1Unité de Recherches sur les Obésités, U586, Institut National de la Santé et de la Recherche Médicale, Institut Louis Bugnard, Centre Hospitalier Universitaire, and 2Laboratoire de Statistiques et Probabilités, Université Paul Sabatier, 31403 Toulouse, France
Submitted 8 April 2003 ; accepted in final form 30 June 2003
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ABSTRACT |
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subcutaneous adipose tissue; microdialysis; catecholamines; 2-adrenoceptor; exercise; lipid oxidation
In normal-weight subjects exercise training has been found to result in increased fat oxidation (3), but whole body lipolysis during exercise was not significantly enhanced by training (20).
Few studies bear on the effect of exercise on adipose tissue lipolysis in obese subjects, whereas in this population an alteration of catecholamine-stimulated lipolysis has been reported (28, 29). In a previous study, we have shown that 3-mo aerobic training in obese men enhanced the lipolytic -adrenergic response of adipose tissue and decreased adipocyte antilipolytic activity of
2-adrenoceptors (AR) in vitro (6); moreover, we have shown in vivo that lipolysis at rest was increased during
-adrenergic stimulation after physical training (31). However, we do not know whether, despite better
-adrenergic response at rest, adipose tissue lipolysis was improved during exercise, inducing release of more nonesterified fatty acids (NEFA) after endurance training than before. If NEFA released by adipose tissue are the main factor of success in weight loss, their oxidation particularly by muscle is also important, decreasing their concentration in the blood, and so enhancing lipid oxidation may prevent cardiovascular disease. Energy restriction is known to induce a decline in fat oxidation. On the other hand, low-intensity exercise training in obese women counteracts, at rest, the decline in fat oxidation occurring after hypocaloric diet (25, 36) and increases the contribution of fat oxidation to total energy expenditure during exercise (34). Most studies concerning training and obesity in females concerned postmenopausal women (25, 33); however, with regard to prevention, it seemed more interesting to explore the young population and overweight subjects who are likely to develop obesity.
The purpose of the present investigation was to explore the effect of endurance training, without modification of diet, on mobilization and oxidation of lipids in overweight men during physiological stimulation of the sympathoadrenal system as induced by physical exercise. The lipid mobilization from abdominal subcutaneous adipose tissue (SCAT) was studied using the microdialysis technique (1, 7); moreover, to delineate the importance of the local 2-AR-mediated pathway in SCAT lipolysis, the effect of
2-AR blockade was explored. Lipid oxidation was evaluated by measuring pulmonary gas exchange ratios (respiratopry exchange ratios, RER) by indirect calorimetry.
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SUBJECTS AND METHODS |
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Exercise training protocol. The training program consisted of practicing aerobic exercise (1 h/day), mainly running and cycling, 5 days/wk, for 4 mo under the control of a physical exercise coach. The exercise intensity and duration were increased progressively. The subjects exercised at a target heart rate corresponding to 5085% of their O2 max, the heart rate being monitored with a Polar Accurex Plus Cardiometer (Monitor; La Varenne, St Hilaire, France). Compliance with training sessions was good, as checked by a training diary including day-to-day activities. Food and calorie intake were unchanged all along the protocol.
Design of the study. Before and after the training program, the following measurements and tests were carried out: laboratory evaluations of blood glucose and lipid fractions. Diet investigation: the amount of food consumed was recorded for 3 days in a food intake diary. The diaries were analyzed with the Regal table and Profil Nutrition software (Acim, Bourges, France). Anthropometric assessment: fat mass and fat-free mass (bone and muscle) were measured using a total body dual-energy X-ray absorptiometer (Hologic, Waltham, MA). Maximum aerobic capacity: 1 wk before the investigations, O2 max was assessed using a graded test conducted on an electromagnetically braked bicycle ergometer (Ergometrics 800s; Ergoline, Jaeger, Germany).
O2 was measured using an Oxycon Pro apparatus (Jaeger, Germany). The initial workload was 60 W, and it was increased by 30 W every 3 min until exhaustion. Heart rate was continuously monitored. The highest
O2 value was considered as
O2 max, and the workload corresponding to 50% of each subject was calculated. Two criteria assessed that the subjects achieved their true
O2 max: maximal heart rate measured at exhaustion (188.6 ± 2.6 beats/min) was not different from age-predicted maximal heart rate (189 ± 2.8 beats/min), and the maximal RER measured at exhaustion was near 1.1. After the
O2 max determination, the subjects were randomly assigned to start the experimental protocol during the following week.
Experimental protocol. Before and after the training, the subjects were investigated at 8:00 AM after an overnight fast. The evening preceding every trial, the subjects were asked to eat a standard meal consisting of 3,350 kJ (55% carbohydrate, 30% fat, 15% protein). Experiments were conducted in a ventilated room at a temperature of 1920°C. At 8:00 AM, two microdialysis probes (Carnegie Medecin, Stockholm, Sweden) of 20 x 0.5 mm and 20,000-molecular-weight cutoff were inserted percutaneously after epidermal anesthesia (200 µl of 1% lidocaine; Astrazeneca, Rueil-Malmaison, France) into the abdominal SCAT at a distance of 10 cm on both sides of the umbilicus. The probes were connected to a microinjection pump (Harvard apparatus, SARL, Les Ulis, France). One probe was perfused with Ringer solution (in nM: 139 sodium, 2.7 potassium, 0.9 calcium, 140.5 chloride) and the second with Ringer plus 0.1 mM phentolamine (-AR antagonist). This nonselective
1-/
2-antagonist, having an efficient
2-AR antagonist action in human fat cells in vitro, was the only agent allowed by the Ethics Committees for use in microdialysis assays in humans. The two perfusate solutions were supplemented with ethanol (2 g/l). Ethanol was added to the perfusate to estimate changes occurring in the local SCAT blood flow, as previously described (2, 7). The perfusion was set at a flow rate of 2.5 µl/min. Two 15-min fractions at rest were kept for basal concentration values.
After the rest period the subjects started a bout of exercise on an electromagnetically braked bicycle ergometer (Ergometrics 800s Ergoline) at a workload corresponding to 50% of their individual O2 max before and after training, i.e., at the same relative power. The exercise duration was 60 min. The heart rate was continuously monitored with the Cardiometer during the exercise. Then, the subjects rested in the semirecumbent position for 60 min. Water intake was allowed ad libitum during the exercise and recovery periods. During the exercise and the recovery period, 15-min fractions of the dialysate were collected.
Just before exercise and every 15 min during exercise and recovery, 5 ml of blood were collected from an indwelling polyethylene catheter inserted into an antecubital vein for glycerol, NEFA, and glucose determinations. Every 30 min, an additional volume of 5 ml of blood was collected for hormonal determinations (catecholamines and insulin). Blood was collected into 50 µl of an anticoagulant and antioxidant cocktail (Immunotech, Marseille, France) to prevent oxidation of catecholamines and was processed immediately in a refrigerated centrifuge. The plasma was stored at 80°C until analysis.
RER values (RER = CO2/
O2) were calculated on-line via open-circuit indirect calorimetry using an Oxycon Pro apparatus coupled to a computer for the RER calculation. Oxygen concentration was analyzed by a paramagnetic analyzer and CO2 concentration by infrared analyzer. Certified calibration gases were used to calibrate the analyzers every day before the beginning of the assay and before the exercising period. Subjects wore the mask for 6 min before each blood sampling, and RER values obtained during the 4 min before mask removal were averaged. For each experimental assay, the lipid oxidation before and during exercise and during the recovery period was calculated assuming that RER is equal to the nonprotein respiratory quotient. From the gas exchange measurements, the part of lipid oxidized during exercise and recovery was evaluated according to the formula described by Ferrannini (12).
For the posttraining test, the experimental design was identical. The subject did not practice the exercise training the day preceding the investigation, to avoid the consequences of the last physical activity bout of the training period on the metabolic responses analyzed.
Drugs and biochemical determinations. Phentolamine methanesulfonate (Regitine) was obtained from Ciba-Geigy (Reuil-Malmaison, France). Glycerol in dialysate (10 µl) and in plasma (20 µl) was analyzed with an ultrasensitive radiometric method (5); the intra-assay and interassay variabilities were 5.0 and 9.2%, respectively. Ethanol in dialysate and perfusate (5 µl) was determined with an enzymatic method; the intra-assay and interassay variabilities were 3.0 and 4.5%, respectively. Plasma glucose and NEFA were determined with a glucose-oxidase technique (Biotrol kit; Merck-Clevenot, Nogent-s-Marne, France) and an enzymatic procedure (WAKO kit;, Unipath, Dardilly, France), respectively. Plasma insulin concentrations were measured using RIA kits from Sanofi Diagnostics Pasteur (Marnes la Coquette, France). Plasma epinephrine and norepinephrine were assayed in 1-ml aliquots of plasma by high-performance liquid chromatography using electrochemical (amperometric) detection. The detection limit was 20 pg/sample. Day-to-day variability was 4% and within-run variability 3%.
Statistical analysis. All the values are means ± SE. The responses to exercise were analyzed using analysis of variance (doubly multivariate repeated-measures design), with statistical software: SAS and proc GLM. During exercise, extracellular glycerol concentration response curves, in µmol · l1 · 60 min, were calculated as the total integrated changes over baseline values [areas under curves (AUC)] by use of a trapezoidal method. P < 0.05 was considered statistically significant.
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RESULTS |
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Plasma catecholamine concentrations. At rest, catecholamine levels were similar before and after training. During exercise, the plasma norepinephrine concentrations were significantly higher than baseline at the 30th minute (P < 0.001) and did not change until the end of exercise either before or after training. After the 60-min recovery period, norepinephrine concentrations decreased but did not reach basal values (Table 1). The plasma epinephrine concentrations increased until the end of exercise (P < 0.001) both before and after training. After the 60-min recovery period, the epinephrine concentrations decreased to a value that did not differ from that measured at rest (Table 1). During exercise and during recovery, plasma norepinephrine and epinephrine concentrations were not different before and after training.
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Plasma glucose and insulin concentrations. No significant variation of plasma glucose level was observed during the exercise bout or the recovery (Table 1). A significant decrease in plasma insulin concentrations was observed during the exercise (P < 0.001 before training, P < 0.01 after training; Table 1). No significant change was noted in plasma glucose concentrations after endurance training, at rest or during exercise. On the other hand, the plasma concentrations of insulin were significantly lower after training than before, at rest (P < 0.001), during exercise (P < 0.01), and after 1-h recovery (P < 0.05; Table 1).
Plasma NEFA and glycerol levels and dialysate glycerol concentrations. At rest, plasma NEFA concentrations were significantly lower (P < 0.001) after training than before (250 ± 25 vs. 494 ± 76 µmol/l). Plasma glycerol concentrations did not differ after endurance training (76 ± 6 vs. 75 ± 6 µmol/l), and the same was also true for the corresponding baseline glycerol concentrations in dialysate in the probe perfused with Ringer solution, i.e., the control probe (54 ± 6 µmol/l and 42 ± 3 µmol/l before and after training, respectively) and in the probe with phentolamine (56 ± 6 µmol/l and 59 ± 10 µmol/l before and after training, respectively). Plasma NEFA concentrations changed weakly along the exercise period, and at the end of exercise NEFA levels were slightly increased compared with baseline values (P < 0.05, before training). After 15 min of recovery, NEFA concentrations greatly increased (962 ± 123 and 538 ± 59 µmol/l before and after training, respectively) and then decreased but remained higher than at rest (P < 0.05 before training). During all exercise and recovery, plasma NEFA concentrations were significantly lower after than before endurance training (P < 0.001; Fig. 1A). Before and after training, plasma glycerol levels increased similarly during the exercise (P < 0.001). Glycerol values showed no significant difference between before and after training (Fig. 1B).
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During the exercise and before training, the glycerol concentrations in the dialysate increased during the first 15 min in the control probe and continued to increase until the end of exercise. In the probe with phentolamine, the glycerol concentrations in the dialysate were significantly higher than in the control probe throughout the exercise period and remained higher during the 45-min recovery period (Fig. 2A). The calculated AUCs during exercise were 152 ± 21 and 244 ± 25 µmol · l1 · 60 min, respectively (P < 0.01). This supports -activity during exercise in SCAT. After training, the exercise-induced increase in dialysate glycerol was significantly higher than before training (P < 0.05) in the control probe. In the probe supplemented with phentolamine, the glycerol concentration in dialysate did not differ from that found in the control probe either during exercise or during the recovery period (Fig. 2B). The calculated AUCs during exercise were 230 ± 29 and 228 ± 33 µmol · l1 · 60 min, respectively. Thus
-activity in SCAT disappeared after training, which explains the higher lipolysis.
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Ethanol outflow-to-inflow ratio in SCAT. Ethanol outflow-to-inflow ratios were expressed as a percentage, i.e., the ethanol concentration in the dialysate divided by the ethanol concentration in the perfusate times 100. At rest, in the control probe, the ethanol ratio was not different before and after training (77.1 ± 1.7 and 78.3 ± 1.6%, respectively); phentolamine did not induce changes in the ethanol ratio (79.6 ± 2.3 and 78.9 ± 2.3% before and after training, respectively; Fig. 3, A and B). A slight but significant decrease in the ethanol outflow/inflow ratio was observed during the first 15 min of exercise in the control probe as well as in the probe with phentolamine. Before and after training, the addition of phentolamine induced a more prolonged decrease in the ethanol outflow/inflow ratio, which remained significantly lower than before exercise during the whole exercise period.
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RER and lipid oxidation. At rest, the RERs were significantly lower (P < 0.05) after training than before (0.75 ± 0.01 vs. 0.77 ± 0.01). During the exercise, the RER increased during the first 15 min (0.89 ± 0.01 and 0.87 ± 0.01, before and after training, respectively) and then decreased during the whole exercise period (Fig. 4). During the 1-h recovery, the RER greatly decreased and reached values lower than those at rest (0.72 ± 0.01 and 0.71 ± 0.01, before and after training, respectively). Exercise and recovery data were not significantly different before and after training with respect to RER (Fig. 4), but the calculated fat oxidation during 1-h exercise was higher after training than before (27.2 ± 1.8 vs. 22.3 ± 1.7 g lipids/h, P < 0.05). On the other hand, calculated fat oxidation during the 1-h recovery was not different before and after training (8.94 ± 0.47 and 8.69 ± 0.38 g lipids/h, respectively).
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DISCUSSION |
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Plasma NEFA concentrations were greatly lowered after exercise training. However, although diet was not modified, loss of fat mass was noticed; this could have contributed slightly to reduced NEFA release. However, NEFA level depends not only on their mobilization from adipose tissue but especially on their utilization by muscle during exercise.
The exercise-induced increase in lipolysis is promoted by increased catecholamine levels and lowered insulinemia. It has been shown that the hormonal response to exercise is determined by relative, and not by absolute, intensity (14). Therefore, in the present study, in both experiments, exercise was performed at the same percentage of O2 max, and the exercise-induced response of catecholamines (epinephrine and norepinephrine) was similar before and after training. Consequently, the increased exercise-induced lipolysis after training was not associated with different plasma catecholamine concentrations.
The catecholamines control lipolysis by a dual action, stimulation via -AR and inhibition via
2-AR. The resulting effect of catecholamines is determined by the relative contributions of the two pathways (22). As demonstrated previously in vivo, perfusing an
-AR antagonist (phentolamine) in situ in SCAT by microdialysis probes increases exercise-induced lipolysis in untrained subjects (8, 30); this demonstrates that, during exercise in sedentary men, antilipolytic
2-AR are activated. This antilipolytic effect is more pronounced in obese subjects (29). In our study, the
2-adrenergic responsiveness found in overweight subjects before training might contribute to a lower lipid mobilization. However, only abdominal SCAT was investigated, and we cannot say whether these data are representative of other adipose tissue sites. For example, an in vitro study with isolated abdominal or gluteal adipose cells in obese women showed an
2-AR affinity and density that differed between abdominal and gluteal subcutaneous fat depots (4). It is also necessary to take sexual dimorphism into account. Indeed, our study was restricted to male subjects, and Hellström et al. (15) have shown a different response of
-adrenergic receptors between nonobese men and women: in nonobese women
2-AR activity was not expressed. However, in a previous study (32), we showed that, in obese women,
-adrenergic receptors are involved in the regulation of lipolysis in SCAT during exercise.
Our in vivo results in overweight men appear to support results of in vitro longitudinal studies (6) in which 12 wk of training elicited a decrease in the 2-antilipolytic action of epinephrine in vitro on fat cells from SCAT of obese men. Moreover, when interpreting the present results, we must consider the differences in fat mass between the two experiments. The higher fat mass before training suggests a higher adipocyte volume, and this could be a reason for the higher
2-AR activity, as it was shown that the
2-AR activity depends on fat cell size (24). Likewise, we found in another experiment (29), that
2-AR activity was more striking in untrained obese subjects with higher adipocyte volume than in untrained nonobese subjects, and Hellström et al. have shown that, after weight loss during very low-calorie diet,
2-AR sensitivity decreased (16). However, when obese subjects were trained for 3 mo, although their weight did not change, the antilipolytic effect of
2-AR in vitro was decreased (6); this illustrates the effect of training on AR activity.
Moreover, as already shown (21), plasma insulin concentrations were lower after training, and this could also have contributed to the increased exercise-induced lipolysis. However, plasma insulin levels decreased subsequently to the improved insulin sensitivity (18, 21). In contrast, when 2-AR were blocked, the exercise-induced rise of extracellular glycerol was not different before and after training, which supports an antilipolytic
2-AR activity rather than an insulin antilipolytic effect in SCAT. Then, after only 4 mo, aerobic training led to the disappearance of antilipolytic
2-AR activity (longitudinal effect), as seen in a transversal experiment comparing exercise-trained with sedentary men (8).
The concentration of glycerol in the extracellular space is not determined solely by the rate of lipolysis in adipocytes; it is also influenced by the local blood flow. Pharmacological studies have shown that local blood flow modifies glycerol levels in adipose tissue, i.e., that vasoconstriction increases and vasodilatation decreases extracellular glycerol concentration in adipose tissue (9). During exercise, the increase in extracellular glycerol concentration could be due to changes in blood flow in the adipose tissue. The measurement of ethanol escape through the dialysis probe is a validated nonquantitative method to estimate the changes in vasomotricity in AT (11). In agreement with some authors (15, 30), the stability of the ethanol outflow/inflow ratio found during the exercise bouts indicated that vasomotricity did not change during exercise. Consequently, exercise-induced changes in extracellular glycerol concentration are not influenced by local blood flow changes and consequently reflect changes in local lipolysis in SCAT.
The decreased plasma NEFA after training can be explained by increased lipid oxidation. Regular physical activity is known to improve NEFA utilization in normal-weight subjects (13) mainly during moderate-intensity exercise (3). On the other hand, in obese subjects, lipid oxidation capacity is impaired (5) subsequent to reduction of carnitine palmitoyltransferase and citrate synthase activity (19). This partly induces high plasma NEFA concentrations. Despite better lipid availability in this population, Ezell et al. (10) did not find differences in lipid oxidation during exercise between obese and nonobese subjects. Few studies have assessed physical exercise effects on lipid oxidation in this population; most experiments associate hypocaloric diet (with great weight loss) with exercise training. In these conditions, exercise training prevents the fall in fat oxidation resulting from diet-induced weight loss in obese men (25). Van Aggel-Leijssen et al. (35) studied the effect of 3-mo training alone at different intensities (40% and 70% of O2 max) in obese men. Unlike in our study, these authors showed that lipid oxidation was unchanged at rest but that RER was lower during a 50%
O2 max exercise, especially after training at 40% of
O2 max. The same data were found in women with upper-body obesity; in lower-body obesity, exercise training did not change lipid utilization (34). In our study, the energy supply during exercise was not shifted toward lipids (equivalent exercise RER before and after training), but the
O2 being increased after training (increased exercise workload) and the amount of oxidized NEFA were higher. The method (indirect calorimetry) that we used did not reveal the origin of oxidized NEFA, but it would seem that the nonplasma NEFA were more oxidized after training (17, 27). Even if endurance training did not raise circulating NEFA utilization during an exercise bout, we have shown that the proportion of lipid being oxidized was increased in the 1-h recovery period (23). Moreover, we can assume that, after exercise, intramuscular triglyceride storage was supplied from plasma NEFA, also contributing to the decrease in plasma circulating lipids.
In summary, the present study demonstrates that 4-mo endurance training in overweight subjects improves lipolysis in SCAT during exercise through decreased 2-AR activity. The NEFA level was also very low at rest and remained decreased throughout the exercise and recovery periods. These data can be explained by an increase in the percentage of lipids oxidized at rest and a greater amount of NEFA oxidation during physical activity. This study underlines the effects of training on the
2-adrenergic pathway sensitivity, a determining event for the regulation of human SCAT lipolysis. It indicates the importance of physical training for the regulation of fat mass and for better lipid utilization in obesity prevention.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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