Table 1.
Sequences of primers used in real-time PCR.
Figure 1.
SR4 decreases body weight and fat mass in HFD obese mice.
Representative mice in each treatment group depicting gross images of whole body shape (top) and abdominal fat (bottom) (panel A). Body weights (panel B), epididymal fat weight (panel C), and daily food intake per animal in each group (n = 8-12 animals per group) (panel D). Data represent mean ± SEM. *p<0.05 vs. LFD **p<0.01 vs. LFD; # p<0.05 vs. HFD vehicle; ## p<0.01 vs. HFD vehicle.
Figure 2.
SR4 improves glycemic control and dyslipidemia in HFD obese mice.
Plasma levels of non-fasting glucose (panel A) and insulin (panel B) were measured at the end of the 6-week treatment period. At week 5, mice were fasted overnight and GTT was performed (panel C) and glucose AUC integrated from 0–120 min for each mouse was calculated using GraphPad Prism software (n = 10 animals per group) (panel D). Plasma levels of cholesterol and triglycerides were also measured at the end of the experimental treatment (panel E). Data represent mean ± SEM. *p<0.05 vs. LFD; **p<0.01 vs. LFD; # p<0.05 vs. HFD vehicle; ## p<0.01 vs. HFD vehicle.
Figure 3.
SR4 decreases adipose tissue hypertrophy and affects circulating adipokine levels in HFD obese mice.
Representative photomicrograph images of sections of WAT (epididymal fat pad) stained with H&E from each treatment group (original magnification, 200×; scale bar, 100 um) (panel A). Cell size distribution of adipose cells from inguinal fat: The mean surface area and the frequency distribution were calculated based on at least 200 random cells from each mice (n = 8 animals per group) (panel B). Levels of plasma leptin and adiponectin at the end of experimental treatment (n = 8 animals per group) (panel C). Each bar represents mean ± SEM. *p<0.05 vs. LFD; **p<0.01 vs. LFD; # p<0.05 vs. HFD vehicle; ## p<0.01 vs. HFD vehicle.
Figure 4.
SR4 prevents hepatic lipid accumulation and fatty liver in HFD obese mice.
Liver weights (panel A), total liver triglyceride contents (panel B), and plasma concentrations of liver enzymes ALT and AST (panel C) at the end of the treatment period (n = 8 mice per group). Representative photomicrographs of H&E staining of liver sections from each treatment group, original magnification, 100×; scale bar, 100 um (upper panel D). Representative photomicrographs of Oil Red O staining of liver sections from mice in each treatment group (lower panel D) and Oil red O staining quantification was done with Image Pro Plus 6.3 software using 3-4 random microscope field sections per animal sample and presented as fold over control (n = 8 animals per group) (panel E). Results represent mean ± SEM. *p<0.05 vs. LFD; **p<0.01 vs. LFD; # p<0.05 vs. HFD vehicle; ## p<0.01 vs. HFD vehicle.
Figure 5.
SR4 regulates expression of genes involved in lipid and glucose metabolism.
Total RNA was isolated from liver of LFD lean or HFD obese mice treated with vehicle or SR4. Relative mRNA levels of liver lipogenic and gluconeogenic genes were determined using real time RT-PCR and quantified using the comparative Ct method with β-actin as internal control. Two independent RT-PCR experiments were performed (n = 4 animals per group). Each bar represents mean ± SEM, *p<0.05 vs. LFD; **p<0.01 vs. LFD; # p<0.05 vs. HFD vehicle; ## p<0.01 vs. HFD vehicle.
Figure 6.
SR4 activates AMPK in liver and WAT.
Representative Western blots analyses of protein lysates from liver and epididymal WAT. Total protein lysates (n = 3 animals in each group) were subjected to SDS-PAGE and immuno-blotted with antibodies specific for phosphorylated and total AMPK, phosphorylated and total ACC or β-actin. The pAMPK/AMPK and pACC/ACC ratios were quantified using a densitometer. At least two independent experiments were performed. Each bar represents mean ± SEM, *p<0.05 vs. LFD; # p<0.05 vs. HFD vehicle; ## p<0.01 vs. HFD vehicle.
Figure 7.
SR4 activates AMPK independent of upstream kinases LKB1 and CaMKKβ.
Representative Western blots analyses showing SR4 dose-dependently activates AMPK in human liver hepatocellular carcinoma (HepG2) (panel A) and human lung adenocarcinoma (A549) cells (panel B). Cells were treated with SR4 (0, 1, 3 5, 10 µM) or the AMPK activator AICAR (2 mM) in the presence or absence of the AMPK inhibitor Compound C or CaMKKβ inhibitor STO-609 (added to the cells 30 min prior to the addition of SR4 or AICAR). After 4 h, cells were collected, lysed, and total cell lysates subjected to Western blot analyses. Densitometric quantitation was performed on each blot and the fold changes were compared with vehicle (DMSO) control. At least three independent experiments were performed, *p<0.05 vs. vehicle control; **p<0.01 vs. vehicle control. Western blots showing the lack of LKB1 protein in A549 cells (panel C). Intracellular AMP: ATP ratios in HepG2 cells following 4 h treatment with various concentrations of SR4 (panel D). Results are shown as mean ± SEM of three independent experiments with three replicates each *p<0.05 vs. DMSO vehicle; **p<0.01 vs. DMSO vehicle.