Ginsenoside Compound K Protects against Obesity through Pharmacological Targeting of Glucocorticoid Receptor to Activate Lipophagy and Lipid Metabolism

Abstract

(1) Background: The glucocorticoid receptor (GR) plays a key role in lipid metabolism, but investigations of GR activation as a potential therapeutic approach have been hampered by a lack of selective agonists. Ginsenoside compound K (CK) is natural small molecule with a steroid-like structure that offers a variety of therapeutic benefits. Our study validates CK as a novel GR agonist for the treatment of obesity. (2) Methods: By using pulldown and RNA interference, we determined that CK binds to GR. The anti-obesity potential effects of CK were investigated in obese mice, including through whole-body energy homeostasis, glucose and insulin tolerance, and biochemical and proteomic analysis. Using chromatin immunoprecipitation, we identified GR binding sites upstream of lipase ATGL. (3) Results: We demonstrated that CK reduced the weight and blood lipids of mice more significantly than the drug Orlistat. Proteomics data showed that CK up-regulated autophagy regulatory proteins, enhanced fatty acid oxidation proteins, and decreased fatty acid synthesis proteins. CK induced lipophagy with the initial formation of the phagophore via AMPK/ULK1 activation. However, a blockade of autophagy did not disturb the increase in CK on lipase expression, suggesting that autophagy and lipase are independent pathways in the function of CK. The pulldown and siRNA experiments showed that GR is the critical target. After binding to GR, CK not only activated lipophagy, but also promoted the binding of GR to the ATGL promoter. (4) Conclusions: Our findings indicate that CK is a natural food candidate for reducing fat content and weight.

Keywords: ATGL; GR; ginsenoside CK; lipid metabolism; lipophagy.

Figure 1

CK treatment ameliorates adiposity and blood glucose in ob/ob mice. Obese mice were orally administered 150 mg/kg of orlistat, or injected with the vehicle (DMSO) or 20 mg/kg of the indicated ginsenosides for 5 weeks (n = 5–8). (a) Body weight. (b) Body weight increase rate and representative photos of mice. (c) Fasting blood glucose levels. (d) Glucose tolerance test and area under the receiver operating characteristic curve (AUC). (e) Insulin tolerance test and AUC. (f) Serum insulin content. Statistical data represent the comparison of each value in the compound-treated groups to the corresponding value in the ob/ob groups. Results are presented as mean ± SD. ##, p < 0.01; ###, p < 0.001; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 2

Metabolic profiles and lipid levels of obese mice after CK treatment. Obese mice were orally administered 150 mg/kg of orlistat, or were injected with the vehicle (DMSO) or 20 mg/kg of the indicated ginsenosides for 5 weeks (n = 4–10). (a) Oxygen consumption. (b) Carbon dioxide production. (c) Heat production. (d) Serum triglyceride levels. (e) Serum non-esterified fatty acid levels. (f) Serum total cholesterol levels. (g) Representative images of fat deposition in liver and white adipose tissue by hematoxylin eosin staining. Scale bar, 100 μm. Statistical data represent the comparison of each value in the compound-treated groups to the corresponding value in the ob/ob groups. Results are presented as mean ± SD. #, p < 0.05; ##, p < 0.01; ###, p < 0.001; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 3

CK suppresses hepatic fat deposit through the enhancement of lipase expression and autophagy. (a) Venn diagrams showing the number of unique or shared genes between ob vs. C57BL/6J and ob + CK vs. ob datasets. The common part (60 genes) likely reflects CK effect. (b) GO enrichment analysis predicted the top 30 enriched pathways of CK-regulated genes. Rich factor is the ratio of the differentially expressed gene number to the total gene number in a certain pathway. Q-value is corrected p-value ranging from 0–1. (c) Heat map of changes in protein levels of autophagy-related and fatty acid catabolism-related factors. (d) Autophagy signaling pathways analyzed using Western blotting of liver tissues (n = 3). (e) Gene expression of lipolysis factors assessed using qRT-PCR (n = 4–10). (f) Protein expression of lipolysis factors assessed using Western blotting (n = 3). (g) cAMP/PKA/HSL signaling pathways were analyzed using Western blotting of liver tissues (n = 3). (h) Gene expression of lipogenesis factors assessed using qRT-PCR (n = 4–10). (i) Protein expression of lipogenesis factors assessed using Western blotting (n = 3). Statistical data represent the comparison of each value in the compound-treated groups to the corresponding value in the ob/ob groups. Results are presented as mean ± SD. #, p < 0.05; ##, p < 0.01; ###, p < 0.001; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 4

Ginsenoside CK induces autophagy in vitro and in vivo. (a) Representative images and quantification of GFP-LC3 puncta in HeLa cells stably expressing GFP-LC3 cultured in normal or starvation medium, or treated with CK in normal medium, in the presence or absence of lysosomal inhibitors 3-MA or BafA1 for the indicated time period (n = 100). Statistical data represent the comparison of each value to the corresponding value under normal conditions. Scale bar, 20 μm. (b) Western blotting detection of p62 and LC3 in HepG2 cells (left) or A549 cells (right) cultured in normal or starvation medium, or treated with CK in normal medium, in the presence or absence of lysosomal inhibitors 3-MA for 3 h (n = 3). (c) Representative images of co-localization of LC3 (green) and FIP200 (red, upper) or WIPI2 (red, lower) in normal or starvation medium, or when treated with CK in normal medium for the indicated time. Scale bar, 10 μm. (d) AMPK and mTOR signaling pathways were analyzed by western blotting in Hepa1-6 cells cultured in normal or starvation medium, or treated with CK in normal medium for 3 h (n = 3). (e) Western blotting detection of p62 in liver of mice injected with CK, in the presence or absence of the lysosomal inhibitor chloroquine (n = 3). Results are presented as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 5

CK activates lipophagy and lipase activity. Hepa1-6 cells were cultured in normal medium, 0.5 mM free fatty acid (FA) medium, or with indicated ginsenosides in FA medium. (a) Insulin-stimulated glucose uptake of the cells cultured in FA medium with the presence or absence of the lysosomal inhibitor BafA1 (n = 3). (b) Representative images of the lipid droplets in cells visualized by oil red staining, in the presence or absence of the lysosomal inhibitor BafA1 (left), and the quantification of lipid droplets (right) (n = 3). Scale bar, 100 μm. (c) The insulin receptor signaling pathways were analyzed using western blotting (n = 3). The cells were transfected with control (NC) or ATG7 siRNAs 36 h prior to FA or CK treatment. (d) Fatty acid oxidation was analyzed in cells treated with FA or CK using an Agilent Seahorse XFp Analyzer (n = 3). (e) Representative images of the co-localization of lipid droplets (green) and autophagosomes (red), and co-localization of lipid droplets (green) and lysosomes (red). Scale bar, 10 μm. (f) Protein expression of lipolysis factors were analyzed using western blotting (n = 3). The cells were transfected with control (NC) or ATG7 siRNAs 36 h prior to FA or CK treatment. Results are presented as mean ± SD. ##, p < 0.01; ###, p < 0.001; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 6

CK enhances lipolysis and autophagy by binding to ATGL promoter through the glucocorticoid receptor (GR). (a) Intracellular transport of CK in hepatocytes was analyzed using UPLC-MS (n = 3). (b) The binding ability of CK to G-protein-coupled receptors were evaluated in the pull-down assay. (c) The competitive combination of CK and dexamethasone with GR was analyzed in the fluorescence quantitative assay (n = 3). (d) Representative images of the nuclear translocation of GR in hepatocytes treated with dexamethasone or CK. Scale bar, 10 μm. (e) qPCR analysis of ATGL mRNA levels treated with CK or RU486 (n = 4–6). (f) ATGL promoter vector dual luciferase statistical results (n = 3–8). (g) ChIP-qPCR analysis showing hepatocytes exposed to CK for 24 h. IgG was used as a negative control (n = 4). (h) The insulin receptor signaling pathways were analyzed using western blotting (n = 3). Cells were transfected with the control (NC) or GR siRNAs 36 h prior to FA or CK treatment. (i) Autophagy signaling pathways were analyzed using western blotting (n = 3). The cells were transfected with the control (NC) or GR siRNAs 36 h prior to FA or CK treatment. (j) The expression of lipolysis factors and lipase expression were analyzed using western blotting (n = 3). The cells were transfected with the control (NC) or GR siRNAs 36 h prior to FA or CK treatment. (k) The expression of lipolysis factors was analyzed using western blotting in the presence or absence of the GR inhibitor RU486 (n = 3). (l) Schematic diagram illustrating the model that CK drives the nucleocytoplasmic transport of GR, subsequently increasing the transcriptional activity of the ATGL gene, resulting in enhanced protein expression and lipolysis activity of ATGL. Results are presented as mean ± SD. ##, p < 0.01; *, p < 0.05; **, p < 0.01; ***, p < 0.001.