Myocellular Triacylglycerol Breakdown in Females but not in Males During Exercise

Transcription

1 AJP-Endo Articles in PresS. Published on November 20, 2001 as DOI /ajpendo E R1 Myocellular Triacylglycerol Breakdown in Females but not in Males During Exercise Charlotte H. Steffensen, Carsten Roepstorff, Marianne Madsen, and Bente Kiens Copenhagen Muscle Research Centre, Department of Human Physiology, University of Copenhagen, DK-2100 Copenhagen, Denmark Running head: Gender differences in MCTG use Address for reprint requests and other correspondence: Bente Kiens Copenhagen Muscle Research Centre Department of Human Physiology August Krogh Institute Universitetsparken 13 DK-2100 Copenhagen Denmark Telephone: Fax: Copyright 2001 by the American Physiological Society.

2 2 ABSTRACT The resting content and the utilization of muscle triacylglycerol during 90 min of submaximal exercise (60% of peak oxygen uptake (VO 2 peak )) were studied in twenty-one eumenorrheic female and twenty-one male subjects at different training levels (untrained (UT), moderately trained (MT), and endurance trained (END)). Males and females were matched according to their VO 2 peak expressed relative to lean body mass (LBM), physical activity level and training history. All subjects ingested the same controlled diet for 8 days and all females were tested in the mid-follicular phase of the menstrual cycle. Resting myocellular triacylglycerol (MCTG), measured by means of the muscle biopsy technique, averaged 48.4±4.2, 48.5±8.4, and 52.2±5.8 mmol/kg dry weight (d.w.) in UT, MT, and END females, respectively, and 34.1±4.9, 31.6±3.3, and 38.4±3.0 mmol/kg d.w. in UT, MT, and END males, respectively (p<0.001, females versus males in all groups). Exercise decreased the content of MCTG in the female subjects by an average of 25%, irrespective of training status, whereas in the male groups the content of MCTG was unaffected by exercise. The arterial plasma concentration of insulin was higher (p<0.05), whereas the arterial plasma concentration of epinephrine was lower (p<0.05) in the females than in the males at rest and during exercise. MCTG utilization was correlated to the resting concentration of MCTG (p<0.001). It is concluded that resting content and utilization of MCTG during exercise are related to gender and, furthermore, are independent of training status. Keywords: Muscle substrate, MCTG, Gender, Training, Triglycerides

3 3 INTRODUCTION It has previously been shown that myocellular triacylglycerol (MCTG) is utilized during the postexercise period (25). The MCTG stores also represent a potentially large energy source during exercise. However, to what extent MCTG is utilized during exercise and whether any differences in MCTG utilization exist between trained and untrained subjects are still under debate. In studies where stable isotope techniques combined with indirect calorimetry have been used, it was estimated that MCTG accounted for 20-25% of the oxidative metabolism during submaximal exercise (27;37). However, when direct measurements of MCTG concentration in muscle biopsies have been used, some studies have found a decrease in MCTG concentration during submaximal exercise (4;19;34), while others have observed no change (1;21;22;25;44). In all of the above mentioned studies, only male subjects have participated. Thus, it is unknown whether gender differences exist in the utilization of MCTG during exercise. Some studies have shown that females utilize lipids to a greater extent than males during submaximal exercise (18;45;46) but to our knowledge it has not been investigated whether this increased lipid utilization in females is primarily from MCTG or other lipid sources. Other studies have not been able to find gender differences in lipid utilization during exercise (3;6;32). This could be due to differences in training status and exercise mode in the experimental designs. The aim of the present study was, therefore, to evaluate the contribution of MCTG during prolonged submaximal exercise, performed at the same relative work load, in female and male subjects at different training levels. The present study is part of a larger project evaluating the metabolism in females and males during exercise at different training levels. The present study, however, focuses only on the aspects of gender and training on MCTG levels and utilization.

4 4 MATERIALS AND METHODS Twenty-one female and twenty-one male subjects were recruited to participate in the study. All subjects were young, healthy (though not screened for family history of type 2 diabetes), and nonsmokers (Table 1). All subjects were fully informed about the nature of the study and the possible risks associated with it before they volunteered to participate, and written consent was given. The study was approved by the Copenhagen Ethics Committee and conformed with the code of ethics of the World Medical Association (Declaration of Helsinki). Pre-experimental protocol. All subjects initially performed an incremental exercise test on a Krogh bicycle ergometer to determine the peak oxygen uptake (VO 2 peak ), and filled out a questionnaire and a training log regarding habitual physical activity, training frequency, intensity and duration as well as competition history to establish these variables. In order to measure lean body mass (LBM,) a three-compartment model (9) was used. Thus, total body composition was assessed by hydrostatic weighing (43), the pulmonary residual volume was determined by the oxygen dilution method (31), and total bone mineral content (BMC) was measured by dual-energy x-ray absorptiometry (DEXA, Lunar Corp, Madison DPX-IQ vs 4.6.6). These measurements were carried out under strictly controlled conditions. The subjects were studied 3-4 h after the last meal and had defecated and urinated beforehand. Furthermore, the subjects refrained from any physical activities on the day prior to the test, which was carried out in the mid-follicular phase of the menstrual cycle in all the female subjects. The female subjects were grouped into either an untrained (UT), a moderately trained (MT), or an endurance trained (END) group based on measured VO 2 peak and their level of physical activity determined from their questionnaire and training log. UT did not participate in any regular physical training but some participated in leisure time physical activity once a week and others occasionally

5 5 biked for local transportation. Their VO 2 peak were less than 45 ml kg -1 BM min -1. MT participated in 2-4 hours of regular exercise training per week and had a VO 2 peak of ml kg -1 BM min -1. Finally, END participated in endurance-type physical training (cross-country skiing, running, rowing, cycling, and swimming) for at least 5-7 hours per week, had trained for and participated in competition for at least two years, and had a VO 2 peak > 55 ml kg -1 BM min -1 (Table 1). All female subjects were eumenorrheic with a normal cycle length of days and did not take oral contraceptives. The groups of male subjects were matched to the respective groups of female subjects according to their VO 2 peak per kg LBM as well as their habitual activity level and training history. In all subjects, the habitual energy and nutrient intake were determined by a 5-day self-reported dietary record (Table 2). All food and beverage intakes were weighed to the accuracy of 1 g and recorded. Subsequently, the energy intake and composition of the habitual diet were calculated by means of a computer database (Dankost 2000, Danish Catering Center, Copenhagen, Denmark). In addition, the expected individual energy intake was determined from the equation for calculation of energy needs provided by the World Health Organization (48). During the 8 days preceding the exercise experiment, each subject consumed a controlled, isoenergetic diet based on the individual food records consisting of 65 energy % (E%) carbohydrate, 20 E% fat and 15 E% protein (Table 2). Between three and seven days before the exercise experiment each subject performed a pretest to determine the work load required to elicit 60% of VO 2 peak. The pretest was performed under conditions similar to those of the exercise experiment (after 12 hours fasting and after having abstained from any physical activity the day before). Exercise experimental protocol. All subjects reported to the laboratory either by bus or car at 8:00 am after an overnight fast. All subjects abstained from any physical activity for 2 days prior to the

6 6 exercise experiment. After 30 min rest in the supine position, resting pulmonary oxygen uptake (VO 2 ) and CO 2 excretion (VCO 2 ) were measured and the respiratory exchange ratio (RER) was calculated. Thereafter, a catheter was inserted into the femoral artery under local anesthesia using aseptic techniques, and the tip was advanced proximally 2 cm above the inguinal ligamentum for blood sampling. After insertion of the catheter the subjects rested for 60 min in the supine position before a second measurement of resting VO 2 and VCO 2 and subsequent calculation of RER. Resting blood samples were drawn, and a muscle biopsy was obtained from the vastus lateralis muscle with suction under local anesthesia before the subjects started to exercise on a Krogh bicycle ergometer for 90 min. All subjects exercised at the same relative work load (60% VO 2 peak ). Expired air was collected in Douglas bags every 15 min during exercise for measurement of VO 2 and VCO 2 and calculation of RER. Blood samples were drawn at 15, 30, 60, 75, and 90 min of exercise. Heart rate was monitored throughout the experiment with a Polar Vantage XL heart rate monitor (Polar Electro OY, Finland). During exercise subjects were offered water ad libitum and were cooled by an electric fan. At termination of exercise another muscle biopsy was obtained through the same skin incision but with the needle pointing in a different direction. All exercise experiments in females were carried out in the mid-follicular phase of the menstrual cycle (between day 7 and 11, mean day 9.0±0.2, 9.1±0.4 and 9.4±0.6 (mean±s.e.m.) in UT, MT, and END, respectively). Pulmonary oxygen uptake and RER were measured and calculated, respectively, during rest and exercise from the collection of expired air in Douglas bags. The volumes of air in the Douglas bags were measured in a Collins bell spirometer (Tissot principle) and the O 2 and CO 2 content of the

7 7 expired air were determined with a paramagnetic (Servomex) and infrared (Beckman LB-2) system, respectively. Two gases of known composition were used to calibrate both systems regularly. Muscle analysis. In the present study, the biopsies were obtained from the same depth of the vastus lateralis muscle to prevent difference in fiber type composition between the two biopsies obtained before and at termination of exercise (30). The biopsy samples were divided in two. One part was frozen in liquid nitrogen within seconds and stored at 80 ºC for subsequent biochemical analysis. The other part was mounted in embedding medium and frozen in precooled isopentane and then stored at 80 ºC for subsequent histochemical analysis. Serial transverse sections (10 µm) were cut and stained for myofibrillar ATPase to identify the different fiber types (2). Before the biochemical analysis, approximately 30 mg wet weight (w.w.) of muscle tissue was freeze-dried, dissected free of all visible adipose tissue, connective tissue, and blood by the use of a stereo microscope leaving the muscle fibers for further analysis. The muscle fibers were mixed and approximately 1 mg dry weight (d.w.) of the pooled fibers was used for measurement of the MCTG concentration according to Kiens and Richter (24). Glycerol from the degraded triacylglycerol was assayed flourometrically as described by Kiens and Richter (24). A coefficient of variation (CV) of the MCTG concentration of 4% has been obtained between five samples from the mixed freeze-dried pool of fibers as described above. This procedure is in contrast to the situation when five small samples of wet muscle have been dissected and analyzed separately resulting in a CV of 31% between these samples. As suggested by Wendling et al. (47), the relatively high variability in MCTG concentration between different locations in the muscle is a concern when detecting expected differences of 20-30%. To circumvent this potential problem two or more biopsies might be obtained. However, the large number of subjects and the consistency in our findings irrespective of training status indicate that our findings are not masked by methodological errors. To further avoid

8 8 the impact of methodological errors as well as day to day variation in the analysis, muscle tissue from both female and male subjects were analyzed in one assay run. Blood analysis. Blood was sampled in heparinized syringes, immediately transferred to plastic test tubes containing EGTA, and centrifuged. Plasma was immediately frozen at 80 ºC until further analysis. Blood for the analysis of progesterone and estradiol was sampled in dry syringes transferred to dry test tubes where the blood coagulated for a few hours before it was centrifuged. Serum was frozen at 80 ºC until further analysis. Insulin in plasma was determined using a radioimmunoassay kit (Pharmacia Insulin RIA 100, Pharmacia & Upjohn Diagnostics, Uppsala, Sweden). The plasma concentrations of epinephrine and norepinephrine were also determined using a radioimmunoassay kit (KatCombi RIA, Immuno-Biological Laboratories GmbH, Hamburg, Germany) as were the serum concentrations of progesterone (Progesterone 125 I RIA, DGR Instruments GmbH, Germany) and estradiol (Estradiol ultrasensitive RIA, DGR Instruments GmbH, Germany). Statistical evaluation. Results are given as means ± S.E.M. A three-way analysis of variance (ANOVA) with repeated measures for the time factor was used to determine whether variables were influenced by gender, training status, or time as well as to test for a possible interaction between these three factors. For the variables independent of time a two-way ANOVA was used to determine any influences of gender and training status and a possible interaction between these two factors. Since females and males were not pairwise matched, gender was not considered a repeated factor. In the case of a significant main effect of one or more factors, a Tukey post hoc test was used to detect pairwise differences between the means. Correlations were evaluated by means of linear regression analysis (Pearson Product Moment Correlations). In all cases an α of 0.05 was used as the level of significance.

9 9 RESULTS Diet. The actual experimental diet averaged 65.5 E% carbohydrates, 19 E% fat and 15.5 E% protein in all groups in line with the intensions (Table 2). There were no differences in energy intake in any groups between the habitual and experimental diet. The energy intake was significantly higher in males than in females and in END compared to UT and MT. In UT and MT females and males the nutrient composition of the habitual diet was similar and slightly but significantly different from the experimental diet. In END females and males the energy percentages of carbohydrates and fat in the habitual diet were similar to the experimental diet. Furthermore, the energy percentage from dietary carbohydrates was higher in END than in UT. Work load. The average work loads during the bicycle exercise test are expressed relative to VO 2 peak and as VO 2 per kg LBM and are provided in Table 1. All subjects completed the 90 min bicycle exercise test at a work load, corresponding to 59% of VO 2 peak. Furthermore, at each training level no gender differences were observed in VO 2 expressed relative to LBM. However, in females as well as males, the UT, MT, and END differed significantly in VO 2 expressed relative to LBM (p<0.01). Respiratory exchange ratio. At rest RER was similar in all groups (0.80±0.02, 0.81±0.02, and 0.79±0.02 in UT, MT, and END females, respectively, and 0.85±0.05, 0.80±0.03, and 0.79±0.01 in UT, MT, and END males, respectively) (Fig. 1). RER remained constant throughout the exercise period in UT and MT, averaging 0.87±0.02 and 0.89±0.02 in UT females and males, respectively, and 0.87±0.02 and 0.89±0.02 in MT females and males, respectively. However, RER remained constant during the first 60 min of exercise in END (0.90±0.02 and 0.91±0.02 at 60 min in females and males, respectively) and subsequently decreased (p<0.05) to 0.87±0.02 and 0.88±0.01 at 90 min

10 10 in females and males, respectively. No gender differences or effects of training status were observed at any time point. Pulmonary oxygen uptake. VO 2 remained constant throughout the exercise period averaging 1.56±0.07, 1.70±0.05, and 2.19±0.11 in UT, MT, and END females, respectively, and 2.17±0.13, 2.31±0.10, and 2.70±0.08 in UT, MT, and END males, respectively. The average VO 2 during exercise was higher in END compared to UT and MT females as well as males (p<0.001). Furthermore, the average VO 2 during exercise was lower in females than males within each training group (p<0.001). Myocellular triacylglycerol (MCTG). At rest the content of MCTG in m. vastus lateralis was significantly higher in the female subjects compared to the male subjects, irrespective of training status (p<0.001) (48.4±4.2, 48.5±8.4, and 52.2±5.8 mmol/kg d.w. in UT, MT, and END females, respectively, and 34.1±4.9, 31.6±3.3, and 38.4±3.0 mmol/kg d.w. in UT, MT, and END males, respectively) (Fig. 2). At termination of exercise a mean decrease (p<0.001) in MCTG content of 25% was observed in the female subjects, irrespective of training status. However, in the male subjects the MCTG content remained unchanged at termination of exercise compared to rest regardless of training level. Thus, a significant gender difference was observed in the MCTG utilization during exercise. A positive correlation existed between MCTG content at rest and the degree of MCTG utilization during exercise (r=0.61, p<0.001, Fig. 3). Fiber type distribution. The fiber type distribution in the vastus lateralis muscle was not different between UT and MT females (Table 3). However, in END females the percentage of type I fibers was greater (p<0.001) and the percentage of type IIB fibers was less (p<0.001) compared to UT and MT females, whereas differences were not observed in the percentage of type IIA fibers.

11 11 In END males the percentage of type I fibers was greater (p<0.001) and the percentage of type IIB fibers was less (p<0.001) compared to UT and MT. The percentages of type I and IIB fibers did not differ between UT and MT males. No effect of training status was observed in the percentage of type IIA fibers in males. Gender differences in fiber type distribution were not observed in MT or END, whereas UT females had a higher percentage of type I fibers than UT males (p<0.01). The area of all fiber types tended to be smaller in END males compared to both UT and MT males (Table 3). Furthermore, the area of the different fiber types was larger in all male groups than in the corresponding female groups (p<0.05). The calculated percentage of fiber types relative to fiber area was larger for type I fibers in females compared to males in UT and END but not in MT subjects, and accordingly the calculated percentages of type IIA and IIB fibers relative to area were smaller in the UT and END female groups compared to the respective male groups (Table 3). The average percentages of the different fiber types did not differ significantly between the biopsy obtained before and at termination of exercise (data not shown). Circulating hormones. At rest the arterial plasma concentration of insulin averaged 8.3±0.9, 8.3±1.2, and 5.9±0.9 µu/ml in UT, MT, and END females, respectively, and 7.1±1.2, 6.2±0.7, and 5.9±0.8 µu/ml in UT, MT, and END males, respectively (Fig. 4A). The arterial plasma insulin concentration decreased continuously throughout exercise to 5.4±0.6, 6.2±2.1, and 4.3±1.1 µu/ml at 90 min in UT, MT, and END females, respectively, and to 3.2±0.4, 3.1±0.4, and 3.1±0.5 µu/ml at 90 min in UT, MT, and END males, respectively (p<0.001). The arterial plasma insulin concentration was significantly higher in females than in males at rest and during exercise irrespective of training status (p<0.05).

12 12 The resting arterial plasma concentration of epinephrine averaged 0.3±0.1, 0.3±.01, and 0.4±0.1 nmol/l in UT, MT, and END females, respectively, and 0.7±0.2, 0.6±0.2, and 0.6±0.2 nmol/l in UT, MT, and END males, respectively (Fig. 4B). In UT and MT the arterial plasma epinephrine concentration did not change during the first 60 min of exercise but then increased throughout the last 30 min of exercise. In END subjects the arterial plasma epinephrine concentration remained unchanged until 30 min of exercise but then increased throughout the rest of the exercise period. At 90 min the arterial plasma epinephrine concentration averaged 1.9±0.4, 2.9±0.8, and 1.9±0.6 nmol/l in UT, MT, and END females, respectively, and 3.6±0.9, 3.9±1.0, and 1.9±0.4 nmol/l in UT, MT, and END males, respectively. The arterial plasma epinephrine concentration was significantly lower in females than in males at rest and during exercise irrespective of training status (p<0.05). At rest the arterial plasma concentration of norepinephrine was similar in females and males irrespective of training status (Fig. 4C). The arterial plasma norepinephrine concentration increased significantly in all groups (p<0.001) after exercise start and remained at a constant level until 60 min whereupon it increased further during the last 30 min of exercise (p<0.05). No gender differences or effects training status were observed. The serum concentration of progesterone averaged 0.19±0.07, 0.12±0.05, and 0.20±0.14 ng/ml in UT, MT, and END females, respectively, and 0.23±0.08, 0.26±0.06, and 0.16±0.06 in UT, MT, and END males, respectively. The serum concentration of progesterone was similar in females and males irrespective of training status. The serum concentration of estradiol averaged 49.1±11.8, 57.6±14.7, and 58.0±10.6 pg/ml in UT, MT, and END females, respectively, and 34.8±4.0, 35.7±3.5, and 30.9±2.8 in UT, MT, and END males, respectively. The serum estradiol concentration was significantly higher in females compared to males irrespective of training status (p<0.01).

13 13 DISCUSSION The results of the present study demonstrated significant gender-based differences in resting content and utilization of MCTG during prolonged submaximal exercise at the same relative work load. Thus, at rest the content of MCTG was significantly higher in females compared to males. During exercise females utilized MCTG whereas males did not. The observations of a higher resting content and utilization during exercise of MCTG in females compared to males were made under conditions where several parameters that could potentially affect substrate oxidation, were standardized and carefully controlled such as the diet prior to testing, the menstrual status of the female subjects, and the phase of their menstrual cycle in which they were tested. MCTG content at rest. It has previously been demonstrated that diet influences the MCTG content in human skeletal muscle, i.e. the consumption of a fat-rich diet increases the MCTG content at rest (23). The higher MCTG content at rest in the female subjects compared to the male subjects in the present study is presumably not ascribed to the diet as all subjects ingested the same carbohydraterich diet for 8 days preceding the exercise experiment. Actually, due to their larger energy intake, males consumed a larger absolute amount of fat compared to females. However, when expressed relative to LBM the fat ingestion was similar in females and males. To explain the finding of a higher resting MCTG content in females compared to males, we examined the muscle fiber composition, since it has previously been shown in male subjects that type I fibers contain more MCTG than type II fibers (8). Furthermore, in a group of females and males it has recently been shown that MCTG content in soleus, tibialis anterior and tibialis posterior muscles varied consistently with the expected fraction of type I fibers in these muscles (20). One might also consider the possibility that females have a higher content of MCTG in type I fibers and/or other

14 14 fiber types than males. To our knowledge, this has not yet been investigated. END females and males had more type I fibers compared to both MT and UT females and males. There was no effect of gender in the percentage of type I fibers in MT and END. However, UT females had a higher percentage of type I fibers than UT males. Previously, a similar fiber type composition in females and males has been found in untrained subjects (5;35;39) as well as in physical education students (40). Others have found that in untrained subjects (42), middle-distance runners (5), trained cyclists (11) and in subjects representing a wide range of physical activity levels (33) females had a higher percentage of type I fibers than males. At all training levels in the present study the area of all the fiber types was smaller in females than in males, and especially the area of type II fibers was considerably smaller, which is in accordance with previous observations (5;35;39;40;42). As a consequence the calculated fiber composition expressed relative to fiber area revealed that type I fibers accounted for a relatively larger area in females than in males. Thus, the higher percentage of type I fibers expressed relative to area might partly explain the higher resting content of MCTG in females. This is further supported by the findings in the present study of a modest but significant correlation between the percentage of type I fibers relative to area and the resting concentration of MCTG (p<0.05, r=0.39). In the present study no effect of training status on MCTG concentrations at rest was observed in females or in males. Previous studies in males have reported inconsistent results, some showing an increase in resting MCTG with training (22;34) whereas others have not (19). The strict dietary control may explain why an effect of training status on the resting MCTG concentrations was not observed in the present study. Thus, the experimental diet was a low-fat diet and differed from the diet in our previous study (22). The possibility that the low fat diet has concealed a training effect on MCTG concentration in the present study can therefore not be excluded. Another possible

15 15 explanation of the increase in MCTG concentration with training in our previous study (22) might be that the subjects had performed exercise the day before the measurements were done. Since MCTG have been shown to be utilized in the post-exercise period (25) the difference in the reported resting concentration between trained and untrained muscle (22) might be influenced by different degrees of post-exercise MCTG utilization in trained and untrained muscle. MCTG utilization during exercise. A major novel finding in the present study was that female subjects utilized a significant amount of MCTG during prolonged exercise irrespective of training status, whereas this was not the case in the male subjects. The fact that male subjects did not utilize MCTG during exercise to any measurable extent is in accordance with previous findings in our laboratory (22;25) and by others (1;12;21;44), but in contrast to some studies (4;19;34) where the MCTG utilization was determined by applying the muscle biopsy technique. In recent studies in females (17;38) where a combination of isotope tracer technique and indirect calorimetry was applied the authors suggested that the additional source of fatty acids oxidized during exercise at the same absolute workload after training, as compared to before training, was provided by MCTG. Applying the same indirect methodology, several studies in males have indicated a similar significant utilization of MCTG during exercise (27;37). However, recent investigations in our laboratory revealed that combining isotope tracer technique and indirect calorimetry does not provide an accurate measure of MCTG utilization in males (13;36) but does in females (36). This suggests that fat utilized during exercise is recruited from different sources in females and males (36). To our knowledge this study is the first to compare MCTG utilization in matched females and males at different training levels during prolonged exercise by applying the muscle biopsy technique. Recently Guo et al. (12) evaluated the kinetics of intramuscular triacylglycerol fatty acids during exercise. Even though both female and male subjects participated in that study (12), a gender

16 16 comparison was not made. Furthermore, net breakdown of MCTG in the vastus lateralis muscle was not observed during exercise supporting our finding in male subjects. However, that study revealed simultaneous esterification of plasma fatty acids to MCTG and MCTG hydrolysis when subjects exercised at 45% of VO 2 peak (12). Whether these events take place concurrently at higher exercise intensities, as in the present study, is not known. If this is the case, however, interpretation of our data might be that the balance between the two processes is displaced towards a higher net breakdown of MCTG in females than in males. The enzymatic regulation of TG breakdown in skeletal muscle is poorly understood. It has been suggested that a hormone sensitive TG lipase (HSL) similar to the adipose tissue HSL might regulate MCTG hydrolysis (41). Support for this hypothesis was provided when HSL protein and mrna were detected in rat skeletal muscle by Western and Northern blotting, respectively (15;16;28;29). Recently, Kjær et al. (26) also demonstrated the existence of HSL in human skeletal muscle, and evidence was provided that β-adrenergic stimulation increased the activity of HSL in skeletal muscle cells (26;28;29). However, in the present study the arterial plasma concentration of epinephrine was significantly lower in females than in males during exercise, while the arterial plasma concentration of norepinephrine was similar in females and males (45). If HSL is responsible for the hydrolysis of MCTG it might be speculated that females have a higher HSL activity compared to males due to a higher content of HSL and/or a higher sensitivity of HSL to potential stimulators. A gender difference in the content of HSL might exist based on the indication of a higher content of HSL in type I fibers (28) and the fact that a greater percentage of type I fibers relative to area was found in females compared to males in the present study. Regarding lipolytic sensitivity to catecholamines, Hellstrøm et al. (14) found, by means of the microdialysis technique, that during exercise females

17 17 have a higher release of glycerol and fatty acids from the abdominal adipose tissue than males despite the fact that both sexes had similar concentrations of norepinephrine and that females had a lower concentration of epinephrine than males. The sensitivity of lipolytic activity towards these two catecholamines might be higher in females than in males in skeletal muscle tissue as well. It has previously been shown that the initial content of MCTG influences the utilization of MCTG during both exercise (7) and infusion of norepinephrine at rest (10). In the present study the gender difference in MCTG utilization during exercise is at least partly explained by the gender difference in resting content of MCTG as we observed a fair (r=0.61) and significant (p<0.001) correlation between these two parameters. Other factors such as sex hormones might, however, also influence MCTG hydrolysis. At rest the circulating level of progesterone was identical in females and males, while the estradiol level was higher and displayed a greater variation in females than in males. Thus, it might be speculated that estradiol exerts an influence on the resting MCTG content as well as on the degree of MCTG utilization during exercise in females. However, in the present study the concentration of estradiol did not correlate with the MCTG content at rest or its utilization during exercise, rendering it unlikely that estradiol is a major regulatory factor acting directly on the storage and breakdown of MCTG in females. It is controversial whether MCTG hydrolysis is increased with training. In studies measuring the concentration of MCTG by the muscle biopsy technique hydrolysis of MCTG has been found to be similar in the trained and untrained leg in males (22) or higher in endurance trained males (19;34) and females (17) than in untrained subjects during exercise at the same absolute work load. However, in the present study where exercise was performed at the same relative work load, training status did not have any effect on the degree of MCTG breakdown. A similar finding was reported in males from the study by Bergman et al. (1).

18 18 In the present study we observed a gender difference in MCTG utilization at all training levels despite RER being similar during exercise in females and males. Even though we observed that RER decreased during the last 30 min of exercise in END but not in UT and MT, we were not able to observe any increased utilization of MCTG in END compared to UT and MT. The higher lipid oxidation during the last 30 min of exercise in END seemed, however, to be covered primarily by plasma FA, since oxidation of plasma FA increased to a greater extent in END than in MT and UT during the last third of the exercise test (unpublished data). A gender difference in MCTG utilization despite similar RER during exercise indicates that females and males obtain lipids from different lipid sources during exercise. It has previously been observed that FA derived from plasma VLDL-TG might contribute to the oxidative metabolism (13;22). Furthermore, it has been suggested that intermyocellular lipids are possibly mobilized during exercise (22;36), and plasma FA are also known to contribute to the oxidative metabolism. Thus, a gender difference might also exist in the utilization of one or more of these three additional lipid sources. In summary, the present study revealed a higher resting content of MCTG in females than in males irrespective of training level. Furthermore, regardless of training status females utilized significant amounts of MCTG during prolonged exercise whereas males did not. Training had no effect on the resting MCTG content or the utilization of MCTG during exercise in either females or males. Females had a lower concentration of epinephrine in plasma than males, i.e. if HSL is responsible for MCTG hydrolysis it might be speculated that a gender difference exists in the concentration of HSL and/or the sensitivity of HSL towards epinephrine. Other factors such as the resting content of MCTG might influence the degree of MCTG hydrolysis as well.

19 19 ACKNOWLEDGMENTS We acknowledge the skilled technical assistance of Irene Bech Nielsen, Betina Bolmgren, and Winnie Taagerup. Professor Erik A. Richter (M.D.) is thanked for performing the invasive procedures. This study was supported by the Danish National Research Foundation, Grant , and by The Danish Sports Research Council. REFERENCES 1. Bergman, B. C., G. E. Butterfield, E. E. Wolfel, G. A. Casazza, G. D. Lopaschuk, and G. A. Brooks. Evaluation of exercise and training on muscle lipid metabolism. Am.J Physiol Endocrinol.Metab 276: E106-E117, Brooke, M. H. and K. K. Kaiser. Three "myosin adenosine triphosphatase" systems: the nature of their ph lability and sulfhydryl dependence. J Histochem.Cytochem. 18: , Burguera, B., D. Proctor, N. Dietz, Z. Guo, M. Joyner, and M. D. Jensen. Leg free fatty acid kinetics during exercise in men and women. Am.J Physiol Endocrinol.Metab 278: E113-E117, Carlson, L. A., L. G. Ekelund, and S. O. Froberg. Concentration of triglycerides, phospholipids and glycogen in skeletal muscle and of free fatty acids and beta-hydroxybutyric acid in blood in man in response to exercise. Eur.J Clin.Invest 1: , Costill, D. L., J. Daniels, W. Evans, W. Fink, G. Krahenbuhl, and B. Saltin. Skeletal muscle enzymes and fiber composition in male and female track athletes. J Appl.Physiol 40: , 1976.

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25 E R1 LEGENDS TO THE FIGURES Figure 1. Respiratory exchange ratio at rest and during 90 min of exercise at 60% of VO 2 peak in UT (circles), MT (triangles), and END (squares) females (filled symbols) and males (open symbols). Values are given as means ± S.E.M., n=7 in each group. * significantly different from exercise, p<0.001; significantly different from 15, 30, and 60 min in END females and males. Figure 2. MCTG concentration in m. vastus lateralis before (black bars) and after (white bars) 90 min of exercise at 60% of VO 2 peak. Values are given as means ± S.E.M., n=7 in each group except in END females where n=6. * significantly different from before exercise, p<0.001; significantly different from female pre- to post-exercise differences within each training level, p<0.01; significantly different from females within each training level, p< Figure 3. The relationship of initial MCTG concentration and the MCTG utilzation during exercise in UT, MT, and END females (filled circles) and UT, MT, and END males (open circles). Figure 4. Arterial plasma concentrations of insulin, epinephrine, and norepinephrine at rest and during 90 min of exercise at 60% of VO 2 peak in UT (circles), MT (triangles), and END (squares) females (filled symbols) and males (open symbols). Values are given as means ± S.E.M., n=7 in each group. A: Arterial insulin concentration. * significant main effect of gender, p<0.05; significantly different from rest, p<0.05, p<0.001; significantly different from previous time point, p<0.05, p<0.001.

26 26 B: Arterial epinephrine concentration. *significant main effect of gender, p<0.05; significantly different from rest and 30 and 60 min in UT and MT, p<0.001; significantly different from rest in END, p<0.01; significantly different from rest and 30 min in END, p<0.01. C: Arterial norepinephrine concentration. significantly different from rest, p<0.001; significantly different from 30 and 60 min, p<0.05.

27 27 Table 1. Subject and testing characteristics Females Males UT MT END UT MT END Age, yr 27±1.4 26±0.6 25±1.0 27±2.2 23±0.9 26±1.0 Height, m 1.69±0.03* 1.66±0.02* 1.75±0.02* 1.85± ± ±0.03 Body mass (BM), kg 65.0±2.85* 59.0±2.49* 65.9±3.3* 82.9± ± ±1.8 Body fat, % 26.2±2.31* 18.6±1.19* 17.5±1.2* 16.7± ± ±1.3 Lean body mass (LBM), kg 46.7±1.83* 47.2±2.07* 54.1±2.5* 69.3± ± ±1.3 VO 2 peak, l min ±0.107* 3.00±0.16* 3.84±0.18* 3.65± ± ±0.11 ml kg -1 BM min ±0.82* 50.7±1.38* 58.1±1.3* 44.8± ± ±0.8 ml kg -1 LBM min ± ± ± ± ± ±0.6 Training Frequency, bouts wk ± ± ± ±0.5 Duration, h wk ± ± ± ±1.2 Work load, % of VO 2 peak 60±2 59±2 58±1 60±2 59±2 58±1 ml O 2 kg -1 LBM min ± ± ± ± ±0.6 Values are means ± S.E.M., n=7 in each group. * significantly different from males, p<0.001; 41.7±0.8 significantly different from UT, p<0.01, p<0.001; significantly different from MT, p<0.001.

28 28 Table 2. Composition of the habitual and experimental diet Habitual Experimental Energy Carbo- Fat, E% Protein, Energy Carbo- Fat, E% Protein, intake, kj hydrate, E% E% intake, kj hydrate, E% E% Female UT 9146±810* 58.7± ± ± ±438* 66.3± ± ±0.2 MT 10255±576* 63.6± ± ± ±500* 66.1± ± ±0.1 END 13644±1229* 69.1± ± ± ±909* 65.7± ± ±0.2 Male UT 13022± ± ± ± ± ± ± ±0.1 MT 13288± ± ± ± ± ± ± ±0.1 END 16240± ± ± ± ± ± ± ±0.1 Values are means ± S.E.M., n=7 in each group. * significantly different from males, p<0.001; significantly different from experimental diet, p<0.01; significantly different from untrained, p< 0.05, p<0.001; significantly different from moderately trained, p<0.001.

29 29 Table 3. Muscle fiber distribution in m. vastus lateralis in females and males. Females Males Untrained Moderately Endurance Untrained Moderately Endurance trained trained trained trained Type I: % per number 58.8±2.3* 53.5± ± ± ± ±2.9 Area (µm 2 ) 4274±720* 3760±242* 4215±248* 5298± ± ±265 % per area 63.9±3.3* 58.9± ±1.7* 44.2± ± ±3.5 Type IIA: % per number 27.9± ± ± ± ± ±3.1 Area (µm 2 ) 3631±540** 3216±314** 3641±258** 6558± ± ±292 % per area 26.1±3.6* 26.4±2.2* 22.2±1.7* 35.6± ± ±3.7 Type IIB: % per number 10.6± ± ± ± ± ±2.7 Area (µm 2 ) 3380±738** 2815±334** 2908±271** 5755± ± ±330 % per area 8.3±0.9* 12.6±1.3* 2.9±0.7 * 18.5± ± ±2.5 Values are means ± S.E.M., n=7 in each group except in END females where n=6. Significantly different from males,* p<0.01, ** p<0.001; significantly different from UT, p<0.01, p<0.001; significantly different from MT, p<0.05, p<0.001.