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. 2023 Jun 29;145(27):14802–14810. doi: 10.1021/jacs.3c03417

Figure 3.

Figure 3

Mechanistic and biological effects of the alternative site PPARγ ligand 2. (a) 2 caused distinguished effects on co-regulator recruitment by PPARγ with activating and inactivating contributions. Pioglitazone (3 μM), 2 (30 μM), GW9662 (10 μM). Data are the mean ± SD ΔHTRF vs DMSO; n = 4. (b) Pioglitazone (10 μM) and the RXR agonist SR11237 (10 μM) increased and 2 (50 μM) diminished heterodimerization of PPARγ with RXR. 2 also blocked the dimer-stabilizing effects of pioglitazone and SR11237. Data are the mean ± SD HTRF vs DMSO; n = 3. (c) In contrast to pioglitazone, 2 induced no differentiation of human adipocyte-derived stem cells (ASC). Data are the mean ± SD relative Oil Red O (ORO) deposition compared to pioglitazone; n = 3. (d) Representative images of ASC differentiation experiments stained with ORO. (e) Differential gene expression in HepG2 cells treated with 2 (20 μM) versus DMSO (0.1%). Volcano plot shows log2(fold change) in the gene expression level (x-axis) versus the statistical significance level (−log10(p-value); y-axis). (f) Compared effects of 2 and GA on gene expression. Heat map shows induced (blue), downregulated (red), and nonregulated (black) genes. (g–i) Selected genes regulated by treatment with 2 (20 μM) associated with FOXO signaling (g), adipo–/lipogenesis (h), TOR signaling (i), cell cycle (i), apoptosis (i), and ATP generation (i). Heat maps show log2(fold change) in the gene expression of significantly (p-value <0.05) regulated genes. (j) Treatment with 2 (20 μM, 16 h) enhanced the inactivating phosphorylation of FOXO3a at Ser253. Data are the mean ± S.E.M.; n = 6. Western blots in the Supporting Information. (k) Pioglitazone enhanced, 2 decreased FOXO activity in HepG2 cells over time. Data are the mean ± S.E.M. FHRE activity; n ≥ 3.