Tongxing Song

Transcriptome comparison between porcine subcutaneous and intramuscular stromal vascular cells during adipogenic differentiation.

Intramuscular fat (IMF) is an important trait influencing meat quality, and preadipocyte differentiation is a key factor affecting IMF deposition. Here we compared the transcriptome profiles of porcine intramuscular and subcutaneous preadipocytes during differentiation to gain insight into specific molecular and cellular events associated with intramuscular stromal vascular cell (MSVC) differentiation. RNA-Seq was used to screen for differentially expressed genes (DEGs) during the in vitro differentiation of MSVC and subcutaneous stromal vascular cell (ASVC) on days 0, 2 and 4. A total of 985 DEGs were identified during ASVC differentiation and 1469 DEGs during MSVC differentiation. Among these DEGs, 409 genes were specifically expressed during ASVC differentiation, 893 genes were specifically expressed during MSVC differentiation, and 576 DEGs were co-expressed during ASVC and MSVC differentiation. The expression profiles of DEGs during ASVC or MSVC differentiation were determined by cluster analysis based on Short Time-series Expression Miner (STEM). Four significant STEM profiles (profiles 1, 4, 5, and 14) were determined during ASVC differentiation, and four significant STEM profiles (profiles 1, 4, 11, and 14) were determined during MSVC differentiation. Gene ontology (GO) analysis indicated that DEGs related to adipocyte differentiation were identified to be significantly enriched in both adipose and muscle profile 14. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs in adipose profile 14 and muscle profiles 11 and 14 (STEM clustered them into one cluster) showed that the PPAR signaling pathway was significantly enriched in these profiles and four signaling pathways were specifically enriched in muscle profiles 11 and 14. Furthermore, analysis of transcription factor binding sites (TFBS) in the gene set revealed two over-represented transcription factors (NR3C4 and NR3C1), which were specifically significantly enriched in the promoter regions of genes within muscle gene expression profiles 11 and 14.

GPR120 promotes adipogenesis through intracellular calcium and extracellular signal-regulated kinase 1/2 signal pathway

Numerous researches have demonstrated that GPR120 (also called FFAR4) exerts novel functions in insulin resistance and adipogenesis. However, the molecular mechanism of GPR120-mediated adipogenic differentiation is still unclear. This study was aimed to interpret the relevant function mechanism of GPR120 in the differentiation of 3T3-L1 adipocytes. The results showed that GPR120 expression was dramatically increased along with the adipogenic differentiation of 3T3-L1 adipocytes and the adipogenic ability was significantly inhibited in shGPR120-transfected cells. TUG-891, a selective agonist of GPR120, promoted the intracellular triglyceride accumulation in a dose-dependent manner and did not enhance adipogenesis in shGPR120-transfected cells. Markedly, TUG-891 increased the activation of PPARγ in a GPR120-dependent pathway as assessed by luciferase reporter assay. Furthermore, in the adipogenic differentiation process of 3T3-L1 adipocytes, TUG-891 increased the [Ca(2+)]i and phosphorylation level of ERK1/2. Pretreatment with inhibitors of either ERK1/2 (U0126) or [Ca(2+)]i (BAPTA-AM) notably attenuated the GPR120-mediated adipogenesis. These results show that GPR120 promotes adipogenesis by increasing PPARγ expression via [Ca(2+)]i and ERK1/2 signal pathway in 3T3-L1 adipocytes.

Cloning and characterization of spliced variants of the porcine G protein coupled receptor 120.

The polyunsaturated fatty acids (PUFAs) receptor GPR120 exerts a significant impact on systemic nutrient homeostasis in human and rodents. However, the porcine GPR120 (pGPR120) has not been well characterized. In the current study, we found that pGPR120 had 3 spliced variants. Transcript 1 encoded 362-amino acids (aa) wild type pGPR120-WT, which shared 88% homology with human short form GPR120. Transcript 1 was the mainly expressed transcript of pGPR120. It was expressed predominantly in ileum, jejunum, duodenum, spleen, and adipose. Transcript 3 (coding 320-aa isoform) was detected in spleen, while the transcript 2 (coding 310-aa isoform) was only slightly expressed in spleen. A selective agonist for human GPR120 (TUG-891) and PUFAs activated SRE-luc and NFAT-luc reporter in HEK293T cells transfected with construct for pGPR120-WT but not pGPR120-V2. However, 320-aa isoform was not a dominant negative isoform. The extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation levels in cells transfected with construct for pGPR120-WT were well activated by PUFAs, especially n-3 PUFA. These results showed that although pGPR120 had 3 transcripts, transcript 1 which encoded pGPR120-WT was the mainly expressed transcript. TUG-891 and PUFAs, especially n-3 PUFA, well activated pGPR120-WT. The current study contributed to dissecting the molecular regulation mechanisms of n-3 PUFA in pigs.

SIRT1 suppresses adipogenesis by activating Wnt/β-catenin signaling in vivo and in vitro.

Sirtuin 1 (SIRT1) regulates adipocyte and osteoblast differentiation. However, the underlying mechanism should be investigated. This study revealed that SIRT1 acts as a crucial repressor of adipogenesis. RNA-interference-mediated SIRT1 knockdown or genetic ablation enhances adipogenic potential, whereas SIRT1 overexpression inhibits adipogenesis in mesenchymal stem cells (MSCs). SIRT1 also deacetylates the histones of sFRP1, sFRP2, and Dact1 promoters; inhibits the mRNA expression of sFRP1, sFRP2, and Dact1; activates Wnt signaling pathways; and suppresses adipogenesis. SIRT1 deacetylates β-catenin to promote its accumulation in the nucleus and thus induces the transcription of genes that block MSC adipogenesis. In mice, the partial absence of SIRT1 promotes the formation of white adipose tissues without affecting the development of the body of mice. Our study described the regulatory role of SIRT1 in Wnt signaling and proposed a regulatory mechanism of adipogenesis.

Methionine Regulates mTORC1 via the T1R1/T1R3-PLCβ-Ca2+-ERK1/2 Signal Transduction Process in C2C12 Cells

The mammalian target of rapamycin complex 1 (mTORC1) integrates amino acid (AA) availability to support protein synthesis and cell growth. Taste receptor type 1 member (T1R) is a G protein-coupled receptor that functions as a direct sensor of extracellular AA availability to regulate mTORC1 through Ca2+ stimulation and extracellular signal–regulated kinases 1 and 2 (ERK1/2) activation. However, the roles of specific AAs in T1R1/T1R3-regulated mTORC1 are poorly defined. In this study, T1R1 and T1R3 subunits were expressed in C2C12 myotubes, and l-AA sensing was accomplished by T1R1/T1R3 to activate mTORC1. In response to l-AAs, such as serine (Ser), arginine (Arg), threonine (Thr), alanine (Ala), methionine (Met), glutamine (Gln), and glycine (Gly), Met induced mTORC1 activation and promoted protein synthesis. Met also regulated mTORC1 via T1R1/T1R3-PLCβ-Ca2+-ERK1/2 signal transduction. Results revealed a new role for Met-regulated mTORC1 via an AA receptor. Further studies should be performed to determine the role of T1R1/T1R3 in mediating extracellular AA to regulate mTOR signaling and to reveal its mechanism.

Identification of zinc finger protein Bcl6 as a novel regulator of early adipose commitment.

Adipose tissue is a key determinant of whole-body metabolism and energy homeostasis. Unravelling the transcriptional regulatory process during adipogenesis is therefore highly relevant from a biomedical perspective. In these studies, zinc finger protein B-cell lymphoma 6 (Bcl6) was demonstrated to have a role in early adipogenesis of mesenchymal stem cells. Bcl6 is enriched in preadipose versus non-preadipose fibroblasts and shows upregulated expression in the early stage of adipogenesis. Gain- and loss-of-function studies revealed that Bcl6 acts as a key regulator of adipose commitment and differentiation both in vitro and ex vivo. RNAi-mediated knockdown of Bcl6 in C3H10T1/2 cells greatly inhibited adipogenic potential, whereas Bcl6 overexpression enhanced adipogenic differentiation. This transcription factor also directly or indirectly targets and controls the expression of some early and late adipogenic regulators (i.e. Zfp423, Zfp467, KLF15, C/EBPδ, C/EBPα and PPARγ). We further identified that Bcl6 transactivated the signal transducers and activators of transcription 1 (STAT1), which was determined as a required factor for adipogenesis. Moreover, overexpression of STAT1 rescued the impairment of adipogenic commitment and differentiation induced by Bcl6 knockdown in C3H10T1/2 cells, thereby confirming that STAT1 is a downstream direct target of Bcl6. This study identifies Bcl6 as a positive transcriptional regulator of early adipose commitment.

Molecular cloning, expression pattern analysis of porcine Rb1 gene and its regulatory roles during primary dedifferentiated fat cells adipogenic differentiation

Adipocytes are the main constituent of adipose tissue and are considered to be a corner stone in the homeostatic control of whole body metabolism. Recent reports evidenced that retinoblastoma 1 (Rb1) gene plays an important role in fat development and adipogenesis in mice. Here, we cloned the partial cDNA sequences of the porcine Rb1 gene which contains the complete coding sequences (CDS) of 2820bp encoding a protein of 939 amino acids. Bioinformatic analysis revealed that the CDS of porcine Rb1 was highly identical with those of cattle, human and mice. The porcine Rb1 has three typical conserved structural domains, including Rb-A pocket domain, CYCLIN domain and C-terminus domain, and the phylogenetic tree indicates a closer genetic relationship with cattle and human. Tissue distribution analysis showed that Rb1 expression appeared to be ubiquitously in various tissues, being higher in heart, liver, muscle, and stomach. Furthermore, significant downregulation of Rb1 was found at the initial stage of dedifferentiated fat (DFAT) cells adipogenic differentiation. With the knockdown of the Rb1 expression by siRNA, the number of DFAT cells recruited to white rather than brown adipogenesis was promoted, and mRNA levels of adipogenic markers, such as PPARγ, aP2, LPL and adiponectin and protein expression of PPARγ and adiponectin were increased after hormone stimulation. The underlying mechanisms may be that knockdown of Rb1 promotes the mitotic clonal expansion and PPARγ expression by derepressing the transcriptional activity of E2F so as to facilitate the first steps of adipogenesis. In summary, we cloned and characterized an important negative regulator in adipogenic commitment of porcine DFAT cells.

Phenotypic and Functional Properties of Porcine Dedifferentiated Fat Cells during the Long-Term Culture In Vitro

It has been proved that terminally differentiated mature adipocytes possess abilities to dedifferentiate into fibroblast-like progeny cells with self-renewal and multiple differentiation, termed dedifferentiated fat (DFAT) cells. However, the biological properties of DFAT cells during long-term culture in vitro have not been elucidated. Here, we obtained fibroblast-like morphology of porcine DFAT cells by ceiling culture. During the dedifferentiation process, round mature adipocytes with single large lipid droplets changed into spindle-shaped cells accompanied by the adipogenic markers PPARγ, aP2, LPL, and Adiponectin significant downregulation. Flow cytometric analysis showed that porcine DFAT cells displayed similar cell-surface antigen profile to mesenchymal stem cells (MSCs). Furthermore, different passages of porcine DFAT cells during long-term culture in vitro retained high levels of cell viabilities (>97%), efficient proliferative capacity including population doubling time ranged from 20 h to 22 h and population doubling reached 47.40 ± 1.64 by 58 days of culture. In addition, porcine DFAT cells maintained the multiple differentiation capabilities into adipocytes, osteoblasts, and skeletal myocytes and displayed normal chromosomal karyotypes for prolonged passaging. Therefore, porcine DFAT cells may be a novel model of stem cells for studying the functions of gene in the different biological events.

MiR-377 promotes white adipose tissue inflammation and decreases insulin sensitivity in obesity via suppression of sirtuin-1 (SIRT1)

Obesity is associated with a wide range of metabolic disorders including inflammation and insulin-resistance. Sirtuin-1 (SIRT1) is an important regulator of metabolic homeostasis and stress response pathways in white adipose tissue. However, involvement of microRNAs (miRNAs) in regulating SIRT1 during obesity-induced inflammation and insulin-resistance remains unclear. Here, we found that miR-377 was upregulated in adipose tissue and showed a negative correlation with SIRT1 in chronic high fat diet (HFD)-fed mice. MiR-377 belongs to a large miRNA cluster and functions as an important tumor suppressor in several human malignancies. Recently, it has also gained considerable attention in oxidative stress and diabetic nephropathy. In our present study, we found that overexpression of miR-377 decreased SIRT1 protein abundance and caused inflammation and insulin-resistance in differentiated 3T3-L1 cells. Conversely, miR-377 inhibition increased SIRT1 mRNA and protein levels, ameliorated inflammation and improved insulin sensitivity. Furthermore, we demonstrated that miR-377 targets the 3′-UTR of SIRT1 mRNA directly, and downregulates SIRT1 protein abundance. Inhibition of SIRT1 by EX527 significantly eliminated the downregulation of the inflammation and insulin-resistance levels induced by the miR-377 inhibitor. Furthermore, SIRT1 deficiency intensified adipose tissue inflammation and insulin-resistance, resulting in hepatic steatosis in chronic-HFD-fed mice. In conclusion, our findings suggest that miR-377 promotes white adipose tissue inflammation and decreases insulin sensitivity in obesity, at least in part, through suppressing SIRT1.

GPR120: a critical role in adipogenesis, inflammation, and energy metabolism in adipose tissue

It is well known that adipose tissue has a critical role in the development of obesity and metabolic diseases and that adipose tissue acts as an endocrine organ to regulate lipid and glucose metabolism. Accumulating in the adipose tissue, fatty acids serve as a primary source of essential nutrients and act on intracellular and cell surface receptors to regulate biological events. G protein-coupled receptor 120 (GPR120) represents a promising target for the treatment of obesity-related metabolic disorders for its involvement in the regulation of adipogenesis, inflammation, glucose uptake, and insulin resistance. In this review, we summarize recent studies and advances regarding the systemic role of GPR120 in adipose tissue, including both white and brown adipocytes. We offer a new perspective by comparing the different roles in a variety of homeostatic processes from adipogenic development to adipocyte metabolism, and we also discuss the effects of natural and synthetic agonists that may be potential agents for the treatment of metabolic diseases.