Gongshe Yang

Modulation of Sirt1 by resveratrol and nicotinamide alters proliferation and differentiation of pig preadipocytes.

Sirt1, a NAD+-dependent histone deacetylase, may regulate senescence, metabolism, and apoptosis. In this study, primary pig preadipocytes were cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS) with or without reagents affecting Sirt1 activity. The adipocyte differentiation process was visualized by light microscopy after Oil red O staining. Proliferation and differentiation of preadipocytes was measured using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and Oil red O extraction. Expression of Sirt1, FoxO1, and adipocyte specific genes was detected with semi-quantitive RT-PCR. The results showed that Sirt1 mRNA was widely expressed in various pig tissues from different developmental stages. Sirt1 mRNA was expressed throughout the entire differentiation process of pig preadipocytes. Resveratrol significantly increased Sirt1 mRNA expression, but decreased the expression of FoxO1 and adipocyte marker gene PPARγ2. Resveratrol significantly inhibited pig preadipocyte proliferation and differentiation. Nicotinamide decreased the expression of Sirt1 mRNA, but increased the expression of FoxO1 and adipocyte specific genes. Nicotinamide greatly stimulated the proliferation and differentiation of pig preadipocytes. In conclusion, these results indicate that Sirt1 may modulate the proliferation and differentiation of pig preadipocytes. Sirt1 may down-regulate pig preadipocytes proliferation and differentiation through repression of adipocyte genes or FoxO1.

MicroRNA identity and abundance in developing swine adipose tissue as determined by solexa sequencing

MicroRNAs (miRNAs) are small ∼22‐nt regulatory RNAs that regulate the stability and translation of cognate mRNAs. MiRNAs participate in the regulation of adipogenesis, and identification of the full repertoire of miRNAs expressed in adipose tissue is likely to significantly increase our understanding of adipose tissue growth and development. Here, we adopted a deep sequencing approach to determine the identity and abundance of miRNAs in developing swine adipose tissue. Via this approach, we identified the sequences and relative expression levels of 227 conserved miRNAs (of which 59 were novel) and 66 potential porcine miRNAs. The expression levels displayed a large range, as reflected by the number of sequence reads, which varied from several counts for rare miRNAs to several million reads for the most abundant miRNAs. The abundant miRNAs principally belonged to 32 miRNA gene families, including miR‐143, miR‐103, let‐7, and miR‐148. Of the conserved miRNAs, 93 miRNAs were up‐regulated and 33 miRNAs were down‐regulated in the adult pig adipose tissue. Moreover, we observed sequence variants and seed edits of the miRNAs. KEGG pathway analysis and GO term enrichment suggested that highly expressed miRNAs are involved in adipose tissue development, signal transduction, cell–cell and cell–extracellular matrix communication, neural development and function, and lipid metabolism including carboxylic acid, oxoacid, fatty acid, steroid, glycerolipid, alcohol and phospholipid metabolism. Our results expand the number of known porcine miRNAs and provide a thorough account of the miRNA transcriptome in porcine adipose tissue. J. Cell. Biochem. 112: 1318–1328, 2011.

Roles of Wnt/β-catenin signaling in adipogenic differentiation potential of adipose-derived mesenchymal stem cells

Wnt/beta-catenin signaling pathway controls differentiation of various cells by regulating the expression of target genes. beta-Catenin plays a central role in Wnt/beta-catenin signaling pathway. To investigate the molecular mechanisms of fate determination in adipose-derived mesenchymal stem cells (AMSCs), we investigated effects of Wnt3a and beta-catenin, two key members of the Wnt/beta-catenin signaling, in adipogenic differentiation of porcine AMSCs. We demonstrated that Wnt3a protein can inhibit the adipogenic differentiation of porcine AMSCs in vitro culture. By stabilization of cytoplasmic beta-catenin with continuous treatment by LiCl, the adipogenic differentiation of AMSCs was also suppressed and the osteogenesis was stimulated. In contrast, a loss of beta-catenin in AMSCs enhanced the adipogenic differentiation and rescued LiCl-induced anti-adipogenesis. In addition, the mutual activation of CCAAT/enhancer-binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor-gamma (PPARgamma) were repressed in the presence of Wnt3a or LiCl, but increased in the gene silencing of beta-catenin. Taken together, our study indicated that Wnt/beta-catenin signaling pathway inhibited the adipogenic differentiation potential and alter the cell fate from adipocytes to osteoblasts.

Osteogenic and adipogenic potential of porcine adipose mesenchymal stem cells

Human, rat, and mouse studies have demonstrated the existence of a population of adipose mesenchymal stem cells (AMSCs) that can undergo multilineage differentiation in vitro. Understanding the clinical potential of AMSCs may require their use in preclinical large-animal models such as pigs. Thus, the objectives of this study were to establish a protocol for the isolation of porcine AMSCs from adipose tissue and to examine their ex vivo differentiation potential to adipocytes and osteoblast. The porcine AMSCs from passage 4 were selected for differentiation analysis. The adipocytes were identified morphologically by staining with Oil Red O, and the adipogenic marker genes were examined by RT-PCR technique. Osteogenic lineage was documented by deposition of calcium stained with Alzarin Red S, visualization of alkaline phosphatase activity, and expression of marker gene. Our result indicates that porcine AMSCs have been successfully isolated and induced differentiation into adipocytes and osteoblasts. This study suggested that porcine AMSCs are also a valuable model system for the study on the mesenchymal lineages for basic research and tissue engineering.

Interleukin-6 stimulates lipolysis in porcine adipocytes

Interleukin (IL)-6 stimulates lipolysis in human and rodents adipocytes. However, the mechanism regulating this process is little known. In this study, we demonstrated that IL-6 increased lipolysis in differentiated porcine adipocytes by activation of extracellular signal-related kinase (ERK), which was inhibited by specific ERK inhibitor PD98059. IL-6 treatment did not elevate intracellular cAMP and specific PKA inhibitor H89 did not affect IL-6-induced lipolysis, which suggested that protein kinase A (PKA) pathway was not involved in IL-6-induced lipolysis. Also, the expressions of perilipin A and PPARγ2 were significantly reduced in response to IL-6 treatment, but the expressions of peroxisome proliferators-activated receptor gamma coactivator-1 alpha (PGC-1α), carnitinepalmitoyl-transferase-1 (CPT-1), and uncoupling protein 2 (UCP2) were significantly elevated. In conclusion, these results suggested that chronic high dose of IL-6 directly stimulated lipolysis in porcine adipocytes through activation of ERK, subsequently repressing perilipin A and promoting PGC-1α expression.

Mitochondrial development and the influence of its dysfunction during rat adipocyte differentiation.

Mitochondrial biogenesis is inherent to adipocyte differentiation. Mitochondrial dysfunction leads to abnormal lipid accumulation or the deterioration of the differentiation process. The aim of this study is to investigate the mitochondrial development during the differentiation of rat primary adipocytes and the effect of mitochondrial dysfunction on this process. We found, for the first time, that the number of mitochondria markedly increased during adipocyte differentiation by transmission electron microscopy. By immunofluorescence staining that the protein content of Cyt c increased in differentiated adipocyte in comparison with preadipocyte. The mRNA expression levels of mitochondrial gene including cytochromes c (Cyt c), malate dehydrogenases (MDH), and peroxisome proliferator activated receptor (PPAR) γ coactivator-1β (PGC-1β) significantly increased along with the proceeding of adipocyte differentiation. The damage to mitochondrial respiratory chain function by rotenone caused significant decrease in gene expressions including mitochondrial MDH and PGC-1β, and PPARγ, CAAT/enhancer binding protein α (C/EBPα) and sterol regulatory element binding protein-1c (SREBP-1c), which are known as transcription factors of differentiation, and differentiation marker gene named fatty acid synthetase. Moreover, an apparent decrease was found in the synthesis of triglyceride and ATP due to the damage to mitochondria by rotenone. Based on the above results, our present study revealed that the density and oxidative capacity of mitochondrial markedly increased during primary adipocyte differentiation, and on the other hand, we suggested that mitochondria dysfunction might inhibit the differentiation process.

Knockdown of PU.1 AS lncRNA inhibits adipogenesis through enhancing PU.1 mRNA translation

PU.1 is an Ets family transcription factor involved in the myelo‐lymphoid differentiation. We have previously demonstrated that PU.1 is also expressed in the adipocyte lineage. However, the expression levels of PU.1 mRNA and protein in preadipocytes do not match the levels in mature adipocytes. PU.1 mRNA level is higher in preadipocytes, whereas its protein is expressed in the adipocytes but not in the preadipocytes. The underlying mechanism remains elusive. Here, we find that miR‐155 knockdown or overexpression has no effect on the levels of PU.1 mRNA and protein in preadipocytes or adipocytes. MiR‐155 regulates adipogenesis not through PU.1, but via C/EBPβ which is another target of miR‐155. We also checked the expression levels of PU.1 mRNA and antisense long non‐coding RNA (AS lncRNA). Interestingly, compared with the level of PU.1 mRNA, the level of PU.1 AS lncRNA is much higher in preadipocytes, whereas it is opposite in the adipocytes. We further discover that PU.1 AS lncRNA binds to its mRNA forming an mRNA/AS lncRNA compound. The knockdown of PU.1 AS by siRNA inhibits adipogenesis and promotes PU.1 protein expression in both preadipocytes and adipocytes. Furthermore, the repression of PU.1 AS decreases the expression and secretion of adiponectin. We also find that the effect of retroviral‐mediated PU.1 AS knockdown on adipogenesis is consistent with that of PU.1 AS knockdown by siRNA. Taken together, our results suggest that PU.1 AS lncRNA promotes adipogenesis through preventing PU.1 mRNA translation via binding to PU.1 mRNA to form mRNA/AS lncRNA duplex in preadipocytes. J. Cell. Biochem. 114: 2500–2512, 2013.

MiRNA-199a-3p Regulates C2C12 Myoblast Differentiation through IGF-1/AKT/mTOR Signal Pathway

MicroRNAs constitute a class of ~22-nucleotide non-coding RNAs. They modulate gene expression by associating with the 3′ untranslated regions (3′ UTRs) of messenger RNAs (mRNAs). Although multiple miRNAs are known to be regulated during myoblast differentiation, their individual roles in muscle development are still not fully understood. In this study, we showed that miR-199a-3p was highly expressed in skeletal muscle and was induced during C2C12 myoblasts differentiation. We also identified and confirmed several genes of the IGF-1/AKT/mTOR signal pathway, including IGF-1, mTOR, and RPS6KA6, as important cellular targets of miR-199a-3p in myoblasts. Overexpression of miR-199a-3p partially blocked C2C12 myoblast differentiation and the activation of AKT/mTOR signal pathway, while interference of miR-199a-3p by antisense oligonucleotides promoted C2C12 differentiation and myotube hypertrophy. Thus, our studies have established miR-199a-3p as a potential regulator of myogenesis through the suppression of IGF-1/AKT/mTOR signal pathway.

Sirt1 AS lncRNA interacts with its mRNA to inhibit muscle formation by attenuating function of miR-34a

Recent studies demonstrate the functions of long non-coding RNAs (lncRNAs) in mediating gene expression at the transcriptional or translational level. Our previous study identified a Sirt1 antisense (AS) lncRNA transcribed from the Sirt1 AS strand. However, its role and regulatory mechanism is still unknown in myogenesis. Here, functional analyses showed that Sirt1 AS lncRNA overexpression promoted myoblast proliferation, but inhibited differentiation. Mechanistically, Sirt1 AS lncRNA was found to activate its sense gene, Sirt1. The luciferase assay provided evidences that Sirt1 AS lncRNA interacted with Sirt1 3′ UTR and rescued Sirt1 transcriptional suppression by competing with miR-34a. In addition, RNA stability assay showed that Sirt1 AS lncRNA prolonged Sirt1 mRNA half-life from 2 to 10 h. Ribonuclease protection assay further indicated that it fully bound to Sirt1 mRNA in the myoblast cytoplasm. Moreover, Sirt1 AS overexpression led to less mouse weight than the control because of less lean mass and greater levels of Sirt1, whereas the fat mass and levels of miR-34a were not altered. Based on the findings, a novel regulatory mechanism was found that Sirt1 AS lncRNA preferably interacted with Sirt1 mRNA forming RNA duplex to promote Sirt1 translation by competing with miR-34a, inhibiting muscle formation.

PU.1 antisense lncRNA against its mRNA translation promotes adipogenesis in porcine preadipocytes.

Antisense long non-coding RNAs (AS lncRNAs) play important roles in refined regulation of animal gene expression. However, their functions and molecular mechanisms for domestic animal adipogenesis are largely unknown. Here, we found a novel AS lncRNA transcribed from the porcine PU.1 gene (also known as SPI1) by strand-specific RT-PCR. Results showed that PU.1 AS lncRNA was expressed and generally lower than the level of PU.1 mRNA in porcine subcutaneous adipose, heart, liver, spleen, lympha, skeletal muscle and kidney tissues. We further found that the levels of PU.1 mRNA and PU.1 protein were significantly lower in subcutaneous and intermuscular adipose than in mesenteric and greater omentum adipose, whereas the levels of PU.1 AS lncRNA showed no difference in porcine adipose tissues from four different parts of the body. During porcine adipogenesis, levels of PU.1 mRNA increased at day 2 and then gradually decreased. Meanwhile, PU.1 AS lncRNA exhibited an expression trend similar to PU.1 mRNA but sharply decreased after day 2. Interestingly, PU.1 protein level rose during differentiation. In addition, at day 6 after differentiation, knockdown of endogenous PU.1 promoted adipogenesis, whereas knockdown of endogenous PU.1 AS lncRNA had the opposite effect. Moreover, peroxisome proliferator-activated receptor gamma (PPARG) and fatty acid synthase (FASN) were significantly upregulated in the PU.1 shRNA treatment group (P < 0.05), whereas they were downregulated in the PU.1 AS shRNA treatment group (P < 0.05). Adipose triglyceride lipase [ATGL; also known as patatin-like phospholipase domain containing 2 (PNPLA2)] and hormone-sensitive lipase [HSL; also known as lipase, hormone-sensitive (LIPE)] contrasted with PPARG and FASN. Finally, the PU.1 mRNA/PU.1 AS lncRNA duplex was detected by an endogenous ribonuclease protection assay combined with RT-PCR. Based on the above results, we suggest that PU.1 AS lncRNA (vs. its mRNA translation) promotes adipogenesis through the formation of a sense-antisense RNA duplex with PU.1 mRNA.