Modulation of intracellular glutathione affects adipogenesis in 3T3-L1 cells

Impairment of redox homeostasis has been extensively associated with obesity, as a consequence of the chronic inflammatory state present in overweight subjects. Deregulation of glutathione (GSH), the most important non‐enzymatic intracellular anti‐oxidant, induces insulin resistance in mature adipocytes, but data are lacking about its effects on adipogenesis. In this report we demonstrate that during adipogenesis of 3T3‐L1 cells the GSH/GSSG ratio decreases, shifting redox status towards oxidizing conditions. Moreover, we demonstrate that inhibition of GSH synthesis, obtained by treatment with L ‐buthionine‐sulfoximine (BSO), enhances C/EBPβ LAP/LIP ratio and PPARγ expression during mitotic clonal expansion (MCE) stimulating adipogenesis. On the contrary, GSH ethyl ester (GSHest) supplementation completely abrogates this process also in the presence of BSO. GSH decrement during the first 24 h of adipogenesis is sufficient to induce higher triglyceride accumulation in differentiated adipocytes with respect to control, whereas GSHest treatment inhibits lipid droplets formation. We further demonstrate that Resveratrol (RV) could exert anti‐adipogenic properties also by increasing GSH content through γ‐glutamyl‐cysteine ligase (GCL) induction. Overall data indicate that in pre‐adipocytes the decrease of GSH accelerates adipogenesis, suggesting that the use of agents able to maintain GSH redox status in adipose tissue, such as RV, could be promising in stopping the lipogenic loop of obesity. J. Cell. Physiol. 226: 2016–2024, 2011. © 2010 Wiley‐Liss, Inc.     

Esculetin induces apoptosis and inhibits adipogenesis in 3T3-L1 cells

Objective: To determine the effects of esculetin, a plant phenolic compound with apoptotic activity in cancer cells, on 3T3‐L1 adipocyte apoptosis and adipogenesis. Research Methods and Procedures: 3T3‐L1 pre‐confluent preadipocytes and lipid‐filled adipocytes were incubated with esculetin (0 to 800 μM) for up to 48 hours. Viability was determined using the Cell Titer 96 Aqueous One Solution cell proliferation assay; apoptosis was quantified by measurement of single‐stranded DNA. Post‐confluent preadipocytes were incubated with esculetin for up to 6 days during maturation. Adipogenesis was quantified by measuring lipid content using Nile Red dye; cells were also stained with Oil Red O for visual confirmation of effects on lipid accumulation. Results: In mature adipocytes, esculetin caused a time‐ and dose‐related increase in adipocyte apoptosis and a decrease in viability. Apoptosis was increased after only 6 hours by 400 and 800 μM esculetin (p < 0.05), and after 48 hours, as little as 50 μM esculetin increased apoptosis (p < 0.05). In preadipocytes, apoptosis was detectable only after 48 hours (p < 0.05) with 200 μM esculetin and higher concentrations. However, results of the cell viability assay indicated a reduction in preadipocyte number in a time‐ and dose‐related manner, beginning as early as 6 hours with 400 and 800 μM esculetin (p < 0.05). Esculetin also inhibited adipogenesis of 3T3‐L1 preadipocytes. Esculetin‐mediated inhibition of adipocyte differentiation occurred during the early, intermediate, and late stages of the differentiation process. In addition, esculetin induced apoptosis during the late stage of differentiation. Discussion: These findings suggest that esculetin can alter fat cell number by direct effects on cell viability, adipogenesis, and apoptosis in 3T3‐L1 cells.     

Co-treatment with dexamethasone and octanoate induces adipogenesis in 3T3-L1 cells

We report here that octanoate, a medium chain fatty acid, induces adipocyte differentiation in 3T3-L1 cells by co-treatment with dexamethasone, although octanoate has been known not to stimulate 3T3-L1 adipogenesis. A low concentration of exogenous glucose prevented 3T3-L1 adipogenesis induced by 1-methyl 3-isobutylxanthine, dexamethasone, and insulin (MDI) treatment (a common protocol for adipocyte differentiation). In contrast, co-treatment with dexamethasone and octanoate (D-OCT) induced adipogenesis under the same conditions. These findings imply that octanoate, rather than glucose, is the source of accumulated lipids in D-OCT-induced adipogenesis. D-OCT increased expression of the differentiation markers peroxisome proliferator-activated receptor (PPAR)gamma2 and caveolin-1. A specific inhibitor of p38 mitogen-activated protein (MAP) kinase inhibited D-OCT-induced adipogenesis. These results suggest that the p38 MAP kinase pathway followed by up-regulation of PPARgamma2 may be involved in 3T3-L1 adipocyte differentiation induced by D-OCT, as well as by MDI.     

Structural requirements of flavonoids for the adipogenesis of 3T3-L1 cells

To search for a new class of antidiabetic compounds, effects of 44 flavonoids on the adipogenesis of 3T3-L1 cells were examined. Among them, 3,4',7-trimethylkaempferol, tetramethylkaempferol, and pentamethylquercetin concentration-dependently enhanced the accumulation of triglyceride, a marker of adipogenesis. With regard to structural requirements of flavonoids for the activity, it was fond that: (1) most flavonoids having hydroxy groups lacked the effect; (2) flavonols with methoxy groups showed stronger effects particularly those with a methoxy group at the 3-position; and (3) a methoxy group of flavonols at the B ring was also important. 3,4',7-Trimethylkaempferol, tetramethylkaempferol, and pentamethylquercetin significantly increased the amount of adiponectin released into the medium and the uptake of 2-deoxyglucose into the cells. Furthermore, tetramethylkaempferol and pentamethylquercetin also increased mRNA levels of adiponectin, glucose transporter 4 (GLUT4), and fatty acid-binding protein (aP2). Both compounds also increased the mRNA levels of peroxisome proliferator-activated receptor (PPAR)γ2 and CCAAT/enhancer-binding protein (C/EBP)α, β, and/or δ, although, different from troglitazone, they did not activate PPARγ directly in a nuclear receptor cofactor assay.     

Effects of amide constituents from pepper on adipogenesis in 3T3-L1 cells

Several amide constituents (piperlonguminine and retrofractamides A, B, and C) from the fruit of Piper chaba promoted adipogenesis of 3T3-L1 cells. Among them, retrofractamide A was the most active and significantly increased the amount of adiponectin released into the medium and the uptake of 2-deoxyglucose into the cells. Retrofractamide A also increased mRNA levels of adiponectin, peroxisome proliferator-activated receptor gamma2 (PPARgamma2), glucose transporter 4 (GLUT4), and insulin receptor substrate 1 (IRS-1), but did not act as a PPARgamma agonist different from troglitazone.     

Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor γ2, CCAAT/enhancer-binding protein alpha (C/EBPα), sterol regulatory element binding protein-1c, and Krüppel-like factor 15, but not those of C/EBPβ or C/EBPδ, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.     

High extracellular calcium attenuates adipogenesis in 3T3-L1 preadipocytes

We studied the effect of extracellular Ca²⁺ concentration ([Ca²⁺]_e) on adipocyte differentiation. Preadipocytes exposed to continuous [Ca²⁺]_e higher than 2.5 mmol/l accumulated little or no cytoplasmic lipid compared to controls in 1.8 mmol/l [Ca²⁺]_e. Differentiation was monitored by Oil Red O staining of cytoplasmic lipid and triglyceride assay of accumulated lipid, by RT-PCR analysis of adipogenic markers, and by the activity of glycerol-3-phosphate dehydrogenase (GPDH). Elevated [Ca²⁺]_e inhibited expression of peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein α, and steroid regulatory binding element protein. High [Ca²⁺]_e significantly inhibited differentiation marker expression including adipocyte fatty acid binding protein, and GPDH. The decrease in Pref-1 expression that accompanied differentiation also was prevented by high [Ca²⁺]_e. Treatment of 3T3-L1 cells with high [Ca²⁺]_e did not significantly affect cell number or viability and did not trigger apoptosis. Levels of intracellular Ca⁺² remained unchanged in various [Ca²⁺]_e. Treatment of 3T3-L1 with pertussis toxin (PTX) partially restored lipid accumulation and increased differentiation markers in cells treated with 5 mmol/l [Ca²⁺]_e. ‘Classical’ parathyroid cell Ca²⁺ sensing receptors (CaSR) were not detected either by RT-PCR or by Western blotting. These results suggest that continuos exposure to high [Ca²⁺]_e inhibits preadipocyte differentiation and that this may involve a G-protein-coupled mechanism mediated by a novel Ca²⁺ sensor or receptor.     

Molecular mechanism of 1,25-dihydroxyvitamin D3 inhibition of adipogenesis in 3T3-L1 cells

We have investigated the molecular mechanism whereby 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibits adipogenesis in vitro. 1,25(OH)2D3 blocks 3T3-L1 cell differentiation into adipocytes in a dose-dependent manner; however, the inhibition is ineffective 24-48 h after the differentiation is initiated, suggesting that 1,25(OH)2D3 inhibits only the early events of the adipogenic program. Treatment of 3T3-L1 cells with 1,25(OH)2D3 does not block the mitotic clonal expansion or C/EBPbeta induction; rather, 1,25(OH)2D3 blocks the expression of C/EBPalpha, peroxisome proliferator-activated receptor-gamma (PPARgamma), sterol regulatory element-binding protein-1, and other downstream adipocyte markers. The inhibition by 1,25(OH)2D3 is reversible, since removal of 1,25(OH)2D3 from the medium restores the adipogenic process with only a temporal delay. Interestingly, although the vitamin D receptor (VDR) protein is barely detectable in 3T3-L1 preadipocytes, its levels are dramatically increased during the early phase of adipogenesis, peaking at 4-8 h and subsiding afterward throughout the rest of the differentiation program; 1,25(OH)2D3 treatment appears to stabilize the VDR protein levels. Consistently, adenovirus-mediated overexpression of human (h) VDR in 3T3-L1 cells completely blocks the adipogenic program, confirming that VDR is inhibitory. Inhibition of adipocyte differentiation by 1,25(OH)2D3 is ameliorated by troglitazone, a specific PPARgamma antagonist; conversely, hVDR partially suppresses the transacting activity of PPARgamma but not of C/EBPbeta or C/EBPalpha. Moreover, 1,25(OH)2D3 markedly suppresses C/EBPalpha and PPARgamma mRNA levels in mouse epididymal fat tissue culture. Taken together, these data indicate that the blockade of 3T3-L1 cell differentiation by 1,25(OH)2D3 occurs at the postclonal expansion stages and involves direct suppression of C/EBPalpha and PPARgamma upregulation, antagonization of PPARgamma activity, and stabilization of the inhibitory VDR protein.     

Role of cyclooxygenases COX-1 and COX-2 in modulating adipogenesis in 3T3-L1 cells

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.