Journal of Applied Physiology AJP: Endocrinology and Metabolism
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J Appl Physiol 93: 1185-1195, 2002; doi:10.1152/japplphysiol.00197.2002
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Vol. 93, Issue 4, 1185-1195, October 2002

INVITED REVIEW
Intramuscular triacylglycerol utilization in human skeletal muscle during exercise: is there a controversy?

Matthew J. Watt1, George J. F. Heigenhauser2, and Lawrence L. Spriet1

1 Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario N1G 2W1; and 2 Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
TRIACYLGLYCEROL STORAGE IN...
EVIDENCE OF ENZYMATIC...
IMTG UTILIZATION DURING...
CONCLUSIONS
REFERENCES

Intramuscular triacylglyerols (IMTGs) represent a potentially important energy source for contracting human skeletal muscle. Although the majority of evidence from isotope tracer and 1H-magnetic resonance spectroscopy (MRS) studies demonstrate IMTG utilization during exercise, controversy regarding the importance of IMTG as a metabolic substrate persists. The controversy stems from studies that measure IMTG in skeletal muscle biopsy samples and report no significant net IMTG degradation during prolonged moderate-intensity (55-70% maximal O2 consumption) exercise lasting 90-120 min. Although postexercise decrements in IMTG levels are often reported from direct muscle measurements, the marked between-biopsy variability (~23%) that has been reported with this technique in untrained subjects is larger than the expected decrease in IMTG content, effectively precluding significant findings. In contrast, recent data obtained in endurance-trained subjects demonstrated reduced variability between duplicate biopsies (~12%), and significant changes in IMTG were detected after 120 min of moderate-intensity exercise. Therefore, it is our contention that the muscle biopsy, isotope tracer, and 1H-MRS techniques report significant and energetically important oxidation of free fatty acids derived from IMTGs during prolonged moderate exercise.

muscle biopsy; isotope tracer; hydrogen-1-magnetic resonance spectroscopy; fat metabolism


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
TRIACYLGLYCEROL STORAGE IN...
EVIDENCE OF ENZYMATIC...
IMTG UTILIZATION DURING...
CONCLUSIONS
REFERENCES

THE CONTRIBUTION OF CARBOHYDRATE and lipid as substrates for energy production in contracting human skeletal muscle has been well defined over the last century (for historical review, see Ref. 1). With the development of precise gas analytic techniques in the late 1800s, the early studies of exercise physiology based their findings on measures of the respiratory quotient (RQ) and focused on whether carbohydrate alone, or a combination of lipid and carbohydrate, was utilized during exercise. Although some maintained that carbohydrate was the sole substrate for energy provision during muscular work, Zuntz and his co-workers found that the RQ was close to resting values during long-duration exercise, thus suggesting a marked contribution from lipid stores (1). This controversy was reconciled by the classic experiments of Krogh and Lindhard (1920) and later Christensen et al. (1939) in Denmark and of Benedict and Cathcart (1913) and Dill and others at the Harvard Fatigue Laboratory (1930) in the United States (1). In a carefully conducted series of experiments, these studies demonstrated a number of fundamental metabolic relationships that still hold true today. They showed that both carbohydrate and lipid are used as fuel for muscular work and that the oxidation of lipid provides less ATP per liter oxygen consumed than carbohydrate. The role of exercise intensity and duration in fuel metabolism was also well defined. During prolonged exercise, lipid oxidation became more important as carbohydrate stores were depleted, whereas carbohydrate metabolism became quantitatively more important with increasing exercise intensity (1).

Although the measurement of RQ provides an estimate of total carbohydrate and lipid oxidation, it does not allow for the determination of the site of fuel utilization. During exercise, carbohydrate and lipid are derived from both extra- and intramuscular locations. Carbohydrate is stored as glycogen in skeletal muscle and liver; the latter is hydrolyzed to glucose and transported to contracting muscle via the circulatory system. Lipids in the form of free fatty acids (FFAs) are derived from the lipolysis of peripheral adipose tissue and possibly from adipose located between skeletal muscle fibers. FFAs can also be hydrolyzed from triacylglycerols bound to circulating lipoproteins (very low-density lipoproteins and chylomicrons) and intramuscular triacylglycerols (IMTGs). New techniques were applied in the period immediately after World War II that enabled investigators to localize and quantify endogenous fuel stores and to determine to what extent these local and exogenous fuels were utilized during exercise. The metabolic fate of blood-borne substrates (glucose, FFAs) were elucidated through the use of labeled isotopes (e.g., 14C) and arterial and venous catheterization techniques, whereas the reintroduction of the muscle biopsy technique in the 1960s (Bergström and Hultman, 1963) allowed for the quantification of endogenous substrates (glycogen, triacylglycerol) and their degradation during exercise (1). In contrast to the extensive research that has led to a comprehensive understanding of the provision of carbohydrate fuel during exercise, few studies have attempted to elucidate and quantitate the various sources of lipid that are metabolized during exercise. To date, the contribution of the various lipid stores to oxidative metabolism during exercise is not completely understood.

Clearly, the mobilization of FFAs from adipose tissue and their eventual uptake and oxidation contribute significantly to oxidative energy production (11, 46, 58, 71). Studies that used [1-14C]palimtate as a tracer and arteriovenous difference measures of FFAs confirmed that approximately one-half of the lipid utilized during moderate-intensity exercise was derived from plasma FFAs, with the remaining lipid (~50%) thought to be derived from "local stores" (37, 38). Although the precise quantification of circulating lipoproteins is difficult to ascertain, evidence from human tracer studies indicates that their maximal contribution to oxidative metabolism is <10% of total lipid metabolism under normal dietary conditions (38, 40). On the basis of these findings, some investigators have suggested that IMTGs supply the "balance" of lipid toward oxidative metabolism and, as such, are an important energy source for contracting skeletal muscle (38). Indeed, morphometric analysis demonstrated an exercise-induced reduction in the size of fat vacuoles in electron micrographs (31), and quantitative ultrastructural analysis revealed reduced IMTG levels immediately after a marathon (76).

Despite these findings, controversy exists (43, 79) as to whether IMTG is oxidized during exercise and, if so, the extent of the contribution. This is based on the observation that IMTG content was not significantly reduced in muscle biopsy samples after prolonged exercise (4, 35, 48, 50, 70, 76, 78, 84). Is it possible that IMTGs do not serve as a source of fatty acids for oxidation during exercise in humans or that are these findings merely a consequence of methodological and experimental limitations? The purpose of this review is to examine the studies that have measured, or estimated, IMTG utilization during exercise in healthy humans. Herein we discuss the potential limitations that underlie the methods commonly used to determine IMTG utilization in an attempt to reconcile the divergent findings observed in the literature. Although we are aware of the literature relating to IMTG accumulation in obseity and its association with insulin resistance (2, 6, 21, 32-34, 39, 63, 65, 82), this review does not discuss IMTG storage and IMTG utilization during exercise in obese and/or patient populations.


    TRIACYLGLYCEROL STORAGE IN HUMAN SKELETAL MUSCLE
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ABSTRACT
INTRODUCTION
TRIACYLGLYCEROL STORAGE IN...
EVIDENCE OF ENZYMATIC...
IMTG UTILIZATION DURING...
CONCLUSIONS
REFERENCES

Although adipose tissue contains the greatest lipid reserve in the body, IMTG and lipid stores located between myofibrils potentially provide an important substrate supply for contracting muscle. Triacylglycerol consists of a glycerol backbone condensed with three fatty acyl-CoA molecules. During exercise the complete breakdown of IMTG is thought to be completed by two lipolytic enzymes. The triacylglycerol lipase, hormone-sensitive lipase (HSL), is a neutral lipase and cleaves the first and second fatty acid from the glycerol backbone. HSL is considered to be the rate-limiting enzyme for IMTG hydrolysis because it is believed to be regulated by intra- (contraction) and extramuscular (epinephrine) factors (53, 54). Additionally, the activities of HSL toward diacylglycerol, and monoacylglycerol lipase for monoacylglycerol, far exceed the hydrolytic activity of HSL for triacylglycerol (28, 54, 80).

All fatty acids released from IMTG into the cytoplasm must be chaperoned by fatty acid-binding proteins, and if destined for oxidation they are transported to the surface of the outer mitochondria membrane. The fatty acids are then activated via binding with CoA, converted to a fatty acyl carnitine compound, and moved across the mitochondrial membranes while bound to carnitine. Inside the mitochondria, the carnitine is removed, the CoA is rebound, and the fatty acyl-CoA molecules are metabolized in the beta -oxidation pathway with the production of reducing equivalents (NADH, FADH2) and acetyl-CoA. The acetyl-CoA is further metabolized in the tricarboxylic pathway with the production of additional reducing equivalents. The electron transport chain, including O2, accepts the reducing equivalents to generate the proton motive force, which provides the chemical energy used to synthesize ATP from Pi and ADP in the process of oxidative phosphorylation.

IMTG are stored as lipid droplets within the cytoplasm of skeletal muscle cells in close proximity to the mitochondria (8), where the free fatty acids released via hydrolysis are ultimately metabolized. The amount of IMTG found within individual fibers, between different fiber types, and between muscle groups is variable. Studies in which individual muscle fibers were dissected out after staining for myofibrillar ATPase revealed that IMTG was positively related to the oxidative capacity of the muscle fiber such that pooled type I muscle fibers contained greater IMTG compared with pooled type II fibers (23). Similar patterns of storage have been observed by quantifying the volume density of lipid droplets by morphometric analysis (44) and with staining for neutral fat by oil red O (23). Studies using 1H-magnetic resonance spectroscopy (MRS) methods suggested that IMTG are highly variable between muscle groups (8, 68). These differences may be related to the function of the specific muscle and the associated fiber type composition, with the soleus (~70% type I) containing larger amounts of IMTG than the gastrocnemius (~35% type II) in humans. Furthermore, estimating IMTG content is also somewhat problematic because of the large variabiltiy of IMTG between individuals (8, 11, 23, 46, 59), with up to a sixfold variance in resting IMTG levels being reported within a homogenous population (24). Evidently, this intra- and interindividual variability places constraints on determining statistical significance when physiological trends may be clear.

With these considerations in mind, the reported preexercise IMTG value obtained from mixed human skeletal muscle samples ranges from 2 to 50 mmol/kg dry mass (dm), with an average of ~30 mmol/kg dm (Table 1) (4, 14, 24, 35, 45, 48, 50, 66, 76, 84). The energy available from the complete oxidation of Delta IMTG in active skeletal muscle during cycle exercise is approximately two-thirds that of glycogen (assuming a glycogen content of 500 mmol/kg dm). In view of the substantial contribution of lipid toward oxidative metabolism during prolonged moderate-intensity [50- 70% peak O2 consumption (VO2 peak)] exercise (31, 70), the potential importance of IMTG as a substrate for energy production is evident.

                              
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  		Table 1.   Pre- and postexercise IMTG content determined in human mixed skeletal muscle by a chemical extraction method


    EVIDENCE OF ENZYMATIC REGULATION OF THE LABILE IMTG POOL
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ABSTRACT
INTRODUCTION
TRIACYLGLYCEROL STORAGE IN...
EVIDENCE OF ENZYMATIC...
IMTG UTILIZATION DURING...
CONCLUSIONS
REFERENCES

Aside from the morphological evidence that shows IMTGs existing in close proximity to the mitochondria, there is considerable evidence to suggest that IMTGs exist as a rapidly turning over pool. Resting IMTG stores are heavily influenced by dietary composition, insomuch that acute (46, 76) and long-term (49) high-fat diets result in a greater capacity for triacylglycerol storage within muscle. Similar increases in IMTGs were observed during lipid and heparin infusion that raised plasma FFA from 0.5 to 1.8 mmol/l (6). The increased IMTGs by consumption of a high-fat diet enhanced triacylglycerol-derived fatty acid oxidation both at rest and during moderate exercise (73). Conversely, when the resting IMTG concentration was reduced by a short-term low-fat (2%) diet, nonplasma fatty acid oxidation was reduced (14). Interestingly, when plasma FFA mobilization and oxidation were reduced by the ingestion of a nicotinic acid analog, IMTG utilization was enhanced in endurance-trained men (16). IMTG stores and utilization during exercise are also influenced by training status. Although equivocal (4, 48, 78), it seems one of the adaptive changes of skeletal muscle to endurance training is increased IMTG storage (32, 44, 59), and this may translate to greater IMTG utilization during whole body exercise (45, 58, 67).

Although some studies suggest that IMTG is not a dynamic fuel store in humans (4, 35, 48, 50, 70, 76, 78, 84), recent studies have demonstrated the presence of a neutral lipase that is acutely regulated in skeletal muscle. HSL protein and mRNA were first identified in rat skeletal muscle by Western and Northern blotting techniques over a decade ago (41, 42), and more recently protein expression was shown to be greater in oxidative compared with glycolytic fibers (54, 64). Moreover, muscle HSL content correlates well with the known concentrations of IMTG storage and the oxidative capacity of skeletal muscle fiber types, thus implying the presence of lipolytic activity in skeletal muscle (23).

Recently, the assay for measuring HSL activity in adipose tissue was modified for skeletal muscle homogenates. Studies conducted in incubated rat soleus demonstrated increased HSL activity when epinephrine was added to the perfusion medium, and this occurred by beta -adrenergic mechanisms via a cAMP-dependent protein kinase pathway (54). Information obtained from humans studies supports such a mechanism. Forearm glycerol release was augmented during intra-arterial infusion of metaprotenolol (beta -agonist) in Type 1 diabetic subjects (85), whereas nonselective beta -adrenergic blockade completely blocked IMTG utilization during prolonged exercise to exhaustion (12). Perfusion of resting skeletal muscle with a beta 2-adrenoceptor agonist in situ increased glycerol levels as measured by use of microdialysis (36), and skeletal muscle HSL activity increased during exercise in epinephrine-deficient adrenalectomized patients with epinephrine infusion, whereas no such increase was observed without infusion (51).

There is also evidence to suggest HSL activity is increased by muscle contractions. Studies conducted in the electrically stimulated rat soleus reported a transient increase in HSL activity that was not affected by prior sympathectomy or the administration of propranolol, a beta -adrenergic receptor antagonist (53). Moreover, addition of a protein phosphatase inhibitor enhanced HSL activity during contraction, suggesting that HSL activation involves phosphorylation (53). HSL activity has recently been shown to increase twofold in humans during moderate-intensity exercise; however, activity did not increase during more intense exercise (51). Whether HSL activity was in fact regulated by contractions is uncertain because HSL activity did not increase in adrenalectomized patients without epinephrine infusion (51). It is not possible to ascertain from these data whether HSL activation occurred via sympathetic- or contraction-mediated mechanisms or via a combination of these factors. Taken together, these data demonstrate the presence of HSL in skeletal muscle and support a role for enzymatic mobilization of IMTG stores by sympathetic stimulation and muscle contraction.


    IMTG UTILIZATION DURING EXERCISE
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ABSTRACT
INTRODUCTION
TRIACYLGLYCEROL STORAGE IN...
EVIDENCE OF ENZYMATIC...
IMTG UTILIZATION DURING...
CONCLUSIONS
REFERENCES

Biopsy studies. In humans, one approach to measuring IMTG is from muscle samples obtained by the needle-biopsy technique and analyzed by chemical extraction. Generally, samples obtained pre- and postexercise are freeze-dried and dissected free of nonmuscle contaminants under magnification. Thereafter, the triacylglycerol is isolated by a chloroform-methanol extraction (27), the triacylglycerol is hydrolyzed, and the final concentration is related to the amount of released glycerol or FFA in solution. Despite the adherence to similar techniques, the data pertaining to IMTG utilization during exercise remain equivocal. Consequently, the importance of IMTG as a substrate during exercise has been questioned. Factors such as the mode and pattern (continuous vs. intermittent) of exercise, preexercise diet, gender, and training status are likely to influence IMTG oxidation; however, detailed discussion of these factors is beyond the scope of this review.

Evidence from studies conducted in the Scandinavian laboratories in the 1970s consistently reported higher resting IMTG concentrations and greater utilization during exercise than did contemporary (1986-2001) investigations. In perhaps the most cited investigation, Fröberg and Mossfeldt (30) studied triacylglycerol depletion in relatively large muscle aliquots (5-7 mg dry muscle, ~30-35 mg wet) and reported a 51% decrease in IMTG (preexercise vs. postexercise: 72.9 vs. 35.6 mmol/kg dm) after 7 h of strenuous skiing. Several other studies have demonstrated time-dependent reductions after moderate-to-heavy, whole body exercise. Those early studies in which exercise duration was <2 h reported an average IMTG reduction of 20% compared with resting values (11, 22, 46), whereas a 45% reduction was evident during more prolonged (>2 h) exercise (5, 13, 30) (Table 1). Additional evidence in favor of IMTG use during exercise was derived from histological studies that demonstrate a reduced size in fat vacuoles after exercise (for review see Ref. 31). When IMTG use was expressed per unit of time, the average rate of depletion in these early studies was ~9 mmol · kg dm-1 · h-1. Assuming that cycle exercise elicits an O2 consumption (VO2) of 2.5 l/min (12.2 kcal/min) and the average molecular mass of a triglyceride is 861 g/mol (26), the amount of energy derived from the complete oxidation of IMTG would provide ~25% of the total energy demand.

In contrast, contemporary research has not generally supported a role for IMTG as a significant substrate for the working muscle. Although there are reports of IMTG utilization during prolonged, moderate exercise in healthy men (12, 67), this has not been a consistent finding. Indeed, several studies have provided evidence that the contribution of IMTG breakdown toward oxidative metabolism is minimal (35, 48, 50, 70, 76, 78, 84), with one group reporting an apparent paradoxical increase (4). These studies have typically studied recreationally active men during 90-120 min of cycle exercise at a power output of 60-70% maximal O2 uptake (VO2 max). On average, IMTG was found to be reduced from resting values by a statistically insignificant 3.2 mmol/kg dm, which corresponded to an oxidation rate of 2.1 mmol · kg dm-1 · h-1. On the basis of the aforementioned example (e.g., cycle exercise at 2.5 l/min), only 5% of the total energy demand would be provided by IMTG oxidation. Thus, in contrast to early reports, there is substantial evidence suggesting that IMTG is of minor importance during contractile activity in humans.

Perhaps the most notable exception to this conclusion is the study conducted by Hurley et al. (45) that demonstrated substantial IMTG utilization during 120 min of moderate-intensity exercise in subjects who underwent 12 wk of endurance training (untrained vs. trained: 12.7 ± 5.5 vs. 26.1 ± 9.3 mmol/kg dm). The rate of IMTG utilization reported in the trained state was sixfold greater than in other studies that employed similar exercise protocols (12, 35, 50, 67, 76, 84). The decline in IMTG in the trained state would have accounted for ~45% of the total energy demand and nearly 80% of the total whole body fat utilization. Not surprisingly, this contribution to energy utilization is substantially higher than any other reported in the literature. To our knowledge, this is the only exercise study in which single muscle fibers were teased out and cleaned free of nonmuscle material by microdissection for the sole purpose of IMTG analysis.

A major criticism of the chemical approach for the determination of IMTG in biopsy samples is the inability to adequately differentiate between IMTG and extramyocellular lipid (EMCL) deposits (i.e., adipocytes between muscle fibers). Thus lipid stored between muscle fibers may be inadvertently sampled with muscle tissue. Evidence from rat soleus muscle suggests that the variable IMTG levels found in different samples obtained from a single muscle is due to the variability of the extramyocellular triacylglycerol (EMTG) content (19). Consistent with this assumption, EMCL measurements as measured by 1H-MRS were highly variable in humans (79). However, contrasting results have recently been reported by Levin et al. (56), who found no EMCL in biopsy samples from obese humans in a study in which >500 muscle samples were counted and analyzed by electron microscopy. Nevertheless, the distinct possibility exists that muscle samples analyzed without individual fibers first being dissected out contain considerable, and varied, undetected lipid depots. Indeed, although the within-biopsy variability of the IMTG measurements is small (6%), the IMTG coefficient of variation (CV) was 23.5% (n = 13) between three biopsies sampled from the same leg at the same time after thorough cleansing in untrained individuals (84). These data suggest a change of >24% (~7 mmol/kg dm) in IMTG content is required for results to be considered meaningful. On the basis of an IMTG breakdown of ~2 mmol · kg dm-1 · h-1, changes in IMTG content would not be detected in samples obtained after 2 h of moderate exercise, the approximate duration used in many contemporary studies. The reality may be that the use of conventional dissection techniques may not allow for the accurate determination of IMTG, and the partitioning of individual muscle fibers and thorough removal of EMCL under greater magnification may be essential for reliable estimates of IMTG content.

To minimize the variability caused by EMTG contamination, Wendling et al. (84) recommended the use of well-trained or recreationally active subjects because these individuals store less EMTG than untrained individuals (79). Our laboratory has recently examined IMTG variability between muscle samples in well-trained men during 4 h of moderate exercise and report a much lower CV of 12.3 ± 9.4% (mean ± SD) in 17 paired muscle samples (83). Furthermore, our laboratory observed an exercise-induced reduction in IMTG (2.8 mmol/kg dm) that was statistically significant but substantially less than that observed in untrained individuals (6 mmol/kg dm) (83). Taken together, these findings highlight the importance of utilizing well-trained individuals when IMTG utilization is examined.

Alternatively, the variability within some muscle samples could be real and not a function of EMTG contamination between fibers. In this regard, a recent study reported a CV of 4% for IMTG concentration when five samples were obtained from the one mixed, freeze-dried pool of fibers (78). In contrast, when a single wet biopsy was divided into five aliquots and freeze-dried and powdered separately before analysis, the CV was 31% (78). This would indicate that there could be high variability between small aliquots of muscle from the same biopsy that is not due to EMTG contamination.

It is also apparent that the divergent results obtained to date are a function of the variable storage of IMTGs between skeletal muscle fiber types. Unlike muscle glycogen, which has nearly equal amounts of glycogen stored in both type I and type II fibers (10-25% higher in type II fibers), triacylglycerol is stored in much greater quantities (2.8-fold) in type I compared with type II fibers (23). A recent study that assessed total neutral lipid content by oil red O staining indicates that these differences are due to the different IMTG content between type I and type IIb fibers (57). Because the pre- and postexercise muscle samples are unlikely to contain the same proportion of fiber types, these differences are likely to induce further variability in IMTG concentration. It is doubtful that this problem can be allayed without control of the composition of the muscle sample being assayed. However, because endurance-trained individuals contain few type IIb fibers, this problem would be evident only in an untrained population.

Finally, when net changes in IMTG are evaluated, it is important to consider the influence of exercise on the incorporation of FFAs into the endogenous lipid pools. The IMTG pool is subject to a continual turnover in resting and exercising skeletal muscle (86), and early studies in exercising men suggest the majority of the FFAs extracted by contracting skeletal muscle are oxidized and have a turnover time of several minutes (37). Whether the fatty acids entered the triacylglycerol pool or directly entered the mitochondria was not determined. Subsequent studies conducted in the contracting forearm have suggested that the IMTG pool is constantly turning over (17).

FFA turnover in the intramuscular compartment was recently assessed by using the dual-tracer, pulse-chase technique in human skeletal muscle (35). IMTG was prelabeled with [14C]palmitate before exercise, [3H]palmitate was infused throughout exercise, and muscle samples were obtained before and after 120 min of exercise. Briefly, IMTG oxidation was estimated by dividing 14CO2 production by the estimated IMTG [14C]palmitate specific activity, 3H2O production was related to FFA oxidation, and the increment in [3H]IMTG specific activity was considered to represent incorporation of FFA into the intramuscular lipid pool. The IMTG pool size measured from a biopsy sample did not change during exercise, and this was apparently a result of simultaneous hydrolysis and esterification of the IMTG. However, IMTG hydrolysis was at least 10-fold higher than esterification of FFA within muscle, and this should have resulted in a 32% reduction of preexercise IMTG. Despite this finding, IMTG as measured by the muscle biopsy technique increased nonsignificantly by 20%. As previously discussed, the sensitivity of the muscle IMTG measurement is not sufficient to detect small changes in the IMTG pool [preexercise vs. postexercise: 8.6 vs. 10.3 mmol/kg dm (35)].

A potential error in the estimation of the IMTG turnover rate is the assumption that IMTG is a homogenous pool turning over at a constant rate. Evidence from histochemical analysis contradicts this assumption (23). Moreover, recent studies of contracting rat skeletal muscle that used a pulse-chase technique report the existence of intramuscular lipid subpools that rapidly turn over (20). It is also apparant that incomplete labeling of the endogenous lipid pool before exercise may result in underestimation of IMTG hydrolysis. Clearly, further studies evaluating IMTG hydrolysis and simultaneous esterification of FFA into the triacylglycerol pool during exercise are warranted.

In summary, the majority of studies that have directly measured IMTG before and after prolonged exercise report small but statistically insignificant reductions in IMTG content. The marked between-biopsy variability (~23%) that has been reported with this technique in untrained subjects is larger than the expected decrease in IMTG content, which effectively precludes the detection of significant changes. In contrast, far less variability is observed between muscle samples of endurance-trained individuals, which enables detection of small changes in IMTG. Therefore, muscle biopsy and chemical extraction techniques may be adequate to detect changes in IMTG concentrations during exercise in endurance-trained individuals. However, an alternative technique is recommended in untrained populations. Although we are unaware of any study that has measured the between-biopsy variability in obese and/or Type 2 diabetic individuals, we predict from work conducted in untrained subjects that the variability would be high. Thus, until the between-biopsy variability is determined, we cannot recommend the use of the biopsy technique for quantification of IMTG during exercise in obese or Type 2 diabetic populations.

Isotope tracer methodology. An alternate approach for estimating IMTG utilization during exercise is by subtracting plasma-derived FFA oxidation obtained by the use of isotopic tracer methodology from total fat oxidation via indirect calorimetry measurements. There are three main methods for measuring the utilization of plasma-derived FFA. Typically, labeled palmitate (e.g., 13C) bound to albumin is infused into a peripheral vein and blood is sampled at rest and throughout exercise. The amount of plasma FFA taken up by the muscle [FFA rate of disappearance (Rd)] is later determined from the changes in plasma FFA and the labeled palmitate enrichment by the use of a one-pool model and non-steady-state equations. This method assumes that 100% of the FFA Rd is oxidized and represents the minimum IMTG contribution to total fat oxidation. However, direct measures suggest that not all of the plasma FFA Rd is actually oxidized in the fasted state (15, 81). An alternative and more accurate determination of FFA oxidation is obtained by measuring the FFA Rd and rate of appearance of labeled CO2 in the breath. It is well known that only a fraction of the labeled CO2 is recovered in the breath because the label can be temporarily fixed during isotopic exchange reactions in the tricarboxylic acid cycle before it is transferred to CO2. Thus subjects are also infused with labeled acetate (e.g., [1-14C]acetate) or bicarbonate, and the recovery of this label is used as a correction factor to improve tracer estimations of plasma FFA oxidation. Finally, other groups have employed direct arteriovenous balance measures across the working limb to calculate FFA turnover and oxidation. Pulmonary gas-exchange measures are concomitantly obtained and standard formulas for the respiratory exchange ratio (RER) or RQ are utilized to calculate total lipid oxidation. Nonplasma fatty acid oxidation is calculated as the difference between total fatty acid oxidation (measured by using indirect calorimetry) and FFA oxidation (or FFA Rd). Most investigators assume that the majority of the nonplasma fatty acid oxidation is derived from IMTG because the oxidation of FFA from circulating very low-density lipoproteins and triglycerides is believed to be negligable under normal dietary (~20% fat) conditions (38, 40).

In contrast to the variable findings from muscle biopsy studies, investigations employing tracer methodology have generally reported IMTG utilization during exercise (Table 2). Nonplasma fatty acid oxidation has been reported across a range (30-80% VO2 max) of exercise intensities in both untrained (29, 47, 58, 67, 73-75) and trained individuals (14, 16, 71, 75, 81) during cycle exercise lasting 30-120 min.

                              
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  		Table 2.   Estimated IMTG utilization during moderate-intensity cycle exercise as estimated by indirect calorimetry and isotope tracer or a-v balance techniques

In the aforementioned studies, nonplasma fatty acid utilization averaged ~20 and 5 µmol · kg body mass-1 · min-1 for trained and untrained individuals, respectively, which equates to 27 and 12% of total fat oxidation. However, when these data are expressed as total IMTG oxidation over a 120-min exercise bout, the average IMTG utilization (assuming body weight = 75 kg, exercising muscle mass = 10 kg) would be 4.2 mmol/kg dm. These calculations show that the "average" values obtained by using both indirect and direct measures of IMTG use are reasonably comparable.

There are numerous assumptions regarding the validity of isotopic tracer and indirect calorimetry measurements and these have been previously debated. One assumption is that all of the plasma FFA taken up by skeletal muscle is oxidized. Direct measures of FFA oxidation indicate that only 77-96% of plasma FFA Rd is oxidized during exercise in the fasted state (15, 69, 73, 80). If it is assumed that IMTG constitute the difference between total lipid and plasma FFA Rd, this overestimation of plasma FFA oxidation by using FFA Rd measures will result in an underestimation of IMTG utilization by 4-23%. Thus this method quantifies the minimum nonplasma fatty acid oxidation. Evidently, this error is avoided when 13CO2 is measured to calculate actual FFA oxidation.

The most critical component of estimating IMTG utilization appears to be the measurement of RER. Indirect calorimetry permits the determination of carbohydrate and lipid oxidation during steady-state exercise conditions. However, both physiological (hyperventilation, storage of CO2, lipogenesis, exhaustive exercise) and technical factors can result in erroneous determinations of the expired gas fractions and volumes and subsequently VO2 and CO2 production (VCO2). Small changes in either VO2 or VCO2 result in large differences in RER and therefore estimates of total fat and total carbohydrate oxidation. For example, a subject may be exercising at a power output where the measured VO2 is 2.5 l/min and the VCO2 is 2.08 l/min, resulting in a RER of 0.83 and the estimation that fat is providing 56.2% of the total oxidative energy. If the VCO2 has been underestimated by only 70 ml, such that the true value is 2.15 l/min, the RER increases to 0.86 and the fat contribution to energy is reduced to 45.9% of the total energy. If the estimate of total fat oxidation is reduced, so is the estimate of IMTG use. Similarly, an overestimation of VO2 by only 89 ml/min, resulting in a true VO2 of 2.41 l/min would also result in an increase of the RER to 0.86 and a lower estimation of reliance on fat and IMTG. If these two errors were combined, the RER would increase to 0.89 and the estimate of IMTG use fall further. Therefore, any underestimation of VCO2 and/or overestimation of VO2 will lead to erroneously high estimates of reliance on fat as a fuel, both at the whole body and muscle levels.

Interestingly, there appears to be a large variation in the exercise RERs reported for aerobically trained subjects at varying power outputs from different laboratories. For example, whereas Romijn et al. (71) reported an RER of 0.83 in their well-trained athletes during exercise at 65% VO2 max, many laboratories commonly report considerably higher RER values (0.87-0.91) in well-trained athletes during exercise at 60-65% VO2 max (3, 10, 25, 62, 70). Moreover, in untrained and recreationally active men, the RER during moderate-intensity exercise is further increased to ~0.89-0.95 (3, 29, 60, 61). It must be remembered that the Romijn et al. data that are often cited as representative of substrate use during exercise were derived from very well-trained subjects after an overnight fast that elevated the plasma FFA concentrations to ~0.8-1.0 mM and predisposed the muscle to the use of fat as a fuel.

In summary, the use of isotope tracer and indirect calorimetry measurements is an accurate and noninvasive method for the estimation of FFA oxidation and, by inference, for IMTG utilization during exercise. Despite there being small differences in the calculated rates of FFA oxidation when the FFA Rd and 13CO2 recovery methods are applied, both techniques consistently estimate significant IMTG utilization during moderate intensity exercise. Most importantly, the magnitude of the IMTG use is very dependent on RER estimates obtained at the mouth.

Proton MRS methodology. There has been a vast increase in the application of MRS in the study of human skeletal muscle metabolism. Recently, a 1H-MRS method has been applied for the noninvasive quantification of IMTG in human skeletal muscle. This technique measures the resonances from methylene and methyl protons of triacylglycerols, which appear as multiple peaks on the proton spectrum of skeletal muscle. Schick et al. (72) observed two compartments of triacylglycerols separated by 0.2 parts/million (ppm) when a voxel was positioned over fat and skeletal muscle. These peaks were later assigned to EMCL (1.6 ppm) and intramyocellular lipid (IMCL; 1.4 ppm) (79). A number of lines of evidence support the notion that the methylene peak at 1.4 ppm is representative of IMCL (IMTG and IMCL are interchangable acronyms). In tissues devoid of adipose cells, a single resonance was observed at 1.3-1.4 ppm (79), whereas a single resonance was seen at 1.5-1.6 ppm in adipose tissue (7). Consistent with these data, a study of patients with congenital generalized lipodystrophy, a condition characterized by almost complete absence of adipose tissue, reported no signal from muscle at 1.6 ppm (78). Also, the IMCL peak was consistent with markers of muscle mass such as creatine and water, whereas the EMCL peak was not (8). Finally, an exercise-induced reduction of IMCL, as indicated by a decrease in the methylene peak at 1.4 ppm, has been reported by several different groups (Table 3), and consumption of a moderate- to high-fat diet allows IMCL to rapidly return to baseline after depletion (7, 55).

                              
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  		Table 3.   IMTG utilization during exercise as determined by 1H-MRS

Evidently, the two major advantages of using 1H-MRS to quantify IMTG over the muscle biopsy and chemical analysis are its noninvasive nature and the ability to adequately differentiate between IMTG and extramuscular lipid stores. Because IMCL resonances are proportional to other intracellular metabolites such as creatine, resizing or slight variations in the positioning of the voxel should not change the IMCL signal. Similar to the 12% variability associated with multiple muscle biopsy measures in trained men (83), the intraindividual CV between three repeated measures obtained from the same trained leg with 1H-MRS was 6.1-16.9% depending on the internal reference standard (8, 55). However, in contrast to the large variability (23%) associated with multiple muscle biopsy measures in untrained men (84), when applied to obese subjects, the IMCL CV was not higher than that for nonobese subjects (78). Thus the noninvasive 1H-MRS technique offers excellent reproducibility, which permits the accurate quantitative assessment of biologically relevant changes in IMTG for all populations. 1H- MRS also permits the determination of IMTG across numerous muscle groups.

Notwithstanding these advantages, 1H-MRS contains a number of limitations and disadvantages. In brief, the technical difficulty associated with signal acquisition, the dependence of the signal on the muscle fiber orientation, and the potential variability caused by large fat layers are perhaps the largest constraints (7). As is the case with all techniques, 1H-MRS cannot distinguish between muscle fiber types and measures only net IMTG changes. Calibration of the obtained signal with actual in vivo muscle IMTG concentrations is also difficult with all MRS technology. Another minor source of error is the contribution of acyl-CoA esters and long-chain acetylcarnitine to the methylene peak. Finally, the ability of 1H-MRS to accurately quantify IMTG may be limited by 1) situations of altered tissue water content (e.g., exercise) because the proton molar density of IMTG per unit volume is converted to proton molar density per unit weight by correcting for tissue density and 2) the assumption for the composition of the fatty acyl chains in IMTG.

To date, few studies have assessed IMTG fluxes with 1H-MRS before and after exercise (Table 3). After 2-3 h of exhaustive running at 65% VO2 peak, IMTG in the soleus was depleted by ~33% from preexercise levels (52) and the utilization of IMTG was consistent with the progressive decrease in RER with exercise. Supporting this observation, Décombaz et al. (18) showed that 2 h of treadmill exercise at ~50% VO2 peak reduced IMTG by 22 and 26% in untrained and trained men, respectively. In another study, IMTG was depleted by ~24% in both the soleus and tibialis anterior after 105 min of running at 64% VO2 max (9). In a cohort that completed a marathon run (225 min at 69% VO2 max), IMTG degradation was twofold greater in the soleus and tibialis anterior, indicating IMTG utilization throughout prolonged running exercise. In contrast, prolonged exercise at a higher power output (85% VO2 max) did not alter IMTG content, suggesting that IMTGs are not important metabolic substrates at higher exercise intensities (9). Although total fat oxidation is reduced at higher exercise intensities, IMTG content was nevertheless unchanged, suggesting that minimal or no IMTG hydrolysis occurred. Taken together, the preliminary studies that used 1H-MRS suggest IMTG is utilized during prolonged, moderate-intensity exercise, whereas energy for more intense exercise is not provided by IMTG hydrolysis.


    CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
TRIACYLGLYCEROL STORAGE IN...
EVIDENCE OF ENZYMATIC...
IMTG UTILIZATION DURING...
CONCLUSIONS
REFERENCES

Although the majority of evidence from isotope tracer and 1H MRS studies demonstrate IMTG utilization during exercise, controversy regarding the importance of IMTG as a metabolic substrate persists because the majority of the studies that used direct measurments in muscle biopsy samples report no significant net IMTG degradation during prolonged, moderate-intensity (55-70% VO2 max) exercise lasting 90-120 min. The marked IMTG variability (~23%) between duplicate or triplicate muscle biopsy samples, when measured in untrained skeletal muscle, is larger than the expected IMTG utilization during exercise of this intensity and duration and most likely accounts for the absence of significant decreases in net IMTG utilization. Our laboratory recently demonstrated reduced IMTG variability (~12%) when duplicate biopsy samples were obtained from aerobically trained subjects (83). The reduced variability resulted in the detection of a significant decrease in IMTG content after 2 h of moderate-intensity cycling. Therefore, it is our contention that all three of the methods currently in use for measuring or estimating net IMTG utilization during prolonged, moderate-intensity exercise report significant and energetically important oxidation of FFA derived from IMTG. We recommend that measuring IMTG concentration in muscle biopsy samples be limited to use in endurance-trained subjects. In untrained and obese subjects, we recommend that either isotope tracer or 1H-MRS techniques be used when examining IMTG utilization during exercise.


    FOOTNOTES

Address for reprint requests and other correspondence: M. J. Watt, Dept. of Human Biology and Nutritional Sciences, Univ. of Guelph, Guelph, Ontario, Canada N1G 2W1 (E-mail: mwatt{at}uoguelph.ca).

10.1152/japplphysiol.00197.2002

Received 11 March 2002; accepted in final form 6 May 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
TRIACYLGLYCEROL STORAGE IN...
EVIDENCE OF ENZYMATIC...
IMTG UTILIZATION DURING...
CONCLUSIONS
REFERENCES

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C. Moro, S. Bajpeyi, and S. R. Smith
Determinants of intramyocellular triglyceride turnover: implications for insulin sensitivity
Am J Physiol Endocrinol Metab, February 1, 2008; 294(2): E203 - E213.
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K. De Bock, T. Dresselaers, B. Kiens, E. A. Richter, P. Van Hecke, and P. Hespel
Evaluation of intramyocellular lipid breakdown during exercise by biochemical assay, NMR spectroscopy, and Oil Red O staining
Am J Physiol Endocrinol Metab, July 1, 2007; 293(1): E428 - E434.
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T. Stellingwerff, H. Boon, R. A. M. Jonkers, J. M. Senden, L. L. Spriet, R. Koopman, and L. J. C. van Loon
Significant intramyocellular lipid use during prolonged cycling in endurance-trained males as assessed by three different methodologies
Am J Physiol Endocrinol Metab, June 1, 2007; 292(6): E