AMP kinase activation with AICAR further increases fatty acid oxidation and blunts triacylglycerol hydrolysis in contracting rat soleus musc
1 Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada
Abstract
Muscle contraction increases glucose uptake and fatty acid (FA) metabolism in isolated rat skeletal muscle, due at least in part to an increase in AMP-activated kinase activity (AMPK). However, the extent to which AMPK plays a role in the regulation of substrate utilization during contraction is not fully understood. We examined the acute effects of 5-aminoimidazole-4-carboxamide riboside (AICAR; 2 mM), a pharmacological activator of AMPK, on FA metabolism and glucose oxidation during high intensity tetanic contraction in isolated rat soleus muscle strips. Muscle strips were exposed to two different FA concentrations (low fatty acid, LFA, 0.2 mM; high fatty acid, HFA, 1 mM) to examine the role that FA availability may play in both exogenous and endogenous FA metabolism with contraction and AICAR. Synergistic increases in AMPK 2 activity (+45%; P < 0.05) were observed after 30 min of contraction with AICAR, which further increased exogenous FA oxidation (LFA: +71%, P < 0.05; HFA: +46%, P < 0.05) regardless of FA availability. While there were no changes in triacylglycerol (TAG) esterification, AICAR did increase the ratio of FA partitioned to oxidation relative to TAG esterification (LFA: +65%, P < 0.05). AICAR significantly blunted endogenous TAG hydrolysis (LFA: –294%, P < 0.001; HFA: –117%, P < 0.05), but had no effect on endogenous oxidation rates, suggesting a better matching between TAG hydrolysis and subsequent oxidative needs of the muscle. There was no effect of AICAR on the already elevated rates of glucose oxidation during contraction. These results suggest that FA metabolism is very sensitive to AMPK 2 stimulation during contraction.
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Introduction
Muscle contraction increases glucose uptake (Hayashi et al. 1998) and fatty acid (FA) metabolism (Dyck & Bonen, 1998; Lau et al. 2001) in isolated rat skeletal muscle. AMP-activated protein kinase (AMPK) was first shown to be activated during treadmill exercise in rat skeletal muscle, leading to decreased malonyl coenzyme A (malonyl-CoA) through the inhibition of acetyl-CoA carboxylase (ACC; Winder & Hardie, 1996) and increased FA oxidation. The coordinated regulation of AMPK, ACC and malonyl-CoA content in rats during treadmill exercise is intensity dependent, with the greatest AMPK activation observed during short-term, high-intensity exercise (Rasmussen & Winder, 1997). In tetanic contraction protocols, AMPK activity increases 3- to 5-fold in glycolytic epitrochlearis muscle (Ai et al. 2002) and 2- to 3-fold in oxidative soleus muscle (Hayashi et al. 1998) during short-term (10 min) protocols.
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Although AMPK is activated during contraction, its exact role in regulating substrate (glucose, exogenous FA, intramuscular triacylglycerol (TAG)) metabolism is controversial. AICAR is a pharmacological activator of AMPK, demonstrating similar effects to exercise for increasing glucose uptake (Merrill et al. 1997; Bergeron et al. 1999; Sakoda et al. 2002) and FA oxidation (Muoio et al. 1999) in skeletal muscle at rest. However, isolated muscle from mice with a dominant inhibitory mutant of AMPK demonstrates full inhibition of AICAR- and hypoxia-stimulated glucose uptake, but only a partial reduction (–40%) in contraction-stimulated glucose uptake (Mu et al. 2001), suggesting that AMPK is only partially involved in the regulation of contraction-induced glucose uptake. Recent evidence in perfused rat hindquarters subjected to a combination of AICAR and low intensity muscle contraction suggests that small increases in AMPK activity (+34%) did not account for the synergistic increase in FA oxidation (+175%) (Raney et al. 2005). This leads to the possibility of AMPK-independent mechanisms regulating substrate oxidation in skeletal muscle.
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Regulation of intramuscular TAG is poorly understood in both resting and contracting skeletal muscle. In isolated muscle preparations, TAG hydrolysis is increased during tetanic contraction in soleus, leading to increased TAG oxidation rates (Dyck & Bonen, 1998). Contraction- and AICAR-stimulated AMPK activation have therefore been suggested to be involved in the regulation of TAG hydrolysis and oxidation in skeletal muscle. However, in adipocytes, AICAR inhibits isoprenaline-induced lipolysis (Sullivan et al. 1994; Corton et al. 1995), apparently by phosphorylating and inhibiting hormone-sensitive lipase (HSL) (Garton et al. 1989). Similarly, AICAR has an anti-lipolytic effect in resting soleus (Alam & Saggerson, 1998) and C2C12 myotubes (Muoio et al. 1999) and recent evidence suggests that activating AMPK with AICAR can override and inhibit adrenaline- (epinephrine)-induced HSL activation in non-contracting L6 myotubes (Watt et al. 2004).
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To gain further insight into the role of AMPK in the regulation of substrate use during contraction, we used tracer methodologies to examine FA metabolism and glucose oxidation in isolated contracting soleus muscle, in the absence or presence of AICAR. With the use of different buffer concentrations of palmitate (low fatty acid (LFA), 0.2 mM; high fatty acid (HFA), 1.0 mM), we also examined the role that FA availability had on both exogenous and endogenous FA metabolism with contraction and AICAR. We wished to determine whether (1) AICAR would increase AMPK activity above the threshold set by high intensity, tetanic contraction; (2) the combination of AICAR and contraction would result in additional increases in FA and glucose oxidation; and (3) AICAR inhibited TAG hydrolysis and oxidation during contraction.
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Methods
Animals and preparation of muscle strips
Female Sprague-Dawley rats (Charles River Laboratory, QC, Canada; weight: 215 ± 2 g) were used for all experiments. Animals were housed in a controlled environment on a 12 h: 12 h reversed light–dark cycle and fed Purina rat chow and water ad libitum. All procedures were approved by the Animal Care Committee at the University of Guelph. Animals were anaesthetized with an intraperitoneal injection of pentobarbital sodium (6 mg (100 g body mass)–1) prior to all experimental procedures. Longitudinal soleus muscle strips were carefully dissected with tendons intact, using a 27-gauge needle. Each strip was sutured, removed and suspended on brass hooks in a 7 ml incubation reservoir, in order to maintain resting tension, as previously described (Dyck & Bonen, 1998). Seven millilitres of warmed (30°C), pre-gassed (95% O2–5% CO2) modified Krebs-Henseleit buffer (KHB) containing 4% FA-free bovine serum albumin (Boehringer, QC, Canada), 10 mM glucose, and 1 mM palmitate, was immediately added to the incubation reservoir. This was the base buffer for all experiments and was maintained at 30°C by an exterior circulating water system, and continuously gassed during all stages except the ‘chase’ phase (see below). A layer of heavy mineral oil was placed on top of the incubation buffer at all stages of the procedure in order to maintain gassing pressures. At the termination of the dissection procedure, the rats were humanely killed with an intracardiac injection of pentobarbital sodium.
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Time course for AMPK activation
Muscle incubations. AMPK 1 and 2 activities were examined at rest and following 10 and 30 min of contraction, with or without the addition of AICAR. Muscle strips were preincubated for 30 min either in KHB or KHB with the addition of 2 mM AICAR (Toronto Research Chemicals, Toronto, ON, Canada). At the end of this preincubation, one muscle strip was removed and freeze-clamped, while other muscles were stimulated to contract (150 ms trains composed of 0.5 ms impulses (20–40 V; 60 Hz) at 20 tetani min–1) for 10 or 30 min, with or without the addition of AICAR in the medium. At the end of the incubation period, the muscle strips were quickly freeze-clamped and stored in liquid nitrogen until further analysis.
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Immunoprecipitation and AMPK activity. Muscle strips (25–30 mg) were homogenized and incubated with AMPK 1 and AMPK 2 (Upstate, Charlottesville, VA, USA) antibody-bound protein A beads (Sigma, St Louis, MO, USA) as previously described (see accompanying paper Smith et al. 2005). Immunocomplexes were used for the AMPK activity assay as previously described (see Smith et al. 2005).
Lipid metabolism (‘pulse–chase’ experiments)
Pulse and wash. After an initial 30 min preincubation period, the buffer was drained from the reservoir and 7 ml KHB with 2 μCi [9,10-3H]palmitate (Amersham Life Science, Oakville, ON, Canada) was added to the reservoir. Muscles were pulsed for 30 min to prelabel the endogenous lipid pools (intramuscular diacylglycerol (DAG) and TAG). After the pulse phase, the buffer was drained and the muscles washed for 30 min in the absence of radiolabelled palmitate to allow for the removal of non-incorporated [3H]palmitate. During the wash, the buffer either remained at 1 mM palmitate (HFA) or was decreased to 0.2 mM palmitate (LFA) (see ‘Chase phase’). During this period, some muscles were exposed to 2 mM AICAR (Toronto Research Chemicals). At the end of the wash phase, one soleus strip from each pair was removed and extracted for endogenous lipids to determine prelabelling.
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Chase phase ('experimental phase'). The remaining muscle strips continued to be incubated for 30 min in pre-gassed, modified KHB containing 0.5 μCi [1-14C]palmitate (Amersham Life Science) and stimulated to contract (150 ms trains composed of 0.5 ms impulses (20–40 V; 60 Hz) at 20 tetani min–1) for 30 min in the presence or absence of 2 mM AICAR, either at HFA or LFA (as in wash phase) and glucose concentration remained at 10 mM. During this phase the gas was turned off to prevent the escape of 14CO2. Exogenous palmitate oxidation and esterification were monitored by the production of 14CO2 and incorporation of [1-14C]palmitate into intramuscular lipids, respectively. Intramuscular lipid hydrolysis and oxidation were monitored simultaneously by measuring the net change in lipid [3H]palmitate content and the production of 3H2O, respectively.
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Palmitate oxidation. Endogenous [9,10-3H]palmitate and exogenous [1-14C]palmitate oxidation was determined as outlined previously (Dyck et al. 2000) with minor modifications (see accompanying paper Smith et al. 2005).
Extraction of muscle lipids. After incubation, the muscles were removed, blotted and weighed, and placed in 13 ml plastic centrifuge tubes containing 5.0 ml ice-cold 2: 1 chloroform–methanol (v/v), homogenized and lipids (intramuscular DAG and TAG) were extracted as previously described (see accompanying paper Smith et al. 2005).
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Muscle triacylglycerol content. In previous studies, we have determined that the endogenous radiolabel tracks only a small portion of the TAG pool in resting muscle (Dyck et al. 1997). Therefore, it is important to determine the relationship between changes in the 3H-radiolabelled TAG pool and enzymatically measured TAG content during contraction. In a separate set of experiments, net TAG utilization was analysed in freeze-dried tissue (5 mg dry) from resting and contracted (30 min) muscles, which had been dissected free of all visible blood and connective tissue. Muscle TAG was measured enzymatically as previously described (Dyck et al. 1997).
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Glucose oxidation
Glucose oxidation was determined in a separate set of experiments. Muscle strips were preincubated as outlined previously (see accompanying paper Smith et al. 2005), in the absence or presence of 2 mM AICAR. After the preincubation, the gas was turned off to prevent the escape of 14CO2, the buffer was drained and 7 ml of pre-gassed, modified KHB containing 2 μCi of [U-14C]glucose (Amersham Life Science) was added to the reservoir. The muscle strips were incubated in the absence or presence of 2 mM AICAR and were stimulated to contract (150 ms trains composed of 0.5 ms impulses (20–40 V; 60 Hz) at 20 tetani min–1). After this phase, the buffer was drained and a 3.5 ml aliquot of the incubation medium was transferred to a 50 ml Erlenmeyer flask, and was acidified as previously described (see accompanying paper Smith et al. 2005) to capture 14CO2.
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Calculations and statistics
The specific activity of the incubation buffer (d.p.m. radiolabelled palmitate (nmol total palmitate)–1) was used to calculate palmitate (nmol (g wet weight)–1) incorporated into lipid pools or oxidized. With enzymatic analysis of net TAG utilization during contraction, we were able to determine the relationship between the changes in the radiolabelled TAG pool and the total (enzymatically determined) TAG pool. Each nanomole of [3H]palmitate represented 83 nanomoles (LFA) and 37 nanomoles (HFA) of total FA in the TAG pool. This ratio was used to calculate the rates of net TAG hydrolysis (net loss of preloaded [3H]palmitate (nmol g–1) from lipid pools (TAG and DAG) between paired soleus strips) and endogenous lipid oxidation during muscle contraction. Glucose oxidation was calculated using the specific activity of labelled glucose in KHB in the same manner as palmitate.
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Results are presented as mean ± S.E.M. Two-way ANOVA followed by Student-Neulman-Keuls post hoc analyses were used to assess statistical significance between time points for AMPK activity, with and without AICAR. Student's unpaired t tests were used to analyse the effect of AICAR on FA and glucose metabolism during contraction. Significance was accepted at P 0.05.
Results
Time course for AMPK activation with contraction and AICAR
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Contraction and AICAR had no effect on AMPK 1 activity (Fig. 1A). However, with contraction alone, AMPK 2 activity (Fig. 1B) was significantly increased at 30 min compared to 10 min (+67%; P < 0.05) and 0 min (+195%; P < 0.05). The combination of contraction and AICAR resulted in significant further increases in AMPK 2 activity (P < 0.01; treatment effect). With contraction and AICAR combined, AMPK 2 activity was significantly increased at 30 min compared to 10 min (+71%; P < 0.05) and 0 min (+154%; P < 0.001). At 30 min of contraction and AICAR, AMPK 2 activity was significantly greater than contraction alone (+45%; P < 0.05).
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Values are means ± S.E.M., pmol min–1 (mg protein)–1, n = 5–8 per group. *Significantly different from 30 min contraction alone (P < 0.05). **Significantly different from 0 and 10 min of same condition. Treatment effect of AICAR significantly different from No AICAR (P < 0.05).
Effects of AICAR on FA metabolism during contraction
Exogenous FA metabolism. The combination of contraction and AICAR significantly increased exogenous FA oxidation at both LFA (+71%; P < 0.05; Fig. 2A) and HFA (+46%; P < 0.05; Fig. 2B). Incorporation into TAG was not significantly different with AICAR (Fig. 2C and D), but there was an increase in the amount of FA partitioned toward oxidation relative to TAG esterification at LFA (+65%; P < 0.05; Fig. 2E), while no significant increase in this ratio was observed at HFA (+28%; P = 0.30; Fig. 2F). There were no significant differences in palmitate esterification into DAG with AICAR (Table 1).
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Effect of AICAR on fatty acid oxidation in low fatty acid (0.2 mM, LFA: A) or high fatty acid (1 mM, HFA: B) modified KHB, on TAG esterification (LFA: C; HFA: D) and on oxidation: TAG esterification ratio (LFA: E; HFA: F). Values are means ± S.E.M., nmol (g wet wt)–1, n = 7 per group. *Significantly different from No AICAR (P < 0.05).
Endogenous FA metabolism. AICAR resulted in significant blunting of TAG hydrolysis during 30 min of contraction at both LFA (–294%; P < 0.001; Fig. 3A), and HFA (–117%; P < 0.05; Fig. 3B) conditions. There were no significant effects of AICAR treatment on endogenous oxidation at LFA or HFA. There were no significant effects on DAG hydrolysis (Table 1) with AICAR treatment during contraction.
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Effect of AICAR on TAG hydrolysis and endogenous fatty acid oxidation in low fatty acid (0.2 mM, LFA: A) or high fatty acid (1 mM, HFA: B) modified KHB. Values are means ± S.E.M., nmol (g wet wt)–1, n = 7 per group. *Significantly different from No AICAR (P < 0.05).
Effects of AICAR on glucose oxidation in isolated contracting rat soleus muscle
AICAR had no significant effect on glucose oxidation during contraction, with either LFA or HFA (Fig. 4).
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There was no significant effect of AICAR on glucose oxidation in low fatty acid (0.2 mM, LFA) or high fatty acid (1 mM, HFA) modified KHB. Values are means ± S.E.M., n = 6–8 per group.
Discussion
With the use of palmitate and glucose tracers, we were able to directly examine the effects of AICAR on FA metabolism and glucose oxidation during high intensity tetanic contraction. Several novel observations were made: (1) incubation with AICAR further increased AMPK 2 activity above that seen with contraction alone; (2) AICAR further increased FA oxidation during contraction, independent of FA availability; (3) AICAR blunted TAG hydrolysis during contraction, independent of FA availability, while (4) having no effect on endogenous oxidation; and (5) AICAR had no effect on glucose oxidation during contraction. Therefore, our data suggest that FA substrate metabolism is much more sensitive than glucose to AMPK 2 stimulation during muscle contraction.
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Effect of AICAR on skeletal muscle lipid metabolism during contraction
To our knowledge, this is the first study to examine AICAR's effect on exogenous and endogenous lipid metabolism during contraction. With AICAR, there was an increase in exogenous FA oxidation above that seen with contraction alone, which could be due to a further increase in AMPK 2 activity. Previous evidence has shown that 15 min of hindlimb perfusion with AICAR-containing medium (at rest; 0.5, 1.0, 2.0 mM) significantly increased ZMP (phosphorylated AICAR) content in gastrocnemius–plantaris muscles, while having no effect on endogenous high-energy phosphates (ATP, ADP, AMP). This was associated with a significant increase in AMPK activity, leading to downstream effects of decreased ACC activity and decreased malonyl-CoA (Merrill et al. 1997). Our previous resting protocol (see accompanying paper Smith et al. 2005) certainly supports this AICAR-mediated increase in AMPK activity, which may be due to an initial increase in ZMP content. This further activation indicates that in our isolated tetanic contraction preparation, AMPK activation is a major regulator of FA oxidation. Recent evidence in AICAR-perfused rat hindquarters using a low intensity contraction protocol demonstrated that the increase in FA oxidation above that seen with contraction could not be attributed to the small increase in AICAR-induced AMPK activity, suggesting that with contraction, there are AMPK-independent mechanisms regulating FA metabolism (Raney et al. 2005). In this study, we used a higher intensity tetanic contraction protocol that has previously shown maximal stimulation of FA metabolism (Dyck & Bonen, 1998). The current data indicate that at high intensity tetanic contraction, AICAR can lead to further activation of AMPK, with further downstream effects on exogenous FA oxidation. Alternatively, AICAR may be targeting other additional regulatory enzymes resulting in an increase in FA oxidation. We also observed an increase in the partitioning of exogenous FA to oxidation at LFA during contraction, demonstrating an increased provision of FA to oxidative processes with AICAR. We did not observe a significant increase in FA partitioning with AICAR at HFA. However, this may not be surprising, as with increased FA substrate available during contraction proportionately more FA is available to be oxidized as well as esterified to TAG (Dyck & Bonen, 1998).
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Regulation of TAG hydrolysis in resting and contracting skeletal muscle is poorly understood. In this study, treatment with AICAR during tetanic contraction significantly blunted TAG hydrolysis, independent of FA availability, while having no effect on endogenously derived TAG oxidation. Indeed, exposing soleus strips to LFA did increase the provision of FA from TAG hydrolysis during contraction (+89% versus HFA), with AICAR attenuating this effect. It appears that AMPK regulation of TAG hydrolysis can occur during contraction and it is possible that AICAR reduced TAG hydrolysis during contraction by inhibiting the activation of HSL. HSL is an important enzyme in the hydrolysis of TAG. Recently, it has been shown that stimulation of AMPK prevents the activation of HSL (Watt et al. 2004). Thus, it is possible that the pharmacological stimulation of AMPK combined with contraction prevented the activation of HSL as would be expected with contraction alone, resulting in a reduction in TAG hydrolysis. However, further work is required to investigate the effect of AMPK activation on the regulation of HSL and TAG hydrolysis.
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Importantly, although AICAR attenuated TAG hydrolysis, there was no significant effect on endogenous oxidation in contracting soleus muscle. It is possible that during contraction, there is an excess of FA released from the endogenous TAG pool which may be reincorporated if the oxidative needs of the cell are met (Dyck & Bonen, 1998). Regulatory mechanisms that hydrolyse the TAG molecule might allow for an excess of FA to be broken down during contraction, but the energy required by the cell would govern actual oxidation. This attenuation with AICAR suggests a better matching of TAG hydrolysis with the oxidative needs of the cell. Even with a slight mismatch between TAG hydrolysis and endogenous oxidation at LFA with AICAR, the results show that AICAR had no effect on the amount of FA oxidized from endogenous stores during contraction.
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Effect of AICAR on muscle glucose oxidation during contraction
AICAR had no further effect on already high rates of glucose oxidation in our tetanic contraction protocol. Much research has demonstrated that AMPK is involved in contraction-stimulated glucose uptake (Hayashi et al. 1998; Musi et al. 2001), but it is probably not the sole mediator (Mu et al. 2001). Indeed, the combination of AICAR and contraction does not have an additive effect on glucose uptake (Hayashi et al. 1998; Bergeron et al. 1999), which supports the current data on glucose oxidation rates. Importantly, it is also possible that factors other than AMPK regulate glucose uptake and oxidation. Muscles from mice with a dominant negative form of AMPK were found to have full inhibition of AICAR- and hypoxia-induced glucose uptake, but only a partial (–40%) reduction in contraction-stimulated glucose uptake (Mu et al. 2001), suggesting that AMPK is only partially involved in the signalling mechanism to regulate glucose uptake and subsequent metabolism during contraction. It should also be noted that only one glucose concentration (10 mM) was used throughout these experiments. Thus, as was the case with FA oxidation, it is possible that an AICAR-induced stimulation of glucose oxidation may have been observed during contraction in the presence of a lower glucose concentration.
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Summary
The results from the present study demonstrate that AICAR-mediated AMPK 2 activation is intimately involved in the regulation of FA metabolism during contraction, while having no effects on the already high rates of glucose oxidation in skeletal muscle. Incubation with AICAR during tetanic contraction (1) leads to synergistic increases in AMPK 2 activity; (2) further increases exogenous FA oxidation; and (3) blunts TAG hydrolysis, both at LFA and HFA. Further studies investigating AMPK regulation of HSL and TAG hydrolysis during contraction are warranted.
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Abstract
Muscle contraction increases glucose uptake and fatty acid (FA) metabolism in isolated rat skeletal muscle, due at least in part to an increase in AMP-activated kinase activity (AMPK). However, the extent to which AMPK plays a role in the regulation of substrate utilization during contraction is not fully understood. We examined the acute effects of 5-aminoimidazole-4-carboxamide riboside (AICAR; 2 mM), a pharmacological activator of AMPK, on FA metabolism and glucose oxidation during high intensity tetanic contraction in isolated rat soleus muscle strips. Muscle strips were exposed to two different FA concentrations (low fatty acid, LFA, 0.2 mM; high fatty acid, HFA, 1 mM) to examine the role that FA availability may play in both exogenous and endogenous FA metabolism with contraction and AICAR. Synergistic increases in AMPK 2 activity (+45%; P < 0.05) were observed after 30 min of contraction with AICAR, which further increased exogenous FA oxidation (LFA: +71%, P < 0.05; HFA: +46%, P < 0.05) regardless of FA availability. While there were no changes in triacylglycerol (TAG) esterification, AICAR did increase the ratio of FA partitioned to oxidation relative to TAG esterification (LFA: +65%, P < 0.05). AICAR significantly blunted endogenous TAG hydrolysis (LFA: –294%, P < 0.001; HFA: –117%, P < 0.05), but had no effect on endogenous oxidation rates, suggesting a better matching between TAG hydrolysis and subsequent oxidative needs of the muscle. There was no effect of AICAR on the already elevated rates of glucose oxidation during contraction. These results suggest that FA metabolism is very sensitive to AMPK 2 stimulation during contraction.
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Introduction
Muscle contraction increases glucose uptake (Hayashi et al. 1998) and fatty acid (FA) metabolism (Dyck & Bonen, 1998; Lau et al. 2001) in isolated rat skeletal muscle. AMP-activated protein kinase (AMPK) was first shown to be activated during treadmill exercise in rat skeletal muscle, leading to decreased malonyl coenzyme A (malonyl-CoA) through the inhibition of acetyl-CoA carboxylase (ACC; Winder & Hardie, 1996) and increased FA oxidation. The coordinated regulation of AMPK, ACC and malonyl-CoA content in rats during treadmill exercise is intensity dependent, with the greatest AMPK activation observed during short-term, high-intensity exercise (Rasmussen & Winder, 1997). In tetanic contraction protocols, AMPK activity increases 3- to 5-fold in glycolytic epitrochlearis muscle (Ai et al. 2002) and 2- to 3-fold in oxidative soleus muscle (Hayashi et al. 1998) during short-term (10 min) protocols.
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Although AMPK is activated during contraction, its exact role in regulating substrate (glucose, exogenous FA, intramuscular triacylglycerol (TAG)) metabolism is controversial. AICAR is a pharmacological activator of AMPK, demonstrating similar effects to exercise for increasing glucose uptake (Merrill et al. 1997; Bergeron et al. 1999; Sakoda et al. 2002) and FA oxidation (Muoio et al. 1999) in skeletal muscle at rest. However, isolated muscle from mice with a dominant inhibitory mutant of AMPK demonstrates full inhibition of AICAR- and hypoxia-stimulated glucose uptake, but only a partial reduction (–40%) in contraction-stimulated glucose uptake (Mu et al. 2001), suggesting that AMPK is only partially involved in the regulation of contraction-induced glucose uptake. Recent evidence in perfused rat hindquarters subjected to a combination of AICAR and low intensity muscle contraction suggests that small increases in AMPK activity (+34%) did not account for the synergistic increase in FA oxidation (+175%) (Raney et al. 2005). This leads to the possibility of AMPK-independent mechanisms regulating substrate oxidation in skeletal muscle.
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Regulation of intramuscular TAG is poorly understood in both resting and contracting skeletal muscle. In isolated muscle preparations, TAG hydrolysis is increased during tetanic contraction in soleus, leading to increased TAG oxidation rates (Dyck & Bonen, 1998). Contraction- and AICAR-stimulated AMPK activation have therefore been suggested to be involved in the regulation of TAG hydrolysis and oxidation in skeletal muscle. However, in adipocytes, AICAR inhibits isoprenaline-induced lipolysis (Sullivan et al. 1994; Corton et al. 1995), apparently by phosphorylating and inhibiting hormone-sensitive lipase (HSL) (Garton et al. 1989). Similarly, AICAR has an anti-lipolytic effect in resting soleus (Alam & Saggerson, 1998) and C2C12 myotubes (Muoio et al. 1999) and recent evidence suggests that activating AMPK with AICAR can override and inhibit adrenaline- (epinephrine)-induced HSL activation in non-contracting L6 myotubes (Watt et al. 2004).
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To gain further insight into the role of AMPK in the regulation of substrate use during contraction, we used tracer methodologies to examine FA metabolism and glucose oxidation in isolated contracting soleus muscle, in the absence or presence of AICAR. With the use of different buffer concentrations of palmitate (low fatty acid (LFA), 0.2 mM; high fatty acid (HFA), 1.0 mM), we also examined the role that FA availability had on both exogenous and endogenous FA metabolism with contraction and AICAR. We wished to determine whether (1) AICAR would increase AMPK activity above the threshold set by high intensity, tetanic contraction; (2) the combination of AICAR and contraction would result in additional increases in FA and glucose oxidation; and (3) AICAR inhibited TAG hydrolysis and oxidation during contraction.
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Methods
Animals and preparation of muscle strips
Female Sprague-Dawley rats (Charles River Laboratory, QC, Canada; weight: 215 ± 2 g) were used for all experiments. Animals were housed in a controlled environment on a 12 h: 12 h reversed light–dark cycle and fed Purina rat chow and water ad libitum. All procedures were approved by the Animal Care Committee at the University of Guelph. Animals were anaesthetized with an intraperitoneal injection of pentobarbital sodium (6 mg (100 g body mass)–1) prior to all experimental procedures. Longitudinal soleus muscle strips were carefully dissected with tendons intact, using a 27-gauge needle. Each strip was sutured, removed and suspended on brass hooks in a 7 ml incubation reservoir, in order to maintain resting tension, as previously described (Dyck & Bonen, 1998). Seven millilitres of warmed (30°C), pre-gassed (95% O2–5% CO2) modified Krebs-Henseleit buffer (KHB) containing 4% FA-free bovine serum albumin (Boehringer, QC, Canada), 10 mM glucose, and 1 mM palmitate, was immediately added to the incubation reservoir. This was the base buffer for all experiments and was maintained at 30°C by an exterior circulating water system, and continuously gassed during all stages except the ‘chase’ phase (see below). A layer of heavy mineral oil was placed on top of the incubation buffer at all stages of the procedure in order to maintain gassing pressures. At the termination of the dissection procedure, the rats were humanely killed with an intracardiac injection of pentobarbital sodium.
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Time course for AMPK activation
Muscle incubations. AMPK 1 and 2 activities were examined at rest and following 10 and 30 min of contraction, with or without the addition of AICAR. Muscle strips were preincubated for 30 min either in KHB or KHB with the addition of 2 mM AICAR (Toronto Research Chemicals, Toronto, ON, Canada). At the end of this preincubation, one muscle strip was removed and freeze-clamped, while other muscles were stimulated to contract (150 ms trains composed of 0.5 ms impulses (20–40 V; 60 Hz) at 20 tetani min–1) for 10 or 30 min, with or without the addition of AICAR in the medium. At the end of the incubation period, the muscle strips were quickly freeze-clamped and stored in liquid nitrogen until further analysis.
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Immunoprecipitation and AMPK activity. Muscle strips (25–30 mg) were homogenized and incubated with AMPK 1 and AMPK 2 (Upstate, Charlottesville, VA, USA) antibody-bound protein A beads (Sigma, St Louis, MO, USA) as previously described (see accompanying paper Smith et al. 2005). Immunocomplexes were used for the AMPK activity assay as previously described (see Smith et al. 2005).
Lipid metabolism (‘pulse–chase’ experiments)
Pulse and wash. After an initial 30 min preincubation period, the buffer was drained from the reservoir and 7 ml KHB with 2 μCi [9,10-3H]palmitate (Amersham Life Science, Oakville, ON, Canada) was added to the reservoir. Muscles were pulsed for 30 min to prelabel the endogenous lipid pools (intramuscular diacylglycerol (DAG) and TAG). After the pulse phase, the buffer was drained and the muscles washed for 30 min in the absence of radiolabelled palmitate to allow for the removal of non-incorporated [3H]palmitate. During the wash, the buffer either remained at 1 mM palmitate (HFA) or was decreased to 0.2 mM palmitate (LFA) (see ‘Chase phase’). During this period, some muscles were exposed to 2 mM AICAR (Toronto Research Chemicals). At the end of the wash phase, one soleus strip from each pair was removed and extracted for endogenous lipids to determine prelabelling.
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Chase phase ('experimental phase'). The remaining muscle strips continued to be incubated for 30 min in pre-gassed, modified KHB containing 0.5 μCi [1-14C]palmitate (Amersham Life Science) and stimulated to contract (150 ms trains composed of 0.5 ms impulses (20–40 V; 60 Hz) at 20 tetani min–1) for 30 min in the presence or absence of 2 mM AICAR, either at HFA or LFA (as in wash phase) and glucose concentration remained at 10 mM. During this phase the gas was turned off to prevent the escape of 14CO2. Exogenous palmitate oxidation and esterification were monitored by the production of 14CO2 and incorporation of [1-14C]palmitate into intramuscular lipids, respectively. Intramuscular lipid hydrolysis and oxidation were monitored simultaneously by measuring the net change in lipid [3H]palmitate content and the production of 3H2O, respectively.
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Palmitate oxidation. Endogenous [9,10-3H]palmitate and exogenous [1-14C]palmitate oxidation was determined as outlined previously (Dyck et al. 2000) with minor modifications (see accompanying paper Smith et al. 2005).
Extraction of muscle lipids. After incubation, the muscles were removed, blotted and weighed, and placed in 13 ml plastic centrifuge tubes containing 5.0 ml ice-cold 2: 1 chloroform–methanol (v/v), homogenized and lipids (intramuscular DAG and TAG) were extracted as previously described (see accompanying paper Smith et al. 2005).
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Muscle triacylglycerol content. In previous studies, we have determined that the endogenous radiolabel tracks only a small portion of the TAG pool in resting muscle (Dyck et al. 1997). Therefore, it is important to determine the relationship between changes in the 3H-radiolabelled TAG pool and enzymatically measured TAG content during contraction. In a separate set of experiments, net TAG utilization was analysed in freeze-dried tissue (5 mg dry) from resting and contracted (30 min) muscles, which had been dissected free of all visible blood and connective tissue. Muscle TAG was measured enzymatically as previously described (Dyck et al. 1997).
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Glucose oxidation
Glucose oxidation was determined in a separate set of experiments. Muscle strips were preincubated as outlined previously (see accompanying paper Smith et al. 2005), in the absence or presence of 2 mM AICAR. After the preincubation, the gas was turned off to prevent the escape of 14CO2, the buffer was drained and 7 ml of pre-gassed, modified KHB containing 2 μCi of [U-14C]glucose (Amersham Life Science) was added to the reservoir. The muscle strips were incubated in the absence or presence of 2 mM AICAR and were stimulated to contract (150 ms trains composed of 0.5 ms impulses (20–40 V; 60 Hz) at 20 tetani min–1). After this phase, the buffer was drained and a 3.5 ml aliquot of the incubation medium was transferred to a 50 ml Erlenmeyer flask, and was acidified as previously described (see accompanying paper Smith et al. 2005) to capture 14CO2.
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Calculations and statistics
The specific activity of the incubation buffer (d.p.m. radiolabelled palmitate (nmol total palmitate)–1) was used to calculate palmitate (nmol (g wet weight)–1) incorporated into lipid pools or oxidized. With enzymatic analysis of net TAG utilization during contraction, we were able to determine the relationship between the changes in the radiolabelled TAG pool and the total (enzymatically determined) TAG pool. Each nanomole of [3H]palmitate represented 83 nanomoles (LFA) and 37 nanomoles (HFA) of total FA in the TAG pool. This ratio was used to calculate the rates of net TAG hydrolysis (net loss of preloaded [3H]palmitate (nmol g–1) from lipid pools (TAG and DAG) between paired soleus strips) and endogenous lipid oxidation during muscle contraction. Glucose oxidation was calculated using the specific activity of labelled glucose in KHB in the same manner as palmitate.
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Results are presented as mean ± S.E.M. Two-way ANOVA followed by Student-Neulman-Keuls post hoc analyses were used to assess statistical significance between time points for AMPK activity, with and without AICAR. Student's unpaired t tests were used to analyse the effect of AICAR on FA and glucose metabolism during contraction. Significance was accepted at P 0.05.
Results
Time course for AMPK activation with contraction and AICAR
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Contraction and AICAR had no effect on AMPK 1 activity (Fig. 1A). However, with contraction alone, AMPK 2 activity (Fig. 1B) was significantly increased at 30 min compared to 10 min (+67%; P < 0.05) and 0 min (+195%; P < 0.05). The combination of contraction and AICAR resulted in significant further increases in AMPK 2 activity (P < 0.01; treatment effect). With contraction and AICAR combined, AMPK 2 activity was significantly increased at 30 min compared to 10 min (+71%; P < 0.05) and 0 min (+154%; P < 0.001). At 30 min of contraction and AICAR, AMPK 2 activity was significantly greater than contraction alone (+45%; P < 0.05).
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Values are means ± S.E.M., pmol min–1 (mg protein)–1, n = 5–8 per group. *Significantly different from 30 min contraction alone (P < 0.05). **Significantly different from 0 and 10 min of same condition. Treatment effect of AICAR significantly different from No AICAR (P < 0.05).
Effects of AICAR on FA metabolism during contraction
Exogenous FA metabolism. The combination of contraction and AICAR significantly increased exogenous FA oxidation at both LFA (+71%; P < 0.05; Fig. 2A) and HFA (+46%; P < 0.05; Fig. 2B). Incorporation into TAG was not significantly different with AICAR (Fig. 2C and D), but there was an increase in the amount of FA partitioned toward oxidation relative to TAG esterification at LFA (+65%; P < 0.05; Fig. 2E), while no significant increase in this ratio was observed at HFA (+28%; P = 0.30; Fig. 2F). There were no significant differences in palmitate esterification into DAG with AICAR (Table 1).
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Effect of AICAR on fatty acid oxidation in low fatty acid (0.2 mM, LFA: A) or high fatty acid (1 mM, HFA: B) modified KHB, on TAG esterification (LFA: C; HFA: D) and on oxidation: TAG esterification ratio (LFA: E; HFA: F). Values are means ± S.E.M., nmol (g wet wt)–1, n = 7 per group. *Significantly different from No AICAR (P < 0.05).
Endogenous FA metabolism. AICAR resulted in significant blunting of TAG hydrolysis during 30 min of contraction at both LFA (–294%; P < 0.001; Fig. 3A), and HFA (–117%; P < 0.05; Fig. 3B) conditions. There were no significant effects of AICAR treatment on endogenous oxidation at LFA or HFA. There were no significant effects on DAG hydrolysis (Table 1) with AICAR treatment during contraction.
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Effect of AICAR on TAG hydrolysis and endogenous fatty acid oxidation in low fatty acid (0.2 mM, LFA: A) or high fatty acid (1 mM, HFA: B) modified KHB. Values are means ± S.E.M., nmol (g wet wt)–1, n = 7 per group. *Significantly different from No AICAR (P < 0.05).
Effects of AICAR on glucose oxidation in isolated contracting rat soleus muscle
AICAR had no significant effect on glucose oxidation during contraction, with either LFA or HFA (Fig. 4).
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There was no significant effect of AICAR on glucose oxidation in low fatty acid (0.2 mM, LFA) or high fatty acid (1 mM, HFA) modified KHB. Values are means ± S.E.M., n = 6–8 per group.
Discussion
With the use of palmitate and glucose tracers, we were able to directly examine the effects of AICAR on FA metabolism and glucose oxidation during high intensity tetanic contraction. Several novel observations were made: (1) incubation with AICAR further increased AMPK 2 activity above that seen with contraction alone; (2) AICAR further increased FA oxidation during contraction, independent of FA availability; (3) AICAR blunted TAG hydrolysis during contraction, independent of FA availability, while (4) having no effect on endogenous oxidation; and (5) AICAR had no effect on glucose oxidation during contraction. Therefore, our data suggest that FA substrate metabolism is much more sensitive than glucose to AMPK 2 stimulation during muscle contraction.
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Effect of AICAR on skeletal muscle lipid metabolism during contraction
To our knowledge, this is the first study to examine AICAR's effect on exogenous and endogenous lipid metabolism during contraction. With AICAR, there was an increase in exogenous FA oxidation above that seen with contraction alone, which could be due to a further increase in AMPK 2 activity. Previous evidence has shown that 15 min of hindlimb perfusion with AICAR-containing medium (at rest; 0.5, 1.0, 2.0 mM) significantly increased ZMP (phosphorylated AICAR) content in gastrocnemius–plantaris muscles, while having no effect on endogenous high-energy phosphates (ATP, ADP, AMP). This was associated with a significant increase in AMPK activity, leading to downstream effects of decreased ACC activity and decreased malonyl-CoA (Merrill et al. 1997). Our previous resting protocol (see accompanying paper Smith et al. 2005) certainly supports this AICAR-mediated increase in AMPK activity, which may be due to an initial increase in ZMP content. This further activation indicates that in our isolated tetanic contraction preparation, AMPK activation is a major regulator of FA oxidation. Recent evidence in AICAR-perfused rat hindquarters using a low intensity contraction protocol demonstrated that the increase in FA oxidation above that seen with contraction could not be attributed to the small increase in AICAR-induced AMPK activity, suggesting that with contraction, there are AMPK-independent mechanisms regulating FA metabolism (Raney et al. 2005). In this study, we used a higher intensity tetanic contraction protocol that has previously shown maximal stimulation of FA metabolism (Dyck & Bonen, 1998). The current data indicate that at high intensity tetanic contraction, AICAR can lead to further activation of AMPK, with further downstream effects on exogenous FA oxidation. Alternatively, AICAR may be targeting other additional regulatory enzymes resulting in an increase in FA oxidation. We also observed an increase in the partitioning of exogenous FA to oxidation at LFA during contraction, demonstrating an increased provision of FA to oxidative processes with AICAR. We did not observe a significant increase in FA partitioning with AICAR at HFA. However, this may not be surprising, as with increased FA substrate available during contraction proportionately more FA is available to be oxidized as well as esterified to TAG (Dyck & Bonen, 1998).
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Regulation of TAG hydrolysis in resting and contracting skeletal muscle is poorly understood. In this study, treatment with AICAR during tetanic contraction significantly blunted TAG hydrolysis, independent of FA availability, while having no effect on endogenously derived TAG oxidation. Indeed, exposing soleus strips to LFA did increase the provision of FA from TAG hydrolysis during contraction (+89% versus HFA), with AICAR attenuating this effect. It appears that AMPK regulation of TAG hydrolysis can occur during contraction and it is possible that AICAR reduced TAG hydrolysis during contraction by inhibiting the activation of HSL. HSL is an important enzyme in the hydrolysis of TAG. Recently, it has been shown that stimulation of AMPK prevents the activation of HSL (Watt et al. 2004). Thus, it is possible that the pharmacological stimulation of AMPK combined with contraction prevented the activation of HSL as would be expected with contraction alone, resulting in a reduction in TAG hydrolysis. However, further work is required to investigate the effect of AMPK activation on the regulation of HSL and TAG hydrolysis.
, 百拇医药
Importantly, although AICAR attenuated TAG hydrolysis, there was no significant effect on endogenous oxidation in contracting soleus muscle. It is possible that during contraction, there is an excess of FA released from the endogenous TAG pool which may be reincorporated if the oxidative needs of the cell are met (Dyck & Bonen, 1998). Regulatory mechanisms that hydrolyse the TAG molecule might allow for an excess of FA to be broken down during contraction, but the energy required by the cell would govern actual oxidation. This attenuation with AICAR suggests a better matching of TAG hydrolysis with the oxidative needs of the cell. Even with a slight mismatch between TAG hydrolysis and endogenous oxidation at LFA with AICAR, the results show that AICAR had no effect on the amount of FA oxidized from endogenous stores during contraction.
, 百拇医药
Effect of AICAR on muscle glucose oxidation during contraction
AICAR had no further effect on already high rates of glucose oxidation in our tetanic contraction protocol. Much research has demonstrated that AMPK is involved in contraction-stimulated glucose uptake (Hayashi et al. 1998; Musi et al. 2001), but it is probably not the sole mediator (Mu et al. 2001). Indeed, the combination of AICAR and contraction does not have an additive effect on glucose uptake (Hayashi et al. 1998; Bergeron et al. 1999), which supports the current data on glucose oxidation rates. Importantly, it is also possible that factors other than AMPK regulate glucose uptake and oxidation. Muscles from mice with a dominant negative form of AMPK were found to have full inhibition of AICAR- and hypoxia-induced glucose uptake, but only a partial (–40%) reduction in contraction-stimulated glucose uptake (Mu et al. 2001), suggesting that AMPK is only partially involved in the signalling mechanism to regulate glucose uptake and subsequent metabolism during contraction. It should also be noted that only one glucose concentration (10 mM) was used throughout these experiments. Thus, as was the case with FA oxidation, it is possible that an AICAR-induced stimulation of glucose oxidation may have been observed during contraction in the presence of a lower glucose concentration.
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Summary
The results from the present study demonstrate that AICAR-mediated AMPK 2 activation is intimately involved in the regulation of FA metabolism during contraction, while having no effects on the already high rates of glucose oxidation in skeletal muscle. Incubation with AICAR during tetanic contraction (1) leads to synergistic increases in AMPK 2 activity; (2) further increases exogenous FA oxidation; and (3) blunts TAG hydrolysis, both at LFA and HFA. Further studies investigating AMPK regulation of HSL and TAG hydrolysis during contraction are warranted.
, 百拇医药
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