Role of cyclooxygenases COX-1 and COX-2 in modulating adipogenesis in 3T3-L1 cells.
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Cyclooxygenase (COX) catalyses the rate-limiting step of prostanoid biosynthesis. Two COX isoforms have been identified, COX-1, the constitutive form, and COX-2, the inducible form. While COX-2 has been implicated in body fat regulation, the underlying cellular mechanism remains to be elucidated. The present study was undertaken to examine the potential role of COX in modulating adipogenesis and to dissect the relative contribution of the two isoenzymes in this process. COX-2 was found to be expressed in undifferentiated 3T3-L1 cells and down-regulated during differentiation, whereas the cellular level of COX-1 remained relatively constant. Abrogating the activity of either of these two isoenzymes by selective COX inhibitors accelerated cellular differentiation, suggesting that both COX isoenzymes negatively influenced differentiation. Tumor necrosis factor-alpha (TNFalpha) significantly up-regulated COX-2 expression ( approximately 2-fold) in differentiating 3T3-L1 cells, whereas similar effect was not observed with COX-1 expression. Abrogating the induced COX-2 activity reversed the TNFalpha-induced inhibition of differentiation by approximately 70%, implying a role for COX-2 in mediating TNFalpha signaling. Hence, both COX isoforms were involved in the negative modulation of adipocyte differentiation. COX-2 appeared to be the main isoform mediating at least part of the negative effects of TNFalpha. (+info)
Isomer-specific regulation of metabolism and PPARgamma signaling by CLA in human preadipocytes.
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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 adipocytes in vitro. Here we investigated the mechanism behind the isomer-specific, 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 gamma (PPARgamma) and several of its downstream target genes when compared with vehicle controls. Transient transfections demonstrated that both CLA isomers antagonized ligand-dependent activation of PPARgamma. 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 PPARgamma expression. (+info)
Regulation of ABCA1 expression and cholesterol efflux during adipose differentiation of 3T3-L1 cells.
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Adipose cells specialized in energy storage, contain large intracellular triglyceride-rich lipid droplets, are enriched with free cholesterol, and express sterol-regulated transcription factors such as liver X receptor (LXR). The recent identification of the LXR-dependent ATP binding cassette transporter A1 (ABCA1) pathway for cholesterol release from peripheral cells has led us to address the question of the expression and function of ABCA1 in adipocytes. In 3T3-L1 adipose cells, we observed a strong induction of ABCA1 mRNA during adipose differentiation, but only limited variations in ABCA1 protein. Lipid efflux onto apolipoprotein A-I (apoA-I), which depends on ABCA1, was comparable in adipocytes and preadipocytes, demonstrating a differential regulation of ABCA1 mRNA and cholesterol efflux. We also found that total cell cholesterol remained stable during differentiation of 3T3-L1 cells, but membrane cholesterol was lower in adipocytes than in preadipocytes, suggesting redistribution of cholesterol to the lipid droplet. Finally, we show that under standard lipolytic stimulation, 3T3-L1 adipocytes do not release cholesterol onto apoA-I, a process that required long exposures to lipolytic agents (24 h). In conclusion, despite large induction of ABCA1 mRNA during differentiation, cholesterol efflux through the ABCA1 pathway remains limited in adipocytes and requires prolonged lipolysis. This is consistent with the view of the adipocyte behaving as a cholesterol sink, with plasma cholesterol-buffering properties. (+info)
Activation of PPARalpha and PPARgamma by environmental phthalate monoesters.
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Phthalate esters are widely used as plasticizers in the manufacture of products made of polyvinyl chloride. Mono-(2-ethylhexyl)-phthalate (MEHP) induces rodent hepatocarcinogenesis by a mechanism that involves activation of the nuclear transcription factor peroxisome proliferator-activated receptor-alpha (PPARalpha). MEHP also activates PPAR-gamma (PPARgamma), which contributes to adipocyte differentiation and insulin sensitization. Human exposure to other phthalate monoesters, including metabolites of di-n-butyl phthalate and butyl benzyl phthalate, is substantially higher than that of MEHP, prompting this investigation of their potential for PPAR activation, assayed in COS cells and in PPAR-responsive liver (PPARalpha) and adipocyte (PPARgamma) cell lines. Monobenzyl phthalate (MBzP) and mono-sec-butyl phthalate (MBuP) both increased the COS cell transcriptional activity of mouse PPARalpha, with effective concentration for half-maximal response (EC50) values of 21 and 63 microM, respectively. MBzP also activated human PPARalpha (EC50=30 microM) and mouse and human PPARgamma (EC50=75-100 microM). MEHP was a more potent PPAR activator than MBzP or MBuP, with mouse PPARalpha more sensitive to MEHP (EC50=0.6 microM) than human PPARalpha (EC50=3.2 microM). MEHP activation of PPARgamma required somewhat higher concentrations, EC50=10.1 microM (mouse PPARgamma) and 6.2 microM (human PPARgamma). No significant PPAR activation was observed with the monomethyl, mono-n-butyl, dimethyl, or diethyl esters of phthalic acid. PPARalpha activation was verified in FAO rat liver cells stably transfected with PPARalpha, where expression of several endogenous PPARalpha target genes was induced by MBzP, MBuP, and MEHP. Similarly, activation of endogenous PPARgamma target genes was evidenced for all three phthalates by the stimulation of PPARgamma-dependent adipogenesis in the 3T3-L1 cell differentiation model. These findings demonstrate the potential of environmental phthalate monoesters for activation of rodent and human PPARs and may help to elucidate the molecular basis for the adverse health effects proposed to be associated with human phthalate exposure. (+info)
Mutational analysis of the hormone-sensitive lipase translocation reaction in adipocytes.
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Lipolysis in adipocytes governs the release of fatty acids for the supply of energy to various tissues of the body. This reaction is mediated by hormone-sensitive lipase (HSL), a cytosolic enzyme, and perilipin, which coats the lipid droplet surface in adipocytes. Both HSL and perilipin are substrates for polyphosphorylation by protein kinase A (PKA), and phosphorylation of perilipin is required to induce HSL to translocate from the cytosol to the surface of the lipid droplet, a critical step in the lipolytic reaction (Sztalryd C., Xu, G., Dorward, H., Tansey, J. T., Contreras, J.A, Kimmel, A. R., and Londos, C. (2003) J. Cell Biol. 161, 1093-1103). In the present paper we demonstrate that phosphorylation at one of the two more recently discovered PKA sites within HSL, serines 659 and 660, is also required to effect the translocation reaction. Translocation does not occur when these serines residues are mutated simultaneously to alanines. Also, mutation of the catalytic Ser-423 eliminates HSL translocation, showing that the inactive enzyme does not migrate to the lipid droplet upon PKA activation. Thus, HSL translocation requires the phosphorylation of both HSL and perilipin. (+info)
Insulin stimulates expression of the pyruvate kinase M gene in 3T3-L1 adipocytes.
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M2-type pyruvate kinase (M2-PK) mRNA is produced from the PKM gene by an alternative RNA splicing in adipocytes. We found that insulin increased the level of M2-PK mRNA in 3T3-L1 adipocytes in both time- and dose-dependent manners. This induction did not require the presence of glucose or glucosamine in the medium. The insulin effect was blocked by pharmacological inhibitors of insulin signaling pathways such as wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), and PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase. A stable reporter expression assay showed that the promoter activity of an about 2.2-kb 5'-flanking region of the rat PKM gene was stimulated by insulin, but the extents of these stimulations were lower than those of the mRNA stimulation. Thus, we suggest that insulin increases the level of M2-PK mRNA in adipocytes by acting at transcriptional and post-transcriptional levels through signaling pathways involving both PI3K and MAPK kinase. (+info)
Syntaxin 6 regulates Glut4 trafficking in 3T3-L1 adipocytes.
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Insulin stimulates the movement of glucose transporter-4 (Glut4)-containing vesicles to the plasma membrane of adipose cells. We investigated the role of post-Golgi t-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in the trafficking of Glut4 in 3T3-L1 adipocytes. Greater than 85% of syntaxin 6 was found in Glut4-containing vesicles, and this t-SNARE exhibited insulin-stimulated movement to the plasma membrane. In contrast, the colocalization of Glut4 with syntaxin 7, 8, or 12/13 was limited and these molecules did not translocate to the plasma membrane. We used adenovirus to overexpress the cytosolic domain of these syntaxin's and studied their effects on Glut4 traffic. Overexpression of the cytosolic domain of syntaxin 6 did not affect insulin-stimulated glucose transport, but increased basal deGlc transport and cell surface Glut4 levels. Moreover, the syntaxin 6 cytosolic domain significantly reduced the rate of Glut4 reinternalization after insulin withdrawal and perturbed subendosomal Glut4 sorting; the corresponding domains of syntaxins 8 and 12 were without effect. Our data suggest that syntaxin 6 is involved in a membrane-trafficking step that sequesters Glut4 away from traffic destined for the plasma membrane. We speculate that this is at the level of traffic of Glut4 into its unique storage compartment and that syntaxin 16 may be involved. (+info)
HDL-mediated cholesterol uptake and targeting to lipid droplets in adipocytes.
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Adipocytes express high levels of the HDL scavenger receptor class B type I in a differentiation-dependent manner. We thus have analyzed the routes of HDL cholesterol trafficking at different phases of adipocyte differentiation in the 3T3-L1 cell line. One novel and salient feature of this paper is the observation of a widespread distribution in the cell cytoplasm of Golgi markers, caveolin-2, and a fluorescent cholesterol analog NBD-cholesterol (NBD-chol), observed in the early phases of adipocyte formation, clearly distinct from that observed in mature fat cells (i.e., with fully formed lipid vesicles). Thus, in cells without visible lipid droplets, Golgi markers (Golgi 58K, Golgin 97, trans-Golgi network 38, Rab 6, and BODIPY-ceramide), caveolin-2, and NBD-chol all colocalize in a widespread distribution in the cell. In contrast, when lipid droplets are fully formed at latter stages, these markers clearly are distributed to distinct cell compartments: a compact juxtanuclear structure for the Golgi markers and caveolin-2, while NDB-chol concentrates in lipid droplets. In addition, disorganization of the Golgi using three different agents (Brefeldin, monensin, and N-ethyl-maleimide) drastically reduces NBD-chol uptake at different phases of adipocyte formation, strongly suggesting that the Golgi apparatus plays a critical role in HDL-mediated NBD uptake and routing to lipid droplets. (+info)