Skeletal muscle cells contain two major energy stores, glycogen and triacylglycerol. It is well established that the intramuscular glycogen serves as a fuel source during exercise. Glycogen utilization increases with intensity of exercise and decreases over time during prolonged exercise, whereas plasma glucose uptake and subsequent oxidation increase with both intensity and duration of exercise. Similar to the breakdown of liver glycogen, the breakdown of muscle glycogen is regulated by the enzyme glycogen phosphorylase. The energy content of the intramuscular triacylglycerol store is two-times that of the glycogen store (460 kJ·kg−1 wet weight vs 230 kJ·kg−1 wet weight). Although the exact contribution to total fat oxidation is still being debated, it is generally accepted that intramuscular triacylglycerol may represent a significant energy source during aerobic exercise. Triacylglycerol oxidation is likely to reach maximal rates during moderate exercise intensity. It may wane with duration of prolonged exercise, whereas combustion of plasma free fatty acids gradually increases. Just like intramuscular glycogen, intramuscular triacylglycerol may be mobilized both in contracting muscle in vitro, indicating regulation by intramuscular mechanisms, and by catecholamines in vivo. The latter finding is in line with the view that the intramuscular triacylglycerol store, in addition to being a local energy source, in parallel with the various adipose tissue triacylglycerol stores may provide fuel for the rest of the body, for instance, during hypoglycemia. Apart from its physiological functions, intramuscular triacylglycerol has been found to directly correlate with insulin resistance, and it may, accordingly, be of pathophysiological importance in diseases within the metabolic syndrome, for instance, type 2 diabetes.
Despite the potential physiological and pathophysiological importance of the intramuscular triacylglycerol depot, the enzymatic regulation of this depot has until recently been poorly understood. Our hypothesis has been that the enzyme hormone-sensitive lipase (HSL), which is known to be the rate limiting enzyme for hydrolysis of triacylglycerol in adipocytes, also regulates the intramyocellular hydrolysis of triacylglycerol to diacylglycerol and further on to monoacylglycerol. This opens the interesting possibility that in analogy to the fact that glycogenolysis in both liver and muscle is regulated by glycogen phosphorylase also lipolysis in extramyocellular and intramyocellular stores is controlled by the same enzyme. Hepatic glycogen phosphorylase and HSL in adipose tissue are stimulated by similar autonomic neurohormonal changes. The features of intramyocellular triacylglycerol metabolism described are compatible with the view that in muscle glycogen, phosphorylase and HSL would also be regulated along the same lines, being under dual control by hormones and contraction-associated intramyocellular factors. So, regarding exercise, the fascinating perspective is that the primary enzymatic setting allows simultaneous mobilization of all major extramuscular and intramuscular energy stores.
DIFFICULTIES IN EVALUATION OF INTRAMYOCELLULAR HSL ACTIVITY
HSL in Adipocytes
HSL protein and mRNA have in fact been found in muscle tissue nearly 20 yrs ago. However, measurements were performed on nondissected muscle, which means that the detected HSL may have originated from adipocytes interlaced between muscle fibers. Colocalization of myocytes and adipocytes represents a general problem in studies of fat metabolism in muscle and may explain that many studies using chemical methods to assess intramyocellular triacylglycerol content have not been able to demonstrate triacylglycerol depletion in skeletal muscle in response to exercise. Illustrating the problem, the triacylglycerol content of one single adipocyte (0.3 nmol) corresponds to approximately 5% of the triacylglycerol content of a 2-mg soleus muscle sample, but the adipocyte volume (250 pl) make up only 0.01% of the sample. It is clear that variation between muscle samples in number of included adipocytes may mask any differences in intramyocellular triacylglycerol content between samples. Accordingly, complete separation of myocytes from adipocytes is critical, but may unfortunately be unrealistic for most purposes.
Neutral Lipases Other Than HSL In Muscle
HSL is a neutral lipase with a pH optimum of 7. Its activity is mostly determined using either radiolabeled triacylglycerol (HSL (TG) activity) or radiolabeled diacyglycerol (HSL (DG) activity) as substrate. HSL (DG) activity is 10-fold higher than HSL (TG) activity, but in adipocytes only the latter is increased by catecholamine stimulation. This is the reason why labeled triacylglycerol is the preferred substrate. In rat muscle, HSL activity measurements in the presence of anti-HSL antibodies as well as immunoprecipitation of HSL have indicated the existence also of neutral lipases other than HSL. Thus, HSL accounts for only 40–90% of overall neutral lipase activity, the percentage being higher for triacylglycerol than for diacylglycerol lipase activity (5,6). Correspondingly, neutral lipase activity is still present in HSL knockout mice, and “new” triacylglycerol lipases are still being discovered. This means that in all conditions, which have not been studied before in muscle, changes in neutral lipase activity cannot be said to represent changes in HSL activity until this has been confirmed by use of anti-HSL antibodies.
HSL Activity Is Measured in Vitro
The activity of HSL is measured during standardized in vitro conditions in the presence of nonsaturating substrate concentrations. The preparation of substrate emulsion is critical and, furthermore, because of unpredictable contribution of endogenous triacylglycerol the final concentration may vary between samples. These facts may disturb the accuracy of the assay. In addition, when applied on tissue samples, the assay is for unknown reasons not linear, a fact that we find particularly pronounced for human muscle. On maximum catecholamine stimulation of adipocytes, an increase in HSL activity of only 50–100% can be measured, although lipolysis, for instance, determined from glycerol release, increases 10 to 20 times. This discrepancy has been ascribed to the fact that in adipocytes chemical activation of HSL is accompanied by translocation of HSL from the cytosol to triacylglycerol droplets as well as by phosphorylation of proteins coating the lipid droplets and possibly representing an adjustable barrier to HSL action in vivo. It follows that in vivo stimulation of HSL is accompanied by an increased access to substrate, which cannot be imitated during in vitro HSL activity measurements. Another fact that weakens the evaluation of in vivo enzyme activities from in vitro measurements is that changes in the latter will only result from covalent enzyme modifications. The activity of purified HSL can be modified allosterically by various metabolites, but changes in allosteric HSL modification in vivo will not be reflected in HSL activities measured during standardized in vitro conditions.
HSL IS PRESENT IN SKELETAL MUSCLE AND CAN BE STIMULATED BY EPINEPHRINE AND CONTRACTIONS
Studies In Rat Muscle
To minimize interpretational problems, we have used young (4-wk-old) rats in which adipocytes interlaced between muscle fibers are rare (6). Using affinity-purified anti-HSL antibody and Western blotting, we demonstrated the presence of HSL in muscle (6). HSL was found in nondissected muscle, in muscle fibers isolated after freeze-drying, and in fibers washed three times after collagenase isolation. Concentrations of immunoreactive HSL differed between muscles and varied directly with neutral lipase activity determined during conditions optimal for HSL. Furthermore, HSL concentrations were directly related to the content of type 1 or 2a fibers in muscle and, accordingly, correlated directly with intramyocellular triacylglycerol concentration and oxidative capacity (Fig. 1). These findings are in accordance with the belief that in muscle, HSL regulates mobilization of the triacylglycerol stores and serve to provide fatty acids for oxidation. Illustrating the size of the enzymatic lipolytic capacity relative to lipid availability, in soleus muscle the ratio between basal HSL activity and triacylglycerol content was 10-times that of epididymal adipose tissue (6).
Epinephrine is a key hormone in exercise and influences most organ systems. Studies in incubated soleus muscle have shown that epinephrine also induces a rapid and prolonged activation of neutral lipase activity in muscle (Fig. 2) (6). The response is abolished if antibody against HSL is present during measurements, a fact showing that the lipase activated by epinephrine is HSL. Experiments using β-adrenergic receptor blockade or incubation of muscle supernatant with the catalytic subunit of cAMP-dependent protein kinase A (PKA) have indicated that epinephrine activates HSL in rat muscle by stimulation of β-adrenergic receptors and PKA. From findings in adipocytes, it is likely that PKA phosphorylates HSL at residues Ser (563), Ser (659), and Ser (660) (3).
Electrically induced contractions, too, cause an increase in neutral lipase activity in incubated soleus muscle (Fig. 2). The increase is rapid but transient. Thus, the time course of activation differs from that seen during stimulation with epinephrine. Also, the increase in neutral lipase activity elicited by electrical stimulation of muscle cannot be blocked by β-adrenergic receptor blockade or sympathectomy showing that it does not result from electrical stimulation of intramuscular sympathetic nerve fibers to myocytes or adipocytes (5). It reflects an increase in HSL activity because it is not seen when measurements are performed in the presence of anti-HSL antibody (5). Furthermore, immunoprecipitation of HSL causes an 80% reduction in HSL protein in muscle lysate, and this reduction is accompanied by a similar reduction in the contraction-induced increase in neutral lipase activity measured in immunoprecipitated lysates (5). The maximum increases in HSL activity in incubated muscle in response to either epinephrine or contractions are similar. They make up 50–100% of overall basal neutral lipase activity, and because basal HSL activity accounts for only approximately 50% of overall neutral lipase activity, the increases make up approximately 100–200% of basal HSL activity (5,6).
Whether elicited by contractions or epinephrine, the time courses of increases in activities of HSL and glycogen phosphorylase, respectively, in vitro in muscle are parallel (Fig. 2). The rapid measurable increase in glycogen phosphorylase activity on contraction is mediated by a feed-forward or early warning Ca2+-mediated kinase activation (4). This is only transient and is thought to be followed by an allosteric feedback phosphorylase activation mediated by, for instance, ADP and accounting for continued glycogenolysis. Ca2+-mediated kinase activation may also elicit HSL activation. Such a role of Ca2+ has not been directly demonstrated. However, in agreement with involvement of kinase activation, the contraction-induced increase in HSL activity is enhanced by protein phosphatase inhibition and rapidly reversed by addition of alkaline phosphatase to lysate from contracted muscle (5). Furthermore, we have found an increase in neutral lipase activity in muscle incubated with caffeine in a concentration known to increase intracellular Ca2+ without causing contraction (1). In contrast, a decrease in HSL activity during incubation of muscle with the same caffeine concentration or with a sarcoplasmic reticulum Ca2+-ATPase inhibitor has also been reported (14). In the latter study, the decrease in HSL activity seemingly was consistent with an accompanying decrease in intramuscular triacylglycerol hydrolysis. However, triacylglycerol hydrolysis was determined from reduction in radioactivity in triacylglycerol prelabeled with radioactive palmitate. Even accepting the unlikely assumption that radiolabeled triacylglycerol solely existed in muscle (and not in, for instance, interspersed adipocytes), it is surprising that this technique was able to detect a fractional triacylglycerol breakdown of only approximately 2 ‰ in control conditions and a change in breakdown of the same magnitude during pharmacological intervention (14).
In accordance with the view that Ca2+ does play a role, PKC inhibitors, which block the Ca2+-activated isoform of the enzyme, have been shown to abolish the contraction-mediated HSL activation in muscle (Fig. 3) (1). Even though use of inhibitors is always associated with uncertainty, apparently contractions activate HSL in muscle by PKC. The effect of PKC seems to be at least partly mediated via extracellular signal-regulated kinase (ERK) (1). Thus, in resting muscle, phorbolester-induced PKC activation is accompanied by ERK phosphorylation and an increase in HSL activity. Furthermore, complete blockade of ERK activation in contracting muscle reduces the increase in HSL activity by 50% (Fig. 3). Finally, activated ERK mimics the effect of contractions on HSL when added to lysate from nonstimulated muscle but has no effect when added to lysate from electrically stimulated muscle. Interestingly, it has been shown that in adipocytes ERK stimulates HSL through phosphorylation of a site, Ser (600), different from but in close proximity to the sites phosphorylated by PKA within the regulatory module. No direct evidence exists about the effect of Ca2+ on HSL activity in adipose tissue. The only study approaching this issue reported that Ca2+/calmodulin-dependent kinase II in vitro phosphorylates HSL at a site that may inhibit subsequent phosphorylation by catecholamines of a neighboring site, which, however, is not essential for PKA-induced lipolysis.
Feedback stimulation of substrate supply in response to lowered intracellular energy state may be mediated by the enzyme 5′AMP-activated protein kinase (AMPK). This enzyme is covalently and allosterically activated by diminished ATP and increased ADP and AMP concentrations and, in turn, enhances fatty acid entrance into mitochondria as well as glucose transport over the muscle cell membrane. In vitro AMPK phosphorylates HSL purified from adipose tissue at Ser (565). Furthermore, in incubated contracting muscle an increase in AMPK activity is accompanied by phosphorylation of HSL at Ser (565) and an increase in HSL activity (2). However, phosphorylation of Ser (565) in HSL is not necessarily caused by AMPK and, furthermore, cannot be a major determinant for the contraction-induced increase in chemically measured HSL activity in muscle. The latter view is based on the finding that the contraction-induced increase in HSL activity can be abolished by pharmacological inhibition of PKC, whereas HSL–Ser (565) is not reduced (Figs. 3 and 4) (2). Rather than enhancing HSL activity, AMPK has also been claimed to attenuate muscle HSL activity during exercise (9,10,13). However, only the indirect evidence that 5-aminoimidazole-4-carboxamide-1-β-D-riboside, which among other effects also causes stimulation of AMPK in some muscle types, inhibits HSL activity in resting incubated rat muscle (3), and abolishes the epinephrine-induced increase in neutral lipase activity in cultured L6 myoblasts (13) exists in favor of this view. The effect of AMPK on HSL in adipose tissue is not clear (2).
Palmitoyl-CoA has been shown to slightly inhibit neutral lipase activity in rat muscle homogenate (11) and, accordingly, may exert allosteric feedback inhibition on HSL in vivo. However, so far no substance has been shown to allosteric stimulate HSL activity. So, epinephrine-induced PKA activation and contraction-induced PKC and ERK activation are the only known mechanisms, which may be involved in stimulation of HSL during exercise. In both slow-twitch oxidative and fast-twitch glycolytic muscle the effects of epinephrine and contractions, respectively, on HSL activity are partially additive (Fig. 5) (4). This probably reflects that the two stimuli phosphorylate different sites in HSL, but this has not been directly demonstrated in muscle and neither has any interaction on the activity of the enzyme between such sites.
Endurance training studies performed in rats have shown that training does not change total HSL activity and protein in either oxidative or in glycolytic muscle fibers (3). However, in response to epinephrine, a diminished increase in HSL activity is seen after training in both fiber types (3). This is in accordance with the finding that in muscle, epinephrine-induced lipolysis is reduced. It is also meaningful in the sense that mobilization of muscle triacylglycerol in response to other stresses than exercise will be lessened, thus saving muscle triacylglycerol for use during exercise. However, a reduced response to epinephrine seems at variance with the widely accepted view that muscle triacylglycerol breakdown during exercise is enhanced by training. The discrepancy is probably explained by the finding that the training-induced adaptations in activation of HSL by epinephrine and contractions, respectively, are oppositely directed, that is, the activation by contractions being enhanced (3). Thus, the findings in trained rats support the view that clearly separate mechanisms account for activation of HSL by epinephrine and contractions, respectively, in muscle (4). Interestingly, also the adaptation of the response of HSL to epinephrine in adipose tissue differs from that in muscle. Thus, training increases the sensitivity to stimulation by epinephrine of HSL as well as the overall amount of the enzyme in various adipose tissues (3). This agrees with the facts that during training the need for substrate delivery is increased in muscle, and that epinephrine-induced lipolysis in adipose tissue is also increased by training.
Studies in Human Muscle
Overall, findings on muscle HSL activity from in vivo human studies are quite consistent with findings from studies on isolated rat muscle. However, available studies on HSL activity in human muscle often mutually disagree and include surprising findings. This may reflect the difficulties in evaluation of intramyocellular HSL and triacylglycerol described. Only in two very recent articles anti-HSL antibody has been used to verify that an increase in muscle neutral lipase activity with exercise reflected an increase in HSL activity (8,13). Other studies on human muscle only report total neutral lipase activities. Exercise intensity was similar in the two mentioned studies, 65 and 70% O2peak, respectively (8,13). In one of the studies, vastus lateralis muscle HSL activity was increased 117% after 30 min of cycling, with the increase tending to be higher when the glycogen content was reduced before exercise compared to control conditions (8). After 60 min of cycling, HSL activity had returned to resting levels (8). In contrast, in the other study, HSL activity in vastus lateralis muscle was increased after 60 min of cycling in control experiments, but not when muscle glycogen had been reduced before exercise (13).
Compatible with a role of Ca2+ in HSL activation, it has been shown that neutral lipase activity in muscle is increased after just 1 min of cycling and that the increase is higher at 90% O2peak than at 30 and 60% (10). At the lower intensities, the increases in lipase activity are maintained (10); however, somewhat surprisingly it was found that at 10 min of cycling the increase is reduced, albeit not abolished, at 90% O2peak despite further increases in epinephrine and decreases in plasma insulin levels. Like neutral lipase activity, ERK1/2 phosphorylation in muscle is increased early in exercise, and it is also increased by epinephrine infusion (15). However, during various conditions, HSL and ERK activities, respectively, are not closely correlated during exercise (8,15). A study of adrenalectomized cortisol substituted patients is in line with dual control of muscle HSL by a transient contraction effect and a prolonged epinephrine effect. Thus, no change in muscle neutral lipase activity was found after 45 and 60 min of moderate cycling in saline-infused patients, whereas increases were seen in control subjects as well as in epinephrine-infused patients.
The two effects are partially additive during short-term submaximal exercise (15). However, this is only true when supraphysiological epinephrine is added after onset of exercise, but, in contrast to findings in incubated muscle, not when exercise is begun during epinephrine infusion. The authors proposed that phosphorylation of Ser (563) by PKA may prevent subsequent phosphorylation of HSL by contraction at other sites. This explanation does not agree with the fact that no phosphorylation of muscle HSL at Ser (563) has so far been demonstrated in exercising humans (8).
A transient increase in muscle neutral lipase activity has been reported in the face of a progressive increase in plasma epinephrine and ongoing decrease in insulin levels (9). During cycling at 60% O2peak, neutral lipase activity peaked at 60 min and had returned to basal levels at 120 min. Considering the hormonal changes, this time course is surprising. It was tentatively ascribed to inhibition of HSL activity by AMPK, although estimated muscle free AMP concentrations increased gradually from the start of exercise. The authors found support for their view in a study in which low preexercise muscle glycogen was accompanied by an increase in AMPK α-2 activity but no change in muscle neutral lipase activity during 60 min of cycling, whereas in experiments with normal preexercise glycogen neutral lipase activity increased in the face of unchanged AMPK α-2 activity and smaller changes in plasma epinephrine and insulin levels (13). However, when the earlier protocol was used again an increase in muscle neutral lipase activity was observed also after 120 min of cycling at 60% O2peak (11). Furthermore, in contrast to the mentioned study, when other investigators studied cycling at 65% O2peak, they found that low preexercise glycogen enhanced the increase in muscle AMPK α-2 activity but did not inhibit the increase in HSL activity compared to findings in experiments with high preexercise muscle glycogen (8). In line with findings in isolated rat muscle, phosphorylation of HSL at the AMPK site Ser (565) did not parallel HSL activity.
Compatible with inhibitory and enhancing effects, respectively, of insulin and epinephrine on muscle HSL, ingestion of a glucose solution during prolonged exercise has been shown to increase insulin levels and diminish epinephrine concentrations, whereas the increase in neutral lipase activity seen at 120 min in control experiments is abolished (12). However, in experiments in which adipose tissue lipolysis was blocked by nicotinic acid and plasma free fatty acid levels in turn were reduced, the epinephrine response to prolonged exercise was increased; nevertheless, muscle HSL activity was not enhanced compared to control experiments (7,11). Despite identical muscle neutral lipase activity, muscle triacylglycerol depletion seemed to be higher in nicotinic acid compared with control experiments. In general, covariation between muscle neutral lipase activity and triacylglycerol breakdown has not been found. This may reflect the aforementioned methodological problems and the fact that triacylglycerol breakdown is accompanied by reesterification of free fatty acids. It should be remembered, too, that glycogen phosphorylase A activity is also not closely associated with muscle glycogen breakdown. In addition to covalent modification, the activity of both enzymes is probably influenced by substrate availability and feedback, as well as allosteric modification.
CONCLUSIONS AND RESEARCH PERSPECTIVES
Findings in humans and rats point at HSL as the enzyme-regulating triacylglycerol breakdown in exercising muscle. Between muscle fiber types HSL concentrations vary directly with triacylglycerol content and inversely with glycogen phosphorylase concentrations. During exercise, the activity of both enzymes in muscle may be enhanced by transient Ca2+-mediated mechanisms, although such a role of Ca2+ on HSL has not been directly demonstrated, as well as by epinephrine (Fig. 6). In addition to covalent enzyme modification, other factors determine the net breakdown of muscle triacylglycerol and glycogen during exercise. However, in response to exercise, the primary enzymatic setting involves feed-forward mechanisms and allows simultaneous mobilization of all major extramuscular and intramuscular energy stores.
Studies on the phosphorylation of HSL in exercise have only recently appeared, and it remains to be clarified whether contractions per se cause phosphorylation of a yet unrecognized site enhancing HSL activity, and, if so, how this interacts with other phosphorylation sites. It is also important to clarify whether translocation of HSL to lipid droplets and modification of proteins coating these droplets operate in muscle, as is the case in adipocytes. Allosteric influence on activated muscle HSL has not been studied and a search for enhancing factors is warranted. Finally, studies using HSL knockout mice and specific HSL blockers are now in progress to definitely demonstrate the importance of HSL in muscle triacylglycerol metabolism.
The authors received financial support from the NOVO NORDIC Foundation, the Danish Diabetes Association, the Lundbeck Foundation, the Danish Medical Research Council, the Swedish Research Council (grant 112 84), and the Swedish Diabetes Association.
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