Fig 2: Graphical abstract. Gyps treatment significantly increased hepatic expression of FXR and its target SHP, and led to the up-regulation of CPT1 and LPL, and down-regulation of SREBP1, FASN and SCD1 protein levels in WT mice but not FXR KO mice. Ultimately, Gyps improves lipid metabolism in a mouse model of NASH through the activation of FXR. Gyps, gypenosides; FXR, farnesoid X receptor; SHP, small heterodimer partner; SREBP1, sterol-regulatory element binding protein 1; SCD1, stearoyl-CoA desaturase 1; FASN, fatty acid synthetase; CPT1A, carnitine palmitoyltransferase 1A; LPL, lipoprotein lipase.

Fig 3: Effects of Gyps on hepatic CPT1A and LPL mRNA and protein expression in WT and FXR KO mice. (A1,A2) Quantification of hepatic CPT1A protein levels in different groups of WT and FXR KO mice. (a1,a2) Relative expression levels of CPT1 mRNA in different groups in WT and FXR KO mice. (B1,B2) Quantification of hepatic LPL protein levels in different groups of WT and FXR KO mice. (b1,b2) Relative expression levels of LPL mRNA in different groups in WT and FXR KO mice. (C1,C2) WB analysis of hepatic CPT1A and LPL protein expression in different groups of WT and FXR KO mice. CPT1A, carnitine palmitoyltransferase 1A; LPL, lipoprotein lipase; ND, normal diet; HFD, high-fat diet; Gyps, gypenosides; WT, wild-type; KO, knockout; WB, western blot. *p < 0.05, **p < 0.01.

Fig 4: Potency of etomoxir binding to cellular CPT1A protein. MCF-7, T47D, and MDA-MB-468 cells treated for 20–24 hours with etomoxir at indicated concentrations were analyzed with immunoprecipitation of native CPT1A protein and immunoblotting of denatured lysates for CPT1A (input) with the same CPT1A antibody. Etomoxir added to intact cells, but not to cellular lysates, prevented CPT1A protein from being immunoprecipitated by the CPA1A antibody. Full-length blots are presented in Supplementary Fig. S4.

Fig 5: Effects of etomoxir, ranolazine and TMZ in primary cells and in mice (A) FAO rates in primary rat hepatocytes, mouse cardiomyocytes, and MEFs treated for 20–24 hours with or without etomoxir (5 µM), ranolazine (25 µM), or TMZ (50 µM) were measured as detailed in Materials and Methods. The predominant expression of tissue-specific CPT1A (liver form) in hepatocytes and CPT1B (muscle form) in cardiomyocytes was confirmed by immunoblotting. Full-length blots are presented in Supplementary Fig. S4. The FAO rates in the metabolically active hepatocytes and cardiomyocytes are presented as % conv/2 × 105 cells/2 hours while those in MEFs were presented as % conv/2 × 105 cells/5 hours. (B) Four groups of adult female mice (n = 3 each group) were treated with etomoxir, ranolazine or TMZ for 3 days before administration of 3H-palmitic acid as detailed in Materials and Methods. The production of 3H2O from in vivo oxidation of 3H-palmitic acid over 1-hour period was assessed by the diffusion analysis of plasma samples of mice. The results were presented as 3H2O-based radioactivity (CPM)/100 µl plasma.