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. 2015 May 1;467(3):461-72.
doi: 10.1042/BJ20141142.

PT-1 selectively activates AMPK-γ1 complexes in mouse skeletal muscle, but activates all three γ subunit complexes in cultured human cells by inhibiting the respiratory chain

Thomas E Jensen  1 , Fiona A Ross  2 , Maximilian Kleinert  1 , Lykke Sylow  1 , Jonas R Knudsen  1 , Graeme J Gowans  2 , D Grahame Hardie  2 , Erik A Richter  1
Affiliations

PT-1 selectively activates AMPK-γ1 complexes in mouse skeletal muscle, but activates all three γ subunit complexes in cultured human cells by inhibiting the respiratory chain

Thomas E Jensen et al. Biochem J. .

Abstract

AMP-activated protein kinase (AMPK) occurs as heterotrimeric complexes in which a catalytic subunit (α1/α2) is bound to one of two β subunits (β1/β2) and one of three γ subunits (γ1/γ2/γ3). The ability to selectively activate specific isoforms would be a useful research tool and a promising strategy to combat diseases such as cancer and Type 2 diabetes. We report that the AMPK activator PT-1 selectively increased the activity of γ1- but not γ3-containing complexes in incubated mouse muscle. PT-1 increased the AMPK-dependent phosphorylation of the autophagy-regulating kinase ULK1 (unc-51-like autophagy-activating kinase 1) on Ser555, but not proposed AMPK-γ3 substrates such as Ser231 on TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1 (TBC1D1) or Ser212 on acetyl-CoA carboxylase subunit 2 (ACC2), nor did it stimulate glucose transport. Surprisingly, however, in human embryonic kidney (HEK) 293 cells expressing human γ1, γ2 or γ3, PT-1 activated all three complexes equally. We were unable to reproduce previous findings suggesting that PT-1 activates AMPK by direct binding between the kinase and auto-inhibitory domains (AIDs) of the α subunit. We show instead that PT-1 activates AMPK indirectly by inhibiting the respiratory chain and increasing cellular AMP:ATP and/or ADP:ATP ratios. Consistent with this mechanism, PT-1 failed to activate AMPK in HEK293 cells expressing an AMP-insensitive R299G mutant of AMPK-γ1. We propose that the failure of PT-1 to activate γ3-containing complexes in muscle is not an intrinsic feature of such complexes, but is because PT-1 does not increase cellular AMP:ATP ratios in the specific subcellular compartment(s) in which γ3 complexes are located.

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Figures

Figure 1
Figure 1
PT-1 dose-dependently increases AMPK Thr172 phosphorylation but slightly suppresses muscle glucose transport. A) C57BL/6 soleus and EDL muscles were incubated with 0, 40, 80 and 100 μM PT-1 for 1h after which AMPK Thr172 was measured by western blotting. Actin was used as a loading control. B) Representative blots and quantifications of AMPK Thr172 in soleus and EDL muscle from wild type vs. kinase-dead (KD) AMPK mice C) 2DG transport in the same muscles. */*** p<0.05/0.001 ANOVA main-effect compared to basal, ### p < 0.001 genotype-effect. n = 2 for optimization and n= 4 for wild type vs. kinase-dead AMPK mice. Results are mean ± SE.
Figure 2
Figure 2
PT-1 stimulates AMPK Thr172, but not ACC2 or TBC1D1 phosphorylation, whereas AICAR stimulates all. Western blotting were performed on C57BL/6 soleus and EDL muscles stimulated with PT-1 (100 μM, 1h), AICAR (2 mM, 1h) or combined PT-1+AICAR treatment to assess A) AMPK Thr172, B) ACC2 Ser212, C) TBC1D1 Ser231 in EDL and D) TBC1D1 Thr590 in EDL. E) Glycogen synthase (GS) Ser8 in soleus and EDL. Representative blots are shown in F). To verify the TBC1D1 Ser231 antibody in G), the top band of the doublet recognized by the phospho-Ser231 antibody in AICAR-stimulated mouse EDL was shown to align with total TBC1D1 and H) to be depleted by immunoprecipitation with total TBC1D1 but not TBC1D4. I) Immunoblot of ACC1, ACC2 and ACC1/2 Ser79/Ser212 in mouse muscle and liver as indicated. J) The GS Ser8 phosphorylation was reduced in resting kinase-dead (KD) AMPK overexpressing soleus muscles compared to wild type. */**/***p <0.05/0.01/0.001 T-test or Tukey post hoc difference compared to control, ǂ/ǂǂǂ p<0.05/0.001 Tukey post hoc difference compared to AICAR. n=8 for PT+AICAR experiments, n=6 for wild type vs. kinase-dead (KD) AMPK muscles. Results are mean ± SE.
Figure 3
Figure 3
In contrast to AICAR, PT-1 does not stimulate muscle glucose transport. C57BL/6 soleus and EDL muscles were stimulated with PT-1 (100 μM, 1h), AICAR (2 mM, 1h) or combined PT-1+AICAR treatment after which 2-deoxyglucose transport was measured in A) soleus and B) EDL. *** p < 0.001 Tukey post hoc difference compared to Basal. n=8. Results are mean ± SE.
Figure 4
Figure 4
PT-1 increases γ1 AMPK, but not α2β2γ3 AMPK associated kinase activity in vitro, whereas AICAR stimulates all complexes. AMPK complexes were sequentially immunoprecipitated from C57BL/6 soleus and EDL muscles (n=8) stimulated with PT-1 (100 μM, 1h), AICAR (2 mM, 1h) or combined PT-1+AICAR treatment to isolate the activities of A) α1β1γ1 and α1β2γ1, B) α2β1γ1 and α2β2γ1, C) α2β2γ3 AMPK. D) Immunoblots and coomassie (loading control) of Raptor Ser792 and ULK1 Ser555 phosphorylation in wild type and kinase-dead (KD) AMPK soleus and EDL ± PT-1 (n=5-7), E) ± AICAR (n=3). E) Quantification of ULK1 Ser555 phosphorylation and F) Quantification of Raptor Ser792 phosphorylation. */**/*** p <0.05/0.01/0.001 Tukey post hoc or T-test difference. †/††/††† p <0.05/0.01/0.001 ANOVA main-effect or interaction (if only above stimulated bars). Results are mean ± SE.
Figure 5
Figure 5
PT-1 fails to fully activate AMPK in a cell line expressing an AMP-insensitive AMPK-γ1 mutant. (A) Characterization by Western blotting of parental HEK-293 cells and cells stably expressing either the wild type (WT) or the R299G mutant (RG) of AMPK-γ1. Duplicate cell lysates were blotted with the indicated antibodies. (B) Effect of incubating WT or RG cells with increasing concentrations of berberine for 1 hr. AMPK was immunoprecipitated using anti-FLAG antibody and immunoprecipitates assays for AMPK activity. Results are expressed as % of the activity in a control without berberine. Mean values significantly different from controls without berberine (1-way ANOVA with Dunnett’s multiple comparison test, n = 4) are indicated: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (C) As (B), but replacing berberine by PT-1 (n = 3). (D) Effect of PT-1 (n = 3) on phosphorylation of Thr172; Western blots for total AMPK-α and FLAG are also shown to confirm equal loadings.
Figure 6
Figure 6
Effect of PT-1 on cellular ADP:ATP and AMP:ATP ratios, and cellular oxygen uptake. (A) Effect on adenine nucleotide ratios; cells were incubated with the indicated concentration of PT-1 for 1hr, perchloric acid extracts prepared, and ADP:ATP ratios estimated by capillary electrophoresis [26]. AMP:ATP ratios were calculated from ADP:ATP by assuming that the adenylate kinase reaction was at equilibrium, so that AMP:ATP = (ADP:ATP)2 [29]. Results are mean ± SD (n = 2). (C) Effect on oxygen uptake; cells were grown in Seahorse™ plates and the baseline rate of oxygen uptake measured in the Seahorse™ XF24 Extracellular Flux Analyzer. At the point shown by the first vertical arrow, PT1 (indicated concentrations), phenformin (3 mM) or vehicle (DMSO) were added and oxygen uptake measured for 75 min. Dinitrophenol (DNP, 100 μM) was then added and measurement of oxygen uptake continued for a further 15 min. results are expressed as percentages of baseline oxygen uptake as mean ± SD (n =3, DMSO/phenformin; n = 4, PT-1).
Figure 7
Figure 7
Effect of PT-1 on the kinase activity in cell-free assays of bacterially expressed constructs derived from rat AMPK-α1 and native AMPK from rat liver. (A) Effect of increasing concentrations of PT-1 on the activity of constructs containing the KD and AID (1-333) and the KD only (1-312). (B) Effect of increasing concentrations of PT-1, and of AMP (200 μM) and A-769662 (1 μM) on the activity of AMPK purified from rat liver.
Figure 8
Figure 8
Phenformin and PT-1 are equally effective in stimulating the activity of AMPK complexes containing γ1, γ2 and γ3 in HEK-293 cells. (A) Characterization by Western blotting of parental HEK-293 cells and cells stably expressing either wild type -γ1, -γ2 or -γ1. Duplicate cell lysates were blotted with the indicated antibodies. (B) Bar chart showing AMPK activity measured in anti-FLAG immunoprecipitates of cells treated for 1 hr with phenformin (10 mM) or PT-1 (100 μM). Results are mean ± SD (n = 3, separate cell incubations), and results that are significantly different from the relevant control without phenformin or PT-1 (1-way ANOVA with Sidak’s multiple comparison test of selected datasets) are shown with asterisks (**** p<0.0001). The lower panels show Western blots of duplicate dishes of cells probed using anti-pT172 (top), anti-AMPK-α (centre) or anti-FLAG (bottom).
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