Beneficial effects of metformin on energy metabolism and visceral fat volume through a possible mechanism of fatty acid oxidation in human subjects and rats - Fig 6

<p><b>The protein expressions of pAMPK and pACC in the liver of SD rats at 15 weeks old.inhibo</b> A; Western blot analysis, B; Densitometric quantification. Open bars denote control group and black bars denote metformin-treated group. Data are presented as mean ± S.E. (n = 3 in each group). *P<0.05 vs. control groups.</p

Pyruvate tolerance test in SD rats at 15 weeks old.

<p>Data are presented as mean ± S.E. (n = 6 in each group). Paired t-test was used for the comparison between groups. Open circle and dotted line; control group, closed circle and solid line; metformin-treated group. *P<0.05 vs. the value of control group at each time point.</p

Fat oxidation-related gene expressions in the liver of SD rats at 15 weeks old.

<p>A; <i>Cpt1</i>, B; <i>Acs</i>, C; <i>Acad</i>, D; <i>Pdk</i> expression corrected by <i>Gapdh</i> expression. Open bars denote control group and black bars denote metformin-treated group. Data are presented as mean ± S.E. (n = 6 in each group). *P<0.05, **P<0.01 vs. control groups.</p

Anthropometric factors, food intake and water consumption in control and metformin-treated rats.

<p>Anthropometric factors, food intake and water consumption in control and metformin-treated rats.</p

Effects of 3-hydroxybutyrate (3-HB) on the endoplasmic reticulum (ER) stress markers.

<p>Expression of ER stress marker genes, <i>Eif2ak3/Perk</i> (A), <i>Ddit3</i> (B), <i>Ppp1r15a</i> (C), <i>Atf6</i> (D), <i>Hsppa5</i> (E), <i>Ire1</i> (F), <i>XBP1u</i> (G) and <i>XBP1s</i> (H) were assessed in HUVECs treated with 5.6 or 0.56 mmol/L glucose in the presence of various concentrations of 3-HB for 6 h. Means and SD, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 (n = 4).</p

Effect of glucose deprivation on endothelial nitric oxide synthase (eNOS).

<p>HUVECs were treated with 5.6 or 0.56 mmol/L glucose in the presence of various concentrations of 3-hydroxybutyrate (3-HB) for 24 h. mRNA (A) and protein levels (B) of eNOS were assessed by real-time PCR and Western blotting, respectively. A representative image of Western blotting is shown (C). Means and SD, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Western blot analysis of ER stress markers.

<p>Protein levels of PERK (A), cleaved ATF6 (cATF6) (B), CHOP (C) and Bip/GRP78 (D) were assessed in HUVECs treated with 5.6 or 0.56 mmol/L glucose in the presence of various concentrations of 3-hydroxybutyrate (3-HB) for 24 h. β-actin was used as a loading control. Representative images of Western blotting are shown. Means and SD, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 (n = 3).</p

Caspase activation by low glucose and suppressive effect of 3-hydroxybutyrate (3-HB).

<p>Caspase-3/7 (A) and caspase-8 (B) activities were measured in HUVECs cultured in low-glucose medium with or without supplementation of 3-HB for 48 h. Results are exppressed as fold increase from control. Means and SD, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 (n = 4). Cell nuclei were visualized by Hoechst 33342 staining (blue) (C-G), and caspase-3/7-positive cells were stained green with a fluorogenic substrate (H-L). Bar, 100μm.</p

Effects of low-glucose exposure on cell viability and protective effects of 3-hydroxybutyrate (3-HB).

<p>Human umbilical vein endothelial cells (HUVECs) were treated with 5.6 or 0.56 mmol/L glucose in the presence of various concentrations of 3-HB (0, 1, 4, 10 mmol/L) for 24 h (A) or 48 h (B). Cell viability was assessed by a CCK-8 assay (A) (B) and an LDH (C) assay. Microscopic images of HUVECs treated for 24 h (D-F). Means and SD, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 (n = 4).</p

Primer sequences.

<p>Primer sequences.</p