This study was undertaken to examine the variability of the IMTG measurement in muscle samples of trained men and to investigate IMTG utilisation and carbohydrate metabolism during 240 min of moderate exercise. The present study demonstrated a substantial reduction in the c.v. of the IMTG measurement in trained men compared with values previously obtained in untrained individuals (Wendling et al. 1996). During exercise from 0 to 120 min IMTG and muscle glycogen were significantly reduced, and considerable utilisation of extramuscular fuels was evident (Fig. 6). No further reduction in IMTG was observed from 120 to 240 min and muscle glycogen degradation was attenuated. It is likely that the origin of almost all of the fat oxidised after 120 min of exercise is from extramyocellular depots and this is consistent with the observed increase in FFA availability and plasma glycerol. PDHa was reduced at 240 min compared with other exercise measures (10, 120 min) and this was in accordance with the reduced whole body carbohydrate oxidation late in exercise. The mechanism underlying the reduction in PDHa is not readily apparent.
Circulating FFAs and IMTG supply the fat fuel required by the muscle for ATP production during exercise. Despite the results from isotope tracer (Romijn et al. 1993; van Loon et al. 2001) and nuclear magnetic resonance (NMR) spectroscopy (Krssak et al. 2000; Décombaz et al. 2001) studies that suggest pronounced IMTG use during exercise, controversy exists regarding whether IMTG is oxidised during exercise. The controversy exists as the bulk of the literature examining IMTG utilisation directly reports no use of IMTG during 90-120 min of exercise at ≈ 65 % V̇O2,max (Keins et al. 1993; Wendling et al. 1996; Starling et al. 1997; Keins & Richter, 1998; Bergman et al. 1999; Guo et al. 2000).
It is commonly perceived that these discrepancies are due to the methodological limitations associated with the muscle biopsy technique. This method does not discriminate between IMTG and fat interlaced between muscle fibres, nor does it account for the variability in IMTG content at different sites resulting from the disparity in fibre type storage (Essén et al. 1975). In a previous study, we reported a between-biopsy (three biopsies) c.v. of 23.5 ± 14.6 % in mainly untrained subjects (Wendling et al. 1996). In that study cycling for 90 min at 65 % V̇O2,max induced a 6 mmol kg−1 d.m. (22.8 %) reduction in IMTG, but the decrease in IMTG was of the same order of magnitude as the between-biopsy variability. Here we report a c.v. of 12.3 ± 9.4 % in 17 paired biopsies. The improved reliability of our measurement is related to the use of trained men as subjects. Trained subjects are less likely to store fat between muscle fibres than untrained individuals (Szczepaniak et al. 1999) and this observation is supported by our finding of no obvious adipose tissue contamination in any of the ≈40 muscle samples. Based on these data in trained men, a 12 % or greater reduction in IMTG (1.9 mmol kg−1 d.m. for the present study) is required for changes to be considered meaningful. Moreover, these conclusions remain consistent when a single (either of the duplicate samples) muscle biopsy is sampled at each time point. These results may explain the controversy of earlier biopsy studies that reported non-significant changes in IMTG use during prolonged moderate exercise.
We recommend that future studies investigating IMTG utilisation recruit trained individuals to serve as subjects to minimise the variability of the IMTG measure and the associated problems observed in untrained subjects. Duplicate samples are not necessary when assessing IMTG utilisation in trained men, whereas the reliability of the IMTG measure in endurance-trained women has not been assessed. Based on previous work that has demonstrated poor repeatability in an untrained population (Wendling et al. 1996), we advocate the use of duplicate biopsies in untrained individuals.
In agreement with previous studies using chemical analysis (Hurley et al. 1986; Cleroux et al. 1989; Phillips et al. 1996a) and more recently 1H-NMR spectroscopy (Krssak et al. 2000; Brechtel et al. 2001; Décombaz et al. 2001) we provide direct evidence of IMTG use during prolonged exercise in trained men during the initial 2 h of cycling at ≈55 % V̇O2,max (Fig. 3). A novel finding of the present study was that further IMTG depletion did not occur in the final 120 min of exercise. Such a finding did not support our original hypothesis that IMTG use would be elevated later in exercise, and was surprising given the progressive increase in whole body fat oxidation (Fig. 1). It has been proposed previously that IMTG is utilised early in exercise to account for the sluggish delivery of adipose-derived FFAs to the contracting muscle (Romijn et al. 1993). Although we have no kinetic measures of glycerol and FFAs, we observed marked and progressive increases in plasma glycerol (Table 2) and plasma FFAs (Fig. 2) after 90 min of moderate exercise. These data support the contention that the progressive increase in peripheral lipolysis and delivery of FFAs to the active muscle results in a shift towards enhanced oxidation of extramuscular fat which would obviate the requirement for IMTG hydrolysis. Indeed, we estimate that exogenous FFAs supplied 39 % (59 g) of the total energy for ATP production in the first 120 min of exercise, and this increased to 59 % (94 g) in the second 120 min when FFA availability was greater (Fig. 6). Thus, trained subjects seem to preferentially utilise plasma FFAs over IMTG when both substrates are available. Interestingly, when plasma FFA mobilisation and oxidation were reduced by the ingestion of a nicotinic acid analogue, IMTG utilisation was enhanced in endurance-trained men (Coyle et al. 1998). Taken together, these data indicate that trained muscle is able to utilise both intra- and extramuscular fat stores and a reciprocal relationship between IMTG and exogenous FFAs use may exist.
Supporting the possibility of enhanced FFA uptake with delivery is the finding of increased FFA uptake when plasma FFAs are increased with Intralipid and heparin infusion (Odland et al. 1998). Also, FFA uptake was shown to increase linearly with FFA delivery in the trained thigh (Turcotte et al. 1992; Kiens et al. 1993), which may be partly due to increased fat transporter content (Kiens et al. 1997; Turcotte et al. 1999). It has been suggested that FFA delivery may be reduced in the trained state because of reduced sympathoadrenal activity (Hartley et al. 1972), but glycerol Ra, an index of whole body lipolysis, was similar before and after endurance training despite ≈50 % reduction in plasma adrenaline after training (Phillips et al. 1996b). Moreover, in rodents, endurance training increases FFA oxidation and esterification and reduces IMTG utilisation during contractions when exogenous FFA availability is adequate (Dyck et al. 2000). Overall, these data are entirely consistent with our findings in trained men that demonstrate reduced IMTG utilisation with concomitant increases in plasma FFA oxidation secondary to greater FFA delivery late in moderate exercise.
A major limitation of estimating IMTG utilisation using any of the basic approaches (e.g. biochemical, isotope tracer kinetics and 1H-NMR spectroscopy) is the distinct possibility of TG/FFA cycling. A recent study using dual-tracer pulse-chase procedures in the contracting rat soleus reported increased palmitate oxidation and an equal concomitant increase in esterification (Dyck & Bonen, 1998). However, in humans, the rate of FFA incorportation into the IMTG pool was only ≈10 % of the IMTG oxidation rate (Guo et al. 2000). This raises the possibility that a small amount of exogenous FFAs entering the cytoplasm is directed towards storage in the cytoplasmic IMTG pool, which would mask the hydrolysis of IMTG, thus resulting in an underestimation of IMTG utilisation.
The mechanisms regulating the reduction in IMTG hydrolysis are unknown. Hormone-sensitive lipase (HSL) was recently identified as the enzyme responsible for IMTG hydrolysis in rat skeletal muscle (Langfort et al. 1999, 2000) and modest increases in HSL activation have been demonstrated during exercise in untrained humans and with adrenaline infusion in adrenalectomised patients (Kjær et al. 2000). Aside from these data, which suggest that HSL is subject to dual control by contractions and adrenaline via increases in intracellular cyclic 5′-AMP, no other information regarding the regulation of HSL in skeletal muscle exists. One attractive hypothesis for the regulation of HSL is feedback inhibition by long-chain fatty acyl CoA (LCFA CoA). In vitro studies have demonstrated direct non-competitive negative feedback of HSL by oleoyl-CoA (Jepson & Yeaman, 1992). The possibility exists that LCFA CoA inhibits HSL activity after binding to a specific site on the enzyme, thus preventing further mobilisation of IMTG stores. Long-chain fatty acyl-CoA synthetase (FACS) catalyses esterification of long-chain fatty acids with coenzyme A, the first step in fatty acid metabolism. Evidence from studies in adipocytes suggest FACS resides in the plasma membrane and long-chain fatty acids are immediately esterified upon entry to the cytoplasm (Gargiulo et al. 1999). Although tenuous, the findings of reduced IMTG hydrolysis and greater plasma-derived FFA oxidation (and presumably LCFA-CoA accumulation) support the possibility of feedback inhibition of HSL. Clearly, further studies investigating LCFA CoA content and the regulation of HSL in human skeletal muscle during exercise are warranted.
The reduced carbohydrate oxidation observed in the second 120 min of exercise was a function of attenuated muscle glycogen use (Table 2). Muscle glycogen utilisation is most rapid in the early stages of exercise and decreases later in exercise secondary to reduced glycogen availability (Gollnick et al. 1974). Moreover, increased availability of plasma FFAs is associated with attenuated glycogen use during whole body exercise (Costill et al. 1977). Our findings of reduced glycogen use and elevated plasma FFAs are entirely consistent with these data. Glucose uptake is reduced late in prolonged exercise as arterial glucose concentrations decline (Ahlborg & Felig, 1974). We estimated no reduction in the contribution of plasma-derived glucose to total energy expenditure (Fig. 6), and this is not surprising given the maintenance of euglycaemia until exercise cessation.
PDH permits entry of carbohydrate into the mitochondria by catalysing the decarboxylation of pyruvate to acetyl CoA. Although numerous studies have previously examined the acute regulation of PDH and its role in intramuscular fuel selection, no study had examined PDH activation during exercise longer than ≈60 min. We observed an increase in PDHa at 10 and 120 min of exercise and although PDHa remained elevated from rest at 240 min, PDHa was lower than at the previous exercise time points. The reduction in PDHa at 240 min was closely linked to the reduction in whole body carbohydrate oxidation, which is consistent with previous studies that report a close match between PDH activation, estimated PDH flux, and carbohydrate oxidation (Constantin-Teodosiu et al. 1991; Gibala et al. 1998; Howlett et al. 1998). PDH activity is controlled by the relative activities of PDH kinase (PDK) and PDH phosphatase (PDP) which inhibit and activate PDH, respectively. During exercise, PDK is inhibited by pyruvate and a high NAD/NADH ratio and stimulated by a high ATP/ADP ratio, whereas PDP is stimulated by Ca2+. In the present study the apparent mechanism(s) mediating the decreased PDHa at 240 min are not readily apparent. The muscle ATP/ADP ratio was unchanged (Table 2) and the NAD/NADH ratio not measured. We have no measure of Ca2+ in the present study, but muscle fatigue in the single mouse fibre is partly caused by failure of the sarcoplasmic reticulum to release Ca2+ (Allen & Westerblad, 2001) whilst mitochondrial Ca2+ uptake is reduced with exhaustive exercise in rodents (Tate et al. 1978). The possibility exists that reduced Ca2+ may contribute to reduced PDH activation late in fatiguing exercise. Based on the reduced glycogen utilisation and negligible lactate accumulation between 120 and 240 min, pyruvate flux was decreased late in exercise despite the absence of change in whole muscle pyruvate concentration (Table 2). Because pyruvate is a substrate for PDH and an inhibitor of PDK (Ki= 0.5-2.0 mm), we attribute the reduction in PDHa to reduced substrate availability. Indeed, the observation that the calculated flux through PDH closely matched the measured PDH activation over the last 120 min (1.47 vs.1.68 mmol kg−1 d.m. min−1, respectively) supports the notion that pyruvate flux is an important regulator of PDH during prolonged exercise. The likelihood is that we were unable to measure the subtle changes in pyruvate that mediate changes in PDH activation.
Alternatively, PDH activity may be reduced late in exercise secondary to rapid increases in PDK protein and activity. Transcriptional activity of PDK4 in response to exercise is rapid, with three- to sevenfold increases occurring immediately after 60-90 min exhaustive knee extensor exercise (Pilegaard et al. 2000). PDK4 mRNA, protein and total activity have been shown to increase within 15-24 h on a low carbohydrate diet or during fasting (Peters et al. 2001; Spriet et al. 2001). Whether the greater PDK4 mRNA translates to increased protein and total activity during a prolonged exercise bout is yet to be determined.
Acetyl group accumulation
Accumulation of acetyl groups is observed during exercise when the formation of acetyl CoA is greater than acetyl CoA utilisation by the tricarboxylic acid (TCA) cycle (Constantin-Teodosiu et al. 1991; Howlett et al. 1998). The excess acetyl CoA units are transferred to carnitine to regenerate free CoA, which is essential for continued PDH activity and many other mitochondrial reactions. In the present study we observed an increase in acetyl group accumulation early in exercise (Fig. 4) that was maintained until 120 min, after which acetylcarnitine decreased at 240 min. Similar levels have previously been reported at fatigue following a low carbohydrate diet (Putman et al. 1993). The decrease in acetylcarnitine coincided with decreased PDHa and whole body carbohydrate oxidation. These data suggest that the reduction in carbohydrate oxidation (flux through PDH) results in an inability to supply sufficient acetyl CoA to fuel oxidative metabolism. Thus, in spite of increased fat metabolism, acetyl units derived from acetylcarnitine were required to fuel oxidative metabolism late in exercise.
In summary, this study describes the time course for fuel utilisation, PDH activation and acetyl group accumulation during 4 h of moderate intensity exercise. The between-biopsy IMTG variability in trained men (12.3 %) was less than the net decrease in IMTG content deeming our results meaningful. During the first 120 min of exercise a significant proportion of the substrate oxidised was derived from IMTG and muscle glycogen, but plasma FFAs and glucose constituted the primary fuel sources. No further reduction in IMTG occurred in the final 120 min of exercise, and this was accompanied by substantial increases in plasma FFA oxidation. It is possible that IMTG was used early in exercise to offset the sluggish delivery of plasma FFAs whereas the increased delivery and oxidation of plasma FFAs obviated the requirement for IMTG hydrolysis late in exercise. Whole body carbohydrate oxidation was decreased in the final 120 min of exercise as a function of reduced muscle glycogen degradation and PDH activation. Although an explanation for the reduced PDH activation was not readily apparent, reduced pyruvate availability secondary to diminished glycolytic flux is the most likely regulator. Despite the compensatory increase in extramuscular fat metabolism late in exercise, ATP derived from acetylcarnitine stores was required to fuel oxidative metabolism.