Our previous investigations demonstrated that four amino acids, E367, F372, D378, and D379, within helix 7 of the ligand-binding domain of PPARγ facilitate a functional interaction between PPARγ and β-catenin (22
). To gain insight into the potential involvement of helix 7 in regulating the transcriptional activity of PPARγ during adipogenesis, we expressed a series of mutant PPARγ proteins in Swiss 3T3 fibroblasts in which E367, F372, D378, or D379 was modified to alanine and assessed their abilities to induce adipogenic gene expression. First, we observed that ectopic expression of the WT PPARγ was capable of inducing the conversion of these fibroblasts into adipocytes simply by exposure to DEX, MIX, and insulin without the need for an exogenous ligand, such as troglitazone (Fig. ). These data suggest that Swiss fibroblasts produce endogenous ligands that can activate the ectopic PPARγ following exposure to the normal cocktail of adipogenic inducers. In fact, exposure to troglitazone appeared to have no additional effect on the morphological features of these Swiss adipocytes (Fig. ). Additionally, the Western blot in Fig. , lanes 1 and 5, shows abundant expression of the adipogenic proteins, C/EBPα, perilipin, and aP2, and a low level of β-catenin production in the Swiss WT PPARγ cells induced to differentiate in the presence or absence of troglitazone. The data also show that the relative abundance of transcriptionally active WT PPARγ was very low due to its rapid turnover. The mutant PPARγ corresponding to E367A (E-PPARγ) retained the ability to induce adipogenesis in the presence or absence of troglitazone (Fig. ), which included degradation of β-catenin (Fig. ). It is interesting, however, that this alteration appeared to stabilize PPARγ in the absence of ligand, while exposure to troglitazone resulted in a significant decrease in its abundance (Fig. , compare lanes 2 and 6). The most interesting data came from analyzing the expression of the mutant PPARγ corresponding to E367A and F372A (EF-PPARγ). Figure demonstrates that mutation of F372 to alanine, in addition to E367A, completely destroyed the ability of PPARγ to respond to an endogenous ligand, since Swiss cells expressing EF-PPARγ remained as fibroblasts (Fig. ) and did not express adipogenic genes or down-regulate β-catenin (Fig. ). Additionally, this mutant PPARγ appeared to be quite stable. More importantly, exposure of the Swiss EF-PPARγ cells to troglitazone induced their conversion into adipocytic cells (Fig. ) and expression of C/EBPα, perilipin, and aP2 (Fig. ). These data are consistent with the notion that F372 and E367 within helix 7 participate in the response of PPARγ to endogenous ligands, whereas responses to exogenous synthetic ligands, such as troglitazone, are less dependent on these amino acids. Mutation of both D378 and D379 to alanine completely destroyed the ability of PPARγ to respond to both endogenous and exogenous ligands, since the corresponding mutant PPARγ was incapable of inducing either morphological conversion or adipogenic gene expression (Fig. , lanes 4 and 8).
FIG. 1. EF-PPARγ does not respond to endogenous ligands but is activated by troglitazone. Swiss cells expressing various forms of PPARγ (WT, E, EF, and DD) were cultured until they were confluent; after 2 days, they were exposed to DEX, MIX, and (more ...)
The data presented in Fig. suggested to us that analysis of mRNA expression in Swiss WT PPARγ versus Swiss EF-PPARγ cells might permit the identification of PPARγ target gene programs responding to endogenous versus exogenous ligands. Consequently, total RNA was harvested from Swiss fibroblasts expressing either WT or EF-PPARγ proteins 5 days following exposure to the adipogenic inducers in the presence or absence of troglitazone and was subjected to oligonucleotide microarray analysis employing Affymetrix chips. The data revealed that the abundances of 1,767 genes of the ~22,690 represented on the array differed at least twofold between the highly differentiated Swiss WT PPARγ cells and the undifferentiated Swiss EF-PPARγ cells (minus troglitazone). A cluster analysis of these genes is shown in Fig. , in which the genes that are highly expressed in WT PPARγ cells (minus troglitazone) relative to their expression in EF-PPARγ cells (minus troglitazone) are arranged in descending order of their relative abundances. Genes that are highly expressed in adipocytes (WT PPARγ) compared to fibroblasts (EF-PPARγ minus troglitazone) cluster together and include those coding for adipogenic, lipogenic, and mitochondrial proteins. In contrast, many genes are expressed at much lower abundance in the adipocytes (WT PPARγ cells) compared to the fibroblasts (EF-PPARγ minus troglitazone) and include components of the Wnt signaling pathway, as well as inflammatory proteins, several of which have previously been reported to be down-regulated during adipogenesis. Figure S1 in the supplemental material represents the relative abundances of selected genes present in each of these clusters and reveals that many of the genes displayed significantly more than a fivefold difference in abundance between the two cell types. In fact, some mRNAs, such as adiponectin and Fsp27, were expressed at least 104-fold more abundantly in the adipocytes (WT PPARγ) than in the fibroblasts (EF-PPARγ minus troglitazone). Figure also illustrates that treatment of the WT PPARγ cells with troglitazone did not significantly alter the overall pattern of gene expression but appeared to enhance the level of adipogenic gene expression while suppressing even further the fibroblastic mRNAs (Fig. , compare lane 2 with lane 1). More importantly, the EF-PPARγ cells that were completely unresponsive to endogenous ligands (minus troglitazone) were extensively induced to express multiple adipogenic, lipogenic, and mitochondrial genes following their exposure to troglitazone (Fig. , compare lane 3 with lane 4). These EF-PPARγ cells also down-regulated expression of the fibroblastic genes in response to troglitazone, consistent with their attaining an adipocyte-like morphology (Fig. ).
Identification of a subset of PPARγ-responsive genes.
To gain more insight into the gene programs regulated by PPARγ in response to endogenous versus exogenous ligands, a more detailed analysis of individual genes was performed, as shown in Tables S1A and S1B in the supplemental material. We also analyzed the profiles of mRNAs expressed during the differentiation of 3T3-L1 preadipocytes for comparison with the mRNAs expressed in Swiss PPARγ cells by performing additional Affymetrix array analysis of mRNAs isolated from the preadipocytes at 0, 4, and 10 days of differentiation. Table S1A in the supplemental material lists a selection of classic adipogenic genes that were induced to various extents during adipogenesis in 3T3-L1 preadipocytes (columns 5, 6, and 7), which include genes coding for proteins involved in lipid storage/metabolism (i.e., FABP4), as well as endocrine functions (i.e., adiponectin). All of these mRNAs were expressed much more abundantly in Swiss fibroblasts expressing WT PPARγ than in cells expressing EF-PPARγ. In fact, the difference in the levels of expression of these mRNAs in EF-PPARγ cells versus WT PPARγ cells was comparable to the difference in their expression levels in preadipocytes versus mature adipocytes (see Table S1A in the supplemental material; compare columns 1 and 3 with columns 5 and 7). It is also relevant to point out that the expression of at least three genes, Resistin (Retn), Hsd11β1, and Orosomucoid (Orm1) genes, was enhanced by WT PPARγ in Swiss cells in response to endogenous ligands (minus troglitazone) and during adipogenesis in 3T3-L1 preadipocytes. Interestingly, troglitazone significantly attenuated the expression of these genes in WT PPARγ (see Table S1A in the supplemental material, Retn, Hsd11b1, and Orm1; compare column 2 with column 1), consistent with reports that TZDs selectively repress the expression of these genes following their dramatic induction during adipogenesis in 3T3-L1 cells (2
). Taken together, the data in Table S1A in the supplemental material are consistent with the notion that PPARγ can induce expression of the majority of the classic adipogenic genes in Swiss fibroblasts in response to an endogenous ligand to the same extent as that occurring during normal adipogenesis in 3T3-L1 preadipocytes. Furthermore, mutation of critical amino acids within helix 7 (EF-PPARγ) prevents PPARγ from responding to endogenous ligand activity (Fig. ; see Table S1A, column 3, in the supplemental material). As shown in Fig. , however, exposure of EF-PPARγ to troglitazone can induce expression of most of these classic adipogenic genes to levels attained in 3T3 adipocytes (see Table S1A in the supplemental material; compare column 4 with column 7).
Following a more extensive analysis of the array data, it was observed that not all genes that are highly expressed in 3T3-L1 or WT PPARγ adipocytes were induced in the EF-PPARγ cells by troglitazone. In fact, it appeared that activation of the mutant EF-PPARγ by the exogenous ligand, while capable of converting these fibroblasts into adipocytic cells that contained small lipid droplets and expressed many of the markers of mature adipocytes (Fig. ), was incapable of inducing the entire adipogenic program (see Table S1B in the supplemental material). More specifically, the data shown in Table S1B in the supplemental material suggest that WT PPARγ induces the expression of a group of responsive genes (columns 1 and 2) that are significantly less responsive to stimulation of EF-PPARγ by troglitazone (column 4). This subset of PPARγ target genes (referred to here as group 2) includes proteins that have not previously been shown to be associated with PPARγ activity, such as the endoplasmic reticulum oxidoreductase Ero1-Lα, FGF21 and genes coding for components of the glycolytic pathway. Table S1B in the supplemental material also shows that some of these genes, including those for Mrap, KLF15, Klb (βKlotho), and Pdxp, are induced severalfold during adipogenesis but are unresponsive to troglitazone activation of EF-PPARγ. Other genes are moderately responsive to adipogenic signals in 3T3-L1 preadipocytes (i.e., the glycolytic genes), but almost all of these genes are induced in response to troglitazone activation of WT PPARγ, but not of EF-PPARγ.
To confirm the oligonucleotide microarray data, a series of reverse transcription (RT)-PCR analyses were performed in which the relative abundances of select mRNAs expressed in the Swiss cell lines were measured. Since the data presented in Fig. suggest that F372 is the amino acid that appears to influence the transcriptional activity of PPARγ, we generated an additional cell line corresponding to Swiss fibroblasts expressing PPARγ in which only F372 was changed to alanine (F-PPARγ cells). We also analyzed Swiss fibroblasts that do not contain an ectopic PPARγ and therefore are completely incapable of adipogenesis even in the presence of troglitazone (control cells). Figure shows the constitutive expression of the corresponding PPARγ mRNAs in each of the Swiss PPARγ cell lines and the absence of any PPARγ mRNA in the control cells. The panel on the left demonstrates expression of select target genes (from Table S1A in the supplemental material) that are induced in the WT PPARγ cells exposed to endogenous (Fig. , lane 3), as well as exogenous (Fig. , lane 4), PPARγ ligands. The expression of most of these genes is unaffected by troglitazone, with the exception of EPHX2, which appears to be enhanced even further by the exogenous ligand. This set of classic adipogenic genes, as expected, is not expressed in the EF- or F-PPARγ cells in response to the endogenous ligands (Fig. , lanes 5 and 7). Furthermore, activation of these mutant cell lines (EF- and F-PPARγ) by exposure to troglitazone significantly induces expression of all of these genes (Fig. , lanes 6 and 8), as shown in Table S1A in the supplemental material. In contrast, the subset of genes (group 2) presented in the panel on the right (selected from Table S1B in the supplemental material) responds quite differently to the action of the mutant PPARγ molecules. These genes are induced to various extents by endogenous ligands in cells expressing WT PPARγ and are enhanced manyfold by exposure to troglitazone (Fig. , compare lanes 11 and 12 with 9 and 10). More importantly, this subset of genes is unresponsive to activation of EF- or F-PPARγ by troglitazone, as well as the endogenous ligands (Fig. , compare lanes 13, 14, 15, and 16 with lanes 11 and 12). We also performed Western blot analysis of C/EBPα, FABP4/aP2, adiponectin (group 1, EF-PPARγ-responsive genes), and Ero1-Lα (a group 2, non-EF-PPARγ-responsive gene) to confirm the RT-PCR data. Figure S2 in the supplemental material shows that troglitazone stimulation of all forms of PPARγ, including WT PPARγ, E-PPARγ, EF-PPARγ, and F-PPARγ, leads to abundant expression of C/EBPα, FABP4/aP2, and adiponectin. In contrast, expression of Ero1-Lα is completely unresponsive to troglitazone stimulation of F-PPARγ or EF-PPARγ but responds to WT and E-PPARγ activities. It is interesting that analysis of the proteins in the culture media showed that adiponectin is secreted from cells expressing WT and E-PPARγ but is absent from the media of F-PPARγ and EF-PPARγ cells. These data suggest that some group 2 proteins likely participate in processes responsible for secretion of adiponectin. In fact, recent studies by others and by us have demonstrated a role for Ero1-Lα in regulating the secretion of adiponectin from adipocytes (30
FIG. 2. (A) Identification of two groups of PPARγ-responsive genes: group 1 (including adiponectin) is responsive, whereas group 2 (including Ero1 and FGF21 genes) is completely unresponsive, to troglitazone activation of EF-PPARγ or F-PPARγ. (more ...) The group 2 subset of genes can be selectively activated in response to troglitazone during the differentiation of Swiss 3T3 fibroblasts into adipocytes.
The data in Fig. and Table S1B in the supplemental material show that many of the group 2 genes are constitutively expressed at a low level during normal adipogenesis in response to endogenous ligands but appear to be responsive to potent exogenous ligands. To gain greater insight into the mechanisms regulating these two programs of gene expression, Swiss WT PPARγ cells were induced to differentiate in the absence or presence of troglitazone, and the expression of selected genes was analyzed each day using RT-PCR technology. Figure demonstrates the constant and abundant expression of the WT PPARγ throughout 7 days of differentiation, which resulted in a robust and sustained induction of the group 1 genes, such as those for adiponectin and C/EBPα, in response to endogenous (minus troglitazone), as well as exogenous (plus troglitazone), ligands. Interestingly, the group 2 genes, including those for Ero1-Lα, Scd3, and FGF21, are transiently expressed at a very low level during the initial 2 to 4 days of adipogenesis and are then down-regulated as differentiation proceeds in the absence of troglitazone. Differentiation of these WT PPARγ cells in the presence of troglitazone has a minimal effect on expression of adiponectin and C/EBPα mRNAs but enhances, as well as maintains, expression of Ero1-Lα, Scd3, and FGF21 throughout the 7-day culture period.
Selected group 2 genes are transiently expressed during the differentiation of brown and white preadipocytes.
The data presented in Fig. were derived from nonadipogenic fibroblasts forced to differentiate into adipocytes by overexpression of PPARγ. We considered it important, therefore, to determine whether this interesting pattern of PPARγ target gene expression occurs in preadipocytes undergoing differentiation into brown, as well as white, adipocytes in response to activation of endogenous adipogenic transcription factors. To this end, we analyzed the expression of genes during the differentiation of 3T3-L1 white preadipocytes and immortalized primary brown preadipocytes. Figure shows the expected induction of PPARγ, C/EBPα, LXRα, and adiponectin mRNAs 2 days after exposure of the preadipocytes to DEX, MIX, insulin, and 10% FBS. Furthermore, expression of these adipogenic genes remained at a high level throughout the differentiation of both brown and white preadipocytes. To confirm that the immortalized primary brown preadipocytes underwent differentiation into brown adipocytes, we also analyzed the expression of PGC-1α and UCP-1, and the data show that the expression of these mRNAs was initiated at 2 days and was maintained throughout brown adipogenesis (Fig. ). In contrast, the group 2 genes that responded poorly to expression of F-PPARγ in the Swiss cells were induced in response to activation of endogenous PPARγ in both the brown and white preadipocytes;, however, the level of expression of the corresponding mRNAs dropped significantly during terminal adipogenesis, as observed in the Swiss WT PPARγ cells differentiated in the absence of troglitazone (Fig. ).
FIG. 3. Selected group 2 PPARγ-responsive genes are transiently induced during the initial phase of adipogenesis in white 3T3-L1 preadipocytes (A) and immortalized primary brown preadipocytes (B). (A) 3T3-L1 white preadipocytes were cultured in 10% (more ...) Differential responses of group 1 and group 2 genes to PPARγ agonists and antagonists.
The fact that mutations within helix 7 rendered PPARγ unresponsive to endogenous ligands and responsive to troglitazone, at least for the group 1 genes, encouraged us to determine the effects of other ligands that had previously been shown to possess a range of activities. Consequently, WT PPARγ and EF-PPARγ cells were induced to differentiate in the presence or absence of 15δ-PGJ2, 9-fluorenylmethoxy carbonyl (FMOC)-leu, troglitazone, rosiglitazone, or GW1929 for 5 days, and the expression of selected genes was analyzed by RT-PCR. Figure shows that all of the exogenous ligands had little or no additional effect on the expression of selected group 1 genes in WT PPARγ cells, since their levels of expression were already at a maximum, due presumably to the stimulation of the ectopic PPARγ by endogenous ligands (compare lanes 2 to 6 with lane 1). In contrast, expression of the group 2 genes was enhanced to various extents by exposure of the WT PPARγ cells to the exogenous ligands. In the case of the EF-PPARγ cells, exposure to the different ligands resulted in a significantly more varied response than that observed in the WT PPARγ cells. Specifically, FMOC-leu was incapable of stimulating the expression of any of the selected group 1 or group 2 genes, and 15δ-PGJ2 activated only FABP4 expression. The TZDs, troglitazone and rosiglitazone, induced the expression of selected group 1 genes, including those for C/EBPα, adiponectin, and FABP4, but had a negligible effect on the expression of the group 2 genes, such as those for Ero1-Lα and Mrap. Interestingly, GW1929, an extremely potent synthetic PPARγ ligand in which N-tyrosine moieties have been substituted for the TZD head group, is capable of inducing expression of the group 2 genes, as well as group 1 genes, in the EF-PPARγ cells. These data clearly show differential responses of the two groups of genes to different ligands; we asked, therefore, whether the genes also showed similar differential responses to a PPARγ antagonist. To address this question, WT PPARγ cells were induced to differentiate in the presence or absence of T0070907 or GW9662 (two PPARγ antagonists) with or without troglitazone, and the corresponding cellular RNAs were analyzed by RT-PCR. Figure demonstrates that T0070907 and GW9662 moderately attenuated the ability of WT PPARγ to induce expression of C/EBPα and adiponectin in response to endogenous ligands. The presence of troglitazone overcame the inhibitory effect of the antagonists (Fig. , compare lanes 5 and 6 with lanes 3 and 4). In contrast, the antagonists almost completely blocked expression of the group 2 genes, including those for Ero1-Lα, Mrap, Elovl3, Egln1, SCD3, and OLR-1, in response to stimulation of WT PPARγ by endogenous ligands, with T0070907 being the most potent. Again, this effect was overcome somewhat by troglitazone. Taken together, the data in Fig. show that activation of the group 2 genes by PPARγ requires more potent ligands and is significantly more sensitive to antagonists than that of the group 1 genes. We also considered it important to determine whether mutation of F372 had simply dampened the ligand-binding affinity of PPARγ and, consequently, had significantly shifted the dose response to troglitazone to higher concentrations. To investigate this possibility, WT PPARγ and EF-PPARγ cells were induced to differentiate for 5 days in the presence of increasing concentrations of troglitazone. At this stage, cells were harvested for analysis of selected genes by RT-PCR. Figure shows abundant expression of selected group 1 genes, including those for C/EBPα, adiponectin, and FABP4/aP2, in WT PPARγ cells due to endogenous ligands (lane 1), with no significant change in expression in response to increasing doses of troglitazone (lanes 2 to 8). As expected, the group 2 genes, encoding FGF21 and OLR-1, are not expressed in WT PPARγ without an exogenous ligand (lane 1) but can be induced by troglitazone in a dose-dependent manner (lanes 2 to 8). Analysis of gene expression in the EF-PPARγ cells showed that the group 1 genes, for C/EBPα, adiponectin, and FABP4/aP2, are not expressed in the absence of exogenous ligand (Fig. , lane 9) but, as expected, are induced in response to doses of troglitazone (250 to 500 nM) previously shown to be specific for PPARγ (Fig. , lanes 10 to 16). Of importance is the observation that expression of FGF21 and OLR1 cannot be activated in the EF-PPARγ cells by doses of troglitazone (10 μM) that far exceed the dose that is specific for PPARγ. These data demonstrate clearly that mutation of F372 within helix 7 prevented PPARγ from responding to endogenous ligands and, additionally, prevented PPARγ from inducing expression of the group 2 genes in response to the TZD troglitazone.
FIG. 4. The differential responses of WT PPARγ versus EF-PPARγ to selected PPARγ ligands and antagonists. (A) Swiss-PPARγ (WT and EF) cells were cultured until they were confluent; after 2 days, they were exposed to DEX, MIX, and (more ...) PPARγ directly regulates expression of FGF21.
It is conceivable that the inability of the mutant PPARγ (EF or F) to induce expression FGF21 and OLR1 by troglitazone, as well as exogenous ligands, is due to the fact that the corresponding genes might not be direct targets of PPARγ. Other studies, however, have shown direct induction of the OLR1 gene promoter by PPARγ (5
). We considered it important and of significant interest to determine whether the FGF21 gene is also a direct target of PPARγ. To this end, we performed two sets of experiments. First, we determined whether the induction of FGF21 gene expression occurred in the absence of ongoing protein synthesis. To do this, WT PPARγ cells were induced to differentiate for 5 days without a synthetic ligand, at which stage troglitazone (5 μM) or cycloheximide (5 μg/ml) was added alone or together for 4, 6, or 8 h, and at each time, RNA was analyzed by RT-PCR. Figure shows significant expression of FABP4/aP2a mRNA at all three times due to its activation by endogenous ligand activity during the 5 days of differentiation of the WT PPARγ cells. In contrast, there are virtually undetectable levels of FGF21 mRNA expression in the absence of an exogenous ligand (Fig. , lanes 1, 5, and 9). Interestingly, exposure to troglitazone rapidly induces FGF21 mRNA expression during the 8-h exposure time (Fig. , lanes 2, 6, and 10), and this event occurs in the presence of cycloheximide (Fig. , lanes 4, 8, and 12), showing that the FGF21 gene is a direct target of PPARγ. Also of interest is the observation that FGF21 mRNA expression is induced simply due to exposure to cycloheximide (Fig. , lanes 3, 7, and 11). This is usually indicative of the existence of a repressor that is removed due to its rapid turnover in the absence of ongoing protein synthesis. To demonstrate further that PPARγ directly activates FGF21 gene expression, we performed a series of FGF21 gene promoter/luciferase reporter assays. To this end, fragments (−500, −1300 and −1500) of the upstream region of the FGF21 gene were cloned into the pGL3 luciferase reporter plasmid as shown in Fig. . Analysis of the sequence of the proximal 1,500 bp of the gene showed the presence of at least five DR-1 response elements, which are highly homologous to a consensus PPRE and therefore have the potential to associate with PPARγ/RXRα heterodimers. Figure shows that transfection of the bp −500 fragment plasmid, which contains two PPREs, into control Swiss fibroblasts in the presence or absence of a potent PPARγ ligand, GW1929, expressed a low basal level of luciferase activity equivalent to a control DR-1/luciferase reporter composed of consensus PPREs. Interestingly, the 1,300-bp and 1,500-bp fragments expressed higher levels of luciferase activity, but the presence of GW1929 had no affect on this activity. Transfection of the reporter plasmids, along with a PPARγ expression plasmid, however, resulted in a significant increase in the activities of all three FGF21 gene fragments, which was enhanced even further in the presence of GW1929. We also analyzed FGF21 promoter activity in control (pBabe-puro) and Swiss WT PPARγ cells by transfecting each of the luciferase reporter plasmids in the presence or absence of GW1929. The results in Fig. are consistent with those in Fig. , showing that the transcriptional activity of the 500-bp fragment of the FGF21 gene was significantly higher in the Swiss cells expressing PPARγ than in control Swiss cells and that this activity was enhanced manyfold by GW1929. The activity of the 1,300- and 1,500-bp fragments in WT PPARγ cells in the presence of GW1929 was only slightly higher than that of the 500-bp region, suggesting that elements within this proximal promoter are likely responsible for the observed PPARγ-dependent activities of all three fragments.
FIG. 5. PPARγ directly activates the FGF21 gene. (A) Troglitazone activates FGF21 gene expression in the absence of ongoing protein synthesis. WT PPARγ cells were induced to differentiate with DEX, MIX, and insulin for 5 days, at which time troglitazone (more ...) Expression of many of the group 2 genes is actively repressed by SIRT1 during terminal adipogenesis.
A recent report demonstrated that activation of SIRT1 in adipocytes triggers lipolysis and loss of fat by mechanisms involving repression of PPARγ activity (28
). We asked, therefore, whether there is a role for SIRT1 in facilitating the differential expression of the group 1 and group 2 genes as preadipocytes become mature fat cells. Consequently, we analyzed the expression of adipogenic genes during differentiation of 3T3-L1 preadipocytes, in which SIRT1 expression is suppressed due to constitutive production of a corresponding SIRT1 RNA interference. The Western blot in Fig. shows the extensive reduction in SIRT1 expression in the RNA interference cells compared to the abundant production in a control line of 3T3-L1 cells expressing the vector alone. It is also relevant that expression of SIRT1 increased severalfold during the early phase of adipogenesis in the control cells but then subsided to preadipocyte levels during the terminal phase. It is also important to point out that there was no significant effect of knockdown of SIRT1 on the production of adiponectin. Total RNA was harvested from SIRT1 knockdown, as well as control, cells at selected times throughout differentiation. RNAs from preadipocytes (day 0) and cells at a middle (day 4) and late (day 10) phase of adipogenesis were subjected to oligonucleotide microarray analysis employing Affymetrix chips, as discussed above. The relative expression levels of selected mRNAs corresponding to both group 1 and group 2 genes were analyzed, as shown in Tables S1A and S1B in the supplemental material, respectively. As discussed above, Table S1A in the supplemental material is a list of classic adipogenic genes (group 1) that are induced during adipogenesis in 3T3-L1 preadipocytes and are differentially responsive to WT PPARγ versus EF-PPARγ. Suppression of SIRT1 activity causes a transient increase (~50%) in the expression levels of most of these genes at day 4 of differentiation in 3T3-L1 cells compared to their levels of expression at this stage of differentiation in control cells (see Table S1A in the supplemental material; compare column 9 with column 6). This difference in expression correlates with a significant increase in SIRT1 expression during early adipogenesis in control 3T3-L1 preadipocytes (Fig. ). Interestingly, these genes appeared to reach a maximum level of expression by day 4 in the knockdown cells, whereas it required 10 days for them to reach this maximum in the control cells (see Table S1A in the supplemental material; compare columns 7 and 10). The data are consistent with the notion that the preadipocytes lacking SIRT1 activity differentiate much faster than control cells, reaching terminal adipogenesis within 4 days compared to 10 days in the control cells. Table S1B in the supplemental material shows the expression profiles of the group 2 mRNAs in control and SIRT1 knockdown 3T3-L1 cells and Swiss Pγ cells. As observed for the group 1 genes in Table S1A in the supplemental material, expression of the group 2 genes also increased at day 4 of differentiation in the knockdown cells, even though most of the genes did not normally show enhanced expression at this stage of differentiation in control cells (see Table S1B in the supplemental material; compare column 9 with column 6). More importantly, most of the group 2 genes were expressed at significantly higher levels at day 10 in the SIRT1 knockdown cells than in control cells (see Table S1B in the supplemental material; compare column 10 with column 7). In fact, some genes, most notably Ero1-Lα (487%), Hig1 (262%), and Trib3 (290%), were enhanced manyfold in response to reduction in SIRT1 abundance. Additionally, all the genes coding for glycolytic enzymes, as well as glucose transporter 1, were also induced in the SIRT1 knockdown cells. It is also worth mentioning that the extent of induction of each of these group 2 genes appeared to correlate with the level of induction by troglitazone in WT PPARγ cells (see Table S1B in the supplemental material; compare columns 1 and 2 with 7 and 10). To confirm the data presented in Tables S1A and S1B in the supplemental material, Fig. shows an RT-PCR analysis of RNAs harvested from control and SIRT1 knockdown 3T3-L1 cells at times throughout differentiation. It is quite apparent that the knockdown of SIRT1 had a selective effect on the group 2 genes compared to the group 1 genes. Specifically, expression of C/EBPα and adiponectin mRNAs (group 1) showed a modest increase at the early stage of adipogenesis in the SIRT1 knockdown cells (Fig. ), as presented in Table S1A in the supplemental material, but no significant increase as these cells matured into adipocytes. In contrast, expression of group 2 genes, including Ero1-Lα, Scd3, FGF21, and Elovl3, was dramatically enhanced in the SIRT1 knockdown cells (Fig. ).
FIG. 6. Suppression of SIRT1 by expression of a corresponding SIRT1 siRNA selectively enhances the expression of group 2 PPARγ-responsive genes during the differentiation of 3T3-L1 preadipocytes. Control and SIRT1 siRNA cells were cultured in 10% (more ...) Group 2 genes are selectively induced in mature adipocytes by exposure to PPARγ ligands.
The data presented in Fig. and Table S1B in the supplemental material suggested that several of the group 2 genes are actively repressed in mature adipocytes by mechanisms involving SIRT1. We asked, therefore, whether exposure of such cells to a synthetic PPARγ ligand could overcome the repression and stimulate their expression. To test this notion, normal 3T3-L1 preadipocytes were induced to differentiate by following standard procedures, and at days 2, 4, and 6, the differentiating cells were exposed to troglitazone for 2 days, at which time total RNA was harvested for analysis by RT-PCR. Figure demonstrates extensive induction of selected group 2 genes at different stages of the differentiation process. Specifically, ELOVL3 was induced by exposure of the 3T3-L1 cells to troglitazone as early as day 4 of adipogenesis, and the corresponding mRNA levels remained elevated throughout differentiation. FGF21 and Ero1-Lα gene expression was also enhanced severalfold but occurred only in more mature adipocytes. Expression of the selected members of the group 1 genes (C/EBPα, adiponectin, and FABP4 genes), however, was essentially unresponsive to the exogenous ligand, since the level of expression was already at a maximum due to their induction by endogenous PPARγ ligands. These data are consistent with the hypothesis that a subset of the group 2 genes, including FGF21 and Ero1-Lα genes, are actively repressed by SIRT1 in mature adipocytes and that this repression can be overcome by exposure to troglitazone. The fact that attenuation of SIRT1 (Fig. ) or exposure to the PPARγ ligand (Fig. ) did not induce expression of the classic adipogenic genes in group 1 suggests that they are distinct from the group 2 genes, since they are presumably in a constant state of optimum transcriptional activity. These data suggest that SIRT1 and PPARγ ligands reciprocally regulate PPARγ activity on group 2 genes; we asked, therefore, whether SIRT1 might attenuate the response of PPARγ to its ligands. To test this notion, we determined the dose of troglitazone required to induce expression of select group 2 genes in control versus SIRT1 knockdown 3T3-L1 preadipocytes at 4 days of differentiation. Specifically, differentiating cells were exposed to increasing doses of troglitazone for 2 days, at which stage RNA was analyzed for expression of selected genes by RT-PCR. Figure shows that expression of FGF21 and Egln1 genes (group 2 genes) was induced in control cells following exposure to doses of troglitazone in the range of 1 to 5 μM; in contrast, induction of these genes in SIRT1 knockdown cells required a significantly lower dose of troglitazone (250 nM). These data suggest that SIRT1 attenuates the response of PPARγ to an exogenous ligand. Additionally, Fig. also confirms that there was a negligible effect of either knockdown of SIRT1 or ligands on the expression of the group 1 genes for adiponectin or FABP4.
FIG. 7. (A) Troglitazone selectively activates expression of group 2 genes in 3T3-L1 adipocytes. 3T3-L1 preadipocytes were differentiated using standard conditions, and at day 2, day 4, and day 6, the differentiating cells were exposed to 5 μM troglitazone (more ...)