Ca²⁺ and AMPK Both Mediate Stimulation of Glucose Transport by Muscle Contractions

2-[1,2-³H] deoxyglucose (2-DG) was purchased from American Radiolabeled Chemicals (St. Louis, MO). [¹⁴C]Mannitol was obtained from ICN Radiochemicals (Irvine, CA). KN62, KN93, and KN92 were obtained from Calbiochem (La Jolla, CA). The anti-phospho CAMK-II and the anti-phospho AMPK antibodies were purchased from Cell Signaling (Beverly, MA). The horseradish peroxidase−conjugated donkey anti-rabbit IgG was purchased from Jackson ImmunoResearch (West Grove, PA). Enhanced chemiluminescence (ECL) reagents were obtained from Amersham (Arlington Heights, IL). All other chemicals were obtained from Sigma (St. Louis, MO).

Male Wistar rats (Charles River) weighing ∼80–120 g were provided with Purina rat chow and water ad libitum. Food was removed at 5:00 p.m. the evening before the experiment. Rats were anesthetized by an intraperitoneal injection of pentobarbital sodium (5 mg/100 g body wt) after which the epitrochlearis muscles were removed. The epitrochlearis is a small, thin muscle of the forelimb that is suitable for studies of glucose transport (14–16). All protocols were approved by the Animal Studies Committee of Washington University.

After dissection, muscles were allowed to recover for 60 min in flasks containing 2 ml of Krebs-Henseleit bicarbonate buffer (KHBB) with 8 mmol/l glucose, 32 mmol/l mannitol, and a gas phase of 95% O₂/5% CO₂. The flasks were placed in a shaking incubator maintained at 35°C. After recovery, some muscles were incubated in the same medium containing 3.5 mmol/l caffeine, 2 mmol/l AICAR, 2 mU/ml insulin, or caffeine plus AICAR. The length of the incubations was 15 min for caffeine (6) and 60 min for insulin and AICAR. (We have found that these durations of incubation induce the maximal effects of these agents on glucose transport.) For measurement of calmodulin-dependent protein kinase (CAMK)-II phosphorylation, the incubation with caffeine was for 2 min. Other muscles were stimulated to contract using a Grass SII stimulator, as previously described (17). Tetanic contractions were produced by stimulating muscles at 100 Hz with 0.2-msec pulses for 10 s at a rate of one contraction per minute for 10 min. In some experiments, the sarcoplasmic reticulum Ca²⁺ release inhibitor dantrolene (5 μmol/l) or the CAMK inhibitors KN62, KN93, or KN92 (10 μmol/l) were included in the incubation medium; in these experiments, the muscles were incubated with or without the inhibitor for 30 min before as well as during treatment with caffeine, contractions, or treatment with insulin. Because these inhibitors are light sensitive, the flasks were wrapped in foil.

After the various treatments, the muscles were rinsed for 10 min at 29°C in 2 ml of oxygenated KHBB containing 40 mmol/l mannitol to remove glucose and treatment agents. After the rinse step, muscles were incubated for 20 min at 29°C in flasks containing 2 ml KHBB with 4 mmol/l 2-DG (1.5 μCi/ml) and 36 mmol/l [¹⁴C]mannitol (0.2 μCi/ml), with a gas phase of 95% O₂/5% CO₂, in a shaking incubator. The muscles were then blotted, clamp frozen, and processed for determination of intracellular 2-DG accumulation and extracellular space, as previously described (15).

Clamp-frozen epitrochlearis muscles were homogenized in a 10:1 vol/wt ratio of ice-cold buffer containing 50 mmol/l Tris-HCL (pH 7.4); 1% NP-40; 0.25% sodium deoxycholate; 150 mmol/l NaCl; 1 mmol/l each of EDTA, phenylmethylsulfonyl fluoride, and NaF; 1 μg/ml each of aprotinin, leupeptin, and pepstatin; 0.1 mmol/l bpV(phen); 25 μmol/l okadaic acid; and 2 mg/ml β-glycerophosphate. Samples were sonicated on ice for 5 s. Homogenized samples were centrifuged for 15 min at 700g at 4°C. Protein concentration was determined by the method of Lowry et al. (18). Samples were prepared in 2 × Laemmli buffer containing 100 mmol/l dithiothreitol and heated in a boiling water bath for 5 min. Next 30 μg of protein from each sample were subjected to SDS-PAGE (10% resolving gel), then transferred to nitrocellulose membranes at 200 mA for 2 h. After the transfer, membranes were reversibly stained with Ponceau S to ensure equal loading and transfer of proteins. Membranes were blocked for 1 h at room temperature in Tris-buffered saline with 0.1% Tween (TBST; 200 mmol/l Tris base, 1.37 mol/l NaCl; pH 7.4) supplemented with 5% nonfat dry milk. Membranes were incubated overnight at 4°C with antibodies specific for phosphorylated CAMK-II (thr-287) at a dilution of 1:1,000 or p-AMPK (thr-172) diluted 1:1,000. TBST/5% BSA was used as a dilutant. Bands were visualized by ECL and quantified using densitometry.

Data are means ± SE. Comparisons of means of multiple groups were made using one-way ANOVA followed by a post hoc comparison using Fisher’s least significant difference method. Statistical significance was established at P < 0.05.

Caffeine causes a release of Ca²⁺ from the sarcoplasmic reticulum into the cytosol in muscle cells (19,20). At a sufficiently high concentration, caffeine causes muscles to contract. However, at the concentration used in this experiment (3.5 mmol/l), the increases in cytosolic Ca²⁺ were too small to cause muscle contraction (6). As shown in Fig. 1, this concentration of caffeine induced an approximate threefold increase in glucose transport activity in epitrochlearis muscles.

The stimulation of glucose transport by caffeine was inhibited by a low concentration (5 μmol/l) of dantrolene, an agent that blocks Ca²⁺ release from the sarcoplasmic reticulum (21), providing evidence that it is mediated by Ca²⁺. The increase in glucose transport induced by caffeine was also blocked by the specific inhibitors of Ca²⁺-CAMKs, KN62 and KN93 (Fig. 2). KN92, the inactive form of KN93 that served as a negative control, had no effect on glucose transport. These results suggest that activation of glucose transport by increases in cytosolic Ca²⁺ is mediated by CAMK.

Numerous previous studies have shown that treatment of striated muscle with AICAR, which is taken up by muscle cells and converted to the AMP analog ZMP, results in activation of AMPK and stimulation of glucose transport (7–10). As shown in Fig. 1, incubation of epitrochlearis muscles with 2 mmol/l AICAR resulted in an approximate threefold increase in glucose transport activity that was similar in magnitude to that induced by a subcontraction concentration of caffeine.

Muscle contractile activity results in increases in both cytosolic Ca²⁺ and AMPK activity. To mimic this response, we treated muscles with caffeine to raise cytosolic Ca²⁺ and with AICAR to activate AMPK. As shown in Fig. 1, the increase in glucose transport activity induced by raising cytosolic Ca²⁺ and activating AMPK were additive. Furthermore, the combined effect on glucose transport of caffeine plus AICAR was not significantly different from that induced by maximally effective electrical stimulation of muscle contractions.

KN62 and KN93, the CAMK inhibitors that block the effects of caffeine on glucose transport, partially inhibited contraction-stimulated glucose transport (Fig. 3). KN92, the inactive form of KN93, had no effect on contraction-stimulated glucose transport. It has been reported that KN62 partially inhibits insulin-stimulated glucose transport (22). This raised the possibility that KN62 has a nonspecific inhibitory effect on glucose transport. However, as shown in Fig. 4, under our experimental conditions, KN62 did not have a significant inhibitory effect on insulin-stimulated glucose transport. KN62 also had no significant inhibitory effect on the stimulation of glucose transport by AICAR. These findings support the interpretation that the mechanism by which raising cytosolic Ca²⁺ stimulates glucose transport in skeletal muscle involves the activation of CAMK.

As shown in Fig. 5, muscle contractions increased CAMK-II phosphorylation approximately threefold. AMPK phosphorylation also increased in response to contractile activity. KN62 completely inhibited the increases in CAMK-II phosphorylation, but had no effect on the increase in AMPK phosphorylation induced by contractions. Similarly, caffeine induced an increase in CAMK-II phosphorylation, but had no effect on AMPK phosphorylation (Fig. 6).

The discovery that AICAR stimulates glucose transport in skeletal muscle has led to the concept that activation of AMPK mediates the activation of glucose uptake by contractile activity (7–11). The evidence that activation of AMPK stimulates glucose transport appeared to argue against our hypothesis that the increases in cytosolic Ca²⁺ during contractile activity provide the signal leading to the exercise-induced increase in glucose uptake (1,5,6). This hypothesis was based, in large part, on the finding that raising cytosolic Ca²⁺ to subcontraction levels that do not cause a decrease in high-energy phosphates results in an increase in muscle glucose transport (6). The present results help to resolve this apparent discrepancy by demonstrating that in rat epitrochlearis muscles, either activation of AMPK with AICAR or raising cytosolic Ca²⁺ with subcontraction concentrations of caffeine results in an increase in glucose transport activity that is ∼50% as great as that induced by maximally effective contractile activity. Furthermore, treatment of muscles with AICAR and caffeine in combination induced as great an increase in glucose transport as maximally effective contractile activity. These findings, which provide evidence that increases in both AMPK activity and Ca²⁺ are involved in mediating the increase in glucose transport, are in keeping with and help to explain the finding by Mu et al. (13) that AMPK activation accounts for only ∼40% of the increase in glucose transport induced by contractions in mouse skeletal muscle.

The stimulation of glucose transport by Ca²⁺ appears to be mediated by activation of Ca²⁺ CAMKs. This is evidenced by the finding that the specific inhibitor of CAMKs, KN62 (23,24), prevented the stimulation of glucose transport induced by raising cytosolic Ca²⁺ using the sarcoplasmic reticulum Ca²⁺-releasing agent caffeine. In light of this evidence that CAMKs mediate the effect of Ca²⁺ on glucose transport, our finding that KN62 inhibits the contraction-induced increase in glucose transport by ∼50% provides further evidence that the increase in cytosolic Ca²⁺ accounts for ∼50% of the effect of contractions on glucose transport. Increases in cytosolic Ca²⁺ result in activation of all three of the CAMKs (i.e., CAMK-I, CAMK-II, and CAMK-IV), and KN62 inhibits all of the CAMKs (23). We found that contractions or caffeine induce phosphorylation of CAMK-II in the epitrochlearis. CAMK-II is the predominant CAMK isoform in the rat epitrochlearis muscle, but this muscle also contains some CAMK-IV and trace amounts of CAMK-I (D.C.W., K.A.H., J.O.H., D.H.H., unpublished observations). Thus, although our results suggest that the next step in the signaling pathway leading from an increase in cytosolic Ca²⁺ to an increase in glucose transport involves activation of CAMK-II, no information is available regarding the relative contributions of CAMK-I and -IV to this process. However, because the CAMKs are closely related enzymes that recognize the same amino acid sequence and have broadly overlapping substrate preferences (23), it seems possible that any of the CAMKs may be able to mediate the increase in glucose transport activity in the muscles in which they are expressed.

Previous studies have shown that raising cytosolic Ca²⁺ stimulates glucose transport (1,5,6) and that AMPK accounts for only part of the contraction-induced increase in glucose transport (13). Viewed in this context, the present results provide evidence that exercise stimulates glucose transport by both lowering high-energy phosphates and raising Pi, thus resulting in activation of AMPK, and by increasing cytosolic Ca²⁺, resulting in activation of CAMKs. If this interpretation is correct, it seems reasonable that increases in cytosolic Ca²⁺ are primarily responsible for the stimulation of glucose transport during mild exercise that results in minimal decreases in high-energy phosphates. Activation of AMPK likely plays a progressively more important role as exercise intensity increases and the high-energy phosphate concentration falls. It is of interest that the signals that appear to be responsible for the acute activation of glucose transport by exercise also appear to mediate the longer term, exercise-induced increase in glucose transport capacity. This is evidenced by the finding that either activation of AMPK with AICAR or raising cytosolic Ca²⁺ with caffeine induces an increase in GLUT4 protein in L6 myotubes and in rat skeletal muscle (25–27).

In conclusion, the results of this study provide evidence that both activation of AMPK and increases in cytosolic Ca²⁺ are responsible for mediating the increase in muscle glucose transport activity induced by muscle contractions.

This research was supported by National Institutes of Health Grant DK-18986. D.C.W. was supported by an American Diabetes Association mentor-based postdoctoral fellowship.

We thank Mark Rheinheimer for excellent technical assistance and Victoria Reckamp for expert assistance in preparation of this manuscript.