Fig 2: Nobiletin alleviated lipid levels and hepatic ANGPTL3 expression in adult zebrafish. Male adult zebrafish were allocated into three groups: normal diet plus vehicle control (ND), high-fat diet plus vehicle (HFDV) and high-fat diet plus nobiletin (HFDN). The zebrafish were administered vehicle or nobiletin (100 mg/kg) by oral gavage (one dose every two days) for two weeks. The (a) body weight, (b) AST activity, (c) triglyceride and (d) cholesterol contents were analyzed as described in Materials and Methods. Values are expressed as the means ± SD. # p < 0.05 and ## p < 0.01 represent significantly statistical differences compared to the ND- or vehicle-treated groups. (e) The protein extracts of livers from 6 individual zebrafish in respective ND, HFD/Vehicle, and HFD/Nobiletin groups (NDV#1~#6, HFDV#1~#6, and HFDN#1~#6) were prepared and the ANGPTL3 and ß-actin proteins were detected by Western blotting analysis. A representative blot is shown. (f) The intensity of the protein signal for ANGPTL3 and ß-actin was measured in different blots from replicate experiments. The normalized values of ANGPTL3 versus ß-actin from 6 individual zebrafish in respective groups (NDV#1~#6, HFDV#1~#6, and HFDN#1~#6) are expressed as the means ± SD. # p < 0.05 represents significantly statistical differences compared to the HFD/Vehicle-treated groups. (g) The protein extracts of livers (10 µg) from zebrafish treated with ND (n = 14), HFD/Vehicle (n = 16) and HFD/Nobiletin (n = 16) groups and LPL enzyme were incubated with substrates to determine the LPL activity via fluorogenic analysis. The fluorescence intensity was measured at 90 min. The data represent the mean ± SD. ## p < 0.01 represents significantly statistical differences compared to the vehicle-treated groups.

Fig 3: Effects of nobiletin on ANGPTL3 promoter activity. (a) HepG2 cells were co-transfected with the pGL3-ANGPTL3 (-980/+20) plasmid and Renilla control plasmid. After 24 h of transfection, the cells were treated with vehicle or nobiletin (20 and 40 µM) for 24 h. Luciferase activity was determined with a reporter assay kit. Data were normalized to Renilla control luciferase activity and represent the mean ± SD from three independent experiments. ** p < 0.01 indicates a statistically significant difference compared to the vehicle-treated group. (b) HepG2 cells were cotransfected with serially deleted ANGPTL3 promoter–luciferase reporter plasmids, including pGL3-ANGPTL3 (-750/+20), pGL3-ANGPTL3 (-500/+20), pGL3-ANGPTL3 (-250/+20), and pGL3-ANGPTL3 (-120/+20), and a control Renilla plasmid followed by treatment with vehicle or nobiletin (40 µM) for 24 h. Data represent the mean ± SD of three independent experiments. ** p < 0.01 indicates a significant difference compared to vehicle-treated pGL3-ANGPTL3 (-980/+20)-transfected cells. ## p < 0.01 indicates a significant difference compared to vehicle treatment of the respective deletion construct-transfected groups. “ns” indicates no significance.

Fig 4: Effects of nobiletin on HFN-1a and LXRa protein expression and LXRa activity. HepG2 cells were treated with vehicle or nobiletin (20 and 40 µM) for 24 h. (a) The nuclear HFN-1a, LXRa and HDAC2 protein levels were detected by Western blot analysis. The normalized intensity of (b) HFN-1a and (c) LXRa versus HDAC2 is presented as the mean ± SD from three independent experiments. (d) HepG2 cells were cotransfected with a pGL3-ANGPTL3 p(-250/+20)) plasmid and a Renilla control plasmid for 24 h. The plasmid-transfected cells were pretreated with vehicle or nobiletin (40 µM) for 1 h followed by incubation with the LXRa agonist T0901317 (1 µM) for an additional 24 h. The luciferase activities were measured with a reporter assay kit and normalized to the respective Renilla luciferase activities. The data represent the mean ± SD from three independent experiments. (e) HepG2 cells were pretreated with vehicle or nobiletin (40 µM) for 1 h followed by treatment with T0901317 (1 µM) for an additional 24 h. ANGPTL3 mRNA was detected via RT–qPCR. Data are shown as the mean ± SD of three independent experiments. ** p < 0.01 indicates a significant difference compared to the vehicle-treated group. ## p < 0.01 indicates a significant difference compared to the group treated only with T0901317.

Fig 5: A hypothetical mechanism by which nobiletin regulates lipid metabolism through modulation of the LXRa-ANGPTL3-LPL axis in vitro and in vivo. In vitro, nobiletin downregulates the mRNA and protein expression of ANGPTL3 in hepatic cell lines by counteracting the activation of LXRa, thereby reducing ANGPTL3 secretion and leading to increased LPL activity in the capillary lumen. In vivo, hepatic ANGPTL3 protein levels are alleviated in HFD-fed, nobiletin-treated zebrafish and potentially enhance the clearance of TGRLs and reduce TG accumulation in circulation.