For many decades, most of the research regarding substrate metabolism during exercise had been conducted predominately with male subjects. Given that an early report had suggested that men and women respond similarly to endurance exercise (12) and have similar muscle fiber types (12), there was little reason to question the generalization of results derived in men to women. Consequently, there was a sex bias in the exercise physiology literature, with a disproportionate number of male subjects, until research began to question the canonical concept that there were no sex differences in metabolism in response to endurance exercise (3,44). At one time, it was even considered that endurance exercise might be harmful for women, and it was not until 1984 that women could officially compete in the Olympic marathon. In fact, data showing that women can oxidize more fat during endurance exercise (see below) would suggest that women would be ideally suited to complete long-duration endurance exercise and, possibly, even outperform men in ultraendurance events. Two studies have found that women outperformed men for a distance of 85 km when matched for their marathon pace (29,42). These data indicate that any exclusion of women from endurance athletics has not been based on scientific evidence. The current review summarizes a presentation from an ACSM/APS symposium focusing on sex differences in substrate metabolism during endurance exercise from a biological and molecular perspective, with emphasis on the potential mediating effects of 17-β-estradiol.
WHOLE-BODY SUBSTRATE OXIDATION
Whole-body substrate metabolism has been evaluated during endurance exercise, using indirect calorimetry for estimation of whole-body fat and carbohydrate (CHO) use (3,9,35,39,44,51). Stable isotope tracers and urea excretion have been used to estimate the relative contribution of amino acid oxidation to intermediary metabolism during endurance exercise (32,35,36). There were several conflicting reports regarding sex differences in metabolism in the 1970s and 1980s (3,12,20). The discrepancies in the findings were not clear; however, there are important potential confounding variables in any between-group study that are magnified by inherent sex differences. Studies evaluating substrate metabolism must control for antecedent diet, habitual training status, time since the last training bout, and body composition. When matching men and women for training status, V˙O2max per kilogram of body mass is higher in men; however, it is usually identical when expressed relative to fat-free mass, given that women have approximately 5-8% body fat more than men (9,32,35,49). It has been suggested that matching on the basis of V˙O2max per kilogram of fat-free mass is the most appropriate way to match men and women for levels of fitness (23,25,35,44). Indeed, our group (9,15,32,35) and others (23,38) have found that when men and women are selected for training background, V˙O2max per kilogram of fat-free mass is the same between the sexes. Furthermore, when men and women are matched for V˙O2max per kilogram of fat-free mass, the lactate threshold is the same between sexes (23,35). Another potential confounding variable is the menstrual cycle phase and menstrual irregularities such as oligoamenorrhea and amenorrhea. For example, amenorrhea reduces the pulsatility of LH and FSH, which lowers total estrogen and progesterone concentrations to those seen in men (testosterone would still be approximately tenfold greater in men). Menstrual cycle phase does have a small influence on substrate selection during endurance exercise; however, these differences are rather small when compared with the differences between the sexes (11,15,18,24). Oral contraceptive use has a slightly greater effect on substrate selection during endurance exercise, with an increase in glycerol rate of appearance (Ra, lipolysis) being observed (18). Together, a failure to consider the aforementioned factors can increase variance and lead to false conclusions regarding sex differences.
When most of the potential confounding factors are considered, and men and women are compared within the same study, there are a number of reports concluding that the RER for women is lower than for men during endurance exercise. A summary from the mean RER for the majority of the studies published in the English literature comparing substrate oxidation in men and women during moderate-duration endurance exercise (at least 60 min) clearly shows that the RER is lower for women than for men (higher fat and lower CHO oxidation) during endurance exercise (Table 1). It is important to note that the data in Table 1 are a compilation of all reported studies, with no weighting for the aforementioned factors that could increase variance and lead to an inability to detect a difference (type II error). Nevertheless, when all of the evidence is considered together, sex differences at the level of whole-body substrate oxidation are a robust finding across studies. In addition to this meta-analysis approach, a recent summary of exercise testing results from 157 men and 147 women in the same laboratory also found higher fat oxidation in women compared with men (51). Finally, the fact that fat oxidation is higher for women than men during endurance exercise before and after a supervised endurance exercise training program (9,32) further suggests that the sex-based differences in substrate selection are attributable to sex, and not matching issues related to unequal training status. Although a higher proportionate area of type I muscle fibers in women compared with men can partially explain the higher fat-oxidation rates seen for women during endurance exercise (10,39), the fact that acute 17-β-estradiol administration to men shifts fuel selection toward higher fat oxidation (14) implies that fiber type is not the only explanation for the sex difference in metabolism.
With respect to glycogen metabolism, there does not seem to be a sex difference in basal muscle glycogen content in trained or untrained individuals (32,49). We have found attenuated glycogen use during treadmill running in women compared with men (44), and similar sex differences have been noted in rats (26,27,40). In contrast, we and others have not found a sex difference in net glycogen use during cycling exercise when using muscle biopsies (32,38). However, estimated glycogen use from data using tracer methodology has suggested glycogen sparing (9), and there was evidence of glycogen sparing during endurance cycling in the luteal phase of the menstrual cycle (15). In contrast to the variable results with respect to muscle glycogen, there is consensus showing a lower glucose Ra and rate of disappearance in women compared with men during endurance exercise (9,15,18,23). This may relate to lower sympathetic activation in women than in men (23,32,43).
Animal studies have robustly demonstrated that the administration of 17-β-estradiol attenuates glycogen use in skeletal muscle, heart, and liver (26,40). Furthermore, ovariectomy leads to an increase in muscle and hepatic glycogen use during endurance exercise in female rats (27). In humans, 17-β-estradiol administration is associated with a reduction in hepatic glucose Ra (8,14,41) and with lower epinephrine (41) during endurance exercise; however, we have not seen any effect on net glycogen use using the muscle biopsy technique (14). We have recently shown that 17-β-estradiol administration to men slightly, but significantly, reduced basal muscle glycogen concentration (14). Importantly, the administration of 17-β-estradiol to men does not seem to work indirectly through a suppression of endogenous testosterone given that short-term testosterone supplementation does not appear to alter substrate oxidation during exercise (4). Finally, one study has used pharmacological suppression of endogenous sex hormone secretion and selective replacement of 17-β-estradiol with and without progesterone in exercising women; the study found that 17-β-estradiol reduced total CHO oxidation by reducing both glucose Ra and estimated muscle glycogen use (13).
We recently compared skeletal muscle mRNA abundance in men and women, and before and after 17-β-estradiol administration, for a variety of transcripts involved in glucose transport (GLUT4), glucose phosphorylation (hexokinase II), glycogen synthesis (glycogenin, glycogen synthase 1, glycogen synthase kinase 3-α), glycogenolysis (glycogen phosphorylase), and glycolysis (phosphofructokinase). We found significantly higher resting skeletal muscle hexokinase II and GLUT4 mRNA content during the follicular phase and lower mRNA abundance for phosphofructokinase during the luteal phase (Table 2). Overall, these mRNA changes were not directionally consistent with any of the observed metabolic substrate-use data described above or with our previous observation that hexokinase II enzyme activity is similar between men and women (49). We also found that 17-β-estradiol administration to men did not alter basal mRNA abundance for any of the aforementioned genes involved in glucose metabolism (Table 2). Overall, it is likely that neither sex nor 17-β-estradiol directly mediates glycogen use in skeletal muscle, and that the sex differences in metabolism are likely responding to a sex difference in fat metabolism occurring during endurance exercise (see below).
Most studies have reported higher intramyocellular lipid (IMCL) in women compared with men (38,39,48). We have used electron microscopy to show that the higher IMCL abundance in women is attributable to more lipid droplets, and not a larger size of individual droplets (48). Biochemical assays have shown a greater use of IMCL during endurance exercise in women (38,39,43). These findings are consistent with the higher hormone-sensitive lipase mRNA and protein (PRO) content reported in women, but not with the fact that the activation of hormone-sensitive lipase after 90 min of exercise was higher in men (39). Given that IMCL content is often reported not to decrease significantly in men in response to endurance exercise (39,43), the role of hormone-sensitive lipase activation in modulating IMCL breakdown during endurance exercise is in question.
There also seems to be a sex difference in whole-body lipolysis, because we (9) and others (18) have found a higher glycerol Ra for women compared with men during endurance exercise. Importantly, the aforementioned sex differences were apparent when the glycerol Ra values were expressed relative to total body mass, and the difference would be even greater if it were expressed relative to the smaller fat-free mass inherent in women. Furthermore, arterial glycerol concentration is higher during endurance cycling in women than in men during the first 30 min (38). Animal studies have shown that 17-β-estradiol administration to male rats led to increased skeletal muscle and lower adipocyte lipoprotein lipase (LPL) activity, which could indicate a preferred storage of lipids in the skeletal muscle for immediate availability during exercise (17). In contrast to the robust effects of 17-β-estradiol on whole-body lipolysis in resting and exercised rats (17), the administration of 17-β-estradiol to men (8) and amenorrheic women (41) was not associated with a higher exercise-stimulated lipolysis (glycerol Ra) during endurance exercise; however, plasma FFA concentrations were higher after 17-β-estradiol in women (41). Studies using testosterone supplementation to postmenopausal women (4) suggest that testosterone may attenuate exercise-stimulated lipolysis, which would make lipolysis higher for women than for men.
In contrast to the lack of directional consistency between the mRNA content for genes involved in CHO metabolism and measured substrate use, the mRNA abundance for a wide variety of genes in skeletal muscle involved in fat metabolism are directionally consistent with the observation of enhanced fat use by women compared with men during endurance exercise. For example, skeletal muscle hormone-sensitive lipase (HSL) mRNA and PRO are all higher in women than in men (39). Women also have much higher skeletal muscle LPL mRNA content compared with men, with no sex differences in LPL activity (28). Fatty acid translocase (FAT/CD36) PRO is about 50% higher in women than in men (28), and women have about twofold more sarcolemmal fatty acid transporter (FATP-1) mRNA compared with men (2). Given the recent observation that FAT/CD36 may also localize to the mitochondrial membrane (22), it will be of interest to see whether there are sex differences in this localization that could indicate greater fatty acyl group transport into mitochondria in women. There does not seem to be a sex difference in carnitine palmitoyl transferase-1 in humans (1); however, 17-β-estradiol supplementation in ovariectomized rats demonstrated an increase in the maximal enzyme activity of carnitine palmitoyl transferase-1 and short-chain β-3-OH-acyl-CoA-dehydrogenase (7). We have not found a sex difference in short-chain β-3-OH-acyl-CoA-dehydrogenase or several components of the electron-transport chain enzyme activity (10,32); however, very-long-chain acyl-CoA dehydrogenase (VLCAD) is significantly higher in women than in men according to Western blot analysis of total PRO content (Fig. 1).
We have recently conducted an evaluation of many of the transcripts involved in fat metabolism in skeletal muscle, and we found a coordinate higher abundance in skeletal muscle of women compared with men (Table 2). Although mRNA abundance may not always indicate PRO abundance or activity, the directional consistency of these findings, and the fact that another laboratory has found identical findings (28,37), suggest that some aspect of sex leads to an increase in fat oxidation through a regulated process at the transcriptome level. From a regulatory perspective, it does not seem that AMP-kinase is involved in the pathway responsible for the increase in fat oxidation in women (39) (Fig. 1). As further evidence that there is some pretranslational control of fat-oxidation pathways, we have administered 17-β-estradiol to men and shown increases in lipid oxidation (14) and in many of the aforementioned mRNA species involved in fat metabolism (Table 2).
There is no question that there is an increase in the oxidation of several amino acids during endurance exercise; however, this rarely represents more than about 5% of the total energy cost. The first evidence that there may be a sex difference in oxidation of amino acids during exercise came from the observation that urinary urea excretion was higher on an exercise day (15.1-km run) versus a rest day for men, but this pattern was not seen in women (32). Subsequently, several other studies have also shown less of an increase in urea excretion for women compared with men in response to endurance exercise (30-32,35,36). There also seems to be a sex difference in amino acid oxidation, with women oxidizing less leucine (30-32,35,36). The increase in leucine oxidation is attributable to dephosphorylation and subsequent activation of branched chain 2-oxo acid dehydrogenase (BCOAD). Our group has shown that the basal activation of BCOAD was lower in women than in men, but the activation after exercise was similar between the sexes (32). Interestingly, after a 2-month period of training, there was an attenuation of BCOAD activation, which corresponded to a suppression of leucine oxidation during exercise for men and women (32). The latter observation would predict that PRO requirements would be lower after a period of training. Consistent with the latter observation, studies in recreationally trained subjects (predominantly men) have found no influence, or a minimal influence, of this type of activity on PRO requirements (50), and modest increases in better-trained athletes (33). In contrast, well-trained to top sport athletes have much higher volumes of training at higher intensities and are not always able to maintain optimal CHO and energy balance. These factors could raise PRO requirements to above that of a sedentary person (5,47).
Three studies used nitrogen balance to determine the dietary PRO requirements for very highly trained male endurance athletes (runners, triathletes, cyclists, nordic skiers), and these estimates came out to approximately 1.4-1.7 g·kg−1·d−1 (5,19,47). We have found that well-trained men and women were both in negative nitrogen balance while consuming a PRO intake that was considered by the Canadian and U.S. government bodies to be adequate to meet the needs of all adults (men = 0.94 g·kg−1·d−1; women 0.8 g·kg−1·d−1) (35). Even though the women were consuming less PRO than the men, they were in a less negative nitrogen balance (35). Carol Meredith and colleagues (33) also have shown that dietary PRO requirements were higher than the U.S. recommended daily intake for young and middle-aged male runners who were training at about the same level as those in our latter study (35).
The most likely candidate for the lower rates of amino acid oxidation and the less negative impact on whole-body PRO balance would be 17-β-estradiol. Consequently, we administered 17-β-estradiol to 12 men in a randomized, double-blind fashion for 8 d, and we found that leucine oxidation during 90 min of cycling was significantly lower by 16%, and this was associated with an increase in fat oxidation (44%) and a reduction in CHO oxidation (16%) (21). We have recently measured the skeletal muscle mRNA content for both BCOAD and its kinase (inactivator), BCOADK, and found that BCOADK was significantly higher for women in both phases of the menstrual cycle, and although mRNA content went up 33% in men on 17-β-estradiol, it was not statistically significant (Fu et al., PhD thesis, 2007).
Nutritional implications of sex differences in metabolism.
On the basis of our observations that women have lower CHO oxidation compared with men during endurance exercise, we evaluated whether this would attenuate their ability to CHO load and derive an ergogenic benefit (45). We randomly allocated athletic men and women to follow a CHO-loading strategy for 4 d while consuming one of two diets (habitual = 55% energy from CHO; high = 75% energy from CHO). Muscle biopsies were taken, and a performance ride (60 min at 75% V˙O2peak → time to exhaustion at 85% V˙O2peak) was completed after each loading session. As expected, the men showed a significant increase in muscle glycogen concentration (+41%), with a corresponding increase in performance (+45%); however, the women showed neither an increase in muscle glycogen concentration (0%) nor an increase in performance (+5%) (45). The mechanism behind the latter result was unclear; however, the relatively low energy intake by the women limited the amount of dietary CHO, such that even on the 75% CHO diet, only 6.5 g·kg−1·d−1 were consumed (45). In contrast, the hallmark study by Sherman and colleagues that set the standard for CHO loading used men where the high-CHO diet represented >8.0 g·kg−1·d−1 (16). For many women, attaining a level of > 8.0 g CHO·kg−1·d−1 would require that 100% of the diet came from CHO; consequently, the only practical method to achieve this would be to increase total energy intake. Consequently, we repeated our CHO-loading study, using men and women with three trials (habitual = 55% energy from CHO; high CHO = 75% energy from CHO; high CHO + extra energy = 75% energy from CHO + 34% more energy (> 8.0 g CHO·kg−1·d−1)). For men, there was a progressive increase in muscle glycogen concentration on each of the three diets, and for women, we again found no muscle glycogen increase in the 75% CHO diet, yet they did show an increase with the high CHO + extra energy diet, albeit to levels that were about 50% that of men (49). Others have found that CHO-loading protocols do not result in the same level of glycogen supercompensation as demonstrated in studies done with men (52). From a practical perspective, attempting to improve exercise performance through CHO loading is futile unless women can get CHO intake to > 8.0 g·kg−1·d−1, which requires an energy intake of 2500 kcal·d−1 with 70% CHO for a 55-kg woman.
The amount of exogenous glucose that is oxidized from sports drinks is similar (53) and, in some cases, slightly higher (7,36), for women compared with men. In contrast to the limited ability for women to CHO load, the dietary recommendations for men and women with respect to sport drink consumption during exercise (36,53) seem to be similar. In the early postexercise recovery period, the rate of muscle glycogen resynthesis from either a CHO or CHO + PRO drink provided immediately and at 1 h after endurance exercise is similar for men and women (46). Given that the rate of muscle glycogen resynthesis during the first 4 h after endurance exercise is negligible (46), it is prudent for both sexes to provide CHO and PRO in the early postexercise period if a subsequent training session or race is scheduled later in the same day, to maximize glycogen resynthesis.
Although no study has specifically calculated PRO requirements for top sport women athletes, the available nitrogen-balance data in men (5,19,47), and the nitrogen-balance data that we have in endurance-trained men and women (35), would suggest that requirements for women are about 25% less than for men (i.e., top sport women = 1.2-1.3 g·kg−1·d−1). It is important to note that most athletes will get this level of dietary PRO intake from an energy-adequate habitual diet containing 10-15% PRO; however, we and others have found that some female and a few male endurance athletes can have intake levels as low as 0.45 g·kg−1·d−1 (32,34,35). Consequently, it is important to evaluate PRO intake on a gram-per-kilogram basis, as opposed to a percentage of the diet, to avoid low intakes that can be seen in energy-restricting athletes. This is particularly important because a low-energy intake is well known to negatively impact PRO requirements (6).
Women oxidize more fat and less CHO and amino acids during endurance exercise as compared with men. At least some of the sex difference is attributable to the higher levels of 17-β-estradiol in women, because administration of 17-β-estradiol to men leads to lower amino acid and CHO (and higher fat) oxidation during endurance exercise. The skeletal muscle mRNA transcript abundance for genes involved in fat metabolism are coordinately consistent with the higher fat oxidation seen in women than in men, and some of the transcripts are altered in a directionally similar pattern with the administration of 17-β-estradiol to men. Given that sex differences in the mRNA abundance for genes involved in CHO and PRO metabolism are not directionally consistent with the observed sex differences in exercise metabolism or the effect of 17-β-estradiol in animals or humans, it is likely that sex/17-β-estradiol primarily influence fat oxidation during exercise and that CHO and PRO metabolism follow by metabolic demand.
The majority of the author's work in the current study was funded by a grant in aid from NSERC Canada. Some of the research was conducted using equipment purchased through a donation from Mr. Warren Lammert and Kathy Corkins. A number of current and former graduate students and postdoctoral fellows have contributed to the work presented from the author's laboratory (Dr. Mazen Hamadeh, Ms. Michaela Devries, Mr. Minghua Fu, Ms. Amy Maher, Mr. Stuart Lowther, Dr. Sherry Carter, Dr. Scott McKenzie, Dr. Stuart Phillips, Dr. Courtney Rennie). None of the work completed in Dr. Tarnopolsky's laboratory involving any aspect of sex differences in metabolism or sex differences in nutrition have been funded by a company.
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