AMP-activated protein kinase activation increases phosphorylation of glycogen synthase kinase 3beta and thereby reduces cAMP-responsive element transcriptional activity and phosphoenolpyruvate carboxykinase C gene expression in the liver

Abstract

AMP-activated protein kinase (AMPK) activation reportedly suppresses transcriptional activity of the cAMP-responsive element (CRE) in the phosphoenolpyruvate carboxykinase C (PEPCK-C) promoter and reduces hepatic PEPCK-C expression. Although a previous study found TORC2 phosphorylation to be involved in the suppression of AMPK-mediated CRE transcriptional activity, we herein present evidence that glycogen synthase kinase 3beta (GSK3beta) phosphorylation induced by AMPK also plays an important role. We initially found that injecting fasted mice with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) markedly increased Ser-9 phosphorylation of hepatic GSK3beta within 15 min. Stimulation with AICAR or the GSK3beta inhibitor SB-415286 strongly inhibited CRE-containing promoter activity in HepG2 cells. Using the Gal4-based transactivation assay system, the transcriptional activity of cAMP-response element-binding protein (CREB) was suppressed by both AICAR and SB415286, whereas that of TORC2 was repressed significantly by AICAR but very slightly by SB415286. These results show inactivation of GSK3beta to directly inhibit CREB but not TORC2. Importantly, the AICAR-induced suppression of PEPCK-C expression was shown to be blunted by overexpression of GSK3beta(S9G) but not wild-type GSK3beta. In addition, AICAR stimulation decreased, whereas Compound C (AMPK inhibitor) increased CREB phosphorylation (Ser-129) in HepG2 cells. The time-courses of decreased CREB phosphorylation (Ser-129) and increased GSK3beta phosphorylation were very similar. Furthermore, AMPK-mediated GSK3beta phosphorylation was inhibited by an Akt-specific inhibitor in HepG2 cells, suggesting involvement of the Akt pathway. In summary, phosphorylation (Ser-9) of GSK3beta is very likely to be critical for AMPK-mediated PEPCK-C gene suppression. Reduced CREB phosphorylation (Ser-129) associated with inactivation of GSK3beta by Ser-9 phosphorylation may be the major mechanism underlying PEPCK-C gene suppression by AMPK-activating agents such as biguanide.

FIGURE 1.

AICAR induced GSK3β phosphorylation in livers of fasted mice. Ten-week-old C57BL6 mice were fasted overnight, then given an intraperitoneal injection of AICAR (250 mg/kg) before sacrifice followed by excision of the liver. Ser-9 phosphorylation (P-) of GSK3β was assessed by Western blotting. The phospho-GSK3β was quantified densitometrically, and the results are presented in the lower panel. The results are presented as the mean ± S.E. of three independent experiments.

FIGURE 2.

Inhibition of PEPCK-C reporter by AICAR in HepG2 cells in the presence and the absence of forskolin. AICAR or the GSK3β inhibitor SB-415286 has the potential to down-regulate CRE-dependent transcription in the presence and in the absence of 10 μm forskolin. HepG2 cells were transfected with the PEPCK-C promoter fused to the luciferase reporter plasmid (pGL4.10-PEPCK-C: 0.5 μg) with an internal reporter, pRL-TK (0.03 μg). HepG2 cells were serum-starved overnight before incubation with AICAR (2 mm) or insulin (10 nm) with or without 10 μm forskolin. After a 6-h incubation, cells were harvested for the reporter assay. A, the PEPCK-C-dependent reporter activities that had been normalized with internal reporter activities were expressed as -fold activities of the empty reporter (pGL4.10). B, HepG2 cells were transfected with the CRE-reporter plasmid (pTAL-CRE: 0.25 μg) with the internal reporter pRL-TK (0.03 μg). HepG2 cells were serum-starved overnight before incubation with AICAR (2 mm), insulin (10 nm), the selective AMPK inhibitor Compound C (20 μm), GSK3β inhibitor, or SB-415286 (30 μm) with or without 10 μm forskolin. After a 6-h incubation, cells were harvested for the reporter assay. C, AMPK (WT, DN) was overexpressed by adenoviral (Ad) gene transfer, and expressions were detected by anti-AMPK antibody (upper blot in the right panel). AMPK activity was monitored by antiphospho-Ser79-ACC (lower blot in the right panel). The cells were transfected with the CRE-reporter plasmid (pTAL-CRE, 0.25 μg) with the internal reporter pRL-TK (0.03 μg), and the reporter assay was performed.

FIGURE 3.

Contributions of CREB and TORC2 to AMPK-, SIK1-, and GSK3β-mediated suppressions of CRE activity. HepG2 cells were cotransfected with the expression plasmids for GAL4-truncated CREB (bZIP domain) (0.25 μg) or GAL4-truncated TORC2 (coiled coil region) (0.25 μg) and a pTAL-5× UAS reporter plasmid (0.25 μg) with the internal reporter pRL-TK (0.03 μg) (A and B). HepG2 cells were serum-starved overnight before incubation with AICAR (2 mm), insulin (10 nm), and GSK3β inhibitor or SB-415286 (30 μm) with or without 10 μm forskolin. Adenovirus (Ad)-mediated overexpressions of SIK1 proteins were detected using an anti-SIK1 immunoblot (lower panel). The specific transactivation activities of TORCs were expressed as the -fold activation of the empty Gal4 vector, pM. Means and S.D. are indicated (n = 3). *, p < 0.05.

FIGURE 4.

Effect of GSK3β inhibitor SB-415286-, AICAR-, or adenovirus-expressed SIK1 (Ad-SIK1) on P-TORC2 levels in primary hepatocytes in the presence and the absence of forskolin. A, stimulation of hepatocytes with GSK3β inhibitor, SB415286, did not shift the mobility of TORC2 in response to the presence or absence of forskolin. Primary hepatocytes were serum-starved overnight before a 2-h incubation with 30 μm SB-415286 (lanes 3 and 4) or 2 mm AICAR (lanes 5 and 6) with or without 10 μm forskolin. TORC2 phosphorylation was detected by Western blotting as a phosphorylation-dependent mobility shift (TORC2, dephosphorylated; P-TORC2, phosphorylated) in primary hepatocytes. AMPK activity was monitored by antiphospho-Ser-79-ACC (lower panel). B, TORC2 was strongly phosphorylated in adenovirus-expressing SIK1. Primary hepatocytes were stimulated with 2 mm AICAR (lanes 3 and 4) and adenovirus-mediated overexpressions of SIK1(lanes 5 and 6) with or without 10 μm forskolin. AMPK activity was monitored by antiphospho-Ser-79-ACC (middle panel). SIK1 proteins were detected using an anti-SIK1 immunoblot (lower panel). Data are representative of at least three independent experiments.

FIGURE 5.

Mutant GSK3β(S9G) overexpression affects PEPCK-C transcriptional activity in HepG2 cells. HepG2 cells were transiently transfected with pGL4.10-PEPCK-C (0.5 μg) with the internal reporter pRL-TK (0.03 μg). After 5 h of transfection, HepG2 cells were infected with LacZ, GSK3β(WT), or GSK3β(S9G) adenovirus. After 48 h of transfection, the cells were stimulated with or without 2 mm AICAR or 10 μm forskolin for 6 h. A, adenovirus-mediated overexpression of GSK3β(WT, S9G) was detected by anti-GSK3β antibody or anti-phospho-(P-)Ser-9-GSK3β. B, the cells were incubated in the presence and in the absence of 2 mm AICAR or 10 μm forskolin, and the reporter assays were then performed. The results are presented as mean relative luciferase (LUC) units (±S.E.), normalized to Renilla LUC activity, derived from three independent experiments. *, p < 0.05.

FIGURE 6.

AMPK activation increased phosphorylation of GSK3β in HepG2 cells. HepG2 cells were treated with AICAR (2 mm) or the selective AMPK inhibitor Compound C (20 μm). A, immunoblot analyses were performed with HepG2 cell extracts prepared using anti-GSK3β, anti-phospho (P-)-GSK3β (Ser-9), anti-CREB, antiphospho-CREB (Ser-129), or anti-phospho-ACC (Ser-79) antibody as indicated. B, Western blot analyses of time-dependent effects were performed with HepG2 cell extracts prepared using anti-GSK3β, anti-phospho-GSK3β (Ser-9), anti-CREB, anti-phospho-CREB (Ser-129), anti-phospho-Akt (Thr-308), or anti-phospho-ACC (Ser-79) antibody. Panels C and D, show the results of densitometric analysis of phospho-GSK3β (Ser-9) and phospho-CREB (Ser-129) as the mean ± S.E. of three samples.

FIGURE 7.

Western blot analysis in AICAR-treated HepG2 cells in the absence or the presence of Akt inhibitor. The Ser-9 phosphorylation (P-) levels of GSK3β were detected by Western blotting using an anti-P-Ser-9-GSk3β antibody (upper panel). HepG2 cells were detected by treatment with AICAR (2 mm) for 1 h or insulin (1 μm) for 15 min after pretreatment with or without 10 μm Akt inhibitor IV. Pretreatment with 10 μm Akt inhibitor IV inhibited AICAR-induced GSK3β phosphorylation (P). The lower panel shows the results of densitometric analysis of phospho-GSK3β (Ser-9) as the mean ± S.E. of three samples. *, p < 0.05.

FIGURE 8.

AICAR stimulation inhibited CREB-CBP/P300 interaction. Amounts of CBP associated with CREB were determined by immunoprecipitation with extracts obtained from transfected HepG2 cells. HepG2 cells transfected with GST-tagged CREB were serum-starved overnight before incubation with AICAR (2 mm) with or without 10 μm forskolin for 1 h. HepG2 cells transfected with GST-tagged CREB were extracted and subjected to immunoprecipitation using GST-specific antibody. Shown is a Western blot of GST-tagged CREB-transfected HepG2 cell lysates with a CBP/P300-specific antibody showing immunoprecipitation of exogenously expressed GST-tagged CREB with endogenous CBP/P300 (upper lanes, immunoprecipitation of endogenous CBP/P300 from GST-tagged CREB transfected cells with the CBP/P300-specific antibody; lower lane, immunoprecipitation (IP) of GST-tagged CREB with the anti-CREB antibody). The results are presented as the mean ± S.E. of three independent experiments.

FIGURE 9.

A model of the role of the AMPK-GSK3β-CREB signaling pathway in gluconeogenesis. PKA, protein kinase A.