Celastrol targets adenylyl cyclase-associated protein 1 to reduce macrophages-mediated inflammation and ameliorates high fat diet-induced metabolic syndrome in mice

Figure 1. Celastrol (Cel) reduces body weight of high fat diet (HFD)-induced metabolic syndrome (MetS) mice. (A) The chemical structure of celastrol. (B)–(D) Analysis of mice on HFD, either without (HFD, n = 7) or treated with celastrol at doses of 0.75 mg/kg/day (HFD+Cel 0.75, n = 7), 1 mg/kg/day (HFD+Cel 1.5, n = 7), or 3 mg/kg/day (HFD+Cel 3, n = 7), including: (B) phenotypic presentation; (C) body weight; (D) food intake (n = 4). Data represent as mean ± standard error of mean (SEM) in (C) (n = 7) and (D) (n = 4). P values are determined by two-tailed Student's t test. ∗P < 0.05, ∗∗P < 0.01.

Figure 2. Celastrol protects mice from HFD-induced insulin resistance. (A)‒(D) Glucose tolerance test and insulin tolerance test were performed on HFD-fed mice treated with celastrol or vehicle. (E) Western blot analysis of insulin signaling in mice (Normal, HFD, HFD+Cel 3 mg/kg) liver. (F) Plasma glucose was determined in HFD-fed mice and celastrol treated mice. Data represent as mean ± SEM in (A) and (B) (n = 7), and (C)–(F) (n = 4). P values are determined by two-tailed Student's t test. ∗P < 0.05, ∗∗P < 0.01. GTT, glucose tolerance; ITT, insulin tolerance; AUC, calculated area under curve.

Figure 3. Celastrol protects against HFD-induced hepatic steatosis. (A) and (B) Representative hematoxylin and eosin (H&E) staining and caveolin-1 staining of liver from HFD-fed mice treated with celastrol or vehicle. (C) and (D) total cholesterol (T-CHO) and triglycerides (TG) in serum from mice were measured. Data represent as mean ± SEM in (C) and (D) (n = 4). P values are determined by two-tailed Student's t test. ∗P < 0.05, ∗∗P < 0.01, ns, not significant.

Figure 4. Celastrol attenuates HFD-induced inflammation. (A)‒(C) Phenotypic presentation, H&E staining, CD11c staining of epididymal adipose tissue from HFD-fed mice treated with celastrol or vehicle. (D) ELISA quantification of protein levels of cytokines in mouse serum. Data represent as mean ± SEM in D (n = 4). P values are determined by two-tailed Student's t test. ∗P < 0.05, ∗∗P < 0.01, ns, not significant.

Figure 5. Celastrol binds adenylyl cyclase-associated protein 1 (CAP1) protein. (A) Schematic representation of the TRAP experiment. (B) Celastrol binding proteins using target-responsive accessibility profiling (TRAP) experiment. (C) and (D) Cells were incubated with celastrol or DMSO for 2 h, and cellular thermal shift assays (CETSA) analyzed the thermal stabilization of CAP1 protein at different temperatures and concentrations. (E) Chemical structure of the biotin labeled celastrol (Cel-biotin). (F) Cel-biotin was added to streptavidin-agarose beads and incubated. Biotin alone was used as a control. Lysates prepared from THP-1 cells were added to the streptavidin-agarose beads with Cel-biotin. Eluent was then loaded on a polyacrylamide gel for Western blot analysis. Total lysates were used as an input control. (G) Immunofluorescence staining of phorbol ester (PMA)-differentiated THP-1 cells were treated with celastrol for 2 h, and then stimulated with biotin or Cel-biotin for 4 h. (H) The interaction of celastrol with CAP1 or vector was measured by microscale thermophoresis (MST). The KD value of celastrol and CAP1 interaction was determined with MO.Affinity Analysis Software. (I)…

Figure 6. Celastrol inhibits resistin-induced cytokines levels. (A) ELISA quantification of resistin level in mouse serum. (B) Resistin staining in epididymal adipose tissue and liver tissue of mice. (C) Resistin expression in mice liver was determined by Western blot. (D) ELISA quantification of cAMP level in cell supernatant. (E) PKA‒NF-κB signaling in PMA-differentiated THP-1 cells were determined by Western blot analysis after celastrol treating. (F) Immunofluorescence staining of PMA-differentiated THP-1 cells were treated with celastrol for 2 h, and then stimulated with resistin for 15 min. (G) ELISA quantification of protein levels of cytokines in cell supernatant. Data represent as mean ± SEM. P values are determined by two-tailed Student's t test (n = 3). ∗P < 0.05, ∗∗P < 0.01, ns, not significant.

Figure 7. Celastrol inhibits CAP1‒resistin interaction. (A) Confocal microscopy imaging of CAP1 in PMA-differentiated THP-1 cells. (B) PMA-differentiated THP-1 cells were untreated or treated with celastrol. Cell lysates underwent immunoprecipitation with CAP1 antibodies and the immunoblotting with indicated antibodies. (C) and (D) CAP1 protein and mRNA levels in PMA-differentiated THP-1 cells infected with shRNA-Control, shRNA-CAP1, vector, or CAP1 lentiviruses. (E) and (F) PKA‒NF-κB signaling in PMA-differentiated THP-1 cells infected with shRNA-Control, shRNA-CAP1, vector, or CAP1 lentiviruses were determined by Western blot analysis after celastrol treating. Data represent as mean ± SEM. P values are determined by two-tailed Student's t test (n = 3). ∗∗P < 0.01.

Figure 8. The graphic illustration of the mechanism of celastrol ameliorating metabolic syndrome. In obesity, adipose tissue generates resistin, which binds to CAP1 on macrophages and activates the PKA‒NF-κB pathway and promotes the expression of pro-inflammatory cytokines. These pro-inflammatory factors further promote inflammation and insulin resistance of adipose tissue on the one hand, and also aggravate liver insulin resistance and hepatic steatosis on the other hand. Celastrol effectively prevents inflammatory response through binding to CAP1 and inhibiting the interaction between resistin and CAP1. Created in BioRender.com.