Expression of p107 and p130 during human adipose-derived stem cell adipogenesis

Within the first 24 h of hormonally stimulated adipocyte differentiation, murine 3T3-L1 preadipocytes undergo a mitotic expansion phase prior to terminal differentiation. During this time, the cell cycle regulatory proteins, p130 and p107 undergo dramatic differential expression and the transient increase in expression of p107 appears to be required for terminal differentiation. Recently, human adipose-derived human stem cells (hASC) of mesenchymal origin have been used as a model of human adipocyte differentiation and we sought to determine if differentiating hASC undergo clonal expansion and if the regulated expression of p130/p107 was similar to that observed during 3T3-L1 adipogenesis. Results indicate that differentiating hASC, unlike 3T3-L1 cells do not undergo clonal expansion and p130 expression gradually diminishes across differentiation. However, p107 expression is transiently increased during hASC differentiation in a manner analogous to 3T3-L1 cells suggesting a similar role for p107 in terminal differentiation in human adipocytes.     

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.     

DNA synthesis and mitotic clonal expansion is not a required step for 3T3-L1 preadipocyte differentiation into adipocytes

Upon differentiation induction of 3T3-L1 preadipocytes by a hormone mixture containing 1-isobutyl-3-methylxanthine, dexamethasone, and insulin, the preadipocytes undergo approximately 2 rounds of mitotic clonal expansion, which just precedes the adipogenic gene expression program and has been thought to be an essential early step for differentiation initiation. By inducing 3T3-L1 preadipocytes with each individual hormone, it was determined that the mitotic clonal expansion was induced only by insulin and not by 1-isobutyl-3-methylxanthine or dexamethasone. Cell number counting and fluorescence-activated cell-sorting analysis indicated that a significant fraction of 3T3-L1 preadipocytes differentiated into adipocytes without mitotic clonal expansion when induced with the combination of 1-isobutyl-3-methylxanthine and dexamethasone. Furthermore, when normally induced 3T3-L1 preadipocytes were treated with PD98059 (an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1) to block the activation of extracellular signal-regulated kinase (Erk) 1 and Erk2, the mitotic clonal expansion was blocked, but adipocyte differentiation was not affected. These observations were confirmed by bromodeoxyuridine labeling. The differentiated adipocytes induced with 1-isobutyl-3-methylxanthine and dexamethasone or standard hormone mixture plus PD98059 were not labeled by bromodeoxyuridine. Thus, it is evident that 3T3-L1 preadipocytes could differentiate into adipocytes without DNA synthesis and mitotic clonal expansion. Our results also suggested that activation of Erk1 and Erk2 is essential to but not sufficient for induction of mitotic clonal expansion.     

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.     

Retinoblastoma protein phosphorylation via PI 3-kinase and mTOR pathway regulates adipocyte differentiation

In the early phase of adipocyte differentiation, transient increase of DNA synthesis, called clonal expansion, and transient hyperphosphorylation of retinoblastoma protein (Rb) are observed. We investigated the role of these phenomena in insulin-induced adipocyte differentiation of 3T3-L1 cells. Insulin-induced clonal expansion, Rb phosphorylation and adipocyte differentiation were all inhibited by the PI 3-kinase inhibitors and rapamycin, but not the MEK inhibitor, whereas the MEK inhibitor, but not PI 3-kinase inhibitors or rapamycin, decreased c-fos induction. We conclude that insulin induces hyperphosphorylation of Rb via PI 3-kinase and mTOR dependent pathway, which promotes clonal expansion and adipocyte differentiation of 3T3-L1 cells.     

Antiadipogenic effect of carnosic acid,a natural compound present in Rosmarinus officinalis, is exerted through the C/EBPs and PPARγ pathways at the onset of the differentiation program

Background

Obesity is a serious health problem all over the world, and inhibition of adipogenesis constitutes one of the therapeutic strategies for its treatment. Carnosic acid (CA), the main bioactive compound of Rosmarinus officinalis extract, inhibits 3T3-L1 preadipocytes differentiation. However, very little is known about the molecular mechanism responsible for its antiadipogenic effect.

Methods

We evaluated the effect of CA on the differentiation of 3T3-L1 preadipocytes analyzing the process of mitotic clonal expansion, the level of adipogenic markers, and the subcellular distribution of C/EBPβ.

Results

CA treatment only during the first day of 3T3-L1 differentiation process was enough to inhibit adipogenesis. This inhibition was accompanied by a blockade of mitotic clonal expansion. CA did not interfere with C/EBPβ and C/EBPδ mRNA levels but blocked PPARγ, and FABP4 expression. C/EBPβ has different forms known as LIP and LAP. CA induced an increase in the level of LIP within 24 h of differentiation, leading to an increment in LIP/LAP ratio. Importantly, overexpression of LAP restored the capacity of 3T3-L1 preadipocytes to differentiate in the presence of CA. Finally, CA promoted subnuclear de-localization of C/EBPβ.

Conclusions

CA exerts its anti-adipogenic effect in a multifactorial manner by interfering mitotic clonal expansion, altering the ratio of the different C/EBPβ forms, inducing the loss of C/EBPβ proper subnuclear distribution, and blocking the expression of C/EBPα and PPARγ.

General significance

Understanding the molecular mechanism by which CA blocks adipogenesis is relevant because CA could be new a food additive beneficial for the prevention and/or treatment of obesity.     

Impact of stress hormone on adipogenesis in the 3T3-L1 adipocytes

Stress hormone is known to play a vital role in lipolysis and adipogenesis in fat cells. The present study was carried out to evaluate the effect of epinephrine on adipogenesis in the 3T3-L1 cells. The investigation on adipogenesis was done in both mono and co-cultured 3T3-L1 cells. 3T3-L1 preadipocytes and C2C12 cells were grown independently on transwell plates and transferred to differentiation medium. Following differentiation, C2C12 cells transferred to 3T3-L1 plate and treated with medium containing 10 μg/ml of epinephrine. Adipogenic markers such as fatty acid binding protein 4, peroxisome proliferator activating receptor, CCAAT/enhancer-binding protein, adiponectin, lipoprotein lipase and fatty acid synthase mRNA expressions were evaluated in the 3T3-L1 cells. Epinephrine treatment reduced adipogenesis, evidenced by reducing adipogenic marker mRNA expression in the 3T3-L1 cells. In addition, glycerol accumulation and oil red-O staining supported the reduced rate of adipogenesis. Taking all together, it is concluded that the stress hormone, epinephrine reduces the rate of adipogenesis in the mono and co-cultured 3T3-L1 cells. In addition, the rate of adipogenesis is much reduced in the co-cultured 3T3-L1 cells compared monocultured 3T3-L1 cells.     

Curcumin pretreatment prevents hydrogen peroxide-induced oxidative stress through enhanced mitochondrial function and deactivation of Akt/Erk signaling pathways in rat bone marrow mesenchymal stem cells

Lead (Pb) is an environmental and industrial contaminant that still represents a public health problem. Elevated Pb exposure has been inversely correlated with femoral bone density and associated with osteoporosis. In the last years, it has been shown that inhibition of osteogenesis from mesenchymal stem cells activates adipogenesis and vice versa. In this paper, we investigated the effect of Pb on the differentiation of 3T3-L1 fibroblasts to adipocytes which is the cell model most used to study adipogenesis. After induction of differentiation, 2 days post-confluent cells re-enter the cell cycle and undergo mitotic clonal expansion (MCE) followed by expression of genes that produce the adipocyte phenotype. The presence of concentrations of Pb up to 10 μM during differentiation of 3T3-L1 fibroblasts did not interfere with MCE but enhanced the accumulation of cytosolic lipids that occur during adipogenesis, as well as, the induction of PPARγ, the master gene in adipogenesis. It is known that PPARγ upregulation is subsequent to induction of C/EBPβ and ERK activation, which are early events in adipogenesis. We found that both events were enhanced by Pb treatment. Our results support a stimulatory effect of Pb on adipogenesis which involves ERK activation and C/EBPβ upregulation prior to PPARγ and adipogenesis activation.     

Lipid rafts/caveolae are essential for insulin-like growth factor-1 receptor signaling during 3T3-L1 preadipocyte differentiation induction

Lipid rafts/caveolae are found to be essential for insulin-like growth factor (IGF)-1 receptor signaling during 3T3-L1 preadipocyte differentiation induction. In 3T3-L1 cells, IGF-1 receptor is located in lipid rafts/caveolae of the plasma membrane and can directly interact with caveolin-1, the major protein component in caveolae. Disruption of lipid rafts/caveolae by depleting cellular cholesterol with cholesterol-binding reagent, beta-methylcyclodextrin or filipin, blocks the IGF-1 receptor signaling in 3T3-L1 preadipocyte. Both hormonal induced adipocyte differentiation and mitotic clonal expansion are inhibited by lipid rafts/caveolae disruption. However, a nonspecific lipid binding reagent, xylazine, does not affect adipocyte differentiation or mitotic clonal expansion. Further studies indicate that lipid rafts/caveolae are required only for IGF-1 receptor downstream signaling and not the activation of receptor itself by ligand. Thus, our results suggest that localization in lipid rafts/caveolae and association with caveolin enable IGF-1 receptor to have a close contact with downstream signal molecules recruited into lipid rafts/caveolae and transmit the signal through these signal molecule complexes.     

Properties of the cysteine-less Pho84 phosphate transporter of Saccharomyces cerevisiae

The murine 3T3-L1 preadipocyte cell line is well characterized for its capacity to undergo differentiation into adipocytes under appropriate hormonal stimulation. p107, a member of the retinoblastoma tumor suppressor gene family has been shown to be dramatically upregulated during the early requisite clonal expansion phase of 3T3-L1 adipogenesis; however, a functional consequence has yet to be described. A phosphorothioate antisense RNA approach was utilized to determine if inhibition of p107 expression would block or perturb adipocyte differentiation. A series of three phosphorothioate oligonucleotides in antisense orientation was generated, designated AS1, AS2, and AS3 along with a sense control oligonucleotide complementary to AS1 and added to postconfluent cells at a concentration of 20 and 50 microM throughout hormonally stimulated differentiation. Treatment of cells with either concentration of the sense, AS1, AS2, or 20 microM AS3 oligonucleotides had little effect on either Oil Red O lipid accumulation or induction of p107 protein levels. In contrast, treatment with 50 microM AS3 inhibited the increase in p107 protein levels and led to a complete block in differentiation as detected by Oil Red O lipid accumulation and inhibition of adipocyte-specific mRNA expression. In addition, treatment with AS3 led to a significant inhibition of cellular proliferation associated with clonal expansion. Combined, these results provide strong evidence supporting a functional role for p107 in 3T3-L1 adipocyte differentiation.     

MicroRNA and 3T3-L1 pre-adipocyte differentiation

MicroRNAs (miRNAs) have been suggested to play important roles in cell proliferation, apoptosis, and differentiation. In this study, we examined miRNA expression profiles during 3T3-L1 pre-adipocyte differentiation. We constructed miRNA libraries from pre- and post-differentiated 3T3-L1 cells, and identified the expression of 77 previously reported miRNAs and 3 new miRNAs. Next, we investigated the expression levels of 102 miRNAs, including those identified in the libraries, during adipogenesis by Northern blot analysis. Sixty-five miRNAs were detected and the expression of 21 miRNAs was up- or down-regulated during adipogenesis. Intriguingly, changes in the miRNA expression pattern were observed at day 9, when lipid droplets were visible, but not at days 1, 2, or 5 after the induction of differentiation. Antisense inhibition of the up-regulated miRNAs did not affect 3T3-L1 pre-adipocyte differentiation. Although these miRNAs may be involved in modulating adipocyte function, mild down-modulations of the up-regulated miRNAs do not appear to affect 3T3-L1 pre-adipocyte differentiation.     

peg10, an imprinted gene, plays a crucial role in adipocyte differentiation

An imprinted gene, paternally expressed gene (peg) 10, was isolated as one of the genes expressed early in adipogenesis. The expression of peg10 was elevated after the addition of inducers, and was detected in adipocyte differentiable 3T3-L1 cells, but not observed in the non-adipogenic cell line NIH-3T3. Moreover, the knockdown of peg10 by RNA interference (RNAi) inhibited the differentiation of 3T3-L1 cells into lipid-laden adipocytes. Interestingly, peg10 RNAi-treatment reduced the expressions of C/EBPbeta and C/EBPdelta, and inhibited mitotic clonal expansion. These findings strongly indicate that peg10 plays a crucial role at the immediate early stage of adipocyte differentiation.     

Stearoyl-CoA desaturase 2 is required for peroxisome proliferator-activated receptor gamma expression and adipogenesis in cultured 3T3-L1 cells

Based on recent evidence that fatty acid synthase and endogenously produced fatty acid derivatives are required for adipogenesis in 3T3-L1 adipocytes, we conducted a small interfering RNA-based screen to identify other fatty acid-metabolizing enzymes that may mediate this effect. Of 24 enzymes screened, stearoyl-CoA desaturase 2 (SCD2) was found to be uniquely and absolutely required for adipogenesis. Remarkably, SCD2 also controls the maintenance of adipocyte-specific gene expression in fully differentiated 3T3-L1 adipocytes, including the expression of SCD1. Despite the high sequence similarity between SCD2 and SCD1, silencing of SCD1 did not down-regulate 3T3-L1 cell differentiation or gene expression. SCD2 mRNA expression was also uniquely elevated 44-fold in adipose tissue upon feeding mice a high fat diet, whereas SCD1 showed little response. The inhibition of adipogenesis caused by SCD2 depletion was associated with a decrease in peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA and protein, whereas in mature adipocytes loss of SCD2 diminished PPARgamma protein levels, with little change in mRNA levels. In the latter case, SCD2 depletion did not change the degradation rate of PPARgamma protein but decreased the metabolic labeling of PPARgamma protein using [(35)S]methionine/cysteine, indicating protein translation was decreased. This requirement of SCD2 for optimal protein synthesis in fully differentiated adipocytes was verified by polysome profile analysis, where a shift in the mRNA to monosomes was apparent in response to SCD2 silencing. These results reveal that SCD2 is required for the induction and maintenance of PPARgamma protein levels and adipogenesis in 3T3-L1 cells.     

Discoidin domain receptor 2 impairs insulin-stimulated insulin receptor substrate-1 tyrosine phosphorylation and glucose uptake in 3T3-L1 adipocytes.

Differentiation of preadipocytes into functional adipocytes depends on early proliferative events (mitotic clonal expansion) and extracellular matrix interactions. We report that discoidin domain receptor (DDR) 2, a novel adhesion receptor, is expressed in 3T3-L1 preadipocytes and is downregulated during the early phase of adipogenesis. DDR2 overexpression (DDR2-L1 preadipocytes) reduced subconfluent proliferation by 56% (p<0.001) and insulin-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS)-1 by 34% (p<0.05). The mitotic clonal expansion phase of differentiating confluent DDR2-L1 preadipocytes was impaired by approximately 25% (p<0.05). Although induction of peroxisome proliferator-activated receptor gamma, fatty acid synthase, and adiponectin was not altered, the resulting adipocytes were 55% larger (p<0.05), and contained 66% more triacylglycerol (p<0.01). The induction of CCAAT/enhancer binding protein alpha was reduced by 37% (p<0.05), correlating with a similar reduction in insulin-stimulated IRS-1 tyrosine phosphorylation and glucose transport in DDR2-L1 adipocytes (decreases of 22% and 27%, respectively; p<0.05 for both). Our data show that DDR2 is expressed in adipose cells and that its overexpression leads to insulin resistance.     

PPARgamma1 synthesis and adipogenesis in C3H10T1/2 cells depends on S-phase progression, but does not require mitotic clonal expansion

Adipogenesis is typically stimulated in mouse embryo fibroblast (MEF) lines by a standard hormonal combination of insulin (I), dexamethasone (D), and methylisobutylxanthine (M), administered with a fresh serum renewal. In C3H10T1/2 (10T1/2) cells, peroxisome proliferator-activated receptor gamma1 (PPARgamma1) expression, an early phase key adipogenic regulator, is optimal after 36 h of IDM stimulation. Although previous studies provide evidence that mitotic clonal expansion of 3T3-L1 cells is essential for adipogenesis, we show, here, that 10T1/2 cells do not require mitotic clonal expansion, but depend on cell cycle progression through S-phase to commit to adipocyte differentiation. Exclusion of two major mitogenic stimuli (DM without insulin and fresh serum renewal) from standard IDM protocol removed mitotic clonal expansion, but sustained equivalent PPARgamma1 synthesis and lipogenesis. Different S-phase inhibitors (aphidicolin, hydroxyurea, l-mimosine, and roscovitin) each arrested cells in S-phase, under hormonal stimulation, and completely blocked PPARgamma1 synthesis and lipogenesis. However, G2/M inhibitors effected G2/M accumulation of IDM stimulated cells and prevented mitosis, but fully sustained PPARgamma1 synthesis and lipogenesis. DM stimulation with or without fresh serum renewal elevated DNA synthesis in a proportion of cells (measured by BrdU labeling) and accumulation of cell cycle progression in G2/M-phase without complete mitosis. By contrast, standard IDM treatments with fresh serum renewal caused elevated DNA synthesis and mitotic clonal expansion while achieved equivalent level of adipogenesis. At most, one-half of the 10T1/2 mixed cell population differentiated to mature adipocytes, even when clonally isolated. PPARgamma was exclusively expressed in the cells that contained lipid droplets. IDM stimulated comparable PPARgamma1 synthesis and lipogenesis in isolated cells at low cell density (LD) culture, but in about half of the cells and with sensitivity to G1/S, but not G2/M inhibitors. Importantly, growth arrest occurred in all differentiating cells, while continuous mitotic clonal expansion occurred in non-differentiating cells. Irrespective of confluence level, 10T1/2 cells differentiate after progression through S-phase, where adipogenic commitment induced by IDM stimulation is a prerequisite for PPARgamma synthesis and subsequent adipocyte differentiation.