The present paper is the introductory paper to a series of brief reviews representing the proceedings of a recent conference on ‘The biochemical basis for the health effects of exercise’ organized by the International Research Group on the Biochemistry of Exercise in conjunction with the Nutrition Society. Here the aim is to briefly review and highlight the main innovations presented during this meeting. The following topics were covered during the meeting: exercise signalling pathways controlling fuel oxidation during and after exercise; the fatty acid transporters of skeletal muscle; mechanisms involved in exercise-induced mitochondrial biogenesis in skeletal muscle; new methodologies and insights in the regulation of fat metabolism during exercise; muscle hypertrophy: the signals of insulin, amino acids and exercise; adipose tissue–liver–muscle interactions leading to insulin resistance. In these symposia state-of-the-art knowledge on how physical exercise exerts its effects on health was presented. The fast-growing number of identified pathways and processes involved in the health effects of physical exercise, which were discussed during the meeting, will help to develop tailored physical-activity regimens in the prevention of inactivity-induced deterioration of health.

Physical inactivity–gene interactions result in changes in gene expression, leading to phenotypic changes in the skeletal muscle cell. A subpopulation of those genes that show changes in expression during physical inactivity are candidates for the environment–gene interactions that cross a threshold of biological significance such that overt clinical disease occurs. AMP kinase, GLUT4 and myosin heavy chain IIx are proposed as candidates for physical inactivity-modulated genes that have an altered function that may trigger a crossing of a threshold to disease. Future experiments will be needed to test the validity of the ideas presented.

Contraction-induced glucose uptake in skeletal muscle is mediated by an insulin-independent mechanism that leads to translocation of the GLUT4 glucose transporter to the muscle surface membrane from an intracellular storage site. Although the signalling events that increase glucose transport in response to muscle contraction are not fully elucidated, the aim of the present review is to briefly present the current understanding of the molecular signalling mechanisms involved. Glucose uptake may be regulated by Ca2+-sensitive contraction-related mechanisms, possibly involving Ca2+/calmodulin-dependent protein kinase II and some isoforms of protein kinase C. In addition, glucose transport may be regulated by mechanisms that reflect the metabolic status of the muscle, probably involving the 5′AMP-activated protein kinase. Furthermore, the p38 mitogen-activated protein kinase may be involved in activating the GLUT4 translocated to the surface membrane. Nevertheless, the picture is incomplete, and fibre type differences also seem to be involved.

The AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is activated during exercise and muscle contraction as a result of acute decreases in ATP:AMP and phosphocreatine:creatine. Physical exercise increases muscle glucose uptake, enhances insulin sensitivity and leads to fatty acid oxidation in muscle. An important issue in muscle biology is to understand whether AMPK plays a role in mediating these metabolic processes. AMPK has also been implicated in regulating gene transcription and, therefore, may function in some of the cellular adaptations to training exercise. Recent studies have shown that the magnitude of AMPK activation and associated metabolic responses are affected by factors such as glycogen content, exercise training and fibre type. There have also been conflicting reports as to whether AMPK activity is necessary for contraction-stimulated glucose transport. Thus, during the next several years considerably more research will be necessary in order to fully understand the role of AMPK in regulating glucose transport in skeletal muscle.

The pyruvate dehydrogenase complex (PDC) has a key position in skeletal muscle metabolism as it represents the entry of carbohydrate-derived fuel into the mitochondria for oxidation. PDC is regulated by a phosphorylation–dephosphorylation cycle, in which the pyruvate dehydrogenase kinase (PDK) phosphorylates and inactivates the complex. PDK exists in four isoforms, of which the PDK4 isoform is predominantly expressed in skeletal and heart muscle. PDK4 transcription and PDK4 mRNA are markedly increased in human skeletal muscle during prolonged exercise and after both short-term high-intensity and prolonged low-intensity exercise. The exercise-induced transcriptional response of PDK4 is enhanced when muscle glycogen is lowered before the exercise, and intake of a low-carbohydrate high-fat diet during recovery from exercise results in increased transcription and mRNA content of PDK4 when compared with intake of a high-carbohydrate diet. The activity of pyruvate dehydrogenase (PDH) is increased during the first 2 h of low-intensity exercise, followed by a decrease towards resting levels, which is in line with the possibility that the increased PDK4 expressed influences the PDH activity already during prolonged exercise. PDK4 expression is also increased in response to fasting and a high-fat diet. Thus, increased PDK4 expression when carbohydrate availability is low seems to contribute to the sparing of carbohydrates by preventing carbohydrate oxidation. The impact of substrate availability on PDK4 expression during recovery from exercise also underlines the high metabolic priority given to replenishing muscle glycogen stores and re-establishing intracellular homeostasis after exercise.

Muscle glycogen is an important fuel for contracting skeletal muscle during prolonged strenuous exercise, and glycogen depletion has been implicated in muscle fatigue. It is also apparent that glycogen availability can exert important effects on a range of metabolic and cellular processes. These processes include carbohydrate, fat and protein metabolism during exercise, post-exercise glycogen resynthesis, excitation–contraction coupling, insulin action and gene transcription. For example, low muscle glycogen is associated with reduced muscle glycogenolysis, increased glucose and NEFA uptake and protein degradation, accelerated glycogen resynthesis, impaired excitation–contraction coupling, enhanced insulin action and potentiation of the exercise-induced increases in transcription of metabolic genes. Future studies should identify the mechanisms underlying, and the functional importance of, the association between glycogen availability and these processes.

Exercise training improves glucose homeostasis through enhanced insulin sensitivity in skeletal muscle. Muscle contraction through physical exercise is a physiological stimulus that elicits multiple biochemical and biophysical responses and therefore requires an appropriate control network. Mitogen-activated protein kinase (MAPK) signalling pathways constitute a network of phosphorylation cascades that link cellular stress to changes in transcriptional activity. MAPK cascades are divided into four major subfamilies, including extracellular signal-regulated kinases 1 and 2, p38 MAPK, c-Jun NH2-terminal kinase and extracellular signal-regulated kinase 5. The present review will present the current understanding of parallel MAPK signalling in human skeletal muscle in response to exercise and muscle contraction, with an emphasis on identifying potential signalling mechanisms responsible for changes in gene expression.

Glycogen synthase (GS) catalyses the rate-limiting step of UDP-glucose incorporation into glycogen. Exercise is a potent regulator of GS activity, leading to activation of GS immediately after exercise promoting glycogen repletion by mechanisms independent of insulin. The incorporation of UDP-glucose is energy demanding, and during intense exercise GS is deactivated, diminishing energy utilization but also increasing the potential for glycogen breakdown. An apparent activation of GS is observed during moderate exercise, which could be considered as a potential waste of energy, although the cellular capacity for glycogen breakdown is considerably higher than that for glycogen synthesis. The understanding of this complex regulation of GS activity in response to exercise is just at its beginning. In the present review potential mechanisms by which exercise regulates GS activity are described, factors that may promote GS activation and factors that may deactivate GS are discussed, pointing to the view that GS activity during exercise is the result of the relative strength of these opposing factors.

The first putative fatty acid transporter identified was plasma membrane fatty acid-binding protein (FABPpm). Later it was demonstrated that this protein is identical to the mitochondrial isoform of the enzyme aspartate aminotransferase. In recent years data from several cell types have emerged, indicating that FABPpm plays a role in the transport of long-chain saturated and unsaturated fatty acids. In the limited number of studies in human skeletal muscle it has been demonstrated that dietary composition and exercise training can influence the content of FABPpm. Ingestion of a fat-rich diet induces an increase in FABPpm protein content in human skeletal muscle in contrast to the decrease seen during consumption of a carbohydrate-rich diet. A similar effect of a fat-rich diet is also observed for cytosolic fatty acid-binding protein and fatty acid translocase/CD36 protein expression. Exercise training up regulates FABPpm protein content in skeletal muscle, but only in male subjects; no significant differences were observed in muscle FABPpm content in a cross-sectional study of female volunteers of varying training status, even though muscle FABPpm content did not depend on gender in the untrained state. A higher utilization of plasma long-chain fatty acids during exercise in males compared with females could explain the gender-dependent influence of exercise training on FABPpm. The mechanisms involved in the regulation of the function and expression of FABPpm protein remain to be clarified.

Fatty acid (FA) translocase (FAT)/CD36 is a key protein involved in regulating the uptake of FA across the plasma membrane in heart and skeletal muscle. A null mutation of FAT/CD36 reduces FA uptake rates and metabolism, while its overexpression increases FA uptake rates and metabolism. FA uptake into the myocyte may be regulated (a) by altering the expression of FAT/CD36, thereby increasing the plasmalemmal content of this protein (i.e. streptozotocin-induced diabetes, chronic muscle stimulation), or (b) by relocating this protein to the plasma membrane, without altering its expression (i.e. obese Zucker rats). By repressing FAT/CD36 expression, and thereby lowering the plasmalemmal FAT/CD36 (i.e. leptin-treated animals), the rate of FA transport is reduced. Within minutes of beginning muscle contraction or being exposed to insulin FA transport is increased. This increase is a result of the contraction- and insulin-induced translocation of FAT/CD36 from an intracellular depot to the cell surface. Neither PPARα nor PPARγ activation alter FAT/CD36 expression in muscle, despite the fact that PPARα activation increases FAT/CD36 by 80% in liver. A novel observation is that FAT/CD36 also appears to be involved in mitochondrial FA oxidation, as this protein is located on the mitochondrial membrane and seems to be required to participate in moving FA across the mitochondrial membrane. Clearly, FAT/CD36 has an important role in FA homeostasis in skeletal muscle and the heart.

Glucose and long-chain fatty acids (LCFA) are two major substrates used by heart and skeletal muscle to support contractile activity. In quiescent cardiac myocytes a substantial portion of the glucose transporter GLUT4 and the putative LCFA transporter fatty acid translocase (FAT)/CD36 are stored in intracellular compartments. Induction of cellular contraction by electrical stimulation results in enhanced uptake of both glucose and LCFA through translocation of GLUT4 and FAT/CD36 respectively to the sarcolemma. The involvement of protein kinase A, AMP-activated protein kinase (AMPK), protein kinase C (PKC) isoforms and the extracellular signal-regulated kinases was evaluated in cardiac myocytes as candidate signalling enzymes involved in recruiting these transporters in response to contraction. The collected evidence excluded the involvement of PKA and implicated an important role for AMPK and for one (or more) PKC isoform(s) in contraction-induced translocation of both GLUT4 and FAT/CD36. The unravelling of further components along this contraction pathway can provide valuable information on the coordinated regulation of the uptake of glucose and of LCFA by an increase in mechanical activity of heart and skeletal muscle.

Efficient uptake and channelling of long-chain fatty acids (LCFA) are critical cell functions. Evidence is emerging that proteins are important mediators of LCFA-trafficking into cells and various proteins have been suggested to be involved in this process. Amongst these proteins is a family of membrane-associated proteins termed fatty acid transport proteins (FATP). So far six members of this family, designated FATP 1–6, have been characterized. FATP 1, 2 and 6 show a highly-conserved AMP-binding region that participates in the activation of very-long-chain fatty acids (VLCFA) to form their acyl-CoA derivatives. The mechanisms by which FATP mediate LCFA uptake are not well understood, but several studies provide evidence that uptake of LCFA across cellular membranes is closely linked to acyl-CoA synthetase activity. It is proposed that FATP indirectly enhance LCFA uptake by activating VLCFA to their CoA esters, which are required to maintain the typical structure of lipid rafts in cellular membranes. Recent work has shown that the structural integrity of lipid rafts is essential for cellular LCFA uptake. This effect might be exerted by proteins, e.g. caveolin-1 and FAT/CD36, that use lipid rafts as platforms and bind or transport LCFA. The proposed molecular mechanisms await further experimental investigation.

For most of the last century, researchers have searched for a muscle contraction-induced factor that mediates some of the exercise effects in other tissues such as the liver and the adipose tissue. It has been called the ‘work stimulus’, the ‘work factor’ or the ‘exercise factor’. In the search for such a factor, a cytokine, IL-6, was found to be produced by contracting muscles and released into the blood. It has been demonstrated that IL-6 has many biological roles such as: (1) induction of lipolysis; (2) suppression of TNF production; (3) stimulation of cortisol production. The IL-6 gene is rapidly activated during exercise, and the activation of this gene is further enhanced when muscle glycogen content is low. In addition, carbohydrate supplementation during exercise has been shown to inhibit the release of IL-6 from contracting muscle. Thus, it is suggested that muscle-derived IL-6 fulfils the criteria of an exercise factor and that such classes of cytokines could be termed ‘myokines’.

Endurance exercise training induces an increase in the respiratory capacity of muscle, resulting in an increased capacity to generate ATP as well as improved efficiency of muscle contraction. Such adaptations are largely the result of a coordinated genetic response that increases mitochondrial proteins, fatty acid oxidation enzymes and the exercise- and insulin-stimulated glucose transporter GLUT4, and shifts the contractile and regulatory proteins to their more efficient isoforms. In recent years a number of the transcriptional regulators involved in this genetic response have been identified and these factors can be classified into two different groups. The first group comprises transcription factors such as nuclear respiratory factors (NRF) 1 and 2 and PPARα that bind DNA in a sequence-specific manner. The second group, referred to as transcriptional co-activators, alter transcription without directly binding to DNA. The PPARγ co-activator (PGC) family of proteins have been identified as the central family of transcriptional co-activators for induction of mitochondrial biogenesis. PGC-1α is activated by exercise, and is sufficient to produce the endurance phenotype through direct interactions with NRF-1 and PPARα, and potentially NRF-2. Furthering the understanding of the activation of PGC proteins following exercise has implications beyond improving athletic performance, including the possibility of providing targets for the treatment of frailty in the elderly, obesity and diseases such as mitochondrial myopathies and diabetes.

Contractile activity induces mitochondrial biogenesis and increases glucose transport capacity in muscle. There has been much research on the mechanisms responsible for these adaptations. The present paper reviews the evidence, which indicates that the decrease in the levels of high-energy phosphates, leading to activation of AMP kinase (AMPK), and the increase in cytosolic Ca2+, which activates Ca2+/calmodulin-dependent protein kinase (CAMK), are signals that initiate these adaptative responses. Although the events downstream of AMPK and CAMK have not been well characterized, these events lead to activation of various transcription factors, including: nuclear respiratory factors (NRF) 1 and 2, which cause increased expression of proteins of the respiratory chain; PPAR-α, which up regulates the levels of enzymes of β oxidation; mitochondrial transcription factor A, which activates expression of the mitochondrial genome; myocyte-enhancing factor 2A, the transcription factor that regulates GLUT4 expression. The well-orchestrated expression of the multitude of proteins involved in these adaptations is mediated by the rapid activation of PPARγ co-activator (PGC) 1, a protein that binds to various transcription factors to maximize transcriptional activity. Activating AMPK using 5-aminoimidizole-4-carboxamide-1-β-D-riboside (AICAR) and increasing cytoplasmic Ca2+using caffeine, W7 or ionomycin in L6 myotubes increases the concentration of mitochondrial enzymes and GLUT4 and enhances the binding of NRF-1 and NRF-2 to DNA. AICAR and Ca-releasing agents also increase the levels of PGC-1, mitochondrial transcription factor A and myocyte-enhancing factors 2A and 2D. These results are similar to the responses seen in muscle during the adaptation to endurance exercise and show that L6 myotubes are a suitable model for studying the mechanisms by which exercise causes the adaptive responses in muscle mitochondria and glucose transport.

Intracellular Ca2+plays an important role in skeletal muscle excitation–contraction coupling and also in excitation–transcription coupling. Activity-dependent alterations in muscle gene expression as a result of increased load (i.e. resistance or endurance training) or decreased activity (i.e. immobilization or injury) are tightly linked to the level of muscle excitation. Differential expression of genes in slow- and fast-twitch fibres is also dependent on fibre activation. Both these biological phenomena are, therefore, tightly linked to the amplitude and duration of the Ca2+transient, a signal decoded downstream by Ca2+-dependent transcriptional pathways. Evidence is mounting that the calcineurin–nuclear factor of activated T-cells pathway and the Ca2+/calmodulin-dependent kinases (CaMK) II and IV play important roles in regulating oxidative enzyme expression, mitochondrial biogenesis and expression of fibre-type specific myofibrillar proteins. CaMKII is known to decode frequency-dependent information and is activated during hypertrophic growth and endurance adaptations. Thus, it was hypothesized that CaMKII, and possibly CaMKIV, are down regulated during muscle atrophy and levels of expression of CaMKIIα, -IIβ, -IIγ and -IV were assessed in skeletal muscles from young, aged and denervated rats. The results indicate that CaMKIIγ, but not CaMKIIα or -β, is up regulated in aged and denervated soleus muscle and that CaMKIV is absent in skeletal but not cardiac muscle. Whether CaMKIIγ up-regulation is part of the pathology of wasting or a result of some adaptational response to atrophy is not known. Future studies will be important in determining whether insights from the adaptational response of muscle to increased loads will provide pharmacological approaches for increasing muscle strength or endurance to counter muscle wasting.

The physiological function of the mitochondrial uncoupling protein (UCP), UCP3, is still under debate. There is, however, ample evidence to indicate that, in contrast to UCP1, the primary function of UCP3 is not the dissipation of energy. Rather, several lines of evidence suggest that UCP3 is associated with cellular fatty acid metabolism. The highest levels of expression of UCP3 have been found in type 2 glycolytic muscle fibres, and fasting and high-fat diets up regulate UCP3. This up-regulation is most pronounced in muscle with a low fat oxidative capacity. Acute exercise also up regulates UCP3, and this effect has been shown to be a result of the exercise-induced increase in plasma fatty acid levels. In contrast, regular physical activity, which increases fat oxidative capacity, reduces UCP3 content. Based on these data it has been postulated that UCP3 functions to export those fatty acids that cannot be oxidized from the mitochondrial matrix, in order to prevent fatty acid accumulation inside the matrix. Several experiments have been conducted to test this hypothesis. Blocking carnitine palmitoyltransferase 1, thereby reducing fat oxidative capacity, rapidly induces UCP3. High-fat diets, which increase the mitochondrial supply of fatty acids, also up regulate UCP. However, feeding a similar amount of medium-chain fatty acids, which can be oxidized inside the mitochondrial matrix and therefore does not need to be exported from the matrix, does not affect UCP3 protein levels. In addition, UCP3 is increased in patients with defective β-oxidation and is reduced after restoring oxidative capacity. In conclusion, it is suggested that UCP3 has an important physiological function in facilitating outward transport from the mitochondrial matrix of fatty acid anions that cannot be oxidized, thereby protecting against lipid-induced mitochondrial damage.

The protein import process of mitochondria is vital for the assembly of the hundreds of nuclear-derived proteins into an expanding organelle reticulum. Most of our knowledge of this complex multisubunit network comes from studies of yeast and fungal systems, with little information known about the protein import process in mammalian cells, particularly skeletal muscle. However, growing evidence indicates that the protein import machinery can respond to changes in the energy status of the cell. In particular, contractile activity, a powerful inducer of mitochondrial biogenesis, has been shown to alter the stoichiometry of the protein import apparatus via changes in several protein import machinery components. These adaptations include the induction of cytosolic molecular chaperones that transport precursors to the matrix, the up-regulation of outer membrane import receptors, and the increase in matrix chaperonins that facilitate the import and proper folding of the protein for subsequent compartmentation in the matrix or inner membrane. The physiological importance of these changes is an increased capacity for import into the organelle at any given precursor concentration. Defects in the protein import machinery components have been associated with mitochondrial disorders. Thus, contractile activity may serve as a possible mechanism for up-regulation of mitochondrial protein import and compensation for mitochondrial phenotype alterations observed in diseased muscle.

The role of intramyocellular triacylglycerol (IMTG) as a substrate source during exercise has recently regained much attention as a result of the proposed functional relationship between IMTG accumulation and the development of insulin resistance. It has been speculated that elevated NEFA delivery and/or impaired fatty acid (FA) oxidation result in intramyocellular accumulation of triacylglycerol and FA metabolites, which are likely to induce defects in the insulin signalling cascade, causing insulin resistance. The progressive accumulation of IMTG in sedentary patients and patients who are obese and/or have type 2 diabetes should therefore form a major therapeutic target, and efforts should be made to develop interventions that prevent excess IMTG accretion by stimulating their rate of oxidation. Although regular exercise is likely to represent such an effective means, there is much controversy about the actual contribution of the IMTG pool as a substrate source during exercise. The apparent discrepancy in the published literature might be explained by differences in the applied research protocol and the selected subject population, but most of all by the techniques that have been employed to estimate IMTG use during exercise. Data obtained in trained-endurance athletes indicate that athletes can substantially reduce their IMTG pool following a single exercise session. With the growing awareness that skeletal muscle has a tremendous potential to oxidise IMTG during prolonged moderate-intensity exercise, more research is warranted to develop combined exercise, nutritional and/or pharmacological interventions that can stimulate IMTG oxidation in sedentary patients and patients who are obese and/or have type 2 diabetes.

Intramyocellular triacylglycerol (TG) is an important energy store, and the energy content of this depot is higher than the energy content of the muscle glycogen depot. It has recently been shown that the mobilization of fatty acids from this TG pool may be regulated by the neutral lipase hormone-sensitive lipase (HSL). This enzyme is known to be rate limiting for intracellular TG hydrolysis in adipose tissue. The presence of HSL has been demonstrated in all muscle fibre types by Western blotting of muscle fibres isolated by collagenase treatment or after freeze-drying. The content of HSL varies between fibre types, being higher in oxidative fibres than in glycolytic fibres. When analysed under conditions optimal for“ HSL, neutral lipase activity in muscle can be stimulated by adrenaline as well as by contractions. These increases are abolished by the presence of anti-HSL antibody during analysis. Moreover, immunoprecipitation with affinity-purified anti-HSL antibody causes similar reductions in muscle HSL protein concentration and in measured neutral lipase responses to contractions. The immunoreactive HSL in muscle is stimulated by adrenaline via β-adrenergic activation of cAMP-dependent protein kinase (PKA). From findings in adipocytes it is likely that PKA phosphorylates HSL at residues Ser563, Ser659and Ser660. Contraction probably also enhances muscle HSL activity by phosphorylation, because the contraction-induced increase in HSL activity is elevated by the protein phosphatase inhibitor okadaic acid and reversed by alkaline phosphatase. A novel signalling pathway in muscle by which HSL activity may be stimulated by protein kinase C (PKC) via extracellular signal-regulated kinase (ERK) has been demonstrated. In contrast to previous findings in adipocytes, in muscle the activation of ERK is not necessary for stimulation of HSL by adrenaline. However, contraction-induced HSL activation is mediated by PKC, at least partly via the ERK pathway. In fat cells ERK is known to phosphorylate HSL at Ser600. Hence, phosphorylation of different sites may explain the finding that in muscle the effects of contractions and adrenaline on HSL activity are partially additive. In line with the view that the two stimuli act by different mechanisms, training increases contraction-mediated HSL activation but diminishes adrenaline-mediated HSL activation in muscle. In conclusion, HSL is present in skeletal muscle and can be activated by phosphorylation in response to both adrenaline and muscle contractions. Training increases contraction-mediated HSL activation, but decreases adrenaline-mediated HSL activation in muscle.