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Evidence for the involvement of CaMKII and AMPK in Ca²⁺-dependent signaling pathways regulating FA uptake and oxidation in contracting rodent muscle

Departments of Kinesiology and Biological Sciences, College of Letters, Arts, and Sciences, University of Southern California, Los Angeles, California

Submitted 5 December 2007 ; accepted in final form 27 February 2008

	ABSTRACT

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Calcium-calmodulin/dependent protein kinase II (CaMKII), AMP-activated protein kinase (AMPK), and extracellular signal-regulated kinase (ERK1/2) have each been implicated in the regulation of substrate metabolism during exercise. The purpose of this study was to determine whether CaMKII is involved in the regulation of FA uptake and oxidation and, if it is involved, whether it does so independently of AMPK and ERK1/2. Rat hindquarters were perfused at rest with (n = 16) or without (n = 10) 3 mM caffeine, or during electrical stimulation (n = 14). For each condition, rats were subdivided and treated with 10 µM of either KN92 or KN93, inactive and active CaMKII inhibitors, respectively. Both caffeine treatment and electrical stimulation significantly increased FA uptake and oxidation. KN93 abolished caffeine-induced FA uptake, decreased contraction-induced FA uptake by 33%, and abolished both caffeine- and contraction-induced FA oxidation (P < 0.05). Caffeine had no effect on ERK1/2 phosphorylation (P > 0.05) and increased ₂-AMPK activity by 68% (P < 0.05). Electrical stimulation increased ERK1/2 phosphorylation and ₂-AMPK activity by 51% and 3.4-fold, respectively (P < 0.05). KN93 had no effect on caffeine-induced ₂-AMPK activity, ERK1/2 phosphorylation, or contraction-induced ERK1/2 phosphorylation (P > 0.05). Alternatively, it decreased contraction-induced ₂-AMPK activity by 51% (P < 0.05), suggesting that CaMKII lies upstream of AMPK. These results demonstrate that regulation of contraction-induced FA uptake and oxidation occurs in part via Ca²⁺-independent activation of ERK1/2 as well as Ca²⁺-dependent activation of CaMKII and AMPK.

fatty acid uptake; erk1/2; caffeine; KN93; electrical stimulation

Studies have shown that pharmacological induction of Ca²⁺ release from the sarcoplasmic reticulum, as a model of the changes associated with the mechanical properties of muscle contraction, results in increased FA uptake in isolated rodent muscle (34) as well as a decrease in malonyl-CoA levels in perfused muscle (14), a change which has been shown repeatedly to be associated with an increase in FA oxidation (17, 21, 23). These results suggest that in addition to or in parallel with AMPK and ERK1/2, Ca²⁺-dependent signaling mechanisms may play an important role in the regulation of FA metabolism during muscle contraction. The multisubunit holoenzyme Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), activated by muscle contraction and caffeine treatment via release of Ca²⁺ from the sarcoplasmic reticulum in rat and human muscle (24, 39, 40), has recently become a target for the study of Ca²⁺-dependent signaling mechanisms in muscle. Indeed, inhibition of CaMKII with KN93 has resulted in prevention of both the caffeine- and contraction-induced increase in glucose uptake in soleus muscle (39) and prevention and partial decrease of caffeine- and contraction-induced increase in glucose uptake, respectively, in epitrochlearis muscle (40), suggesting that CaMKII activation is critical in the regulation of glucose uptake in skeletal muscle in response to an increase in intracellular Ca²⁺ level. However, the involvement of Ca²⁺-dependent signaling mechanisms via activation of CaMKII in the regulation of FA uptake and oxidation is unknown.

It has been suggested that Ca²⁺/calmodulin-dependent signaling molecules may be involved in signaling pathways that also include ERK1/2 (1) and AMPK (5, 7, 36). Studies indicate that the upstream kinase of CaMK, Ca²⁺/calmodulin-dependent protein kinase kinase (CaMKK), has the ability to phosphorylate and activate AMPK independently of the conventional upstream AMPK kinase, LKB1 (7). Other data collected in pulmonary endothelial cells suggest that ERK1/2 might lie downstream of CaMKII in a Ca²⁺-dependent signaling pathway during contraction (1). However, it is not known whether AMPK and/or ERK1/2 are activated by Ca²⁺-dependent signaling pathways in skeletal muscle and if so, whether their relationship with CaMKII signaling persists in the regulation of contraction-induced FA metabolism. Therefore, the purpose of this study was to determine whether CaMKII is involved in the regulation of contraction-induced FA uptake and oxidation and, if it is involved, whether it does so independently of AMPK and/or ERK1/2. We chose to treat skeletal muscle with exogenous caffeine to examine the effect of an increase in intracellular Ca²⁺ level similar to that measured with muscle contraction (18, 28, 41) on cellular signaling and FA metabolism. We also chose to use the inactive and active CaMKII inhibitors, KN92 and KN93, respectively, to better characterize the signaling mechanisms activated by CaMKII during muscle contraction, inhibitors that are commonly used in muscle metabolism studies (6, 27, 38, 40). We hypothesized that both moderate-intensity muscle contraction and caffeine treatment would result in increased FA uptake and oxidation in perfused muscle and be accompanied by increases in CaMKII, AMPK, and ERK1/2 activity. We further hypothesized that inhibition of CaMKII activity with KN93 would significantly decrease both contraction- and caffeine-induced FA uptake and oxidation. Because it has been suggested that ERK1/2 may lie downstream of CaMKII in contraction-induced signaling cascades (1), and because KN93 is a specific inhibitor of CaMKII (26) and has not to our knowledge been shown to inhibit CaMKK, the Ca²⁺-dependent signaling molecule shown to stimulate AMPK activity (5, 7, 36), we expected that treatment with KN93 would result in a significant decrease in ERK1/2 activity and no change in AMPK activity during caffeine treatment and contraction in skeletal muscle.

	MATERIALS AND METHODS

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Animal preparation.   Male Wistar rats (300–350 g) were housed in pairs and kept on a 12:12-h light-dark cycle. They received standard rat chow and water ad libitum. Rats were randomly assigned to rest (R), caffeine (C), or moderate-intensity muscle contraction (MC) groups, and each group was subdivided into treatment with dimethyl sulfoxide and either KN92 or KN93, inactive and active CaMKII inhibitors, respectively: R+KN92 (n = 5), R+KN93 (n = 5), C+KN92 (n = 8), C+KN93 (n = 8), MC+KN92 (n = 7), MC+KN93 (n = 7). Ethical approval for the present study was obtained from the Institutional Animal Care and Use Committee at the University of Southern California.

Hindquarter perfusion.   On the day of the experiment, rats were anesthetized with an intraperitoneal injection of a ketacet/xylazine mixture (80 and 12 mg/kg body wt, respectively). Rats were then prepared surgically for hindquarter perfusion as previously described (32). Before the perfusion catheters were inserted, heparin (150 IU) was administered into the inferior vena cava. The rats were killed with an intracardial injection of pentobarbital sodium (0.4 mg/g body wt), and arterial and venous catheters were inserted immediately into the descending aorta and vena cava, respectively. The preparation was placed in a perfusion apparatus where the left iliac vessels were tied off, and a clamp was fixed tightly around the proximal part of the leg to prevent bleeding (32).

The initial perfusate (250 ml) consisted of Krebs-Henseleit solution, 5% bovine serum albumin (Bovuminar pH 7; Serologicals, Norcross, GA), 550 µM albumin-bound palmitate, 8 µCi of albumin-bound [1-¹⁴C] palmitate, 6 mM glucose, 10 µM KN92 or KN93 dissolved in dimethyl sulfoxide, and 3 mM caffeine dissolved in Krebs-Henseleit buffer in the C groups. Previous studies have shown that caffeine at concentrations below 3 mM stimulates the release of Ca²⁺ from the sarcoplasmic reticulum (18, 28, 41) and mimics the metabolic effects of contraction in skeletal muscle without inducing force production (18, 28, 41). Insulin was not included to isolate contraction effects on muscle metabolism. The perfusate (37°C) was continuously gassed with a mixture of 95% O₂-5% CO₂, which yielded arterial pH values of 7.4–7.6 and arterial PO₂ and PCO₂ values that were typically 250–450 and 33–48 mmHg, respectively. Mean perfusion pressures were not affected by caffeine, muscle contraction, KN92, or KN93 and averaged 93.8 ± 19.2, 92.7 ± 19.3, 87.0 ± 12.9, 97.8 ± 16.8, 80.0 ± 15.4, and 88.4 ± 13.5 mmHg in the R+KN92, R+KN93, C+KN92, C+KN93, MC+KN92, and MC+KN93 groups, respectively (P > 0.05).

The first 25 ml of perfusate that passed through the right hindquarter were discarded, whereupon the hindquarter was equilibrated for 20 min. Animals were then perfused for an additional 20 min at a perfusate flow of 15 ml/min (average for all groups: 0.80 ± 0.01 ml·min^–1·g^–1 perfused muscle). The right leg of animals in the MC groups was immobilized at the tibiopatellar ligament, and a hook electrode was placed around the sciatic nerve and connected to a S48 Grass stimulator (Grass Telefactor, West Warwick, RI). The resting length of the gastrocnemius-soleus-plantaris muscle group was adjusted to obtain maximum active tension upon stimulation. Isometric muscle contractions were induced by stimulating the sciatic nerve electrically with supramaximal 15-V trains of 100 Hz with impulse duration of 1 ms, delivered every 2 s and lasting 50 ms, which is considered moderate-intensity muscle contraction (12). We chose this train duration because a previous study in our lab demonstrated that FA metabolism is maximized during this moderate-intensity stimulation protocol (19). During the 20-min muscle stimulation, the tension developed by the gastrocnemius-soleus-plantaris muscle group was recorded with a modular chart recorder (Cole Parmer, Vernon Hills, IL). The decrease in tension development over the stimulation period was used as an indicator of performance.

Postequilibration, arterial and venous perfusate samples were taken at 5, 10, 15, and 20 min for analysis of [¹⁴C]FA and ¹⁴CO₂ radioactivities, as well as FA, glucose, and lactate concentrations. Arterial and venous perfusate samples for determinations of PCO₂, PO₂, and pH were taken at 10 and 20 min. At the end of the 20-min experimental period, the gastrocnemius-soleus-plantaris muscle group of the right leg was freeze-clamped in situ with aluminum clamps precooled in liquid N₂, taken out, and stored for later analysis.

Blood sample analyses.   YSI-1500 was used to analyze both plasma glucose and lactate concentrations (Yellow Springs Instruments, Yellow Springs, OH). Plasma FA concentrations were determined spectrophotometrically using the WAKO NEFA-C test (Biochemical Diagnostics, Edgewood, NY). Plasma [¹⁴C]FA and ¹⁴CO₂ radioactivities were determined in duplicate as previously described in detail (29, 31, 32). PCO₂, PO₂, and pH were measured with an ABL-5 analyzer (Radiometer America, Westlake, OH).

Muscle sample analyses.   To quantify dual phosphorylation of ERK1/2, muscle samples (90 mg) were powdered under liquid N₂, homogenized with 1 ml of ice-cold RIPA buffer, and prepared for Western blot analysis as previously described (15). For AMPK activity determinations, powdered muscle (400 mg) was homogenized in a buffer containing 210 mM sucrose, 1 mM EDTA, 5 mM sodium pyrophosphate, 50 mM NaF, 1 mM DTT, 2 mM PMSF, and 50 mM HEPES, pH 7.4, and centrifuged for 45 s at 15,000 g. The supernatant was used to determine total AMPK activity, while isoform-specific AMPK and CaMKII activity was determined in immunoprecipitates from 200–400 µg of supernatant protein after overnight incubation at 4°C with 1.5 µg of affinity-purified isoform-specific goat IgG against ₂-AMPK and CaMKII, respectively, in 20–40 µl of protein A/G-agarose beads (Santa Cruz Biotechnology). ³²P-ATP incorporation into SAMS peptide was used to measure total AMPK and ₂-AMPK activity from the respective preparations (19, 35). We have shown previously that the moderate-intensity muscle contraction protocol used in the current study does not significantly increase ₁-AMPK activity (19), and therefore ₁-AMPK activity was not determined. CaMKII autonomous activity was determined with an assay kit purchased from Upstate Cell Signaling (Lake Placid, NY). Briefly, 10 µl of immunoprecipitated CaMKII homogenate was added to 10 µl of assay dilution buffer (20 mM MOPS, 25 mM β-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, pH 7.2), 10 µl of CaMKII substrate cocktail (500 µM autocamtide, 40 µg/ml calmodulin in assay dilution buffer), 10 µl each of PKA and PKC inhibitor cocktail, and 10 µl of ³²P-ATP dissolved in magnesium/ATP cocktail. The solution was then incubated for 10 min at 30°C with constant agitation before 25 µl was spotted on P81 paper. The filter papers were then washed 3 times for 10 min in 0.75% phosphoric acid and once in acetone, then analyzed for radioactivity, an indication of CaMKII phosphotransferase activity, with 3 ml of Budget Solve in a Hewlett Packard scintillation counter.

Calculations and statistics.   Palmitate delivery, fractional and total palmitate uptake, and percent and total palmitate oxidation were calculated as previously described in detail (29, 32). Both percent and total palmitate oxidation values were corrected for label fixation by means of previously calculated acetate correction factors (32). The arterial specific activity for palmitate did not vary over time and was not significantly different between groups, averaging 79.2 ± 3.0, 75.1 ± 8.8, 79.8 ± 5.6, 75.7 ± 8.0, 83.0 ± 2.3, and 79.4 ± 4.0 µCi/mmol in R+KN92, R+KN93, C+KN92, C+KN93, MC+KN92, and MC+KN93 groups, respectively (P > 0.05). Oxygen and glucose uptake as well as lactate release were calculated as previously described (32) and are expressed per gram of perfused muscle, which was previously determined to be 5.6% of body wt for unilateral hindquarter perfusion (33). At rest and during caffeine treatment or muscle contraction, time effects for glucose, lactate, and FA concentrations and kinetic data in both control (KN92) and KN93 groups were analyzed by means of a two-way ANOVA with repeated measures. Because there was no significant difference in values measured after 10, 15, and 20 min of perfusion, average values were used for each animal in subsequent analyses. The effects of KN93 and caffeine treatment or muscle contraction on the same variables as well as on muscle CaMKII and AMPK activity and ERK1/2 phosphorylation were analyzed by means of a two-way ANOVA (StatSoft Statistica v 5.0, Tulsa, OK). Scheffé's test for post hoc multiple comparisons was performed when appropriate. A significance level of 0.05 was chosen for all statistical methods.

	RESULTS

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Muscle performance and O₂ uptake.   Oxygen uptake did not vary over time in any of the groups (P > 0.05). Average values including all time points are expressed in table 1. Neither caffeine nor KN93 had any effect on oxygen uptake compared with the rates measured in the resting groups (P > 0.05). Muscle contraction significantly increased oxygen uptake, and the increase was maintained in the group treated with KN93 (P < 0.05 for R vs. MC groups). The initial amount of tension developed by the contracting muscles was not affected by CaMKII inhibition (P > 0.05; Table 1). Muscle tension development decreased markedly during the first 10–15 min of electrical stimulation and was followed by a more gradual decrease. After 10 min of electrical stimulation, muscle tension development had decreased to 69–77% of initial tension development in both MC groups.

View larger version (9K): [in this window] [in a new window]   Fig. 1. Effect of CaMKII inhibition on fatty acid (FA) uptake in rat hindquarters perfused during moderate-intensity muscle contraction (A) or in presence of caffeine (B). Values are means ± SE for R+KN92 (n = 5), R+KN93 (n = 5), MC+KN92 (n = 7), MC+KN93 (n = 7), C+KN92 (n = 8), C+KN93 (n = 8). R, rest; MC, muscle contraction; C, caffeine. Open bars represent control groups perfused with inactive CaMKII inhibitor KN92. Solid bars represent experimental groups perfused with active CaMKII inhibitor KN93. Because there was no significant difference in values measured after 10, 15, and 20 min of perfusion, average values were used for each animal. *P < 0.05 compared with respective R groups; #P < 0.05 compared with similarly treated KN92 groups.

View larger version (9K): [in this window] [in a new window]   Fig. 2. Effect of CaMKII inhibition on total FA oxidation in rat hindquarters perfused during moderate-intensity muscle contraction (A) or in presence of caffeine (B). Values are means ± SE for R+KN92 (n = 5), R+KN93 (n = 5), MC+KN92 (n = 7), MC+KN93 (n = 7), C+KN92 (n = 8), C+KN93 (n = 8). Open bars represent control groups perfused with the inactive CaMKII inhibitor KN92. Solid bars represent experimental groups perfused with active CaMKII inhibitor KN93. Because there was no significant difference in values measured after 10, 15, and 20 min of perfusion, average values were used for each animal. Total palmitate oxidation was corrected for label fixation, as described in MATERIALS AND METHODS. *P < 0.05 compared with respective R groups; #P < 0.05 compared with similarly treated KN92 groups.

View larger version (9K): [in this window] [in a new window]   Fig. 3. Effect of CaMKII inhibition on CaMKII activity in rat hindquarters perfused during moderate-intensity muscle contraction (A) or in presence of caffeine (B). CaMKII activity is calculated per gram of muscle present in assay preparation. Values are means ± SE for R+KN92 (n = 5), R+KN93 (n = 5), MC+KN92 (n = 7), MC+KN93 (n = 7), C+KN92 (n = 8), C+KN93 (n = 8). Open bars represent control groups perfused with inactive CaMKII inhibitor KN92. Solid bars represent experimental groups perfused with active CaMKII inhibitor KN93. *P < 0.05 compared with respective R groups; #P < 0.05 compared with similarly treated KN92 groups.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Effect of CaMKII inhibition on ERK1/2 phosphorylation in rat hindquarters perfused during moderate-intensity muscle contraction (A) or in presence of caffeine (B). ERK1/2 phosphorylation results are expressed after standardization with phosphorylation of preperfused gastrocnemius muscle standard. Values are means ± SE for R+KN92 (n = 5), R+KN93 (n = 5), MC+KN92 (n = 7), MC+KN93 (n = 7), C+KN92 (n = 8), C+KN93 (n = 8). Open bars represent control groups perfused with inactive CaMKII inhibitor KN92. Solid bars represent experimental groups perfused with active CaMKII inhibitor KN93. *P < 0.05 compared with respective R groups.

View larger version (9K): [in this window] [in a new window]   Fig. 5. Effect of CaMKII inhibition on 2-AMPK activity in rat hindquarters perfused during moderate intensity muscle contraction (A) or in the presence of caffeine (B). 2-AMPK activity is calculated per gram of protein present in assay preparation. Values are means ± SE for R+KN92 (n = 5), R+KN93 (n = 5), MC+KN92 (n = 7), MC+KN93 (n = 7), C+KN92 (n = 8), C+KN93 (n = 8). Open bars represent control groups perfused with inactive CaMKII inhibitor KN92. Solid bars represent experimental groups perfused with active CaMKII inhibitor KN93. *P < 0.05 compared with respective R groups; #P < 0.05 compared with similarly treated KN92 groups.

	DISCUSSION

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View larger version (16K): [in this window] [in a new window]   Fig. 6. Proposed Ca2+-dependent and Ca2+-independent signaling mechanisms involved in regulation of contraction-induced FA metabolism. Mechanisms are based on results from previous studies performed in our lab (19, 30) and results from current study. In the diagram, it is proposed that FA uptake is regulated by calcium-dependent and calcium-independent pathways during moderate-intensity muscle contraction. Calcium-dependent pathways include CaMKII and CaMKK activation of AMPK and subsequent downstream upregulation of FA uptake and oxidation. In addition, CAMKII and AMPK activation lead to upregulation of FA oxidation independent of FA uptake. Calcium-independent pathways include activation of AMPK, which may or may not lie upstream of ERK1/2. Calcium-independent activation of AMPK and ERK lead to upregulation of FA uptake and oxidation in sequence as well as independently of one another.

More importantly, our results, which show that CaMKII inhibition downregulates FA uptake and oxidation with both caffeine treatment and muscle contraction, suggest that CaMKII activation is part of Ca²⁺-dependent signaling under these conditions in muscle. Since CaMKII inhibition was accompanied by different relative changes in contraction-induced FA uptake and oxidation, our data further suggest that CaMKII, like many other signaling intermediates, may have different relative effects on the cellular events that mediate the changes in FA metabolism. Although KN93, the CaMKII inhibitor used in this study, has been shown to inhibit other CaMKs (2), recent studies demonstrate that CaMKII is the primary functional CaMK expressed in rat skeletal muscle (24, 25). In addition, we are unaware of any evidence suggesting that KN93 has the ability to inhibit CaMKK. The specificity of KN compounds for CaMK pathways in general has also been questioned based on their ability to inhibit insulin-stimulated glucose uptake in rat soleus and epitrochlearis muscle in some (37), but not all (40) studies. To compensate for this possibility, perfusions in the current study were completed without the use of insulin, ensuring that insulin-stimulated pathways were not activated. Therefore, we are confident that the metabolic changes associated with KN93 treatment in the current study were a result of a KN93-induced decrease in CaMKII activity.

While our caffeine data provide evidence for a direct role of CaMKII in the regulation of FA uptake and oxidation, our experiments also uncovered several other important points. First, our data showed that Ca²⁺-dependent signaling includes direct activation of AMPK. Indeed, we found that caffeine-induced Ca²⁺ release significantly increased AMPK activity in perfused muscle. Because 3 mM caffeine increases cellular Ca²⁺ levels without inducing changes in force production or cellular energy charge (41), we feel confident that the caffeine-induced activation of total and ₂-AMPK activity measured in our study was the result of increased cellular Ca²⁺ levels, not a change in the AMP/ATP ratio. Our data collected in perfused muscle are in agreement with previous studies performed in L6 myotubes incubated with elevated intracellular Ca²⁺ levels, induced by treatment with the Ca²⁺ ionophore A-23187 (4, 8), and studies performed with isolated rodent skeletal muscle treated with 3 mM caffeine (10). The decrease in total and ₂-AMPK activity with CaMKII inhibition during muscle contraction demonstrated another important point, namely that the well-documented increase in AMPK activity during muscle contraction is partially driven by Ca²⁺-dependent signaling via CaMKII activation. However, since CaMKII inhibition did not affect the caffeine-induced increase in total and ₂-AMPK activity and only partially decreased the contraction-induced increase in total and ₂-AMPK activity, this further indicated to us that Ca²⁺-dependent activation of AMPK includes both CaMKII-dependent and CaMKII-independent components. Lastly, because CaMKII inhibition prevented the caffeine-induced increase in FA uptake and oxidation but did not affect AMPK activity, and because we have repeatedly shown that contraction-induced FA uptake and oxidation can be manipulated despite no change in AMPK activity (20, 21, 30), our current data also suggest that AMPK activation is not always sufficient to increase FA uptake and oxidation in muscle.

These conclusions are in line with data showing that AMPK can be activated directly by different CaMKK isozymes (5, 7, 36). They are also in agreement with data showing that CaMKII inhibition decreased Ca²⁺-dependent activation of AMPK in pancreatic β-cells (13) and inhibited AMPK phosphorylation and activity during ex vivo, low-intensity muscle contraction in mouse skeletal muscle (11). Thus, our results suggest that CaMKII lies upstream of AMPK under some experimental conditions that include moderate-intensity muscle contraction. However, our results do not eliminate the possibility that CaMKII regulates FA uptake and oxidation independently of its effects on AMPK activity (see Fig. 6). It has been suggested that Ca²⁺-dependent activation of AMPK is due to an increase in ₁-AMPK rather than ₂-AMPK activity (10). We chose not to measure ₁-AMPK activity in our study because the overall purpose of our experiments was to delineate contraction-induced signaling pathways in muscle contracting at a moderate intensity. Because we have shown that ₁-AMPK activity is not affected by this moderate-intensity muscle contraction protocol in perfused muscle (19), we chose not to measure ₁-AMPK activity. We feel confident that ₁-AMPK activity would not have been affected by the different treatment conditions employed in this study, because as reported previously (19, 21), the relative changes in total and ₂-AMPK activity were similar, implying that ₁-AMPK did not change. The discrepancy in caffeine-induced changes in ₁-AMPK and ₂-AMPK activity between our study and the one cited above are uncertain but may be due to differences in caffeine treatment duration (15 vs. 40 min), muscle preparation (incubated vs. perfused), and fiber type selection (slow-twitch oxidative vs. mixed).

The greater increase in FA uptake and oxidation during muscle contraction compared with caffeine treatment and the partial decrease in FA uptake with CaMKII inhibition in contracting muscle provide some evidence for the existence of Ca²⁺-independent signaling mechanisms in the regulation of contraction-induced FA metabolism. In line with previous results (3, 19, 20, 30), we found that during muscle contraction ERK1/2 phosphorylation was increased and was not affected by CaMKII inhibition. Combined with the fact that caffeine treatment did not increase ERK1/2 phosphorylation, our new data suggest that Ca²⁺-induced activation of ERK1/2 is not predominant in skeletal muscle, especially during muscle contraction. Since both ERK1/2 phosphorylation and FA uptake remained elevated above basal levels with CaMKII inhibition, our data are in line with others suggesting that ERK1/2 plays a role in the Ca²⁺-independent signaling mechanism (3, 9, 16). The prevention of contraction-induced FA oxidation with KN93 treatment despite no change in ERK1/2 phosphorylation may at first seem contradictory to our previously published work demonstrating a role for ERK1/2 activity in the regulation of contraction-induced FA oxidation (20). However, as shown in that study, FA oxidation is very sensitive to changes in enzyme activity, more sensitive than FA uptake (20). This is likely due to tighter regulation of cellular substrate utilization relative to cellular substrate availability. Therefore, the changes in CaMKII activity with the use of KN93 during muscle contraction in the current study may have been sufficient to bring FA oxidation rates back to rates measured at rest. The data from both studies indicate that CaMKII and ERK1/2 may regulate contraction-induced FA oxidation via distinct signaling pathways and that activation of either may be sufficient to maximize FA oxidation during moderate-intensity muscle contraction.

As shown previously in our lab, it is likely that the calcium-dependent and calcium-independent signaling pathways involved in the regulation of FA uptake during moderate-intensity muscle contraction include the translocation of the FA transporter FAT/CD36 from intracellular stores to the plasma membrane (19, 20, 30). We have also shown repeatedly that although an increase in FA uptake provides additional substrate for mitochondrial FA oxidation, FA oxidation is upregulated by additional mechanisms independent of FA uptake (30). These mechanisms likely involve manipulation of CPT-1 activity and subsequent entry of long chain FA into the mitochondria, perhaps via a decrease in malonyl CoA levels (14, 21). Additional studies must be performed to verify the connection between the calcium-dependent and calcium-independent signaling mechanisms discovered in the current study with the potential downstream FA transporters CD36 and CPT-1 in the regulation of contraction-induced FA metabolism.

In summary, the results from this study provide critical information regarding the signaling pathways regulating FA uptake and oxidation in muscle. Our results provide evidence for the existence of both Ca²⁺-dependent and Ca²⁺-independent signaling mechanisms in the regulation of FA uptake and oxidation in muscle. Our data show that Ca²⁺-dependent signaling mechanisms occur in part via CaMKII activation and that Ca²⁺-dependent activation of AMPK occurs in part via CaMKII activation. Conversely, our data also show that AMPK activation is not always sufficient to induce an increase in FA uptake and oxidation. Lastly, our data show that Ca²⁺-independent regulation of FA uptake and oxidation may include ERK1/2 activation.

	GRANTS

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The present study was supported by grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR-45168) and the University of Southern California Women in Science and Engineering (WiSE) program.

	FOOTNOTES

 

Address for reprint requests and other correspondence: Lorraine P. Turcotte, Depts. of Kinesiology and Biological Sciences, College of Letters, Arts, and Sciences, Univ. of Southern California, 3560 Watt Way, PED 107, Los Angeles, CA 90089-0652 (e-mail: turcotte{at}usc.edu)

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.

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