Figure 1.
Effects of T0901317 on insulin function.
A–F, Body weight (A, n = 14–16 mice per group), glucose levels and glucose level area under the curve (AUC) during an intraperitoneal glucose test (IPGTT, 1.5 g glucose per kg body weight) (B), insulin levels and insulin level area under the curve (AUC) during IPGTT (C, n = 7–8 mice per group), glucose levels and glucose level area under the curve (AUC) during an insulin tolerance test (ITT, 0.5 IU insulin per kg body weight, n = 7–8 mice per group) (D), free fatty acid levels (E) and triglyceride levels in serum after an overnight fast (n = 7–8 mice per group). All values are presented as mean ± SEM, *P<0.05 and **P<0.01 vs. control mice treated with DMSO.
Figure 2.
Effect of T0901317 on fat mass and adipokines.
A–E, the percent of epididymal fat mass (A, % as body weight) and inguinal fat mass (B, % as body weight, n = 14–16 mice per group), H&E staining of EP fat (C, ×200 magnification), adiponectin levels in serum (D) and leptin levels in serum (E, n = 7–8 mice per group). All values are presented as mean ± SEM, *P<0.05 and **P<0.01 vs. control mice treated with DMSO.
Figure 3.
Effect of T0901317 on adiponectin signaling in EP fat.
A–D, quantitative real-time RT-PCR analyses of adiponectin mRNA (A) and its receptors 1 (AdipoR1) and 2 (AdipoR2) mRNAs (B) in EP fat; Western blot analyses of phosphorylation of AMPK, phosphorylation of ACC, IRS-1 and PPARα protein levels using β-actin as a loading control (C); quantitative real-time RT-PCR analysis of Glut-4 mRNA in EP fat (D). All values are presented as means ± SEM, n = 4–6 mice per group. *P<0.05, **P<0.01 vs. control mice treated with DMSO. E–F, both basal and insulin-stimulated Glut-4 membrane translocations detected by Western blotting using β-actin as a loading control (E); and immunofluorescence detection of membrane Glut-4 (F, ×1000 magnification). Values are presented as means ± SEM, n = 4 mice per group injected i.p. with insulin or saline, *P<0.05 as indicated.
Figure 4.
Effects of T0901317 adiponectin activity in liver.
A–B, quantitative real-time RT-PCR analyses of adiponectin receptors 1 (AdipoR1) and 2 (AdipoR2) mRNAs in liver (A); Western blotting analyses of phosphorylation of AMPK, phosphorylation of ACC, IRS-1 and PPARα protein levels using β-actin as a loading control in liver (B). All values are presented as means ± SEM, n = 4–6 mice per group. *P<0.05, **P<0.01 vs. control mice treated with DMSO.
Figure 5.
Effect of LXR activation on adiponectin expression in vitro.
A–E, adiponectin mRNA by quantitative real-time RT-PCR (A-1) and adiponectin protein levels by Western blotting using β-actin as a loading control (A-2) in differentiated adipocytes treated with different doses of T0901317 for 24 h; analysis of adiponectin mRNA by quantitative real-time RT-PCR (B-1) and adiponectin protein levels by Western blotting using β-actin as a loading control (B-2) in differentiated adipocytes treated with different doses of GW3965 for 24 h; quantitative real-time RT-PCR analysis of LXRα mRNA (C-1) and Western blotting analysis of LXRα protein levels using LaminB1 as a loading control (C-2) in LXRα silenced 3T3-L1 adipocytes at 0, 5 and 10 days during the differentiation periods; quantitative real-time RT-PCR analysis of adiponectin mRNA (D-1) and Western blotting analysis of adiponectin protein using β-actin as a loading control (D-2) in differentiated adipocytes with LXRα silencing and treated with 10 µM T0901317; ChIP-qPCR analysis of the binding of PPARγ to adiponectin PPRE in differentiated adipocytes treated with different doses of T0901317 (E, data were normalized to input samples for the amount of chromatin). The results are presented as the mean ± SD of three replicates in three separate experiments.*P<0.05, **P<0.01, NS non-significant as indicated.
Figure 6.
Effects of T0901317 on PPARγ transcriptional activity.
A–C, quantitative real-time RT-PCR analyses of PPARγ and adiponectin expressions in differentiated adipocytes treated with 3 µM Pioglitazone in the presence or absence 10 µM T0901317 (A, white bar-DMSO treatment, grey bar-3 µM Pioglitazone treatment, black bar- 3 µM Pioglitazone and 10 µM T0901317 treatment); double promoter luciferase reporter assay of PPRE-Luc activity in HEK293 cells co-transfected with LXRα or PPARγ or both and treated with different drugs (B, cells subjected to the same basic co-transfection () with 0.4 µg PPRE-Luc +0.02 µg phRL CMV in each group); double promoter luciferase reporter assay of PPRE-Luc activity in HEK293 cells subjected to multiple plasmid co-transfections with (
+0.2 µg pcDNA PPARγ+0.2 µg pcDNA LXRα) and treated with different doses of T0901317 in the presence or absence of 3 µM Pioglitazone (C). The results are presented as the mean ± SD of three replicates in three separate experiments.*P<0.05 as indicated.