Role In Adipocyte Differentiation Research Articles

Understanding the adipose tissue acetylome in obesity and insulin resistance

Obesity is a widely prevalent pathology with a high exponential growth worldwide. Altered lipid accumulation by adipose tissue is one of the main causes of obesity and exploring lipid homeostasis in this tissue may represent a source for the identification of possible therapeutic targets. The study of the proteome and the post-translational modifications of proteins, specifically acetylation due to its involvement in energy metabolism, may be of great interest to understand the molecular mechanisms involved in adipose tissue dysfunction in obesity. The objective of this study was to characterize the subcutaneous and omental adipose tissue acetylome in conditions of obesity and insulin resistance and to describe the importance of acetylation of key molecules in adipose tissue to use them as therapeutic targets. The results describe for the first time the acetylome of subcutaneous and omental adipose tissue under physiological and physiopathological conditions such as obesity and insulin resistance. New evidence showed different acetylation patterns between two main depots and highlight the molecular complexity of adipose tissue. Results showed changes in FABP4 acetylation in subcutaneous fat in relation to insulin resistance, thus unveiling a potential marker of depot-specific dysfunctional expansion in obesity-associated metabolic disease. Furthermore, it is shown that the acetylation of FABP4 affects its function, modulating the capacity of differentiation in adipocytes. In conclusion, this study demonstrates a profound, depot-specific alteration of adipose tissue acetylome, wherein the acetylation of FABP4 may play a key role in adipocyte differentiation and lipid accumulation.

Long noncoding RNA XIST regulates brown preadipocytes differentiation and combats high-fat diet induced obesity by targeting C/EBP\u03b1

BackgroundActivation of brown adipose tissue (BAT) increases energy expenditure, which makes it an attractive therapeutic strategy for obesity. LncRNAs play an important role in adipocyte differentiation and regulation. Here we assessed the effect of lncRNA XIST on brown preadipocytes differentiation and metabolic regulation.MethodsXIST expression levels were detected in human perirenal (peri-N) and subcutaneous adipose tissues (sub-Q), brown preadipocytes and 3T3-L1 preadipocytes. XIST overexpression and knockdown experiments were performed in brown preadipocytes. XIST overexpression mouse model was established by plasmid injection through tail vein.ResultsIn human adipose tissues, XIST expression was significantly higher in female than in male individuals. In vitro, XIST expression was significantly up-regulated during brown adipocyte differentiation. XIST knockdown inhibited differentiation of brown preadipocytes, while overexpression of XIST promotes brown preadipocytes to fully differentiation. RNA Binding Protein Immunoprecipitation (RIP) experiment revealed that XIST could directly bind to C/EBPα. In vivo, XIST overexpression prevents high-fat diet induced obesity and improves metabolic dysorder in male mice.ConclusionOur results suggest that XIST combats obesity through BAT activation at least partly by combination with transcription factor C/EBPα.

High expression of P4HA3 in obesity: a potential therapeutic target for type 2 diabetes.

The aims of the present study were to evaluate the expression of prolyl 4-hydroxylase subunit alpha 3 (P4HA3) in adipocytes and adipose tissue and to explore its effect on obesity and type 2 diabetes mellitus (T2DM). We initially demonstrated that P4HA3 was significantly upregulated in the subcutaneous adipose tissue of obesity and T2DM patients, and its functional roles in adipocyte differentiation and insulin resistance were investigated using in vitro and in vivo models. The knockdown of P4HA3 inhibited adipocyte differentiation and improved insulin resistance in 3T3-L1 cells. In C57BL/6J db/db mice fed with a high fat diet (HFD), silencing P4HA3 significantly decreased fasting blood glucose and triglycerides (TG) levels, with concomitant decrease of body weight and adipose tissue weight. Further analysis showed that P4HA3 knockdown was correlated with the augmented IRS-1/PI3K/Akt/FoxO1 signaling pathway in the adipose and hepatic tissues of obese mice, which could improve hepatic glucose homeostasis and steatosis of mice. Together, our study suggested that the dysregulation of P4HA3 may contribute to the development of obesity and T2DM.

3T3-L1 세포에서 김치유산균 Weissella koreensis DB1 추출물의 지방분화 억제효과

Weissella koreensis (W. koreensis) DB1 with high-ornithine-producing capacity was isolated from kimchi and shows the anti-adipogenic activity via its production of ornithine. This study was carried out the effects of W. koreensis DB1 cell extracts on the inhibition of adipogenesis in 3T3-L1 cells. MTT results showed that 0-0.75 μg/mL W. koreensis DB1 cell extracts had no significant effect on cell vability. The cell extracts of W. koreensis DB1 at non-cytotoxic concentrations significantly suppressed the differentiated 3T3-L1 cells by decreasing triglyceride level and intracellular lipid accumulation compared to that associated with the untreated control group. The major transcriptional factors involved in adipocyte differentiation, such as peroxisome proliferator-activated receptorγ (PPARγ), CCAAT/enhancer-binding proteinα (C/EBPα), and sterol regulatory element-binding transcription factor-1c (SREBP-1c), were down-regulated by the cell extracts of W. koreensis DB1 treatments compared with the untreated control group. Furthermore, the expressions of lipogenesis-related enzymes including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) genes were also decreased by the cell extracts of W. koreensis DB1 treatment. Accordingly, these results indicate that the cell extracts of W. koreensis DB1 treatment inhibited the adipogenesis and lipogenesis of 3T3-L1 cells, suggesting a protective role in adipocyte differentiation.

PPARs-Orchestrated Metabolic Homeostasis in the Adipose Tissue.

It has been more than three decades since peroxisome proliferator-activated receptors (PPARs) were first discovered. Many investigations have revealed the central regulators of PPARs in lipid and glucose homeostasis in response to different nutrient conditions. PPARs have attracted much attention due to their ability to improve metabolic syndromes, and they have also been proposed as classical drug targets for the treatment of hyperlipidemia and type 2 diabetes (T2D) mellitus. In parallel, adipose tissue is known to play a unique role in the pathogenesis of insulin resistance and metabolic syndromes due to its ability to “safely” store lipids and secrete cytokines that regulate whole-body metabolism. Adipose tissue relies on a complex and subtle network of transcription factors to maintain its normal physiological function, by coordinating various molecular events, among which PPARs play distinctive and indispensable roles in adipocyte differentiation, lipid metabolism, adipokine secretion, and insulin sensitivity. In this review, we discuss the characteristics of PPARs with special emphasis on the roles of the different isotypes in adipocyte biology.

JAZF1 heterozygous knockout mice show altered adipose development and metabolism

BackgroundJuxtaposed with another zinc finger protein 1 (JAZF1) is associated with metabolic disorders, including type 2 diabetes mellitus (T2DM). Several studies showed that JAZF1 and body fat mass are closely related. We attempted to elucidate the JAZF1 functions on adipose development and related metabolism using in vitro and in vivo models.ResultsThe JAZF1 expression was precisely regulated during adipocyte differentiation of 3T3-L1 preadipocyte and mouse embryonic fibroblasts (MEFs). Homozygous JAZF1 deletion (JAZF1-KO) resulted in impaired adipocyte differentiation in MEF. The JAZF1 role in adipocyte differentiation was demonstrated by the regulation of PPARγ—a key regulator of adipocyte differentiation. Heterozygous JAZF1 deletion (JAZF1-Het) mice fed a normal diet (ND) or a high-fat diet (HFD) had less adipose tissue mass and impaired glucose homeostasis than the control (JAZF1-Cont) mice. However, other metabolic organs, such as brown adipose tissue and liver, were negligible effect on JAZF1 deficiency.ConclusionOur findings emphasized the JAZF1 role in adipocyte differentiation and related metabolism through the heterozygous knockout mice. This study provides new insights into the JAZF1 function in adipose development and metabolism, informing strategies for treating obesity and related metabolic disorders.

MicroRNAs mediated regulation of glutathione peroxidase 7 expression and its changes during adipogenesis

Glutathione peroxidase 7 (GPx7) acts as an intracellular stress sensor/transmitter and plays an important role in adipocyte differentiation and the prevention of obesity related pathologies. For this reason, finding the regulatory mechanisms that control GPx7 expression is of great importance. As microRNAs (miRNAs) could participate in the regulation of GPx7 expression, we studied the inhibition of GPx7 expression by four selected miRNAs with relation to obesity and adipogenesis. The effect of the transfection of selected miRNAs mimics on GPx7 expression was tested in three cell models (HEK293, SW480, AT-MSC). The interaction of selected miRNAs with the 3′UTR of GPx7 was followed up on using a luciferase gene reporter assay. In addition, the levels of GPx7 and selected miRNAs in adipose tissue mesenchymal stem cells (AT-MSC) and mature adipocytes from four human donors were compared, with the changes in these levels during adipogenesis analyzed. Our results show for the first time that miR-137 and miR-29b bind to the 3′UTR region of GPx7 and inhibit the expression of this enzyme at the mRNA and protein level in all the human cells tested. However, no negative correlation between miR-137 nor miR-29b level and GPx7 was observed during adipogenesis.Despite the confirmed inhibition of GPx7 expression by miR-137 and miR-29b, the action of these two molecules in adipogenesis and mature adipocytes must be accompanied by other regulators.

Whole-Transcriptome Analysis of Preadipocyte and Adipocyte and Construction of Regulatory Networks to Investigate Lipid Metabolism in Sheep.

Many local sheep breeds in China have poor meat quality. Increasing intramuscular fat (IMF) content can significantly improve the quality of mutton. However, the molecular mechanisms of intramuscular adipocyte formation and differentiation remain unclear. This study compared differences between preadipocytes and mature adipocytes by whole-transcriptome sequencing and constructed systematically regulatory networks according to the relationship predicted among the differentially expressed RNAs (DERs). Sequencing results showed that in this process, there were 1,196, 754, 100, and 17 differentially expressed messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs), respectively. Gene Ontology analysis showed that most DERs enriched in Cell Part, Cellular Process, Biological Regulation, and Binding terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the DERs primarily focused on Focal adhesion, phosphoinositide 3-kinase (PI3K)-Akt, mitogen-activated protein kinase (MAPK), peroxisome proliferator-activated receptor (PPAR) signaling pathways. Forty (40) DERs were randomly selected from the core regulatory network to verify the accuracy of the sequence data. The results of qPCR showed that the DER expression trend was consistent with sequence data. Four novel promising candidate miRNAs (miR-336, miR-422, miR-578, and miR-722) played crucial roles in adipocyte differentiation, and they also participated in multiple and important regulatory networks. We verified the expression pattern of the miRNAs and related pathways’ members at five time points in the adipocyte differentiation process (0, 2, 4, 6, 8, 10 days) by qPCR, including miR-336/ACSL4/LncRNA-MSTRG71379/circRNA0002331, miR-422/FOXO4/LncRNA-MSTRG54995/circRNA0000520, miR-578/IGF1/LncRNA-MSTRG102235/circRNA0002971, and miR-722/PDK4/LncRNA-MSTRG107440/circ RNA0002909. In this study, our data provided plenty of valuable candidate DERs and regulatory networks for researching the molecular mechanisms of sheep adipocyte differentiation and will assist studies in improving the IMF.

Role of GATA2 in the Maintenance of the Bone Marrow Microenvironment

(Background) The bone marrow (BM) microenvironment comprises multiple stem cell niches derived from BM mesenchymal stem cells (BM-MSCs). Previous in vitro analyses have suggested that transcription factor GATA2 plays an important role in adipocyte differentiation and hematopoietic supporting activity of BM-MSCs (Kamata et al. Haematologica 2014). However, the role of GATA2 in BM-MSCs in vivo remains uncharacterized.(Method) Mice harboring a Gata2 exon 5 flanked by loxP sites (Gata2 -floxed mice, designated Gata2flox) were a kind gift from Dr. Camper (Charles et al. Molecular Endocrinology 2006). Gata2 was deletedin under the MSC-related promoters (Nestin , Prx1 , and Lepr ) (Matsuzaki et al. Cell Stem Cell 2014) based on the Cre-loxP system (Gata2f/f/ Nes -ER-Cre, Gata2f/f/ Prx1 -Cre, and Gata2f/f/ Lepr -Cre mice) (The Jackson Laboratory). For transplantation analysis, 2x105 BM cells from SJL (CD45.1+; The Jackson Laboratory) mice were intravenously injected into lethally irradiated CD45.2+Gata2flox/flox/ER-Cre or CD45.2+Gata2flox/flox mice (a total of 10 Gy). The recipient mice were injected intraperitoneally with 1 mg of tamoxifen (Sigma, USA) on days 29-31 and 36-38 after transplantation and subsequently analyzed on days 50-51. For the analysis, we focused on the CD45.1 donor cell fraction. To isolate MSCs, the compact bones were crushed with a pestle and treated with collagenase (Wako, Japan), after which Ter119-CD45- MSC fraction was separated using EasySep Mouse Mesenchymal Stem/Progenitor Cell Enrichment Kit (StemCell Technologies, Canada). BM hematopoietic progenitors were separated and evaluated with both MACS (Miltenyi Biotech) and BD FACSAria II (BD Biosciences). For transcription profiling, a GeneChip Mouse Gene 2.0 ST Array (Affymetrix, USA) was used and the data was subsequently analyzed with the DAVID Bioinformatics Program (http://david.abcc.ncifcrf.gov/).(Results) After confirming Gata2 deletion under each BM-MSC-related promoter (Nestin , Prx1 , and Lepr ), we evaluated the hematological profile, total BM cell counts, BM progenitor frequency (LSK, CMP, GMP, MEP, and CLP), colony assay with sorted LSK fraction, BM histology, and colony-forming cell assay of BM-MSCs. Whereas we found a significant decrease in the colony number of Gata2flox/flox/ Prx1 -Cre mice, the phenotypic alterations in BM-MSC-specific conditional Gata2 knockout mice are somewhat weak, implying that Gata2 deletion with a single marker might be insufficient. Thus, we next transplanted normal BM cells into irradiated Gata2flox/flox/ER-Cre mice, after which Gata2 was knocked out by tamoxifen administration. In this case, Gata2 would be deleted in all cell types constituting the BM microenvironment but not the hematopoietic cells. Based on the analyses of the CD45.1+ donor cell fraction, we found a significant decrease in the percentage of the CMP fraction, whereas that of CLP was marginally increased (p = 0.081) by Gata2 knockout, suggesting that GATA2 expression in BM-MSCs might function in the differentiation of LSKs to CMPs.To elucidate the underlying molecular mechanisms, we performed expression profiling with the MSCs obtained from Gata2flox/flox/ER-Cre mice. Prior to the profiling analysis, RT-PCR analysis confirmed that the separated MSCs abundantly expressed MSC-related genes, including Nestin , Prx1 , Lepr , Cd29 , and Sca-1 . Based on the average of 2 independent datasets, we demonstrated that GATA2 knockout activated 110 and repressed 141 genes (> 2-fold). The repressed gene ensemble included adhesion molecules (Itga11 , Itgb3 ), extracellular matrix (Prg3 ), and genes involved in hematopoietic progenitor mobilization (Mmp9 , Elane , Ctsg ). Activated genes included Fabp5 , involved in fatty acid metabolism, suggesting that GATA2 knockout might accelerate adipocyte differentiation in vivo . Gene Ontology analysis revealed significant enrichment of genes related to cell adhesion (P = 4.8E−05) and chemotaxis (P = 2.7E−08), suggesting that the expressions for extracellular signals in BM-MSCs might be regulated by GATA2.(Conclusion) Besides its critical role in hematopoietic stem cells, GATA2 could also affect the function of BM-MSCs in vivo . Our data might offer a better understanding of the pathophysiology of bone marrow failure syndrome. DisclosuresNo relevant conflicts of interest to declare.

Molecular Cloning, Expression and Characterization of Peroxisome Proliferators-Activated Receptors Gamma in the Sea Urchin (Strongylocentrotus intermedius)

Peroxisome proliferators-activated receptor gamma (PPARγ) plays important regulatory roles in adipocyte differentiation. In this study, we cloned the full-length sequence of the PPARγ gene and analyzed its expression profile in different developmental stages and tissues of Strongylocentrotus intermedius. The full-length cDNA of PPARγ contains 2286 base pairs (bp) with a putative open reading frame of 1755 bp, and the gene encodes encoding a polypeptide of 584 amino acid residues. The predicted molecular mass of the protein is 67.27 kDa, and its theoretical isoelectric point (pI) is 10.07. The protein contains conserved motifs, including an RRM (RNA recognition motif) domain. PPARγ expression with the highest level was observed in the gonad, and the lowest was observed in the tube feet of S. intermedius. Time-course expression measurements at different developmental stages showed that the highest expression level of PPARγ is in the eggs and its weakest expression level is in the 32-cells stage. Knock-down of PPARγ by specific siRNA revealed that UCP2 expression is significantly decreased in the gonads and intestines 48 h post-transfection, indicating that the UCP2 is a downstream target gene of PPARγ. This finding suggests that PPARγ and UCP2 have positive regulatory effects in regulating adipocyte differentiation. Changes in fatty acid levels in the gonads before and after PPARγ interference were assessed, and decreased C18:2(trans, n™6) and C20:3(n−6) levels were observed 48 h after siRNA transfection. The results showed the function of PPARγ in fatty acid anabolism, The data are helpful to improve the current understanding of the fatty acid synthesis pathways and regulatory mechanisms in S. intermedius. They also provide an experimental basis for improving fatty acid synthesis in sea urchins, which is important for cultivating sea urchins with high nutritional value.

Hematopoietically-expressed homeobox protein HHEX regulates adipogenesis in preadipocytes

Obesity is a key health problem and is associated with a high risk of type 2 diabetes and other metabolic diseases. Increased weight as well as dysregulation of adipocyte homeostasis are the main drivers of obesity. Pathological adipogenesis plays a central role in obesity-related complications such as type 2 diabetes, hypertension and others. Thus, an understanding of the molecular mechanisms involved in physiological and pathogenic adipogenesis can help to develop new strategies to prevent or cure obesity and related diseases. Previously, genetic polymorphisms in the HHEX gene that encodes the homeobox transcription factor HEX (PRH) were found to be associated with type 2 diabetes and high body mass index at birth by GWAS in distinct human populations. To understand whether HHEX has a regulatory function in adipogenesis, we performed RNAi-mediated knockdown of Hhex in preadipocyte cell line 3T3-L1 in vitro, and studied changes in the efficacy of adipogenesis. We found that Hhex knockdown blocks adipogenesis in preadipocytes in a dose-dependent manner and leads to a significant decrease of PPAR-gamma protein – the main regulator of adipogenesis. We also propose that Hhex can play an important role in adipocyte differentiation by affecting the level of the PPAR-gamma protein. Our study supports the claim that Hhex plays an important role in adipocyte differentiation program and can contribute to fat tissue homeostasis.

PPAR-gamma induced AKT3 expression increases levels of mitochondrial biogenesis driving prostate cancer

Peroxisome Proliferator-Activated Receptor Gamma (PPARG) is one of the three members of the PPAR family of transcription factors. Besides its roles in adipocyte differentiation and lipid metabolism, we recently demonstrated an association between PPARG and metastasis in prostate cancer. In this study a functional effect of PPARG on AKT serine/threonine kinase 3 (AKT3), which ultimately results in a more aggressive disease phenotype was identified. AKT3 has previously been shown to regulate PPARG co-activator 1 alpha (PGC1α) localisation and function through its action on chromosome maintenance region 1 (CRM1). AKT3 promotes PGC1α localisation to the nucleus through its inhibitory effects on CRM1, a known nuclear export protein. Collectively our results demonstrate how PPARG over-expression drives an increase in AKT3 levels, which in turn has the downstream effect of increasing PGC1α localisation within the nucleus, driving mitochondrial biogenesis. Furthermore, this increase in mitochondrial mass provides higher energetic output in the form of elevated ATP levels which may fuel the progression of the tumour cell through epithelial to mesenchymal transition (EMT) and ultimately metastasis.

PPARgamma in Metabolism, Immunity, and Cancer: Unified and Diverse Mechanisms of Action

The proliferator-activated receptor γ (PPARγ), a member of the nuclear receptor superfamily, is one of the most extensively studied ligand-inducible transcription factors. Since its identification in the early 1990s, PPARγ is best known for its critical role in adipocyte differentiation, maintenance, and function. Emerging evidence indicates that PPARγ is also important for the maturation and function of various immune system-related cell types, such as monocytes/macrophages, dendritic cells, and lymphocytes. Furthermore, PPARγ controls cell proliferation in various other tissues and organs, including colon, breast, prostate, and bladder, and dysregulation of PPARγ signaling is linked to tumor development in these organs. Recent studies have shed new light on PPARγ (dys)function in these three biological settings, showing unified and diverse mechanisms of action. Classical transactivation—where PPARγ activates genes upon binding to PPAR response elements as a heterodimer with RXRα—is important in all three settings, as underscored by natural loss-of-function mutations in FPLD3 and loss- and gain-of-function mutations in tumors. Transrepression—where PPARγ alters gene expression independent of DNA binding—is particularly relevant in immune cells. Interestingly, gene translocations resulting in fusion of PPARγ with other gene products, which are unique to specific carcinomas, present a third mode of action, as they potentially alter PPARγ’s target gene profile. Improved understanding of the molecular mechanism underlying PPARγ activity in the complex regulatory networks in metabolism, cancer, and inflammation may help to define novel potential therapeutic strategies for prevention and treatment of obesity, diabetes, or cancer.

PAR2 Deficiency Induces Mitochondrial ROS Generation and Dysfunctions, Leading to the Inhibition of Adipocyte Differentiation.

Protease-activated receptor 2 (PAR2) is a member of G-protein-coupled receptors and affects ligand-modulated calcium signaling. Although PAR2 signaling promotes obesity and adipose tissue inflammation in high fat- (HF-) fed conditions, its role in adipocyte differentiation under nonobesogenic conditions needs to be elucidated. Here, we used several tissues and primary-cultured adipocytes of mice lacking PAR2 to study its role in the development of adipose tissues. C57BL/6J mice with PAR2 deficiency exhibited a mild lipodystrophy-like phenotype in a chow diet-fed condition. When adipocyte differentiation was examined using primary-cultured preadipocytes, PAR2 deficiency led to a notable decrease in adipocyte differentiation and related protein expression, and PAR2 agonist treatment elevated adipocyte differentiation. Regarding the mechanism, PAR2-deficient preadipocytes exhibited impaired mitochondrial energy consumption. Further studies indicated that calcium-related signaling pathways for mitochondrial biogenesis are disrupted in the adipose tissues of PAR2-deficient mice and PAR2-deficient preadipocytes. Also, a PAR2 antagonist elevated mitochondrial reactive oxygen species and reduced the MitoTracker fluorescent signal in preadipocytes. Our studies revealed that PAR2 is important for the development of adipose tissue under basal conditions through the regulation of mitochondrial biogenesis and adipocyte differentiation.

Deletion of SREBF1, a Functional Bone-Muscle Pleiotropic Gene, Alters Bone Density and Lipid Signaling in Zebrafish.

Through a genome-wide analysis of bone mineral density (BMD) and muscle mass, identification of a signaling pattern on 17p11.2 recognized the presence of sterol regulatory element-binding factor 1 (SREBF1), a gene responsible for the regulation of lipid homeostasis. In conjunction with lipid-based metabolic functions, SREBF1 also codes for the protein, SREBP-1, a transcription factor known for its role in adipocyte differentiation. We conducted a quantitative correlational study. We established a zebrafish (ZF) SREBF1 knockout (KO) model and used a targeted customized lipidomics approach to analyze the extent of SREBF1 capabilities. For lipidomics profiling, we isolated the dorsal muscles of wild type (WT) and KO fishes, and we performed liquid chromatography-tandem mass spectrometry screening assays of these samples. In our analysis, we profiled 48 lipid mediators (LMs) derived from various essential polyunsaturated fatty acids to determine potential targets regulated by SREBF1, and we found that the levels of 11,12 epoxyeicosatrienoic acid (11,12-EET) were negatively associated with the number of SREBF1 alleles (P = 0.006 for a linear model). We also compared gene expression between KO and WT ZF by genome-wide RNA-sequencing. Significantly enriched pathways included fatty acid elongation, linoleic acid metabolism, arachidonic acid metabolism, adipocytokine signaling, and DNA replication. We discovered trends indicating that BMD in adult fish was significantly lower in the KO than in the WT population (P < 0.03). These studies reinforce the importance of lipidomics investigation by detailing how the KO of SREBF1 affects both BMD and lipid-signaling mediators, thus confirming the importance of SREBF1 for musculoskeletal homeostasis.

Isolation, identification, expression and subcellular localization of PPARG gene in buffalo mammary gland.

Peroxisome proliferator-activated receptor gamma (PPARG), as a member of the nuclear receptor superfamily, plays an important role in adipocyte differentiation and regulation of lipid and glucose metabolism. In this study, the transcripts of PPARG gene were isolated and identified in buffalo mammary gland. The results showed that two types of transcripts (PPARG1 and PPARG2) of PPARG gene produced by alternative 5' end use were expressed in buffalo mammary gland, and each of them had four different alternative splicing variants. The PPARG1 includes PPARG1a, PPARG1b, PPARG1c and PPARG1d, while the PPARG2 contains PPARG2a, PPARG2b, PPARG2c and PPARG2d. Among them, only PPARG1a, PPARG2a and PPARG2d can encode complete functional proteins with three complete functional domains, and the rest encode truncated proteins with incomplete functional domains. All the eight variants of PPARG protein do not contain transmembrane regions and signal peptides, but their conserved domain, secondary and tertiary structure and subcellular localization were different. Subcellular localization confirmed that the main transcripts PPARG1a and PPARG2a played a functional role in the nucleus, which was consistent with the results by in silico prediction. RT-qPCR analysis of buffalo mammary tissue showed that the mRNA expression levels of PPARG1 and PPARG2 in lactation were higher than those in non-lactation, and the expression levels of transcripts PPARG2d and PPARG1b+PPARG2b in lactating stage were also higher than those in non-lactating stage, but the mRNA abundance of transcripts PPARG1c, PPARG1d and PPARG2c in non-lactating period was higher than that in lactating period. The results of this study suggest that PPARG1 and PPARG2 may play important role in buffalo milk fat synthesis, and the eight alternative splicing variants found here are likely to be related to the post-transcriptional regulation of lactation.

Release of the Non-Steroidal Anti-Inflammatory Drug Flufenamic Acid by Multiparticulate Delivery Systems Promotes Adipogenic Differentiation of Adipose-Derived Stem Cells.

Engineered tissue-like structures often instigate an inflammatory response in the host that can inhibit wound healing and ultimately lead to the rejection of the implant. In our previous study, we have characterized the properties and biocompatibility of novel multiparticulate drug delivery systems (MDDS), based on collagen matrix with gradual release of anti-inflammatory drug flufenamic acid, we evaluated their anti-inflammatory potential and demonstrated their efficiency against burns and soft tissue lesions. In addition to these results, FA was previously described as a stimulant for adipogenesis, therefore we hypothesized that MDDS might also be appropriate for adipose tissue engineering. After the cell-scaffold constructs were obtained, cell morphology, adhesion and spreading on the systems were highlighted by scanning electron microscopy, immunostaining and confocal microscopy. The effect of FA-enriched materials on adipogenesis was evaluated at gene and protein level, by RT-qPCR, confocal microscopy and immunohistochemistry. Our current work indicates that flufenamic acid plays a beneficial role in adipocyte differentiation, with a direct effect upon the gene and protein expression of important early and late markers of adipogenesis, such as PPARγ2 and perilipin.

A Novel Regulatory Circuit “C/EBPα/miR-20a-5p/TOB2” Regulates Adipogenesis and Lipogenesis

Recent studies have identified growing importance of microRNAs as key regulators of adipocyte differentiation. We have previously reported that miR-20a-5p is able to induce adipogenesis of established adipogenic cell lines and bone marrow derived mesenchymal stem cells (BMSCs). However, the molecular mechanisms by which miR-20a-5p controls adipogenesis and by which miR-20a-5p expression is regulated need to be further explored. In the current study we found that miR-20a-5p expression was induced during adipocyte differentiation from preadipocyte 3T3-L1 and was increased in epididymal white adipose tissue from either ob/ob mice or high fat diet-induced obese mice. Functional studies identified miR-20a-5p as a positive regulator of adipocyte differentiation and lipogenesis in 3T3-L1 by using either synthetic mimics to supplement miR-20a-5p, or using synthetic inhibitor or sponge lentivirus to inactivate endogenous miR-20a-5p. Luciferase activity assay revealed that TOB2 is a novel target of miR-20a-5p and functional experiment demonstrated its negative regulatory role in adipocyte differentiation. Moreover, Tob2 overexpression significantly attenuated adipocyte formation induced by miR-20a-5p supplementation. In-depth investigation of mechanisms that govern miR-20a-5p expression clarified that C/EBPα transcriptionally activated miR-20a-5p expression via binding to the promoter of miR-20a-5p. Taken together, we conclude that a novel C/EBPα/miR-20a-5p/TOB2 circuit exists and regulates adipogenesis and lipogenesis.

MiR-340-5p inhibits sheep adipocyte differentiation by targeting ATF7.

Several microRNAs (miRNAs) have been identified to play roles in adipocyte differentiation. However, little is known about their involvement in the differentiation of ovine intramuscular adipocytes. Here, the role of one such miRNA, miR-340-5p, in ovine adipocyte differentiation was investigated. Stromal vascular (SV) cells were isolated from skeletal muscle tissues of 1-month-old lambs and induced to differentiate into mature adipocytes. miRNA mimics and inhibitors were used for miR-340-5p overexpression and knockdown assays. For overexpression and knockdown of activating transcription factor 7 (ATF7), lentivirus infection was performed. Luciferase reporter assay was performed to determine the relationship between miR-340-5p and ATF7. The expression of adipogenesis marker genes, PPARγ, C/EBPα, FABP4, ADIPOQ, and ACC, and formation of lipid droplets were detected after the overexpression and inhibition of miR-340-5p, or upon overexpression or knockdown of ATF7. miR-340-5p inhibited the expression of the marker genes and the formation of lipid droplets. ATF7 positively regulated the expression of the marker genes and the formation of lipids. Thus, ATF7 is the target of miR-340-5p in sheep. Overall, these findings indicate that miR-340-5p acts as an inhibitor of the differentiation of intramuscular adipocytes by targeting ATF7. Our study provides a new theoretical basis for improving sheep meat quality.

Acer okamotoanum inhibits adipocyte differentiation by theregulation of adipogenesis and lipolysis in 3T3‑L1 cells.

Acer okamotoanum is reported to have various antioxidant, anti‑inflammatory and beneficial immune system effects. The anti‑adipocyte differentiation effects and mechanisms of the ethyl acetate (EtOAc) fraction of an A.okamotoanum extraction was investigated in 3T3‑L1 adipocyte cells. Treatment with differentiation inducers increased the level of triglycerides (TGs) in 3T3‑L1 adipocyte cells compared with an untreated control. However, the EtOAc fraction of A.okamotoanum significantly decreased TGs. Treatment with 1, 2.5 and 5µg/ml showed weak activity, but TG production was inhibited at 10µg/ml compared with the control. In addition, A.okamotoanum caused a significant downregulation of proteins related to adipogenesis, such as γ‑cytidine‑cytidine‑adenosine‑adenosine‑thymidine/enhancer binding protein‑α, ‑β and peroxisome proliferator‑activated receptor‑γ, compared with the untreated control. Furthermore, A.okamotoanum significantly upregulated lipolysis related protein, hormone‑sensitive lipase and the phosphorylation of adenosine monophosphate‑activated protein kinase (AMPK). Therefore, these results indicate that A.okamotoanum suppressed adipogenesis and increased lipolysis and the activation of AMPK, suggesting a protective role in adipocyte differentiation.