Departments of Kinesiology and Biological Sciences, Diabetes Research Center, University of Southern California, Los Angeles, California
Submitted 8 July 2004 ; accepted in final form 11 November 2004
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ABSTRACT |
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electrical stimulation; perfused hindquarter; acetyl-coenzyme A carboxylase; malonyl-coenzyme A; cellular signaling; 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside; fatty acids
It has been proposed that the signaling molecule AMP-activated protein kinase (AMPK) may be involved in the regulation of substrate utilization during muscle contraction and exercise (11, 30). In rats, AMPK activity is upregulated during muscle contraction induced by electrical stimulation or treadmill running and after treatment with the pharmacological agent 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside (AICAR) (13, 20, 31). Studies have demonstrated that the increase in AMPK activity measured during muscle contraction and after AICAR treatment parallels the increase in GLUT4 translocation to the plasma membrane and glucose uptake in skeletal muscle (3, 12, 16), suggesting that AMPK may be involved in the regulation of glucose uptake under these conditions. In a manner similar to GLUT4, the contraction-induced increase in FA uptake has been shown to be mediated in part by the translocation of the FA transporter protein FAT/CD36 to the plasma membrane (6). Furthermore, studies in rat cardiac myocytes have shown that both AICAR treatment and muscle contraction activate AMPK and are associated with an increase in FAT/CD36 translocation to the plasma membrane and FA uptake, suggesting that AMPK activation under these experimental conditions may also be important for the regulation of FA uptake in heart (18). However, it is not known whether AMPK activation induced by either muscle contraction or AICAR treatment plays a similarly important role in the regulation of FA uptake in skeletal muscle. Additionally, the effect of AICAR treatment in combination with muscle contraction on FA uptake in skeletal muscle has never been studied.
In skeletal muscle, AMPK is most widely known for its role in the upregulation of FA oxidation during muscle contraction (30). It has been shown that, once activated, AMPK phosphorylates and inactivates acetyl-CoA carboxylase (ACC) and is associated with a reduction in malonyl-CoA levels, a release of the inhibition on carnitine palmitoyltransferase I, and a subsequent increase in -oxidation (13, 20). Treatment with AICAR has resulted in significant increases in FA oxidation in perfused and isolated rodent skeletal muscle (1, 20, 21). However, it has yet to be determined whether muscle contraction and AICAR treatment have additive effects on FA oxidation in skeletal muscle.
Consequently, the purpose of this study was twofold: 1) to determine whether AMPK activation via AICAR treatment increases FA uptake in skeletal muscle at rest and 2) to determine whether muscle contraction and AICAR treatment together increase AMPK activity, FA uptake, and FA oxidation more than either treatment alone in skeletal muscle. To achieve the second purpose, we used a low-intensity stimulation protocol that increases oxygen uptake and muscle force production one-half as much as our commonly used stimulation protocol, thus allowing for further activation of AMPK with AICAR treatment. We hypothesized that if AMPK activation plays a critical role in the regulation of FA uptake, then AICAR treatment alone would increase FA uptake. We further hypothesized that if the effects of AICAR treatment and muscle contraction are additive on AMPK activation, then, in combination, both treatments would result in additive increases in FA uptake and FA oxidation.
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MATERIALS AND METHODS |
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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 (27). 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 (27).
The initial perfusate (300 ml) consisted of Krebs-Henseleit solution, 1-day-old washed bovine erythrocytes (hematocrit, 30%), 5% bovine serum albumin (Cohn fraction V; Sigma Chemical, St. Louis, MO), 550 µM albumin-bound palmitate, 4 µCi of albumin-bound [1-14C]palmitate, 6 mM glucose, and 10 µU/ml insulin in the absence (R, ES) or presence (A, ES+A) of 2 mM AICAR. The perfusate (37°C) was continuously gassed with a mixture of 95% O2-5% CO2, which yielded arterial pH values of 7.27.3 and arterial PO2 and PCO2 values that were typically 110130 and 4864 Torr, respectively. Mean perfusion pressures were not affected by muscle contraction or AICAR treatment and averaged 61.8 ± 6.3, 82.0 ± 3.5, 90.6 ± 17.1, and 94.4 ± 24.5 mmHg in the R, A, ES, and ES+A groups, respectively (P > 0.05).
The first 25 ml of perfusate that passed through the right hindquarter were discarded, whereupon the hindlimb was equilibrated for 20 min at a perfusate flow of 5.0 ml/min (average for all groups: 0.26 ± 0.10 ml·min1·g1 perfused muscle). R and A animals were perfused for an additional 40 min at a perfusate flow of 5.0 ml/min (0.26 ± 0.08 and 0.26 ± 0.11 ml·min1·g1 perfused muscle, respectively, P > 0.05). In the ES and ES+A animals, the right leg was immobilized at the tibiopatellar ligament, and a hook electrode was placed around the sciatic nerve and connected to an S48 Grass stimulator (Grass Telefactor, West Warwick, RI). Perfusate flow was increased to 12.5 ml/min (0.64 ± 0.1 and 0.66 ± 0.3 ml·min1·g1 perfused muscle in the ES and ES+A groups, respectively, P > 0.05). The resting length of the gastrocnemius-soleus-plantaris muscle group was adjusted to obtain maximum active tension upon stimulation. Low-intensity isometric muscle contractions were induced by stimulating the sciatic nerve electrically with supramaximal (20 V) trains of 100 ms and 50 Hz, with an impulse duration of 1 ms, delivered every 10 s. During the 40-min muscle stimulation, the tension developed by the gastrocnemius-soleus-plantaris muscle group was recorded with Lab VIEW v. 5.0 (National Instruments, Austin, TX) software. 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 10, 20, 30, and 40 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 40 min. Arterial perfusate samples were taken for determination of hematocrit and hemoglobin before equilibration. At the end of the 40-min experimental period, the gastrocnemius-soleus-plantaris muscle group of the right leg was freeze-clamped in situ with aluminum clamps precooled in liquid nitrogen, taken out, and stored for later analysis. As previously determined by dissection of the hindlimb and by histochemistry, the hindlimb is composed of 76% type IIb, 19% type IIa, and 5% type I fibers (2).
Blood sample analyses. Blood samples for glucose and lactate were put into 200 µM EGTA (pH 7) and immediately analyzed using the YSI-1500 glucose and lactate analyzers (Yellow Springs Instrument, Yellow Springs, OH), respectively. Plasma FA concentrations were determined spectrophotometrically with the WAKO NEFA-C test (Biochemical Diagnostics, Edgewood, NY). Plasma [14C]FA and 14CO2 radioactivities were determined in duplicate as previously described in detail (25, 26, 27). PCO2, PO2, and pH were measured with an ABL-5 analyzer (Radiometer America, Westlake, OH), and hemoglobin was determined spectrophotometrically (Sigma Chemical).
Muscle sample analyses.
Muscle samples (200 mg) were powdered under liquid nitrogen, and perchloric acid extracts were neutralized for measurement of malonyl-CoA using the [3H]acetyl-CoA method (28). Total AMPK and ACC activities were determined using ammonium sulfate precipitates from homogenates prepared from the powdered muscles, as described previously (31). Isoform-specific AMPK activity was determined in immunoprecipitates from 200 µg of supernatant protein in a total volume of 1 ml of AMPK buffer (210 mM sucrose, 1 mM EDTA, 5 mM sodium pyrophosphate, 50 mM NaF, 1 mM DTT, 2 mM PMSF, 50 mM HEPES, pH 7.4) after overnight incubation at 4°C with 1.5 µg of affinity-purified isoform-specific goat IgG against either AMPK
1 or AMPK
2 in 20 µl of protein A/G-agarose beads (Santa Cruz Biotechnology). AMPK activity was determined by measuring the incorporation of [32P]ATP into SAMS, the peptide substrate for AMPK, and ACC activity was determined by measuring the incorporation of [14C]bicarbonate into acid-stable compounds (malonyl-CoA) in the presence of the allosteric activator citrate (20 mM) (31).
Calculations and statistical analyses. Palmitate delivery, fractional and total palmitate uptake, and percent and total palmitate oxidation were calculated as previously described in detail (25, 27). Both percent and total palmitate oxidation values were corrected for label fixation by using previously calculated acetate correction factors (27). The arterial specific activity for palmitate did not vary over time and was not significantly different between groups, averaging 42.0 ± 1.0, 46.0 ± 2.0, 39.0 ± 3.0, and 46.0 ± 2.0 µCi/mmol in R, A, ES, and ES+A groups, respectively (P > 0.05). Oxygen and glucose uptake as well as lactate release were calculated as previously described (27) and are expressed per gram of perfused muscle, which was previously determined to be 5.6% of body weight for unilateral hindquarter perfusion (28). Malonyl-CoA concentrations were calculated from the radioactive incorporation of [3H]acetyl-CoA into palmitic acid and corrected for unspecific labeling (19).
At rest and during muscle contraction, time effects for glucose, lactate, and FA concentrations and kinetic data in both control and AICAR groups were analyzed using a two-way ANOVA with repeated measures. Because there was no significant difference in values measured after 20, 30, and 40 min of perfusion, average values were used for each animal in subsequent analyses. The effects of AICAR treatment and muscle contraction on the same variables as well as on muscle malonyl-CoA level, ACC, and AMPK activities were analyzed using a two-way ANOVA (StatSoft Statistica v. 5.0, Tulsa, OK). Scheffé's test for post hoc multiple comparisons was performed when appropriate. In all instances, an value of 0.05 was used to determine significance.
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RESULTS |
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Muscle metabolite levels and enzyme activities.
Low-intensity muscle contraction did not significantly increase total, 1-subunit, or
2-subunit AMPK activity (P > 0.05; Fig. 3). AICAR treatment did not have a significant effect on AMPK
1-subunit activity during rest or muscle contraction (P > 0.05). However, AICAR treatment increased total AMPK activity by 3482% during rest and muscle contraction (R: 950.5 ± 35.9 vs. A: 1,726.3 ± 141.0 and ES: 1,067.7 ± 58.8 vs. ES+A: 1,429.4 ± 89.9 nmol·min1·g1, P < 0.05) as well as AMPK
2-subunit by 84128% (P < 0.05). In line with the AMPK data, low-intensity muscle contraction did not affect ACC activity (Fig. 4A). However, AICAR treatment decreased ACC activity by 39% both at rest and during muscle contraction (P < 0.05). Malonyl-CoA levels were significantly lower in the ES than in the R group (P < 0.05; Fig. 4B). AICAR treatment resulted in a significant decrease in malonyl-CoA levels at rest (R: 0.81 ± 0.10 vs. A: 0.17 ± 0.03 pmol/mg wet wt, P < 0.05) but had no effect during muscle contraction (ES: 0.34 ± 0.08 vs. ES+A: 0.20 ± 0.05 pmol/mg wet wt, P > 0.05).
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DISCUSSION |
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Our study demonstrates for the first time that AICAR treatment at rest results in an increase in FA uptake in skeletal muscle. At rest, the AICAR-induced increase in FA uptake was accompanied by a parallel increase in total and AMPK2 activity, suggesting that, under resting conditions, AMPK activation may play a role in the regulation of FA uptake. This notion is in agreement with data collected in quiescent cardiac myocytes in which AMPK activation, as assessed by ACC phosphorylation, was directly correlated with FAT/CD36 translocation and FA uptake (18). As shown by others (3, 12, 16), we measured parallel increases in glucose uptake and AMPK activity at rest with AICAR treatment. Recent data collected in AMPK
2 knockout mice in which AICAR-induced glucose uptake in skeletal muscle was completely abolished suggest that the AMPK
2 subunit may be critical in this regulation of glucose uptake at rest (15).
As we have shown previously (25, 27), muscle contraction was associated with an increase in FA uptake. However, the stimulation protocol did not increase total, 1-subunit, or
2-subunit AMPK activity. It is important to note that, for this study, we chose a stimulation protocol that can be characterized as low intensity compared with other published protocols of moderate and high intensity used by others and us (27, 32). This can be ascertained by the fact that, compared with the stimulation protocol commonly used in our lab, the protocol used in this study was associated with rates of oxygen uptake and force production that were at least 50% lower (25, 27). Because one of the goals of this study was to study the impact of AICAR treatment during muscle contraction and because research has shown that contraction-induced AMPK activity is intensity dependent (7, 14), we chose this low-intensity protocol to ensure that AMPK activity would not be maximally stimulated by the stimulation protocol, allowing for a possible increase in AMPK activity when muscle contraction was combined with AICAR treatment. Our results show that low-intensity muscle contraction increased both glucose and FA uptake without significantly increasing AMPK activity, providing evidence that AMPK activation may not always be necessary to measure contraction-induced increases in FA and glucose uptake. In agreement with other data (8, 15, 17), our results suggest that AMPK-independent mechanisms may be involved in the regulation of contraction-induced FA and glucose uptake under some conditions. Indeed, in quiescent rat cardiac myocytes, dipyridamole, an inhibitor of nucleoside transport, was shown to induce FAT/CD36 translocation and FA uptake without stimulating AMPK activity (17). In slow oxidative muscle it has been shown that, although a high precontraction glycogen level prevented the contraction-induced activation of AMPK, it did not reduce the rate of contraction-induced glucose uptake (8). Furthermore, it has been shown that contraction-induced glucose uptake is not significantly different in muscle of AMPK
1 or -
2 knockout mice compared with wild-type animals (15). Although it is not known which other cellular mechanisms may be involved in the regulation of contraction-induced glucose uptake, we have shown that, in perfused contracting muscle, the addition of the mitogen-activated protein kinase inhibitor PD-98059 completely abolished the contraction-induced increase in FA uptake, suggesting that the contraction-induced increase in FA uptake may be mediated in part by the ERK1/2 signaling pathway (24). Put together, our results and those of others show that, although AMPK activation may be an important factor in the regulation of glucose and FA uptake under some muscle contraction protocols, other cellular mechanisms of regulation must exist.
As expected, our results show that low-intensity muscle contraction and AICAR treatment individually increased FA oxidation and that this was associated with a decrease in malonyl-CoA level measured under both conditions. The decrease in malonyl-CoA level induced by AICAR treatment alone was also associated with an increase in total and AMPK2 activity and a decrease in ACC activity, providing a mechanism for the AICAR-induced decrease in malonyl-CoA level. However, the low-intensity muscle contraction protocol did not affect the activity of total AMPK, AMPK
2, or ACC. Although it has been suggested by some (29) that ACC phosphorylation is a more sensitive measurement of ACC activation, we are confident that our data reflect a true lack of response of ACC activity to low-intensity muscle contraction, because AMPK
2 activity determined from AMPK immunoprecipitates has been shown to reciprocally mirror ACC phosphorylation (29). Thus, if ACC activation had been measured using the phosphorylation assay, we would expect to show a similar lack of change in ACC activity with low-intensity muscle contraction because of the reciprocal lack of change in AMPK
2 activity. It is therefore likely that the contraction-induced decrease in malonyl-CoA level is not due to covalent modification of ACC. Alternatively, the contraction-induced decrease in malonyl-CoA level could have been due to a decrease in ACC activity induced by allosteric mechanisms of regulation or to an increase in malonyl-CoA decarboxylase (MCD) activity. Whereas a contraction-induced decrease in malonyl-CoA level (22) and an increase in muscle MCD activity that parallels previous measurements of AMPK activity have been observed by some (23), other studies have shown that MCD is not a phosphorylation substrate of active AMPK in skeletal muscle (10). Hence, it is possible that the contraction-induced decrease in malonyl-CoA level measured in our study could be accounted for by means of an AMPK-independent activation of MCD. Thus, in agreement with the FA uptake data, these results on FA oxidation suggest that, at rest, AMPK activation may be an important factor in the regulation of FA uptake and oxidation, whereas during low-intensity muscle contraction, other AMPK-independent mechanisms may regulate changes in FA uptake and oxidation.
Our results also show that low-intensity muscle contraction and AICAR treatment in combination were associated with a further increase in FA oxidation but no change in FA uptake compared with muscle contraction alone. If, as described above, FA uptake and oxidation are regulated by AMPK-dependent and AMPK-independent mechanisms, then an additive effect might be expected when AICAR and low-intensity muscle contraction are combined. The lack of change in contraction-induced FA uptake with AICAR treatment suggests that the cellular mechanism regulating FA uptake induced by this stimulation protocol prevailed over those associated with AMPK activation. Conversely, AICAR treatment in contracting muscle was associated with a synergistic effect on FA oxidation but no further increase in AMPK activity compared with AICAR treatment alone. These results suggest that AICAR treatment has the ability to enhance contraction-induced FA oxidation via an AMPK-independent mechanism.
In summary, our results show that AMPK activation may play an important role in the regulation of glucose uptake, FA uptake, and FA oxidation at rest in skeletal muscle. In contrast, our results suggest that regulation of these same variables during low-intensity muscle contraction occurs via AMPK-independent mechanisms. We have also shown that the AMPK-independent mechanisms regulating FA oxidation during low-intensity muscle contraction can be amplified by AICAR-induced AMPK activation, suggesting that, when combined, muscle contraction and AICAR have the potential to maximize FA oxidation in skeletal muscle. Our study is the first to demonstrate that, even though muscle contraction is associated with an increase in AMPK activity under some stimulation protocols, AMPK activation is not critical in the regulation of FA uptake and FA oxidation during low-intensity muscle contraction.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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