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. 2021 Jul 25;17(12):3173-3187.
doi: 10.7150/ijbs.62556. eCollection 2021.

Berberine modulates deacetylation of PPARγ to promote adipose tissue remodeling and thermogenesis via AMPK/SIRT1 pathway

Yingxi Xu  1   2 , Tianhao Yu  3 , Guojing Ma  2 , Lixia Zheng  2 , Xuehan Jiang  2 , Fan Yang  2 , Zhuo Wang  2 , Na Li  2 , Zheng He  4 , Xiaoyu Song  2 , Deliang Wen  5 , Juan Kong  1 , Yang Yu  2   5 , Liu Cao  2
Affiliations

Berberine modulates deacetylation of PPARγ to promote adipose tissue remodeling and thermogenesis via AMPK/SIRT1 pathway

Yingxi Xu et al. Int J Biol Sci. .

Abstract

Pharmacological stimulation of adipose tissue remodeling and thermogenesis to increase energy expenditure is expected to be a viable therapeutic strategy for obesity. Berberine has been reported to have pharmacological activity in adipose tissue to anti-obesity, while the mechanism remains unclear. Here, we observed that berberine significantly reduced the body weight and insulin resistance of high-fat diet mice by promoting the distribution of brown adipose tissue and thermogenesis. We have further demonstrated that berberine activated energy metabolic sensing pathway AMPK/SIRT1 axis to increase the level of PPARγ deacetylation, which leads to promoting adipose tissue remodeling and increasing the expression of the thermogenic protein UCP-1. These findings suggest that berberine that enhances the AMPK/SIRT1 pathway can act as a selective PPARγ activator to promote adipose tissue remodeling and thermogenesis. This study proposes a new mechanism for the regulation of berberine in adipose tissue and offers a great prospect for berberine in obesity treatment.

Keywords: AMPK/SIRT1 axis; Adipose tissue; Berberine; Deacetylation; PPARγ.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
BBR significantly suppresses high-fat induced weight gain by increasing energy metabolism. Male C57BL/6J mice aged 6 weeks were randomly divided into four groups as NCD, HF, BBR-25, BBR-100 group. Mice in HF, BBR-25, and BBR-100 were fed by high-fat diet from 6 weeks. After 8 weeks, the HF, BBR-25, and BBR-100 group were respectively treated with 0.9% sterile saline, BBR (25 mgkg-1), and BBR (100 mgkg-1) by gavage until 20 weeks of age. (A) Body weight. (B) Food intake after BBR treatment. (C) Blood glucose levels during the oral glucose tolerance test (OGTT) after BBR treatment 5 weeks. (D) Blood glucose levels during the intraperitoneal injection of insulin tolerance test (IPITT) after BBR treatment 5 weeks. (E) The areas under curve (AUC) of OGTT. (F) AUC of IPITT. (G-I) The values of biochemical indicators including total cholesterol (T-CHO), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) were measured in each group after 12 h fasting at BBR treatment 6th week. The data are presented as the mean± SEM (n=10), *P<0.05 and **P<0.01 compared with NCD, #P<0.05 and ##P<0.01 compared with HF. A 5-day indirect calorimetry study was performed using metabolic cages after a 2-day acclimation. The rate of (J-K) VO2, (L-M) VCO2, and (N-O) energy expenditure were measured in each group after 8 weeks treatment under basal condition, *P<0.05 and **P<0.01 compared with NCD, #P<0.05 and ##P<0.01 compared with HF.
Figure 2
Figure 2
BBR increases the content of BAT and promotes the emergence of characteristics of adipose tissue browning in HF mice. The ratio of (A) inguinal white adipose tissue (I-WAT) (B) visceral white adipose tissue (V-WAT), and (C) brown adipose tissue (BAT) to total body weight, values were expressed as means± SEM (n=10), *P<0.05 and **P<0.01 compared with NCD, #P<0.05 and ##P<0.01 compared with HF. (D) Micro-CT scanning and 3D reconstruction of scapular area of mice in each group. Red areas represent interscapular BAT, Green areas represent interscapular WAT. (E) The core temperatures and (F) AUC of core temperature levels under cool exposure test were recorded every 30 min at 4°C for 2 h. Values were expressed as means± SEM (n=10), *P<0.05 and **P<0.01 compared with NCD, #P<0.05 and ##P<0.01 compared with HF.
Figure 3
Figure 3
BBR promotes the thermogenesis in WAT and BAT of HF mice. (A) Representative HE staining and immunohistochemistry staining of UCP1 (yellow) in I-WAT and BAT of mice, Scale bar=50 μm. Western blot analyzes the key protein changes in (B) I-WAT and (C) BAT of HF mice treated with vehicle or BBR (25 mgkg-1, 100 mgkg-1). Relative mRNA levels of thermogenesis and fatty acid oxidation gene in (D) I-WAT and (E) BAT of HF mice after BBR treatment. Values were expressed as means± SEM (n=10), *P<0.05 and **P<0.01 compared with HF.
Figure 4
Figure 4
The regulation of adipose tissue remodeling and thermogenesis by BBR is dependent on SIRT1. After 8 days of differentiation, 3T3-L1 and HIB1b cells were treated with vehicle or BBR (5 μM) for 24 h. (A) The fully differentiated cells were fixed and subjected to Mito-tracker Red staining, Scale bar=100μm. (B) Representative transmission electron microscopy images from fully differentiated 3T3-L1 cells treated with vehicle and BBR (5 μM), Scale bar=1μm (×12000). L: lipid droplets, M: mitochondria. The proteins of SIRT1, PPARγ, and UCP1 were expressed in fully differentiated (C) 3T3-L1 and (D) HIB1b cells treated with vehicle or BBR (0.5 μM and 5 μM, respectively) for 24 h. 3T3-L1 and HIB1b cells with stable knockdown SIRT1 were established by lentivirus transfection. (E) 3T3-L1 and (F) HIB1b cells transfected with SIRT1 plasmid for 36 h or treated with BBR for 24 h. SIRT1 knockdown 3T3-L1 cells were treated with or without BBR for 24 h. The expressions of SIRT1, PPARγ, and UCP1 were detected by Western blot. (G) Relative mRNA levels of thermogenesis genes and fatty acid oxidation genes in NC or SIRT1 knockdown 3T3-L1 cells after differentiation treated with vehicle or BBR 12 h. *P<0.05 and **P<0.01 compared with NC-Veh, ##P<0.01 compared with NC-BBR. (H) After full differentiation, 3T3-L1 and shSIRT1 3T3-L1cells were treated with or without BBR for 24 h and stained Oil red O.
Figure 5
Figure 5
BBR increases the deacetylated level of PPARγ by SIRT1 activation. The levels of PPARγ acetylation were measured in fully differentiated (A, B) 3T3-L1 and (C, D) HIB1b cells treated with BBR (5 μM) for 24 h. The levels of PPARγ acetylation were examined via immunoprecipitation (IP) with anti-acetylated lysine antibody and anti-PPARγ antibody. The effects of BBR on PPARγ acetylation were determined in WT and SIRT1 knockout (E) 3T3-L1 and (F) HIB1b cells, respectively. (G) WT and Sirt1-/- mouse embryonic fibroblasts (MEFs) were treated with or without BBR (5 μM) for 24 h. (H) Flag-SIRT1 plasmid or catalytically inactive mutant 363 HY plasmid was transfected into stably SIRT1 knockdown 3T3-L1 cells. WT and SIRT1 knockdown 3T3-L1 cells were treated with BBR (5 μM) for 24 h. The levels of PPARγ acetylation were examined by IP with anti-acetylated lysine antibody.
Figure 6
Figure 6
BBR promotes the interaction between SIRT1 and PPARγ. (A) Representative immunofluorescence images demonstrating colocalization of SIRT1 (green) and PPARγ (red) in differentiated 3T3-L1 cells treated with BBR (5 μM) for 24 h. (B, C) 3T3-L1 cells were transfected with Flag-SIRT1 plasmid for 36 h and treated with vehicle or BBR (5 μM). The expressions of SIRT1 and PPARγ were respectively examined by IP with anti-PPARγ antibody and anti-SIRT1 antibody.
Figure 7
Figure 7
Berberine affects SIRT1 protein levels and activity to regulate PPARγ deacetylation levels in an AMPK-dependent manner. Western blot analyzes the expressions of P-AMPK and total-AMPK in (A) I-WAT and (B) BAT of HF mice treated with vehicle or BBR (25 mgkg-1, 100 mgkg-1). (C-E) 3T3-L1 cells were pretreated with BBR (5μM), AMPK activator AICAR (0.5mM), AMPK inhibitor Compound C (20μM), and Compound C+ BBR (20μM) for 4h. The levels of NAD+ and NADH were measured with the kits, and their ratios were calculated. (F)3T3-L1 cells were treated with AICAR (0.5 mM), Compound C (20 μM), and BBR (5 μM) for 12 h. (G) WT and SIRT1 knockdown 3T3-L1 cells were treated with BBR (5 μM) or AICAR (0.5 mM) for 12 h. Representative proteins were detected by Western blot. (H) 3T3-L1 cells with stable knockdown AMPK were established using lentivirus transfection. WT and AMPK knockdown 3T3-L1 cells were treated with vehicle or BBR (5 μM) for 12 h. (I) WT and SIRT1 knockdown 3T3-L1 cells were treated with vehicle or BBR (5 μM) for 12 h. The levels of PPARγ acetylation were examined by IP with anti-acetylated lysine antibody.
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