Fig 2: Effects of nobiletin on ANGPTL3 gene expression and LPL activity. HepG2 and Huh7 cells were treated with vehicle or nobiletin (20 and 40 µM) for 24 h. (a,d) The mRNA levels of ANGPTL3 and GAPDH were determined using RT–qPCR. (b,e) The protein levels of ANGPTL3 and actin were detected via Western blotting analysis. A representative blot is shown. (c,f) The normalized intensity of the signal for ANGPTL3 protein versus actin protein. (g) The culture medium from vehicle- or nobiletin (40 µM)-treated HepG2 cells was collected, and the extracellular ANGPTL3 protein was detected via ELISA. (h) The concentrated extracellular proteins (50 µg) from vehicle- or nobiletin (40 µM)-treated HepG2 cells and LPL enzyme were incubated with substrates to determine the catalytic activity of LPL via fluorogenic analysis. The fluorescence intensity was measured over 90 min. The data represent the mean ± SD of three independent experiments. * p < 0.05 and ** p < 0.01 indicate significant differences compared to the vehicle-treated group.

Fig 3: 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.

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: 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.