Apigenin targets fetuin-A to ameliorate obesity-induced insulin resistance

Abstract

Fetuin-A, a hepatokine secreted by hepatocytes, binds to insulin receptors and consequently impairs the activation of the insulin signaling pathway, leading to insulin resistance. Apigenin, a flavonoid isolated from plants, has beneficial effects on insulin resistance; however, its regulatory mechanisms are not fully understood. In the present study, we investigated the molecular mechanisms underlying the protective effects of apigenin on insulin resistance. In Huh7 cells, treatment with apigenin decreased the mRNA expression of fetuin-A by decreasing reactive oxygen species-mediated casein kinase 2α (CK2α)-nuclear factor kappa-light-chain-enhancer of activated B activation; besides, apigenin decreased the levels of CK2α-dependent fetuin-A phosphorylation and thus promoted fetuin-A degradation through the autophagic pathway, resulting in a decrease in the protein levels of fetuin-A. Moreover, apigenin prevented the formation of the fetuin-A-insulin receptor (IR) complex and thereby rescued the PA-induced impairment of the insulin signaling pathway, as evidenced by increased phosphorylation of IR substrate-1 and Akt, and translocation of glucose transporter 2 from the cytosol to the plasma membrane. Similar results were observed in the liver of HFD-fed mice treated with apigenin. Collectively, our findings revealed that apigenin ameliorates obesity-induced insulin resistance in the liver by targeting fetuin-A.

Keywords: CK2α; apigenin; fetuin-A; insulin receptor; insulin resistance.

Figure 1

Apigenin decreases palmitic acid (PA)‐induced expression of fetuin‐A protein in hepatocytes. (A) Huh7 cells were treated with PA (300 μM) for the indicated times (0, 3, 6, 9, and 18 h). (B) Huh7 cells were pretreated with PA for 6 h, and then incubated with the indicated concentrations (0, 5, 10, and 20 μM) of apigenin (Api) for an additional 12 h. (C) Huh7 cells were pretreated with PA (300 μM) for 6 h, and then with Api (20 μM) for the indicated times (0, 6, 9, and 12 h). Western blot analysis of fetuin-A and β-actin. Data from 5 independent experiments are expressed as mean ± standard error of the mean (SEM). *p < 0.05 vs. the 0 h or the vehicle group. #p < 0.05 vs. the PA alone group.

Figure 2

Apigenin decreases the fetuin-A mRNA expression by decreasing palmitic acid (PA)-induced phosphorylation of nuclear factor kappa B (NF-κB) and promotes the degradation of fetuin‐A protein by activating the autophagy pathway in hepatocytes. (A) Huh7 cells were pretreated with PA (300 μM) for 6 h, and then incubated with apigenin (Api, 20 μM) for the indicated times (0, 3, 6, 9, and 18 h). The fetuin-A mRNA expression was assessed by real-time PCR analysis. (B) Huh7 cells were treated with PA (300 μM) for the indicated times (0, 5, 10, 15, 30, and 60 min). The levels of NF-κB phosphorylation and total NF-κB were examined by western blot analysis. (C) Huh7 cells were pretreated with Api (20 μM) for 12 h, and then incubated with or without PA for 5 min. The levels of NF-κB phosphorylation and total NF-κB were evaluated by western blot analysis. (D-F) Western blot analysis of fetuin-A and β-actin. (D) Huh7 cells were pretreated with PA (300 μM) for 18 h, and then treated with Api (20 μM) in the presence of cycloheximide (CHX, 20 μg/mL) for the indicated times (0, 2, 4, and 8 h). (E) Huh7 cells were pretreated with PA (300 μM) for 6 h, and then incubated with autophagy inhibitors (bafilomycin A1, BafA1, 100 nM or 3-methyladenine, 3-MA, 5 μM) for 2 h, followed by Api (20 μM) for an additional 12 h. (F) Huh7 cells were pretreated with or without PA (300 μM) for 6 h, and then incubated with lysosome pathway inhibitor (chloroquine, CQ, 40 μM) or late endosomal pathway inhibitor 4-bromobenzaldehyde N-(2,6-dimethylphenyl) semicarbazone (EGA) (10 μM) for 2 h, followed by Api (20 μM) for an additional 12 h. Data from 5 independent experiments are expressed as mean ± standard error of the mean (SEM). *p < 0.05 vs. the vehicle group. #p < 0.05 vs. the PA alone group.

Figure 3

Apigenin inhibits casein kinase 2α (CK2α)-mediated fetuin-A phosphorylation and prevents the palmitic acid (PA)-induced deregulation of insulin signaling. (A and B) Huh7 cells were pretreated with PA (300 μM) for the indicated times (0, 3, 6, 9, and 18 h). Cellular lysates or cultured medium were immunoprecipitated (IP) with an anti-fetuin-A antibody and immunoblotting (IB) was performed with an anti-phosphor (p-Ser) antibody. IgG was used as a control for the fetuin-A antibody. (B) Western blot analysis of casein kinase 2α (CK2α) and β-actin. (C and D) Huh7 cells were pretreated with PA (300 μM) for 6 h and then incubated with Api (20 μM) for 12 h. Cellular lysates or cultured medium were IP with anti-fetuin-A antibody and IB was performed with anti-phosphos (p-Ser) antibody. IgG was used as a control for the fetuin-A antibody. (D) Cellular lysates were IP with anti-fetuin-A antibody and IB was then performed with anti-IR or anti-fetulin-A antibody. IgG was used as a control. (E) Representative confocal microscopy images of proximity ligation assay (PLA) performed using anti-fetuin-A and anti-insulin receptor (IR) antibodies. 4',6-diamidino-2-phenylindole (DAPI) (blue), proximity ligation assay (PLA) signals (green). (F and G) Western blot analysis of phosphorylated levels of insulin receptor substrate (IRS) (p-IRS) and Akt (p-Akt), and total levels of IRS and Akt. (F) Huh7 cells were treated with insulin (100 nM) for the indicated times (0, 5, 10, 15, 30, and 60 min). (G) Huh7 cells were pretreated with PA (300 μM) for 6 h and then incubated with Api (20 μM) for 5 min. (H) Cells were pretreated with PA (300 μM) for 6 h and then incubated with Api (20 μM) for 12 h, followed by insulin (100 nM) for 20 min in the presence of 2-deoxy-D-glucose (2-NBDG) (50 μM). Representative images of 2-NBDG (green) and DAPI (blue) by confocal microscopy. Glucose uptake was assessed using the assay kit. Data are expressed as mean ± standard error of the mean (SEM) from 5 independent experiments. *p < 0.05 vs. the vehicle group. #p < 0.05 vs. the PA alone group.

Figure 4

Apigenin prevents the palmitic acid (PA)‐induced increase in generation of reactive oxygen species in hepatocytes. (A and B) The intracellular level of superoxide was evaluated with hydroethidine (HE)/ethidium (ETH) fluorescent probe assay. (A) Huh7 cells were treated with PA (300 μM) for indicated times (0, 5, 10, 15, 30, and 60 min). (B) Huh7 cells were pretreated with apigenin (Api, 20 μM) for 2 h, and then with PA (300 μM) for 30 min. (C and D) The intracellular level of hydrogen peroxide was evaluated using dichlorodihydrofluorescein diacetate (DCFH-DA)/2', 7'-dichlorofluorescein (DCF) fluorescent probe assay. (C) Huh7 cells were treated with PA (300 μM) for the indicated times (0, 5, 10, 15, 30, and 60 min). (D) Huh7 cells were pretreated with Api (20 μM) for 2 h, and then with PA (300 μM) for 30 min. (E) Huh7 cells were pretreated with N-acetylcysteine (NAC, 10 mM) or apocynin (Apo, 50 μM) for 2 h, and then incubated with PA (300 μM) for an additional 6 h. Western blot analysis of CK2α, fetuin-A, and β-actin. (F) Huh7 cells were pretreated with NAC (10 mM) or Apo (50 μM) for 2 h, and then incubated with PA (300 μM) for an additional 12 h. Cellular lysates were immunoprecipitated (IP) with an anti-fetuin-A antibody and then immunoblotted with anti-phospho (p-Ser) antibody. IgG was used as a control for fetuin-A antibody. Data are expressed as mean ± standard error of the mean (SEM) from 5 independent experiments. *p < 0.05 vs. the vehicle group. #p < 0.05 vs. the PA alone group.

Figure 5

Effects of apigenin on body weight, tissue weight, and plasma lipids of HFD-fed mice. Eight-week-old C57BL/6 mice were fed with HFD and orally treated daily with apigenin (Api, 20 mg/kg) or vehicle (oil) for 12 weeks. (A) Weekly body weight changes in vehicle- and apigenin-treated groups. (B) The images of body appearance and the distribution of white adipose tissue (WAT) were indicated by arrows. (C) The body weight. (D) The weight gained. (E) The weight of the liver. (F) The weight of WAT. (G-J) Serum levels of triglycerides, total cholesterol, non-high-density lipoprotein cholesterol (non-HDL-c), and HDL cholesterol (HDL-c). (K-M) The appearance of the liver and representative histological images by hematoxylin and eosin (H&E) staining and Oil Red O staining. (N-S) The hepatic levels of total cholesterol, free cholesterol, cholesteryl ester, triglycerides, free fatty acids, and glycerol. Data are expressed as mean ± standard error of the mean (SEM) from 7 mice. *p < 0.05 vs. the vehicle-treated group.

Figure 6

Apigenin decreases hepatic inflammation in the liver of HFD-fed mice. Eight-week-old C57BL/6 mice were fed with HFD and orally treated daily with apigenin (Api, 20 mg/kg) or vehicle (oil) for 12 weeks. (A) The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and the ratio of AST/ALT. (B) The hepatic levels of interleukin 1β (IL-1β), IL-4, tumor necrosis factor-alpha (TNFα) and IL-6. (C) Western blot analysis of F4/80, myeloperoxidase (MPO), and β-actin. (D) Immunohistochemistry of F4/80 and MPO in the liver sections. Data are expressed as mean ± standard error of the mean (SEM) from 7 mice. *p < 0.05 vs. the vehicle group.

Figure 7

Apigenin decreases the expression of fetuin-A protein by regulating CK2α -NF-κB and autophagy pathways in the liver of HFD-fed mice. Eight-week-old C57BL/6 mice were fed with HFD and treated daily with apigenin (Api, 20 mg/kg) or vehicle (oil) for 12 weeks. (A) Western blot analysis and ELISA analysis of the serum and hepatic of fetuin-A. (B and C) Western blot analysis of CK2α, phosphorylated and total NF-κB, LC3, p62, and β-actin in the liver. Data are expressed as mean ± standard error of the mean (SEM) from 7 mice. *p < 0.05 vs. the vehicle-treated group.

Figure 8

Apigenin reduces oxidative stress in the liver of HFD-fed mice. Eight-week-old C57BL/6 mice were fed with HFD and treated daily with apigenin (Api, 20 mg/kg) or vehicle (oil) for 12 weeks. (A) The level of lipid peroxidation in the liver. (B) Western blot analysis of 4-hydroxynonenal (4-HNE) and β-actin. (C) Immunohistochemistry of 4-HNE in the liver sections. (D) Western blot analysis of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), superoxide dismutase (SOD)1, SOD2, glutathione peroxidase (GPx), and β-actin in the liver. Data are expressed as mean ± standard error of the mean (SEM) from 7 mice. *p < 0.05 vs. the vehicle group.

Figure 9

Apigenin attenuates HFD-induced insulin resistance by inhibiting fetuin-A phosphorylation and the formation of the fetuin-A-insulin receptor complex in the liver of mice. Eight-week-old C57BL/6 mice were fed with HFD and treated daily with apigenin (Api, 20 mg/kg) or vehicle (oil) for 12 weeks. (A) Liver lysates were immunoprecipitated (IP) with an anti-fetuin-A antibody, and immunoblotting (IB) was performed with anti-insulin receptor (IR) or anti-p-Ser antibodies. IgG was used as a control. (B) Representative confocal microscopy images of proximity ligation assay (PLA) were performed using anti-fetuin-A and anti-IR antibodies. The formation of fetuin-A-IR in liver sections was indicated by arrows. 4',6-diamidino-2-phenylindole (DAPI) (blue), PLA signals (green). (C) Western blot analysis of phosphorylated and total and of the IR substrate (IRS) and Akt. (D) Representative confocal microscopy images of glucose transporter 2 (GLUT2) in liver sections; GLUT2 located in cytosol is indicated by stars, GLUT2 located around cell membrane is indicated by arrows. (E) Results of oral glucose tolerance test (OGTT). Mice were orally administrated with glucose (1.0 g/kg of body weight) and the levels of blood glucose were measured at the indicated time points (0, 30, 60, 90, and 120 min). The area under curve (AUC) for OGTT was calculated. (F) Results of insulin tolerance test (ITT). Mice were intraperitoneally injected with insulin (0.75 unit/kg of body weight) and the levels of blood glucose were measured at the indicated time points (0, 30, 60, 90, and 120 min). AUC for ITT was calculated. Data are expressed as mean ± standard error of the mean (SEM) from 7 mice. *p < 0.05 vs. the vehicle-treated group.

Figure 10

Schematic illustration of the proposed molecular mechanisms by which apigenin protects the liver from obesity-induced insulin resistance. As shown, treatment with apigenin prevents lipid-induced deregulation of glucose metabolism by inhibiting ROS-induced CK2α expression and CK2α-mediated activation of signaling pathways including the phosphorylation of NF-κB and fetuin-A, resulting in reduction in the expression and stability of fetuin-A protein thereby preventing the formation of fetuin-A-insulin receptor (IR) complex, and ultimately improving obesity-induced insulin resistance.