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. 2021 Jun;42(6):964-974.
doi: 10.1038/s41401-020-00524-0. Epub 2020 Sep 15.

Pyrazolone derivative C29 protects against HFD-induced obesity in mice via activation of AMPK in adipose tissue

Bo-Han Li  1   2 , Mei Zhang  1 , Ya-Nan Duan  1 , Lin Shuai  1 , Hao-Wen Jiang  1 , Jia Li  1   2 , Fa-Jun Nan  3 , Jing-Ya Li  4
Affiliations

Pyrazolone derivative C29 protects against HFD-induced obesity in mice via activation of AMPK in adipose tissue

Bo-Han Li et al. Acta Pharmacol Sin. 2021 Jun.

Abstract

Beige adipocytes have been considered as a potential strategy in anti-obesity therapy because of its thermogenic capacity. AMP-activated protein kinase (AMPK) plays important roles in regulating adipose tissue function. C29 is a novel pyrazolone derivative with AMPK activity. In the current study, we investigated the role of C29 in the regulation of thermogenesis using differentiated adipocytes and diet-induced obese mice, and explored the mechanisms that might be involved in energy expenditure via adipocyte AMPK activation. We showed that treatment with C29 (2.5-10 μM) concentration-dependently increased thermogenesis in differentiated preadipocytes separated from inguinal white adipose tissue (iWAT), evidenced by increased expression levels of thermogenesis markers such as Ucp1, Pgc-1α, Dio2, Prdm16, Cox7a1, Cox8b, Elovl3, and Cidea, fatty acid oxidation (FAO) genes including Cpt1a, Lcad and Pparα, as well as beige-selective genes such as Cd137, Tmem26, Slc27a1, and Tbx1. In high-fat diet (HFD)-fed mice, oral administration of C29 (30 mg·kg-1·day-1) for 9 weeks alleviated HFD-induced obesity, promoted energy expenditure and modulated iWAT browning. However, these effects were not observed in adipose-specific AMPKα1/α2 knockout (AKO) mice following C29 administration. Together, this study demonstrates that C29 regulates energy balance via adipocyte AMPK. Our findings show that the discovery of AMPK activators that specifically target adipose tissue may have therapeutic potential for treating obesity-related metabolic diseases.

Keywords: AMP-activated protein kinase; energy expenditure; inguinal white adipose tissue; obesity; pyrazolone derivative C29; thermogenesis; white adipose browning.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. C29 increased thermogenesis in differentiated iWAT-SVF cells.
iWAT-SVF cells were induced to differentiate towards brown-like adipocytes and then treated with C29 at the indicated concentration for 24 h on day 7. Relative mRNA level of the indicated genes (a); Western blot analysis of the indicated proteins (b, c); β-actin was used as the loading control; basal and uncoupled cellular oxygen consumption rate (OCR) (d, e). n = 3. Data are the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, C29 group versus vehicle group by one-way ANOVA.
Fig. 2
Fig. 2. Chronic C29 treatment reduced high-fat diet-induced obesity.
Body weight (a) and average food intake per day (b) in high-fat diet-fed mice during 9 weeks of treatment. Fat and lean mass (c) and relative weight of the liver and different adipose tissue depots (d) after treatment. n = 6. Data are the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, C29 group versus vehicle group by Student’s t test.
Fig. 3
Fig. 3. C29 promoted energy expenditure and adaptive thermogenesis.
The change in O2 consumption (VO2) (a), change in energy expenditure (EE) (c), average O2 consumption (VO2) (b) and energy expenditure (EE) (d) under basal and CL316,243 stimulation conditions after 7 weeks of treatment. Body temperature change (e), representative thermal images of mice (f) and interscapular BAT skin temperature (g) during cold exposure at 4 °C for 6 h after 8 weeks of treatment. n = 6. Data are the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, C29 group versus vehicle group by Student’s t test.
Fig. 4
Fig. 4. C29 induced the browning of iWAT in HFD-induced mice.
Representative H&E-stained images of iWAT (top), eWAT (middle) and BAT (bottom) after treatment, scale bar, 100 μm (a). The average adipocyte area of iWAT (b) and eWAT (c) and average lipid droplet area of BAT (d) after treatment, n = 6. Relative mRNA levels of the indicated genes (e) and Western blot analysis of the indicated proteins (f, g) in iWAT; β-actin was used as the loading control, n = 4–6. Relative mRNA levels of the indicated genes in BAT (h) and eWAT (i) after treatment. n = 6. Data are the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, C29 group versus vehicle group by Student’s t test.
Fig. 5
Fig. 5. C29 protected against HFD-induced obesity in an AMPK-dependent manner.
Body weight (a) and average food intake per day (b) in HFD-fed AKO mice and age-matched floxed littermates during 14 weeks of treatment. Fat and lean mass (c) and relative weight of the liver and different adipose tissue depots (d) after treatment. n = 6. Data are the means ± SEM. *P < 0.05, **P < 0.01, C29 group versus vehicle group; #P < 0.05, ##P < 0.01, ###P < 0.001 AKO group versus floxed group by Student’s t test.
Fig. 6
Fig. 6. C29 improved energy expenditure and cold tolerance through the activation of adipocyte AMPK.
The change in O2 consumption (VO2) (a), change in energy expenditure (EE) (c), average O2 consumption (VO2) (b) and energy expenditure (EE) (d) under basal and CL316,243 stimulation conditions in HFD-fed AKO mice and age-matched floxed littermates after 10 weeks of treatment. Body temperature change (e), representative thermal images of mice (f) and interscapular BAT skin temperature (g) during cold exposure at 4 °C for 6 h after 12 weeks of treatment, n = 5. Data are the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, C29 group versus vehicle group; #P < 0.05, ##P < 0.01, ###P < 0.001, AKO group versus floxed group by Student’s t test.
Fig. 7
Fig. 7. AMPK activation was essential for C29 in the browning of iWAT in HFD-induced mice.
Representative H&E-stained images of iWAT after treatment; scale bar, 100 μm (a). Average adipocyte area of iWAT in HFD-fed AKO mice and age-matched floxed littermates after treatment (b), n = 6. Relative mRNA levels of the indicated genes (c) and Western blot analysis of the indicated proteins (d, e) in iWAT; β-actin was used as the loading control, n = 3–5. Data are the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, C29 group versus vehicle group; #P < 0.05, ##P < 0.01, ###P < 0.001, AKO group versus floxed group by Student’s t test.
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