Peroxisome proliferator-activated receptor delta (PPARdelta )-mediated regulation of preadipocyte proliferation and gene expression is dependent on cAMP signaling

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a key regulator of terminal adipocyte differentiation. PPARdelta is expressed in preadipocytes, but the importance of this PPAR subtype in adipogenesis has been a matter of debate. Here we present a critical evaluation of the role of PPARdelta in adipocyte differentiation. We demonstrate that treatment of NIH-3T3 fibroblasts overexpressing PPARdelta with standard adipogenic inducers led to induction of PPARgamma2 expression and terminal adipocyte differentiation in a manner that was strictly dependent on simultaneous administration of a PPARdelta ligand and methylisobutylxanthine (MIX) or other cAMP elevating agents. We further show that ligands and MIX synergistically stimulated PPARdelta-mediated transactivation. In 3T3-L1 preadipocytes, simultaneous administration of a PPARdelta-selective ligand and MIX significantly enhanced the early expression of PPARgamma and ALBP/aP2, but only modestly promoted terminal differentiation as determined by lipid accumulation. Finally, we provide evidence that synergistic activation of PPARdelta promotes mitotic clonal expansion in 3T3-L1 cells with or without forced expression of PPARdelta. In conclusion, our results suggest that PPARdelta may play a role in the proliferation of adipocyte precursor cells, whereas activation of endogenous PPARdelta in 3T3-L1 cells appears to have only minor impact on the processes leading to terminal adipocyte differentiation.     

Crucial role of TCL/TC10beta L,a subfamily of Rho GTPase,in adipocyte differentiation

The events at the beginning of adipocyte differentiation are not well known. We previously cloned the genes expressed early in the differentiation of mouse 3T3-L1 preadipocyte cells. One of them, similar in sequence to human TC10, was identified as TC10-like/TC10betaLong (TCL/TC10betaL), a new Rho GTPase by the cloning of full-length cDNA. The expression of TCL/TC10betaL increased rapidly right after the addition of inducers for differentiation, whereas the levels of other Rho family genes were unchanged at this stage. The antisense TCL/TC10betaL-expressing experiment revealed that the differentiation of 3T3-L1 cells into adipocytes was inhibited. Moreover, the sense TCL/TC10betaL-expressing experiment using NIH-3T3 cells, which do not usually differentiate into adipocytes, clearly showed the accumulation of oil droplets as well as the elevated expression of various adipogenic marker genes in the presence of the ligand for peroxisome proliferator-activated receptor gamma (PPARgamma). These results strongly indicated that TCL/TC10betaL has a crucial role in the early stage of adipocyte differentiation, probably linked to the PPARgamma pathway. Using a subtraction protocol, the genes specifically regulated by TCL/TC10betaL were also isolated. The expression pattern of some of them was similar to TCL/TC10betaL expression in adipogenesis, suggesting that the expression of these genes would be regulated by TCL/TC10betaL.     

SLC39A14, a LZT protein, is induced in adipogenesis and transports zinc

During adipocyte differentiation, there is an underlying complex series of gene expressions. We have previously isolated many genes whose expression levels are quickly elevated by the addition of inducers to mouse 3T3-L1 preadipocyte cells. Here we report the isolation and characterization of SLC39A14, a member of the LZT proteins, one of the subfamilies of ZIP transporters. The expression of the SLC39A14 gene was strongly and rapidly induced at the early stages of differentiation. Moreover, it was highly restricted to the potential differentiation state of 3T3-L1 cells and the expression level was quite low in the nonadipogenic NIH-3T3 cells, indicating a dominant expression in adipocyte differentiation. The zinc uptake assay revealed that SLC39A14 functions as a zinc transporter. Taken together, these results suggest that SLC39A14 plays a role as a zinc transporter during the early stages of adipogenesis.     

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.     

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.     

Regulation of adipocyte differentiation and insulin action with rapamycin

Here, we demonstrated that inhibition of mTOR with rapamycin has negative effects on adipocyte differentiation and insulin signaling. Rapamycin significantly reduced expression of most adipocyte marker genes including PPARgamma, adipsin, aP2, ADD1/SREBP1c, and FAS, and decreased intracellular lipid accumulation in 3T3-L1 and 3T3-F442A cells, suggesting that rapamycin would affect both lipogenesis and adipogenesis. Contrary to the previous report that suppressive effect of rapamycin on adipogenesis is limited to the clonal expansion, we revealed that its inhibitory effect persisted throughout the process of adipocyte differentiation. Thus, it is likely that constitutive activation of mTOR might be required for the execution of adipogenic programming. In differentiated 3T3-L1 adipocytes, chronic treatment of rapamycin blunted the phosphorylation of AKT and GSK, which is stimulated by insulin, and reduced insulin-dependent glucose uptake activity. Taken together, these results suggest that rapamycin not only prevents adipocyte differentiation by decrease of adipogenesis and lipogenesis but also downregulates insulin action in adipocytes, implying that mTOR would play important roles in adipogenesis and insulin action.     

Distinct stages in adipogenesis revealed by retinoid inhibition of differentiation after induction of PPARgamma.

Retinoic acid (RA) inhibits adipocyte differentiation of 3T3-L1 preadipocytes but is effective only early in adipogenesis. RA prevented induction of the adipogenic factors PPARgamma and C/EBPalpha. Using receptor-specific ligands, we determined that the effects of RA were mediated by liganded RA receptors (RARs) rather than retinoid X receptors. Preadipocytes expressed primarily RARalpha and RARgamma; during adipocyte differentiation, RARalpha gene expression was nearly constant, whereas RARgamma1 mRNA and protein levels dramatically decreased. Ectopic expression of RARgamma1 extended the period of effectiveness of RA by 24 to 48h; RARalpha expression had a similar effect, suggesting functional redundancy of RAR subtypes. Remarkably, RA inhibited differentiation when added after PPARgamma1 and PPARgamma2 proteins had already been expressed and resulted in the loss of PPARgamma proteins from cells. By 72 to 96 h after the induction of differentiation, RA failed to prevent differentiation of even ectopic-RAR-expressing cells. Thus, the unresponsiveness of 3T3-L1 preadipocytes to RA after the induction of differentiation is initially due to the reduction in cellular RAR concentration rather than to the induction of PPARgamma. At later times cells continue along the differentiation pathway in a manner which is RA and RAR independent.     

Orphan nuclear receptor Nur77 accelerates the initial phase of adipocyte differentiation in 3T3-L1 cells by promoting mitotic clonal expansion

Nur77 is an orphan member of the nuclear receptor superfamily that is expressed in various types of cells and mediates diverse biological processes. Although Nur77 mRNA is induced in the early stage of adipogenesis of 3T3-L1 cells, its roles are not known. To address this issue, we closely inspected the expression of Nur77 mRNA and protein during differentiation of 3T3-L1 cells. Nur77 was induced rapidly and transiently at both mRNA and protein levels only in the initial phase of differentiation induction, and localized almost exclusively in the nuclei. Isobutylmethylxanthine was essential for the induction of Nur77 protein, acting by at least in part protecting the protein from rapid degradation by proteasome. Nur77 siRNA resulted in delayed adipogenesis in 3T3-L1, accompanied by retarded mitotic clonal expansion. These effects were mediated at least partly by decreased expression of cyclins D and E. Constitutive expression of Nur77 inhibited adipogenesis of 3T3-L1, accompanied by enhanced expression of cyclin D1 and prolonged mitotic clonal expansion. Moreover, constitutive expression of Nur77 inhibited, but transient induction of Nur77 promoted, adipogenesis in NIH-3T3 cells. These results suggest that Nur77 accelerates adipocyte differentiation by regulating cell cycle progression and the rapid and transient induction is crucial for its action.