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Am J Physiol Endocrinol Metab 291: E656-E665, 2006. First published May 9, 2006; doi:10.1152/ajpendo.00020.2006
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Endurance training has little effect on active muscle free fatty acid, lipoprotein cholesterol, or triglyceride net balances

Kevin. A. Jacobs,1 Ronald M. Krauss,1 Jill A. Fattor,1 Michael A. Horning,1 Anne L. Friedlander,2 Timothy A. Bauer,3 Todd A. Hagobian,2 Eugene E. Wolfel,3 and George A. Brooks1

1University of California, Berkeley; 2Clinical Studies Unit, Veterans Affairs Palo Alto Health Care System, Palo Alto, California; and 3University of Colorado Health Sciences Center, Division of Cardiology, Denver, Colorado

Submitted 13 January 2006 ; accepted in final form 1 May 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We evaluated the hypothesis that net leg total FFA, LDL-C, and TG uptake and HDL-C release during moderate-intensity cycling exercise would be increased following endurance training. Eight sedentary men (26 ± 1 yr, 77.4 ± 3.7 kg) were studied in the postprandial state during 90 min of rest and 60 min of exercise twice before (45% and 65% VO2 peak) and twice after 9 wk of endurance training (55% and 65% posttraining VO2 peak). Measurements across an exercising leg were taken to be a surrogate for active skeletal muscle. To determine limb lipid exchange, femoral arterial and venous blood samples drawn simultaneously at rest and during exercise were analyzed for total and individual FFA (e.g., palmitate, oleate), LDL-C, HDL-C, and TG concentrations, and limb blood flow was determined by thermodilution. The transition from rest to exercise resulted in a shift from net leg total FFA release (–44 ± 16 µmol/min) to uptake (193 ± 49 µmol/min) that was unaffected by either exercise intensity or endurance training. The relative net leg release and uptake of individual FFA closely resembled their relative abundances in the plasma with ~21 and 41% of net leg total FFA uptake during exercise accounted for by palmitate and oleate, respectively. Endurance training resulted in significant changes in arterial concentrations of HDL-C (49 ± 5 vs. 52 ± 5 mg/dl, pre vs. post) and LDL-C (82 ± 9 vs. 76 ± 9 mg/dl, pre vs. post), but there was no net TG or LDL-C uptake or HDL-C release across the resting or active leg before or after endurance training. In conclusion, endurance training favorably affects blood lipoprotein profiles, even in young, healthy normolipidemic men, but muscle contractions per se have little effect on net leg LDL-C, or TG uptake or HDL-C release during moderate-intensity cycling exercise. Therefore, the favorable effects of physical activity on the lipid profiles of young, healthy normolipidemic men in the postprandial state are not attributable to changes in HDL-C or LDL-C exchange across active skeletal muscle.

crossover concept; cholesterol; lipid metabolism; exertion

PLASMA FREE FATTY ACIDS (FFAs) represent a fuel source whose turnover, availability, and rate of oxidation are highly adaptable. FFA concentration [FFA] can rise two- to fourfold during 60 min of moderate-intensity exercise (5, 10, 11, 21) and is accompanied by a two- to threefold rise in turnover (10, 11, 21). FFAs represent an important whole body fuel source contributing 10–60% of whole body lipid oxidation and 5–20% of total energy expenditure during rest and exercise (10, 11, 21). The importance of FFAs to active skeletal muscle metabolism has been investigated, with the inclusion of the arteriovenous (a-v) balance technique to distinguish between active limb skeletal muscle from whole body lipid metabolism. It appears that FFAs contribute to active skeletal muscle metabolism to some degree, because studies employing the a-v balance technique have demonstrated a shift from net leg FFA release at rest to uptake during one-legged knee extension (23, 45) and two-legged cycle ergometry (2, 5, 40, 41) performed at a variety of durations and intensities (45–240 min, 30–65% VO2 peak).

Although considerable attention has been directed toward describing changes in whole body lipid metabolism with endurance training, far less is known about training-induced changes in FFA, lipoprotein, or plasma triglyceride (TG) metabolism across the active limb. Cross-sectional studies indicate that endurance-trained subjects exhibit greater net leg FFA uptake than untrained subjects during prolonged one-legged knee extension exercise (45). As well, longitudinal studies have shown that endurance training results in an increase in net leg FFA uptake at the same absolute intensity during prolonged one-legged knee extension exercise (23) and at the same relative intensity during two-legged cycle ergometry (5). To date, a detailed examination of the influence of endurance training on net leg individual FFA balance has not been reported. Except for a slight preference for the uptake of the unsaturated fatty acids oleate and linoleate over that of the saturated fatty acid palmitate (16), the relative net uptake of individual FFAs across the exercising forearm has been shown to be highly related to FFA concentrations in the plasma. However, issues related to the effects of endurance training on individual and total FFA and lipoprotein exchange across large muscle groups responsible for the majority of energy flux during exercise have not been explored in detail.

Long-term (8 mo) endurance training has been shown to result in significant improvements in the plasma lipid and lipoprotein profiles of sedentary, overweight subjects with mild to moderate dyslipidemia (26). Additionally, 4 mo of endurance training has been shown to significantly increase HDL cholesterol (HDL-C) in healthy, sedentary men (24). However, little is known about the potential role of skeletal muscle in training-induced changes in plasma lipid and lipoprotein profiles. In the only study to date to examine net leg lipoprotein balance, Kiens and Lithell (25) demonstrated that 8 wk of one-legged knee extension training resulted in significantly higher rates of net VLDL-TG uptake at rest and net HDL2-C release at rest and after 110 min of exercise in the trained vs. untrained leg. Results of that study suggest that active skeletal muscle plays a significant role in training-induced improvements in plasma lipoprotein profiles. Moreover, because of constancy of the hormonal environment presented to trained and untrained legs in the same individuals, the results can be interpreted to implicate intramuscular as opposed to hormonally-mediated regulation of lipoprotein metabolism.

The purpose of this study was to employ a longitudinal study design to evaluate the effect of two-legged endurance training on net leg individual FFAs, lipoprotein, and TG balances at rest and during moderate-intensity exercise. We tested the hypothesis that net leg FFA, LDL-C, and TG uptake and HDL-C release during moderate-intensity cycling exercise would increase following endurance training.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Detailed descriptions of many of the methods employed in this study have been published previously (5, 21), but relevant information is reiterated for the convenience of the reader. As well, results in the present report are ultimately to be considered with those contained in another report from the same study (Freidlander AL, Jacobs KA, Fattor JA, Horning MA, Hagobian TA, Bauer TA, Wolfel EE, and Brooks GA, unpublished observations).

Subjects. Eight healthy sedentary men age 18–32 yr were recruited from the University of California, Berkeley campus, by posted notices. Subjects were considered sedentary if they engaged in no more than 2 h of regular strenuous physical activity per week for 2 yr and had a peak oxygen consumption (VO2 peak) of <50 ml·kg–1·min–1. Subjects were included in the study if they had <25% body fat, were nonsmokers, were diet and body weight stable, had a 1-s forced expiratory volume (FEV1) that was >70% of forced vital capacity (FVC), and were injury and disease free as determined by health history questionnaires and physical examination. The procedures and risks were thoroughly explained to the subjects, and their written, informed consent was obtained. The study was approved by the Committee for the Protection of Human Subjects at the University of California, Berkeley (CPHS no. 2002-3-21).

Experimental design. After being screened, subjects completed a pair of experimental trials before and after 9 wk of endurance training. Each experimental trial consisted of 90 min of rest and 60 min of exercise. Prior to training, subjects were tested in random order at 45% (45% Pre) and 65% (65% Pre) VO2 peak with ~1 wk between trials. After being trained, subjects were tested in random order at 65% pretraining VO2 peak [same absolute intensity (ABT)] and 65% posttraining VO2 peak [same relative intensity (RLT)] with ~1 wk between trials, during which subjects continued training.

Screening tests. VO2 peak was determined before training, after 4–5 wk of training, and at the end of the 9-wk training program using a continual graded exercise test on an electronically braked cycle ergometer (Monark Ergometric 839E, Vansbro, Sweden). An initial power output of 50 W was increased by 25–50 W every 3 min until volitional exhaustion. Respiratory gases were analyzed by an open-circuit indirect calorimetry system (ParvoMedics TrueMax 2400; ParvoMedics, Sandy, UT). Body composition was determined by the skinfold method before and after training (20). Subjects were asked to maintain regular dietary regimens, and body weight was recorded on a daily basis to evaluate the need for adjustments in caloric intake to maintain baseline body weight. Three-day dietary records were collected to assess habitual dietary habits and monitor each subject's caloric intake and macronutrient composition at baseline and every 2–3 wk during training using the Nutritionist III program (N-Squared Computing, Salem, OR). FEV1 and FVC were determined with a 9-liter spirometer, and a 12-lead electrocardiogram (ECG) was recorded to screen for any cardiac arrhythmias.

Testing protocol. Subjects were admitted to the metabolic ward at the Clinical Studies Unit at the Palo Alto Veterans Affairs Health Care System the night before each experimental trial and remained there until testing was completed the following day. The night before each experimental trial, subjects were fed a standardized dinner [1,180 kcal: 69% carbohydrate (CHO), 21% fat, and 10% protein]. Two subjects were tested per day, and morning and afternoon testing was randomly assigned and then replicated in the remaining trials. Morning subjects were fed a standardized pretrial meal (434 kcal: 74% CHO, 10% fat, and 16% protein) 1 h before procedures started and 4–5 h before exercise. Afternoon subjects were fed a standardized breakfast (661 kcal: 55% CHO, 34% fat, and 11% protein) and the same standardized pretrial meal as the morning subjects 1 h before procedures started and 4–5 h before exercise. We chose to test our subjects in the 3- to 4-h postprandial state to control for the effects of meal size, composition, and timing and to mimic the eating practices of active individuals in a nonlaboratory environment. Although the ATP III lipoprotein analysis guidelines (1) recommend the use of blood samples from overnight-fasted subjects, recent reports (43) conclude that fasting conditions are not necessary for the direct assessment of total cholesterol, LDL-C, and HDL-C.

Catheterizations. After local anesthesia with Lidocaine, the femoral artery and vein of the same leg were cannulated using standard percutaneous techniques, as previously described (50), with the following modifications. Localization and cannulation of the femoral artery and vein were performed using vascular ultrasound (Site-Rite 3, Bard Access Systems; Dymax, Pittsburgh, PA). A 5.0-French, 65-cm angiographic catheter (model 451-501V5; Cordis, Miami, FL) was inserted 25 cm and positioned in the distal abdominal aorta via the femoral artery. A 6-French theromdilution nonballoon venous catheter (model F06TNN001; American Edwards Laboratory, Irvine, CA) was placed with the tip in the distal iliac vein through a venous sheath. After insertion, the venous sheath and catheter were withdrawn such that the distal tip of the catheter was 15 cm and the proximal port (10 cm from the tip) was 5 cm from the skin insertion site. The proximal port, which was used for all femoral venous blood sampling and the cold saline injection for thermodilution blood flow measurements, was positioned ~2 cm from the entry site into the femoral vein. Both catheters were sutured to the skin and further secured by an Ace bandage wrap. The external portions of each catheter were directed toward the hip for easy access during exercise. Alternate legs were used for the two tests both pretraining and posttraining. One subject experienced pain in the groin related to the catheter placement when he was positioned on the cycle ergometer for exercise, and so that trial (45% Pre) was discontinued and the catheters were removed. A cardiologist experienced in vascular catheterization techniques performed all catheterizations.

Blood sampling. Arterial and venous blood samples were drawn simultaneously and anaerobically after 75 and 90 min of rest and 30, 45, and 60 min of exercise. Blood for FFA and lipoprotein analyses was placed in tubes containing EDTA and a preservative cocktail, respectively, and was centrifuged at 2,800 g for 18 min at 4°C. The lipoprotein preservative cocktail contained EDTA (0.15%), enzyme inhibitors (D-phenylalanyl-L-prolyl-L-arginyl-CHCl2, 1 µM; aprotinin, 50 KU/ml), antibiotics (gentamicin sulfate, 50 µg/ml; chloramphenicol sodium succinate, 0.05 mg/ml), and a bacterioside (sodium azide, 0.01% wt/vol). Exactly 1 ml of plasma for FFA analysis was mixed with 4 ml of heptane-isopropanol (30:70) extraction solution, 2 ml of 3.3 mM H2SO4, and 100 nmol of pentadecanoic acid (PDA; 15:0) as an internal standard. Plasma for lipoprotein analysis was flushed with nitrogen prior to capping, and all plasma samples were stored at –20°C until analysis. Hematocrit measurements were performed on both arterial and venous blood using a circular microcapillary tube reader (International Equipment, no. 2201).

Hemodynamics. Heart rate and ECG were continuously recorded and displayed using a three-lead ECG connected to a MacLab analog-to-digital converter (AD Instruments, Castle Hill, Australia). Arterial blood pressure was continuously recorded and displayed using a Transpac pressure inducer (Baxter) positioned at the level of the heart that was connected to the MacLab system and calibrated before trials. Blood temperature was recorded from a thermister at the end of the venous thermodilution catheter. Iliac venous blood flow was determined by the thermodilution technique using a cardiac output computer (model 9520; American Edwards Laboratory), with a 10-ml bolus injection of sterile saline cooled to 0°C with an ice slurry (Co-Set II, model 93600; American Edwards Laboratory). Blood flow measurements were made in triplicate or quadruplicate during rest and exercise immediately after blood sampling. The validity and precautions associated with the thermodilution technique have been described previously (4).

Training protocol. Subjects exercised on stationary bicycle ergometers in the laboratory 5 days/wk for a total of 9 wk. Additionally, they were asked to perform an activity of their choosing on 1 day during the weekend. Exercise duration and intensity was gradually increased during the first 2 wk of training until subjects were exercising for 1 h at 75% VO2 peak. Intensity was monitored by heart rate telemetry on the basis of VO2 peak data collected at baseline and after 4–5 wk of training. During the last 2 wk of training, two of the steady-state training sessions per week were replaced with interval training to maximize increases in VO2 peak. Personal trainers monitored and recorded each subject's workout performed in the laboratory and encouraged subjects to warm up, cool down, and stretch to prevent injury.

FFA analysis. The FFAs in the extracted plasma samples were isolated by thin-layer chromatography using an oleate (18:1 n-9) standard. Samples were derivatized to their fatty acid methyl esters to allow volatilization by gas chromatography, and individual FFA abundances were determined by flame ionization detection (GC model 6890; Hewlett Packard). Individual FFA concentrations were calculated relative to the abundance of the internal PDA standard and a physiological range of external standards, as described previously (10). Total FFA concentration was determined by summing the individual FFA concentrations.

Plasma lipoprotein analyses. Plasma was analyzed for concentrations of total cholesterol (3), TG (34), and HDL-C measured directly after precipitation of apolipoprotein B (apoB) containing lipoproteins in plasma (48). LDL-C was circulated from the Friedewald equation by subtraction of estimated VLDL and measured HDL-C from the measured total cholesterol and TG in plasma (9). Lipid assays were enzymatic end-point measurements utilizing enzyme reagent kits (Ciba-Corning Diagnostics, Oberlin, OH) and a Ciba-Corning Express 550 automated analyzer. The measurements were standardized through the CDC-NHLBI Lipid Standardization Program.

Measurement of LDL peak particle size was performed on whole plasma with the use of nondenaturing 2–14% polyacrylamide gradient gel electrophoresis and standardized conditions (35). Following electrophoresis, lipoproteins were lipid stained with Sudan Black, and the protein calibration standards were stained with Coomassie R-250. Gels were analyzed using computer-automated densitometry, and calculations of peak particle sizes were based on the migration of reference standards of known particle size. Coefficient of variation for measurements made in the LDL size range was within 3%. Lipid-stained area of 7 LDL subclasses was measured within particle-size boundaries and calculated as percent area/subclass.

An immunoturbidimetric assay (39, 44) was used to measure apolipoprotein A-I (apoA-I) and apoB. Reagents, standards, and reference plasma controls, with and without elevated lipids, were included in the immunoturbidimetric assay reagent kit (Bacton Assay Systems, San Marcos, CA). Measurements were performed using the Express Plus 550 analyzer according to kit instructions. Calibrators and reference controls were assigned concentration values with the use of International Federation of Clinical Chemistry standard reference materials SP1 for apoA-I and SP3-07 for apoB. In-house controls measured in each group of 20 unknowns had a coefficient of variation <3%.

Calculations. Net leg individual FFA and lipoprotein balances were calculated as the product of leg plasma flow and a-v differences where arterial and venous hematocrit values were used to correct for changes in plasma volume:

Formula

Formula
where Q is leg plasma flow, Hct is hematocrit, FFA is free fatty acid, lipo is lipoprotein, and the subscripts a and v represent arterial and venous plasma, respectively.

Net leg FFA fractional extraction was calculated from the ratio of a-v [FFA] and arterial [FFA] with correction of venous [FFA] for blood volume shifts, as determined from hematocrit measurement:

Formula

Statistics. Data are represented as means ± SE. Results of Shapiro-Wilks tests indicated that the data were normally distributed. The significance of within- and between-condition mean differences was assessed by ANOVA, with repeated measures followed by post hoc analyses using the Least Significant Difference test. The 95% confidence intervals of net leg FFA fractional extraction and balance data were examined to determine whether or not they contained zero. Those data points whose 95% confidence intervals did not contain zero were ascertained to have reached significant levels of net fractional extraction, release, or uptake. The adequacy of the estimation of net leg total FFA balance from net leg palmitate balance was evaluated with multiple approaches. Regression analyses were performed to examine the strength of the relationship between measured and estimated net leg total FFA balance. The error associated with the estimation of net leg total FFA balance was quantified with the calculation of residuals (estimated minus measured values) that were then standardized by dividing by the appropriate SD of the residuals to account for the much smaller net leg total FFA balance values at rest compared with exercise. Finally, Cohen's d scores (average residual/SD) were calculated to categorize the magnitude of the error of estimation as large (>0.8), moderate (0.5–0.8), or small (<0.5) (6) at rest, 30, 45, and 60 min of exercise. Significance was set a priori at {alpha} < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
One subject did not complete a 45% Pre trial due to discomfort experienced at rest following catheterization. As a result, the data from the remaining seven subjects were used in comparisons made between trials, whereas the data from all subjects were used in comparisons made before and after training (subject characteristics) and when data were averaged across all four trials (individual FFA concentrations, net leg fractional extraction, and net leg balance).

Subject characteristics, hemodynamics, and pulmonary gas exchange. Dietary macronutrient composition (46 ± 2% CHO, 35 ± 2% fat, and 16 ± 1% protein) did not change due to training. Subjects remained body weight and composition stable during the study despite an increase in energy expenditure (Table 1). Heart rate at rest was lower after training than before and increased from rest to exercise in a RLT-dependent manner (45% Pre < ABT < 65% Pre and RLT, P < 0.05; Table 2). VO2 and VCO2 increased significantly from rest to exercise in an ABT-dependent manner (45% Pre < 65% Pre and ABT < RLT, P < 0.05). Respiratory exchange ratio increased from rest to exercise in all conditions and was lower at the same absolute workload after training (ABT) than at 65% Pre (P < 0.05). As a result of the significant 15% increase in relative VO2 peak with endurance training (P < 0.05), the workload that elicited 66% of VO2 peak before training (156 ± 6 W) elicited only 55% of the new VO2 peak after training in the ABT trial (Tables 1 and 2). Leg blood flow increased from rest to exercise in all trials and was significantly higher during exercise at 65% Pre, ABT, and RLT than at 45% Pre (P < 0.05).


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  		Table 1. Physical characteristics of subjects before and after 9 wk of endurance training

 

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  		Table 2. Workload, heart rate, respiratory gas exchange, and blood flow at rest and during exercise before and after 9 wk of endurance training

 
[FFA]. The plasma concentrations of total and individual FFA tended to be higher during exercise at 45% Pre than all other trials, but these differences never reached statistical significance, and therefore, the data were pooled (Table 3). Consequently, as assessed over the course of a 60-min exercise bout, neither exercise intensity nor endurance training significantly affected total [FFA]. However, plasma stearate, oleate, and total [FFA] decreased significantly from rest to 30 min of exercise (P < 0.05), whereas plasma myristate, palmitate, palmitoleate, oleate, linoleate, linolenate, and total [FFA] increased ~26% between 30 and 60 min of exercise (P < 0.05). Oleate and palmitate were the most abundant FFAs (41 and 28% of total FFAs, respectively). The percentage of oleate did not change from rest to exercise (41.4 ± 0.5 vs. 40.7 ± 0.5% of total FFA), whereas the percentage of palmitate increased significantly (27.2 ± 0.3 vs. 28.7 ± 0.2% of total FFA, P < 0.05). Unsaturated FFA (palmitoleate, oleate, linoleate, linolenate, and arachidonate; 62% of total FFA pool) concentration increased ~30% from 30 to 60 min of exercise, whereas the concentration of saturated FFA (myristate, palmitate, and stearate; 38% of total FFA pool) increased only ~22%. Correspondingly, the ratio of unsaturated to saturated fatty acids increased significantly from rest to the last 30 min of exercise (Table 3).


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  		Table 3. Individual arterial [FFA] and ratio of unsaturated to saturated fatty acids at rest and during exercise across all trials

 
Net leg FFA fractional extraction and balance. The plasma concentrations of myristate, palmitoleate, linolenate, and arachidonate were too small (together representing 7% of total FFA concentration) to accurately determine [a-v] differences, net leg fractional extraction, or balance. Therefore, only the net leg fractional extraction and balance data of palmitate, stearate, oleate, linoleate, and total FFA are presented (Table 4 and Fig. 1). Net leg plasma palmitate, stearate, oleate, and total FFA fractional extraction increased significantly from rest throughout exercise (P < 0.05) (Table 4). Net leg plasma palmitate, stearate, oleate, and total FFA fractional extraction during exercise at 30 and 45 min were significantly greater than zero and the corresponding values at 60 min of exercise (P < 0.05). Net leg plasma linoleate fractional extraction did not change significantly from rest but decreased significantly at 45 min of exercise to the point that it was significantly different from 60 min of exercise (P < 0.05). Net leg plasma palmitate, oleate, and total FFA balance increased significantly from rest through 45–60 min of exercise (P < 0.05; Table 4). Net leg plasma palmitate, oleate, and total FFA balance during exercise at 45 min were significantly greater than 60 min of exercise, and values from 30 to 60 min of exercise were significantly greater than zero net balance (P < 0.05). Although the net leg balance of plasma stearate and linoleate tended to increase from rest to exercise, they never reached statistical significance.


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  		Table 4. Net leg individual FFA fractional extraction and balance at rest and during exercise across all trials

 

Figure 1
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  		Fig. 1. Effect of exercise intensity and endurance training on arteriovenous (a-v) differences in total free fatty acid [FFA]; (A), net leg total FFA fractional extraction (B), and net leg total FFA balance (C). Values are means ± SE; n = 7 subjects. *Significantly different from zero at 45% pretraining (Pre); {dagger}significantly different from zero at 65% Pre; #significantly different from zero at absolute intensity (ABT); {ddagger}significantly different from zero at relative intensity (RLT). P < 0.05. Main effects: min 30, 45, and 60 > rest; min 45 > min 60; P < 0.05.

 
Plasma lipid and lipoprotein concentrations and net leg balance. All subjects were normolipidemic with TG, total cholesterol, and LDL-C concentrations meeting the ATP III normal/optimal classifications set by the National Cholesterol Education Program (Table 5) (1). Nine weeks of endurance training significantly increased plasma HDL-C concentration by ~6%, decreased plasma LDL-C concentration by ~7%, and decreased LDL peak particle size by 0.7% (P < 0.05; Table 5). Only minor changes were noted between rest and exercise in some of the lipoprotein variables, and neither exercise intensity nor endurance training significantly influenced plasma lipid or lipoprotein concentrations during exercise. The net leg balances of plasma TG, HDL-C, and LDL-C did not change from rest to exercise, were not affected by endurance training, and were not different from zero at rest or during exercise.


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  		Table 5. Plasma lipid and lipoprotein parameters at rest and during exercise before and after 9 wk of endurance training

 
Estimation of net leg total FFA balance from net leg palmitate balance. Studies employing a combination of fatty acid tracer infusion and a-v balance techniques commonly estimate net leg total FFA balance by dividing the measured net leg palmitate balance by the proportion of total [FFA] accounted for by palmitate (46, 47). This estimation assumes that the net leg balances of the remaining individual FFA (72% of total FFA pool) will be in the same direction as palmitate (i.e., negative or positive) with a magnitude that is in direct proportion to their respective concentrations. Data from the present study indicated that relative individual arterial [FFA] accounted for ~91% of the variance in relative net leg uptake of individual FFA during exercise (Fig. 2). The measured net leg total FFA balance data of the present study were highly correlated to those estimated by dividing net leg palmitate balance by the proportion of total [FFA] accounted for by palmitate (0.28) both at rest (r = 0.9622; P < 0.01) and during exercise (r = 0.8899, P < 0.01; Fig. 3A). The absolute residuals between measured and estimated net leg total FFA balance were smaller at rest than during exercise (Fig. 3B and Table 6). The average residual (estimated minus measured net leg total FFA balance) was +6.3 ± 7.3 µmol/min (+13.9%) at rest and ranged from –14.2 ± 47.8 to –29.4 ± 50.0 µmol/min (–6.8 to –21.3%) during exercise (Table 6). Examination of Cohen's d values indicated a small effect on peak particle size at rest and during exercise (Cohen's d = +0.31 and –0.11 to –0.25, respectively). The residuals were standardized by dividing rest and exercise values by their respective average SD of the residuals to account for the much smaller net leg total FFA balance values at rest compared with exercise (–44 vs. 193 µmol/min; Fig. 3C). These standardized residuals were similar at rest and during exercise.


Figure 2
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  		Fig. 2. Relationship between relative individual arterial [FFA] and relative net leg individual FFA uptake during exercise. Plotted values are means across all 4 trials during the last 30 min of exercise.

 

Figure 3
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  		Fig. 3. A: relationship between measured and estimated net leg total FFA balance at rest and during exercise. B: absolute difference (estimated minus measured) at rest and during exercise. C: standardized difference (absolute difference divided by either average rest or exercise SD of the absolute difference) at rest and during exercise.

 

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  		Table 6. Error in estimation of net leg total FFA balance at rest and during exercise

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
As part of our effort to describe the magnitude and adaptability of human lipid metabolism, we employed a longitudinal study design to examine the effects of endurance training on net leg lipid and lipoprotein balances at rest and during moderate-intensity exercise. Despite significant improvements in aerobic capacity and arterial HDL-C and LDL-C concentrations, the net leg balances of individual FFAs, lipoproteins, and TG at rest and during moderate-intensity exercise were unaffected by endurance training. The results led to the following conclusions that will be subsequently discussed. 1) Relative availability is the predominant determinant of individual FFA use by active skeletal muscle, 2) endurance training-induced improvements in the lipoprotein profiles of normolipidemic men are not attributable to changes in HDL-C or LDL-C exchange across active skeletal muscle, and 3) the measurement of net leg palmitate balance provides a reasonably accurate means for estimating net leg total FFA balance at rest and during exercise.

Dietary controls. Individuals with odd dietary habits were excluded from the subject pool, and participants were counseled on appropriate dietary habits. Evening and morning meals were standardized and provided in the metabolic unit before trials. Three-day food records were kept before and after training, and subjects were weight stable across the training period. As already noted, analysis of dietary macronutrient composition indicated no change during training. Surprisingly, dietary energy intake did not rise with training, so subjects either compensated by altering discretionary physical activity or they underreported food consumption.

Dynamics of individual FFA availability, net leg fractional extraction, and balance. The percent contribution of each individual FFA to the total concentration at rest agrees well with results of other studies on both men (13, 17, 19, 30, 32) and women (14, 15, 21, 33). Additionally, the percent contribution of each individual FFA to the total concentration during exercise agrees well with a previous study in our laboratory (21) of women exercising at the same relative intensities for the same duration. The absolute concentrations of total and individual FFA at rest are two- to threefold higher than other reports of 3- to 4-h postprandial men (32) and women (21) and agree more closely with those of overnight-fasted subjects (13, 15, 19, 30, 33) and 3- to 4-h postprandial men studied in our laboratory following nearly identical femoral catheterization procedures (5). These results are likely explained by an elevation in the rate of lipolysis due to the psychological stress experienced by the subjects during the femoral catheterization performed 75–90 min prior to the blood draws. In support of this contention, standardized 20–30 min mental stress protocols have been shown to result in pronounced increases in FFA and glycerol concentrations (18, 31, 49) that are similar in magnitude to those seen during moderate-intensity exercise (31).

Net leg FFA fractional extraction was not affected by exercise intensity or endurance training and averaged 7.6% across all trials and time points (Table 4 and Fig. 1B) and is similar to previously reported values calculated without isotope tracers during one-legged knee extension (6–7%) (23) and two-legged cycle ergometry (6.0–6.4%) (5). Hagenfeldt and Wahren (16) performed the first study on individual FFA net limb fractional extraction and balance and determined that the exercising forearm has a slight preference for the uptake of the unsaturated fatty acids oleate and linoleate over that of the saturated fatty acid palmitate. However, in a subsequent investigation in the same laboratory, no differences in individual FFA net forearm fractional extraction were found during exercise (17). The present study is the first to evaluate the influence of relative individual FFA availability on uptake across large muscle groups responsible for the majority of energy flux during exercise. It appears that the unsaturated fatty acids oleate and linoleate are above the line of best fit, whereas the saturated fatty acid palmitate is below the line of best fit (Fig. 2) supporting the initial results of Hagenfeldt and Wahren (16). However, regardless of the effect of saturation status, it is clear that relative availability is the predominant determinant of individual FFA use by active skeletal muscle. These observations are consistent with the interpretation that FFA disposal in active muscle is limited by mitochondrial translocation (42), but that for a given rate of muscle FFA uptake, sarcolemmal fatty acid translocators do not discriminate among FFA moieties. Individual FFA appear to compete for sarcolemmal binding and uptake by virtue of relative abundances. With regard to the effect of availability on muscle FFA uptake and utilization, it is appropriate to reiterate that, although arterial [FFA] rises during exercise (Table 4), fractional extraction by active skeletal muscle is small compared with delivery that rose ~8- to 10-fold during exercise due to increases in blood flow and arterial [FFA]. Hence, in healthy young men, the use of circulating FFA by muscle is clearly limited by events in muscle and not by vascular delivery.

Exercise resulted in a shift from net leg total FFA release to uptake (Table 4 and Fig. 1C), as has been consistently reported in studies employing one-legged knee extension (23, 45) and two-legged cycle ergometry (2, 5, 40) during 45–240 min of exercise at 30–65% VO2 peak. Net leg total FFA uptake during exercise was not affected by an increase in exercise intensity either before or after endurance training, as we found previously (5). Our laboratory previously demonstrated that 9 wk of endurance training increased net leg total FFA uptake during exercise at the same relative intensity (65% VO2 peak). The difference between the present results and those of our previous study is due to larger net leg total FFA uptake during the first 30 min of exercise in the previous study. In the present study, we did not determine net leg total FFA balance during the first 30 min of exercise, but values for net leg total FFA uptake were similar to those in our previous report at 45 and 60 min of exercise.

In comparing our results with those of others, the absence of a training effect in active net leg total FFA balance during exercise was also observed in a cross-sectional report comparing endurance-trained and sedentary subjects (22). Others have reported that endurance training increases net leg total FFA uptake during 2–3 h of one-legged knee extension exercise in the 9- to 12-h-fasted state using both cross-sectional (45) and longitudinal research designs (23). The disparity in the present findings with those of previous studies is likely explained by differences in nutritional status (3–4 h postprandial vs. 9–12 h fasted), exercise modality (two-legged cycle ergometry vs. one-legged knee extension), exercise duration (60 vs. 120–180 min), absolute power output (156–193 vs. 23–34 W), relative exercise intensity (65 vs. ~15% whole body VO2 peak), and magnitude of autonomic stimulation. In combination, results of our two studies as well as the results of others lead to the conclusion that the training effect on active skeletal muscle net total FFA uptake is small and easily overridden by recent CHO nutrition, high muscle power output, and other factors favoring CHO utilization. However, the results obtained as a consequence of the present investigation do not preclude a role of skeletal muscle for lipid oxidation during recovery from exercise (27).

Role of skeletal muscle in endurance training-induced improvements in the lipoprotein profile. We chose to test our subjects in the 3- to 4-h postprandial state to mimic nonlaboratory conditions despite the ATP III recommendation to use blood samples from overnight-fasted subjects for lipoprotein analyses (1). It has recently been reported (43) that concentrations of total cholesterol, LDL-C, and HDL-C 4 h after a large, high-fat meal (880 kcal, 57% fat) were only 2–5% lower compared with a 12-h fast. Therefore, we feel confident that the standardized pretrial low-fat meals (434 kcal, 10% fat) fed to our subjects had little influence on our lipoprotein data or our conclusions that endurance training significantly decreased LDL-C and increased HDL-C concentrations (Table 5).

Recent meta-analyses have revealed that endurance exercise training is most commonly associated with moderate (4.6%), but inconsistent (range of –5.8 to +25%), increases in HDL-C and little or no change in either total or LDL-C (8, 28). The variability of the HDL-C response may be due to methodological differences, such as exercise intensity and duration (26) as well as various biological, behavioral, and lifestyle characteristics (28). Regardless, the 6% increase reported in the present study agrees well with other training interventions on healthy, sedentary males (25). Modest increases in HDL-C with endurance training are also likely due to a shift in HDL subspecies characterized by an increase in HDL2 and a decrease in HDL3 in both normal-weight (37) and obese subjects (7). The importance of this exercise-induced shift in HDL subspecies is highlighted by the fact that HDL2 is the final cholesterol carrier in reverse cholesterol transport and, therefore, plays a pivotal role in reducing cardiovascular disease risk.

Although the mechanisms for endurance training-induced increases in concentrations of total HDL-C and HDL2-C are unclear, cross-sectional studies of active and sedentary males and females (36) have found that HDL-C concentration is highly correlated to the activity of both adipose tissue and skeletal muscle lipoprotein lipase (LPL). Patsch et al. (38) first described the potential mechanism for the shift in HDL subspecies with in vitro evidence that hydrolysis of VLDL-TG by LPL resulted in the transfer of protein, phospholipids, and cholesterol from VLDL to HDL3, leading to its transformation into HDL2. Taken together, these results highlight the potential important role of peripheral tissue, such as skeletal muscle in endurance training-induced improvements in the plasma lipoprotein profile. In the only study to date to examine net leg lipoprotein balance, Kiens and Lithell (25) demonstrated that 8 wk of one-legged knee extension training resulted in significantly higher rates of net VLDL-TG uptake at rest and net HDL2-C release at rest and after 110 min of exercise in the trained vs. untrained leg. The higher VLDL-TG uptake in the trained leg was associated with a 70% higher muscle LPL activity and greater capillary density than the untrained leg, thereby increasing the capacity and surface area for TG hydrolysis, respectively. However, the applicability of the results is limited due to the use of a one-legged training model in which the hormonal environment is not greatly altered and power output is low compared with two-legged exercise. Additionally, the importance of skeletal muscle as a site for VLDL-TG hydrolysis and HDL2-C release may have been magnified by the 10- to 12-h-fasted state of the subjects, which has previously been shown (29) to increase skeletal muscle LPL activity while decreasing adipose tissue LPL activity. Similar to our findings, Kiens and Lithell (25) reported no evidence of an endurance training-induced increase in net total HDL-C release at rest or during exercise. Taken together, the results of the present study and those of others (25) can be interpreted to indicate that endurance training-induced adaptations in skeletal muscle are responsible in part for the migration in the HDL subspecies from HDL3 to HDL2 without any great net changes in total HDL-C production across the limb. The impact of endurance training on lipoprotein balance across other extrahepatic tissues, such as adipose and cardiac tissue during exercise and postexercise recovery (27), warrants investigation.

Adequacy of the estimation of net leg total FFA balance from net leg palmitate balance. The study of limb fatty acid kinetics is complicated by the fact that fatty acids undergo simultaneous uptake and release. This leads to an underestimation of leg total FFA uptake in studies relying solely on limb a-v balance techniques. More recently, this methodological shortcoming has been overcome with the combination of fatty acid tracer infusion and a-v balance techniques allowing for the measurement of tracer-measured leg FFA uptake and release. A potential shortcoming of this approach, however, is that it relies on the estimation of net leg total FFA balance, uptake, and release by dividing the measured net leg balance, uptake, and release of an individual FFA (most commonly palmitate) by the proportion of total FFA concentration accounted for by the measured individual FFA (46, 47). This estimation assumes that the flux of the remaining individual FFAs (72% of total FFA pool) will be in the same direction as palmitate with a magnitude that is in direct proportion to their respective concentrations. The methodology of the present study allowed for the assessment of the error associated with the prediction of net leg total FFA balance by examining the residuals, standardized residuals, and Cohen's d scores at rest and during exercise. Examination of the residuals and standardized residuals (Fig. 3, B and C, and Table 6) indicated that estimation resulted in an overprediction at rest (+13.9%) and an underprediction during exercise (–12.2%). These differences were evident when comparing the present results with those of a companion manuscript on this study that used tracer methodology to compare whole body and leg lipid metabolism (Friedlander AL, Jacobs KA, Fattor JA, Horning MA, Hagobian TA, Bauer TA, Wolfel EE, and Brooks GA, unpublished observations). The average Cohen's d scores (0.31 at rest and –0.18 during exercise; Table 6) were small, with the implication that the estimation of net leg total FFA balance from net leg palmitate balance is reasonably accurate. As a means of comparison, net leg total FFA balance was also estimated from net leg oleate balance. Although oleate has a greater relative concentration than palmitate (41 vs. 28% of total FFA pool), the error in the estimation was not improved, resulting in Cohen's d scores of –0.29 and 0.16 at rest and during exercise, respectively. The error involved in the estimation of tracer-measured leg total FFA uptake and release from palmitate data could be examined with the infusion of multiple isotopically labeled fatty acids. However, the results of the present study can likely be extended to assume that the error involved in the estimation of tracer-measured leg total FFA uptake and release from leg palmitate uptake and release is also small.

In conclusion, endurance training favorably affects blood lipoprotein profiles even in young, healthy normolipidemic men, but muscle contractions per se have little effect on net leg LDL-C, or TG uptake or HDL-C release during moderate-intensity cycling exercise. Therefore, the favorable effects of physical activity on the lipid profiles of young, healthy normolipidemic men in the postprandial state are not attributable to changes in HDL-C or LDL-C exchange across active skeletal muscle.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-042906, by gifts from the Brian and Jennifer Maxwell Foundation and CytoSport, and by the Veterans Affairs Palo Alto Health Care System.


   ACKNOWLEDGMENTS
 
We thank the participants for their time and efforts and Monika Bautista for providing most of the laboratory support for this study. In addition, we are grateful to Sharon Moynihan and the nurses at the Clinical Studies Unit for their assistance with the dietary controls and study procedures, respectively.

Present address for K. A. Jacobs: University of Miami, Department of Exercise and Sport Sciences, 5202 University Drive, Merrick Building 317-1, Coral Gables, FL 33146.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. A. Brooks, Dept. of Integrative Biology, 3060 VLSB, Univ. of California, Berkeley, Berkeley, CA 94720–3140 (e-mail: gbrooks{at}berkeley.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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