The Citrus Flavonoid Nobiletin Downregulates Angiopoietin-like Protein 3 (ANGPTL3) Expression and Exhibits Lipid-Modulating Effects in Hepatic Cells and Adult Zebrafish Models

Figures

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  Figure 1. Effects of nobiletin on the viability of hepatic cells. (a) The chemical structure of nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone). (b) HepG2 and (c) Huh 7 cells were treated with vehicle (0.1% DMSO) or nobiletin (5, 10, 20, 40 and 60 μM) for 24 h. Cell viability was analyzed with MTT assays. The data are shown as the mean ± SD of three independent experiments.

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  Figure 2. Microarray data and GSEA of DEGs for determination of gene ontology (GO) in response to nobiletin treatment in hepatic cells. HepG2 cells were treated with vehicle or nobiletin (40 μM) for 24 h, and the differential mRNA expression profiles were analyzed using a human cDNA microarray. (a) Flowchart of the transcriptome analysis and identification of DEGs in nobiletin-treated HepG2 cells. (b) GSEA demonstrating that the GO BP signature “Enrichment plot: GOBP Regulation of Lipid Metabolic Process” (GO: 0019216) gene set is enriched and downregulated with the DEGs in nobiletin-treated cells. The barcode shows the position of the genes involved in the gene set. (c) The fold changes of 28 significantly downregulated expression genes associated with GO:0019216 in response to nobiletin treatment (p < 0.05).

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  Figure 3. 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.

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  Figure 4. 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…

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  Figure 5. Effects of nobiletin on HFN-1α and LXRα protein expression and LXRα activity. HepG2 cells were treated with vehicle or nobiletin (20 and 40 μM) for 24 h. (a) The nuclear HFN-1α, LXRα and HDAC2 protein levels were detected by Western blot analysis. The normalized intensity of (b) HFN-1α and (c) LXRα 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 LXRα 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.…

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  Figure 6. The poses of nobiletin docked into the ligand-binding domain (LBD) of LXRα. (a) The LXRα receptor is shown as a gray ribbon, and nobiletin is shown in green. (b) Superposition of both nobiletin and T0901317 (blue) bound to the LBD of LXRα. (c) Two-dimensional interaction map of LXRα and nobiletin. Nobiletin is surrounded by hydrophobic residues (green) and hydrophilic residues (pink) within the LBD of LXRα. Dotted line indicates a stacking interaction between the heterocyclic ring of nobiletin and the aromatic group of the phenylalanine (Phe315) residue.

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  Figure 7. 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 β-actin proteins were detected by Western blotting analysis. A representative blot is shown. (f) The intensity of the protein signal for ANGPTL3 and β-actin was measured in different blots from replicate experiments. The normalized values of ANGPTL3 versus β-actin from 6 individual zebrafish in respective groups (NDV#1~#6,…

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  Figure 8. A hypothetical mechanism by which nobiletin regulates lipid metabolism through modulation of the LXRα-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 LXRα, 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.