ASFT is not only an energy storage organ, but also an endocrine organ. However, the specific functional roles of ASFT are still poorly understood. In this study, we systematically analyzed the gene expression profiles from differing ASFTs to investigate the transcriptome characteristics using bioinformatics methods. Finally, ten hub genes, some key pathways, three key regulated genes and some regulatory miRNAs were identified.
Among identified key pathways, three pathways involved in lipid metabolism and adipocyte differentiation were found, including p53 signaling pathway, MAPK signaling pathway and fatty acid metabolism. A previous study showed that p53 pathway was activated in mature obese adipocytes [19], and p53, the core gene in p53 signaling pathway, played a key role in regulating cellular metabolism [20]. Moreover, p53 activation in mice during high-fat diet (HFD) feeding could lead to HFD-induced obesity by regulating systemic metabolism [21]. In our study, the DEGs enriched in this pathway were basically significantly upregulated, such as MDM2, which targeted p53 for nuclear export and proteasomal degradation through attaching ubiquitin moieties, played a critical role in the maintenance of adipocyte homeostasis [22]. MAPK signaling pathway consists of ERK, JNK and p38 signaling pathways. ERK-MAPK signaling pathway affects adipogenic differentiation [23], and P38-MAPK was identified to be a positive regulator of intramuscular fat (IMF) deposition in pigs [24]. JNK-MAPK signaling pathway was associated with obesity in broilers muscle, and its inactivation could effectively resist obesity [25]. Fatty acid metabolism signal pathway was involved in fatty acid biosynthesis and fatty acid beta oxidation, and played an important role in regulating fatty acid metabolism and growth traits in pigs [26]. Several studies have shown that the significantly upregulated genes enriched in the fatty acid metabolism signal pathway, such as ACAT1 [27], HADH [28], ACADSB [29], were associated with energy metabolism. In our study, it was worth noting that the upregulated genes(such as ACAT1 and HADH)of the fatty acid metabolism pathway were enriched in BPs such as fatty acid β-oxidation and negative regulation of lipid localization. This study further demonstrated that p53 signaling pathway, MAPK signaling pathway and fatty acid metabolism might play important roles in regulating ASFD in pigs.
Ten hub genes (ABL1, HDAC1, CDC42, HDAC2, MRPS5, MRPS10, MDM2, JUP, RPL7L1 and UQCRFS1) were identified by PPI network analysis with the algorithm of degree centrality. ABL1, a member of the ABL interacting protein family, is a protooncogene that encodes a protein tyrosine kinase involved in various cellular processes, including cell division, adhesion, differentiation, and response to stress. A published study has reported that ABL1 is accountable for fat metabolism, lean body mass and fat deposition, and transport in mice [30]. Furthermore, ABL1 (c-Abl) was highly expressed in SCF from obese humans and HFD induced obese mice [31]. In this study, the upregulated expression of ABL1 was observed in obese pigs, which was consistent with previous studies indicating that a high expression of ABL1 was closely related to fat deposition. HDAC1 is histone acetylation and deacetylation, catalyzed by multisubunit complexes, which plays a key role in the regulation of eukaryotic gene expression. Previous studies have shown that high expression levels of HDAC1 were correlated with obesity and overweight in HFD-fed mice [32], while there was also a study showing the expression of HDAC1 in adipose tissues from obese women is lower in comparison with normal-weight individuals [33]. HDAC2, which plays an important role in transcriptional regulation, cell cycle progression and developmental events, is an important paralog of HDAC1 [34]. Previous studies have demonstrated that HDAC2 contributed to obesity [35]. A previous study reported that gene expression of HDAC2 in subcutaneous adipose tissue (SAT) was comparable between obese and non-obese women, and HDAC2 mRNA gene expression in SAT was inversely correlated with waist circumference (WC) [36]. However, some studies also reported that HDAC2, as the mediator of MKP-3 action in liver lipid metabolism, might be associated with reducing adiposity by repressing adipocyte differentiation in mice [37]. In our study, significant upregulation of HDAC1 and HDAC2 was observed in the MAs of obese pigs compared to lean pigs. Overall, our results were consistent with previous studies, which indicated that HDAC1 and HDAC2 might play a critical role in fat deposition. CDC42 is a member of the Rho family and is closely associated with biological processes such as cell transformation, cell division, and enzyme activity [38]. CDC42, as a signaling molecule in the insulin action pathway, contributes to glucose transporter-4 translocation and glucose transport [39]. Published studies reported that the mRNA expression of CDC42 was significantly upregulated in both subcutaneous and visceral adipose tissues of mice fed the HFD [40]. Here, we also observed the upregulation of CDC42 in the MAs of obese pigs compared to lean pigs. This result was consistent with previous studies, which indicates that a high expression of CDC42 might be closely associated with the fat deposition[41]. MRPS5 is closely related to oxidative phosphorylation, and uncoupling protein decoupling of mitochondrial oxidative phosphorylation can strongly reduce mitochondrial reactive oxygen species [42]. MRPS10, related to peptide chain elongation and mitochondrial translation, has been reported to be associated with various diseases, such as breast cancer and rheumatoid arthritis [43, 44]. Presently, the function of MRPS5 and MRPS10 in fat deposition remains little known. MDM2 gene encodes a nuclear-localized E3 ubiquitin ligase and MDM2 targeted p53 is associated with adipocyte homeostasis for nuclear export and proteasomal degradation through attaching ubiquitin moieties [45, 46]. Some published studies showed that MDM2 played a pivotal role in the early steps of adipocyte differentiation [47, 48]. Recently, a study showed that the levels of MDM2 expression significantly increased in the white adipose tissue (WAT) of diet-induced obese mice and genetically obese mice [22]. Our results further confirmed that MDM2 might contribute to fat deposition. JUP, encoding a major cytoplasmic protein, is the only constituent common to submembranous plaques of both desmosomes and intermediate junctions, and plays a role in the regulation of insulin signaling and glucose uptake in adipocytes [49, 50]. It has also been shown that the expression level of JUP was controlled by SIK2, and its expression in human SAT is positively correlated with the expression level of SIK2 [51, 52]. Our study further identified that JUP expression was associated with fat deposition, and its upregulation contributed to fat deposition in obese pigs. RPL7L1 enables RNA binding activity, and is predicted to be involved in the maturation of LSU-rRNA from tricistronic rRNA transcript. However, no published studies reported that the function of RPL7L1 was associated with fat deposition. Our results observed that low mRNA expression of RPL7L1 contributed to obesity in pigs, which provided a novel insight into the role of RPL7L1 involved in fat deposition, and this needs to be further explored and confirmed. UQCRFS1 is synthesized as a pre-protein in the cytosol, and involves in mitochondrial respiratory chain complex III assembly and respiratory electron transport chain [53]. Little is known about the function of UQCRFS1 in fat deposition. In this study, we found that UQCRFS1 was dysregulated expressed between differing types of ASFTs, and might serve as a potential candidate gene associated with fat deposition.
KEGG pathway analysis indicated that ten hub genes were involved in immune response, fatness, and metabolism. Published studies showed that some genes in notch signaling pathway modulated the adipogenesis process. For example, Notch1 included the proliferation and differentiation of adipocyte progenitor cells in adipocyte progenitor cells [54, 55]. Furthermore, Notch1 was identified to play a crucial role in the development and functions of MAs, beige adipocyte formation, and energy metabolism [56, 57]. Our study identified that the other HDAC1 and HDAC2 in notch signaling pathway were differentially expressed between differing types of ASFTs which indicates that notch signaling pathway might play a key role in ASFD.
Although ten hub genes were identified and some genes have been validated by previous studies, some limitations must be noted in the current study. First, the results were obtained by a bioinformatics method, the expression of hub genes must be validated using more accurate methods, such as real-time PCR or (and) western blot. Second, the specific functions of hub genes need to be revealed in ASFD by overexpression or knockdown methods.