Obesity is characterized by increased adipose tissue mass that results from increased fat cell size (hypertrophy) and number (hyperplasia), suggesting that the main contributor to obesity is an adipose tissue [10]. Hypertrophy and hyperplasia are two possible growth mechanisms of adipose tissue. Hypertrophy (energy storage) occurs prior to hyperplasia to meet the need for additional fat storage capacity in the progression of obesity [37]. Hyperplastic growth (adipocyte differentiation) appears at early stage in adipose tissue development and late stage of obesity [38, 39]. Therefore, understanding the molecular mechanisms of hypertrophy and hyperplasia for modulation of adipocyte has been the subject of intense investigation, which could help to find novel approaches for preventing and combating human obesity. In the present study, we provided several lines of evidences that many genes were up- or down-regulated during adipocyte differentiation. Most of the gene expression changes were occurred in the early four days of differentiation. GO and KEGG analysis demonstrated that these altered genes were mainly involved in metabolic process, lipid metabolism and oxidation-reduction process. Moreover, our results demonstrated the mRNA expression levels of several TRP channels were altered during adipocyte differentiation, suggested that these TRP channels might be involved in adipocyte differentiation.
Transcriptome changes result the proteome alteration in cells, which subsequently affect the molecular and cellular functions. 3T3-L1 cells are a model cell line which could be inducted from pre-adipocytes (fibroblast-like cells) to the differentiated adipocytes (round cells with lipid droplets) (Fig. 1). 3T3-L1 adipocyte differentiation was conducted in a Petri dish in vitro, this allows us to investigate the mechanism of adipocyte differentiation. Therefore, we performed RNA sequencing experiment to detect the transcriptomic changes during adipocyte differentiation. As shown in Fig. 1, we chose pre-adipocytes, 4-day differentiated adipocytes (middle stage) and 8-day differentiated adipocytes (matured adipocytes) for RNA sequencing. The morphological changes in early phase of adipocyte differentiation were dramatically. Lipid droplets were already observed in 4-day-differentiated adipocytes. The lipid droplets and cell size were further enlarged in the late phase of adipocyte differentiation (Fig. 1). In parallel with the morphological changes, the RNAseq results showed that there were 1295 genes up-regulated, 1114 genes down-regulated in the early phase of adipocyte differentiation. Moreover, there were 523 genes up-regulated, 325 genes down-regulated in the late phase of adipocyte differentiation (Fig. 2). Taken together, our results revealed that the major gene expressional and functional changes might occur in the early phase of adipocyte differentiation, suggested that the early 4 days differentiation might play a decisive role in adipocyte differentiation.
GO analysis demonstrated that the DEGs during the whole process of adipocyte differentiation were significantly enriched in the classifications of “lipid metabolic process”, “oxidation − reduction process”, “metabolic process”, and “oxidoreductase activity” (Fig. 3). KEGG analysis revealed that the differential genes were significantly involved in the classifications of “metabolic pathways” (Fig. 4). Moreover, the DEGs during the late phase of adipocyte differentiation were enriched in the classification of “cell adhesion”, “hormone activity”, “metabolic pathways”, “HIF − 1 signaling pathway” and “PI3K − Akt signaling pathway”. These results revealed that the metabolic and oxidation-reduction process were the major process during adipocyte differentiation, especially in the early 4 days of differentiation. On the other hand, signaling pathways changes and cell adhesion, proliferation were mainly happened in the late phase of adipocyte differentiation. Our results suggested that lipid metabolism and oxidation − reduction reaction are the major processes in the early phase of adipocyte differentiation. In addition to the lipid metabolism, cell aging and signaling pathway are mainly involved in the late phase of adipocyte differentiation. These results were in coincidence with the morphological changes which we observed during adipocyte differentiation.
Birsoy K et al. has compared the gene expression of adipogenesis in vivo and in vitro using 3T3-L1 cells in culture [14]. The results demonstrated that 3T3-L1 adipocyte differentiation in culture share similar expression patterns with the development of WAT in vivo, provided direct evidences that differentiation of adipocyte in culture recapitulates many of the transcriptional programs that are functional during development of WAT in vivo. Moreover, a transcriptome analysis of adipose tissue from pigs revealed DEGs related to adipose growth, lipid metabolism, extracellular matrix and immune response [40]. Jiang MK et al. performed RNA sequencing during adipogenesis using the primary cultured brown adipocyte, they found 6668 DEGs during adipogenesis but without GO and KEGG analysis [41]. Our present study examined the transcriptional profile changes during adipocyte differentiation using a 3T3-L1 cell line. We performed KEGG, Go analysis and hierarchical clustering for the first time, demonstrated the cellular functions during adipocyte differentiation are phase-dependent, although lipid metabolism and metabolic process are involved throughout the whole process of adipocyte differentiation.
RNA sequencing results revealed 8 divergent gene expression patterns during adipocyte differentiation. We therefore performed a hierarchical clustering to generate a heatmap of the 8 divergent gene expression patterns (Fig. 5). The most significant patterns were profile 2 and 7, which are first increase and decrease, respectively. GO analysis revealed that these 2 patterns were mainly involve an increased metabolism ability, decreased immune response and cellular functions. Moreover, increased fat cell differentiation and decreased mRNA transcriptomic function were also enriched in profile 2 and 7. These results clearly demonstrated the distinct expression patterns involve different cellular functions, which further revealed the differentiation mechanisms of adipocyte.
To date, calcium signaling is poorly known in adipocyte differentiation. Most of the TRP channels are calcium-permeable channels. Bishnoi M et al. reported that TRPV1, TRPV3, TRPM8, TRPC4, TRPC6 were differentially expressed in preadipocytes and adipocytes [32]. We previously reported that Trpv1 and Trpv3 mRNA were significantly decreased, whereas Trpv2 and Trpv4 mRNA were significantly increased in WAT of either db/db or diet-induced obesity mice [42]. It has been reported that TRPV1 and TRPV3 were significantly decreased in WAT of obesity mice, and involved in adipogenesis of WAT [20, 43]. TRPV2 is up-regulated in matured brown adipocyte and involved in brown adipocyte differentiation [21, 44]. Moreover, TRPC1 regulates brown adipose tissue activity in a PPARγ-dependent manner [45]. TRPV4 is decreased in differentiated adipocyte, and involved in the regulation of adipose oxidative metabolism, inflammation, and energy homeostasis [22]. TRPM4, but not TRPM5, has been reported to be required for adipogenesis [46]. In addition, TRPM7 has been reported to be involved in osteogenic differentiation of mesenchymal stromal cells through osterix pathway [47]. Our present results demonstrated that the mRNA expression levels of Trpv4, Trpm4, Trpm5 and Trpm7 were significantly decreased in the differentiated adipocytes. On the other hand, the mRNA expression levels of Trpv1, Trpv2, Trpv6 and Trpc1 were significantly increased, compared with pre-adipocytes (Fig. 6). Taken together, our results suggested that these altered TRP channels, including TRPV1, TRPV2, TRPV4, TRPV6, TRPM4, TRPM5 and TRPM7 and TRPC1, might be involved in adipocyte differentiation.