR), we detected the expression of c-fos gene in the heart, liver, spleen, lung, kidney, subcutaneous fat, longissimus dorsi and subcutaneous adipocytes of goat upon induced differentiation for 0 h to 120 h. The goat overexpression vector pEGFP-c-fos was constructed and transfected into the subcutaneous preadipocytes for induced differentiation. The morphological changes of lipid droplet accumulation were observed by oil red O staining and bodipy staining. Furthermore, qPCR was used to test the relative mRNA level of the c-fos overexpression on adipogenic differentiation marker genes. The results showed that the cloned goat c-fos gene was 1 477 bp in length, in which the coding sequence was 1 143 bp, encoding a protein of 380 amino acids. Protein structure analysis showed that goat FOS protein has a basic leucine zipper structure, and subcellular localization prediction suggested that it was mainly distributed in the nucleus. The relative expression level of c-fos was higher in the subcutaneous adipose tissue of goats (P < 0.05), and the expression level of c-fos was significantly increased upon induced differentiation of subcutaneous preadipocyte for 48 h (P < 0.01). Overexpression of c-fos significantly inhibited the lipid droplets formation in goat subcutaneous adipocytes, significantly decreased the relative expression levels of the AP2 and C/EBPß lipogenic marker genes (P < 0.01). Moreover, AP2 and C/EBPß promoter are predicted to have multiple binding sites. In conclusion, the results indicated that c-fos gene was a negative regulatory factor of subcutaneous adipocyte differentiation in goats, and it might regulate the expression of AP2 and C/EBPß gene expression.

PDZK1-Interacting Protein 1(PDZKIP1) Inhibits Goat Subcutaneous Preadipocyte Differentiation through Promoting Autophagy.

PDZK1IP1 is highly expressed in tumor tissue and has been identified as a tumor biomarker. However, the role of PDZK1IP1 in goat subcutaneous preadipocyte differentiation remains largely unknown. The molecular mechanism of autophagy in regulating the differentiation of goat subcutaneous preadipocytes has not been clarified yet. In our study, PDZK1IP1 gain of function and loss of function were performed to reveal its functions in preadipocyte differentiation and autophagy. Our results showed that the overexpression of PDZK1IP1 inhibited the differentiation of goat subcutaneous preadipocytes, whereas it promoted autophagy. Consistently, the knockdown of PDZK1IP1 demonstrated the opposite tendency. Next, we investigated whether PDZK1IP1 inhibited the differentiation of goat preadipocytes by regulating autophagy. We found that inhibiting autophagy can rescue the PDZK1IP1-induced differentiation restraint in goat subcutaneous preadipocytes. In conclusion, PDZK1IP1 acts as a regulator of adipogenesis, and inhibits goat subcutaneous preadipocyte differentiation through promoting autophagy. Our results will contribute to further understanding the role and mechanism of PDZK1IP1 in controlling adipogenesis.