1Sections of Cardiovascular Medicine, Endocrinology, and Metabolism, Department of Internal Medicine and 2Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510; and 3Faculty of Life Sciences, Division of Molecular Physiology, University of Dundee DD1 5EH, Scotland, United Kingdom
Submitted 16 April 2003 ; accepted in final form 15 May 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
energy metabolism; signal transduction; glucose
Exercise is known to increase the uptake and utilization of free fatty acids and glucose in the heart (18, 52), as it does in skeletal muscle (54). During exercise, heart contractile function and metabolism are regulated in a complex fashion by a number of signaling pathways, including adrenergic receptor and calcium-activated mechanisms. In skeletal muscle, activation of AMPK appears to have a role in mediating the enhanced glucose uptake (36) and fatty acid oxidation (48) that occur with increased contractile activity, although additional pathways may be involved. However, in the heart, it remains uncertain whether AMPK has a role in mediating the cardiac response to exercise.
When activated pharmacologically with the compound
5-aminoimidazole-4-carboxamide-1--D-ribofuranoside, increased
free fatty acid and glucose uptake occur in skeletal muscle
(35) and heart
(4). One of the best
characterized downstream targets of AMPK is acetyl-CoA carboxylase (ACC; see
Refs. 8,
12, and
53). The ACC-1 and ACC-2
isoforms both contain serine residues that are phosphorylated by AMPK
(2,
14). ACC phosphorylation
inhibits malonyl-CoA synthesis, enhancing carnitine palmitoyltransferase I
activity and free fatty acid oxidation
(31,
32,
54). AMPK may also modulate
free fatty acid oxidation through the activation of malonyl-CoA decarboxylase
(43). In addition, AMPK
increases cellular glucose uptake through translocation of the GLUT4
transporter to the sarcolemma in skeletal muscle
(33) and heart
(42). Although the downstream
targets of AMPK responsible for GLUT4 translocation are unknown, AMPK
signaling (7,
27,
42) is distinct from the
phosphatidylinositol 3-kinase (PI 3-kinase) pathway that mediates
insulin-activated glucose transport. AMPK also increases glucose utilization
through activation of 6-phosphofructo-2-kinase, which leads to the production
of fructose 2,6-bisphosphate, an activator of glycolysis
(34).
AMPK is a heterotrimeric complex comprised of a catalytic -subunit
as well as regulatory
- and
-subunits
(23,
28,
46). In most tissues,
including the heart and skeletal muscle, there are two isoforms of the
catalytic subunit,
1 and
2. In skeletal
muscle, there is evidence that the two
-isoforms may be differentially
activated during contraction and exercise, with a greater degree of activation
of
2 (9,
17,
48,
58). These findings are
consistent with the observation that the
2-isoform may have
a greater dependence on the cellular AMP concentration
(45). Although both
-isoforms are activated during ischemia
(10), the response of the
heart AMPK
-isoforms to exercise is unknown.
Therefore, the objectives of the present study were 1) to
determine whether cardiac AMPK activity increases during exercise; 2)
to examine whether in vivo activation of AMPK in the heart depends on the
exercise intensity; 3) to evaluate the degree of activation of the
two -isoforms of AMPK in the heart in response to exercise; and
4) to relate activation of AMPK during exercise with potential
downstream actions, including phosphorylation of ACC and GLUT4
translocation.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue homogenization. Myocardial tissue samples were homogenized in Tris buffer (125 mM Tris, 10 mM EDTA, 10 mM EGTA, 250 mM mannitol, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM DTT, 1 mM benzamidine, 0.004% trypsin inhibitor, and 3 mM sodium azide, pH 7.5) with a Polytron as previously described (31, 42). The homogenate was centrifuged, and the supernatant was subjected to polyethylene glycol (PEG) precipitation. The 2.5-6% PEG fraction was suspended in homogenization buffer without mannitol for measurement of AMPK activity. All procedures were performed at 4°C. Protein concentration was determined spectrophotometrically using the Bio-Rad reagent.
AMPK immunoprecipitation. The AMPK activity associated with
specific -subunit isoforms was examined after immunoprecipitation with
polyclonal sheep antibodies raised against synthetic peptides derived from the
1 (TSPPDSFLDDHHLTR)- or
2
(MDDSAMHIPPGLKPH)-isoforms
(45,
59). Antibodies were prebound
in excess to protein G-Sepharose beads before incubation with 50 µg of
PEG-precipitated homogenate overnight at 4°C. The immunoprecipitates were
washed extensively with homogenization buffer containing 0.1% Igepal, and then
with assay buffer (40 mM HEPES, 80 mM NaCl, 8% glycerol, and 0.8 mM EDTA, pH
7.0) before kinase assay.
AMPK assay. AMPK activity was measured using the SAMS peptide
HMRSAMSGLHLVKRR phosphorylation assay. PEG extracts or -subunit
immunoprecipitates were resuspended in homogenization buffer containing 0.8 mM
DTT and 0.2 mM AMP, with or without 0.2 mM SAMS peptide (see Refs.
31,
42, and
45). The kinase assay was
performed in the presence of 5 mM MgCl2, 0.2 mM ATP, and
[32P]ATP (New England Nuclear, Boston, MA) for 10 min at 37°C.
Aliquots of the reaction mixture supernatant were spotted on Whatman filter
paper (P81). The filters were washed with cold 150 mM phosphoric acid for 40
min and with acetone for 20 min and then were allowed to dry before
scintillation counting. AMPK activity was calculated as picomoles per
milligram PEG-precipitated protein per minute, and results were expressed as
the degree of increase compared with resting rats.
Immunoblotting. Western blot analyses of -isoforms of AMPK,
pThr172 AMPK, and phosphorylated ACC (pACC) were performed on 40
µg of PEG-precipitated heart protein (2.5-6% fraction) after SDS-PAGE on 8%
polyacrylamide for AMPK and 5% gels for pACC. AMPK immunoprecipitates were
suspended in sample buffer, boiled for 10 min, and then used for SDS-PAGE.
Proteins were subjected to electrophoresis and transferred to PVDF membranes.
For immunoblotting, antibodies were diluted as follows:
anti-
1 (45)
at 1:2,000, anti-
2-AMPK
(45) at 1:3,000,
anti-pan-
-AMPK (Cell Signaling, Beverly, MA) at 1:5,000,
anti-pThr172 AMPK (Cell Signaling) at 1:5,000, anti-pACC that
recognizes both the Ser79 of ACC-1 and the equivalent
Ser218 of ACC-2 (Upstate, Waltham, MA) at 1:5,000,
anti-pThr308 Akt (Upstate) at 1:1,000, and anti-pSer473
Akt and anti-pan Akt1/2 (Cell Signaling) at 1:1,000.
Membrane fractionation. Membrane fractions were prepared from hearts as described previously (42, 61). In brief, crude homogenates were prepared with a Polytron. The supernatant containing the crude membrane fraction was pelleted by ultracentrifugation, and the membrane fractions were separated on a discontinuous sucrose gradient (25, 30, and 35% wt/vol) at 150,000 g for 20 h. The sarcolemma and the intracellular membranes were harvested and stored at -80°C. GLUT4 immunoblots were performed on 40-µg membrane protein in low-ionic-strength Laemli buffer on 8% polyacrylamide gels.
Measurement of high-energy phosphates and glycogen. Heart samples were extracted with 6% perchloric acid, and the supernatants were neutralized with 3 M K2CO3. Myocardial nucleotide contents were measured by reverse-phase HPLC, whereas creatine phosphate was measured using spectrophotometric methods, as previously described (6, 7). Glycogen was measured after KOH extraction and ethanol precipitation as previously described (6, 7). Results are expressed as micromoles per gram wet weight.
Statistical analysis. All data are reported as means ± SE. The number of rats in each group is presented in Figs. 1, 2, 3, 4, 5, 6, 7. Data were analyzed by ANOVA, and contrasts were used for planned comparisons between groups using Statistical Analysis Software (SAS Institute, Cary, NC). Differences were considered significant at P < 0.05.
|
|
|
|
|
|
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Heart -isoform AMPK activities during exercise. To
examine whether there was differential activation of the catalytic subunits of
AMPK, SAMS kinase activity was also measured in
1- or
2-immunoprecipitates from hearts after exercise.
Immunoprecipitation procedures were isoform specific with no demonstrable
cross-immunoreactivity of precipitated proteins when subjected to
immunoblotting (Fig. 2). AMPK
activity in
1-immunoprecipitates tended to increase after
both moderate (2.1 ± 0.6-fold)- and high (1.9 ±
0.4-fold)-intensity exercise compared with that in resting rats
(Fig. 3). However, more
pronounced increases in AMPK activity were observed in cardiac
2-immunoprecipitates: 2.8 ± 0.4-fold after
moderate-intensity exercise (P < 0.02) and 4.5 ± 0.6-fold
after high-intensity exercise (P < 0.001;
Fig. 3). The increase in heart
2 activity was significantly greater (P < 0.02)
in rats exercising at the faster treadmill speed, indicating that there was a
graded activation of the
2-isoform in the heart with
exercise. In addition, high-intensity exercise increased the activity of
2-AMPK to a significantly greater extent than
1 activity (P < 0.05), suggesting that this
isoform is more responsive to the physiological stress of exercise in the
heart, as it is in skeletal muscle
(17,
48).
AMPK phosphorylation. AMPK activity measured with the SAMS kinase
assay largely reflects the extent to which the -subunit was
phosphorylated in vivo, since the assay conditions do not replicate the in
vivo concentrations of AMP that were present in the heart during exercise. The
primary phosphorylation site responsible for regulating AMPK activity is the
Thr172 residue of both the
1- and
2-catalytic subunits
(22,
23,
25). Thus we assessed the
degree of phosphorylation of Thr172, using a
phosphopeptide-specific antibody, and expressed the amount of
pThr172 relative to the total amount of AMPK in the samples. There
was a twofold increase (P < 0.001) in the pThr172
content in PEG precipitates of hearts from rats running at high intensity
(Fig. 4); no significant
increase was apparent after moderate-intensity exercise.
Recent evidence suggests that cellular stress may activate noninsulin receptor-linked PI 3-kinase (13) and its downstream kinase Akt (44). To examine whether AMPK activation might be associated with Akt activation, we evaluated the effects of exercise on Akt phosphorylation by immunoblotting heart homogenates with antibodies against the pThr308 and pSer473 residues of Akt. In contrast to the increased phosphorylation of AMPK observed during exercise, there was no apparent increase in either Thr308 or Ser473 Akt phosphorylation (Fig. 5).
Total heart nucleotide and glycogen content. The activity of AMPK
is modulated by several factors in muscle tissues. Increases in the ratio of
the cellular contents of AMP/ATP are known to activate AMPK through enhanced
phosphorylation, decreased dephosphorylation, and allosteric activation of the
-subunit (22,
23). The total contents of
adenine nucleotides were measured in neutralized acid extracts of
freeze-clamped hearts, but no significant changes in either total ATP or AMP
were apparent after exercise (Table
1). These measurements do not exclude the possibility that
increases in the free concentration of AMP might have occurred. Free AMP
concentration typically increases when creatine phosphate decreases
(15), as it did after both
moderate- and high-activity exercise (Table
1). In addition, the cardiac glycogen content fell in proportion
to exercise activity, with 31 and 62% reductions after 10 min of moderate- and
high-intensity exercise, respectively
(Table 1).
|
Downstream effects of AMPK activation during exercise. Free fatty acid oxidation is an important source of ATP generation in the heart during exercise (52). We assessed whether the increase in AMPK during exercise is associated with downstream phosphorylation of ACC, a key mediator of free fatty acid oxidation in the heart (31, 32). Although both AMPK and protein kinase A (PKA) phosphorylate multiple sites on ACC (11, 20), both Ser79 on ACC-1 and the equivalent Ser218 on ACC-2 are phosphorylated by AMPK but not by PKA. Immunoblots of PEG heart precipitates with an antibody that recognizes these specific phosphorylated serine residues showed a twofold increase in phosphorylation of both ACC-1 (265 kDa) and ACC-2 (280 kDa) after exercise (Fig. 6).
AMPK is also known to increase glucose uptake in both heart (42) and skeletal (7, 16, 27, 35) muscle by translocating GLUT4 transport proteins to the sarcolemma (33, 42). To evaluate whether AMPK activation was associated with GLUT4 translocation, we immunoblotted GLUT4 in sarcolemma and intracellular membranes from hearts after rest or high-intensity exercise. After high-intensity exercise, there was an increase in the sarcolemma GLUT4 content and a reduction in the intracellular membrane GLUT4 content, indicating the translocation of transporters during exercise (Fig. 7).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
AMPK is activated by exercise in skeletal muscle, where it has an important role in regulating substrate metabolism (30, 54). Although AMPK is known to be activated in the ischemic (31, 32) and hypertrophied heart (49), it has been unclear whether AMPK serves as a physiological regulator in the normal heart. The current findings indicate that heart AMPK is activated during normal exercise. In previous experimental models, AMPK activation was not evident when cardiac workload was manipulated by dobutamine infusion in pigs (21) or by increasing afterload in isolated working rat hearts (5). However, confounding comparisons with the current results were the effects of surgery in the former study (21) and the relatively lower workloads examined in the latter report (5). During exercise, several determinants of cardiac work increase, including blood pressure, heart rate, and cardiac contractility, although it is possible that additional factors may be involved in the exercise activation of AMPK in vivo.
These results also indicate a greater degree of activation of the
2-compared with the
1-isoform in the heart
during exercise. The
2-associated activity increased
2.8-fold during moderate-intensity and 4.5-fold during high-intensity
exercise. Although treadmill running also appeared to be associated with a
trend to an increase in
1-AMPK activity, it was less
consistent than that in
2-AMPK activity. These observations
parallel those in skeletal muscle with regard to greater
2
activation during moderate acute exercise
(17,
48,
58). However, activation of
the
1-isoform has been shown only after sprinting in humans
(9) and during electrically
stimulated contraction of isolated rat skeletal muscles
(26). Thus, although
1 may be somewhat more readily activated in heart than in
skeletal muscle during exercise, heart
2 is significantly
more responsive than
1. The role of each of these isoforms
in modulating the cardiac exercise response remains to be determined.
AMPK activity is modulated by the phosphorylation state of the
Thr172 residue of the -subunits, which is determined by the
activities of upstream AMPK kinase(s) and protein phosphatases
(22-24).
The Thr172 site lies within the critical kinase activation domain
of the
-catalytic subunit
(23). We observed a
significant increase in phosphorylation of Thr172 in heart
homogenates from rats run at high intensity, using a phosphopeptide-specific
AMPK antibody directed against this domain. Similarly, exercise appears to
increase Thr172 phosphorylation in skeletal muscle after exercise
(48). We did not detect a
significant increase in Thr172 phosphorylation in hearts from rats
run at moderate-intensity exercise, despite a modest increase in AMPK
activity. This most likely reflects the lesser sensitivity of the
phosphopeptide immunoblots compared with the enzymatic AMPK assay, rather than
activation of the kinase through alternate phosphorylation sites.
Several mechanisms may contribute to the activation of heart AMPK during
exercise. A rise in the intracellular concentration of AMP (or the AMP-to-ATP
ratio) increases the activity of upstream AMPK kinase(s)
(25,
47), enhances the sensitivity
of AMPK to phosphorylation, and decreases its susceptibility to
dephosphorylation (24). The
total cardiac AMP concentrations measured by HPLC in freeze-clamped hearts are
two orders of magnitude higher than the intracellular concentration of free
AMP (as estimated by NMR spectroscopy in vitro), which regulates AMPK activity
(15). Although it is not
feasible to make such measurements in the heart during exercise, it is likely
that the free AMP concentration increases during exercise, given the decrease
observed in the creatine phosphate concentration
(15). In addition, the
decrease in the creatine phosphate concentration may also regulate AMPK
through allosteric mechanisms that would operate in vivo
(38) but may not be reflected
by the in vitro measurements of kinase activity. An additional mechanism
regulating the activity of AMPK is the concentration of glycogen
(48,
56), through glycogen binding
to the -subunit of AMPK
(28,
37). Thus the finding that
cardiac glycogen content was reduced significantly in rats, as in previous
reports (19), is of interest
in terms of AMPK activation in the heart during exercise.
In these studies, we found that exercise was associated with phosphorylation of ACC in the heart. ACC is a well-recognized downstream target of AMPK (22) and is an important regulator of malonyl-CoA concentrations that modulate carnitine palmitoyltransferase-1 activity in both heart (31, 32) and skeletal muscle (40). In skeletal muscle, AMPK phosphorylates ACC during acute exercise (54, 55), although the role of AMPK in maintaining high levels of free fatty acid oxidation during more prolonged exercise remains somewhat uncertain (57). We observed exercise-induced phosphorylation of both ACC-1 (265 kDa) and ACC-2 (280 kDa). ACC-2 is associated with the mitochondria (1) and has an important role in modulating fatty acid oxidation (3), which is an important metabolic pathway in the heart during exercise. The pACC antibody that was utilized detects phosphorylation of Ser79 on ACC-1 and the equivalent Ser218 site on ACC-2, neither of which is thought to be a target for PKA (11, 14, 20). Catecholamines have a well-recognized role in modulating both the cardiac metabolic and contractile responses to exercise by increasing circulating free fatty acid supply to the heart as well as through direct PKA-mediated effects. These observations suggest that the AMPK pathway and catecholamines may have distinct but complementary effects on the heart during exercise.
These results also provide evidence that GLUT4 translocation to the
sarcolemma occurs in association with AMPK activation in the heart during
high-intensity treadmill running. Low levels of GLUT4 translocation are
difficult to detect, and these measurements were not performed during
moderate-intensity exercise. Pharmacological activation of AMPK is also known
to increase GLUT4 translocation and glucose uptake in heart
(4,
42) and skeletal
(27,
33,
35,
36) muscle. In addition, AMPK
appears to have a critical role in modulating glucose transport in both
hypoxic skeletal muscle (36)
and ischemic heart (41).
However, the exact role of AMPK in mediating glucose utilization during
exercise remains uncertain. In skeletal muscle, AMPK appears to be only
partially responsible for contraction-mediated GLUT4 translocation, based on
evidence from transgenic mice expressing a kinasedeficient -isoform of
AMPK (36). Catecholamines and
increased intracellular calcium concentrations
(39) also cause GLUT4
translocation in the heart, and it is possible that these additional
mechanisms play a role in enhancing cardiac glucose uptake during
exercise.
Although the downstream targets of AMPK that mediate GLUT4 vesicular trafficking have not been identified, AMPK stimulation of glucose transport does not require activation of PI 3-kinase, a key lipid kinase in the pathway of insulin-stimulated glucose transporter translocation (27, 42). Although PI 3-kinase does not mediate glucose transport during contraction in skeletal muscle (60), there is some evidence that it may be involved in glucose transport in isolated cardiac myocytes during electrical stimulation-induced contraction (50). In the current studies, we examined the effect of exercise on the phosphorylation state of the serine-threonine kinase Akt, which is distal to PI 3-kinase and appears to be activated by contraction in isolated skeletal muscle (44). However, we found no evidence that exercise increased the phosphorylation of either of the two key regulatory sites (Thr308 and Ser473) that mediate Akt activity in the heart. Thus these results suggest that, to the extent that AMPK stimulates glucose transporter translocation during exercise, it does so through a mechanism that does not involve downstream activation of the Akt pathway in the heart.
In conclusion, this study is the first to demonstrate that AMPK is activated by exercise in the normal heart, consistent with the hypothesis that AMPK may have a role in the cardiac response to physiological stress. Further studies will help to elucidate the extent to which AMPK activation is required for regulation of key physiological pathways in the normal heart.
![]() |
DISCLOSURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
Current address for R. Bergeron: Merck Research Laboratories, Rahway, NJ 07065.
![]() |
FOOTNOTES |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|