HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 540/1/387    most recent 2001.013912v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sacchetti, M. Articles by van Hall, G. Search for Related Content PubMed PubMed Citation Articles by Sacchetti, M. Articles by van Hall, G.

Journal of Physiology (2002), 540.1, pp. 387-395
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.013912

Intramuscular fatty acid metabolism in contracting and non-contracting human skeletal muscle

M. Sacchetti, B. Saltin, T. Osada and G. van Hall

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

The present study was undertaken to investigate the fate of blood-borne non-esterified fatty acids (NEFA) entering contracting and non-contracting knee extensor muscles of healthy young individuals. [U-¹³C]-palmitate was infused into a forearm vein during 5 h of one-legged knee extensor exercise at 40 % of maximal work capacity and the NEFA kinetics, oxidation and rate of incorporation into intramuscular triacylglycerol (mTAG) were determined for the exercising and the non-exercising legs. During 4 h of one-legged knee extensor exercise, mTAG content decreased by 30 % (P < 0.05) in the contracting muscle, whereas it was unchanged in the non-contracting muscle. The uptake of plasma NEFA, as well as the proportion directed towards oxidation, was higher in the exercising compared to the non-exercising leg, whereas the rate of palmitate incorporation into mTAG was fourfold lower (0.70 ± 0.14 vs. 0.17 ± 0.04 µmol (g dry wt)^-1 h^-1; P < 0.05), resulting in fractional synthesis rates of 1.0 ± 0.2 and 3.8 ± 0.9 % h^-1 (P < 0.01) for the contracting and non-contracting muscle, respectively. These findings demonstrate that mTAG in human skeletal muscle is continuously synthesised and degraded and that the metabolic fate of plasma NEFA entering the muscle is influenced by muscle contraction, so that a higher proportion is directed towards oxidation at the expense of storage in mTAG.

(Resubmitted 16 November 2001; accepted after revision 14 January 2002)
Corresponding author M. Sacchetti: The Copenhagen Muscle Research Centre, Rigshospitalet Section 7652, 9 Blegdamsvej, DK-2100 Copenhagen Ø, Denmark. Email: cmrc{at}rh.dk

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

Fatty acids represent an important energy source for skeletal muscle. During exercise, skeletal muscle becomes the most important tissue for fatty acid utilisation and therefore plays a crucial role for the homeostasis of fatty acids in the body.

Circulating non-esterified fatty acids (NEFA), as well as fatty acids originating from the hydrolysis of triacylglycerol transported in plasma lipoproteins, are extracted by skeletal muscle where they can be directed towards oxidation, esterified into complex lipids or remain free in the muscle (Gorski, 1992). In addition to plasma NEFA, fatty acids originating from intramuscular triacylglycerol (mTAG) hydrolysis can also be utilised by the muscle. Therefore, mTAG represents both the intracellular store of fatty acids and a potential source of fatty acids for energy generation. In response to exercise, mTAG content in men has been reported either to decrease (Carlson et al. 1971; Froberg & Mossfeld, 1971; Costill et al. 1973; Hurley et al. 1986) or to remain unchanged (Kiens et al. 1993; Kiens & Richter, 1998; Starling et al. 1997). One of the limitations of measuring only mTAG concentration is that no information is obtained relating to the kinetics (i.e. synthesis vs. degradation) of this lipid pool. The available evidence for the dynamic characteristics of mTAG comes mostly from studies on isolated rat muscle preparations (Hopp & Palmer, 1990; Dyck et al. 1997; Gorski & Bonen, 1997; Dyck & Bonen, 1998). These studies demonstrate that electrical stimulation increases the absolute amount of fatty acid incorporated into mTAG (Gorski & Bonen, 1997; Dyck & Bonen, 1998), and induces a change in the partitioning of intracellular fatty acids, favouring oxidation over esterification (Dyck & Bonen, 1998; Dyck et al. 2001). Although this view of the dynamic characteristics of the mTAG pool has recently been supported by observations in humans (Guo et al. 2000), no direct comparison between active and inactive muscle has been made and, hence, the effect of muscle contraction on intramuscular fatty acid metabolism in humans remains to be explored.

The aim of the present study was, therefore, to investigate the metabolic fate of plasma NEFA entering both contracting and non-contracting human skeletal muscle. To this end, the knee extensor exercise model was used, since it allows comparison between contracting and non-contracting muscles receiving blood with both the same NEFA concentration and the same hormonal levels. Using a combination of femoral arterial-venous difference, muscle biopsy and stable isotope methodology, plasma palmitate kinetics and oxidation in the leg, as well as the rate of palmitate incorporation into mTAG were determined.

We hypothesised that mTAG synthesis decreases in response to muscle contraction and that a larger proportion of the fatty acids entering the muscle from the circulation are utilised to support the augmented energy requirement of the contracting muscle.

	METHODS

Top Abstract Introduction Methods Results Discussion References

Subjects

Six healthy, active male volunteers (age 26 ± 2 years; weight 77 ± 1 kg; height 183 ± 1 cm) participated in the study. They were informed about the aim and possible risks of the study and gave their written consent to participate. The study was conducted according to Declaration of Helsinki Principles and the experimental protocol was approved by the Ethical Committee of Copenhagen-Frederiksberg.

Preliminary testing and diet

One week before the experiment, subjects visited the laboratory to familiarise themselves with the modified Krogh bicycle ergometer for the one-legged dynamic knee extensor exercise model. After a period of familiarisation, the subjects underwent an incremental test to determine their maximal leg work capacity (W_max). After 1 h of rest, the volunteers performed 3 h of knee extensor exercise at 40 % W_max,. On the same occasion, anthropometrical measurements of the thigh were taken in order to estimate the mass of the quadriceps muscle (Rådegran et al. 1999). In preparation for the experiment, subjects refrained from consuming food with high natural abundance of ¹³C, such as carbohydrate derived from C₄ plants (maize, cane sugar etc.) for 7 days in order to reduce the probability of shift in ¹³CO₂ enrichment during exercise (Wagenmakers et al. 1993). Subjects also refrained from participating in any strenuous physical activity for the 24 h preceding the trial.

Experimental procedure

On the day of the experiment, the subjects reported to the laboratory at 08.00 after an overnight fast. They rested for 10 min in a supine position then Teflon catheters (20G, Ohmeda, Wiltshire, UK) were inserted, under local anaesthesia (lidocaine 20 mg ml^-1), in a proximal direction in the femoral artery of one leg and in a distal direction in the femoral vein of both legs using the Seldinger technique. Distal cannulation of the femoral veins was performed as it provides better estimates than proximal positioning of the actual substrate exchange across the quadriceps muscles (van Hall et al. 1999). An additional catheter was placed in a forearm vein for isotope infusion. Throughout the experiment, the catheters were kept patent by intermittent flushing with 0.9 % saline. After resting for an additional 30 min in a supine position, subjects mounted the ergometer and blood samples were drawn simultaneously from the femoral artery and both femoral veins for background measurements. At the same time, expired pulmonary breath samples were collected (CPX, Medical Graphics, Spiropharma, Denmark) and blood flow in the femoral artery of both legs was measured by ultrasound Doppler (Rådegran, 1997). The subjects then commenced one-legged knee extensor exercise and continued for 5 h at the workload determined in the familiarisation trial. Care was taken to maintain the non-exercising leg inactive. Ten minutes after the start of exercise, a bolus of NaH¹³CO₃ (Cambridge Isotope Laboratories, Andover, USA; 1 µmol kg^-1) was administered to prime the bicarbonate pool and a constant infusion of [U-¹³C]-palmitate (Cambridge Isotope Laboratories, Andover, USA; 0.0174 µmol kg^-1 min^-1) was started using a volumetric infusion pump (model PC-1, IMEC, San Diego, CA, USA). The 10 min delay in administering the tracer infusion was adopted in order to reduce the consequences of CO₂ washout from the bicarbonate pools at the beginning of exercise (van Hall, 1999). Blood and breath sampling, indirect calorimetry and blood flow measurements were carried out after 1 h and every subsequent 30 min until the termination of exercise. At each sampling time point, a pneumatic cuff placed under the knee was inflated to a suprasystolic pressure in order to avoid mixing of femoral venous blood with the blood from the lower leg and shunting (van Hall et al. 1999). After the first 60 min, and at the end of the exercise, a muscle biopsy was obtained from the middle portion of the vastus lateralis muscle of both legs. The muscle specimens were immediately frozen (within 10 s of cessation of kicking) in liquid nitrogen and stored at -80 °C until further processing.

Blood analysis

Blood was collected into pre-chilled tubes containing 0.3 M EDTA (10 µl ml^-1 blood) and immediately centrifuged at 4 °C for 10 min to obtain plasma, which was stored at - 80 °C until analysis. Plasma NEFA concentration was determined using a Wako NEFA-C test kit (Wako Chemical, Neuss, Germany) on an automated analyser (Cobas Fara, Roche, Switzerland). Additional blood was collected anaerobically into heparinised syringes for the measurement of blood pH, P_CO2 and P_O2 (ABL5, Radiometer, Denmark), haemoglobin, oxygen saturation (OSM3 hemoximeter, Radiometer, Denmark) and haematocrit.

Plasma palmitate enrichment and concentration

For the measurement of plasma palmitate concentration and enrichment, NEFA were extracted and derivatised according to the method of Patterson et al. (1999). Briefly, heptadecanoic acid (30 nmol) was added, as an internal standard, to 200 µl plasma and the proteins were precipitated with ice-cold acetone. After centrifugation, lipids were extracted with hexane and the fatty acids were methylated with iodomethane (CH₃I). The samples were finally purified by solid phase extraction (SPE) chromatography, the eluted solution dried under a stream of nitrogen and the sample finally re-dissolved in hexane. Individual plasma NEFA concentration was measured with a gas chromatograph (Autosystem XL, Perkin-Elmer, Northwalk, CT, USA). Plasma palmitate ¹³C enrichment was measured by gas chomatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS, Hewlett Packard 5890-Finnigan GC combustion III-Finnigan Delta Plus, Finnigan MAT, Germany). Palmitate enrichment was corrected by a factor of 17/16 to account for the extra methyl group of the methyl palmitate derivative.

Muscle tissue analysis

Muscle samples were freeze-dried and dissected free of blood and visible connective tissue under a stereomicroscope. As recently shown in rat muscle, this procedure allows pure muscle samples to be obtained without contamination from extramyocellular fat (Guo et al. 2001). Shortly after dissection, heptadecanoic acid and tripentadecanoin were added to the muscle fibres (9-15 mg dry wt) as internal standards for the determination of intramuscular non-esterified fatty acid and TAG-fatty acid concentration, respectively. Lipids were then extracted twice with 2:1 chloroform-methanol (Folch, 1957) and the organic phase divided into two portions: two thirds were used for the analysis of intramuscular non-esterified fatty acids, which were methylated and purified as described for plasma NEFA, whereas the remaining one third was used for the analysis of fatty acids in mTAG (mTAG-FA). This aliquot was evaporated at 45 °C under a stream of nitrogen, re-dissolved in 1:1 chloroform-methanol solution and applied on a silica gel 60 plate (Merk, Germany). The lipids were separated with a mobile phase composed of petroleum ether : diethyl ether : acetic acid (120 : 25 : 1.5), then the plates were left to dry under nitrogen and sprayed with rhodamine 6G solution (0.01 % in methanol). The different lipid fractions were visualised under long-wave ultraviolet light and the TAG band identified by means of TAG standards run on separate lanes of the same plate and scraped off into glass tubes. TAG was saponified with ethanolic KOH (1 M in 95 % ethanol) at 70 °C for 1 h and then acidified with 2.5 M H₂SO₄. TAG fatty acids (TAG-FA) were extracted twice with hexane, which was then evaporated under a stream of nitrogen. Methylation of the fatty acids was carried out with methanol iso-octane solution (4:1 v/v) and acetyl chloride at 100 °C for 60 min. Six per cent potassium carbonate was then added to stop the methylation reaction and neutralise the mixture. After centrifugation, the upper layer was transferred to a new tube, evaporated under nitrogen and the fatty acid methyl esters were finally re-dissolved in hexane and transferred into vials for concentration and enrichment analysis.

GC-C-IRMS analysis

Palmitate ¹³C-enrichment in mTAG and enrichment of non-esterified palmitate in muscle was measured by GC-C-IRMS (Hewlett Packard 5890-Finnigan GC combustion III-Finnigan Delta Plus, Finnigan MAT, Germany) equipped with a programmed temperature vaporisation (PTV) inlet (model G2617A, Agilent Technologies, Wilmington, DE, USA). Measurement of the concentration of non-esterified palmitate in muscle was carried out by measuring the ratio between the area of the CO₂ curve originating from the combustion of palmitate and that generated by the combustion of the internal standard heptadecanoic acid.

For assessment of ¹³C enrichment in arterial and venous blood CO₂, 0.5 ml of blood were collected into 10 ml vacutainer tubes and the CO₂ liberated into the headspace by addition of 0.5 ml of 1 M H₂SO₄. The ¹³C/¹²C ratio in blood CO₂ was determined by GC-C-IRMS.

Fatty acid profile in mTAG and mTAG concentration

Fatty acid profile in mTAG was determined by GC (Autosystem XL, Perkin-Elmer, Nothwalk, CT, USA). The concentration of TAG-FA was determined by comparing the area of the peak of each individual fatty acid with that generated by the tripentadecanoin-derived fatty acid standard. Intramuscular TAG concentration was determined by adding the concentrations of the six major fatty acids in the TAG molecule (14 : 0, 16 : 0, 16 : 1, 18 : 0, 18 : 1, 18 : 2) which represent more than 98 % of the total TAG-FA pool (Andersson et al. 2000).

Calculations

The [U-¹³C]-palmitate infusion rate was determined by multiplying the infusion rate by the measured concentration of palmitate in the infusate. Enrichment was expressed as tracer to tracee ratio (TTR), calculated as:

TTR = (¹³C/¹²C)_sa - (¹³C/¹²C)_bk,

where 'sa' and 'bk' are the sample and background (pre-exercise) values, respectively.

Leg NEFA balance, kinetics and oxidation

Net NEFA balance across the leg was calculated by multiplying the femoral arterial-venous concentration difference by plasma flow, computed as blood flow (1 - haematocrit)/100. Leg fractional extraction of palmitate was computed as:

where C_a and E_a, and C_v and E_v are the concentration and the tracer enrichment of palmitate (TTR) in the femoral artery and the femoral vein, respectively. Palmitate uptake was then calculated as:

Uptake = fractional extraction C_a plasma flow,

and palmitate release as:

Release = uptake - net balance.

By knowing the concentration and the ¹³C enrichment of CO₂ in arterial and femoral venous blood it was possible to calculate the portion of the leg palmitate uptake that was oxidized. This was computed as:

% palmitate uptake oxidised =

where C_a,CO2 and E_a,CO2, and C_v,CO2 and E_v,CO2, represent the concentration and the ¹³C enrichment of blood CO₂ in the femoral artery and femoral vein, respectively, and a_r is the fractional recovery of acetate across the leg. ¹³CO₂ production is divided by 16 in order to account for the fact that 1 mol of [U-¹³C]-Palmitate when oxidised gives 16 mol of ¹³CO₂. For the correction of plasma palmitate oxidation in the leg for isotopic exchange reactions, an acetate carbon recovery of 100 % for the exercising leg and 60 % for the non-exercising leg was assumed. The choice of these values was based on the results of a series of experiments performed in our laboratory where acetate recovery was assessed for both legs during one-legged knee extensor exercise using an unprimed [1,2-¹³C]acetate infusion (G. van Hall, M. Sacchetti & G. Rådegran, unpublished observations).

Finally, leg palmitate oxidation was calculated as the product of palmitate uptake and percentage palmitate uptake oxidised.

Intramuscular TAG fractional synthesis rate (FSR)

Fractional synthesis rate of mTAG was calculated as the ratio between the difference in ¹³C-palmitate enrichment in mTAG at time 5 h (t₂) and 1 h (t₁) and the average enrichment of the intramuscular non-esterified palmitate over the same period of time. This assumes the intramuscular NEFA is the precursor pool for TAG synthesis (Guo & Jensen, 1998). Therefore:

where E_TAG-palm and E_NEpalm are the ¹³C enrichments of palmitate in mTAG and of intramuscular non-esterified palmitate, respectively.

In order to verify the effect of choosing different precursors for the determination of mTAG FSR the calculation was repeated using the enrichment of venous plasma palmitate, which was therefore substituted in the denominator of the above formula.

Absolute rate of palmitate incorporation into m TAG and TAG synthesis

The absolute rate of incorporation of plasma palmitate into mTAG was calculated as the product of the TAG FSR and the TAG-palmitate pool size.

Statistics

Data are expressed as means ± S.E.M. Differences between values at the different time points and between the two legs were analysed by using a two-factor repeated-measures analysis of variance (ANOVA). When ANOVA revealed a significant effect a Student-Newman-Keuls test was used to isolate the differences. Significance was accepted at P < 0.05.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Characterization of the exercise stimulus

During the 5 h of one-legged knee extensor exercise, the workload was set at 40 % of the maximal exercise capacity and averaged 30 W (range 25-35 W). In response to exercise, the blood flow in the exercising leg increased (P < 0.01) from the resting value of 0.39 ± 0.08 l min^-1 to an average of 2.5 l min^-1. The blood flow in the non-exercising leg was similar to that measured in the exercising leg at rest and doubled while the contralateral leg was exercising. The oxygen uptake across the exercising leg remained stable throughout the exercise at ~330 ml min^-1. This was five times higher than that observed in the non-exercising leg (P < 0.01).

Arterial palmitate concentration, leg palmitate kinetics and leg palmitate oxidation

The post-absorptive arterial palmitate concentration was 97 ± 17 µmol l^-1, and increased three-fold throughout the 5 h of exercise (Fig. 1A). The fractional extraction of plasma palmitate in the non-exercising leg decreased from 41 ± 5.9 % after 1 h to 21.9 ± 4.4 % after 5 h, while it was maintained in the non-exercising leg at a value of ~20 %(Fig. 2A).

		View larger version [in this window] [in a new window]
Figure 1. Arterial concentration and net palmitate balance Arterial palmitate concentration (A) and net palmitate uptake in the exercising and non-exercising leg (B) during 5 h of one-legged knee extensor exercise at 40 % Wmax. # P < 0.05 vs. inactive leg (n = 6).

		View larger version [in this window] [in a new window]
Figure 2. Leg palmitate kinetics Leg palmitate fractional extraction (A), palmitate oxidation (B) and tracer calculated uptake (C) and release (D) in the exercising and non-exercising leg during one-legged knee extensor exercise at 40 % Wmax. # P < 0.05 vs. inactive leg ; * P < 0.05 vs. 1 h (n = 6).

A net palmitate uptake was observed in both legs and was about four times higher in the exercising leg than in the non-exercising leg at the end of the exercise (Fig. 1B).

The unidirectional palmitate uptake increased in the exercising leg throughout the last 4 h of exercise while it remained constant in the non-exercising leg (Fig. 2C). Leg palmitate release was also higher in the exercising leg compared to the non-exercising leg, although the difference was substantially lower than that observed for the uptake (Fig. 2D).

The rate of plasma palmitate oxidation in the exercising leg was 34 ± 5 µmol min^-1 after 60 min of exercise and reached 79 ± 9 µmol min^-1 (P < 0.01) at the end (Fig. 2B). Palmitate oxidation in the non-exercising leg was substantially lower compared to the exercising leg, and did not change significantly over time. The fraction of the palmitate taken up directed towards oxidation was about 85 % after 60 min and showed a tendency to a slight increase towards the end of exercise. Similarly, the proportion of the palmitate taken up and oxidised in the non-exercising leg showed a tendency to a slight increase as the exercise proceeded, and was approximately 50 % towards the end of the exercise period.

Intramuscular non-esterified palmitate

The concentration of non-esterified palmitate in vastus lateralis muscle was lower in the exercising compared to the non-exercising leg (P < 0.05) and was augmented at the termination of the exercise compared with the concentration after 60 min (P < 0.05) (Table 1). The intramuscular non-esterified palmitate enrichment (Table 1) was similar in the two legs after 60 min and was significantly increased at the end of the 5 h period (P < 0.05).

mTAG concentration and ¹³C-enrichment in mTAG-palmitate

During the last 4 h of exercise mTAG content in the vastus lateralis muscle of the exercising leg decreased by 30 % (P < 0.05), whereas no significant change was observed in the non-exercising leg (Table 1). Labelled palmitate was found in mTAG of the vastus lateralis of both legs, indicating that part of the circulating NEFA taken up by skeletal muscle were directed towards esterification. The ¹³C-enrichment in intramuscular TAG-palmitate was approximately the same in both legs after 1 h of exercise and significantly augmented at the end of the study period (Table 1), although the rate of increase between 1 and 5 h was greater in the non-exercising than in the exercising leg. Palmitate ¹³C-enrichment in mTAG in both legs was substantially lower than that measured in the non-esterified fatty acid pool, which, in turn, was lower than that observed in the femoral vein (Table 1).

Intramuscular TAG fractional synthesis rate and absolute rate of palmitate incorporation into mTAG

The fractional synthesis rate of mTAG was determined between 1 and 5 h of exercise (Fig. 3). We have assumed that the intramuscular palmitate enrichment represents the appropriate enrichment of the precursor pool. However, in order to compare the effect of using different precursor pools, mTAG FSR was calculated using either the enrichment of the intramuscular non-esterified palmitate or the enrichment of plasma palmitate in the femoral vein. When considering the intramuscular NEFA pool as the precursor for mTAG synthesis, FSR was approximately four times higher in the non-exercising than in the active muscle (P < 0.001), which translated into a four-fold increase in absolute rate of palmitate incorporation into mTAG in the non-contracting compared to the contracting muscle (Fig. 4). Fractional synthesis rate of mTAG calculated by using the venous palmitate enrichment was substantially lower than that obtained with the intramuscular palmitate enrichment either for the exercising (0.33 ± 0.11 % h^-1 vs. 1.03 ± 0.27 % h^-1) or the non-exercising muscle (0.91 ± 0.24 % h^-1 vs. 3.81 ± 0.91 % h^-1). However, despite the different FSR values obtained when using the different precursor pools, the relative difference between the mTAG FSR in the two legs was only slightly affected.

		View larger version [in this window] [in a new window]
Figure 3. mTAG fractional synthesis rate Fractional synthesis rate (FSR) of intramuscular TAG in vastus lateralis muscle of the exercising and the non exercising leg, between the end of the first hour and the end of 5 h one-legged knee extensor exercise at 40 % Wmax. The FSR values were calculated using either the enrichment of the non-esterified palmitate in muscle (mNE-Palm) or the plasma palmitate enrichment in the femoral vein for enrichment of the presursor pool. # P < 0.05 vs. non-exercising leg.

		View larger version [in this window] [in a new window]
Figure 4. Rate of palmitate incorporation into mTAG Absolute rate of palmitate incorporation into mTAG in vastus lateralis muscle of the exercising and the non-exercising leg between the end of the first hour and the end of 5 h of one-legged knee extensor exercise at 40 % Wmax. # P < 0.05 vs. inactive leg.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The present study demonstrates that the intramuscular TAG pool in human skeletal muscle is simultaneously synthesised and degraded and that muscle contraction results in a reduction of the rate of plasma fatty acid esterification into mTAG.

In order to investigate the effect of muscle contraction on the intramuscular fate of circulating NEFA entering a muscle it is important to reduce the influence of factors other than muscle activity. Indeed, hormonal factors affect both mTAG synthesis (Coleman et al. 2000) and degradation (Holm et al. 2000). In fact, adrenaline activates hormone-sensitive lipase (HSL)-mediated mTAG hydrolysis in both rat (Langfort et al. 1999) and human (Kjær et al. 2000) skeletal muscle and insulin increases fatty acid esterification into mTAG in non-contracting and electrically stimulated isolated rat skeletal muscle (Dyck et al. 2001). In addition to the effect of hormones, the level of plasma NEFA has also been suggested to influence the rate of mTAG synthesis, as indicated by an increase in mTAG content following acute elevation of plasma NEFA concentration (Boden et al. 2001). Although we have removed these confounding effects, by using the one-legged knee extensor exercise model, it should be noted that the observations made in the non-exercising leg do not represent the situation occurring in a purely resting muscle, as indicated by the doubling of leg blood flow and oxygen uptake in response to exercise performed by the contralateral leg. Nonetheless, the data provide novel information regarding intramuscular fatty acid metabolism in humans.

The presence of ¹³C-labelled palmitate detected in TAG of the vastus lateralis muscle indicates that circulating NEFA are used as a substrate for mTAG synthesis. Furthermore, the comparison of the rate of palmitate incorporation into mTAG in the vastus lateralis muscle of the two legs shows this process to be significantly influenced by muscle contraction, being four times higher in the non-contracting than the contracting muscle. The lower rate of plasma NEFA esterification in the contracting muscle is consistent with previously reported observations in rat muscle preparations (Hopp & Palmer, 1990). More recent investigations in isolated rat skeletal muscle have also indicated that electrical stimulation decreases the proportion of fatty acids taken up by the muscle directed toward esterification into TAG while the percentage directed toward oxidation increases (Dyck & Bonen, 1998; Dyck et al. 2001).

We have estimated the percentage of palmitate taken up from the circulation and esterified into mTAG by extrapolating the palmitate incorporation rate measured in the muscle biopsies to the total muscle mass of the upper leg, since the circulation was occluded below the knee during the measurements. During knee extensor exercise, the hamstring muscles are nearly inactive, especially at the low intensity utilised in the present study, whereas the knee extensor muscles are nearly fully activated (van Hall et al. 1999). In order to account for the difference in metabolic activation of the different muscles in the exercising leg, the palmitate incorporation rate as measured in the active vastus lateralis muscle was used to extrapolate the total palmitate incorporation into the active muscles, whereas the rate of palmitate incorporation into the non-contracting muscles was used for the remaining upper leg muscles (hamstrings, adductors, sartorius). With this approach, the total amount of palmitate incorporated into mTAG of the active muscle during 4 h of knee extensor exercise could account for ~10 % of the total leg palmitate uptake. This is consistent with the average amount of labelled palmitate recovered as ¹³CO₂ during the same period, which represented ~85 % of the total amount of label taken up. In contrast, in the non-contracting muscle, the rate of palmitate esterification into mTAG could account for ~50 % of the palmitate taken up from the circulation, which is consistent with the percentage of palmitate uptake directed toward oxidation. Therefore, the present results demonstrate that, in response to muscle contraction, the metabolic fate of the fatty acids entering skeletal muscle changes, so that a higher proportion is directed towards oxidation at the expense of storage in mTAG.

The content of mTAG in the contracting muscle decreased by 30 % during the last 4 h of exercise, whereas it was unchanged in the non-contracting muscle. The decline in mTAG content in the vastus lateralis of the exercising leg was observed in all subjects. The evidence of an active incorporation of fatty acids into TAG, coupled with no net change in size of this lipid pool in the non-contracting muscle, implies that mTAG was simultaneously synthesised and hydrolysed and that these two processes were occurring at the same rate. In contrast, in the contracting muscle the process of degradation prevailed over synthesis, and the content of mTAG decreased. It appears, therefore, that in response to an augmented energy requirement, mTAG stores are controlled to function as a source of fatty acids. This is evidenced by a decrease in synthesis and, potentially, by an increase in degradation. The inhibition of the process of NEFA esterification during muscle contraction is illustrated by the difference in fractional synthesis rate of mTAG observed between the two legs. This was 3.8 % h^-1 in the non-contracting muscle and 1 % h^-1 in the contracting muscle. These values give an indication of the dynamic characteristics of this pool, as also indicated by previous studies in rat muscle preparations (Budohoski et al. 1996; Dyck et al. 1997; Guo & Jensen, 1998) and in exercising humans (Guo et al. 2000).

Importantly, however, we have considered the pool of non-esterified fatty acids as a precursor for mTAG synthesis, whereas in the previous study conducted in humans the plasma NEFA pool was used (Guo et al. 2000).

It is crucial when calculating the synthesis rate of mTAG that the labelling of the appropriate precursor pool is considered. For this reason, and in accordance with previous work in rat muscle (Guo & Jensen, 1998), we have characterised the change in enrichment of the intramuscular non-esterified palmitate, since it reflects the immediate precursor for mTAG synthesis (palmitoyl-Coa) more appropriately. It is clear that using plasma palmitate to calculate FSR would underestimate the values because of the difference in enrichment between plasma and intramuscular non-esterified palmitate. In the present study using the palmitate enrichment in the femoral vein as enrichment of the precursor pool would have resulted in a fourfold reduction of TAG FSR. However, even when using the less appropriate venous palmitate enrichment, the relative difference in mTAG synthesis rate between the two legs was still apparent, although reduced by ~30 %. This lower difference is probably a reflection of the fact that during exercise, shunting and a reduced NEFA fractional extraction by the leg muscles exaggerate the difference in palmitate enrichment between the intracellular and the venous compartments. The difference observed in mTAG FSR when either the venous or the intracellular enrichment were considered reinforces the finding of a lower plasma NEFA esterification in contracting versus non-contracting skeletal muscle.

Another assumption when calculating mTAG FSR it is that once labelled palmitate is incorporated into the mTAG pool, it does not reappear via mTAG hydrolysis. Should this actually occur, the amount of label incorporated into TAG would be underestimated and so would the calculated FSR. The problem related to reappearance of the label is more consistent for the higher the rates of turnover of the pool. This could give cause for concern in the present study due to the observation of a net TAG degradation in the contracting but not in the non-contracting muscle, which may imply that label reappearance was more likely to occur in the former than in the latter. On the other hand, the fourfold increase of mTAG FSR in the non-contracting muscle enhances the probability of more label reappearing from mTAG hydrolysis in the non-exercising than the exercising leg. Therefore, the absolute FSR values should be taken to be minimum estimates.

The analysis of NEFA in skeletal muscle provides information on the kinetics and metabolic fate of the plasma NEFA during and after crossing the plasma membrane. It is acknowledged that, by influencing the concentration gradient between the vascular space and the cytosol of the myocyte, the intracellular NEFA concentration is partly responsible for the degree to which NEFA cross the plasma membrane (van Der Vusse & Reneman, 1996). When comparing the results from the present work with those obtained in the few previous studies in humans, it appears that the concentration of intramuscular non-esterified fatty acids in the present investigation is markedly lower than that reported by Morgan et al. (1969), whereas it is only slightly higher than the values reported by Kiens et al. (1999). The latter difference, however, is no longer apparent when the data is normalised for the higher arterial plasma NEFA concentration. In keeping with the theory that a positive concentration gradient has to be present between the artery and the intracellular compartment in order to promote an uptake of NEFA by the muscle, our data indicate that this gradient was present in both legs after 1 h, and that this was higher in the exercising compared to the non-exercising leg. After 5 h of exercise, the intracellular concentration of palmitate in muscle was significantly increased compared to the level after 1 h; however, this increase was smaller than that recorded in the artery. This implies that the driving force for passive diffusion from the circulation into the muscle increased over time, which is in accordance with the augmented NEFA uptake observed.

It has been proposed that the intracellular concentration of non-esterified fatty acid mirrors the balance between fatty acid supply and metabolism (Kiens et al. 1999). In fact, the intramuscular non-esterified pool reflects the result of several processes, namely, the influx of fatty acid from the circulation, the rate of fatty acid oxidation, the rate of fatty acid deposition into complex lipids and finally the rate at which fatty acids are made available from mTAG hydrolysis. The intramuscular palmitate content was significantly lower in the exercising than in the non-exercising leg, after both one and after 5 h. It would therefore appear that the higher fatty acid oxidation in the contracting muscle induces a reduction of the intramuscular NEFA pool, which in turn results in a higher plasma NEFA uptake due to the enhancement of the concentration gradient between the vascular and the intracellular compartment. In addition, the reduced intramuscular NEFA concentration may induce a reduction in mTAG synthesis rate and an increase in mTAG hydrolysis by mass action.

General overview and conclusions

Our data provide clear evidence that mTAG synthesis and hydrolysis are influenced by the metabolic requirement of the muscle. In the non-contracting skeletal muscle, about half of the plasma NEFA entering the muscle are utilised for esterification into mTAG and half are oxidised. In these conditions, a relatively high rate of mTAG synthesis is balanced by an equal rate of degradation, resulting in a maintained mTAG content. In response to contraction and as the exercise continues, the uptake of NEFA from the circulation increases, in part due to the increasing concentration gradient between the vascular compartment and the muscle. With muscle contraction a greater proportion of the fatty acids taken up from the circulation is directed toward oxidation, whereas the proportion esterified into mTAG is markedly reduced. The decrease of mTAG synthesis rate in the contracting muscle is not accompanied by a proportional reduction in mTAG hydrolysis, leading to a diminution of mTAG content. As a result, additional fatty acids are made available for -oxidation, supporting the augmented energy request of the muscle.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

ANDERSSON, A., SJODIN, A., HEDMAN, A., OLSSON, R. & VESSBY, B. (2000). Fatty acid profile of skeletal muscle phospholipids in trained and untrained young men. American Journal of Physiology 279, E744-751
BODEN, G., LEBED, B., SCHATZ, M., HOMKO, C. & LEMIEUX, S. (2001). Effects of acute changes of plasma free fatty acids on intramyocellular fat content and insulin resistance in healthy subjects. Diabetes 50, 1612-1617	[Abstract/Full Text]
BUDOHOSKI, L., GORSKI, J., NAZAR, K., KACIUBA-USCILKO, H. & TERJUNG, R. L. (1996). Triacylglycerol synthesis in the different skeletal muscle fiber sections of the rat. American Journal of Physiology 271, E574-581	[Medline]
CARLSON, L. A., EKELUND, L. G. & FRÖBERG, S. O. (1971). Concentration of triglycerides, phospholipids and glycogen in skeletal muscle and of free fatty acids and beta-hyroxybutyric acid in blood in man in response to exercise. Acta Physiologica Scandinavica 89, 374-383
COLEMAN, R. A., LEWIN, T. M. & MUOIO, D. M. (2000). Physiological and nutritional regulation of enzymes of triacylglycerol synthesis. Annual Review of Nutition 20, 77-103
COSTILL, D. L., GOLLNICK, P. D., JANSSON, E. D., SALTIN, B. & STEIN, E. M. (1973). Glycogen depletion pattern in human muscle fibres during distance running. Acta Physiologica Scandinavica 89, 374-383	[Medline]
DYCK, D. J. & BONEN, A. (1998). Muscle contraction increases palmitate esterification and oxidation and triacylglycerol oxidation. American Journal of Physiology 275, E888-896	[Medline]
DYCK, D. J., PETERS, S. J., GLATZ, J., GORSKI, J., KEIZER, H., KIENS, B., LIU S., RICHTER, E. A., SPRIET, L. L., VAN DER VUSSE, G. J. & BONEN, A. (1997). Functional differences in lipid metabolism in resting skeletal muscle of various fiber types. American Journal of Physiology 272, E340-351	[Medline]
DYCK, D. J., STEINBERG, G. & BONEN, A. (2001). Insulin increases FA uptake and esterification but reduces lipid utilization in isolated contracting muscle. American Journal of Physiology 281, E600-607
FOLCH, J., LEES, M. & STANLEY, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497-510
FROBERG, S. O. & MOSSFELDT, F. (1971). Effect of prolonged strenuous exercise on the concentration of triglycerides, phospholipids and glycogen in muscle of man. Acta Physiologica Scandinavica 82, 167-171	[Medline]
GORSKI, J. (1992). Muscle triglyceride metabolism during exercise. Canadian Journal of Physiology and Pharmacology 70, 123-131
GORSKI, J. & BONEN, A. (1997). Palmitate incorporation into lipids pools of contracting red and white muscles. Molecular and Cellular Biochemistry 166, 73-83	[Medline]
GUO, Z., BURGUERA, B. & JENSEN, M. D. (2000). Kinetics of intramuscular triglyceride fatty acids in exercising humans. Journal of Applied Physiology 89, 2057-2064
GUO, Z. & JENSEN, M. D. (1998). Intramuscular fatty acid metabolism evaluated with stable isotopic tracers. Journal of Applied Physiology 84, 1674-1679
GUO, Z., MISHRA, P. & MACURA, S. (2001). Sampling the intramyocellular triglycerides from skeletal muscle. Journal of Lipid Research 42, 1041-1048	[Abstract/Full Text]
HOLM, C., OSTERLUND, T., LAURELL, H. & CONTRERAS, J. A. (2000). Molecular mechanisms regulating hormone-sensitive lipase and lipolysis. Annual Review of Nutrition 20, 365-393	[Abstract/Full Text]
HOPP, J. F. & PALMER, W. K. (1990). Electrical stimulation alters fatty acid metabolism in isolated skeletal muscle. Journal of Applied Physiology 68, 2473-2481
HURLEY, B. F., NEMETH, P. M., MARTIN, W. H. 3RD, HAGBERG, J. M., DALSKY, G. P. & HOLLOSZY, J. O. (1986). Muscle triglyceride utilization during exercise: effect of training. Journal of Applied Physiology 60, 562-567
KIENS, B., ESSEN-GUSTAVSSON, B., CHRISTENSEN, N. J. & SALTIN, B. (1993). Skeletal muscle substrate utilization during submaximal exercise in man: effect of endurance training. Journal of Physiology 469, 459-478	[Abstract]
KIENS, B. & RICHTER, E. A. (1998). Utilization of skeletal muscle triacylglycerol during postexercise recovery in humans. American Journal of Physiology 275, E332-337
KIENS, B., ROEMEN, T. H. & VAN DER VUSSE, G. J. (1999). Muscular long-chain fatty acid content during graded exercise in humans. American Journal of Physiology 276, E352-357	[Medline]
KJAER, M., HOWLETT, K., LANGFORT, J., ZIMMERMAN-BELSING, T., LORENTSEN, J., BULOW, J., IHLEMANN, J., FELDT-RASMUSSEN, U. & GALBO, H. (2000). Adrenaline and glycogenolysis in skeletal muscle during exercise: a study in adrenalectomised humans. Journal of Physiology 528, 371-378	[Abstract/Full Text]
LANGFORT, J., PLOUG, T., IHLEMANN, J., SALDO, M., HOLM, C.& GALBO, H. (1999). Expression of hormone-sensitive lipase and its regulation by adrenaline in skeletal muscle. Biochemical Journal 340, 459-465	[Medline]
MORGAN, T. E., SHORT, F. A. & COBB, L. A. (1969). Effect of long-term exercise on skeletal muscle lipid composition. American Journal of Physiology 216, 82-86	[Medline]
PATTERSON, B. W., ZHAO, G., ELIAS, N., HACHEY, D. L. & KLEIN, S. (1999). Validation of a new procedure to determine plasma fatty acid concentration and isotopic enrichment. Journal of Lipid Research 40, 2118-2124	[Abstract/Full Text]
RÅDEGRAN, G. (1997). Ultrasound Doppler estimates of femoral artery blood flow during dynamic knee extensor exercise in humans. Journal of Applied Physiology 83, 1383-1388	[Abstract/Full Text]
RÅDEGRAN, G., BLOMSTRAND, E. & SALTIN, B. (1999). Peak muscle perfusion and oxygen uptake in humans: importance of precise estimates of muscle mass. Journal of Applied Physiology 87, 2375-2380	[Abstract/Full Text]
STARLING, R. D., TRAPPE, T. A., PARCELL, A. C., KERR, C. G., FINK, W. J. & COSTILL, D. L. (1997). Effects of diet on muscle triglyceride and endurance performance. Journal of Applied Physiology 82, 1185-1189	[Abstract/Full Text]
VAN DER VUSSE, G. J. & RENEMAN, R. S. (1996). Lipid metabolism in muscle. In Handbook of Physiology, section 12, Exercise: Regulation and Integration of Multiple Sytems, ed. ROWELL, L. B. & SHEPARD, J. T., pp. 952-994. Oxford Univeristy Press, New York
VAN HALL, G. (1999). Correction factors for 13C-labelled substrate oxidation at whole-body and muscle level. Proceedings of the Nutrition Society 58, 979-86	[Medline]
VAN HALL, G., GONZALEZ-ALONSO, J., SACCHETTI, M. & SALTIN, B. (1999). Skeletal muscle substrate metabolism during exercise: methodological considerations. Proceedings of the Nutrition Society 58, 899-912	[Medline]
WAGENMAKERS, A. J., REHRER, N. J., BROUNS, F., SARIS, W. H. & HALLIDAY, D. (1993). Breath 13CO2 background enrichment during exercise: diet related differenced between Europe and America. Journal of Applied Physiology 74, 2353-2357	[Medline]

Acknowledgements

We thank Dr Mark Febbraio for his help in the preparation of the manuscript. The Copenhagen Muscle Research Centre is funded by a grant from the Danish National Research Foundation (grant 504-14).

This article has been cited by other articles:

				L. Yao, M. J. Delmonico, S. M. Roth, B. D. Hand, J. Johns, J. Conway, L. Douglass, and B. F. Hurley Adrenergic Receptor Genotype Influence on Midthigh Intermuscular Fat Response to Strength Training in Middle-Aged and Older Adults J. Gerontol. A Biol. Sci. Med. Sci., June 1, 2007; 62(6): 658 - 663. [Abstract] [Full Text] [PDF]

				T. J. Horton, E. K. Miller, and K. Bourret No effect of menstrual cycle phase on glycerol or palmitate kinetics during 90 min of moderate exercise J Appl Physiol, March 1, 2006; 100(3): 917 - 925. [Abstract] [Full Text] [PDF]

				B. Kiens Skeletal Muscle Lipid Metabolism in Exercise and Insulin Resistance Physiol Rev, January 1, 2006; 86(1): 205 - 243. [Abstract] [Full Text] [PDF]

				T. W. Zderic and M. T. Hamilton Physical inactivity amplifies the sensitivity of skeletal muscle to the lipid-induced downregulation of lipoprotein lipase activity J Appl Physiol, January 1, 2006; 100(1): 249 - 257. [Abstract] [Full Text] [PDF]

				K De Bock, E. A Richter, A. P Russell, B. O Eijnde, W Derave, M Ramaekers, E Koninckx, B Leger, J Verhaeghe, and P Hespel Exercise in the fasted state facilitates fibre type-specific intramyocellular lipid breakdown and stimulates glycogen resynthesis in humans J. Physiol., April 15, 2005; 564(2): 649 - 660. [Abstract] [Full Text] [PDF]

				M. Sacchetti, B. Saltin, D. B Olsen, and G. van Hall High triacylglycerol turnover rate in human skeletal muscle J. Physiol., December 15, 2004; 561(3): 883 - 891. [Abstract] [Full Text] [PDF]

				L. J. C. van Loon Use of intramuscular triacylglycerol as a substrate source during exercise in humans J Appl Physiol, October 1, 2004; 97(4): 1170 - 1187. [Abstract] [Full Text] [PDF]

				M. J. Watt, G. J. F. Heigenhauser, P. J. LeBlanc, J. G. Inglis, L. L. Spriet, and S. J. Peters Rapid upregulation of pyruvate dehydrogenase kinase activity in human skeletal muscle during prolonged exercise J Appl Physiol, October 1, 2004; 97(4): 1261 - 1267. [Abstract] [Full Text] [PDF]

				R. J. Sigal, G. P. Kenny, D. H. Wasserman, and C. Castaneda-Sceppa Physical Activity/Exercise and Type 2 Diabetes Diabetes Care, October 1, 2004; 27(10): 2518 - 2539. [Full Text] [PDF]

				C. Roepstorff, B. Vistisen, K. Roepstorff, and B. Kiens Regulation of plasma long-chain fatty acid oxidation in relation to uptake in human skeletal muscle during exercise Am J Physiol Endocrinol Metab, October 1, 2004; 287(4): E696 - E705. [Abstract] [Full Text] [PDF]

				M. J. Watt, A. G. Holmes, G. R. Steinberg, J. L. Mesa, B. E. Kemp, and M. A. Febbraio Reduced plasma FFA availability increases net triacylglycerol degradation, but not GPAT or HSL activity, in human skeletal muscle Am J Physiol Endocrinol Metab, July 1, 2004; 287(1): E120 - E127. [Abstract] [Full Text] [PDF]

				S. R. Stannard and N. A. Johnson Insulin resistance and elevated triglyceride in muscle: more important for survival than 'thrifty' genes? J. Physiol., February 1, 2004; 554(3): 595 - 607. [Abstract] [Full Text] [PDF]

				M. J. Watt, P. Krustrup, N. H. Secher, B. Saltin, B. K. Pedersen, and M. A. Febbraio Glucose ingestion blunts hormone-sensitive lipase activity in contracting human skeletal muscle Am J Physiol Endocrinol Metab, January 1, 2004; 286(1): E144 - E150. [Abstract] [Full Text]

				V. B. Schrauwen-Hinderling, L. J. C. van Loon, R. Koopman, K. Nicolay, W. H. M. Saris, and M. E. Kooi Intramyocellular lipid content is increased after exercise in nonexercising human skeletal muscle J Appl Physiol, December 1, 2003; 95(6): 2328 - 2332. [Abstract] [Full Text]

				J. W. Helge and F. Dela Effect of Training on Muscle Triacylglycerol and Structural Lipids: A Relation to Insulin Sensitivity? Diabetes, August 1, 2003; 52(8): 1881 - 1887. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 540/1/387    most recent 2001.013912v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sacchetti, M. Articles by van Hall, G. Search for Related Content PubMed PubMed Citation Articles by Sacchetti, M. Articles by van Hall, G.