The activity of AMP-activated protein kinase (AMPK) increases during muscle contractions as a result of elevated AMP concentration. We tested whether activation of AMPK would be altered during contractions in adenylate kinase (AK) 1-deficient (AK1−/−) mice, because they have a reduced capacity to form AMP. The right gastrocnemius-soleus-plantaris muscle group was stimulated via the sciatic nerve at 2 Hz for 30 min in both wild-type (WT) and AK1−/− animals. Initial force production was not different between the two groups (129.2 ± 3.3 g vs. 140.9 ± 8.5 g for WT and AK1−/−, respectively); however, force production by AK1−/− mice was significantly greater over the 30-min stimulation period, and final tension was 85 ± 4.5% of initial in WT and 102 ± 3.2% of initial in AK1−/− mice. Western blot analysis showed that AMPK phosphorylation with contractions was clearly increased in WT muscles (4.0 ± 1.1 above resting values), but did not change noticeably with AK deficiency (1.6 ± 0.4 above WT resting values). However, increases in phosphorylation of acetyl CoA carboxylase were robust in both WT and AK1−/− muscles and not different between the two groups. These results suggest that reduced formation of AMP during contractions in skeletal muscle of AK1−/− mice results in reduced phosphorylation of AMPK. However, altered AMPK signaling was not apparent in the phosphorylation status of acetyl CoA carboxylase, a typical marker of AMPK activity.
- acetyl CoA carboxylase
- muscle contraction
in skeletal muscle, the rate of ATP consumption is matched by the rate of ATP synthesis throughout a wide range of submaximal energy demands (32, 45). The ATP synthesis occurs predominantly through the oxidation of carbohydrate sources (glycogen, blood glucose, and lactate) and fatty acids (3). Although the precise control of substrate utilization is not well understood, recent work has identified some important signals involved in this process. For example, activation of AMP-activated protein kinase (AMPK) is a recognized signal, sensitive to increased energy demands, that is involved in the regulation of glucose uptake and fatty acid oxidation with muscle contractions (2, 7, 16, 57). However, recent work on transgenic models of AMPK deficiency suggests that AMPK may not be the sole regulator of these actions (25, 35). In addition to an acute role in balancing energy supply to demand, AMPK has been implicated as an important signal for the adaptive increase in mitochondrial capacity that occurs with endurance training (60, 64). An increase in mitochondria increases resistance to muscle fatigue, improving the capacity to manage changes in energy demands. Thus AMPK appears to be an integral part of the metabolic design in which ATP demands from muscle contractions are balanced by the supply of ATP from substrate oxidation.
The regulation of AMPK activity in skeletal muscle is complex and involves both allosteric and covalent modulation in response to the increased energy demands of muscle contractions. Covalent activation occurs by phosphorylation of AMPK by an upstream kinase, AMPK kinase (AMPKK); recently, the tumor suppressor protein LKB1 has been identified as an upstream kinase capable of phosphorylating and thereby activating AMPK (5, 19, 21, 26, 47, 63). Allosteric regulation of AMPK determined from studies in vitro includes activation by AMP and creatine (Cr), as well as inhibition by ATP and phosphocreatine (PCr) (6, 40). Increases in AMP concentration also may allosterically activate the upstream kinase AMPKK (15, 57, 59), as well as inhibit the removal of the phosphate from AMPK by protein phosphatase-2A, facilitating sustained AMPK activation (9, 49). Thus AMPK activation and regulation are sensitive to changes in metabolites related to the cellular energy state (AMP, Cr, ATP, and PCr) through the combined effects of allosteric activation and direct phosphorylation by an upstream kinase.
The formation of AMP during muscle contractions occurs primarily by the enzyme-catalyzed reaction of adenylate kinase (AK). The AK reaction consists of the phosphotransfer of a terminal phosphate from one ADP to another ADP, resulting in the formation of AMP and ATP. The AK reaction is expected to be in equilibrium, even at high energy demands, owing to the very high activity of AK in skeletal muscle (27, 31, 33). Thus the formation of AMP through AK becomes a function of the concentration of ADP, which is directly related to the cellular energy state. Furthermore, PCr serves as an intracellular buffer for ATP during transitions in energy demands, through the creatine kinase reaction. To maintain the concentration of ATP as energy demands increase with contractions, the phosphate group on PCr can be transferred to ADP, resulting in increased Cr formation and a decline in the concentration of PCr. Like AK, the activity of creatine kinase in skeletal muscle is very high, and the reaction is expected to be in equilibrium under most energy demands. Therefore, the regulation of AMPK by AMP and Cr (activators) as well as PCr and ATP (inhibitors) makes its activity sensitive to changes in energy demands and a logical regulator of cellular processes important in maintaining the cellular energy balance (15, 17, 57).
Although the regulation of AMPK has been detailed from in vitro experiments, the relative importance of the different factors involved is difficult to determine in intact muscle. A direct assessment of the degree of AMPK activation by phosphorylation can be made by assaying AMPK activity from muscle homogenates and/or evaluation of the AMPK phosphorylation state (39). The allosteric activation of AMPK by AMP cannot be directly measured from muscle, because the concentration of free AMP is too small and remains unknown (31); however, the AMPK-mediated phosphorylation of acetyl CoA carboxylase (ACC), and coordinate reduction in ACC activity, reflects the combined allosteric and covalent activation of AMPK (23, 39, 54, 58). Thus allosteric activation may be inferred by evaluating the phosphorylation state of ACC (39). Recently, a knockout mouse with AK deficiency (AK1−/−) has been developed (24) that exhibits a markedly diminished AMP production with muscle contractions (13, 14). The differences in AMP formation with AK deficiency may be useful in examining the AMP-dependent activation of AMPK. Therefore, the purpose of this study was to examine the consequences of AK deficiency on the phosphorylation state of AMPK and its downstream target ACC after a period of moderately demanding twitch contractions. We hypothesized that AK deficiency would result in a tempered increase in phosphorylation of AMPK and ACC due to the reduction in AMP formation with contractions. As hypothesized, an increase in AMPK phosphorylation was not observed after muscle contractions in AK-deficient muscle; however, a robust increase in ACC phosphorylation was observed, similar to that of normal wild-type (WT) controls. Thus these results further illuminate the role of AMP in the regulation of AMPK activity in muscle.
The design of this study was aimed at evaluating the phosphorylation of AMPK and ACC after 30 min of moderately demanding muscle contractions in AK1-deficient (AK1−/−) and WT control muscles. Isometric twitch contractions were elicited in situ by electrical stimulation in anesthetized animals, and muscle sections were taken after contractions for the evaluation of enzyme phosphorylation and metabolite concentrations.
AK1−/− mice bred on a C57BL/6 × 129/Ola background and WT control mice of similar genetic background were used in this study (24). AK1 is the predominant isoform of AK expressed in skeletal muscle (24). AK activity is found in most tissues, owing to the expression of one or more of five isozymes identified as AK1 through AK5 (38, 51, 53). The highest expression and activity in any tissue is found in skeletal muscle, owing almost entirely to the AK1 isoform and a relatively insignificant expression of AK3 localized in the mitochondria (AK3 activity is ∼1% of the activity of AK1 in skeletal muscle) (51). Although AK1 is also expressed in other tissues, AK1−/− animals have not been shown to exhibit any overt phenotypic abnormalities (14, 24, 41).
Animals were housed in a temperature-controlled environment (22°C) with a 12:12-h light-dark cycle. Mice were fed ad libitum on standard rodent chow. Animal use and care conformed to the National Institutes of Health guidelines and were approved by the Animal Care and Use Committee at the University of Missouri-Columbia.
Mice were anesthetized with ∼70 mg/kg pentobarbital sodium by intraperitoneal injection, surgically prepared, and placed in a prone position so that the femur and the foot could be secured, and the Achilles tendon was tied to a Cambridge 305B force transducer-lever arm (Aurora Scientific), as described previously (14). Isometric twitch contractions of the gastrocnemius-plantaris-soleus complex were elicited in situ via direct stimulation of the sciatic nerve at a rate of 2 s−1 for 30 min (3–5 V stimulation, 0.05-ms square wave using a Grass S48 stimulator). The voltage used was that which elicited the maximal tetanic contraction in a series of tetanic contractions before the beginning of the twitch contractions. These contractions were spaced out by a minimum of 10 s, and a few minutes of time were allowed before the beginning of the twitch-contraction protocol. The tension elicited and the temperature (controlled at 37°C) were monitored by using a Cambridge 305B lever system and MacLab software, respectively. Mixed gastrocnemius muscle sections from the stimulated (right) and rested (left) hindlimbs were excised and quick frozen by using aluminum tongs cooled in liquid nitrogen, after 30 min of stimulation. Samples were stored at −80°C until use. Mixed gastrocnemius muscle was used because the gastrocnemius accounted for ∼85–90% of the muscle mass of the gastrocnemius-plantaris-soleus complex and thus is the most representative of the metabolic response to the energy demands of the contractions monitored.
Western blot analysis was performed on both muscles for pan-α-AMPK, phosphorylated α-AMPK, and phosphorylated acetyl CoA carboxylase. Frozen muscle samples were homogenized in 5 mM Tris·HCl, pH 7.4, 1 mM EDTA, and 0.2 mM phenylmethylsulfonyl fluoride. Samples were then centrifuged for 10 min at 1,000 g, and the supernatant was used for protein analysis. The protein concentration of each sample was measured by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL), and 40 μg of total protein were separated by SDS-PAGE (8%). Proteins were then transferred from the gel to a nitrocellulose membrane, which was blocked with milk for 1 h. Membranes were then incubated overnight in diluted solutions of the respective antibodies [AMPK-α 1:1,000 and phospho-AMPK-α (T172) 1:500, from Cell Signaling Technology, and phospho-ACC (S79) 1:500, from Upstate Cell Signaling Solutions]. These commercially available antibodies have been used to characterize AMPK phosphorylation and AMPK-dependent phosphorylation of ACC (1, 10, 25, 39, 50). After incubation with the primary antibody, membranes were washed three times and then incubated with the secondary antibody (goat anti-rabbit 1:5,000 for phospho-AMPK-α and AMPK-α, or goat anti-mouse 1:2,500 for phospho-ACC) in 50 mM Tris·HCl buffer, pH 7.5, with 0.2% Tween 20 and milk. After additional washing, membranes were placed in solution with a chemiluminescent substrate (ECL Plus: Amersham Parmacia Biotech) and exposed to film (30 s to 2 min). Protein bands were analyzed with NIH Image version 1.62. Each gel contained both unstimulated and stimulated samples from WT and AK1−/− groups, and all bands were normalized to the density of the resting WT muscle sample within each gel.
The adenine nucleotide (ATP, ADP, and AMP), IMP, PCr, and Cr contents of muscle samples were measured. Frozen muscle samples from rested and stimulated limbs were homogenized in cold 3.5% perchloric acid, the protein pellet was spun down, and the supernatant was neutralized with tri-n-octylamine and 1,1,2-trichlorotrifluoroethane (8). Reverse-phase HPLC was used to measure ATP, ADP, AMP, and IMP, as described previously (52). The muscle contents of PCr and Cr were measured by using ion-exchange HPLC (61). Metabolites were corrected to a constant water content of rested muscle using total creatine (46), based on values we obtained in previous work [26.9 ± 0.8 (n = 49) in WT and 27.1 ± 0.9 (n = 41) in AK1−/− (14)].
Analyses of variance, with repeated measures in one factor, were used to evaluate differences between WT and AK−/− mice and the influence of contractions within groups. A t-test was used to evaluate a difference in muscle force development at 4 min of contractions. A P value of <0.05 was considered statistically significant.
Active force development of nonfatigued AK1−/− muscle is similar to WT control muscle (14, 24). Likewise, the initial force development during our twitch-contraction protocol was similar for both WT and AK1−/− muscle (129.2 ± 3.3 vs. 140.9 ± 8.5 g for WT and AK1−/−, respectively). The AK-deficient mice, however, exhibited a moderately elevated force production compared with the WT control group after ∼4 min of 2-Hz twitch contractions; this was sustained through 30 min of contractions (Fig. 1). This suggests that the energy demands placed on the AK-deficient muscles were likely greater than those on the WT control muscle, because the energy cost per contraction is a function of force development (22). We previously observed that there were no significant shifts in muscle fiber type in AK−/− mice (14).
The content of ATP, ADP, and IMP was not different in rested WT and AK-deficient muscles (Table 1). However, as previously observed (14), the total AMP measured in AK1−/− muscle was higher than in WT muscle at rest (Table 1). The concentration of ATP did not decline significantly after contractions in either group, consistent with the moderate energy demands of the contraction frequency employed (2 s−1). There was no significant effect of contractions on the concentration of total measured ADP or AMP in either AK-deficient or WT muscles. This is not unexpected, however, because the vast majority of the measured ADP and AMP is bound, and the metabolically free concentrations are too small to directly measure under the conditions used in this study (14). A modest increase in IMP concentration was observed in stimulated WT control muscles (Table 1), whereas no increase was observed in AK1−/− muscles. The decline in PCr and the increase in Cr with contractions was significant and similar in both WT and AK-deficient muscles. Thus the distribution of high-energy phosphates after 30 min of twitch contractions was similar in WT and AK-deficient muscles.
AMPK-α content and phosphorylation.
The content of AMPK-α in the gastrocnemius muscle of WT and AK-deficient muscles was evaluated by Western blot analysis. Although no significant difference in the total AMPK-α was found in AK-deficient muscle compared with WT control muscles, AK-deficient muscles tended to exhibit (P < 0.10) reduced levels of AMPK (Fig. 2). Therefore, significant alteration in the expression of AMPK due to AK1 deficiency is not apparent in these muscles. The degree of AMPK-α T172 phosphorylation is a measure of AMPK activation by the upstream kinase AMPKK (1, 10, 25, 39, 50). Thus Western blot analysis was performed for the phosphorylation of the AMPKK phosphorylation site (T172) on the α-subunit of AMPK of rested and after 30 min of twitch contractions in both WT and AK-deficient muscles. The AMPK phosphorylation in WT muscle increased approximately fourfold above resting values. In contrast, there was no increase in AMPK phosphorylation in AK-deficient muscles, because the stimulated muscle was not different from the rested muscle (Fig. 3). Therefore, modulation of AMPK activity by phosphorylation, in response to contractions, was not apparent in AK-deficient muscle.
The in vivo activity of AMPK is not directly measurable by assay of muscle homogenates, because AMPK is modulated by allosteric (AMP, ATP, PCr, and Cr) and covalent (phosphorylation by AMPKK) mechanisms. Because ACC-β is a substrate of AMPK, the phosphorylation of ACC-β likely reflects AMPK activity in vivo. Furthermore, the phosphorylation of ACC-β has been found to parallel the phosphorylation of AMPK-α T172 in rat fast-twitch skeletal muscle (39). Therefore, we investigated the phosphorylation of ACC-β in WT and AK-deficient muscle after 30 min of moderately demanding contractions. Phosphorylation of ACC-β in WT muscle after contractions exhibited a robust increase of ∼11-fold over resting values (Fig. 4). Interestingly, the increase in phospho-ACC-β in AK-deficient muscle after contractions was also robust (∼15-fold) and not different from that observe in contracting WT muscles.
The activation of AMPK is thought to be an important factor involved in signaling changes in energy demands that occur with muscle contractions. AMPK has been implicated as a signal for an acute increase in glucose uptake, by the recruitment of glucose transporters, and for an increase in fatty acid oxidation, by the phosphorylation of ACC-β (7); in addition, it may also be involved in more chronic adaptations seen with exercise training such as mitochondrial biogenesis (20, 60, 64). The regulation of AMPK activity is complex and involves allosteric activation by metabolites, which are elevated when the cellular energy state is challenged (AMP and Cr), and phosphorylation by an upstream kinase AMPKK, which is enhanced by elevations in AMP (34, 56). Thus AMP appears to be central in determining the activity of AMPK from both allosteric modulation and enhancing covalent modulation through phosphorylation (48, 56). In this study, we have sought to determine the extent of this control by AMP, using muscle with a deficiency in AK. We found that the typical increase in AMPK-α T172 phosphorylation did not occur in AK1−/− muscle after contractions, consistent with an absent or tempered production of AMP. This absence of AMPK phosphorylation cannot easily be explained by an abbreviated duration of phosphorylation, because the stability of phosphorylated AMPK extends well beyond the duration of our experiment (42). Rather, our findings imply that elevations in AMP are critical in the activation of AMPK in contracting skeletal muscle. The absence of AMPK-α phosphorylation would presumably reduce downstream events typical of AMPK activation. However, we observed an apparent inconsistency, as there was a robust elevation in ACC phosphorylation, a response that is normally coincident with elevated AMPK phosphorylation (39, 54). Thus a more careful consideration of the response within contracting AK1−/− muscle is warranted.
AK deficiency and AMP formation.
The increase in measured AMPK activity with muscle contractions reflects an increase in the phosphorylation of AMPK, which is sensitive to elevations in AMP (34, 56). Using a model of muscle AK deficiency, we sought to determine whether the AMPK phosphorylation that normally occurs with contractions would be depressed when AMP production is likely to be limited. Previous work on AK1−/− muscle at very high energy demands has demonstrated an exceptionally elevated ADP accumulation and a markedly reduced IMP production (suggesting low AMP concentrations) (13, 14). The AK1−/− mouse is a knockout model of the predominant AK isoform in skeletal muscle (24, 51). The remaining AK activity in AK1−/− muscle is exceptionally low (24), presumably owing to the presence of other AK isoforms expressed in skeletal muscle. These are thought to be localized to the inner mitochondrial membrane (AK2) and the mitochondrial matrix (AK3) (37, 51). If AK activity in skeletal muscle were completely absent, one may expect the capacity to form ATP by de novo purine synthesis and purine salvage pathways to be limited and the nucleotide contents to be severely reduced. The measured concentrations of ATP and ADP in resting muscle, however, are not different between AK1−/− and WT muscles. Thus even with the marked AK1 deficiency, the AK localized to the mitochondrial inner membrane apparently has access to cytosolic nucleotides and, given enough time, could result in the normal ATP and ADP measured. In contrast, the total AMP measured is significantly elevated in AK1−/− muscle, as reported in Table 1 and previously (14). The vast majority of the AMP measured from muscle extracts is bound or restricted to a compartment where it is not metabolically active (27, 31, 55). Thus one cannot infer alterations in the physiologically significant concentration of AMP from changes in total muscle AMP content. Furthermore, if the difference in AMP measured from muscle extracts of rested AK1−/− and WT muscles [∼0.05 μmol/g wet wt; cf. Table 1 or our previous work (14)] represented an increase in free AMP, this would be well in excess of two orders of magnitude greater than the free-AMP concentration calculated in WT muscles (assuming CK and AK are in equilibrium, as described by Refs. 27, 31, 55). This increase seems highly unreasonable, considering that the extent of AMPK phosphorylation in rested AK1−/− muscle was not elevated over that observed in WT muscle. Therefore, we submit that changes in measured AMP do not reflect alterations in the concentration of free AMP present in the cytosol that are germane to AMP-mediated signaling. How then can we have any confidence that free AMP was not increased as much during contractions in the AK1−/−, compared with WT muscle?
Normally, the free-AMP concentration can be calculated by using the AK and creatine kinase reactions, because these reactions are expected to be in equilibrium (11, 27). As is obvious, the assumption of equilibrium kinetics of AK is not applicable in the AK1−/− model employed in this study. This undermines our ability to estimate free AMP and have confidence in possible changes with contractions. However, some insight as to elevations in the physiological AMP concentration is available from the production of IMP. As energy demands become excessive, a decline in ATP is observed that is matched by an increase in IMP (11, 33). IMP formation occurs by the removal of the six-amino group from AMP through the reaction catalyzed by AMP deaminase (AMPD). AMPD activity is very sensitive to elevations in AMP (29, 30). Previous work on AK1−/− muscle has shown that AMPD activity muscle is ∼65% that of WT muscle (24). This is not likely to impact the IMP production in AK1−/− muscle based on the previous findings that muscle with only 20% of normal AMPD activity does not have impaired IMP formation (44). In this study, IMP formation in WT control muscles was real, but modest (Table 1), as expected with the moderate energy demands of this contraction frequency (2-Hz twitch contractions). This would occur with an elevation in AMP during contractions. In contrast, there was no accumulation of IMP in the AK−/− muscle (Table 1). This is consistent with a minimal to absent production of AMP during contractions and supports our conclusion that AMP was not meaningfully increased in the AK−/− muscle. We believe that these findings are useful in illuminating the role that AMP has in activating AMPK through phosphorylation and highlight the importance of AMP in facilitating the covalent modulation of AMPK in contracting muscle.
Allosteric regulation of AMPK.
Although the absence of AMPK-α phosphorylation in contracting AK−/− muscle is, we believe, reasonably explained by a reduced AMP concentration, the downstream event of a robust phosphorylation of ACC is not. However, it is important to recognize that the regulation of AMPK in response to muscle contractions involves both phosphorylation and allosteric activation (15, 17, 18). Although the activity of AMPK by phosphorylation is readily apparent in enzyme activity measured in vitro, there are no such means of detecting allosteric modulation of AMPK activity short of knowing reaction conditions in vivo. Therefore, the phosphorylation of ACC by AMPK and/or its attendant large decline in ACC activity are often measured to indicate AMPK activity in vivo (4, 28). Indeed, phosphorylation and inactivation of ACC are normally accompanied by an increase in phosphorylated AMPK-α. Park et al. (39) demonstrated a clear linear relationship between AMPK phosphorylation, AMPK activity, and the phosphorylation of ACC with increasing contraction frequency in rat skeletal muscle. However, covalent modulation of AMPK activity by phosphorylation is an unlikely candidate to account for ACC phosphorylation in our study. This implies that the increased AMPK activity observed in vivo (increased ACC phosphorylation) was due to the allosteric activation of AMPK. Experiments on purified AMPK complexes in vitro have shown that AMP has a direct activating effect on AMPK (12, 48). Thus, considering that the concentration of AMP is likely tempered in AK1−/− muscle, we would expect the direct allosteric activation of AMPK by AMP in AK1−/− muscles to be reduced compared with the WT controls. Other allosteric modulators include PCr, which at concentrations found in resting muscle is expected to exert an inhibitory effect on AMPK (estimated to be ∼50%), but released by the increase in Cr that occurs with contractions (40). The observed values of PCr and Cr in both WT and AK1−/− muscles (Table 1) are in the apparent asymptotic range of this effect in vitro (40); therefore, we would expect that any possible activation of AMPK due to changes in PCr and Cr in the contracting muscles would be similar in magnitude in both WT and AK1−/− groups. Thus the decline in PCr, increase in Cr, and the potentially tempered concentration of AMP with AK deficiency is consistent with a reduced allosteric activation of AMPK, relative to WT muscle. This, however, is not consistent with the robust phosphorylation of ACC-β observed in Fig. 4, which would require normal AMPK activity in AK-deficient muscle.
There are several possibilities that could contribute to this robust ACC-β phosphorylation in the absence of compelling evidence for equivalent covalent and allosteric control of AMPK activity among the WT and AK−/− muscle. First, AK1−/− muscle may possess enhanced sensitivity to allosteric activation. This could conceivably be accomplished through a much higher expression of the more allosterically sensitive α2-subunit of AMPK (40); however, this is not likely to be the sole reason for the discrepancy, because the α2-subunit is already the predominant isoform expressed in skeletal muscle and already accounts for most of the measured AMPK activity (48). Second, ACC could be phosphorylated by a yet-to-be-recognized kinase, independent of AMPK. Alternatively, an enhanced activity of AMPK could have been mediated by some allosteric activators that have not been characterized. Finally, there could be altered rates of dephosphorylation in the AK1−/− muscles that may account for this apparent discrepancy. These possibilities are speculation and remain to be supported by any evidence to our knowledge.
The dichotomy between ACC phosphorylation (reduced activity in vitro) and AMPK phosphorylation (increased activity in vitro) is not without precedent. Winder et al. (60) measured AMPK and ACC activity in muscle homogenates after a single injection of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), and after 4 wk of daily AICAR injections. AICAR is an analog of adenosine that is taken up by muscle and phosphorylated, forming an analog of AMP, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl-5′-monophosphate (ZMP), which activates AMPK (17). After a single injection of AICAR, AMPK activity increased and ACC activity was markedly inhibited (via ACC phosphorylation) (60). Interestingly, after 4 wk of daily injections of AICAR, there was no evidence for enhanced AMPK phosphorylation in the muscle (increased activity in vitro); nonetheless, the decline in in vitro ACC activity (increased ACC phosphorylation) was similar to that observed previously with acute allosteric modulation by AICAR. The lack of an increase in the AMPK activity measured from the muscle homogenate indicates that AMPK was not phosphorylated. In contrast, the decline in ACC activity presumably owing to ACC-β phosphorylation by AMPK demonstrated enhanced AMPK activity due to allosteric activation by ZMP, without an increase in AMPK phosphorylation (60). Furthermore, Roepstorff et al. (43) demonstrated a similar pattern of tempered AMPK activation with normal ACC phosphorylation after 60 min of moderate bicycle exercise in human vastus lateralis when muscle glycogen remained elevated. In addition, in human muscle, a study by Wojtaszewski et al. (62) demonstrated that phosphorylation of ACC-β occurred at an earlier time point than the increase in AMPK-T172 phosphorylation during exercise. This is consistent with our findings that the normal increase in AMPK phosphorylation was not required for the increase in ACC-β phosphorylation that occurred after moderately demanding contractions. Interestingly, the pattern of ACC and AMPK activity after exercise in rat muscle does not exhibit this dissociation (42). This leads to the final possibility that a modest increase in AMPK activity not reflected in the phosphorylation status of AMPK-α, brought about by even the expectedly reduced allosteric modulation, was sufficient to permit accumulation of the same amount of ACC-β phosphorylation over the 30-min contraction period.
Although the purpose of this study was to examine the response of AMPK to contractions in the context of AK deficiency, we also report here that the AK-deficient muscle was able to sustain greater force production than the wild type over the course of the 30 min of moderately demanding contractions (Fig. 1). This is the first report of enhanced performance with AK deficiency. Although the reason for this increase is not known, it may be due an enhanced aerobic capacity, evident by a higher mitochondrial content and greater capillary density (14, 24), that would better support the muscle’s ability to meet the moderate energy demands of our stimulation conditions (2 Hz). Therefore, one interpretation of the reduction in AMPK phosphorylation may be that AMPK is less activated owing to the reduced AMP formation that occurs because of the enhanced mitochondrial content, similar to what occurs in trained muscle (36). However, this would not be consistent with the observed robust increase in ACC phosphorylation (Fig. 4).
In conclusion, an increase in phosphorylation of AMPK-α after contractions was not apparent in AK-deficient muscle, consistent with limited AMP production and highlighting the role that AMP has facilitating AMPK-α phosphorylation by AMPKK. In contrast, the phosphorylation of ACC-β increased in AK-deficient muscle similar to WT controls in the absence of compelling evidence to account for allosteric modulation of AMPK activity. Future work is necessary to clarify this apparent inconsistency and to determine whether other downstream effects of AMPK activation are altered in AK1-deficient muscle.
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases grant AR-21617 and Netherlands NWO-GMW (901-01-095).
We would like to thank Dr. Bé Wieringa for providing the AK1−/− mouse model used in this study. The technical assistance of Catharine Clark, Yuhua Xiao, and Kirk Abraham is also greatly appreciated.
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.
- Copyright © 2006 the American Physiological Society