The Wnt/beta-catenin signalling pathway appears to operate to maintain the undifferentiated state of preadipocytes by inhibiting adipogenic gene expression. To define the mechanisms regulating suppression of Wnt/beta-catenin signalling, we analysed the beta-catenin expression in response to activation of transcription factors that regulate adipogenesis. The results show an extensive down-regulation of nuclear beta-catenin that occurs during the first few days of differentiation of 3T3-L1 preadipocytes and coincides with the induction of the adipogenic transcription factors, C/EBPbeta (CCAAT-enhancer-binding protein) and PPARgamma (peroxisome-proliferator-activated receptor). To assess the role of each of these factors in this process, we conditionally overexpressed C/EBPbeta in Swiss mouse fibroblasts using the TET-off system. Abundant expression of C/EBPbeta alone had minimal effect on beta-catenin expression, whereas expression of C/EBPbeta, in the presence of dexamethasone, induced PPARgamma expression and caused a measurable decrease in beta-catenin. In addition, exposure of cells expressing both C/EBPbeta and PPARgamma to a potent PPARgamma ligand resulted in an even greater decrease in beta-catenin by mechanisms that involve the proteasome. Our studies also suggest a reciprocal relationship between PPARgamma activity and beta-catenin expression, since ectopic production of Wnt-1 in preadipocytes blocked the induction of PPARgamma gene expression. Moreover, by suppressing beta-catenin expression, ectopic expression of PPARgamma in Wnt-1-expressing preadipocytes rescued the block in adipogenesis after their exposure to the PPARgamma ligand, troglitazone.

Molecular mechanisms coupling growth arrest and cell differentiation were examined during adipogenesis. Data are presented that document a cascade expression of members of two independent families of cyclin-dependent kinase inhibitors that define distinct states of growth arrest during 3T3-L1 preadipocyte differentiation. Exit from the cell cycle into a pre-differentiation state of post-mitotic growth arrest was characterized by significant increases in p21 and p27. During onset of irreversible growth arrest associated with terminal differentiation, the level of p21 declined with a concomitant, dramatic increase in p18 and a sustained level of p27. The expression of p18 and p21, regulated at the level of protein and mRNA accumulation, was directly coupled to differentiation. Stable cell lines were engineered to express adipogenic transcription factors to examine the active role of trans-acting elements in regulating these cell cycle inhibitors. Ectopic expression of peroxisome proliferator-activated receptor (PPAR) ␥ in non-precursor fibroblastic cell lines resulted in conversion to adipocytes and a coordinated increase in p18 and p21 mRNA and protein expression in a PPAR␥ ligandassociated manner. These data demonstrate a role for PPAR␥ in mediating the differentiation-dependent cascade expression of cyclin-dependent kinase inhibitors, thereby providing a molecular mechanism coupling growth arrest and adipocyte differentiation.

Trans-10,cis-12 conjugated linoleic acid (CLA) has previously been shown to be the CLA isomer responsible for CLA-induced reductions in body fat in animal models, and we have shown that this isomer, but not the cis-9,trans-11 CLA isomer, specifically decreased triglyceride (TG) accumulation in primary human adiopcytes in vitro. Here we investigated the mechanism behind the isomerspecific, CLA-mediated reduction in TG accumulation in differentiating human preadipocytes. Trans-10,cis-12 CLA decreased insulin-stimulated glucose uptake and oxidation, and reduced insulin-dependent glucose transporter 4 gene expression. Furthermore, trans-10,cis-12 CLA reduced oleic acid uptake and oxidation when compared with all other treatments. In parallel to CLA's effects on metabolism, trans-10,cis-12 CLA decreased, whereas cis-9,trans-11 CLA increased, the expression of peroxisome proliferator-activated receptor γ (PPARγ) and several of its downstream target genes when compared with vehicle controls. Transient transfections demonstrated that both CLA isomers antagonized ligand-dependent activation of PPARγ. Collectively, trans-10,cis-12, but not cis-9, trans-11, CLA decreased glucose and lipid uptake and oxidation and preadipocyte differentiation by altering preadipocyte gene transcription in a manner that appeared to be due, in part, to decreased PPARγ expression. Supplementary key wordsconjugated linoleic acid; fatty acids; lipid metabolism; glucose metabolism; triglycerides; peroxisome proliferator-activated receptor gamma Abbreviations ACBP, acyl-CoA binding protein; ACC, acetyl-CoA carboxylase; aP2/FABP, adipocyte fatty acid binding protein; BCA, bicinchoninic acid; BMI, body mass index; BSA, bovine serum albumin; CD-36, fatty acid translocase; C/EBPα, CAAT/enhancer binding protein α; CLA, conjugated linoleic acid; GC, gas chromatography; GLUT4, insulin-dependent glucose transporter 4; GPDH, glycerol-3-phosphate dehydrogenase; HSL, hormone-sensitive lipase; IBMX, isobutylmethylxanthine; LA, linoleic acid; LPL, lipoprotein lipase; MUFA, monounsaturated fatty acid; ORO, oil red O; PPAR, 1 To whom correspondence should be addressed. e-mail:mkmcinto@uncg.edu. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator response element; SCD-1, stearoyl-CoA desaturase-1; SFA, saturated fatty acid; SV, stromal vascular; TG, triglyceride Conjugated linoleic acid (CLA) refers to a group of geometric and positional dienoic isomers of linoleic acid (LA) [18:2(n-6)]. The two predominant isomers of CLA found in food and commercial preparations are cis-9,trans-11 CLA and trans-10,cis-12 CLA. CLA is found in ruminant meats, pasteurized cheeses, and dairy products, and therefore is a natural part of the diet. CLA has been extensively studied due to its potentially beneficial effects on carcinogenesis (1-3), diabetes (4,5), atherosclerosis (6,7), immune function (8-10), and body composition (11)(12)(13)(14)(15)(16).Collectivel...

To define the molecular mechanisms that control GLUT4 expression during adipogenesis, NIH-3T3 fibroblasts ectopically expressing different adipogenic transcription factors (C/EBPbeta, C/EBPdelta, C/EBPalpha, and PPARgamma) under the control of a tetracycline-responsive inducible (C/EBPs) or a constitutive retroviral (PPARgamma) expression system were used. Enhanced production of C/EBPbeta (beta2 cell line), C/EBPbeta together with C/EBPdelta (beta/delta39 cell line), C/EBPalpha (alpha1 cell line), or PPARgamma (Pgamma2 cell line) in cells exposed to dexamethasone and the PPARgamma ligand ciglitazone (a thiazolidinedione) resulted in expression of GLUT4 mRNA as well as other members of the adipogenic gene program, including aP2 and adipsin. Focusing our studies on the beta/delta39 cells, we have demonstrated that C/EBPbeta along with C/EBPdelta in the presence of dexamethasone induces PPARgamma, adipsin, and aP2 mRNA production; however, GLUT4 mRNA is only expressed in cells exposed to ciglitazone. In addition, enhanced expression of a ligand-activated form of PPARgamma in the beta/delta39 fibroblasts stimulates synthesis of GLUT4 protein and gives rise to a population of adipocytic cells that take up glucose in direct response to insulin. C/EBPalpha is not expressed in the beta/delta39 cells under conditions that stimulate the adipogenic program. This observation suggests that PPARgamma alone or in combination with C/EBPbeta and C/EBPdelta is capable of activating GLUT4 gene expression.

Recent advances regarding the biology of adipose tissue have identified the adipocyte as an important mediator in many physiologic and pathologic processes regarding energy metabolism. Consideration for a central role of adipose tissue in the development of obesity, cardiovascular disease and noninsulin-dependent diabetes mellitus has resulted in new incentives toward understanding the complexities of adipocyte differentiation. Current knowledge of this process includes a cascade of transcriptional events that culminate in the expression of peroxisome proliferator-activated receptor-gamma (PPARgamma) and CCAAT/enhancer binding protein-alpha (C/EBPalpha). These prominent adipogenic transcription factors have been shown to regulate, directly or indirectly, the gene expression necessary for the development of the mature adipocyte. Hormonal and nutritional signaling that impinges on these trans-acting factors provides a molecular link between lipids and lipid-related compounds and the gene expression important for glucose and lipid homeostasis. Knowledge concerning the transcriptional events mediating adipocyte differentiation provides a basis for understanding the physiologic processes associated with adipose tissue as well as for the development of therapeutic interventions in obesity and its related disorders.

The 3T3-L1 cell line differentiates under the controlled conditions of cell culture from fibroblasts, or preadipocytes, into cells with the morphological and biochemical properties of adipocytes (Green and Kehinde, 1974;Green and Kehinde, 1976) in a process that closely resembles the development of adipose tissue in vivo. Upon differentiation, these cells acquire sensitivity to hormones and exhibit a coordinate increase in the activities of numerous enzymes in the lipolytic, lipogenic, and glycolytic pathways (Smas and Sul, 1995). To date, members of two transcription factor families, C/EBP (C/AAAT Enhancer Binding Proteins) and PPAR (Peroxisome Proliferator Activated Receptors) have been shown to be induced during adipocyte differentiation and are thought to play a significant role in the regulation of fat-specific gene expression.The STAT (Signal Transducers and Activators of Transcription) family of transcription factors is comprised of six family members (STATs 1-6) that, in response to stimulation of various receptors, mainly those for cytokines, are phosphorylated on tyrosine residues, which causes their translocation to the nucleus. Each STAT family member shows a distinct pattern of activation by cytokines, has a unique tissue distribution, and upon nuclear translocation can regulate the transcription of particular genes Ihle, 1995). The likely order of events for STAT activation can be described as follows: 1) ligand binding of cell surface receptor; 2) receptor association with a JAK (Janus kinase) kinase family member; 3) JAK tyrosine phosphorylation of STAT proteins; 4) dimerization of the STATs; 5) translocation to the nucleus; and 6) DNA binding. STATs have been shown to bind at least three different consensus sequences, and this binding regulates the transcription of specific genes Ihle, 1995).One of the first identified inhibitors of adipocyte differentiation was tumor necrosis factor-␣ (TNF␣), 1 a cytokine that elicits a wide range of biological effects including the regulation of growth and differentiation. In addition, TNF␣ has been shown to down-regulate the insulin responsiveness of fully differentiated adipocytes (Stephens and Pekala, 1991;Hotamisligil et al. 1993). Because regulation of the STATs is mainly cytokine-mediated, TNF␣ could be a mediator of STAT expression during and/or after adipocyte differentiation. Most of the studies on the STAT family of transcription factors have focused on their tyrosine phosphorylation and DNA binding. In this report, we demonstrate that another level of regulation of these proteins exists as they are induced during the differentiation of adipose cells in culture. Moreover, we demonstrate that inhibition of differentiation by TNF␣ completely suppresses the expression of two STAT family members. We interpret these data to indicate that STAT family members may play a role in the regulation of genes that contribute to the phenotype of the mature adipocyte. EXPERIMENTAL PROCEDURESCell Culture-Murine 3T3-L1 preadipocytes were cultured, maintained, and diffe...

These data indicate that WAT macrophages are a source of OSM and that OSM levels are significantly induced in murine and human obesity/type 2 diabetes mellitus. These studies suggest that OSM produced from immune cells in WAT acts in a paracrine manner on adipocytes to promote a proinflammatory phenotype in adipose tissue.

BackgroundWith the current rise in obesity-related morbidities, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become a widely used method for assessment of genes expressed and regulated by adipocytes. In order to measure accurate changes in relative gene expression and monitor intersample variability, normalization to endogenous control genes that do not change in relative expression is commonly used with qRT-PCR determinations. However, historical evidence has clearly demonstrated that the expression profiles of traditional control genes (e.g., β-actin, GAPDH, α-tubulin) are differentially regulated across multiple tissue types and experimental conditions.Methodology/Principal FindingsTherefore, we validated six commonly used endogenous control genes under diverse experimental conditions of inflammatory stress, oxidative stress, synchronous cell cycle progression and cellular differentiation in 3T3-L1 adipocytes using TaqMan qRT-PCR. Under each study condition, we further evaluated the impact of reference gene selection on experimental outcome using examples of target genes relevant to adipocyte function and differentiation. We demonstrate that multiple reference genes are regulated in a condition-specific manner that is not suitable for use in target gene normalization.Conclusion/SignificanceData are presented demonstrating that inappropriate reference gene selection can have profound influence on study conclusions ranging from divergent statistical outcome to inaccurate data interpretation of significant magnitude. This study validated the use of endogenous controls in 3T3-L1 adipocytes and highlights the impact of inappropriate reference gene selection on data interpretation and study conclusions.