Nutr Res Pract.  2024 Aug;18(4):479-497. 10.4162/nrp.2024.18.4.479.

Cryptotanshinone promotes brown fat activity by AMPK activation to inhibit obesity

• Ni J
• Ye A
• Gong L
• Zhao X
• Fu S
• Guo J

• KMID: 2558487
• DOI: http://doi.org/10.4162/nrp.2024.18.4.479

Abstract

BACKGROUND/OBJECTIVES
Activating brown adipose tissue (BAT) and browning of white adipose tissue (WAT) can protect against obesity and obesity-related metabolic conditions. Cryptotanshinone (CT) regulates lipid metabolism and significantly ameliorates insulin resistance. Adenosine-5'-monophosphate (AMP)-activated protein kinase (AMPK), a receptor for cellular energy metabolism, is believed to regulate brown fat activity in humans.
MATERIALS/METHODS
The in vivo study included high-fat-fed obese mice administered orally 200/400 mg/kg/d CT. They were evaluated through weight measurement, the intraperitoneal glucose tolerance test (IPGTT), intraperitoneal insulin tolerance test (IPITT), cold stimulation test, serum lipid (total cholesterol, triglycerides, and low-density lipoprotein) measurement, hematoxylin and eosin staining, and immunohistochemistry. Furthermore, the in vitro study investigated primary adipose mesenchymal stem cells (MSCs) with incubation of CT and AMPK agonists (acadesine)/inhibitor (Compound C). Cells were evaluated using Oil Red O staining, Alizarin red staining, flow cytometry, and immunofluorescence staining to identify and observe the osteogenic versus adipogenic differentiation. Quantitative real-time polymerase chain reaction and the Western blot were used to observe related gene expression.
RESULTS
In the diet-induced obesity mouse model mice CT suppressed body weight, food intake, glucose levels in the IPGTT and IPTT, serum lipids, the volume of adipose tissue, and increased thermogenesis, uncoupling protein 1, and the AMPK pathway expression. In the in vitro study, CT prevented the formation of lipid droplets from MSCs while activating brown genes and the AMPK pathway. AMPK activator enhanced CT’s effects, while the AMPK inhibitor reversed the effects of CT.
CONCLUSION
CT promotes adipose tissue browning to increase body thermogenesis and reduce obesity by activating the AMPK pathway. This study provides an experimental foundation for the use of CT in obesity treatment.

Keyword

Obesity; salvia miltiorrhiza; adipocytes; insulin resistance; brown fat

Figure

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  Fig. 1 CT reduces body weight and improves glucose intolerance and insulin resistance in mice. (A) Mice body weight and (B) food intake were examined (n = 6). (C) IPGTT was used to examine the effect of CT on glucose tolerance in mice on HF group. (D) AUC was calculated for IPGTT (n = 6). (E) IPITT was used to evaluate the effect of CT on insulin tolerance in mice on HF group (n = 6). (F) AUC was calculated for IPITT (n = 6). Data from A, B, D and F were analyzed by 1-way ANOVA. Data from C and E were analyzed by 2-way ANOVA.Mice were randomly divided into 4 groups: ND, normal diet; HF, high-fat diet; CT-200, received 200 mg/kg/d CT; and CT-400, received 400 mg/kg/d CT (CT groups received via gavage for 6 weeks, respectively).CT, Cryptotanshinone; IPGTT, intraperitoneal glucose tolerance test; AUC, area under the curve; IPITT, intraperitoneal insulin tolerance test; ANOVA, analysis of variance.*P < 0.05, **P < 0.01 vs. ND; ##P < 0.01 vs. HF.

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  Fig. 2 CT increases adaptive thermogenesis and suppresses dyslipidemia. (A) The cold stimulation test was used to examine the regulation of body temperature in mice by CT and the data were analyzed by 2-way ANOVA (n = 6). (B-D) Serum TC, TG, and LDL-C levels were measured in each group of mice, and the data were analyzed by 1-way ANOVA (n = 6).Mice were randomly divided into 4 groups: ND, normal diet; HF, high-fat diet; CT-200, received 200 mg/kg/d CT; and CT-400, received 400 mg/kg/d CT (CT groups received via gavage for 6 weeks, respectively).CT, Cryptotanshinone; ANOVA, analysis of variance; TC, total cholesterol; TG, triglycerides; LDL-C, low-density lipoprotein cholesterol; ANOVA, analysis of variance.**P < 0.01 vs. ND; ##P < 0.01 vs. HF.

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  Fig. 3 CT improved adipose tissue morphology and promoted UCP1 levels. (A) Morphological effects of CT on WAT and BAT in mice as observed by H&E staining (magnification, 200×). (B) Average adipocyte diameter for quantitative analysis. (C, D) Immunohistochemistry was used to detect the effect of CT on UCP1 expression levels in WAT and BAT (magnification, 200×), and the data were analyzed by 1-way ANOVA (n = 3).Mice were randomly divided into 4 groups: ND, normal diet; HF, high-fat diet; CT-200, received 200 mg/kg/d CT; and CT-400, received 400 mg/kg/d CT (CT groups received via gavage for 6 weeks, respectively).CT, Cryptotanshinone; UCP1, uncoupling protein 1; WAT, white adipose tissue; BAT, brown adipose tissue; H&E, hematoxylin and eosin; ANOVA, analysis of variance.**P < 0.01 vs. ND; #P < 0.05, ##P < 0.01 vs. HF.

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  Fig. 4 CT promotes brown gene expression in adipose tissue. qRT-PCR was used to examine the effect of CT on the brown genes (A) PGC-1α, (B) UCP1, (C) CIDEA, (D) Prdm16, (E) Tmem26, (F) PPARγ, and (G) CD137 mRNA levels in the WAT and BAT tissues. Data were analyzed by one-way ANOVA (n = 3).Mice were randomly divided into 4 groups: ND, normal diet; HF, high-fat diet; CT-200, received 200 mg/kg/d CT; and CT-400, received 400 mg/kg/d CT (CT groups received via gavage for 6 weeks, respectively).CT, Cryptotanshinone; qRT-PCR, quantitative real-time polymerase chain reaction; PGC-1α, proliferator-activated receptor-gamma coactivator-1α; UCP1, uncoupling protein 1; CIDEA, cell death inducing DFFA like effector A; Prdm16, prdomain-containing 16; Tmem26, transmembrane protein 26; PPARγ, peroxisome proliferator-activated receptor γ; CD, cluster of differentiation; WAT, white adipose tissue; BAT, brown adipose tissue; ANOVA, analysis of variance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.*P < 0.05, **P < 0.01 vs. ND; #P < 0.05, ##P < 0.01 vs. HF.

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  Fig. 5 CT promotes brown gene protein expression and improves insulin sensitivity and lipolysis in adipose tissue. Western blot was used to detect the effect of CT on protein expression levels of p-AKT/AKT, p-HSL/HSL, UCP1, Complex III, Complex V, and p-AMPK/AMPK in (A) WAT and (B) BAT. (C) qRT-PCR was used to examine the effect of CT on protein expression levels above. Data were analyzed by one-way ANOVA (n = 3).Mice were randomly divided into 4 groups: ND, normal diet; HF, high-fat diet; CT-200, received 200 mg/kg/d CT; and CT-400, received 400 mg/kg/d CT (CT groups received via gavage for 6 weeks, respectively).CT, Cryptotanshinone; AKT, protein kinase B; HSL, hormone-sensitive triglyceride lipase; UCP1, uncoupling protein 1; AMPK, adenosine-5'-monophosphate (AMP)-activated protein kinase; WAT, white adipose tissue; BAT, brown adipose tissue; ANOVA, analysis of variance.*P < 0.05, **P < 0.01 vs. ND; #P < 0.05, ##P < 0.01 vs. HF.

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  Fig. 6 CT inhibits lipid droplet formation and mRNA levels of brown genes in MSCs through activation of AMPK. (A, B) Immunofluorescence assays were used to detect lipid droplet formation in MSCs (magnification, 200×). (C) qRT-PCR was used to examine the effect of CT on the brown genes PGC-1α, UCP1, CIDEA, Prdm16, Tmem26, PPARγ, CD137 mRNA levels in MSCs. Data were analyzed by one-way ANOVA (n = 3).Mice were randomly divided into 4 groups: ND, normal diet; HF, high-fat diet; CT-200, received 200 mg/kg/d CT; and CT-400, received 400 mg/kg/d CT (CT groups received via gavage for 6 weeks, respectively).CT, Cryptotanshinone; MSC, mesenchymal stem cell; AMPK, adenosine-5'-monophosphate (AMP)-activated protein kinase; qRT-PCR, quantitative real-time polymerase chain reaction; PGC-1α, proliferator-activated receptor-gamma coactivator-1α; UCP1, uncoupling protein 1; CIDEA, cell death inducing DFFA like effector A; Prdm16, prdomain-containing 16; Tmem26, transmembrane protein 26.PPARγ, peroxisome proliferator-activated receptor γ; CD, cluster of differentiation; ANOVA, analysis of variance.*P < 0.05, **P < 0.01 vs. ND; #P < 0.05, ##P < 0.01 vs. HF.

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  Fig. 7 CT promotes brown gene expression and improves insulin sensitivity and lipolysis in adipose tissue through activation of AMPK. (A, B) Western blot was used to detect the effect of CT on protein expression levels of p-AKT/AKT, p-HSL/HSL, UCP1, Complex III, Complex V, p-AMPK/AMPK in MSCs. Data were analyzed by one-way ANOVA (n = 3).Mice were randomly divided into 4 groups: ND, normal diet; HF, high-fat diet; CT-200, received 200 mg/kg/d CT; and CT-400, received 400 mg/kg/d CT (CT groups received via gavage for 6 weeks, respectively).CT, Cryptotanshinone; AMPK, adenosine-5'-monophosphate (AMP)-activated protein kinase; AKT, protein kinase B; HSL, hormone-sensitive triglyceride lipase; UCP1, uncoupling protein 1; MSC, mesenchymal stem cell; ANOVA, analysis of variance.*P < 0.05, **P < 0.01 vs. ND; #P < 0.05, ##P < 0.01 vs. HF.

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