Exercise produces a multitude of time- and intensity-dependent physiological, biochemical and molecular changes within skeletal muscle. With the onset of contractile activity, cytosolic and mitochondrial [Ca2+] levels are rapidly increased and, depending on the relative intensity of the exercise, metabolite concentrations change (i.e. increases in [ADP] and [AMP], decreases in muscle creatine phosphate and glycogen). These contraction-induced metabolic disturbances activate several key kinases and phosphatases involved in signal transduction. Important among these are the calcium dependent signalling pathways that respond to elevated Ca2+ concentrations (including Ca2+/calmodulin-dependent kinase, Ca2+-dependent protein kinase C and the Ca2+/calmodulin-dependent phosphatase calcineurin), the 5′-adenosine monophosphate-activated protein kinase, several of the mitogen-activated protein kinases and protein kinase B/Akt. The role of these signal transducers in the regulation of carbohydrate and fat metabolism in response to increased contractile activity has been the focus of intense research efforts during the past decade.
Skeletal muscle ATP stores are small and would be consumed in several seconds of maximal contraction if not rapidly replenished. Without a rapid resynthesis of ATP, skeletal muscle would be incapable of initiating any further contraction. Hence, the metabolic pathways that synthesize ATP must have the capacity to respond immediately to an increased demand for ATP that can increase 100-fold above resting requirements during intense physical activity. In most situations a precise matching of ATP demand and resynthesis is accomplished to maintain skeletal muscle ATP concentrations. To accomplish this, marked changes in metabolism occur in skeletal muscle including increased glycogen breakdown, glycolysis, glucose uptake and fatty acid oxidation. A fundamental goal of exercise biochemistry is to understand the mechanism(s) by which perturbations in energy status are monitored inside contracting muscle cells and to identify molecules that are regulated to increase fuel substrate supply in order to maintain ATP levels. In addition, many of these (acute) signals initiate responses that form the basis of the metabolic adaptations to regular exercise training. This chapter will focus on the cellular and molecular mechanisms governing fuel utilization and metabolic adaptations in human skeletal muscle during and after endurance exercise.
Integration of the metabolic responses to exercise
Important intramuscular metabolic signals responsible for the activation and coordination of the various energy-producing pathways during exercise include alterations in [Ca2+], changes in the concentrations of metabolites related to the cytoplasmic phosphorylation potential of the muscle cell and the mitochondrial reduction/oxidation (redox) state of [NAD]/[NADH] . Ca2+ release plays an essential role in initiating muscle contraction and activating metabolism via a ‘feed-forward’ mechanism. Calcium provides the trigger for force development and ATP hydrolysis and contributes to the activation of glycogenolysis and possibly oxidative phosphorylation. A rise in intracellular [Ca2+] levels also contributes to the regulation of glucose uptake during muscle contraction, by activating a signal transduction pathway leading to GLUT 4 (glucose transporter 4) translocation. Several other contraction induced signalling intermediates [i.e. PKC (protein kinase C)] and the MAPK (mitogen activated protein kinase) cascades] are also sensitive to changes in [Ca2+] and are likely to mediate exercise-induced responses, that may include glucose and lipid metabolism or gene regulatory events.
Whilst Ca2+-activated signals are important for early events in excitation–contraction coupling and the stimulation of metabolic pathways, the concentrations of metabolites related to the cytoplasmic phosphorylation potential of skeletal muscle (i.e. [ATP]/[ADP][Pi]) provide feedback signals necessary to balance ATP production with ATP consumption (Figure 1). Changes in the concentrations of metabolites in the phosphagen pool provide the main stimulus for increased oxidative phosphorylation and are necessary (but not sufficient) for full activation of glycolytic ATP synthesis during intense (≥85% of maximal aerobic power [VO2max]) sustained exercise. The redox state of the skeletal muscle activates numerous reactions in substrate/product or activator/inhibitor capacities. All three types of signal are important in coordinating the expression of enzymes that are vital for ATP production in mitochondrial and cytoplasmic compartments during exercise . Along with systemic factors, such as increased blood flow leading to increased delivery of oxygen and hormonal factors, intramuscular and extramuscular substrate availability and the local release of growth factors and cytokines from contracting muscle, these signals provide the framework in which fuel mobilization and utilization are increased and intracellular signal transduction to the transcriptional machinery in the nucleus is enhanced, thereby modulating gene expression and ultimately protein turnover.
Signalling pathways during exercise
The relative importance of various putative signalling pathways in the acute regulation of metabolism in response to exercise and chronic metabolic adaptations to exercise training has been the focus of considerable research efforts in recent years. Some of the major pathways are summarized in Figure 2 and are discussed subsequently.
There has also been much interest in the interactions between the insulin and contraction-signalling pathways within skeletal muscle. Whilst these pathways independently activate metabolic processes (e.g. GLUT4 protein translocation and glucose uptake, Figure 3), enhanced insulin action is a well described effect of acute and chronic exercise, suggesting communication between these pathways. Further work is required to elu cidate the underlying mechanisms to explain such phenomena.
Calcium dependent signalling
The increase in sarcoplasmic [Ca2+] levels during exercise has a fundamental role in excitation–contraction coupling, stimulation of ATP-generating metabolic pathways and activation of transcriptional responses (Figure 4). Calcium-dependent signalling pathways include CaMK (Ca2+/calmodulin-dependent kinase), PKC (Ca2+-dependent protein kinase C) isoforms and the Ca2+/calmodulin-dependent phosphatase calcineurin. A number of CaMK isoforms have been identified, but CaMK II appears to be the dominant isoform expressed in skeletal muscle . Exercise increases CaMK II activity in human skeletal muscle , with the magnitude of the increase during very high-intensity (approx. 100% Vo2max) exercise being greater than that during moderately high-intensity (approx. 75% VO2max) exercise. Early studies in rat muscle demonstrated increased PKC activity with electrical stimulation, consistent with activation of conventional and novel PKC isoforms by contraction-induced increases in Ca2+ and diacylglycerol . Recent studies in humans suggest that the increase in total PKC activity in skeletal muscle following exercise may be the result of activation of the atypical PKC isoforms [5,6]. We are unaware of any studies that have examined the effects of exercise on calcineurin activity in skeletal muscle.
Calcium binds directly to and activates phosphorylase kinase, which in turn transforms phosphorylase to an active form, thereby contributing to enhanced glycogenolysis in skeletal muscle during exercise. Further stimulation of glycogenolysis is provided by increases in inorganic phosphate, AMP and ADP. Calcium has long been recognized to also increase glucose uptake, although the specific signalling intermediates are unresolved . Inhibition of CaMK reduced glucose uptake by approx. 50% during contraction of epitrochlearis muscles in vitro  and completely abolished the increase in glucose uptake during tetanic contractions in soleus muscle . The PKC inhibitor calphostin C attenuates the contraction-induced increase in skeletal muscle glucose uptake . In addition, PKC is believed to regulate the activation of HSL (hormone-sensitive lipase), a key enzyme in the degradation of intramuscular triglyceride . Finally, increased Ca2+ results in transcriptional activation of numerous genes involved in mitochondrial biogenesis and muscle hypertrophy . All of the Ca2+-dependent signalling pathways are believed to be involved in metabolic and gene regulatory events to some extent. For example, CaMK inhibition abolishes Ca2+-induced mitochondrial biogenesis , PKC has been implicated in increased cytochrome c expression following Ca2+ stimulation  and calcineurin has a role in determining skeletal muscle fibre type .
AMP-activated protein kinase
A key pathway in skeletal muscle that responds to changes in the concentrations of metabolites related to the cytoplasmic phosphorylation potential is AMPK (AMP-activated protein kinase). This kinase appears to be a key sensor of skeletal muscle energy status . AMPK exists as a heterotrimer consisting of α, β and γ subunits. The α subunit of AMPK, of which there are two known isoforms (α1 and α2), contains the catalytic domain that transfers a high-energy phosphate from ATP to serine and threonine residues on a number of different target proteins. In addition, the α subunit contains a specific threonine residue (Thr172) that functions as an activating phosphorylation site for several upstream kinases. The α2 isoform is abundant in skeletal muscle and is more sensitive to changes in AMP than the α1 isoform. The β and γ regulatory subunits are essential for full enzymatic activity and multiple isoforms of β (β1 and β2) and γ (γ1, γ2 and γ3) exist. Submaximal exercise in humans primarily increases AMPK α2 activity in skeletal muscle [14,15], although intense exercise can increase AMPK α1 activity . The increased AMPK activity during exercise is attenuated by elevated pre-exercise muscle glycogen availability , further emphasizing the role of AMPK as a fuel/energy sensor.
There has been considerable interest in the potential role of AMPK in stimulating muscle glucose uptake during exercise/contractions [4,7,18]. Such interest arises from observations that AICAR (5-aminoimidazole-4-carboxamide riboside), a compound taken up by skeletal muscle and metabolized to ZMP (an analogue of AMP), which is an activator of AMPK, enhances glucose transport in rat skeletal muscle [7,8]. AICAR has been used as a pharmacological tool to activate the ‘exercise-responsive’ pathway to glucose transport. Approaches in functional genomics reveal that AMPK is necessary and sufficient for AICAR induced glucose transport . Ablation of either the AMPK α2 or γ3 subunit, or overexpression of a kinase dead AMPK α2 mutant rendered skeletal muscle insensitive to AICAR on glucose uptake . Consistent with this, skeletal muscle specific deletion of LKB1, the putative upstream AMPK kinase, largely abolished the contraction-induced increase in AMPK activation and glucose uptake . However, contraction-induced glucose transport was only reduced approx. 40% in skeletal muscles from transgenic mice with the kinase dead AMPK α2 mutant and was normal in both α1 and α2 whole-body knockout mice  and AMPK γ3 knockout mice  despite complete abolition of the AICAR response in all models. Finally, in rats AMPK was shown not to be necessary for slow twitch soleus muscle glucose transport during contractions in vitro . Collectively, these results suggest that there are factors in addition to AMPK that regulate skeletal muscle uptake during exercise.
AMPK promotes fatty acid oxidation in skeletal muscle during exercise by inhibiting ACC-β (acetyl-CoA carboxylase) and activating malonyl-CoA, thus removing inhibition of mitochondrial fatty acyl-CoA translocation by CPT-1 (carnitine palmitoyltransferase-1). A number of studies have reported that these exercise-induced effects on ACC-β and malonyl-CoA are closely paralleled by activation of AMPK [19,20] especially the α2 isoform when isoform-specific activity has been determined . Effects of exercise intensity on AMPK signalling and whole-body rates of fat oxidation in humans have been determined . With low- (approx. 40% VO2max) to moderate- (approx. 60% VO2max) intensity cycling, AMPK α1 (1.5-fold) and AMPK α2 (5-fold) activities were increased, with a further increase in AMPK α2 activity during high-intensity compared with moderate-intensity exercise. Rates of whole-body fat oxidation increased from rest to low intensity exercise and paralleled the increases in ACC-β phosphorylation. As expected, rates of fat oxidation declined during high-intensity exercise, despite a further increase in AMPK activity and ACC-β phosphorylation . AMPK opposes the adrenergic stimulation of skeletal muscle HSL activity during exercise, but HSL activity is dissociated from intramuscular triglyceride hydrolysis, suggesting the possible involvement of other skeletal muscle lipases . Following short-term exercise training, there remains a robust increase in glucose uptake and fat oxidation during exercise, despite an almost complete blunting of the increase in AMPK α2 activity . These findings challenge the role of AMPK in the regulation of carbohydrate and fat metabolism during exercise in humans.
Pharmacological activation of AMPK by AICAR increases protein expression of GLUT4, hexokinase and several oxidative enzymes, as well as mitochondrial density and muscle glycogen content [23,24]. However, the exercise-induced increase in transcription or mRNA abundance of selected metabolic genes was unchanged between α-AMPK knockout mice and wild-type mice , suggesting that AMPK is insufficient and that other pathways also contribute to transcriptional activation by exercise (e.g. CaMK, MAPK).
The MAPK signal transduction cascade has been identified as a candidate system that converts contraction-induced biochemical perturbations into appropriate intracellular responses [1,26]. Exercise is a powerful and rapid activator of several MAPK isoforms including the ERK (extracellular signal-regulated kinases)1/2 and the two stress-activated protein kinases, p38 MAPK and JNK (c-Jun NH2-terminal kinase) . Local and systemic factors mediate phosphorylation of MAPK signalling cascades  that are implicated in exercise metabolism and transcriptional regulation of important genes . The p38 MAPK pathway has been implicated in the contraction-induced activation of muscle glucose uptake, whilst the role of ERK is equivocal . Increased ERK activity may contribute to increased skeletal muscle HSL activity during exercise . Exercise-induced activation of the p38 MAPK pathway has recently been demonstrated to play a role in skeletal muscle adaptation by promoting specific co-activators involved in mitochondrial biogenesis and slow muscle fibre formation . MAPK activation can result not only in the production of transcription factors mediating gene expression, but can also stimulate the activity of the translational stage of protein synthesis.
The protein kinase B/Akt is a serine/threonine kinase with three isoforms (Akt1, Akt2 and Akt3) that share >80% homology. Akt 1 and Akt 2 are the predominant isoforms expressed in skeletal muscle. This protein kinase family has been implicated as an important target for mediating insulin action on glycogen synthesis, GLUT4 translocation and glucose transport and gene regulatory responses . Many, but not all studies have reported that skeletal muscle contraction increases Akt activity or phosphorylation . The time course of contraction-stimulated glucose transport and Akt activation are similar, suggestive of a role for Akt in signalling glucose transport into exercising muscle. However, when contraction-stimulated Akt phosphorylation and activity are inhibited, glucose transport is unaffected, suggesting that Akt does not function to increase contraction-induced glucose transport . Several Akt substrates have been identified as putative links between insulin signalling and metabolic or gene regulatory responses. A novel 160 kDa signalling-protein has been identified and characterized as an Akt substrate (AS160) in 3T3-L1 adipocytes and skeletal muscle. AS160 is phosphorylated in rat skeletal muscle in response to contraction  and also in human skeletal muscle in response to exercise . Whilst contraction-induced AS160 phosphorylation in isolated skeletal muscle was completely abolished by wortmannin , glucose uptake was not inhibited by this agent. Future studies are warranted to identify and characterize the multiple exercise-responsive Akt substrates in skeletal muscle.
The increase in sympathoadrenal activity that occurs during exercise, particularly at higher exercise intensities, has an important role in substrate mobilization and utilization (Figure 5). Adrenaline stimulates muscle glycogenolysis  and HSL activity  via the β-adrenergic receptor and activation of protein kinase A. The importance of adrenaline and sympathetic noradrenergic activity in the mobilization of liver glycogen during exercise is equivocal, but they are crucial for adipose tissue lipolysis and the mobilization of fatty acids into the blood stream . A blunting of sympathoadrenal activity during exercise is a key factor mediating altered substrate metabolism following exercise training . Adrenergic blockade is associated with increased fatigue and reduced exercise tolerance, but does not appear to prevent the training-induced increase in muscle oxidative capacity .
One fundamental goal of exercise biochemistry is to characterize the mechanism(s) by which changes in energy status are monitored in skeletal muscle during periods of increased demand and to identify the specific molecular pathways (and downstream targets) that are regulated to increase fuel provision and maintain ATP levels. Linking specific signalling cascades to defined metabolic responses and changes in gene expression that occur after exercise will be complicated because there is a high degree of cross-talk between many of these pathways, with feedback regulation, transient activation and redundancy.
• The main intramuscular metabolic signals responsible for the activation and coordination of the various energy-producing pathways during exercise are alterations in [Ca2+], changes in the concentrations of metabolites related to the cytoplasmic phosphorylation potential of the muscle cell and the mitochondrial reduction/oxidation (redox) state of [NAD]/[NADH].
• Calcium-dependent signalling pathways include CaMK, PKC isoforms and the Ca2+/calmodulin-dependent phosphatase calcineurin. A number of CaMK isoforms have been identified, but CaMK II appears to be the dominant isoform expressed in skeletal muscle.
• A key pathway in skeletal muscle that responds to changes in the concentrations of metabolites related to the cytoplasmic phosphorylation potential is AMPK. This kinase appears to be a key sensor of skeletal muscle energy status.
• Submaximal exercise in humans increases AMPK α2 activity in skeletal muscle whereas intense exercise also increases AMPK α1 activity.
• Exercise is a powerful and rapid activator of several MAPKs including ERK1/2 and the two stress-activated protein kinases, p38 MAPK and JNK. Local and systemic factors mediate phosphorylation of MAPK signalling cascades implicated in exercise metabolism and transcriptional regulation of important genes.
• The protein kinase B/Akt is a serine/threonine kinase with two isoforms (Akt1 and Akt2) that have been implicated as an important target for mediating insulin action on glycogen synthesis, GLUT4 translocation and glucose transport and gene regulatory responses in skeletal muscle.
- © 2006 The Biochemical Society, London