Understanding Of Adipogenesis Research Articles

Prmt6 represses the pro-adipogenic Ppar-gamma–C/ebp-alpha transcription factor loop

The feed-forward loop between the transcription factors Ppar-gamma and C/ebp-alpha is critical for lineage commitment during adipocytic differentiation. Ppar-gamma interacts with epigenetic cofactors to activate C/ebp-alpha and the downstream adipocytic gene expression program. Therefore, knowledge of the epigenetic cofactors associated with Ppar-gamma, is central to understanding adipocyte differentiation in normal differentiation and disease. We found that Prmt6 is present with Ppar-gamma on the Ppar-gamma and C/ebp-alpha promoter. It contributes to the repression of C/ebp-alpha expression, in part through its ability to induce H3R2me2a. During adipocyte differentiation, Prmt6 expression is reduced and the methyltransferase leaves the promoters. As a result, the expression of Ppar-gamma and C/ebp-alpha is upregulated and the adipocytic gene expression program is established. Inhibition of Prmt6 by a small molecule enhances adipogenesis, opening up the possibility of epigenetic manipulation of differentiation. Our data provide detailed information on the molecular mechanism controlling the Ppar-gamma–C/ebp-alpha feed-forward loop. Thus, they advance our understanding of adipogenesis in normal and aberrant adipogenesis.

Novel Insights into Adipogenesis from the Perspective of Transcriptional and RNA N6-Methyladenosine-Mediated Post-Transcriptional Regulation.

Obesity is a critical risk factor causing the development of metabolic diseases and cancers. Its increasing prevalence worldwide has aroused great concerns of the researchers on adipose development and metabolic function. During adipose expansion, adipogenesis is a way to store lipids as well as to avoid lipotoxicity in other tissues, and may be an approach to offset the negative metabolic effects of obesity. In this Review, the transcriptional regulation of adipogenesis is outlined to characterize numerous biological processes in research on the determination of adipocyte fate and regulation of adipogenic differentiation. Notably, one of the post‐transcriptional modifications of mRNA, namely, N6‐methyladenosine (m6A), has been recently found to play a role in adipogenesis. Here, the roles of m6A‐related enzymes and proteins in adipogenesis, with a particular focus on how these m6A‐related proteins function at different stages of adipogenesis, are mainly discussed. The Review also highlights the coordination role of the transcriptional and post‐transcriptional (RNA m6A methylation) regulation in adipogenesis and related biological processes. In this context, a better understanding of adipogenesis at both the transcriptional and post‐transcriptional levels may facilitate the development of novel strategies to improve metabolic health in obesity.

The Role of Pref-1 during Adipogenic Differentiation: An Overview of Suggested Mechanisms.

Obesity contributes significantly to the global health burden. A better understanding of adipogenesis, the process of fat formation, may lead to the discovery of novel treatment strategies. However, it is of concern that the regulation of adipocyte differentiation has predominantly been studied using the murine 3T3-L1 preadipocyte cell line and murine experimental animal models. Translation of these findings to the human setting requires confirmation using experimental models of human origin. The ability of mesenchymal stromal/stem cells (MSCs) to differentiate into adipocytes is an attractive model to study adipogenesis in vitro. Differences in the ability of MSCs isolated from different sources to undergo adipogenic differentiation, may be useful in investigating elements responsible for regulating adipogenic differentiation potential. Genes involved may be divided into three broad categories: early, intermediate and late-stage regulators. Preadipocyte factor-1 (Pref-1) is an early negative regulator of adipogenic differentiation. In this review, we briefly discuss the adipogenic differentiation potential of MSCs derived from two different sources, namely adipose-derived stromal/stem cells (ASCs) and Wharton’s Jelly derived stromal/stem cells (WJSCs). We then discuss the function and suggested mechanisms of action of Pref-1 in regulating adipogenesis, as well as current findings regarding Pref-1’s role in human adipogenesis.

Adipogenesis for soft tissue reconstruction.

It has been increasingly common to use adipose tissue for regenerative and reconstructive purposes. Applications of autologous fat transfer and different stem cell therapies have significant limitations and adipose tissue engineering may have the potential to be an important strategy in the reconstruction of large tissue defects. A better understanding of adipogenesis will help to develop strategies to make adipose tissue more effective for repairing volumetric defects. We provide an overview of the current applications of adipose tissue transfer and cellular therapy methods for soft tissue reconstruction, cellular physiology, and factors influencing adipogenesis, and adipose tissue engineering. Furthermore, we discuss mechanical properties and vascularization strategies of engineered adipose tissue, and its potential applications in the clinical settings. Autologous fat tissue transfer is the standard of care technique for the majority of surgeons; however, high resorption rates, poor perfusion within a large volume fat graft and widely inconsistent graft survival are the main limitations. Adipose tissue engineering is a promising field to reach the first goal of producing adipose tissue which has more predictable survival and higher graft retention rates. Advancements of scaffold and vascularization strategies will contribute to metabolically and functionally more relevant adipose tissue engineering.

A Novel lnc-RNA, Named lnc-ORA, Is Identified by RNA-Seq Analysis, and Its Knockdown Inhibits Adipogenesis by Regulating the PI3K/AKT/mTOR Signaling Pathway.

Obesity is closely associated with numerous adipogenic regulatory factors, including coding and non-coding genes. Long noncoding RNAs (lncRNAs) play a major role in adipogenesis. However, differential expression profiles of lncRNAs in inguinal white adipose tissue (iWAT) between wild-type (WT) and ob/ob mice, as well as their roles in adipogenesis, are not well understood. Here, a total of 2809 lncRNAs were detected in the iWAT of WT and ob/ob mice by RNA-Sequencing (RNA-Seq), including 248 novel lncRNAs. Of them, 46 lncRNAs were expressed differentially in WT and ob/ob mice and were enriched in adipogenesis signaling pathways as determined by KEGG enrichment analysis, including the PI3K/AKT/mTOR and cytokine–cytokine receptor interaction signaling pathways. Furthermore, we focused on one novel lncRNA, which we named lnc-ORA (obesity-related lncRNA), which had a seven-fold higher expression in ob/ob mice than in WT mice. Knockdown of lnc-ORA inhibited preadipocyte proliferation by decreasing the mRNA and protein expression levels of cell cycle markers. Interestingly, lnc-ORA knockdown inhibited adipocyte differentiation by regulating the PI3K/AKT/mTOR signaling pathway. In summary, these findings contribute to a better understanding of adipogenesis in relation to lncRNAs and provide novel potential therapeutic targets for obesity-related metabolic diseases.

Now UCP(rotein), Now You Don’t: UCP1 Is Not Mandatory for Thermogenesis

Adipocyte-targeted therapies could potentially combat obesity and metabolic disorders; however, our understanding of adipogenesis and the characterization of the various adipose tissues are incomplete. In this issue, Bertholet etal. (2017) report the existence of two types of heat-producing beige-like adipocytes, those with and those without the mitochondrial uncoupling protein 1 (UCP1).

Cell Models and Their Application for Studying Adipogenic Differentiation in Relation to Obesity: A Review.

Over the last several years, the increasing prevalence of obesity has favored an intense study of adipose tissue biology and the precise mechanisms involved in adipocyte differentiation and adipogenesis. Adipocyte commitment and differentiation are complex processes, which can be investigated thanks to the development of diverse in vitro cell models and molecular biology techniques that allow for a better understanding of adipogenesis and adipocyte dysfunction associated with obesity. The aim of the present work was to update the different animal and human cell culture models available for studying the in vitro adipogenic differentiation process related to obesity and its co-morbidities. The main characteristics, new protocols, and applications of the cell models used to study the adipogenesis in the last five years have been extensively revised. Moreover, we depict co-cultures and three-dimensional cultures, given their utility to understand the connections between adipocytes and their surrounding cells in adipose tissue.

18O-Tracer Metabolomics Reveals Protein Turnover and CDP-Choline Cycle Activity in Differentiating 3T3-L1 Pre-Adipocytes

The differentiation of precursor cells into mature adipocytes (adipogenesis) has been an area of increased focus, spurred by a rise in obesity rates. Though our understanding of adipogenesis and its regulation at the cellular level is growing, many questions remain, especially regarding the regulation of the metabolome. The 3T3-L1 cell line is the most well characterized cellular model of adipogenesis. Using a time course metabolomics approach, we show that the 3T3-L1 preadipocyte metabolome is greatly altered during the first 48 hours of differentiation, where cells go through about two rounds of cell division, a process known as mitotic clonal expansion. Short-chain peptides were among several small molecules that were increased during mitotic clonal expansion. Additional indicators of protein turnover were also increased, including bilirubin, a degradation product of heme-containing proteins, and 3-methylhistidine, a post-translationally modified amino acid that is not reutilized for protein synthesis. To study the origin of the peptides, we treated differentiating preadipocytes with 18O labeled water and found that 18O was incorporated into the short chain peptides, confirming them, at least in part, as products of hydrolysis. Inhibitors of the proteasome or matrix metalloproteinases affected the peptide levels during differentiation, but inhibitors of autophagy or peptidases did not. 18O was also incorporated into several choline metabolites including cytidine 5'-diphosphocholine (CDP-choline), glycerophosphocholine, and several phosphatidylcholine species, indicative of phosphatidylcholine synthesis/degradation and of flux through the CDP-choline cycle, a hallmark of proliferating cells. 18O-Tracer metabolomics further showed metabolic labeling of glutamate, suggestive of glutaminolysis, also characteristic of proliferating cells. Together, these results highlight the utility of 18O isotope labeling in combination with metabolomics to uncover changes in cellular metabolism that are not detectable by time-resolved metabolomics.

Comparative proteome analysis of 3T3-L1 adipocyte differentiation using iTRAQ-coupled 2D LC-MS/MS

Adipose tissue is critical in obesity and type II diabetes. Blocking of adipocyte differentiation is one of the anti-obesity strategies targeting on strong rise in fat storage and secretion of adipokine(s). However, the molecular basis of adipocyte differentiation and its regulation remains obscure. Therefore, we exposed 3T3-L1 cell line to appropriate hormonal inducers as adipocyte differentiation model. Using iTRAQ-coupled 2D LC-MS/MS, a successfully exploited high-throughput proteomic technology, we nearly quantitated 1,000 protein species and found 106 significantly altered proteins during adipocyte differentiation. The great majority of differentially expressed proteins were related to metabolism enzymes, structural molecules, and proteins involved in signal transduction. In addition to previously reported differentially expressed molecules, more than 20 altered proteins previously unknown to be involved with adipogenic process were firstly revealed (e.g., HEXB, DPP7, PTTG1IP, PRDX5, EPDR1, SPNB2, STEAP3, TPP1, etc.). The partially differential proteins were verified by Western blot and/or real-time PCR analysis. Furthermore, the association of PCX and VDAC2, two altered proteins, with adipocyte conversion was analyzed using siRNA method, and the results showed that they could contribute considerably to adipogenesis. In conclusion, our data provide valuable information for further understanding of adipogenesis.

Cellular signaling pathways regulating the initial stage of adipogenesis and marbling of skeletal muscle

Due to extensive efforts to increase lean growth, intramuscular fat (marbling) is reducing in beef, pork and chicken breast, which impairs the eating quality of meat. Because fat is the major contributor to meat flavor, the presence of intramuscular fat is indispensible for the high eating quality of meat. However, up to now, our understanding of adipogenesis (formation of fat cells) in skeletal muscle is limited. Adipocyte differentiation in skeletal muscle initiates from multipotent mesenchymal stem cells, which are abundant in skeletal muscle at early developmental stages. In this review, the known cellular mechanisms regulating adipogenesis from multipotent cells are summarized, which include hedgehog, Wingless and Int (Wnt)/beta-catenin, and bone morphogenesis protein (BMP) mediated signaling pathways, as well as AMP-activated protein kinase. Promoting adipogenesis within skeletal muscle will effectively increase intramuscular fat, improving the quality of meat.

Impact of Stimulatory Pathways on Adipogenesis and HIV-Therapy Associated Lipoatrophy

Current understanding of adipogenesis derives mainly from studies with in vitro cell culture systems with divergent experimental requirements. We aimed to investigate the discrepancy between the anti-adipogenic effects of the HIV protease-inhibitor indinavir (IDV) in vitro and the lack of evidence that IDV inhibits adipogenesis in humans. We studied cell viability and adipogenesis in murine 3T3-F442A, 3T3-L1 and primary human subcutaneous preadipocytes (phsPA). Differentiation was studied after activation of the established four signalling pathways in different combinations. We analyzed CCAAT/enhancer-binding protein (C/EBP) alpha and peroxisome proliferator-activated receptor (PPAR) gamma expression and triacylglyceride accumulation. Cells were exposed to IDV at concentrations around therapeutic C(max) levels and higher (10 muM and 20 muM) for up to 30 days. Under insulin and fetal calf serum (FCS) input, IDV inhibited 3T3-F442A differentiation, an effect that was partially rescued by the addition of 3-isobutyl-1-methylxanthine (IBMX) stimulation. Combined stimulation with FCS, insulin, dexamethasone (DEX) and IBMX led to normal 3T3-L1 differentiation even in the presence of IDV. However, omission of IBMX rendered this cell line sensitive to IDV's anti-adipogenic effects. Differentiation of phsPA requiring complete adipogenic stimulation was not affected by IDV presence. Our data suggest that the potency of IDV to impair differentiation under partial stimulation disappears when all of the differentiation pathways are activated. Such compensatory mechanisms might be responsible for the inability of the drug to affect adipogenesis in vivo.

Altered expression of CCAAT/enhancer binding protein and FABP11 genes during adipogenesisin vitroin Atlantic salmon (Salmo salar)

The study of CCAAT/enhancer binding proteins (C/EBPs) is important in the understanding of adipogenesis, but little is known about their regulation in fish. Here, we report three Atlantic salmon orthologs of c/ebp, and their expression in different tissues and in adipogenesis in vitro. During differentiation the expression of c/ebpα and fatp1 were upregulated in early differentiation stage with continuing high expression level in mature adipocytes, whereas c/ebpδ and fabp11 expression were elevated in mature adipocytes. Furthermore, the fatty acids eicosapentaenoic (EPA) and docosahexaenoic (DHA), suppressed the expression of the c/ebps, pparβ, and fatty acid transport protein (fatp1) during terminal adipocyte differentiation. The study indicates that C/EBPs are induced upon the differentiation of primary-cultured adipocytes from Atlantic salmon and that marine n-3 highly unsaturated fatty acids (HUFAs) affect the c/ebps expressions in mature adipocytes. Therefore, the established cell model described here appears to be valuable for studying modulation of fat content in farmed Atlantic salmon.

How much progress have we made over the last few decades?

To recognize the 20th anniversary of the opening of the Pennington Biomedical Research Center in Baton Rouge, Louisiana, USA, a conference was held featuring some of the most prominent obesity researchers worldwide, addressing the most significant advances in obesity research. The Center had been founded with a gift from wealthy oilman Claude B Pennington with the mission of 'promoting healthier lives through research and education in nutrition and preventive medicine.' The purpose of the conference was to provide a cohesive presentation of the key events and steps that led to the major discoveries or advances in obesity science and give insight into new research directions. Only 20 of many possible topics could be adequately covered in the program of the conference. Their increasing importance is reflected in the increasing number of related publications as extracted from PubMed. The broad general areas in which these topics fall include measurement and evaluation of obesity, measurement of body composition and fat distribution, adipose tissue biology, genetics, bariatric surgery and public policy. Advances in the areas covered at the conference have had a major impact on our understanding of adipogenesis, central and peripheral regulation of body weight, health consequences of excess body weight, challenges of weight loss, magnitude of the worldwide epidemic and impact of public health policies.

A New Preadipocyte Cell Line, AP-18, Established from Adult Mouse Adipose Tissue

Establishing preadipocyte cell lines from mature adipose tissues could help lead to a better understanding of adipogenesis. We have established a unique preadipocyte cell line, AP-18, derived from the subepidermal layer of ear skin from an adult C3H/HeM mouse. AP-18 cells exhibit fibroblast-like morphology, slow growth, and contact inhibition. The doubling time of AP-18 cells is 50-60 h, which is about 2-fold longer than that of well-known 3T3-L1 cells derived from mouse embryos. A small population of AP-18 cells spontaneously differentiates into adipocytes by 8 days after confluence, as judged by the accumulation of triglyceride droplets. Treatment of confluent AP-18 preadipocytes with adipogenic agents, containing dexamethasone, 3-methyl-1-isobutylxanthine, and insulin, increased triglyceride contents about 5-fold compared to the contents in untreated cells. We also analyzed mRNA expression profiles for key transcription factors involved in adipocyte differentiation, peroxisome proliferator-activated receptor (PPAR)gamma and the CCAAT/enhancer binding protein (C/EBP) family, and for differentiation markers, aP2, adipocyte-specific fatty acid-binding protein and adipsin, adipocyte-specific serine protease. AP-18 preadipocytes express mRNAs for C/EBPbeta, C/EBPalpha, PPARgamma, and aP2 before differentiation, but not adipsin mRNA. Expression of aP2 mRNA was increased in fully differentiated AP-18 cells. Likewise, expression of adipsin mRNA was increased after induced differentiation of AP-18 cells and reached the highest level in fully differentiated adipocytes. Thus, differentiation of AP-18 cells is associated with the increased expression of aP2 and adipsin mRNAs. The newly established AP-18 cell line provides a useful model for investigating adipocyte differentiation and adipogenesis.

Dueling Transcription Factors in Adipogenesis

The nuclear hormone receptor family member PPARγ (peroxisome proliferator-activated receptor γ) has important insulinomimetic functions in lipid homeostasis. Dowell et al. used a yeast two-hybrid screen to detect interacting proteins and identified Foxo1, a transcription factor that regulates multiple biological processes including insulin action and metabolic control. Foxo proteins are phosphorylated in response to insulin, which results in decreased transcriptional activity, and constitutively active Foxo1 is antiadipogenic. PPARγ, on the other hand, has insulin-like proadipogenic actions. Transcriptional reporter assays in human embryonic kidney 293T cells showed that PPARγ and Foxo1 each had inhibitory effects on transcription mediated by the other factor. In C. elegans , genetic interactions occur between genes encoding an insulin receptor-like gene, daf-2 ; a Foxo-like gene, daf-16 ; and a nuclear hormone receptor, daf-12 . In vitro, DAF-16 and DAF-12 interacted, as did their mammalian counterparts. DAF-12 also interacted, although more weakly, with mammalian Foxo proteins. The authors propose that there may be an evolutionarily conserved mechanism by which insulin and PPARγ antagonize actions of Foxo proteins and vice versa. Further elucidation of such mechanisms would enhance our understanding of adipogenesis and insulin sensitivity. P. Dowell, T. C. Otto, S. Adi, M. D. Lane, Convergence of peroxisome proliferator-activated receptor γ and Foxo1 signaling pathways. J. Biol. Chem. 278 , 45485-45491 (2003). [Abstract] [Full Text]

Microarray Analyses during Adipogenesis: Understanding the Effects of Wnt Signaling on Adipogenesis and the Roles of Liver X Receptor α in Adipocyte Metabolism

Wnt signaling maintains preadipocytes in an undifferentiated state. When Wnt signaling is enforced, 3T3-L1 preadipocytes no longer undergo adipocyte conversion in response to adipogenic medium. Here we used microarray analyses to identify subsets of genes whose expression is aberrant when differentiation is blocked through enforced Wnt signaling. Furthermore, we used the microarray data to identify potentially important adipocyte genes and chose one of these, the liver X receptor alpha (LXR alpha), for further analyses. Our studies indicate that enforced Wnt signaling blunts the changes in gene expression that correspond to mitotic clonal expansion, suggesting that Wnt signaling inhibits adipogenesis in part through dysregulation of the cell cycle. Experiments designed to uncover the potential role of LXR alpha in adipogenesis revealed that this transcription factor, unlike CCAAT/enhancer binding protein alpha and peroxisome proliferator-activated receptor gamma, is not adipogenic but rather inhibits adipogenesis if inappropriately expressed and activated. However, LXR alpha has several important roles in adipocyte function. Our studies show that this nuclear receptor increases basal glucose uptake and glycogen synthesis in 3T3-L1 adipocytes. In addition, LXR alpha increases cholesterol synthesis and release of nonesterified fatty acids. Finally, treatment of mice with an LXR alpha agonist results in increased serum levels of glycerol and nonesterified fatty acids, consistent with increased lipolysis within adipose tissue. These findings demonstrate new metabolic roles for LXR alpha and increase our understanding of adipogenesis.