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 insulin, with or without 5 μM troglitazone (TROG). (A) Day 5 cells were fixed, stained with Oil Red O, and photographed. (B) Total cellular proteins were collected at day 5 and subjected to Western blot analysis with the indicated antibodies. (C) Profile of ∼1,700 mRNAs expressed in Swiss fibroblasts in response to WT or EF-PPARγ. Total RNAs of WT-PPARγ and EF-PPARγ cells at day 5 (with or without troglitazone) were isolated using Trizol Reagent (Invitrogen), and microarray analysis was performed as described in Materials and Methods. The colors correspond to signal intensities.

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% calf serum until they reached confluence. Two days postconfluence, the cells were induced to differentiate by exposure to DEX, MIX, insulin, and 10% FBS. At the indicated days of differentiation, cells were harvested for RT-PCR analysis as described in Materials and Methods. (B) Immortalized brown preadipocytes were grown to confluence in differentiation medium composed of DMEM containing 10% FBS supplemented with 20 nM insulin and 1 nM 3,3′,5-triiodo-l-thyronine. At 2 days postconfluence, the cells were induced to differentiate by exposure to DEX, MIX, insulin, 0.125 mM indomethacin, and 10% FBS. On the indicated days of differentiation, cells were harvested for RT-PCR analysis as described in Materials and Methods.

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 (5 μM) was added in the presence or absence of cycloheximide (5 μg/ml) for the indicated times. Cells were then harvested for extraction of RNA, followed by RT-PCR analysis of FGF21 and FABP4/aP2 mRNAs as described in Materials and Methods. (B) Reporter assays were performed in control Swiss fibroblasts following transfection of individual FGF21 luciferase (Luc) plasmids (bp −1500, −1300, and −500 fragments), along with a PPARγ (pBabe-PPARγ) or control (pBabe-Puro) expression plasmid and a Renilla (Ren)-based pGL3 reporter as a control in the presence or absence of the potent PPARγ ligand GW1929. The scheme at the top shows the presence of putative PPREs in the upstream region of the gene that have the following DR-1 sequences: 1, AGACCAAGGAGCA; 2, AGACCCAAGGCCC; 3, TGGCCTGTGGCCA; 4, TGAGCACAAGGCCC; and 5, AGTTCCAGGGCCA. (C) Reporter assays were also performed in Swiss fibroblasts stably expressing a WT PPARγ or a pBabe-puro empty vector (control cells) following transfection of the FGF21 promoter reporter plasmids, along with the Renilla control vector, in the presence or absence of GW1929. In both assays B and C, a set of cells were also transfected with a reporter plasmid consisting of the PPRE from the aP2 gene upstream of luciferase within pGL3 (DR-1). The transcriptional activity of each of the fragments of the FGF21 gene promoter is shown as the ratio of luciferase activity to Renilla activity (Luc/Ren) as described in Materials and Methods.

(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 for 2 days. Untreated and treated cells were harvested for analysis of select mRNAs using RT-PCR as described in Materials and Methods. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) Knockdown of SIRT1 in 3T3-L1 preadipocytes enhances the expression of FGF21 in response to exposure to troglitazone. Control and SIRT1 knockdown 3T3-L1 preadipocytes were differentiated for 4 days, at which time the cells were exposed to the indicated doses of troglitazone for 2 days. Total RNA of the cells was isolated at day 6 and subjected to RT-PCR for analysis of the indicated group 1 and group 2 genes as described in Materials and Methods. (C) Model proposing interplay between PPARγ and SIRT1 in controlling adipocyte function. PPARγ functions to regulate adipocyte formation and function. Endogenous ligands activate PPARγ, requiring participation of both helices 7 and 12 to orchestrate adipogenesis. In mature adipocytes, SIRT1 mediates hormonal and nutrient control of select PPARγ target genes that are involved in controlling metabolism by suppressing the actions of endogenous ligands. The TZD family of synthetic PPARγ ligands can overcome the suppressive effects of SIRT1 acting through helix 7, as well as helix 12, to induce the metabolic genes.

(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. Swiss fibroblasts (lane C) and Swiss-PPARγ (lanes WT, EF, and F) cells were cultured until they were confluent; after 2 days, they were exposed to DEX, MIX, and insulin, with or without 5 μM troglitazone, for 5 days. Total RNAs of the cells were isolated using Trizol reagent (Invitrogen) and subjected to RT-PCR analysis as described in Materials and Methods. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) Troglitazone selectively enhances expression of the group 2 PPARγ-responsive genes during the differentiation of Swiss fibroblasts expressing WT PPARγ. Swiss WT PPARγ cells were cultured in 10% FBS until they reached confluence. Two days postconfluence, the cells were induced to differentiate by exposure to DEX, MIX, insulin, and 10% FBS with or without troglitazone. At days 0, 1, 2, 3, 4, 5, 6, and 7 of differentiation, cells were harvested for RT-PCR analysis as described in Materials and Methods.

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 insulin, with or without the following PPARγ ligands: FMOC-leu (15 μM), 15δ-PGD2 (7 μM), troglitazone (5 μM), rosiglitazone (10 μM), and GW1929 (10 μM). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) WT PPARγ cells were differentiated as in panel A by exposure to DEX, MIX, and insulin in the presence or absence of troglitazone with or without either T0070907 (10 μM) or GW9662 (10 μM) (PPARγ antagonists). (C) WT PPARγ and EF-PPARγ cells were induced to differentiate with DEX, MIX, insulin, and the indicated doses of troglitazone (Trog). In panels A, B, and C, total RNA of the cells was isolated at day 5 using Trizol reagent (Invitrogen) and subjected to RT-PCR analysis of the indicated group 1 and group 2 genes as described in Materials and Methods.

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% calf serum until they reached confluence. At 2 days postconfluence, the cells were induced to differentiate by exposure to DEX, MIX, insulin, and 10% FBS. On the indicated days of differentiation, cells were harvested for Western blot analysis (A) and RT-PCR analysis (B) of the indicated gene products as described in Materials and Methods. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.