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First published online 28 March 2006
doi: 10.1242/jcs.02865

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Extracellular adenosine activates AMP-dependent protein kinase (AMPK)

¹ Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona, 08071 Barcelona, Spain
² Institut Biomédical des Cordeliers, U465 INSERM, Paris, France

View larger version (20K): [in a new window]   Fig. 1. Adenosine activates AMPK in IEC-6 cells. (A) IEC-6 cells were incubated either in the presence or absence of 10 µM adenosine (Ado) or 500 µM AICAR (Aicar) for 10 minutes and 1 hour, respectively; C, control. AMPK complexes were isolated from cell lysates by immunoprecipitation with antibodies to either anti-1AMPK (open bars) or anti-2AMPK antibody (solid bars). 1AMPK and 2AMPK complex activities were measured by phosphorylation of AMARA peptide. Activities shown are the mean and s.e. of five and three independent experiments for 1AMPK and 2AMPK, respectively, with each assayed in duplicate. The statistical significance of the adenosine and AICAR effects was established by Student's t-test (**P<0.01; ***P<0.001). (B) Cells were treated with either adenosine or AICAR or control (Ctrl), as detailed above. Equal amounts of protein (40 µg) were loaded to allow direct comparisons of phosphorylated AMPK (P-AMPK) levels. ß-actin was used as a loading control. A representative western blot is shown.

View larger version (18K): [in a new window]   Fig. 2. Dose dependence of the adenosine effect on AMPK phosphorylation and activity. (A) Phosphorylated AMPK (P-AMPK) protein levels were monitored by western blot at the indicated times after the addition of 10 µM adenosine. (B) The dose dependence of the adenosine effect was assessed after treatment for 10 minutes at the adenosine concentrations shown. Representative western blots are shown. The magnitude of the induction triggered in four independent experiments was quantified densitometrically. Results (mean ± s.e.m.) are shown as arbitrary units normalized to control values (non-treated cells). (C) Endogenous 1AMPK activity was measured 10 minutes after the addition of adenosine at the indicated concentrations. Non-linear fitting of the data was used for the estimation of the K0.5 value. Results are the mean and s.e. from four independent experiments. The statistical significance of the adenosine effect was assessed by analysis of variance (P<0.05) combined with the Student's t-test: *P<0.05; **P<0.01; ***P<0.001. Ado, adenosine; Ctrl, control.

View larger version (21K): [in a new window]   Fig. 3. Adenosine promotes phosphorylation of the AMPK downstream target acetyl-CoA carboxylase (ACC) in IEC-6 cells. (A) Western blot analysis of phosphorylated ACC (P-ACC) was performed after treatment for 10 minutes and 60 minutes with 10 µM adenosine (Ado) and 500 µM AICAR (Aicar), respectively; Ctrl, control. A representative immunoblot is shown. The densitometric analysis corresponds to the mean ± s.e.m. of three independent experiments. The statistical significance was assessed by Student's t-test: *P<0.05; ***P<0.001. (B) Cells were infected with either Ad.Null or Ad.1DN adenoviral vectors (30 pfu/cells). At 24 hours post-infection, cells were incubated for 10 minutes either in the presence (Ado) or the absence (C) of 10 µM adenosine. Total AMPK activity was measured in cell lysates, without prior immunoprecipitation, using the AMARA peptide assay and ACC regulation was monitored by western blot under the same conditions. As a control for infection efficiency, total 1AMPK subunit protein was measured by western blot in both Ad.Null- and Ad.1DN-infected cultures.

View larger version (18K): [in a new window]   Fig. 4. Adenosine phosphorylation is required for AMPK activation in IEC-6 cells. The effect of adenosine kinase inhibition on the increase in AMPK activity triggered by adenosine (Ado) was monitored after pre-incubating the cells with 0.2 µM 5'-iodotubercidin (5'-ITU) for 30 minutes. (A) Endogenous 1AMPK activity (mean ± s.e.m.) from four independent experiments. (B) Two representative western blots of phosphorylated AMPK (P-AMPK) and phosphorylated ACC (P-ACC), and corresponding densitometric analysis (means ± s.e.m.). (C) The effect of 5'-ITU on AICAR-triggered AMPK stimulation was determined. A representative western blot and corresponding densitometric analysis is shown. The statistical significance was assessed by Student's t-test: A: *P<0.05, **P<0.01; B: *P<0.05; C: **P<0.01. Ctrl or C, control.

View larger version (21K): [in a new window]   Fig. 5. Basal CNT2 expression and activity in intestinal and liver cells. (A) Reverse transcriptase (RT)-PCR was performed on RNA from intestinal mucosa (I), IEC-6 cells, liver (L), FAO cells and rat liver parenchymal cells (Hep). (B) CNT2 protein expression was analyzed in crude extracts (H) and total membrane (M) fractions from IEC-6 cells, FAO cells and rat hepatocytes (Hep) by western blot. (C) CNT2-mediated uptake (open bars) was measured, as indicated in the Materials and Methods, using 1 µM adenosine as a substrate and 1 minute incubation time. Na+-independent adenosine uptake (solid bars) corresponds to the sum of ENT1- and ENT2-related transport activities, plus putative binding. Results are the mean ± s.e.m. of four experiments.

View larger version (20K): [in a new window]   Fig. 6. CNT2-mediated adenosine transport is required for AMPK activation in IEC-6 cells. Cells were treated with 1 µM adenosine (Ado), either in the absence or presence of 100 µM formycin B (FB) for 10 minutes, and then processed as indicated previously. (A) 1AMPK activity (mean ± s.e.m.) from four independent experiments. (B) A representative western blot of phosphorylated ACC (P-ACC) and corresponding densitometric analysis of the gels (mean ± s.e.m.). (C) Cells were incubated with 1 µM adenosine, either in the absence or the presence of 1 µM NBTI and 1 µM dipyridamole (Dip) for 10 minutes. A representative western blot of phosphorylated ACC and the corresponding densitometric analysis (mean ± s.e.m.) is shown. The statistical significance was assessed by Student's t-test: A: *P<0.05, **P<0.01; B: *P<0.05, ***P<0.001; C: **P<0.01. Ctrl or C, control.

View larger version (28K): [in a new window]   Fig. 7. Adenosine activates AMPK and phosphorylates its downstream target in the hepatoma cell line FAO and primary culture of rat hepatocytes. (A) FAO cells (Fao) were treated with either 10 µM adenosine (Ado) or 500 µM AICAR (Aicar) for 30 minutes and 60 minutes, respectively. A representative western blot of the phosphorylated form of ACC (P-ACC) and densitometric analysis of four independent experiments are shown. (B) Primary cultures of rat hepatocytes were incubated in the presence of 10 µM adenosine and protein extracts were analyzed at the indicated times for ACC phosphorylation. A western blot and the corresponding densitometric analysis of three independent experiments are shown. ß-actin is used as a loading control. The statistical significance was assessed by Student's t-test: A: ***P<0.001; B: **P<0.01. Ctrl, control.

View larger version (19K): [in a new window]   Fig. 8. Adenosine phosphorylation is necessary for AMPK and ACC phosphorylation in FAO cells. The effect of adenosine kinase inhibition on the increase in AMPK and ACC phosphorylation triggered by adenosine (Ado) was monitored after pre-incubating the cells with 0.2 µM 5'-iodotubercidin (ITU) for 30 minutes. Two representative western blots of phosphorylated AMPK (P-AMPK) (A) and phosphorylated ACC (P-ACC) (B), and corresponding densitometric analysis (means ± s.e.m.) of six independent experiments are shown. Statistical significance was assessed by Student's t-test: **P<0.01; *P<0.05. Ctrl or C, control.

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