Fig 2: Validation of the inducible system. (A) C6-rtTA-AMPK-DN cells were exposed to increasing concentrations of DOX for 24, 48, and 72 h and the expression of the myc-tagged α1DN was evaluated by immunoblotting. (B,C) To assess for the stability of the α1DN, cells were either exposed to 10 µg/mL of cycloheximide (B), or the culture medium was renewed to remove DOX after 24 h (C). Cells were collected every 2 h, and the expression of the myc-tagged α1DN was evaluated by immunoblotting. Histograms show means ± SEM normalized to GAPDH expression. Blots shown are representative of three independent experiments. Blots in panel C were cropped to remove nonessential experimental conditions. Statistical analyses were performed by one-way ANOVA followed by Dunnett’s multiple comparison test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Fig 3: Thermogenic effects induced by Rut are attenuated by absence of AMPK. (A) Body weight of the indicated mice group. (B) Rectal temperature changes of four groups mice were recorded at 4 °C. (C) Representative infrared thermal images of floxed or AKO mice treated with vehicle or Rut after 6 h of exposure to 4 °C. (D) Statistical graphs of interscapular BAT skin heat. (E) Glucose levels were plotted versus times post insulin injection. (F) Analysis of AUC during ITT. (G) Testing of O2 consumption. (H) Average of O2 consumption under basal and CL316,243 stimulation conditions. (I) Assessment of EE. (J) Average of EE under basal and CL316,243 stimulation conditions. (K,L) Expression level of thermogenic genes by RT-qPCR analysis in iBAT (K) and iWAT (L). (M,N) Western blot analysis of AMPK, PGC-1a and UCP1 expression level in iBAT (M) and iWAT (N) of different groups mice. n = 5–6 per group. Data are presented as the means ±SEM. Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, Rut group versus vehicle group; # p < 0.05, ## p < 0.01, ### p < 0.001, AKO group versus floxed group.

Fig 4: Effects of a single dose of metformin on blood glucose (A) and hepatic AMPK activation (B) in C57BL/6J mice. Blood glucose measurement and liver sampling were conducted at 2 h after administration of metformin. Data are presented as the mean + SD of 4 mice. *** p < 0.001 vs. vehicle group.

Fig 5: CTRP9 inhibits the VSMC switch to macrophage-like cells, activates the AMPK pathway and suppresses cholesterol-induced NF-kB pathway activation VSMCs were left untreated (control) or were treated with 5 µg/ml cholesterol in the absence or presence of 10 µg/ml CTRP9 for 72 hrs. (A) Representative fluorescence images showing ACTA2 expression levels in VSMCs under the different treatment conditions. (Red: ACTA2 Blue: DAPI) (200×) (B) Representative flow cytometry analysis of CD68-positive cells. (C) Representative Western blot to examine ACTA2, TLR4, MYD88, phospho-p65 (p-p65), total p65 (t-p65), phospho-AMPK (p-AMPK) and total AMPK (AMPK) in VSMCs from each treatment. (C–H) Quantification of Western blot images. Values represent the means ± S.D. of triplicate reactions and are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.