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Am J Physiol Heart Circ Physiol 290: H2472-H2479, 2006. First published January 20, 2006; doi:10.1152/ajpheart.01206.2005
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Activation of cardiac AMP-activated protein kinase by LKB1 expression or chemical hypoxia is blunted by increased Akt activity

Carrie-Lynn M. Soltys, Suzanne Kovacic, and Jason R. B. Dyck

Cardiovascular Research Group, Departments of Pediatrics and Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada

Submitted 15 November 2005 ; accepted in final form 17 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
AMP-activated protein kinase (AMPK) plays a major role in the regulation of cardiac energy substrate utilization and can be negatively regulated by Akt activation in the heart. It has recently been shown that Akt directly phosphorylates AMPK{alpha}1/{alpha}2 on Ser485/491 in vitro and prevents the AMPK kinase (AMPKK) LKB1 from phosphorylating AMPK{alpha} at its primary activation site, Thr172 (S Horman, D Vertommen, R Heath, D Neumann, V Mouton, A Woods, U Schlattner, T Wallimann, D Carling, L Hue, and MH Rider. J Biol Chem 281: 5335–5340, 2006). To determine whether this is also the case in the cardiac myocyte, neonatal rat cardiac myocytes (NRCM) were infected with a recombinant adenovirus expressing a constitutively active mutant of Akt1 (myrAkt1) and then with or without adenoviruses expressing the active LKB1 complex. Expression of myrAkt1 blunted LKB1-induced phosphorylation of AMPK{alpha} at Thr172, which resulted in a dramatic decrease in phosphorylation of AMPK's target, acetyl CoA-carboxylase. This decrease in AMPK activity was associated with prior Akt1-dependent phosphorylation of AMPK{alpha}1/{alpha}2 at Ser485/491. To investigate whether Akt1 activation was also able to prevent other AMPKKs from phosphorylating AMPK{alpha}, we subjected NRCM to chemical hypoxia and noted a marked increase in phosphorylation of AMPK{alpha} at Thr172, despite no change in LKB1 activity. NRCM expressing myrAkt1 demonstrated increased phosphorylation of AMPK{alpha}1/{alpha}2 at Ser485/491 and a complete inhibition of chemical hypoxia-induced phosphorylation of AMPK{alpha} at Thr172. Taken together, our data show that activation of Akt1 is able to prevent activation of cardiac AMPK by LKB1 and at least one other AMPKK, likely by prior phosphorylation of AMPK{alpha}1/{alpha}2 at Ser485/491.

cardiac myocyte; metabolism; insulin; ischemia; AMP-activated protein kinase kinase

AMP-ACTIVATED PROTEIN KINASE (AMPK), a serine/threonine protein kinase (consisting of {alpha}-, beta-, and {gamma}-subunits), is a major regulator of cardiac energy substrate utilization. AMPK is activated by metabolic stresses that deplete cellular ATP (see Ref. 10 for review) or by a fall in the phosphocreatine-to-creatine ratio (28). When ATP levels fall, there is a corresponding increase in intracellular AMP levels, and AMPK is activated allosterically by AMP and by phosphorylation of the catalytic subunit ({alpha}) by at least one upstream AMPK kinase (AMPKK). This phosphorylation occurs at amino acid 172 (Thr172) of the {alpha}-subunit of AMPK and significantly increases the activity of the kinase (12). Once activated, cardiac AMPK increases energy production by 1) increasing fatty acid oxidation (21), 2) accelerating glucose uptake (29), and 3) stimulating glycolysis (24, 35). At the same time, cardiac AMPK switches off energy-consuming pathways, such as protein synthesis (5, 7, 15, 18), in an attempt to conserve intracellular ATP. As AMPK controls a number of cellular processes, an understanding of the complex regulatory mechanisms of AMPK activity is essential for the complete understanding of the role of AMPK in the heart.

LKB1 has recently been identified as an AMPKK that phosphorylates AMPK at its activating phosphorylation site (Thr172) (22). Although the role of LKB1 has been expanded to include the regulation of a number of AMPK-related kinases (23), LKB1 was originally identified as a tumor suppressor that was mutated in patients with Peutz-Jeghers syndrome (14). LKB1 is a serine/threonine kinase that complexes with two other regulatory proteins (2, 4): MO25 (4) and Ste20-related adaptor protein (STRAD) (2). STRAD is considered to be a pseudokinase (2), and MO25 appears to be a scaffolding protein. Together, the LKB1-MO25{alpha}-STRAD{alpha} heterotrimeric complex forms an active AMPKK (11). Although LKB1 is abundantly expressed in the cardiac myocyte (A. Noga et al., unpublished observations), its precise role in the heart is unknown. Because AMPK is significantly phosphorylated during myocardial ischemia, it was originally hypothesized that LKB1 might also be activated during ischemia. However, recent work has shown that stimulation of AMPK phosphorylation by myocardial ischemia is not dependent on increased LKB1 activity, suggesting the existence of at least one other AMPKK in the heart (1). Although an additional AMPKK has recently been identified as the Ca2+/calmodulin-dependent protein kinase kinase (13, 17, 34), the identities or existence of the others has not yet been published.

Although phosphorylation by LKB1 and other proposed AMPKKs appear to be major regulators of AMPK activity by phosphorylating AMPK{alpha} at Thr172, additional sites on AMPK may also be targets for phosphorylation by other kinases. Indeed, our previous work suggested that activated Akt directly phosphorylates AMPK on a separate site, other than Thr172, and that this alternative phosphorylation site may prevent subsequent phosphorylation by AMPKKs under steady-state conditions (20). Although the ability of activated Akt to decrease the phosphorylation of AMPK at its primary activation site of the catalytic subunit is a novel pathway that adds to the already complex mechanisms involved in AMPK regulation, the mechanism by which this occurred was unknown. Horman et al. (16) showed in an in vitro setting that Akt phosphorylates AMPK{alpha}1/{alpha}2 on Ser485/491, which prevents bacterially expressed recombinant LKB1 from phosphorylating AMPK{alpha} at Thr172. They also showed in ex vivo perfused rat hearts that insulin antagonizes ischemia-induced activation of AMPK, presumably by preventing LKB1 from phosphorylating AMPK{alpha} at Thr172. However, because pharmacological agents or physiological stimuli known to increase AMPK phosphorylation have not been shown to activate LKB1, it is not known whether Akt can prevent LKB1 phosphorylation of AMPK in the intact cardiac myocyte. Also, whether the effect of Akt is specific to LKB1 or whether Akt activation is also able to prevent or blunt LKB1-independent phosphorylation of AMPK{alpha} in the cardiac myocyte is unknown. Understanding how Akt regulates AMPK activation by its upstream kinases will assist in further characterization of the complex mechanisms controlling AMPK activity in the cardiac myocyte.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal care. The University of Alberta Animal Policy and Welfare Committee approved all experimental procedures involving animals. The University of Alberta adheres to the principles for biomedical research involving animals developed by the Council for International Organizations of Medical Sciences and complies with the National Institutes of Health animal care guidelines.

Materials. Primary antibodies, such as rabbit anti-phospho-Akt (Ser473), rabbit anti-Akt, rabbit anti-phospho-AMPK{alpha} (Thr172), rabbit anti-AMPK{alpha}, rabbit anti-phospho-AMPK{alpha}1/{alpha}2 (Ser485/491), and mouse anti-Myc, were purchased from Cell Signaling Technology; mouse anti-FLAG M2 antibody from Sigma; rabbit anti-phospho-acetyl CoA-carboxylase (ACC) (Ser79) antibody from Upstate Biotechnology; peroxidase-labeled streptavidin from Kirkegaard and Perry Labs; goat anti-actin (I-19), goat anti-LKB1 (M18), goat anti-rabbit, goat anti-mouse, and donkey anti-goat secondary antibodies from Santa Cruz Biotechnology; DNase, collagenase, and trypsin from Worthington; Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham (DMEM/F-12), fetal bovine serum, insulin-transferrin-selenium (ITS) liquid media supplement, cytosine beta-D-arabinofuranoside, mammalian protease inhibitor cocktail, 2-deoxy-D-glucose, and phosphatase inhibitor cocktail I from Sigma; gentamicin and horse serum and all other tissue culture solutions from Invitrogen; and sodium cyanide from Fisher Scientific.

Cell culture and treatment. Cardiac myocytes were isolated from the hearts of 1- to 3-day-old neonatal rat pups and plated on Primeria dishes (Falcon) at a density of 2.0 x 106 cells/plate, essentially as described previously (6, 20). After 18 h of culture, neonatal rat cardiac myocytes were rinsed twice with serum-free DMEM/F-12 containing 50 µg/ml gentamicin and cultured in serum-free DMEM/F-12 containing 50 µg/ml gentamicin supplemented with 1x ITS liquid media supplement and 10 µM cytosine beta-D-arabinofuranoside to prevent the growth of fibroblasts.

For adenoviral infections, neonatal rat cardiac myocytes were infected with adenovirus expressing green fluorescent protein (Ad.GFP) or the constitutively active mutant of Akt1 (Ad.myrAkt1) at the multiplicity of infection (MOI) of 6 in serum-free DMEM/F-12 containing 50 µg/ml gentamicin supplemented with 1x ITS liquid media supplement and 10 µM cytosine beta-D-arabinofuranoside. After infection for 4 h, the cells were infected again with a combination of Ad.GFP and adenoviruses expressing MO25{alpha} (Ad.MO25{alpha}) and STRAD{alpha} (Ad.STRAD{alpha}) or adenoviruses expressing LKB1 (Ad.LKB1), Ad.MO25{alpha}, and Ad.STRAD{alpha}, each at the MOI of 6 for 24 h.

For chemical hypoxia experiments, neonatal rat cardiac myocytes were infected with Ad.GFP or Ad.myrAkt1 at the MOI of 10 in serum-free DMEM/F-12 containing 50 µg/ml gentamicin supplemented with 1x ITS liquid media supplement and 10 µM cytosine beta-D-arabinofuranoside. After 24 h, the cells were washed three times with serum-free medium and treated with or without 3 mM 2-deoxyglucose and 2 mM sodium cyanide for 3 h in glucose-free, serum-free medium. After 3 h, the cells were harvested as described below.

Immunoblot analysis. For immunoblot analysis, the cells were rinsed twice with ice-cold 1x PBS and 150 µl of lysis buffer [20 mM Tris·HCl (pH 7.4), 50 mM NaCl, 50 mM NaF, 5 mM sodium pyrophosphate, 0.25 M sucrose, 0.1% Triton X-100, mammalian protease inhibitor cocktail, phosphatase inhibitor cocktail I, and 1 mM dithiothreitol] was added to each plate. The cells were scraped and lysed for 10 min on ice and then centrifuged at 800 g for 10 min at 4°C. The protein concentration of the supernatant was determined with the Bradford protein assay (Bio-Rad).

Cell homogenates were subjected to SDS-PAGE in gels containing 5% or 10% acrylamide and transferred to nitrocellulose membranes as previously described (32). The membranes were blocked in 5% milk-1x Tris-buffered saline (TBS)-0.1% Tween 20 and then immunoblotted overnight at 4°C with rabbit anti-phospho-AMPK{alpha} (Thr172), rabbit anti-AMPK{alpha}, rabbit anti-phospho-Akt (Ser473), rabbit anti-Akt, rabbit anti-phospho-AMPK{alpha}1/{alpha}2 (Ser485/491), rabbit anti-phospho-ACC (Ser79), mouse anti-Myc, mouse anti-FLAG M2, or goat anti-actin in 5% BSA-1x TBS-0.1% Tween 20 overnight at 4°C. After they were washed extensively, the membranes were incubated with goat anti-rabbit, goat anti-mouse, or donkey anti-goat secondary antibody or with peroxidase-labeled streptavidin in 5% milk-1x TBS-0.1% Tween 20. After the membranes were washed again, the antibodies were visualized using the Amersham Pharmacia enhanced chemiluminescence Western blotting detection system.

LKB1 activity assay. Endogenous LKB1 was immunoprecipitated from 100 µg of cellular protein using 10 µl of LKB1 (M18), and LKB1 activity was measured using the LKBtide (Alberta Peptide Institute) substrate assay as described elsewhere (23).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of constitutively active Akt1 on AMPK{alpha} phosphorylation status in cultured neonatal rat cardiac myocytes. We previously showed reduced phosphorylation of AMPK{alpha} at Thr172 in neonatal rat cardiac myocytes infected with Ad.myrAkt1 (6, 20). Because it has been shown that insulin inhibits the activation of AMPK during ischemia in ex vivo heart perfusions (3, 16) and because in vitro studies have also shown that Akt is able to phosphorylate AMPK{alpha}1/{alpha}2 at Ser485/491 and prevent subsequent phosphorylation by LKB1 at Thr172 (16), we examined whether this was also the case in the intact cardiac myocyte. Neonatal rat cardiac myocytes were infected with Ad.GFP or Ad.myrAkt1 recombinant adenoviruses. Immunoblot analysis of cell lysates confirmed that expression of myrAkt1 in the heart induced a dramatic increase in total Akt1 protein levels and phospho-Akt1 levels (Fig. 1A). We previously showed that increased phosphorylation of Akt1 at Ser473 is indicative of Akt1's activation status (20). Immunoblot analysis also confirmed a significant decrease in phosphorylation of AMPK{alpha} at Thr172 in the presence of activated Akt1 (Fig. 1A). To determine whether myrAkt1 expression increased phosphorylation of AMPK at alternative amino acids, an antibody recognizing phosphorylation at Ser485 (AMPK{alpha}1) or Ser491 (AMPK{alpha}2) was utilized. Immunoblot analysis of cell lysates with this phosphospecific antibody revealed that expression of constitutively active Akt1 resulted in increased phosphorylation of AMPK{alpha}1/{alpha}2 at Ser485/491 (Fig. 1B). These data suggest that Akt1 phosphorylates AMPK{alpha} directly at this site and that this phosphorylation prevents upstream kinases from subsequently phosphorylating AMPK{alpha} at its major activating phosphorylation site (Thr172).


Figure 1
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  		Fig. 1. Expression of constitutively active Akt1 alters phosphorylation of the {alpha}-subunit of AMPK (AMPK{alpha}). Neonatal rat cardiac myocytes were infected with adenovirus expressing green fluorescent protein (Ad.GFP) or constitutively active mutant of Akt1 (Ad.myrAkt1) at multiplicity of infection (MOI) of 6. At 24 h after infection, cells were harvested and cellular extracts were subjected to immunoblot analysis. A: representative immunoblots and densitometry from 4–8 independent experiments performed on cellular extracts probed with anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-AMPK{alpha} (Thr172), anti-AMPK{alpha}, and anti-actin antibodies. Values are means ± SE. *P < 0.05 vs. control (by Student's 2-tailed t-test). B: immunoblots of cellular extracts from neonatal rat cardiac myocytes infected as described in A and probed with anti-phospho-AMPK{alpha}1/{alpha}2 (Ser485/491), anti-AMPK{alpha}, and anti-actin antibodies.

 
Expression of LKB1-STRAD{alpha}-MO25{alpha} complex increases AMPK{alpha} phosphorylation at Thr172. To better understand the relation between phosphorylation of AMPK{alpha}1/{alpha}2 at Ser485/491 and phosphorylation at Thr172 by upstream kinases, we utilized our recently developed Ad.LKB1, Ad.STRAD{alpha}, and Ad.MO25{alpha} recombinant adenoviruses. To confirm the effect of LKB1 activation on AMPK{alpha} phosphorylation status in the cardiac myocyte, we infected neonatal rat cardiac myocytes with recombinant adenoviruses harboring FLAG-tagged LKB1 and STRAD{alpha} and Myc-tagged MO25{alpha}. Immunoblot analysis of cell lysates from neonatal rat cardiac myocytes infected with the LKB1-STRAD{alpha}-MO25{alpha} complex showed increased expression of all three proteins (Fig. 2A). This increase in expression of the LKB1-STRAD{alpha}-MO25{alpha} complex also produced a significant increase in LKB1 activity as determined using an in vitro peptide substrate assay (A. Noga et al., unpublished observations). In accordance with this increase in LKB1 activity, phosphorylation of AMPK{alpha} at Thr172 was also significantly increased (Fig. 2B). Thus expression of LKB1, STRAD{alpha}, and MO25{alpha} is sufficient to produce an active LKB1 complex able to phosphorylate AMPK{alpha} at its activation loop at Thr172 in neonatal rat cardiac myocytes.


Figure 2
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  		Fig. 2. Expression of LKB1-Ste20-related adaptor protein (STRAD{alpha})-MO25{alpha} complex results in increased phosphorylation of AMPK{alpha} at Thr172. Neonatal rat cardiac myocytes were infected with Ad.GFP or adenovirus expressing LKB1 (Ad.LKB1), STRAD{alpha} (Ad.STRAD{alpha}), and MO25{alpha} (Ad.MO25{alpha}) at the MOI of 6 for each virus. In all experiments the MOI was kept constant by use of equivalent amounts of adenovirus encoding for GFP (+). At 24 h after infection, cells were harvested and cellular extracts were subjected to immunoblot analysis. A: immunoblot analysis performed on cellular extracts using anti-FLAG or anti-Myc antibodies. Note high level of expression of all 3 tagged proteins in the LKB1-STRAD{alpha}-MO25{alpha} complex. B: representative immunoblots from 4 independent experiments performed on cellular extracts from neonatal rat cardiac myocytes infected as described in A and probed with anti-phospho-AMPK{alpha} (Thr172) and anti-AMPK{alpha} antibodies. Note increased phosphorylation of AMPK{alpha} by LKB1 complex.

 
Expression of constitutively active Akt1 reduces phosphorylation of AMPK{alpha} by LKB1 in cultured neonatal rat cardiac myocytes. To investigate the ability of Akt1 to prevent phosphorylation of AMPK{alpha} at Thr172 in the cardiac myocyte, neonatal rat cardiac myocytes were infected with Ad.GFP or Ad.myrAkt1 recombinant adenoviruses and then with or without the LKB1-STRAD{alpha}-MO25{alpha} complex. As in previous experiments (Fig. 1B), expression of myrAkt1 significantly increased the phosphorylation of AMPK{alpha}1/{alpha}2 at Ser485/491, even in the presence of increased LKB1 activity (Fig. 3A). Interestingly, expression of the LKB1-STRAD{alpha}-MO25{alpha} complex also results in a modest, but significant, increase in phosphorylation of AMPK{alpha}1/{alpha}2 at Ser485/491 compared with control treated cells. This is consistent with the findings of Horman et al. (16), who suggest that an LKB1-induced activation of AMPK results in AMPK autophosphorylation at Ser485. We suspect that the increase in phosphorylation of AMPK{alpha}1/{alpha}2 at Ser485/491 in cells expressing the LKB1 complex may be due to this autophosphorylation event. Although this is only speculation, we know that expression of the LKB1-STRAD{alpha}-MO25{alpha} complex does not alter Akt phosphorylation (Ser473), which suggests that LKB1-induced alterations in Akt activity are not responsible for the increase in phosphorylation of AMPK{alpha}1/{alpha}2 at Ser485/491 in cells expressing the LKB1 complex (Fig. 3B).


Figure 3
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  		Fig. 3. Expression of constitutively active Akt1 alters phosphorylation of AMPK{alpha} in the presence of the LKB1-STRAD{alpha}-MO25{alpha} complex. Neonatal rat cardiac myocytes were infected with Ad.GFP or Ad.myrAkt1 at MOI of 6 for each virus for 4 h. After initial infection, myocytes were infected with or without an active LKB1 complex. In all experiments, MOI was kept constant by use of equivalent amounts of adenovirus encoding for GFP (+). At 24 h after infection, cells were harvested and cellular extracts were subjected to immunoblot analysis. A: representative immunoblots from 4 independent experiments on cellular extracts using anti-phospho-AMPK{alpha}1/{alpha}2 (Ser485/491), anti-AMPK{alpha}, and anti-actin antibodies. B: cellular extracts from neonatal rat cardiac myocytes infected as described in A and probed with anti-phospho-AMPK{alpha} (Thr172), anti-AMPK{alpha}, anti-phospho-Akt (Ser473), anti-Akt, and anti-actin antibodies. Note blunted phosphorylation of AMPK by LKB1 complex in the presence of constitutively active Akt1. C: cellular extracts from A and B also subjected to immunoblot analysis using anti-phospho-acetyl CoA-carboxylase (ACC) (Ser79) antibody. Note phosphorylation of ACC, with peroxidase-labeled streptavidin serving as loading controls for total ACC protein levels. For all immunoblots, densitometry and subsequent statistical analysis are also shown. Values are means ± SE. *P < 0.05 vs. control; tP < 0.05 vs. LKB1 complex (by Student's 2-tailed t-test).

 
In addition to increased phosphorylation of AMPK{alpha}1/{alpha}2 at Ser485/491, myrAkt1 expression also blunted LKB1-induced phosphorylation of AMPK{alpha} at Thr172 (Fig. 3B). Although this significant decrease in phosphorylation of AMPK{alpha} at Thr172 in the presence of activated Akt1 is not dramatic (20%), the effects on AMPK activity are more pronounced, as shown with decreased phosphorylation of ACC at its AMPK phosphorylation site (Fig. 3C). Thus activation of Akt1 is able to partially prevent activation of cardiac AMPK by one of its major upstream kinases, possibly by prior phosphorylation at AMPK{alpha}1/{alpha}2 at Ser485/491.

Increased phosphorylation of AMPK{alpha} induced by chemical hypoxia is blunted by expression of constitutively active Akt1 in cultured neonatal rat cardiac myocytes. Recent evidence has suggested the existence of at least two AMPKKs in the heart (1). Although one of these kinases has been identified as LKB1, it has been shown that another kinase is responsible for activating AMPK during myocardial ischemia (1). Therefore, to investigate whether Akt1 activation was also able to prevent phosphorylation of AMPK{alpha} at Thr172 by an AMPKK other than LKB1, we subjected cells to a major component of ischemia, namely, hypoxia. Neonatal rat cardiac myocytes were infected with Ad.GFP or Ad.myrAkt1 and then to chemical hypoxia by treatment with a glucose-free medium supplemented with 3 mM 2-deoxyglucose and 2 mM sodium cyanide for 3 h. Expression of myrAkt1 significantly increased the phosphorylation of AMPK{alpha}1/{alpha}2 at Ser485/491 during control and chemical hypoxia (Fig. 4A). In addition, expression of constitutively active Akt1 significantly reduced phosphorylation of AMPK{alpha} at Thr172 (Fig. 4B) and AMPK activity (Fig. 4C), suggesting that phosphorylation of AMPK{alpha}1/{alpha}2 at Ser485/491 by Akt1 prevents upstream kinases from subsequently phosphorylating AMPK{alpha} at its major activating phosphorylation site. Because LKB1 activity was not altered by chemical hypoxia (Fig. 4D), our data suggest that expression of activated Akt1 either prevents other AMPKK(s) from phosphorylating AMPK{alpha} at its major activating phosphorylation site (Thr172) or alters the interaction of AMPK{alpha} with LKB1.


Figure 4
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  		Fig. 4. Expression of constitutively active Akt1 alters chemical hypoxia-induced phosphorylation of AMPK{alpha} without altering LKB1 activity. Neonatal rat cardiac myocytes were infected with Ad.GFP or Ad.myrAkt1 at the MOI of 10 for each virus. At 24 h after infection, cells were exposed to control medium or chemical hypoxia for 3 h. After 3 h of incubation, cells were harvested and cellular extracts were subjected to immunoblot analysis. A: representative immunoblots from 6 independent experiments performed on cellular extracts using anti-phospho-AMPK{alpha}1/{alpha}2 (Ser485/491), anti-AMPK{alpha}, anti-phospho-Akt (Ser473), and anti-Akt antibodies. B: immunoblots of cellular extracts from neonatal rat cardiac myocytes infected as described in A and probed with anti-phospho-AMPK{alpha} (Thr172) and anti-AMPK{alpha} antibodies. Note blunted phosphorylation of AMPK{alpha} by chemical hypoxia in the presence of constitutively active Akt1. C: immunoblots of cellular extracts from neonatal rat cardiac myocytes infected as described in A and B and probed with anti-phospho-ACC (Ser79) to demonstrate activation status of AMPK, with peroxidase-labeled streptavidin serving as loading controls for total ACC protein levels. D: antibodies of cell lysates from neonatal rat cardiac myocytes infected as described in A were used to confirm that chemical hypoxia did not increase LKB1 activity. For all immunoblots, densitometry and subsequent statistical analysis are also shown. Values are means ± SE. *P < 0.05 vs. all other groups; #P < 0.05 vs. GFP + control; tP < 0.05 vs. GFP + chemical hypoxia (by Student's 2-tailed t-test).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We recently showed that activation of Akt can reduce phosphorylation and activity of AMPK in the heart (20). Although all the mechanism(s) by which this occurs have not been clearly defined, it appears that at least one pathway may occur via a direct phosphorylation of AMPK{alpha} by Akt. Indeed, in an in vitro system, Horman et al. (16) showed that Akt directly phosphorylates AMPK{alpha}1/{alpha}2 on Ser485/491 and that this phosphorylation can prevent subsequent activation of AMPK{alpha} at Thr172 by LKB1. In addition, Horman et al. (16) showed in ex vivo perfused rat hearts that insulin antagonizes ischemia-induced activation of AMPK and, combined with their in vitro data, provided compelling evidence that direct phosphorylation of AMPK{alpha}1/{alpha}2 on Ser485/491 by Akt can prevent LKB1 from phosphorylating AMPK{alpha} at Thr172. The experiments described here confirm that this is likely the case in the cardiac myocyte and provide further evidence that activation of Akt1 is a major negative regulator of AMPK in the heart. Interestingly, the ability of Akt1 to negatively regulate AMPK{alpha} phosphorylation is less pronounced in cardiac myocytes expressing the LKB1-STRAD{alpha}-MO25{alpha} complex than in the cell-free system of Horman et al. (16). We suggest that this may be due to the dramatic and prolonged increase in LKB1 activity and more dynamically regulated phosphorylation/dephosphorylation processes expected in our cellular system. Despite this subtle decrease in phosphorylation of AMPK{alpha} at Thr172 in the presence of constitutively active Akt1 in our studies, the effect on AMPK activity (as determined by phosphorylation of ACC at Ser79) is much more pronounced and is more consistent with the in vitro data presented by Horman et al. (16).

Although we hypothesize that phosphorylation of AMPK{alpha}1/{alpha}2 at Ser485/491 prevents other upstream kinases from subsequently phosphorylating AMPK{alpha} at its major activating phosphorylation site, it is also possible that phosphorylation at Ser485/491 alters the interaction of AMPK{alpha} with its upstream kinases. Indeed, Zou et al. (36) showed that AMPK{alpha} coimmunoprecipitates with LKB1, suggesting that AMPK associates with LKB1 and that this association may be involved in AMPK activation. On the basis of the study of Zou et al. (36) and the data presented here, it is possible that phosphorylation of AMPK{alpha}1/{alpha}2 at Ser485/491 alters the physical association of AMPK{alpha} with LKB1, such that phosphorylation at Thr172 occurs less readily. Alternatively, increased Akt1 activity may inhibit the activity of upstream AMPK kinases, which could lead to a decrease in phosphorylation of AMPK{alpha} at Thr172, rather than directly modulate AMPK through phosphorylation of AMPK{alpha}1/{alpha}2 at Ser485/491. Although this latter hypothesis is a possibility, it is less likely given the recent in vitro data by Horman et al. (16) demonstrating that phosphorylation of AMPK{alpha} at Ser485 by Akt is necessary to block LKB1-induced activation of AMPK.

To demonstrate that Akt1 activation prevents phosphorylation of AMPK{alpha} by an AMPKK distinct from LKB1, we subjected cells to chemical hypoxia. Our data show that our model of chemical hypoxia does not result in LKB1 activation (Fig. 4D) and reflect the findings that have been reported during myocardial ischemia (1). In addition, we show that Akt1 activation completely prevents chemical hypoxia-induced phosphorylation of AMPK{alpha} at Thr172 (Fig. 4B). Because it has been reported that a kinase other than LKB1 is responsible for activating AMPK during myocardial ischemia (1), our data suggest that the effect of activated Akt1 on AMPK is not specific to LKB1 and may involve other AMPKKs. However, in contrast to the initial findings by Altarejos et al. (1), it was recently shown that LKB1 is necessary for ischemia-induced activation of cardiac AMPK (31). Therefore, although LKB1 activity does not increase during hypoxia, it is equally likely that Akt1 activation can still prevent hypoxia-induced phosphorylation of AMPK{alpha} via LKB1, possibly by altering the interaction of AMPK{alpha} with LKB1.

Of additional interest is our finding that the ability of Akt1 to negatively regulate AMPK{alpha} phosphorylation at Thr172 is more dramatic in myocytes subjected to chemical hypoxia (Fig. 4B) than in cardiac myocytes expressing the LKB1-STRAD{alpha}-MO25{alpha} complex (Fig. 3B). This is also reflected in the activity of AMPK as determined by ACC phosphorylation (Figs. 3C and 4C). Although the absolute decrease in phosphorylated ACC in both experiments is approximately the same, activated Akt1 in cardiac myocytes expressing the LKB1-STRAD{alpha}-MO25{alpha} complex decreases phosphorylated ACC only to twice baseline levels, whereas activated Akt1 in myocytes subjected to chemical hypoxia completely restores phosphorylated ACC to baseline values. Although we have no data to explain why the magnitude of the effects on AMPK activity are different, perhaps the extent of activation of the AMPKKs or the kinetic properties of the chemical hypoxia-induced AMPKK are responsible.

With the in vitro data provided by Horman et al. (16) and the data provided here, Akt1 has emerged as an additional AMPKK. Whereas the significance of this interaction in the heart has yet to be fully explored, the ability of Akt1 to negatively regulate AMPK suggests a more expanded role in the regulation of cardiac energy metabolism. Although Akt1 activation has been shown to increase glucose uptake in cultured cardiac myocytes (25), it is not clear whether increased Akt activity can dramatically alter substrate utilization in the heart, nor is it known whether Akt activation can also alter fatty acid utilization. Indeed, AMPK activation is associated with increased fatty acid oxidation in the heart (21), and it is possible that Akt1 may also regulate fatty acid oxidation rates secondary to inhibiting AMPK activity.

The ability of Akt1 to negatively regulate AMPK activity becomes especially relevant in the setting of ischemia-reperfusion. For instance, in vivo adenoviral gene transfer of constitutively active Akt1 in rat and mouse hearts has been shown to be beneficial by protecting against apoptosis and reducing infarct size after myocardial ischemia-reperfusion (8, 25, 26). Although the protective effects of increased Akt1 activity have been attributed to the inhibition of apoptosis, it is possible that Akt1 can also exert protective effects by altering cardiac energy metabolism. Indeed, it has been shown that activated Akt improves coupling of glycolysis to glucose oxidation and preserves mitochondrial integrity in fibroblasts (9). An improved coupling of glycolysis to glucose oxidation is beneficial to the functional recovery of the heart after ischemia (see Ref. 19 for review). Associated with this is the proposal that, in the reperfused ischemic heart, AMPK activation increases fatty acid oxidation rates, which may contribute to ischemic injury (21). In this setting, Akt1 activation may be able to protect the heart from ischemic injury by inhibiting fatty acid oxidation secondary to decreasing AMPK activity. In support of the potential beneficial effects of Akt negatively regulating AMPK, Beauloye et al. (3) showed in the perfused rat heart that insulin pretreatment can blunt the normal AMPK response induced by ischemia. An insulin-mediated Akt activation and subsequent inhibition of AMPK may provide a rationale for why insulin has been shown to be cardioprotective during ischemia-reperfusion (33). Therefore, it is possible that Akt1 expression can protect the heart from ischemic injury by improving the coupling of glycolysis to glucose oxidation, directly by altering glucose utilization and indirectly by inhibiting fatty acid oxidation. Although this is an attractive hypothesis, the roles of AMPK and Akt in myocardial ischemia are controversial (27, 30). Indeed, AMPK activation has been proposed to be essential for ischemic tolerance, possibly by increasing glucose utilization (30), whereas chronic activation of Akt1 has been shown to be detrimental to the mouse heart subjected to ischemia-reperfusion (27). Together, these findings argue against the notion that Akt activation and subsequent AMPK inactivation would be beneficial for the heart subjected to ischemia-reperfusion. To help clarify this issue, future studies are needed to investigate the role(s) of these two kinases in the hypoxic and/or ischemic cardiac myocyte.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
J. R. B. Dyck is a Scholar of the Alberta Heritage Foundation for Medical Research and a Canadian Institutes of Health Research (CIHR) New Investigator. This research was supported by CIHR Grant MOP53088.


   ACKNOWLEDGMENTS
 
The authors thank Dr. D. R. Allessi for kindly providing the cDNAs for LKB1, STRAD{alpha}, and MO25{alpha}.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. R. B. Dyck, 474 Heritage Medical Research Centre, Univ. of Alberta, Edmonton, AB, Canada, T6G 2S2 (e-mail: Jason.Dyck{at}UAlberta.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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