Adipocyte Differentiation Process Research Articles

Proteome analysis for 3T3-L1 adipocyte differentiation.

Adipose tissue is an important endocrine organ involved in the control of whole body energy homeostasis and insulin sensitivity. Considering the increased incidence of obesity and obesity-related disorders, including diabetes, it is important to understand thoroughly the process of adipocyte differentiation and its control. Therefore, we performed a differential proteome mapping strategy using two-dimensional gel electrophoresis combined with peptide mass fingerprinting to identify intracellular proteins that are differentially expressed during adipose conversion of 3T3-L1 pre-adipocytes in response to an adipogenic cocktail. In the current study, we identified 46 differentially expressed proteins, 6 of which have not been addressed previously in 3T3-L1 cell differentiation. Notably, we found that phosphoribosyl pyrophosphate synthetase (PRPS), a regulator of cell proliferation, was preferentially expressed in pre-adipocytes than in fully differentiated adipocytes. In conclusion, our results provide valuable information for further understanding of the adipogenic process.

Functional expression of glucose-dependent insulinotropic polypeptide receptors is coupled to differentiation in a human adipocyte model

To establish that human adipocytes express functional glucose-dependent insulinotropic peptide (GIP) receptors and in particular the regulation of GIP receptor (GIPR) expression in the context of the dynamic process of adipocyte differentiation. A combination of semiquantitative real-time PCR and measurement of GIP-stimulated cAMP accumulation was used to establish the expression and functional coupling of GIPRs during in vitro differentiation of human Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes. Semiquantitative real-time PCR revealed that GIPR expression was substantially increased by day 4 of differentiation, reaching a maximum around 6-8 days (approximately 200-fold increase above undifferentiated cells, n=2). We also analysed the expression of the adipocyte fatty acid binding protein (FABP4) to relate GIPR expression to a molecular differentiation marker of adipogenesis. FABP4 expression was barely detectable in undifferentiated cells. However, following exposure to adipogenic medium, FABP4 expression gradually increased, with a maximal expression level around 10 days (approximately 1,600,000-fold increase above undifferentiated cells, n=2). Thus, the increases in GIPR mRNA during adipogenesis occur earlier than FABP4, suggesting that it might represent a gene expressed early in terminal differentiation and thus plays a role in fat droplet formation. A unit of 1 microM GIP failed to raise intracellular cAMP levels above basal levels in undifferentiated cells (n=3). In stark contrast, the 9-day differentiated cells produced a robust concentration-dependent increase in cAMP accumulation following stimulation with GIP, with an EC(50) value of 2.3 nM (n=3). The maximal response represented a 9-34-fold increase in cAMP accumulation above basal levels. This study demonstrates that GIPRs are expressed by human adipocytes, both GIPR mRNA and functional receptor expression being present in differentiated adipocytes but not in preadipocytes. Further investigation into the functional effects of GIP on differentiated SGBS cells could help towards understanding exactly how GIP regulates fat accumulation in human adipocytes.

Rehmannia inhibits adipocyte differentiation and adipogenesis

Rehmannia glutinosa, a Traditional Chinese Medicine (TCM), has been used to increase physical strength. Here, we report that Rehmannia glutinosa extract (RE) inhibits adipocyte differentiation and adipogenesis. RE impairs differentiation of 3T3-L1 preadipocytes in a dose-dependent manner. At the molecular level, treatment with RE inhibits expression of the key adipocyte differentiation regulator C/EBPβ, as well as C/EBPα and the terminal marker protein 422/aP2, during differentiation of preadipocytes into adipocytes. Additionally, RE inhibits the mitotic clonal expansion (MCE) process of adipocyte differentiation, and RE prevents localization of C/EBPβ to the centromeres. RE also prevents high fat diet (HFD) induced weight gain and adiposity in rats. Taken together, our results indicate that Rehmannia glutinosa extract inhibits preadipocyte differentiation and adipogenesis in cultured cells and in rodent models of obesity.

Hedgehog Signaling Alters Adipocyte Maturation of Human Mesenchymal Stem Cells

Human stem cells are powerful tools by which to investigate molecular mechanisms of cell growth and differentiation under normal and pathological conditions. Hedgehog signaling, the dysregulation of which causes several pathologies, such as congenital defects and cancer, is involved in several cell differentiation processes and interferes with adipocyte differentiation of rodent cells. The present study was aimed at investigating the effect of Hedgehog pathway modulation on adipocyte phenotype using different sources of human mesenchymal cells, such as bone marrow stromal cells and human multipotent adipose-derived stem cells. We bring evidence that Hedgehog signaling decreases during human adipocyte differentiation. Inhibition of this pathway is not sufficient to trigger adipogenesis, but activation of Hedgehog pathway alters adipocyte morphology as well as insulin sensitivity. Analysis of glycerol-3-phosphate dehydrogenase activity and expression of adipocyte marker genes indicate that activation of Hedgehog signaling by purmorphamine impairs adipogenesis. In sharp contrast to reports in rodent cells, the maturation process, but not the early steps of human mesenchymal stem cell differentiation, is affected by Hedgehog activation. Hedgehog interferes with adipocyte differentiation by targeting CCAAT enhancer-binding protein alpha and peroxisome proliferator-activated receptor (PPAR) gamma2 expression, whereas PPARgamma1 level remains unaffected. Although Hedgehog pathway stimulation does not modify the total number of adipocytes, adipogenesis appears dramatically impaired, with reduced lipid accumulation, a decrease in adipocyte-specific markers, and acquisition of an insulin-resistant phenotype. This study indicates that a decrease in Hedgehog signaling is necessary but not sufficient to trigger adipocyte differentiation and unveils a striking difference in the adipocyte differentiation process between rodent and human mesenchymal stem cells.

Human adenovirus Ad-36 induces adipogenesis via its E4 orf-1 gene

Understanding the regulation of adipocyte differentiation by cellular and extracellular factors is crucial for better management of chronic conditions such as obesity, insulin resistance and lipodystrophy. Experimental infection of rats with a human adenovirus type 36 (Ad-36) improves insulin sensitivity and promotes adipogenesis, reminiscent of the effect of thiozolinediones. Therefore, we investigated the role of Ad-36 as a novel regulator of the adipogenic process. Even in the absence of adipogenic inducers, infection of 3T3-L1 preadipocytes and human adipose-derived stem cells (hASC) by Ad-36, but not Ad-2 that is another human adenovirus, modulated regulatory points that spanned the entire adipogenic cascade ranging from the upregulation of cAMP, phosphatidylinositol 3-kinase and p38 signaling pathways, downregulation of Wnt10b expression, and increased expression of CCAAT/enhancer binding protein-beta and peroxisome proliferator-activated receptor gamma2 and consequential lipid accumulation. Next, we identified that E4 open reading frame (orf)-1 gene of the virus is necessary and sufficient for Ad-36-induced adipogenesis. Selective knockdown of E4 orf-1 by RNAi abrogated Ad-36-induced adipogenic signaling cascade in 3T3-L1 cells and hASC. Compared to the null vector, selective expression of Ad-36 E4 orf-1 in 3T3-L1 induced adipogenesis, which was abrogated when the PDZ-binding domain of the protein was deleted. Thus, Ad-36 E4 orf-1 is a novel inducer of rodent and human adipocyte differentiation process.

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 PPARgamma2. 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.

Brg1 chromatin remodeling factor is involved in cell growth arrest, apoptosis and senescence of rat mesenchymal stem cells

Self-renewal, proliferation and differentiation properties of stem cells are controlled by key transcription factors. However, their activity is modulated by chromatin remodeling factors that operate at the highest hierarchical level. Studies on these factors can be especially important to dissect molecular pathways governing the biology of stem cells. SWI/SNF complexes are adenosine triphosphate (ATP)-dependent chromatin remodeling enzymes that have been shown to be required for cell cycle control, apoptosis and cell differentiation in several biological systems. The aim of our research was to investigate the role of these complexes in the biology of mesenchymal stem cells (MSCs). To this end, in MSCs we caused a forced expression of the ATPase subunit of SWI/SNF (Brg1 - also known as Smarca4) by adenoviral transduction. Forced Brg1 expression induced a significant cell cycle arrest of MSCs in culture. This was associated with a huge increase in apoptosis that reached a peak 3 days after transduction. In addition, we observed signs of senescence in cells having ectopic Brg1 expression. At the molecular level these phenomena were associated with activation of Rb- and p53-related pathways. Inhibition of either p53 or Rb with E1A mutated proteins allowed us to hypothesize that both Rb and p53 are indispensable for Brg1-induced senescence, whereas only p53 seems to play a role in triggering programmed cell death. We also looked at the effects of forced Brg1 expression on canonical MSC differentiation in adipocytes, chondrocytes and osteocytes. Brg1 did not induce cell differentiation per se; however, this protein could contribute, at least in part, to the adipocyte differentiation process. In conclusion, our results suggest that whereas some ATP-dependent chromatin remodeling factors, such as ISWI complexes, promote stem cell self-renewal and conservation of an uncommitted state, others cause an escape from 'stemness' and induction of differentiation along with senescence and cell death phenomena.

Suppression of PPAR-γ attenuates insulin-stimulated glucose uptake by affecting both GLUT1 and GLUT4 in 3T3-L1 adipocytes

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) plays a critical role in regulating insulin sensitivity and glucose homeostasis. In this study, we identified highly efficient small interfering RNA (siRNA) sequences and used lentiviral short hairpin RNA and electroporation of siRNAs to deplete PPAR-gamma from 3T3-L1 adipocytes to elucidate its role in adipogenesis and insulin signaling. We show that PPAR-gamma knockdown prevented adipocyte differentiation but was not required for maintenance of the adipocyte differentiation state after the cells had undergone adipogenesis. We further demonstrate that PPAR-gamma suppression reduced insulin-stimulated glucose uptake without affecting the early insulin signaling steps in the adipocytes. Using dual siRNA strategies, we show that this effect of PPAR-gamma deletion was mediated by both GLUT4 and GLUT1. Interestingly, PPAR-gamma-depleted cells displayed enhanced inflammatory responses to TNF-alpha stimulation, consistent with a chronic anti-inflammatory effect of endogenous PPAR-gamma. In summary, 1) PPAR-gamma is essential for the process of adipocyte differentiation but is less necessary for maintenance of the differentiated state, 2) PPAR-gamma supports normal insulin-stimulated glucose transport, and 3) endogenous PPAR-gamma may play a role in suppression of the inflammatory pathway in 3T3-L1 cells.

Phosphorylation of the large subunit of replication factor C is associated with adipocyte differentiation

Adipocyte differentiation is an ordered multistep process requiring the sequential activation of several groups of adipogenic transcription factors, including CCAAT/enhancer-binding protein-alpha and peroxisome proliferator-activated receptor-gamma, and coactivators. Here we show that replication factor C 140, which was known to act as a coactivator for CCAAT/enhancer-binding protein-alpha in our previous study, was phosphorylated on the proliferating cell nuclear antigen-bindng domain during the adipocyte differentiation process. Calmodulin-dependent protein kinase II was responsible for phosphorylating replication factor C 140 in the process of adipocyte differentiation. Ser518 of replication factor C 140 was identified as a major target of calmodulin-dependent protein kinase II phosphorylation in vitro. Calmodulin-dependent protein kinase II inhibitor attenuated phosphorylation of replication factor C 140 by differentiation inducers and blocked replication factor C 140-derived transcriptional activation. Taken together, these findings demonstrate that calmodulin-dependent protein kinase II signaling leads the cooperative transactivation of CCAAT/enhancer-binding protein-alpha and replication factor C 140 through an increase in replication factor C 140 phosphorylation, and subsequently enhances the transcriptional activation of target genes involved in adipocyte differentiation.

Fat-specific FUS-DDIT3-transgenic mice establish PPAR inactivation is required to liposarcoma development

FUS-DDIT3 is a chimeric oncogene generated by the most common chromosomal translocation t(12;16)(q13;p11) associated to liposarcomas. The application of transgenic methods and the use of primary mesenchymal progenitor cells to the study of this sarcoma-associated FUS-DDIT3 gene fusion have provided insights into their in vivo functions and suggested mechanisms by which lineage selection may be achieved. These studies indicate that FUS-DDIT3 contributes to differentiation arrest acting at a point in the adipocyte differentiation process after induction of peroxisome proliferator-activated receptor gamma (PPARgamma) expression. To test this idea within a living mouse, we generated mice expressing FUS-DDIT3 within aP2-positive cells, because aP2 is a downstream target of PPARgamma expressed at the immature adipocyte stage. Here, we report that FUS-DDIT3 expression was successfully induced at the aP2 stage of differentiation both in vivo and in vitro. aP2-FUS-DDIT3 mice do not develop liposarcomas and exhibit an increase in white adipose tissue size. Consistent with in vivo data, mouse embryonic fibroblasts (MEFs) obtained from aP2-FUS-DDIT3 mice not only were capable of terminal differentiation but also showed an increased capacity for adipogenesis in vitro compared with wild-type MEFs. Taken together, this study provides genetic evidence that the presence of FUS-DDIT3 in an aP2-positive cell is not enough to cause liposarcoma development and establishes that PPARgamma inactivation is required for liposarcoma development.

Skp2 Controls Adipocyte Proliferation during the Development of Obesity

The increase in the mass of adipose tissue during the development of obesity can arise through an increase in cell size, an increase in cell number, or both. Here we show that long term maintenance of C57BL/6 mice on a high fat diet (for approximately 25 weeks) induces an initial increase in adipocyte size followed by an increase in adipocyte number in white adipose tissue. The latter effect was found to be accompanied by up-regulation of expression of the gene for the F-box protein Skp2 as well as by downregulation of the cyclin-dependent kinase inhibitor p27(Kip1), a principal target of the SCF(Skp2) ubiquitin ligase, in white adipose tissue. Ablation of Skp2 protected mice from the development of obesity induced either by a high fat diet or by the lethal yellow agouti (A(y)) mutation, and this protective action was due to inhibition of the increase in adipocyte number without an effect on adipocyte hypertrophy. The reduction in the number of adipocyte caused by Skp2 ablation also inhibited the development of obesity-related insulin resistance in the A(y) mutant mice, although the reduced number of beta cells and reduced level of insulin secretion in Skp2-deficient mice resulted in glucose intolerance. Our observations thus indicate that Skp2 controls adipocyte proliferation during the development of obesity.

Cytokines Promote Wnt Signaling and Inflammation and Impair the Normal Differentiation and Lipid Accumulation in 3T3-L1 Preadipocytes

Obesity with enlarged fat cells is associated with high local concentrations of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) in the adipose tissue. We examined the effects of this inflammatory state on 3T3-L1 preadipocyte development and differentiation to mature adipose cells. Both IL-6 and TNFalpha impaired the normal differentiation pattern and lipid accumulation. However, IL-6 allowed a normal early induction of differentiation with inhibition of Wnt10b and Pref-1, whereas expression of CCAAT/enhancer-binding protein alpha, in contrast to peroxisome proliferator-activated receptor gamma, was markedly reduced. TNFalpha also allowed a normal early induction of differentiation, whereas the terminal differentiation to adipose cells was completely prevented. However, both cytokines induced an inflammatory phenotype of the cells but with different profiles. Remarkably, both IL-6 and TNFalpha maintained and augmented the canonical Wnt signaling associated with low axin and high low density lipoprotein receptor-related protein (LRD), Dishevelled, and beta-catenin levels. TNFalpha, but not IL-6, activated Wnt10b expression, whereas IL-6 increased the apparent phosphorylation of Dishevelled. Thus, both IL-6 and TNFalpha prevent the normal development of preadipocytes to fully differentiated adipose cells and, instead, promote an inflammatory phenotype of the adipocytes. These results provide an explanation as to why obesity and diabetes are associated with both local and systemic inflammation, insulin resistance, and ectopic lipid accumulation.

Acetate and Propionate Short Chain Fatty Acids Stimulate Adipogenesis via GPCR43

It has recently been discovered that G protein-coupled receptors (GPCR) 41 and 43 are characterized by having the short chain fatty acids acetate and propionate as their ligands. The objective of this study was to investigate the involvement of GPCR41, GPCR43, and their ligands in the process of adipogenesis. We measured the levels of GPCR41 and GPCR43 mRNA in both adipose and other tissues of the mouse. GRP43 mRNA expression was higher in four types of adipose tissue than in other tissues, whereas GPCR41 mRNA was not detected in any adipose tissues. A high level of GPCR43 expression was found in isolated adipocytes, but expression level was very low in stromal-vascular cells. Expression of GPCR43 was up-regulated in adipose tissues of mice fed a high-fat diet compared with those fed a normal-fat diet. GPCR43 mRNA could not be detected in confluent and undifferentiated 3T3-L1 adipocytes; however, the levels rose with time after the initiation of differentiation. GPCR41 expression was not detected in confluent and differentiated adipocytes. Acetate and propionate treatments increased lipids present as multiple droplets in 3T3-L1 adipocytes. Propionate significantly elevated the level of GPCR43 expression during adipose differentiation, with up-regulation of PPAR-gamma2. Small interfering RNA mediated a reduction of GPCR43 mRNA in 3T3-L1 cells and blocked the process of adipocyte differentiation. In addition, both acetate and propionate inhibited isoproterenol-induced lipolysis in a dose-dependent manner. We conclude that acetate and propionate short chain fatty acids may have important physiological roles in adipogenesis through GPCR43, but not through GPCR41.

Adipogenesis: cellular and molecular aspects

Obesity and lipoatrophy are major risks for insulin resistance, non-insulin-dependent diabetes and cardiovascular disease. In the past three decades, significant advances have been made in delineating the key transcription factors of adipogenesis, as well as extracellular effectors and intracellular signalling pathways that regulate fat cell formation. This review focuses on in vitro models of adipocyte differentiation, and on the balance between pro- and anti-adipogenic factors that drive the adipocyte differentiation process. Full understanding of the mechanisms of adipose tissue differentiation represents a major issue to develop a comprehensive strategy to prevent and treat obesity.

Genistein, EGCG, and capsaicin inhibit adipocyte differentiation process via activating AMP-activated protein kinase

Phytochemicals such as soy isoflavone genistein have been reported to possess therapeutic effects for obesity, diabetes, and cardiovascular diseases. In the present study, the molecular basis of selective phytochemicals with emphasis on their ability to control intracellular signaling cascades of AMP-activated kinase (AMPK) responsible for the inhibition of adipogenesis was investigated. Recently, the evolutionarily conserved serine/threonine kinase, AMPK, emerges as a possible target molecule of anti-obesity. Hypothalamic AMPK was found to integrate nutritional and hormonal signals modulating feeding behavior and energy expenditure. We have investigated the effects of genistein, EGCG, and capsaicin on adipocyte differentiation in relation to AMPK activation in 3T3-L1 cells. Genistein (20–200 μM) significantly inhibited the process of adipocyte differentiation and led to apoptosis of mature adipocytes. Genistein, EGCG, and capsaicin stimulated the intracellular ROS release, which activated AMPK rapidly. We suggest that AMPK is a novel and critical component of both inhibition of adipocyte differentiation and apoptosis of mature adipocytes by genistein or EGCG or capsaicin further implying AMPK as a prime target of obesity control.

Up-regulation of adipogenin, an adipocyte plasma transmembrane protein, during adipogenesis

Until now, the various proteins highly expressed in adipose tissues have been identified and characterized by traditional gene cloning techniques. However, methods of computer analysis have been developed to compare the levels of expression among various tissues, and genes whose expression levels differ significantly between tissues have been found. Among these genes, we report on the possible function of a new adipose-specific gene, showed higher expression in adipose tissue through 'Search Expression' on Genome Institute of Norvartis Research Foundation (GNF) SymAtlas v0.8.0. This database has generated and analyzed gene expression of each gene in diverse samples of normal tissues, organs, and cell lines. This newly discovered gene product was named adipogenin because of its role in stimulating adipocyte differentiation and development. Adipogenin mRNA was highly expressed in four different fat depots, and exclusively expressed in adipocytes isolated from adipose tissues. The level of adipogenin mRNA was up-regulated in the subcutaneous and visceral adipose tissues of mice fed a high-fat diet compared to those on the control diet. The expression of adipogenin mRNA is dramatically elevated during adipocyte differentiation of 3T3-L1 cells. Troglitazone, which up-regulated peroxisome proliferators-activated receptor gamma2 (PPAR-gamma2) expression, increased adipogenin mRNA expression, although this gene was down-regulated by retinoic acid. Confocal image analyses of green-fluorescent protein-adipogenin (pEGFP-adipogenin) transiently expressed in 3T3-L1 adipocytes showed that adipogenin was strictly localized to membranes and was absent from the cytosol. Moreover, small interfering RNA (siRNA) mediated a reduction of adipogenin mRNA in 3T3-L1 cells and blocked the process of adipocyte differentiation. These results indicate that adipogenin, an adipocyte-specific membrane protein, may be involved with adipogenesis, as one of the regulators of adipose tissue development.

Efficient adenoviral transduction of 3T3-F442A preadipocytes without affecting adipocyte differentiation

The preadipocyte cell lines 3T3-L1 and 3T3-F442A are widely used to study the cellular mechanisms of preadipocyte differentiation and mature adipocyte functions. However, transfection with naked DNA is inefficient in these cell lines. Adenoviral gene transfer is a powerful technique to induce high levels of transgene expression. After failing to obtain 3T3-F442A stable transfectants, we studied different techniques designed to enhance the efficiency of adenoviral transduction in fat cells. First, we compared the effects of two agents known to significantly enhance adenoviral transgene transduction, namely the cationic lipid lipofectamine and the cationic polymer polylysine. We show here that lipofectamine-assisted adenoviral transduction was more efficient in 3T3-F442A than in 3T3-L1 preadipocytes at all tested multiplicity of infection. Lipofectamine, and more efficiently polylysine, yielded high and sustained levels of adenoviral transgene expression in 3T3-F442A preadipocytes. Adenoviral transgene expression was maintained throughout the differentiation process. Furthermore, the two agents also efficiently enhanced adenoviral transduction in mature 3T3-F442A adipocytes. Interestingly, neither protocol affected the differentiation process, morphological features or protein expression of mature adipocytes. These approaches could be of interest to study fat cell differentiation and the functions of mature adipocytes.

Grape-seed derived procyanidins interfere with adipogenesis of 3T3-L1 cells at the onset of differentiation

Our group's previous results on the effects of a grape seed procyanidin extract (GSPE) on adipose metabolism showed that peroxisome proliferator-activated receptor-gamma (PPARgamma) plays a central role in the lipolytic effects of GSPE on adipocytes. Since PPARgamma2 is a main regulator of the differentiation process of adipocytes, we investigated whether GSPE affects the adipogenesis of 3T3-L1 cells. We performed a time point screening by treating 3T3-L1 cells with GSPE during the differentiation process for 24 h. Differentiation markers and differential gene expression due to GSPE treatment (using the microarray technique). Twenty four hour-GSPE treatment at the onset of differentiation reduces adipose-specific markers and maintains the expression of preadipocyte marker preadipocyte factor-1 (Pref-1) significantly elevated. These effects were not found in other time points. Microarray analysis of gene expression after GSPE treatment at the early stage of differentiation showed a modified gene expression profile in which cell cycle and growth-related genes were downregulated by GSPE. These results suggest that GSPE affects adipogenesis, mainly at the induction of differentiation, and that procyanidins may have a new role in which they impede the formation of adipose cells.

Changes in mRNA expression of regulatory factors involved in adipocyte differentiation during fatty acid induced adipogenesis in chicken

The adipocyte differentiation process involves a cascade of transcriptional events that culminates in the expression of peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer binding protein-α (C/EBPα). These adipogenic transcription factors regulate the expression of genes necessary for the development of mature adipocytes in mammals. The current study was undertaken to identify regulatory factors that affect adipogenesis and to analyze species-specific mRNA expression of factors involved in chicken adipocyte differentiation. We developed a system for differentiation of chicken ( Gallus gallus) adipocytes in culture using medium containing 500 nM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 20 μg/mL bovine insulin, 300 μM oleate, and 10% fetal bovine serum. The rapid differentiation of cells to mature adipocytes in this culture system was verified by observed increases in adipocyte fatty acid-binding protein (aP2) expression, glycerol-3-phosphate dehydrogenase (GPDH) activity and intracellular triglyceride accumulation. In contrast, cells cultured in a differentiation medium without fatty acids did not differentiate into mature adipocytes. The expression profiles of genes involved in the regulation of adipocyte differentiation, such as PPARγ, C/EBPα, β, δ, sterol regulatory element binding protein-1 (SREBP-1), fatty acid synthase (FAS), lipoprotein lipase (LPL), and glucose transporters 1 and 8 (GLUT1 and GLUT8) were studied. Rapid increases in PPARγ and aP2 expression were observed after 9 and 12 h of culture in differentiation medium, respectively. In contrast, the expression patterns of the other adipogenic genes only differed slightly from those previously determined for mammalian adipocytes. These results suggest that exogenous fatty acid is essential for adipocyte differentiation in chickens, and that PPARγ is possibly a key regulator in the early stages of chicken preadipocyte differentiation.

Deletion of PPARγ in adipose tissues of mice protects against high fat diet-induced obesity and insulin resistance

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a crucial role in adipocyte differentiation, glucose metabolism, and other physiological processes. To further explore the role of PPARgamma in adipose tissues, we used a Cre/loxP strategy to generate adipose-specific PPARgamma knockout mice. These animals exhibited marked abnormalities in the formation and function of both brown and white adipose tissues. When fed a high-fat diet, adipose-specific PPARgamma knockout mice displayed diminished weight gain despite hyperphagia, had diminished serum concentrations of both leptin and adiponectin, and did not develop glucose intolerance or insulin resistance. Characterization of in vivo glucose dynamics pointed to improved hepatic glucose metabolism as the basis for preventing high-fat diet-induced insulin resistance. Our findings further illustrate the essential role for PPARgamma in the development of adipose tissues and suggest that a compensatory induction of hepatic PPARgamma may stimulate an increase in glucose disposal by the liver.