Fig 1.
Effect of fetuin-A (Fet-A) on insulin signaling and insulin mediated suppression of gluconeogenesis and glucose production in HepG2.
[A] HepG2 cells were pre-treated with recombinant Fet-A in the presence or absence of insulin, and cell lysates were subjected to immunoblotting for AKT and GSK3 phosphorylation status (n = 3). [B] Quantified data of the ratio of cellular pAKT/GAPDH immunoblots (n = 3) and C] ratio of cellular pGSK/GAPDH immunoblots (n = 3) are shown. [D] HepG2 cells were serum-starved for 6 h, followed by treatment with dexamethasone (Dexa), insulin, or insulin and Fet-A for 12 h. Real-time gene expression of Pepck were analyzed (n = 4). [E] To analyze glucose production, HepG2 cells (n = 4) were treated with 0.5 μM dexamethasone and 0.1 mM 8-CTP-cAMP (Dex/cAMP), various concentrations of Fet-A or 100 nM insulin (Ins) in glucose free DMEM medium (pH 7.4 supplied with 20 mM sodium lactate and 2 mM sodium pyruvate) for 5 h. Glucose production was assayed by measuring glucose concentration in the medium as described previously [33]. Data are shown as Means ± SEM. P values were determined by one-way ANOVA followed by Tukey’s multiple comparison tests (A-C). Data are representative of at least three independent experiments performed in replicates. * Indicates p < 0.05.
Fig 2.
Activation of AMPK downregulates high glucose-induced Fet-A expression in HepG2 cells.
[A] HepG2 cells were incubated in a media containing either mannitol, low glucose or high glucose for 12 hours, and cell lysates/media were subjected to immunoblotting for Fet-A and pFet-A (n = 3). [B] HepG2 cells were incubated with either low- or high-glucose in the absence or presence of AICAR for 12 h and cell lysates were analyzed by Western blotting. The blots were analyzed with antibodies against Fet-A (n = 4). [C] HepG2 cells were incubated with increasing concentrations (0.5, 1, 2 mM) of AICAR for 12 h. Cell lysate or media were analyzed by Western blotting for indicated proteins (n = 3) and [D] level of Fet-A and pFet-A in media, as a ratio of GAPDH were expressed. [E] HepG2 cells were incubated in low or high glucose in the absence or presence of AICAR/metformin for 12 hours, and media was used to detect Fet-A by ELISA technique (n = 4). [F] Real-time gene expression analysis was carried out for Fet-A after AICAR and metformin treatment (n = 4). [G] HepG2 cells were incubated with AICAR/metformin in the presence or absence of Compound C, an AMPK inhibitor. Cell lysates were immunoblotted for pAMPK, Fet-A as well as p-Fet-A and [H] Fet-A levels, as a ratio to GAPDH are depicted (n = 4). Data are shown as Means ± SEM. P values were determined by one-way ANOVA followed by Tukey’s multiple comparison tests (C-F). Data are representative of at least three independent experiments performed in replicates. * Indicates p < 0.05.
Fig 3.
AMPK activation downregulates Fet-A expression through p38 MAPK.
[A] HepG2 cells were incubated with either low- or high-glucose in the absence or presence of AICAR for 12hr and used for Western blot analysis for ERK1/2 phosphorylation expression (n = 3). [B] HepG2 cells were incubated with different concentration of AICAR for 12 h and cell lysates were analyzed by Western blotting for ERK1/2, p38MAPK and JNK phosphorylation (n = 3). [C] Cells were treated with p38 MAPK inhibitor [SB202190, n = 4] before treatment of AICAR for 12 hr. Cell lysate or media were analyzed by Western blotting for indicated proteins and [D] Fet-A levels, as a ratio to GAPDH were determined. [E] Cells were treated with AMPK inhibitor [Comp C, n = 4] before treatment of AICAR for 12hr. Cell lysates were analyzed by Western blotting for phosphorylated p38 MAPK (pP38MAPK) and [F] pP38MAPK levels, as a ratio to GAPDH were determined. [G] Knockdown of p38 MAPK was performed using MAPK14 [p38 MAPK] small interfering RNA [siRNA] in HepG2 cells. Following AICAR treatment for 12 h, cell lysates were analyzed by Western blotting for expression of p38 MAPK, phosphorylated p38 MAPK, Fet-A, and pAMPK. [H] Efficiency of p38MAPK siRNA in HepG2 cells were determined by immunoblotting transfected cells for p38MAPK and levels were expressed as a ratio to GAPDH. [I] Effect of AICAR on Fet-A expression in scrambled or p38MAPK siRNA transfected cells were determined by expressing Fet-A levels, as a ratio to GAPDH (n = 4). [J] Effect of protein synthesis inhibitors, cycloheximide and puromycin, were compared with anisomycin, also a protein synthesis inhibitor, for effects on Fet-A and phosphorylated p38 MAPK expression (n = 3). Data are shown as Means ± SEM. P values were determined accordingly by either by unpaired two-tailed test (E) or one-way ANOVA followed by Tukey’s multiple comparison tests (C-F). * Indicates p < 0.05.
Fig 4.
Effect of AICAR or anisomycin treatment on Fet-A expression in Hep3B cells and primary rat hepatocytes.
[A] Hep3B cells were incubated in low or high glucose media with or without AICAR for 12 hours, and cell lysates were subjected to immunoblotting for Fet-A and phosphorylated Fet-A (n = 4). [B] High glucose-mediated changes in Fet-A level in Hep3B cells was expressed as a ratio to GAPDH. [C] Hep3B cells were incubated with different concentration of AICAR for 12 h and cell lysates were analyzed by Western blotting for indicated proteins (n = 3). [D] Effect of AICAR on Fet-A level in Hep3B cells was expressed as a ratio of GAPDH. [E] Hep3B cells were treated with various concentrations of anisomycin for 0.5 h to analyze phosphorylated p38 MAPK (pP38MAPK) and Fet-A expression (n = 3). [F] Effect of anisomycin on Fet-A level in Hep3B cells was expressed as a ratio of GAPDH [G] Primary rat hepatocytes were incubated with different concentration of AICAR for 12 h and cell lysate or media were analyzed by Western blotting for indicated proteins (n = 4). [H] Effect of AICAR in primary rat hepatocytes on Fet-A levels were expressed as ratio to GAPDH [I] primary rat hepatocytes were treated with anisomycin for 0.5 h to analyze phosphorylated p38 MAPK (pP38MAPK), Fet-A and p38MAPK expression (n = 4). [J] Effect of anisomycin in primary rat hepatocytes on Fet-A levels were expressed as ratio to GAPDH (n = 4). Data are shown as mean ± SEM. P values were determined accordingly by either by unpaired two-tailed test (E) or one-way ANOVA followed by Tukey’s multiple comparison tests (A-D). * Indicates p < 0.05.
Fig 5.
AMPK regulates Fet-A expression by proteosomal degradation and decreased C/EBP beta expression.
[A] HepG2 cells were treated with AICAR [2mM] at 2 and 4 hr in the presence or absence of MG-132 and used for Western blot analysis of pAMPK, phosphorylated p38MAPK (pP38MAPK), and Fet-A expression (n = 3). [B] Effect of AICAR and MG-132 treatment for 2 and 4 hr on Fet-A levels were expressed as a ratio to GAPDH in HepG2 cells [C] HepG2 cells were treated with AICAR [2mM, 2, 4 and 12 hr] and immunoprecipitated with anti-Fet-A antibody. The immunoprecipitates were analyzed by Western blotting for anti-ubiquitin and Fet-A antibodies (n = 3). [D] HepG2 cells were treated with AICAR [2mM] in the presence or absence of different concentration of MG-132 for 12 hr and used for Western blot analysis for Fet-A expression (n = 2). [E] Effect of AICAR and MG-132 treatment for 12 hr on Fet-A levels were expressed as a ratio to GAPDH in HepG2 cells. [F] HepG2 cells were incubated with different concentration of AICAR or metformin for 12 hr. Cell lysates were analyzed by Western blotting for C/EBP beta (n = 4) and [G] levels were expressed as a ratio to GAPDH. [H] Primary rat hepatocytes were treated with different concentration of AICAR for 12 hr, lysates were used to immunoblotting for C/EBP beta (n = 4) and [I] levels were expressed as a ratio to GAPDH. Data are shown as Means ± SEM. P values were determined accordingly by one-way ANOVA followed by Tukey’s multiple comparison tests (A, C-E). * Indicates p < 0.05.
Fig 6.
Working model for AICAR-mediated regulation of hepatic Fet-A expression.
Metformin or AICAR increases the phosphorylation of AMPK, which activates p38 MAPK. As a result, AMPK-p38MAPK signaling suppresses high-glucose induced Fet-A expression either by increasing proteasomal degradation and/or by decreasing hepatic expression of C/EBP beta.