Effect of ATP on preadipocyte migration and adipocyte differentiation by activating P2Y receptors in 3T3-L1 cells.
ABSTRACT: The effect of extracellular ATP on adipogenesis was investigated using the mouse 3T3-L1 cell line. Incubation of cells with ATP (1-100 microM) for 5 min induced actin filament reorganization and membrane ruffling mediated through P2Y receptors. Enhancement of preadipocyte migration into fat cell clusters is one of the essential processes of adipose tissue development in vivo and cell migration assays revealed that stimulation of P2Y receptors enhanced chemokinesis (migration) in a concentration dependent manner. In this cell line, growth arrest is required before initiation of differentiation and growth-arrested post-confluent cells can be converted into adipocytes by the presence of the adipogenic hormones dexamethasone, 3-isobutyl-1-methylxanthine and insulin. On the other hand, those hormones alone do not trigger differentiation in proliferating cells. ATP did not induce differentiation when applied alone to either proliferating or postconfluent cells. By contrast, proliferating cells (density <50%) preincubated with ATP for 5 min and subsequently given the adipogenic hormones in the continued presence of ATP, underwent adipocyte differentiation mediated through phospholipase C-coupled P2Y receptors. These adipocytes were found to show very similar characteristics, including morphology and intracellular triacylglycerol accumulation compared with adipocytes differentiated from post-confluent preadipocytes with those adipogenic hormones. When proliferating cells were preincubated with ATP before the addition of the adipogenic hormones, gene expression of aP2 (adipose protein 2) was markedly increased within 6 days, whereas without ATP pretreatment the expression level stayed very low. These results suggest that extracellular ATP renders preadipocytes responsive to adipogenic hormones during the growth phase.
Project description:Oleuropein, the major phenolic compound found in olive leaves and oil, exerts antioxidant, anti-inflammatory and anti-atherogenic effects and suppresses the adipocyte differentiation in vitro. Herein, we characterized molecular mechanisms underlying the anti-adipogenic effects of oleuropein on 3T3-L1 cells and adipocytes derived from stromal-vascular fraction of dorsolumbar and gonadal fat dissected from mice. We found that oleuropein (>100 ?M) decreased viability of preadipocytes proliferating in vitro and did not exerted any cytotoxic effects in post-confluent cells after induction of differentiation. Oleuropein (>100 ?M) inhibited adipocyte differentiation, suppressed gene expression of peroxisome proliferator-activated receptor ? (PPAR?), CCAAT-/enhancer-binding protein ?, sterol regulatory element-binding transcription factor 1c and fatty acid synthase. Furthermore, we tested ability of oleuropein to regulate of PPAR?-, PPAR?- or PPAR?-/PPAR?-mediated ?-lactamase expression in appropriate reporter gene assays. Oleuropein between 10 and 400 ?M concentrations did not affect activity of PPAR? or PPAR?/?. Contrary, PPAR? activity, either basal or rosiglitazone activated, was inhibited by oleuropein. Our data suggest that oleuropein exerts anti-adipogenic effect through direct inhibition of PPAR? transcriptional activity.
Project description:Differentiation of preadipocytes into mature adipocytes capable of efficiently storing lipids is an important regulatory mechanism in obesity. Here, we examined the involvement of histone deacetylases (HDACs) and histone acetyltransferases (HATs) in the regulation of adipogenesis. We find that among the various members of the HDAC and HAT families, only HDAC9 exhibited dramatic down-regulation preceding adipogenic differentiation. Preadipocytes from HDAC9 gene knock-out mice exhibited accelerated adipogenic differentiation, whereas HDAC9 overexpression in 3T3-L1 preadipocytes suppressed adipogenic differentiation, demonstrating its direct role as a negative regulator of adipogenesis. HDAC9 expression was higher in visceral as compared with subcutaneous preadipocytes, negatively correlating with their potential to undergo adipogenic differentiation in vitro. HDAC9 localized in the nucleus, and its negative regulation of adipogenesis segregates with the N-terminal nuclear targeting domain, whereas the C-terminal deacetylase domain is dispensable for this function. HDAC9 co-precipitates with USF1 and is recruited with USF1 at the E-box region of the C/EBP? gene promoter in preadipocytes. Upon induction of adipogenic differentiation, HDAC9 is down-regulated, leading to its dissociation from the USF1 complex, whereas p300 HAT is up-regulated to allow its association with USF1 and accumulation at the E-box site of the C/EBP? promoter in differentiated adipocytes. This reciprocal regulation of HDAC9 and p300 HAT in the USF1 complex is associated with increased C/EBP? expression, a master regulator of adipogenic differentiation. These findings provide new insights into mechanisms of adipogenic differentiation and document a critical regulatory role for HDAC9 in adipogenic differentiation through a deacetylase-independent mechanism.
Project description:The rapid increase of obesity during the last decades and its future prospects are alarming. Besides the general discussed causes of obesity, the 'Developmental Origins of Health and Disease' (DOHaD) hypothesis received more attention in recent years. This hypothesis postulates an adverse influence during early development that programs the unborn child for metabolic dysfunctions later in life. Childhood obesity - an as much increasing problem - can be predisposed by maternal overweight and diabetes. Both, obesity and hyperinsulinemia are major causes of female hyperandrogenemia. As predicted by the DOHaD hypothesis and shown in animal models, developmental androgen excess can lead to metabolic abnormalities in offspring. In this study, we investigated, if androgen exposure adversely affects the adipogenic differentiation of preadipocytes and the endocrine function of adult adipocytes. The human SGBS preadipocyte model was used to affirm the de novo biosynthesis of steroid hormones under normal adipogenesis conditions. Normal adipogenesis was paralleled by an increase of corticosteroids and androgens, whereas estrogen remained at a steady level. Treatment with androstenedione had no effect on SGBS proliferation and differentiation, but adult adipocytes exhibited a significant higher accumulation of triglycerides. Progesterone (up to 2-fold), testosterone (up to 38-fold) and cortisone (up to 1.4-fold) - but not cortisol - were elevated by androstenedione administration in adult adipocytes. Estrogen was not altered. Data suggest that androgen does not negatively influence adipogenic differentiation, but steroidogenic function of SGBS adipocytes.
Project description:Tetrabromobisphenol A (TBBPA) is the most common flame retardant used in electrical housings, circuit boards, and automobiles. High-throughput screening and binding assays have identified TBBPA as an agonist for human peroxisome proliferator-activated receptor gamma (PPAR?), the master regulator of adipogenesis. TBBPA has been suggested to be an obesogen based on in vitro cellular assays and zebrafish data. We hypothesized that exposing preadipocytes to TBBPA could influence adipogenesis via genes other than those in the PPAR? pathway due to its structural similarity to bisphenol A, which demonstrates varied endocrine disrupting activities. Mouse-derived 3T3-L1 preadipocytes were induced to differentiate and continually treated with TBBPA for 8?days. High-content imaging of adipocytes displayed increased adipocyte number and lipid accumulation when treated with TBBPA. TBBPA exhibited weak induction of mPPAR?, with an AC50 of 397?µM. Quantitative PCR revealed that TBBPA exposure increased early expression of genes involved in glucocorticoid receptor (GR) signaling and PPAR? transcriptional activation, as well as upregulating downstream genes needed for adipocyte maintenance and nontraditional ER signaling, such as Gpr30. Additionally, Pref1 and Thy1, inhibitors of differentiation, were downregulated by some concentrations of TBBPA. Furthermore, proliferating preadipocytes treated with TBBPA, only prior to differentiation, exhibited increased adipocyte number and lipid accumulation after 8?days in normal culture conditions. In conclusion, TBBPA influenced gene expression changes in GR, nontraditional ER, and known adipogenic regulatory genes, prior to PPAR? expression; effects suggesting early programming of adipogenic pathways.
Project description:Adipose tissue plays central role in determining the gustatory quality of beef, but traditional Chinese beef cattle have low levels of fat content. We applied RNA-seq to study the molecular mechanisms underlying adipocyte differentiation in Qinchuan cattle. A total of 18,283 genes were found to be expressed in preadipocytes and mature adipocytes, respectively. 470 of which were significantly differentially expressed genes (DEGs) [false discovery rate (FDR) values < 0.05 and fold change ? 2]. In addition, 4534 alternative splicing (AS) events and 5153 AS events were detected in preadipocytes and adipocytes, respectively. We constructed a protein interaction network, which suggested that collagen plays an important role during bovine adipogenic differentiation. We characterized the function of the most down-regulated DEG (P < 0.001) among genes we have detected by qPCR, namely, the transthyretin (TTR) gene. Overexpression of TTR appears to promote the expression of the peroxisome proliferator activated receptor ? (PPAR?) (P < 0.05) and fatty acid binding Protein 4 (FABP4) (P < 0.05). Hence, TTR appears to be involved in the regulation of bovine adipogenic differentiation. Our study represents the comprehensive approach to explore bovine adipocyte differentiation using transcriptomic data and reports an involvement of TTR during bovine adipogenic differentiation. Our results provide novel insights into the molecular mechanisms underlying bovine adipogenic differentiation.
Project description:BACKGROUND:The distribution and deposition of fat tissue in different parts of the body are the key factors affecting the carcass quality and meat flavour of chickens. Intramuscular fat (IMF) content is an important factor associated with meat quality, while abdominal fat (AbF) is regarded as one of the main factors affecting poultry slaughter efficiency. To investigate the differentially expressed genes (DEGs) and molecular regulatory mechanisms related to adipogenic differentiation between IMF- and AbF-derived preadipocytes, we analysed the mRNA expression profiles in preadipocytes (0d, Pre-) and adipocytes (10d, Ad-) from IMF and AbF of Gushi chickens. RESULTS:AbF-derived preadipocytes exhibited a higher adipogenic differentiation ability (96.4% + 0.6) than IMF-derived preadipocytes (86.0% + 0.4) (p < 0.01). By Ribo-Zero RNA sequencing, we obtained 4403 (2055 upregulated and 2348 downregulated) and 4693 (2797 upregulated and 1896 downregulated) DEGs between preadipocytes and adipocytes in the IMF and Ad groups, respectively. For IMF-derived preadipocyte differentiation, pathways related to the PPAR signalling pathway, ECM-receptor interaction and focal adhesion pathway were significantly enriched. For AbF-derived preadipocyte differentiation, the steroid biosynthesis pathways, calcium signaling pathway and ECM-receptor interaction pathway were significantly enriched. A large number of DEGs related to lipid metabolism, fatty acid metabolism and preadipocyte differentiation, such as PPARG, ACSBG2, FABP4, FASN, APOA1 and INSIG1, were identified in our study. CONCLUSION:This study revealed large transcriptomic differences between IMF- and AbF-derived preadipocyte differentiation. A large number of DEGs and transcription factors that were closely related to fatty acid metabolism, lipid metabolism and preadipocyte differentiation were identified in the present study. Additionally, the microenvironment of IMF- and AbF-derived preadipocyte may play a significant role in adipogenic differentiation. This study provides valuable evidence to understand the molecular mechanisms underlying adipogenesis and fat deposition in chickens.
Project description:Brown adipocytes are a primary site of energy expenditure and reside not only in classical brown adipose tissue but can also be found in white adipose tissue. Here we show that microRNA 155 is enriched in brown adipose tissue and is highly expressed in proliferating brown preadipocytes but declines after induction of differentiation. Interestingly, microRNA 155 and its target, the adipogenic transcription factor CCAAT/enhancer-binding protein ?, form a bistable feedback loop integrating hormonal signals that regulate proliferation or differentiation. Inhibition of microRNA 155 enhances brown adipocyte differentiation and induces a brown adipocyte-like phenotype ('browning') in white adipocytes. Consequently, microRNA 155-deficient mice exhibit increased brown adipose tissue function and 'browning' of white fat tissue. In contrast, transgenic overexpression of microRNA 155 in mice causes a reduction of brown adipose tissue mass and impairment of brown adipose tissue function. These data demonstrate that the bistable loop involving microRNA 155 and CCAAT/enhancer-binding protein ? regulates brown lineage commitment, thereby, controlling the development of brown and beige fat cells.
Project description:Adipocytes are differentiated by various transcriptional cascades integrated on the master regulator, Ppar?. To discover new genes involved in adipocyte differentiation, preadipocytes were treated with three newly identified pro-adipogenic small molecules and GW7845 (a Ppar? agonist) for 24 hours and transcriptional profiling was analyzed. Four genes, Peroxisome proliferator-activated receptor ? (Ppar?), human complement factor D homolog (Cfd), Chemokine (C-C motif) ligand 9 (Ccl9), and GIPC PDZ Domain Containing Family Member 2 (Gipc2) were induced by at least two different small molecules but not by GW7845. Cfd and Ccl9 expressions were specific to adipocytes and they were altered in obese mice. Small hairpin RNA (shRNA) mediated knockdown of Cfd in preadipocytes inhibited lipid accumulation and expression of adipocyte markers during adipocyte differentiation. Overexpression of Cfd promoted adipocyte differentiation, increased C3a production, and led to induction of C3a receptor (C3aR) target gene expression. Similarly, treatments with C3a or C3aR agonist (C4494) also promoted adipogenesis. C3aR knockdown suppressed adipogenesis and impaired the pro-adipogenic effects of Cfd, further suggesting the necessity for C3aR signaling in Cfd-mediated pro-adipogenic axis. Together, these data show the action of Cfd in adipogenesis and underscore the application of small molecules to identify genes in adipocytes.
Project description:Growth of adipose tissue involves differentiation of preadipocytes into mature lipid-containing adipocytes. The matrix metalloproteinases (MMPs) are known regulators of adipose tissue biology, and previous studies suggested a key role for gelatinase B (MMP-9) in adipogenesis. In the present study we have evaluated a potential functional role of MMP-9 by performing gene silencing in 3T3-F442A preadipocytes. At the end of a 12-day differentiation period, no significant differences were observed between MMP-9 knockdown cells and the control cells with respect to intracytoplasmatic lipid content, or expression of the adipogenic markers aP2, PPAR?, Lpl, and adiponectin, or of the preadipocyte marker Pref-1. Thus, in vitro differentiation of 3T3-F442A preadipocytes into mature adipocytes is not significantly affected by the loss of MMP-9.
Project description:OBJECTIVE: Obesity is an enormous burden for patients and health systems world-wide. Brown adipose tissue dissipates energy in response to cold and has been shown to be metabolically active in human adults. The type I transforming growth factor ? (TGF?) receptor Activin receptor-like kinase 7 (Alk7) is highly expressed in adipose tissues and is down-regulated in obese patients. Here, we studied the function of Alk7 in brown adipocytes. METHODS: Using pharmacological and genetic tools, Alk7 signaling pathway and its effects were studied in murine brown adipocytes. Brown adipocyte differentiation and activation was analyzed. RESULTS: Alk7 is highly upregulated during differentiation of brown adipocytes. Interestingly, Alk7 expression is increased by cGMP/protein kinase G (PKG) signaling, which enhances brown adipocyte differentiation. Activin AB effectively activates Alk7 and SMAD3 signaling. Activation of Alk7 in brown preadipocytes suppresses the master adipogenic transcription factor PPAR? and differentiation. Stimulation of Alk7 during late differentiation of brown adipocytes reduces lipid content and adipogenic marker expression but enhances UCP1 expression. CONCLUSIONS: We found a so far unknown crosstalk between cGMP and Alk7 signaling pathways. Tight regulation of Alk7 is required for efficient differentiation of brown adipocytes. Alk7 has differential effects on adipogenic differentiation and the development of the thermogenic program in brown adipocytes.