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. 2003 Mar 21;548(Pt 3):919–927. doi: 10.1113/jphysiol.2002.031179

Skeletal muscle fat and carbohydrate metabolism during recovery from glycogen-depleting exercise in humans

Nicholas E Kimber *, George J F Heigenhauser *, Lawrence L Spriet *, David J Dyck *
PMCID: PMC2342904  PMID: 12651914

Abstract

The primary aim of the present study was to determine whether intramuscular triacylglycerol (IMTG) utilization contributed significantly to the increase in lipid oxidation during recovery from exercise, as determined from the muscle biopsy technique. In addition, we also examined the regulation of pyruvate dehydrogenase (PDHa) and changes in muscle acetyl units during an 18 h recovery period after glycogen-depleting exercise. Eight endurance-trained males completed an exhaustive bout of exercise (∼90 min) on a cycle ergometer followed by ingestion of carbohydrate (CHO)-rich meals (64–70 % of energy from carbohydrate) at 1, 4 and 7 h of recovery. Duplicate muscle biopsies were obtained at exhaustion, and 3, 6 and 18 h of recovery. Despite the large intake of CHO during recovery (491 ± 28 g or 6.8 ± 0.3 g kg−1), respiratory exchange ratio values of 0.77 to 0.84 indicated a greater reliance on lipid as an oxidative fuel. However, there was no net IMTG utilization during recovery. IMTG content at exhaustion was 23.5 ± 3.5 mmol (kg dry wt)−1, and remained constant at 24.6 ± 2.6, 25.7 ± 2.8 and 28.4 ± 3.0 mmol (kg dry wt)−1 after 3, 6 and 18 h of recovery. Muscle glycogen increased significantly from 37 ± 11 mmol (kg dry wt)−1 at exhaustion, to 165 ± 13, 250 ± 18, and 424 ± 22 mmol (kg dry wt)−1 at 3, 6 and 18 h of recovery, respectively. PDHa was reduced at 6 and 18 h when compared to exhaustion, but did not change during the recovery period. Acetyl-CoA, acetylcarnitine and pyruvate contents declined significantly after 3 h of recovery compared to exhaustion, and thereafter remained unchanged. We conclude that IMTG has a negligible role in contributing to the enhanced fat oxidation during recovery from exhaustive exercise. Despite the elevation of glucose and insulin following high-CHO meals during recovery, CHO oxidation and PDH activation were decreased, supporting the hypothesis that glycogen resynthesis is of high metabolic priority. Plasma fatty acids, very low density lipoprotein triacylglycerols, as well as intramuscular acetylcarnitine stores are likely to be important fuel sources for aerobic energy, particularly during the first few hours of recovery.

Fat is an important source of energy for resting and contracting muscle, and is available exogenously from the circulation (fatty acids, chylomicrons, very low density lipoproteins (VLDL)), as well as within the muscle fibre. Intramuscular triacylglycerol (IMTG) is found in the form of lipid droplets adjacent to mitochondria (Hoppeler et al. 1999) and is believed to be an energy substrate during long-term endurance activities (Havel et al. 1967; Essen, 1977; Staron et al. 1989; Romijn et al. 1993; Klein et al. 1994; Romijn et al. 1995; Watt et al. 2002a). However, the contribution of the IMTG pool as a metabolic substrate during prolonged submaximal exercise has been debatable. The majority of evidence from isotopic tracer and 1H-magnetic resonance spectroscopy (MRS) studies demonstrate a net utilization of IMTG during 90–120 min of exercise, while the findings of studies measuring IMTG content directly from skeletal muscle biopsies have been controversial (for review, see Watt et al. 2002b).

Relatively little attention has been given to lipid utilization during the recovery period following exhaustive, glycogen-depleting exercise in humans. The data from several studies indicate an increase in whole-body fat oxidation after exercise as demonstrated by a decrease in the respiratory exchange ratio (RER) (Krzentowski et al. 1982; Bielinski et al. 1985; Kiens & Richter, 1998). Recently, it was also demonstrated that IMTG content decreased significantly during the initial 18 h of recovery in well-trained individuals (Kiens & Richter, 1998). These authors suggested that following glycogen-depleting exercise, muscle glycogen resynthesis is of high metabolic priority, resulting in the preferential utilization of IMTG and circulating lipids by the recovering skeletal muscle. Paradoxically, the reduction in IMTG content occurred during a period of high-CHO feeding when circulating insulin, an antilipolytic hormone, was elevated. However, recent 1H-MRS studies in endurance-trained subjects have failed to demonstrate a reduction in IMTG content during recovery, with reports of either unchanged (Decombaz et al. 2000, 2001) or increased (Larson-Meyer et al. 2002) IMTG content. It should be noted that there are several differences between the biopsy and MRS studies which may result in disparate findings: (1) sampling site, i.e. soleus/ tibialis anterior (Type I fibres) in the MRS studies, vastus lateralis (mixed) in the biopsy studies; (2) differences in muscle recruitment patterns (running, MRS; cycling, biopsy); (3) timing of post-exercise sampling, i.e. IMTG content was not determined until 22 h of recovery in the study by Larson-Meyer et al. (2002) as opposed to within the first 6 h of recovery in the study by Kiens & Richter (1998).

Since the existing MRS data suggest that IMTG are not utilized as an energy source in the post-exercise recovery period, we felt that it was important to verify these findings with direct measurements in human biopsy samples. Furthermore, potential mechanisms underlying altered substrate utilization following glycogen-depleting exercise, when combined with the standard practice of high-CHO feedings to replenish muscle glycogen stores, remain unknown. Thus, the objectives of this study were to: (1) examine the utilization of IMTG as an energy substrate after exhaustive exercise, and (2) provide a more thorough examination of the metabolic changes which occur in skeletal muscle during the recovery period following exhaustive, glycogen-depleting exercise. In order to facilitate comparison to the only previous study to measure IMTG content in muscle biopsies during recovery, we essentially replicated the experimental protocol of Kiens & Richter (1998). There were two major differences in design between these studies. First, in the present study, muscle biopsies were sampled in duplicate at each time point, which should permit a more accurate determination of IMTG content (our coefficient of variation between duplicate biopsies in trained individuals is 12 % (Watt et al. 2002a) compared to 24 % in untrained individuals (Wendling et al. 1996)). Second, we included additional metabolic measurements, namely pyruvate dehydrogenase activation (PDHa), and changes in muscle pyruvate, acetyl CoA and acetylcarnitine contents during recovery. Based on the premise that elevated insulin levels would be expected to inhibit IMTG lipolysis, and in view of the recent MRS data that do not support the use of IMTG following exercise, we hypothesized that IMTG content would remain unchanged during the recovery phase. Furthermore, we hypothesized that PDHa would decrease during recovery to facilitate the partitioning of glucose towards glycogen resynthesis.

METHODS

Subjects

Eight endurance-trained males, age, 25 ± 3 years; body mass, 72.7 ± 2.1 kg; and maximal oxygen uptake (Inline graphic), 63.1 ± 2.6 ml kg−1 min−1 (mean ±s.e.m.) volunteered to participate in this study. Six of the subjects were competitive athletes (three swimmers, two runners, one canoeist) and two were highly active. All subjects were considered to be healthy, did not smoke or take any medication, and had no evidence of cardiovascular or metabolic diseases. The experimental procedures and possible risks of the study were explained orally and in writing to all participants before obtaining their informed, written consent to participate. The study protocol (Fig. 1) was approved by the ethics committees of both institutions, and was performed according to the Declaration of Helsinki.

Figure 1.

Figure 1

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Schematic representation of protocol

Pre-experimental protocol

Daily energy intake and composition of each subject's habitual diet was assessed using a 4-day food record (3 weekdays and 1 weekend day). Dietary data (Table 1) were analysed for energy intake and macronutrient composition (Food Processor Nutrition Analysis Software, version 7, ESHA Research, Salem OH, USA). Subjects' Inline graphic was determined on an electromagnetically braked cycle ergometer (Lode Excalibur, Quinton Instruments, Seattle, WA, USA). Inline graphic was determined from a plateau in Inline graphic (increase < 2 ml kg−1 min−1) with a further increase in power output. From the Inline graphic test, power outputs corresponding to submaximal Inline graphic values to be used during the experimental trial (see below) were determined using linear regression analysis for each subject. All subjects were familiarized with the intensity of the glycogen-depleting protocol and the experimental power outputs verified ≈1 week before the experiments. For 2 days prior to the experimental trials, all subjects consumed a high-CHO diet to maximize glycogen stores (Table 1) and abstained from exercise, and consumption of caffeine and alcohol.

Table 1.

Daily energy intake, dietary composition and glycaemic index before and during post-exercise recovery

Pre-exercise Recovery
2 days Meal 1 Meal 2 Meal 3
Habitual before Breakfast 1 h 4 h 7h Average
Energy (kcal) 2987 ± 229 3005 ± 300 205 ± 0 1446 ± 94 690 ± 33 815 ± 57 984 ± 78
CHO(%) 54 ± 1.6 60 ± 1.8* 84 ± 0 70 ± 0.5 64 ± 0.8 65 ± 1.7 66 ± 0.9
Fat(%) 32 ± 1.2 26 ± 1.6* 12 ± 0 19 ± 0.3 22 ± 0.4 22 ± 1.9 21 ± 0.8
Protein (%) 14 ± 0.7 14 ± 0.9 4 ± 0 11 ± 0.2 14 ± 0.4 13 ± 0.3 13 ± 0.3
CHO (g (kg body wt)−1) ND ND 0.63 ± 0.02 3.5 ± 0.2 1.5 ± 0.1 1.8 ± 0.1 2.3 ± 0.2
Glycaemic index (GI) ND ND 68.7 ± 0.0 64.1 ± 0.3 62.3 ± 0.3 55.8 ± 0.5 64.5 ± 0.6
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Values are means ±s.e.m., n = 8. CHO, carbohydrate; ND, not determined.

*

Significantly different (P < 0.05) from habitual.

Experimental protocol

Subjects reported to the laboratory at 09.30 h on the experiment day after a 10–12 h overnight fast. After voiding, body mass was recorded and a teflon catheter inserted into an antecubital vein for blood sampling. Following 30 min of rest in a supine position, resting Inline graphicO2 and Inline graphicCO2 were measured (for determination of the respiratory exchange ratio (RER)), and a 5 ml venous blood sample was drawn prior to exercise. Subjects then consumed a light breakfast consisting of CHO-rich, high glycaemic index (GI) foods (Table 1). During a subsequent 2 h rest period, two incisions were made over the vastus lateralis muscle under local anaesthesia (2 % lignocaine (lidocaine) without adrenaline (epinephrine)). Respiratory and blood samples were then obtained and exercise initiated on a Lode Excalibur cycle ergometer. The glycogen-depleting protocol was performed as previously described (Kuipers et al. 1987; Kiens & Richter, 1998). This consisted of cycling at 75 % Inline graphic for 20 min, followed by alternating 2 min bouts of 90 % and 50 % of Inline graphic for 4–5 intervals, then decreasing intensity to 80 % and 50 % Inline graphic > 2 min bouts for another 4–5 intervals, and finishing with 2 min 70 % and 50 % Inline graphic bouts until exhaustion. The mean exercise duration was 85.8 ± 3.2 min. During exercise, subjects ingested water ad libitum and a blood sample was taken just prior to exhaustion. After cessation of exercise (exhaustion), subjects were moved from the cycle ergometer to an examination table, at which point two percutaneous needle muscle biopsies were obtained from the same thigh. Approximately 3–4 min lapsed between the termination of the exercise and the procurement of the muscle samples. During the recovery period, duplicate muscle biopsy samples were obtained at 3, 6 and 18 h by alternating between left and right thighs through different incisions spaced 2–3 cm apart. Blood and breath samples were obtained after 30 min, and every hour during the initial 6 h of recovery, and again after 18 h the following morning. Inline graphic, Inline graphic, and RER were determined at rest and during exercise using a metabolic cart (Quinton Q-plex 1, Quinton Instruments). RER was calculated by averaging expired air samples for the final 5 min of a 10 min collection period.

Recovery diet

Energy and macronutrient intake for the three meals consumed during the recovery period (Table 1) were calculated for each subject and were similar to those used previously (Kiens & Richter, 1998). All subjects ate the same type of common food items and each meal was carefully prepared and weighed to the nearest gram prior to each trial. The first meal was ingested after 1 h of recovery and provided sufficient energy to replace that used during the exercise bout (≈1450 kcal; 1 kcal = 4.184 kJ). A much lighter second meal was eaten after 4 h of recovery, followed by a third meal 7 h into the recovery period. After the final meal, subjects were only permitted to drink water until their final two muscle biopsies the next morning at 18 h of recovery. Total energy intake during recovery was matched closely to the average energy intake reported from individual 4-day food records (Table 1). The composition of the post-exercise recovery diet was calculated to provide 65–70 % of energy from CHO, 20 % from fat, and 10–15 % from protein, with an average glycaemic index (GI) of 60–65 using glucose as a reference (Table 1).

Analyses

Blood samples were collected in heparinized collection tubes and placed on ice. A 200 μl aliquot was deproteinized in 1.0 ml of 0.6 m HClO4 and the supernatant stored at −80 °C for subsequent fluorometric determination of whole blood glycerol, lactate and glucose (Bergmeyer, 1974). A second portion of whole blood was centrifuged, and 800 μl of plasma was added to 200 μl of 5 m NaCl at 56 °C for 30 min, to inactivate lipoprotein lipase. This treated plasma was analysed for fatty acids (Wako NEFA C test kit, Wako Chemicals, Richmond, VA, USA). The remaining untreated plasma was assayed in duplicate for insulin using an RIA specific kit (LINCO Research, St Charles, MO, USA).

Muscle biopsies were immediately frozen in liquid N2, removed from the needle while frozen, and stored in liquid N2 until analysed. A small (10–20 mg) piece of muscle was chipped from one of the duplicate biopsies under liquid N2 and analysed for PDH activation (PDHa), as previously described (Putman et al. 1993). The remaining muscle was freeze-dried, dissected free of blood, connective tissue and visible fat, and powdered for subsequent metabolite analyses. One aliquot of powdered muscle (2–3 mg) was assayed enzymatically for glycogen as previously described (Harris et al. 1974). Total muscle triacylglycerol (IMTG) was extracted from a 4–8 mg sample of powdered muscle using chloroform-methanol (Frayn & Maycock, 1980). After phospholipid removal using silicic acid, the extracts were saponified with ethanolic KOH at 60 °C for 1 h, and neutralized with MgSO4. Triacylglycerol concentration was then determined by assaying the liberated glycerol fluorometrically. IMTG content was calculated from the mean of the paired biopsies at each time point. The remaining powdered muscle was extracted in a solution of 0.5 m HClO4 and 1 m EDTA and neutralized with 2.2 m KHCO3. These extracts were assayed by spectrophotometry for ATP, phosphocreatine (PCr) and creatine as previously described (Bergmeyer, 1974; Harris et al. 1974). Pyruvate was determined flurometrically (Passoneau & Lowry, 1993) and acetyl CoA and acetylcarnitine were determined by radiometric assays (Cederblad et al. 1990).

Statistics

Results were analysed using a one-way ANOVA with repeated measures for the time factor to test for changes in each variable measured during the recovery period. A Newman-Keuls post hoc test was used if differences between time points were detected. Significance was accepted at P≤ 0.05. Values are presented as means ±s.e.m.

RESULTS

Diet analysis

Total energy intake (Table 1) during the trial day (breakfast + recovery meals) averaged 3156 ± 164 kcal and was closely matched to daily habitual caloric intake (2987 ± 229 kcal). Dietary composition during recovery consisted of an average of 67 ± 0.9 % of kcal from CHO, 21 ± 0.8 % from fat and 13 ± 0.3 % from protein, and an average GI of 64.5 ± 0.6. Total CHO intake during the recovery period was 491 ± 28 g or 6.8 ± 0.3 g kg−1. Total fat intake was 73 ± 3 g during the recovery period. The first meal consumed during recovery provided an average of 46 ± 0.9 and 52 ± 0.5 %, respectively, of the total energy and CHO intake for the trial day.

Respiratory and blood responses

Resting RER was 0.77 ± 0.02 at 2 h prior to exercise, and 0.80 ± 0.02 immediately prior to exercise. These were not statistically significant (Table 2). During the hour following exhaustive exercise, RER was significantly lower than baseline (P < 0.001), and returned to basal levels 2 h after exercise.

Table 2.

RER, blood glucose, plasma insulin, fatty acids, glycerol and lactate before exercise, near exhaustion and during an 18 h post-exercise recovery period

Time RER Glucose Insulin Fatty acids Glycerol Lactate
(h) (mmol l−1) (μU ml−1) (mmol l−1) (mmol l−1) (mmol l−1)
Rest 0.77 ± 0.02 4.2 ± 0.12 8.9 ± 0.9 0.26 ± 0.04 0.08 ± 0.01 0.3 ± 0.05
Pre-exercise 0.80 ± 0.02 4.1 ± 0.10 14.3 ± 2.0 0.22 ± 0.03 0.09 ± 0.003 0.5 ± 0.04
End-exercise ND 3.6 ± 0.15 4.5 ± 0.6 0.40 ± 0.06 0.23 ± 0.02‡ 5.2 ± 0.6‡
0.5 0.62 ± 0.03‡ 3.6 ± 0.10 5.5 ± 0.4 1.27 ± 0.16‡ 0.20 ± 0.02‡ 1.7 ± 0.3†
1 0.66 ± 0.03‡ 3.7 ± 0.18 5.5 ± 1.3 1.54 ± 0.17‡ 0.19 ± 0.02‡ 0.8 ± 0.15
2 0.77 ± 0.02 6.3 ± 0.30‡ 68.3 ± 13.3‡ 0.40 ± 0.07 0.11 ± 0.01 1.2 ± 0.2
3 0.77 ± 0.02 5.7 ± 0.31‡ 65.7 ± 1.6‡ 0.19 ± 0.03 0.09 ± 0.01 1.2 ± 0.1
4 0.80 ± 0.02 5.3 ± 0.30‡ 61.9 ± 9.4‡ 0.18 ± 0.03 0.09 ± 0.01 0.9 ± 0.08
5 0.84 ± 0.02 4.8 ± 0.25‡ 66.6 ± 8.3‡ 0.18 ± 0.02 0.09 ± 0.01 0.9 ± 0.10
6 0.79 ± 0.02 4.2 ± 0.21 41.7 ± 3.1* 0.22 ± 0.07 0.08 ± 0.01 0.8 ± 0.09
18 0.76 ± 0.02 4.3 ± 0.11 10.2 ± 1.0 0.44 ± 0.06 0.10 ± 0.04 0.4 ± 0.05
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Values are means ±s.e.m., n = 8. RER, respiratory exchange ratio; ND, not determined. Fatty acids are free fatty acids. Significantly different from before exercise

*

P < 0.05

†

P < 0.01.

‡

P < 0.001.

Resting whole blood glucose was 4.1 ± 0.12 mm (Table 2) immediately prior to the commencement of exercise. After 2 h of recovery (1 h after the first high-CHO meal), blood glucose concentrations rose significantly above baseline and remained elevated for another 3 h (P < 0.001). Plasma insulin concentrations increased markedly above baseline after meal 1 to reach a peak of 68.3 ± 13.3 μU ml−1 (P < 0.001) at 2 h of recovery (Table 2). For the following 4 h, plasma insulin concentration remained significantly elevated and returned to baseline levels by the following morning at 18 h after exercise. Plasma fatty acid levels were 0.22 ± 0.03 mm immediately prior to exercise and increased significantly (P < 0.001) during the first hour of recovery to 1.54 ± 0.17 mm (Table 2). After ingestion of the first meal, fatty acid concentration rapidly declined at 2 h (0.40 ± 0.07 mm) and remained near baseline throughout the recovery period. Plasma glycerol concentrations were highest near exhaustion (0.23 ± 0.02 mm), and similar to fatty acid responses, showed a marked reduction at 2 h and throughout recovery (Table 2). The exercise protocol produced a significant elevation in plasma lactate concentration (5.2 ± 0.6 mm, P < 0.001), although levels quickly returned to baseline after 1 h and remained unchanged during the recovery (Table 2).

Intramuscular triacylglycerol (IMTG)

At the termination of exercise, IMTG content was 23.5 ± 3.5 mmol (kg dry wt)−1 (Fig. 2), and remained constant at 24.6 ± 2.6, 25.7 ± 2.8 and 28.4 ± 3.0 mmol (kg dry wt)−1 after 3, 6 and 18 h of recovery, respectively.

Figure 2. Muscle triacylglycerol (IMTG) contents in the vastus lateralis muscle immediately after exercise and during an 18 h recovery period.

Figure 2

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Values are means ±s.e.m. for 8 subjects.

Muscle glycogen and metabolites

Muscle glycogen content was 37 ± 11 mmol (kg dry wt)−1 upon completion of the exercise bout and increased significantly throughout the recovery period to reach a maximal content of 424 ± 22 mmol (kg dry wt)−1 (P < 0.001) after 18 h of recovery (Fig. 3). Muscle ATP, PCr and creatine were generally unchanged throughout the recovery period (Table 3). Acetyl CoA and acetylcarnitine content decreased significantly during the first 3 h of recovery (P < 0.001) and remained significantly below post-exercise concentrations throughout the recovery period (Table 3). Pyruvate (P < 0.01) was also significantly reduced compared to levels at exhaustion and remained low throughout the post-exercise period (Table 3).

Figure 3. Muscle glycogen contents in the vastus lateralis muscle immediately after exercise and during an 18 h recovery period.

Figure 3

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Values are means ±s.e.m. for 8 subjects. * Significantly different (P < 0.001) from each previous time point.

Table 3.

Muscle metabolite concentrations immediately after exercise and during an 18 h post-exercise recovery period

Metabolite 0 h 3h 6h 18 h
ATP 23.5 ± 0.9 25.0 ± 1.0 26.2 ± 1.1 27.0 ± 1.6†
PCr 64.4 ± 3.0 68.0 ± 3.0 69.2 ± 3.1 69.1 ± 4.8
Creatine 48.4 ± 3.1 44.8 ± 2.5 43.7 ± 2.0 43.8 ± 2.6
Acetyl CoA 22.0 ± 1.8 3.7 ± 1.0‡ 9.5 ± 2.0† 7.3±0.8‡
Acetyl-carnitine 17.5 ± 2.7 1.9 ± 0.4‡ 2.3 ± 0.6‡ 3.0 ± 0.5‡
Pyruvate 0.34 ± 0.07 0.15 ± 0.03† 0.14 ± 0.05† 0.10 ± 0.01†
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Values are means ±s.e.m., n = 8. All measurements are given in mmol (kg dry wt)−1, except for acetyl CoA (μmol (kg dry wt)−1). Significantly different from 0 h,

†

P < 0.01

‡

P < 0.001.

Pyruvate dehydrogenase (PDHa)

PDHa was 1.37 ± 0.17 mmol min−1 (kg wet wt)−1 after exhaustive exercise (Fig. 4), and decreased during recovery, being significantly different from exhaustion at 6 h (0.66 ± 0.10 mmol min−1 (kg wet wt)−1; P < 0.01) and 18 h (0.71 ± 0.15 mmol min−1 (kg wet wt)−1; P < 0.05) of recovery (Fig. 4).

Figure 4. Muscle pyruvate dehydrogenase activation (PDHa) in the vastus lateralis muscle immediately after exercise and during an 18 h recovery period.

Figure 4

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Values are means ±s.e.m. for 8 subjects. # Significantly different (P < 0.01) from 0 h; * significantly different (P < 0.05) from 0 h.

DISCUSSION

Consistent with previous studies (Krzentowski et al. 1982; Bielinski et al. 1985; Kiens & Richter, 1998), our results indicate a greater contribution from fat oxidation during recovery in the presence of reduced muscle glycogen. This is in support of the hypothesis that a metabolic priority of recovering muscle is to resynthesize glycogen stores, resulting in a relative shift from CHO to fat oxidation. Indeed, activation of glycogen synthase in exercised human muscle has been recently demonstrated (Wojtazsewski et al. 2001), supporting the contention that glycogen resynthesis is of high priority early in recovery. However, our data do not support the previous findings of Kiens & Richter (1998) that IMTG are a significant source of lipid to be utilized during recovery. Rather, our data support the more recent findings of 1H-MRS studies that IMTG content does not decrease during recovery from exhaustive exercise in well-trained individuals even when low fat meals are ingested after exercise (Decombaz et al. 2000, 2001; Larson-Meyer et al. 2002). This suggests that the primary lipid source during recovery may be derived from the circulation (i.e. plasma fatty acids and triacylglycerol (TG)), which is in agreement with our observation of a rapid reduction of plasma fatty acids during the first 2 h of recovery. However, this reduction may also be due in part to reduced lipolysis as indicated by the low glycerol. Furthermore, the large reduction in muscle acetylcarnitine stores early in recovery may have provided additional acetyl units for oxidation, and therefore have contributed to energy provision during this period. Despite the significant increase of insulin in response to the high-CHO feedings during recovery, muscle PDHa decreased. This reduction in activity is probably due, at least in part, to reduced substrate (pyruvate) availability as a result of the preferential partitioning of glucose towards glycogen resynthesis.

Metabolic response during recovery

Responses during the first hour of recovery

During the first hour of recovery, we observed very low RER values (i.e. below 0.70). The explanation for this is uncertain, but is likely to be due to a shift towards predominantly fat oxidation, as well as the replenishment of the body's CO2 stores within the bicarbonate pool (Jones & Heigenhauser, 1996). This would lead to a reduction in CO2 release at the mouth and consequently, an underestimation of the true RER. The rapid increase in plasma fatty acids post-exercise, subsequent to the release of fatty acid trapped within adipose tissue (Hodgetts et al. 1991; Romijn et al. 1993) would have provided a large source of energy for the recovering muscle. In addition, the reduced insulin and glucose levels, i.e. factors that are anti-lipolytic and inhibit fat oxidation, are likely to contribute to the increase in fat utilization during the immediate recovery period. Muscle acetyl-CoA and acetylcarnitine accumulation was also evident post-exercise as previously reported (Howlett et al. 1998; Watt et al. 2002a).

Responses during 1–6 h of recovery

After ingestion of the first high CHO meal at 1 h, insulin and glucose levels were increased markedly while fatty acids and glycerol concentrations were rapidly reduced. Despite these responses, RER values remained low (0.77–0.80) until the following meal at 4 h, indicating predominantly fat oxidation, as previously observed during the early phase of recovery from prolonged (Krzentowski et al. 1982; Bielinski et al. 1985; Horton et al. 1998) or exhaustive exercise (Kiens & Richter, 1998). The source of increased fat oxidation after glycogen-depleting exercise has previously been attributed to the utilization of IMTG (Kiens & Richter, 1998). However, in this study, IMTG content remained unchanged during the first 6 h of recovery, suggesting that circulating sources such as plasma fatty acids, and possibly plasma TG, are major sources of fat oxidation during recovery. Indeed, previous studies have found an increase in the activity of the muscle lipoprotein lipase (LPL) activity after exhaustive exercise (Lithell et al. 1981; Kiens et al. 1989; Kiens & Richter, 1998), as well as increased muscle LPL mRNA at 4 h and protein content at 8 h post-exercise (Seip et al. 1997).

Glycogen content increased rapidly after 3 and 6 h of recovery (4.5- and ≈7-fold respectively) in the presence of high circulating insulin and glucose. The average rate of glycogen resynthesis was ≈40 mmol (kg dry wt)−1, which is very similar to that reported previously by Kiens & Richter (1998) using a nearly identical protocol to ours. This rapid rate of glycogen resynthesis was probably influenced by the extremely low glycogen content immediately following exercise, the well-trained status of the subjects, and the large amount of CHO consumed during the first meal.

In spite of the elevated insulin, PDHa declined after 3 h and showed a further downward trend at 6 h to a value significantly (P < 0.01) below that observed immediately post-exercise (Fig. 4). Coincident with this reduction in PDHa was a reduction in muscle acetylcarnitine stores, indicating that this pool may have been a significant source of acetyl units for oxidation early in recovery. Taken together, these data indicate a relative partitioning of glucose uptake during recovery towards glycogen resynthesis and an increased reliance on fatty acids as an oxidative fuel. Approximately 365 g of glucose were consumed during the first 6 h of recovery. During this period, muscle glycogen content increased by ≈200 mmol (kg dry wt)−1, which corresponds to a total of ≈100 g of glucosyl units, assuming that ≈12 kg of muscle mass was utilized during the exhausting exercise and that the muscle was actively resynthesizing glycogen during recovery. Thus, assuming complete absorption, ≈40 % of the ingested glucose was directed towards muscle glycogen resynthesis during the first 6 h of recovery. Of the remaining glucose absorbed (365–100 = 265 g), ≈210 g can be accounted for by (1) whole-body glucose oxidation (≈30 g) based on an average RER and Inline graphic of 0.78 and 0.3 l min−1, respectively, and (2) liver glycogen resynthesis (≈180 g), assuming a liver mass of ≈2 kg, and near repletion of its glycogen stores. The remaining unaccounted glucose (55 g) may be due to a combination of factors, including an underestimation of the working muscle mass, and glucose oxidation if our measured RER values are slightly low.

Responses during 6–18 h of recovery

The main finding during this period of recovery was that there was no change in IMTG content. Glycogen content continued to increase significantly during this period, increasing from 250.2 ± 17.7 to 423.8 ± 22.2 mmol (kg dry wt)−1. The RER remained low, as expected, following the overnight fast (0.76 ± 0.02), indicating a continued reliance on fat. In addition, plasma fatty acid levels were slightly elevated in response to low levels of circulating insulin. In further support of a shift in metabolic priority towards muscle glycogen resynthesis and away from CHO oxidation during recovery, PDHa activity, muscle pyruvate and acetyl-CoA concentrations were similar to levels found at 6 h of recovery.

Mechanisms for increased lipid metabolism during recovery

The mechanism for increased fat oxidation during recovery, in the presence of elevated insulin and glucose, is not readily apparent. IMTG content was unchanged during recovery, suggesting muscle lipid stores are not an appreciable source of fatty acids for increasing fat oxidation. Therefore, it is likely that elevated plasma fatty acids during the first hour of recovery resulted in an increased uptake and oxidation of fatty acids within glycogen-depleted muscle. This is supported by the rapid decline in plasma fatty acid levels early in recovery, which can probably be attributed to both the rapid clearance by peripheral tissues, including skeletal muscle, as well as an insulin-mediated reduction in peripheral lipolysis (Horowitz et al. 1997; Mittendorfer et al. 2002) following the first high-CHO meal during recovery. It is paradoxical, though, that fat oxidation remained elevated in the presence of elevated insulin levels. It should be noted, however, that resistance to insulin's inhibition of fatty acid oxidation during recovery from a marathon has previously been reported (Tuominen et al. 1996).

Regulation of IMTG content during post-exercise recovery

Enzymatic regulation of muscle TG esterification and hydrolysis during resting and contracting conditions is poorly understood. Recently, a neutral hormone-sensitive lipase (HSL) was identified in skeletal muscle (Langfort et al. 1998) and appears to be under dual control by adrenaline and contraction-related mechanisms (Langfort et al. 2000). Plasma adrenaline concentrations were not determined in the present study, but would be expected to decrease rapidly (i.e. within 30 min) during recovery. Regulation of HSL activity may also result from allosteric feedback inhibition by long chain fatty acyl CoA (Jepson & Yeaman, 1992). Thus, the large increase in plasma fatty acids observed early in recovery may have elevated cytosolic long chain fatty acid- (LCFA)-CoA concentrations and reduced IMTG hydrolysis by inhibiting HSL. For example, during a resting hyperinsulinaemic glucose clamp with elevated fatty acid levels, a rapid increase in the IMTG pool was found during a 5 h time period (Brechtel et al. 2001). It is therefore probably not surprising that IMTG content did not decrease during the early stages of recovery, given the marked elevation of insulin, and the possible accumulation of cytosolic LCFA-CoA in the recovering muscle. However, in fairness, it should be pointed out that during the first hour of recovery in the study by Kiens & Richter (1998), plasma fatty acids were also significantly elevated (≈2 mm), at a time when IMTG stores were being utilized.

In spite of our conclusions regarding IMTG use, the possibility that we were simply unable to detect a small, but physiologically meaningful contribution from IMTG hydrolysis and/or oxidation must be acknowledged. Estimation of IMTG utilization, using biochemical analysis or other established techniques such as isotope tracer kinetics and 1H-MRS, is limited by the inability to account for triacylglycerol (TG) and/or fatty acid cycling. Using the dual label pulse-chase technique in isolated rat soleus muscle, it has been demonstrated that the IMTG pool undergoes simultaneous esterification and hydrolysis during both rest and contraction (Dyck & Bonen, 1998). This raises the possibility that TG resynthesis during recovery may have masked the utilization of IMTG as an oxidative fuel during recovery from exhaustive exercise. A recent pulse-chase study in exercising humans (Guo et al. 2000) indicated that fatty acid incorporation into IMTG was relatively small compared to the rates of IMTG oxidation i.e. ≈8 %, making this possibility unlikely. However, it is possible that the incorporation of fatty acids into TG might be greater during recovery. Furthermore, determining the specific contribution of IMTG as an oxidative fuel during post-exercise recovery is complicated by the methodological limitations associated with the muscle biopsy technique. While the use of trained subjects reduces the coefficient of variation between biopsies to 12.3 ± 9.4 % (Watt et al. 2002a), compared to 23.5 ± 14.6 % in untrained subjects (Wendling et al. 1996), this does not explain the discrepancy between the present findings and those of Kiens & Richter (1998), who used equally well-trained subjects in their study.

Regulation of PDHa during post-exercise recovery

While the regulation of PDH activity during acute and prolonged exercise has been widely examined, to our knowledge this study is the first to report PDH activation in human skeletal muscle during CHO feeding after exhaustive exercise. PDH activity is controlled by the relative activities of PDH kinase (PDK), which phosphorylates and deactivates PDH, and PDH phosphatase (PDP), which dephosphorylates and activates PDH. PDK is stimulated by high ATP/ADP, acetyl-CoA/CoA-SH and NADH/ NAD+ ratios and is inhibited by elevated pyruvate concentrations, whereas PDP is stimulated by Ca2+ and increased by pyruvate. Muscle ATP, acetyl-CoA and pyruvate concentrations were unchanged during recovery, while Ca2+ and the NADH/NAD+ ratio were not measured in this study. However, since pyruvate is a substrate for PDH and an inhibitor of PDK, the possibility exists that a decline in pyruvate availability contributed to the trend in PDHa reduction between 3 and 6 h of recovery. In addition, it is also well documented that PDK4 expression is increased during recovery from prolonged, exhaustive exercise (Pilegaard et al. 2000, 2002), which may also account for the observed decrease in PDHa.

Conclusions

In summary, we report that IMTG content remains unchanged during recovery from glycogen-depleting exercise in the presence of elevated glucose and insulin levels. It appears that the partitioning of exogenous glucose towards glycogen resynthesis is of high metabolic priority during immediate post-exercise recovery, and is supported by the trend towards reduced PDH activity and increased fat oxidation. Plasma fatty acids (and possibly plasma TG), as well as intramuscular acetylcarnitine stores are likely to be important fuels for muscle metabolism in the immediate recovery period. However, IMTG appears to have a negligible role in contributing to the enhanced fat oxidation during recovery from exhaustive exercise.

Acknowledgments

This study was supported by the Natural Sciences and Research Council of Canada and the Canadian Institute of Health Research.

REFERENCES

  1. Methods of Enzymatic Analysis. New York: Academic Press; 1974. [Google Scholar]
  2. Bielinski R, Schutz Y, Jequier E. Energy metabolism during the postexercise recovery in man. Am J Clin Nutr. 1985;42:69–82. doi: 10.1093/ajcn/42.1.69. [DOI] [PubMed] [Google Scholar]
  3. Brechtel K, Dahl DB, Machann J, Bachmann OP, Wenzel I, Maier T, Claussen CD, Haring HU, Jacob S, Schick F. Fast elevation of the intramyocellular lipid content in the presence of circulating free fatty acids and hyperinsulinemia: a dynamic 1H-MRS study. Magn Reson Med. 2001;45:179–183. doi: 10.1002/1522-2594(200102)45:2<179::aid-mrm1023>3.0.co;2-d. [DOI] [PubMed] [Google Scholar]
  4. Cederblad G, Carlin JI, Constantin-Teodosiu D, Harper P, Hultman E. Radioisotopic assays of CoASH and carnitine and their acetylated forms in human skeletal muscle. Anal Biochem. 1990;185:274–278. doi: 10.1016/0003-2697(90)90292-h. [DOI] [PubMed] [Google Scholar]
  5. Decombaz J, Fleith M, Hoppeler H, Kreis R, Boesch C. Effect of diet on the replenishment of intramyocellular lipids after exercise. Eur J Nutr. 2000;39:244–247. doi: 10.1007/s003940070002. [DOI] [PubMed] [Google Scholar]
  6. Decombaz J, Schmitt B, Ith M, Decarli B, Diem P, Kreis R, Hoppeler H, Boesch C. Postexercise fat intake repletes intramyocellular lipids but no faster in trained than in sedentary subjects. Am J Physiol Regul Integr Comp Physiol. 2001;281:R760–769. doi: 10.1152/ajpregu.2001.281.3.R760. [DOI] [PubMed] [Google Scholar]
  7. Dyck DJ, Bonen A. Muscle contraction increases palmitate esterification and oxidation and triacylglycerol oxidation. Am J Physiol. 1998;275:E888–896. doi: 10.1152/ajpendo.1998.275.5.E888. [DOI] [PubMed] [Google Scholar]
  8. Essen B. Intramuscular substrate utilization during prolonged exercise. Ann N Y Acad Sci. 1977;301:30–44. doi: 10.1111/j.1749-6632.1977.tb38183.x. [DOI] [PubMed] [Google Scholar]
  9. Frayn KN, Maycock PF. Skeletal muscle triacylglycerol in the rat: methods for sampling and measurement, and studies of biological variability. J Lipid Res. 1980;21:139–144. [PubMed] [Google Scholar]
  10. Guo Z, Burguera B, Jensen MD. Kinetics of intramuscular triglyceride fatty acids in exercising humans. J Appl Physiol. 2000;89:2057–2064. doi: 10.1152/jappl.2000.89.5.2057. [DOI] [PubMed] [Google Scholar]
  11. Harris RC, Hultman E, Nordesjo LO. Glycogen, glycolytic intermediates and high-energy phosphates determined in biopsy samples of musculus quadriceps femoris of man at rest. Methods and variance of values. Scand J Clin Lab Invest. 1974;33:109–120. [PubMed] [Google Scholar]
  12. Havel RJ, Pernow B, Jones NL. Uptake and release of free fatty acids and other metabolites in the legs of exercising men. J Appl Physiol. 1967;23:90–99. doi: 10.1152/jappl.1967.23.1.90. [DOI] [PubMed] [Google Scholar]
  13. Hodgetts V, Coppack SW, Frayn KN, Hockaday TD. Factors controlling fat mobilization from human subcutaneous adipose tissue during exercise. J Appl Physiol. 1991;71:445–451. doi: 10.1152/jappl.1991.71.2.445. [DOI] [PubMed] [Google Scholar]
  14. Hoppeler H, Billeter R, Horvath PJ, Leddy JJ, Pendergast DR. Muscle structure with low- and high-fat diets in well-trained male runners. Int J Sports Med. 1999;20:522–526. doi: 10.1055/s-1999-8838. [DOI] [PubMed] [Google Scholar]
  15. Horowitz JF, Mora-Rodriguez R, Byerley LO, Coyle EF. Lipolytic suppression following carbohydrate ingestion limits fat oxidation during exercise. Am J Physiol. 1997;273:E768–775. doi: 10.1152/ajpendo.1997.273.4.E768. [DOI] [PubMed] [Google Scholar]
  16. Horton TJ, Pagliassotti MJ, Hobbs K, Hill JO. Fuel metabolism in men and women during and after long-duration exercise. J Appl Physiol. 1998;85:1823–1832. doi: 10.1152/jappl.1998.85.5.1823. [DOI] [PubMed] [Google Scholar]
  17. Howlett RA, Parolin ML, Dyck DJ, Hultman E, Jones NL, Heigenhauser GJ, Spriet LL. Regulation of skeletal muscle glycogen phosphorylase and PDH at varying exercise power outputs. Am J Physiol. 1998;275:R418–425. doi: 10.1152/ajpregu.1998.275.2.R418. [DOI] [PubMed] [Google Scholar]
  18. Jepson CA, Yeaman SJ. Inhibition of hormone-sensitive lipase by intermediary lipid metabolites. FEBS Lett. 1992;310:197–200. doi: 10.1016/0014-5793(92)81328-j. [DOI] [PubMed] [Google Scholar]
  19. Jones NL, Heigenhauser GJ. Getting rid of carbon dioxide during exercise. Clin Sci. 1996;90:323–335. doi: 10.1042/cs0900323. [DOI] [PubMed] [Google Scholar]
  20. Kiens B, Lithell H, Mikines KJ, Richter EA. Effects of insulin and exercise on muscle lipoprotein lipase activity in man and its relation to insulin action. J Clin Invest. 1989;84:1124–1129. doi: 10.1172/JCI114275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kiens B, Richter EA. Utilization of skeletal muscle triacylglycerol during postexercise recovery in humans. Am J Physiol. 1998;275:E332–337. doi: 10.1152/ajpendo.1998.275.2.E332. [DOI] [PubMed] [Google Scholar]
  22. Klein S, Coyle EF, Wolfe RR. Fat metabolism during low-intensity exercise in endurance-trained and untrained men. Am J Physiol. 1994;267:E934–940. doi: 10.1152/ajpendo.1994.267.6.E934. [DOI] [PubMed] [Google Scholar]
  23. Krzentowski G, Pirnay F, Luyckx AS, Pallikarakis N, Lacroix M, Mosora F, Lefebvre PJ. Metabolic adaptations in post-exercise recovery. Clin Physiol. 1982;2:277–288. doi: 10.1111/j.1475-097x.1982.tb00032.x. [DOI] [PubMed] [Google Scholar]
  24. Kuipers H, Keizer HA, Brouns F, Saris WH. Carbohydrate feeding and glycogen synthesis during exercise in man. Pflugers Arch. 1987;410:652–656. doi: 10.1007/BF00581327. [DOI] [PubMed] [Google Scholar]
  25. Langfort J, Ploug T, Ihlemann J, Enevoldsen LH, Stallknecht B, Saldo M, Kjaer M, Holm C, Galbo H. Hormone-sensitive lipase (HSL) expression and regulation in skeletal muscle. Adv Exp Med Biol. 1998;441:219–228. doi: 10.1007/978-1-4899-1928-1_20. [DOI] [PubMed] [Google Scholar]
  26. Langfort J, Ploug T, Ihlemann J, Holm C, Galbo H. Stimulation of hormone-sensitive lipase activity by contractions in rat skeletal muscle. Biochem J. 2000;351:207–214. doi: 10.1042/0264-6021:3510207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Langfort J, Ploug T, Ihlemann J, Saldo M, Holm C, Galbo H. Expression of hormone-sensitive lipase and its regulation by adrenaline in skeletal muscle. Biochem J. 1999;340:459–465. [PMC free article] [PubMed] [Google Scholar]
  28. Larson-Meyer DE, Newcomer BR, Hunter GR. Influence of endurance running and recovery diet on intramyocellular lipid content in women: a 1H NMR study. Am J Physiol Endocrinol Metab. 2002;282:E95–106. doi: 10.1152/ajpendo.2002.282.1.E95. [DOI] [PubMed] [Google Scholar]
  29. Lithell H, Cedermark M, Froberg J, Tesch P, Karlsson J. Increase of lipoprotein-lipase activity in skeletal muscle during heavy exercise. Relation to epinephrine excretion. Metabolism. 1981;30:1130–1134. doi: 10.1016/0026-0495(81)90059-7. [DOI] [PubMed] [Google Scholar]
  30. Mittendorfer B, Horowitz JF, Klein S. Effect of gender on lipid kinetics during endurance exercise of moderate intensity in untrained subjects. Am J Physiol Endocrinol Metab. 2002;283:E58–65. doi: 10.1152/ajpendo.00504.2001. [DOI] [PubMed] [Google Scholar]
  31. Passoneau JA, Lowry OH. Enzymatic Analysis: A Practical Guide. Totawa, NJ, USA: Humana; 1993. [Google Scholar]
  32. Pilegaard H, Keller C, Steensberg A, Helge JW, Pedersen BK, Saltin B, Neufer PD. Influence of pre-exercise muscle glycogen content on exercise-induced transcriptional regulation of metabolic genes. J Physiol. 2002;541:261–271. doi: 10.1113/jphysiol.2002.016832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pilegaard H, Ordway GA, Saltin B, Neufer PD. Transcriptional regulation of gene expression in human skeletal muscle during recovery from exercise. Am J Physiol Endocrinol Metab. 2000;279:E806–814. doi: 10.1152/ajpendo.2000.279.4.E806. [DOI] [PubMed] [Google Scholar]
  34. Putman CT, Spriet LL, Hultman E, Lindinger MI, Lands LC, McKelvie RS, Cederblad G, Jones NL, Heigenhauser GJ. Pyruvate dehydrogenase activity and acetyl group accumulation during exercise after different diets. Am J Physiol. 1993;265:E752–760. doi: 10.1152/ajpendo.1993.265.5.E752. [DOI] [PubMed] [Google Scholar]
  35. Romijn JA, Coyle EF, Sidossis LS, Gastaldelli A, Horowitz JF, Endert E, Wolfe RR. Regulation of endogenous fat and carbohydrate metabolism in relation to exercise intensity and duration. Am J Physiol. 1993;265:E380–391. doi: 10.1152/ajpendo.1993.265.3.E380. [DOI] [PubMed] [Google Scholar]
  36. Romijn JA, Coyle EF, Sidossis LS, Zhang XJ, Wolfe RR. Relationship between fatty acid delivery and fatty acid oxidation during strenuous exercise. J Appl Physiol. 1995;79:1939–1945. doi: 10.1152/jappl.1995.79.6.1939. [DOI] [PubMed] [Google Scholar]
  37. Seip RL, Mair K, Cole TG, Semenkovich CF. Induction of human skeletal muscle lipoprotein lipase gene expression by short-term exercise is transient. Am J Physiol. 1997;272:E255–261. doi: 10.1152/ajpendo.1997.272.2.E255. [DOI] [PubMed] [Google Scholar]
  38. Staron RS, Hikida RS, Murray TF, Hagerman FC, Hagerman MT. Lipid depletion and repletion in skeletal muscle following a marathon. J Neurol Sci. 1989;94:29–40. doi: 10.1016/0022-510x(89)90215-3. [DOI] [PubMed] [Google Scholar]
  39. Tuominen JA, Ebeling P, Bourey R, Koranyi L, Lamminen A, Rapola J, Sane T, Vuorinen-Markkola H, Koivisto VA. Postmarathon paradox: insulin resistance in the face of glycogen depletion. Am J Physiol. 1996;270:E336–343. doi: 10.1152/ajpendo.1996.270.2.E336. [DOI] [PubMed] [Google Scholar]
  40. Watt MJ, Heigenhauser GJ, Dyck DJ, Spriet LL. Intramuscular triacylglycerol, glycogen and acetyl group metabolism during 4 h of moderate exercise in man. J Physiol. 2002a;541:969–978. doi: 10.1113/jphysiol.2002.018820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Watt MJ, Heigenhauser GJ, Spriet LL. Intramuscular triacylglycerol utilization in human skeletal muscle during exercise: is there a controversy? J Appl Physiol. 2002b;93:1185–1195. doi: 10.1152/japplphysiol.00197.2002. [DOI] [PubMed] [Google Scholar]
  42. Wojtazsewski JF, Nielsen P, Kiens B, Richter EA. Regulation of glycogen synthase kinase-3 in human skeletal muscle: effects of food intake and bicycle exercise. Diabetes. 2001;50:265–269. doi: 10.2337/diabetes.50.2.265. [DOI] [PubMed] [Google Scholar]

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