Regulation of Peroxisome Proliferator-Activated Receptor γ Expression by Adipocyte Differentiation and Determination Factor 1/Sterol Regulatory Element Binding Protein 1: Implications for Adipocyte Differentiation and Metabolism

Cholesterol depletion induces PPARγ expression. (A) Western blot analysis of nuclear extracts of 3T3-L1 preadipocytes with an anti-PPARγ antibody. Preconfluent cells (lane 1) were incubated for 24 h (lane 2) in cholesterol-depleted medium. After 24 h of incubation in cholesterol-depleted medium, a mixture containing 10 μM cholesterol and 1 μM 25-OH-cholesterol was added to the medium for 6 additional h (24 + 6 chol) (lane 3). The fold induction of PPARγ or SREBP as determined by densitometric quantification of the results is shown in parentheses underneath the number of the lane. (B) Similar Western blot experiments as described for panel A, but with HepG2 nuclear extracts instead of 3T3-L1 nuclear extracts. (C) Expression of SREBP-1 protein as detected after Western blot analysis of the 3T3-L1 nuclear extracts used in panel A. Western blotting was performed with an anti-SREBP-1 antiserum. (D) Western blot analysis of nuclear extracts of CCL-39 cells transfected with the constitutively active form of ADD-1, ADD-1 403. Cells were exposed to the same cholesterol depletion as specified for panel A.

Inhibition of de novo cholesterol synthesis by statins induces PPARγ expression. (A) Analysis of the PPARγ protein by Western blotting of cell extracts from HepG2 cells incubated with medium supplemented with simvastatin (0.5 μM) for either 6 or 12 h. Fold induction of PPARγ protein levels is shown in parentheses. (B) Quantification of PPARγ protein levels in nuclear extracts from 3T3-L1 preadipocytes. Cells were lipid starved as in Fig. 2, and a mixture of linoleic acid (150 μM) and linolenic acid (150 μM) was added to the medium for a period of 12 h. An anti-PPARγ specific antibody was used for Western blot analysis. Fold induction of PPARγ protein levels is shown in parentheses.

ADD-1/SREBP-1 and SREBP-2 transactivate the PPARγ1 and -3 promoters. (A) ADD-1/SREBP-1 and SREBP-2 transactivate the PPARγ1 and -3 promoters, but not the PPARγ2 promoter. Relative luciferase activity as determined after transfection of CCL-39 cells with the reporter constructs pGL3γ1p2000, pGL3γ2p1000, and pGL3γ3p800. Cells were either cotransfected with an empty expression vector (control) and exposed to medium containing cholesterol (10 μM) and 25-hydroxycholesterol (1 μM), cotransfected with an empty expression vector and maintained in cholesterol-depleted medium (Chol depletion), or cotransfected with an expression plasmid for SREBP-1 (SREBP-1) or SREBP-2 (SREBP-2) in medium containing cholesterol (10 μm) and 25-hydroxycholesterol (1 μM). Results are expressed as fold induction and represent the mean ± standard deviation of three independent experiments. Statistically significant differences by Student’st test (P < 0.05) are indicated. (B) A scheme of the genomic structure of the 5′ end of the human PPARγ gene and of the approximate location of the response elements. Exons 1 to 6 are shared by all three subtypes. PPARγ1 contains in addition the untranslated exons A1 and A2; PPARγ2 contains exon B, which is translated; and PPARγ3 contains only the untranslated exon A2. The respective hPPARγ promoters are indicated by arrows. The approximate locations of the E-boxes are indicated.

Binding of ADD-1/SREBP-1 to the PPARγ1 and -3 promoters. (A) EMSA with a partially purified baculovirus-produced SREBP-1a protein, a different splice variant of ADD-1, and a labelled double-stranded oligonucleotide representing the PPARγ1-E-box. Competition experiments were performed with cold oligonucleotides representing either the PPARγ1-E-box (γ1-E-box; LF-141), the consensus HMG-CoA synthase SRE site (SREcons) (42), or the mutated PPARγ1-E-box (γ1-E-box_mut; LF-143) at either 10-, 50-, or 100-fold molar excess. (B) Sequence of the wild-type and mutated PPARγ1-E-box. The consensus E-box is indicated in bold characters. The three bases which are mutated are indicated underneath the original bases. (C) EMSA performed under exactly the same conditions as described for panel A with the PPARγ3-E-box (LF-102) as a labelled double-stranded oligonucleotide and the mutated PPARγ3-E-box (γ3 E-box_mut; LF-106) as a competitor instead of the PPARγ1-E-box and γ1-E-box_mut, respectively. (D) Sequence of the wild-type and mutated PPARγ3 E-boxes. The consensus E-box is underlined, whereas a potential SRE is indicated in boldface. The three bases mutated are indicated underneath the original bases.

The PPARγ1-E-box and the PPARγ3-E-box mediate the induction of the PPARγ gene by ADD-1/SREBP-1 and SREBP-2. Relative luciferase activity as determined after transfection of CCL39 cells with the reporter constructs pGL3γ1p2000, pGL3γ1p2000-E-box_mut, pGL3γ3p800, and pGL3γ3p800-E-box_mut. Cells were cotransfected with either an empty expression vector (control) or an expression plasmid for SREBP-1 or SREBP-2. Values are the mean ± standard deviation of three independent experiments. Statistically significant differences (P < 0.05) by Student’s t test are indicated by asterisks.

Transactivation of PPARγ is enhanced under conditions of cholesterol depletion. (A and B) Promoter activity of the PPRE-driven luciferase reporter vector J3-TK-Luc after addition of different doses of BRL 49,653 in cells maintained in medium with (dashed squares) or without added cholesterol (10 μM) and 25-hydroxycholesterol (1 μM) (open squares). The results in RK-13 cells (A) and in 3T3-L1 preadipocytes (B) are shown. The results represent the mean ± standard deviation of three independent experiments. Differences between the two conditions were statistically significant. RLU, relative light units. (C) Activity of the J3-TK-Luc reporter gene is stimulated by linolenic acid (C_18:3 [400 μM]) in RK-13 cells. Cells were transfected with J3-TK-Luc reporter constructs and maintained for an additional 16 h in medium with (open bars; upper part of the graph) or without added cholesterol (10 μM) and 25-hydroxycholesterol (1 μM) (hatched bars; lower part of the panel). Cells grown under these basal conditions (control cells [C]) were compared with cells treated with BRL 49653 (1 μM [BRL]) or linolenic acid (400 μM [FA]). The results represent the mean ± standard deviation of three independent experiments. The asterisks are indicative of significant differences between the stimulated cells and controls by Student’st test (P < 0.05). (D) Activity of both the J3-TK-Luc and ACO-TK-Luc reporter genes is stimulated by cholesterol depletion in undifferentiated 3T3-L1 cells. Cells were transfected with J3-TK-Luc or ACO-TK-Luc reporter constructs and incubated under the same conditions than in panel C. The results represent the mean ± standard deviation of three independent experiments. The asterisks are indicative of significant differences by Student’s t test (P < 0.05).