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Fatty acid reesterification but not oxidation is increased by oral contraceptive use in women

Exercise Physiology Laboratory, Department of Integrative Biology, University of California, Berkeley, California

Submitted 1 July 2004 ; accepted in final form 17 December 2004

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
We evaluated the hypothesis that fatty acid reesterification would be increased during rest and exercise in the midluteal menstrual cycle phase and during oral contraceptive use, when ovarian hormone concentrations are high, compared with the early follicular phase. Subjects were eight moderately active, weight-stable, eumenorrheic women (24.8 ± 1.2 yr, peak oxygen consumption = 42.0 ± 2.3 ml·kg^–1·min^–1) who had not taken oral contraceptives for at least 6 mo. Plasma free fatty acid (FFA) kinetics were assessed in the 3-h postprandial state by continuous infusion of [1-¹³C]palmitate and [1,1,2,3,3-²H]glycerol during 90 min of rest and 60 min of exercise at 45% and 65% peak oxygen consumption in the early follicular and midluteal menstrual cycle phases and during the inactive- and high-dose phases following 4 mo of oral contraceptive use. Plasma FFA rates of appearance, disappearance, and oxidation increased significantly from rest to exercise with no differences noted between menstrual cycle or oral contraceptive phases or exercise intensities. Compared with either menstrual cycle phase, oral contraceptive use resulted in an increase in plasma-derived fatty acid reesterification and a decrease in the proportion of plasma FFA rate of disappearance that was oxidized at rest and during exercise. Endogenous and exogenous synthetic ovarian hormones do not exert a measurable influence on plasma FFA turnover or oxidation at rest or during moderate-intensity exercise in the 3-h postprandial state when carbohydrate use predominates. The increase in whole body lipolytic rate during exercise noted previously with oral contraceptive use is not matched by an increase in fatty acid oxidation and results in an increase in reesterification. Synthetic ovarian hormones contained in oral contraceptives increase lipolytic rate, but fatty acid oxidation during exercise is determined by exercise intensity and its metabolic and endocrine consequences.

estrogen; progesterone; menstrual cycle; exertion

Longitudinal study designs to assess menstrual cycle and oral contraceptive effects on lipid metabolism avoid the subject-matching problems faced by gender comparison studies. Fluctuations in ovarian hormones between the follicular (FP) and luteal phases (LP) of the menstrual cycle do not influence glycerol turnover (14) or whole body fatty acid R_ox at rest or during prolonged exercise (7, 37, 40, 56). Additionally, plasma FFA turnover at rest was similar between the FP and LP (34). Studies reporting subtle increases in whole body fatty acid R_ox during exercise in the luteal compared with the FP also demonstrated that these effects were mitigated at lower exercise intensities (63) or by carbohydrate feeding (10). Intersubject variability in ovarian hormone concentrations during the different phases of the menstrual cycle often makes the results of studies on humans difficult to interpret. The only study to tightly control ovarian hormones in eumenorrheic women with pharmacological suppression and replacement of estradiol and progesterone found that conditions of high estradiol-low progesterone resulted in greater whole body fatty acid R_ox during prolonged exercise than either low estradiol-low progesterone or high estradiol-high progesterone (17). To date, a detailed examination of the influence of ovarian hormones across the normal menstrual cycle on plasma FFA kinetics at rest and during exercise has not been reported.

The influence of synthetic ovarian hormone supplementation in amounts typical of low-dose oral contraceptives on human lipid metabolism has received little attention. Synthetic estradiol supplementation in men (13) and amenorrheic women (50) did not alter glycerol turnover or whole body fatty acid R_ox at rest or during prolonged exercise. Synthetic estradiol and progesterone supplementation through the use of oral contraceptives is common and has been shown to increase plasma cortisol concentration at rest and during exercise (1, 14, 60) and plasma FFA concentration during low-intensity exercise (6). However, 3 mo of oral contraceptive use did not alter plasma oleate turnover at rest (26). Cross-sectional investigations have found no difference in whole body fatty acid R_ox during 30–90 min of exercise at 45–85% peak oxygen consumption (O_{2 peak}) between women using mono- or multiphasic oral contraceptives for 12–98 mo and those not taking oral contraceptives (4, 6). Recently, our laboratory (14) reported that oral contraceptive use increases plasma glycerol turnover and, therefore, whole body lipolytic rate, during exercise. To date, a detailed examination of the influence of oral contraceptive use on parameters of plasma FFA kinetics, including fatty acid reesterification, at rest and during exercise has not been reported.

The existing body of research suggests that, while ovarian hormones may promote fatty acid mobilization, their effects on whole body fatty acid R_ox and plasma FFA kinetics are unclear. The purpose of this investigation was to employ a longitudinal study design to examine the influences of endogenous ovarian hormones and synthetic ovarian hormones contained in oral contraceptives on whole body fatty acid R_ox and plasma FFA turnover and R_ox at rest and during moderate-intensity exercise. Assuming that substrate use during exercise is primarily regulated by exercise intensity and its metabolic and endocrine consequences (9), we anticipated that whole body fatty acid R_ox and plasma FFA turnover and R_ox would be unaffected by either endogenous or synthetic ovarian hormones. Given our expectation that ovarian hormones would promote fatty acid mobilization (14) without affecting oxidation, we tested the hypothesis that fatty acid reesterification would be increased during rest and exercise in the LP and during oral contraceptive use, when ovarian hormone concentrations are high, compared with the early FP.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Detailed descriptions of the methods employed in this study have been previously published (14, 15, 55, 56), but relevant information is reiterated for the convenience of the reader.

Subjects.   Eight healthy, nonsmoking, weight-stable women were recruited from the University of California, Berkeley community by posted notices and E-mails for participants in a series of experiments to examine the effects of ovarian hormones on maximal aerobic capacity (O_{2 peak}) and substrate use during submaximal exercise. Subjects were nulliparous, reported regular menstrual cycles (24–32 days in duration), and had not taken oral contraceptives for 6 mo. Subjects habitually exercised 2–6 h/wk (3.7 ± 0.7 h/wk), but were not trained endurance athletes. Health history questionnaires and physical examination revealed that the women were injury and disease free. The procedures and risks were thoroughly explained to the subjects, and their written, informed consent was obtained. The University of California Committee for the Protection of Human Subjects approved the study protocol (CPHS no. 2001–8-132).

Experimental design.   After screening, subjects performed eight stable isotope tracer infusion trials: four trials during the menstrual cycle phases and four trials following oral contraceptive use (Fig. 1). Stable isotope tracer infusion trials consisted of 90 min of rest followed by 60 min of bicycle ergometer exercise at either 45 or 65% O_{2 peak} and were separated by 3 days. Before oral contraceptive (BOC) use, trials were conducted within two sequential menstrual cycles in a randomized order during the early FP (3–9 days after the start of menses), and the mid-LP (17–25 days after the start of menses and 4–10 days after ovulation). Ovulation was predicted using urine ovulation predictor kits (First Response, Carter Products). FP and LP were confirmed by the determination of plasma estradiol and progesterone concentrations from blood samples taken before exercise, and estradiol levels >50 pg/ml and progesterone levels >3 ng/ml were used as verification of LP (52). Luteinizing hormone surges were detected from urinary analysis of every menstrual cycle studied. However, post hoc analyses of plasma from three subjects did not show anticipated rises in estradiol and progesterone concentrations following luteinizing hormone surges. After completion of the menstrual cycle-phase testing, each subject began taking the same triphasic oral contraceptive (Ortho Tri-Cyclen, 1 pill/day) for four complete cycles (28 days/cycle), starting on the first Sunday after the start of menses. Each pill taken on days 1–21 contained 0.035 mg of ethinyl estradiol and a norgestimate dose that increased weekly (0.180, 0.215, and 0.250 mg norgestimate on days 1–7, 8–14, and 15–21, respectively). On days 22–28, the pills contained no synthetic ovarian hormones. Four stable isotope tracer infusion trials were conducted within two sequential pill cycles in randomized order during the third week of high-dose phase of active pill ingestion (HP) and during the week of inactive-dose phase of pill ingestion (IP).

View larger version (9K): [in this window] [in a new window]   Fig. 1. Experimental design. FP, follicular phase; LP, luteal phase; IP, inactive-dose phase; HP, high-dose phase; O2 peak, peak oxygen consumption. Order of trials before and during oral contraceptive use was randomized.

Tracer protocol.   Subjects were instructed to refrain from exercise, caffeine, and medications 24 h before each stable isotope tracer infusion trial. The stable isotope tracer infusion trials were conducted in a 3-h postprandial state in the morning, and dietary intake was controlled for the 24 h immediately preceding each of the eight stable isotope tracer infusion trials (2,183 kcal, 63% carbohydrate, 22% fat, and 15% protein). Each subject consumed a standardized low-glycemic index breakfast (308 kcal, 75% carbohydrate, 9% fat, and 16% protein) in the laboratory 3 h before the start of exercise. We chose to test our subjects in a rested and recently fed 3-h postprandial state to control for the effects of meal size, composition, and timing, and to mimic conditions in a nonlaboratory environment. On the morning of a trial, a catheter was placed in a hand or wrist vein to obtain "arterialized" blood samples using the heated hand vein technique, and a forearm venous catheter was placed in the contralateral arm for continuous stable isotope tracer infusion. In previous studies in our laboratory (36), radial artery and heated hand vein blood samples were drawn simultaneously for determination of glucose, glycerol, and palmitate isotopic enrichments (IE) and were not found to be significantly different. After collection of background blood and breath samples, subjects rested supine or semisupine for 90 min while [1-¹³C]palmitate was continuously infused (0.61 mg/min; Baxter Travenol 6300 infusion pump). The resting palmitate tracer infusion rate was doubled (1.22 mg/min) during exercise at 45 and 65% O_{2 peak} to maintain isotopic equilibrium (20, 23). [1-¹³C]palmitate was obtained from Cambridge Isotope Laboratories (Nashua, NH), combined with 100 ml of 25% human albumin, diluted in 400-ml 0.9% sterile saline, and pharmacologically tested for sterility and pyrogenicity (School of Pharmacy, University of California, San Francisco). All palmitate/albumin infusates were used within 5 days of completion of sterility testing. Subjects were simultaneously infused with [6,6-²H]glucose and [1,1,2,3,3-²H]glycerol to assess glucose and glycerol kinetics, and these results have been previously published (14, 55, 56).

At each of the blood sampling points, respiratory gas exchange was determined, and an aliquot of expired air was collected in duplicate in 10-ml vacuum Exetainer tubes for the determination of ¹³CO₂ IE. The expired air samples were stored at room temperature and were analyzed by Metabolic Solution (Merrimack, NH) using isotope ratio mass spectrometry (MS). Heart rate was recorded throughout rest and exercise using an electrocardiograph (model Q750, Quinton, Seattle, WA), and blood pressure was measured by auscultation.

Blood sampling and analysis.   Blood samples were taken at 0, 60, 75, and 90 min of rest and 15, 30, 45, and 60 min of exercise, thoroughly mixed, spun in a refrigerated centrifuge at 2,800 g for 13 min, decanted, and stored at –20 or –80°C until analysis. The sampling and analytic procedures for plasma total FFA (enzymatic assay), glycerol, hormones and blood glucose, and lactate have been previously published (14, 55, 56). Importantly, the estrogen and progesterone kits used detected only endogenous ovarian hormones and not the synthetic ovarian hormones found in the oral contraceptives. Samples for palmitate-stable IE analysis were prepared by thoroughly mixing exactly 1 ml of plasma with a heptane-isopropanol (30:70) extraction solution containing H₂SO₄ (0.0033 M) and 100 nmol of pentadecanoic acid (15:0) as an internal standard. Extracted plasma samples were stored at –20°C for later analysis. Hematocrit was measured at each sampling point using circular microcapillary tube reader (no. 2201, International Equipment) to assess any changes in plasma volume due to exercise. Subjects drank tap water ad libitum during each trial to maintain normal hydration status.

Plasma catecholamine analysis.   Blood for catecholamine analysis was collected in chilled tubes containing glutathione and EGTA to prevent oxidation. Blood was immediately centrifuged at 3,000 g, and the plasma was transferred to a storage tube and was frozen at –80°C until analysis. Catecholamines were extracted from the plasma using methods adapted from Anton and Sayre (2) using acid-washed WA-4 Alumina (Sigma, St. Louis, MO) and 1.5 M Tris buffer containing 2% EGTA at a pH of 8.6. An internal standard of 500 pg/ml of dihydroxybenzylamine (ESA, Clemsford, MA) was added to each standard and sample before extraction. Standard catecholamine solutions were purchased from ESA (Clemsford, MA). After vigorous shaking for 15 min, samples were rinsed three times with 2 ml deionized water and then transferred to filter tubes and centrifuged at 1,200 rpm in a table-top centrifuge (Microfuge 12, Beckman, Fullerton, CA), and perchloric acid (0.1 M) was used to elute the catecholamines. Finally, 100 µl of the eluent were injected into the HPLC system (ESA Coulochem LC/EC, 5200A, Clemsford, MA). The flow rate was 1.0 ml/min, the mobile phase was Cataphase 2 (ESA, Cambridge, MA), the column was C-18 4.6 x 80 mm, and the electrodes were set at +350, 50, and –350 mV for the 5021 Conditioning cell and 5011 Dual Channel High Sensitivity Analytical cell, respectively. Chromatographs were analyzed using EZChrom Elite data analysis system (ESA).

IE analysis.   The fatty acids of the extracted plasma samples were isolated by thin-layer chromatography using an oleate [18:1(n-9)] standard. Isolated samples were derivatized to their fatty acid methyl esters to allow easy volatilization by gas chromatography (GC). Individual plasma long-chain fatty acid concentrations and palmitate IE were determined simultaneously by flame ionization detection (FID) and GC-MS, respectively (GC model 6890 and MS model 5973N; Hewlett-Packard). Following separation by GC, a microcontroller was used to split the column and deliver appropriate loads to the FID and MS. For GC-MS analysis, the injector temperature was set at 250°C, and initial oven temperature was set at 55°C. The oven temperature was held at 55°C for 30 s and gradually increased by 25°C/min until it reached a temperature of 195°C and was then ramped at 3°C/min to 205°C and then 8°C/min to 230°C. The column used was a DB225, 30 m x 0.25 µm (Agilent). Helium was used as the carrier gas in constant flow mode for all analyses with an average velocity of 60 cm/s. The source temperature was set at 230°C, and the quadrupole temperature was set at 150°C. Electron impact ionization was performed with selected ion monitoring to monitor ion mass-to-charge ratios of 270 and 271 for [¹²C]palmitate and [¹³C]palmitate, respectively. Glycerol IE was determined as described previously (14).

Calculations.   Individual plasma long-chain fatty acid concentrations were calculated relative to the abundance of the internal pentadecanoic acid standard and a physiological range of external standards. Palmitate IE values were corrected for baseline enrichments from background blood samples taken before the infusion of the isotopes. Palmitate rate of appearance (R_a) and disappearance (R_d) and metabolic clearance rate were calculated using equations defined by Steele and modified for use with stable isotopes (61), as described previously (21). Palmitate kinetics were converted to FFA kinetics by dividing by the fraction of plasma palmitate to total FFA concentration (0.30) as determined by FID, assuming that the relative concentration of each individual plasma FFA is directly related to its relative release from adipose tissue. This assumption has been supported by recent work (46), and palmitate and oleate have long been considered optimal for modeling plasma FFA kinetics (25, 28, 29, 33, 46). EE values were derived from respiratory data averaged over the last 5 min of each 10-min collection period. The proportion of EE derived from carbohydrate and lipid and the rates of EE, whole body carbohydrate, and lipid oxidation were calculated using the following stochiometric equations as described previously (21, 42, 64):

where CHO is carbohydrate; RER is the respiratory exchange ratio; and O₂ is oxygen uptake expressed in l/min. Rates of carbohydrate and lipid oxidation were converted to body weight relative units (µmol·kg^–1·min^–1) using the molecular weights of glucose (180 g/mol) and a representative triglyceride (860 g/mol), and lipid R_ox was converted to fatty acid R_ox by multiplying by 3 (3 mol fatty acids/mol triglyceride). Plasma FFA R_ox was calculated as:

where IE_breath is the IE of ¹³C in the breath, CO₂ is CO₂ production in µmol·kg^–1·min^–1, assuming 22.4 l/mol CO₂, IE_plasma is the IE of [¹³C]palmitate in the plasma, CF is the bicarbonate correction factor [0.65 for rest and 0.90 for exercise determined previously (43)], and %palmitate is the fraction of plasma palmitate to total FFA concentration as determined by FID. The R_ox of other fatty acids (purported to primarily represent IMTG) was calculated by subtracting plasma FFA R_ox from whole body fatty acid R_ox. The rate of lipolysis was calculated as three times glycerol R_a assuming that released glycerol enters the plasma pool only as a result of lipolysis, glycerol cannot be reincorporated into triglyceride within adipose tissue or skeletal muscle, and triglyceride molecules are completely hydrolyzed (62). The rates of reesterification (R_s) were calculated as:

Local fatty acid R_s (often termed "intracellular R_s") represents the reesterification of fatty acids that presumably never left their site of origin, whereas plasma-derived fatty acid R_s (often termed "extracellular R_s") represents the reesterification of fatty acids that exited their site of origin and were reesterified in another tissue (adipose, liver, or skeletal muscle). Local fatty acid R_s has previously been calculated by subtracting plasma FFA R_a from the rate of lipolysis (59, 62). We feel that this approach may have overestimated local fatty acid R_s by ignoring the reesterification of fatty acids within skeletal muscle that originated from the hydrolysis of IMTG and never entered the plasma pool (A. L. Friedlander, personal communication). The equations of the present investigation attempt to correct this oversight by incorporating the oxidation of other fatty acids purported to primarily represent IMTG.

Statistics.   Data are represented as means ± SE. While individual plasma long-chain fatty acid data were analyzed over time, steady-state FFA kinetic data were calculated using the last two (75 and 90 min) and three (30, 45, and 60 min) IE values obtained during rest and exercise, respectively. Results of Shapiro-Wilks tests indicated that the data were normally distributed. The significance of mean differences was assessed by ANOVA with repeated measures followed by post hoc analyses using the least significant difference test. Significance was set a priori at P < 0.05.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Despite concerted efforts to test subjects in the intended menstrual cycle phase, three women did not meet the ovarian hormone concentration criteria (one in FP and two in LP). Menstrual cycle phase comparisons (FP vs. LP) were performed in the remaining five women with complete sets of data. No significant differences were noted in any variable measured between FP and LP, and the data from all eight subjects collected BOC were pooled by averaging each subject's FP and LP values (excluding the individual trials where ovarian hormone criteria were not met). Likewise, data on resting women within each condition (BOC, IP, and HP) were not different and were pooled. Although not statistically different from each other, FP and LP fatty acid turnover and R_ox data are described where appropriate for the purpose of comparison with previous studies.

Subject characteristics.   The physical characteristics of the subjects have been presented previously (14, 15, 55, 56) and are repeated in Table 1 for convenience of the reader. Oral contraceptive use was associated with a small, but significant increase in body weight, body fat percent, and fat mass and an 11% decrease in O_{2 peak} compared with BOC (15).

Individual plasma long-chain fatty acid concentrations.   Plasma myristate, palmitate, palmitoleate, stearate, oleate, linoleate, and total plasma FFA concentration increased approximately two- to fivefold during exercise compared with rest in all conditions and both exercise intensities (Table 2). Changes in linolenate and arachidonate could not be tested statistically due to missing values when their concentrations were below the level of detection, but collectively they represented <2% of the total plasma FFA pool. Plasma palmitate concentration was higher in HP than BOC at 45 and 60 min of exercise at 45% O_{2 peak} (P < 0.05). Oleate and palmitate were the most abundant plasma FFA (42 and 28% of total FFA, respectively), and the percentage of palmitate increased significantly from rest (25.7 ± 0.5% of total FFA) to the last 15 min of exercise (28.2 ± 0.6% of total FFA) in all conditions and both exercise intensities. The percentage of unsaturated plasma FFA (palmitoleate, oleate, linoleate, linolenate, and arachidonate) increased from rest (57.1 ± 1.2% of total FFA) to the last 30 min of exercise (62.4 ± 0.9% of total FFA) at both intensities for BOC only. Correspondingly, the percentage of saturated plasma FFA (myristate, palmitate, and stearate) decreased significantly from rest (42.9 ± 1.2% of total FFA) to the last 30 min of exercise (37.6 ± 0.9% of total FFA) at both intensities for BOC only. The ratio of unsaturated to saturated fatty acids (U/S) of BOC increased significantly from rest (1.39 ± 0.07) to exercise in the last 30 min at 45% O_{2 peak} (1.70 ± 0.06) and the last 15 min at 65% O_{2 peak} (1.67 ± 0.06). The U/S of IP and HP was not different from that determined in women BOC but did not change significantly from rest to exercise (1.54 ± 0.09 vs. 1.66 ± 0.09, P > 0.10).

View larger version (25K): [in this window] [in a new window]   Fig. 2. Isotopic enrichment of plasma [13C]palmitate (A) and breath 13CO2 (B). MPE, molar percent excess; APE, atom percent excess; BOC, before oral contraceptives. Values are means ± SE; n = 8. Significantly different than *rest at 45% O2 peak for all conditions, rest at 65% O2 peak for all conditions, and #corresponding values at 45% O2 peak for all conditions except HP at 60 min: P < 0.05.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Effect of exercise intensity and oral contraceptive use on plasma free fatty acid (FFA) rate of appearance (A), plasma FFA rate of disappearance (B), plasma FFA metabolic clearance rate (C), and plasma FFA rate of oxidation (Rox; D). Values are means ± SE of the last 15 and 30 min of rest and exercise, respectively; n = 8. Significantly different than *rest and #corresponding value at 45% O2 peak: P < 0.05.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Effect of exercise intensity and oral contraceptive use on whole body fatty acid (FA) Rox and lipolytic rate (from Ref. 14) (A), FA reesterification (B), and oxidative energy sources (C). CHO, carbohydrates. Values are means ± SE of the last 15 and 30 min of rest and exercise, respectively; n = 8. Significantly different than *rest, #corresponding value at 45% O2 peak, and BOC: P < 0.05.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
As part of our overall effort to describe the effects of exercise intensity and other factors on substrate partitioning, we employed a longitudinal study design to examine the influences of endogenous ovarian hormones and synthetic ovarian hormones contained in oral contraceptives on whole body fatty acid R_ox and plasma FFA turnover and R_ox at rest and during exercise. The results suggest that neither fluctuations in endogenous ovarian hormones across the normal menstrual cycle nor supplementation with synthetic ovarian hormones through oral contraceptive use exert any measurable influence on whole body fatty acid R_ox or plasma FFA turnover or R_ox at rest or during moderate-intensity exercise in the 3-h postprandial state when carbohydrate use predominates. Rather, energy flux as determined by exercise intensity had the greatest influence on substrate partitioning. Oral contraceptive use did, however, result in an increase in total and plasma-derived fatty acid R_s and a decrease in the proportion of plasma FFA R_d that was oxidized compared with BOC. These results lead to general conclusions regarding 1) the availability and dynamics of individual plasma FFA in women during exercise, 2) the importance of endogenous ovarian hormones in the regulation of human lipid metabolism, and 3) the ability of oral contraceptive use to alter lipid metabolism. These general conclusions are subsequently discussed.

Availability and dynamics of individual plasma FFA in women.   For the first time that we are aware, individual plasma FFA concentrations at multiple time points at rest and during controlled exercise bouts in young women are presented. The percent contribution of each individual plasma FFA to the total concentration agrees well with results of studies of men (25, 29, 33, 46, 47) and women (26, 27, 48). The absolute concentrations of the individual plasma FFA at rest agree closely with another report of 3- to 4-h postprandial subjects (47) and are 40–60% lower than seen in overnight fasted subjects (25, 27, 33, 46, 48), likely due to suppression of lipolysis by the prestudy dietary treatment.

The two- to fivefold increase in plasma FFA concentration from rest to exercise in all conditions was accompanied by a significant 21% increase in the plasma U/S in the BOC condition. This rise in the plasma U/S to approximately 1.69 closely resembles the adipose tissue triglyceride U/S of 1.70–3.20 (30, 46–48) and suggests that -adrenergic stimulation of lipolysis during exercise results in the liberation of FFA primarily from adipose tissue triglyceride. In fact, Mittendorfer et al. (46) found that the relative increase in individual fatty acid R_a during epinephrine infusion in resting subjects closely matched the relative fatty acid content of adipose tissue triglyceride. Alternatively, a preferential uptake of saturated fatty acids during exercise could result in the observed rise in the plasma U/S. However, the only published study to examine working skeletal muscle individual fatty acid uptake to date has shown that exercising human forearm has a small preference for the uptake of the unsaturated fatty acids oleate and linoleate over that of the saturated fatty acid palmitate (28). This issue needs to be examined in large muscle groups, such as the leg, that are responsible for a greater proportion of whole body fatty acid uptake and oxidation during exercise. Preliminary evidence suggests that relative net leg individual fatty acid uptake during moderate-intensity exercise is proportional to relative individual plasma fatty concentration (Jacobs KA, Krauss RM, Fattor, JA, Horning MA, Friedlander AL, Bauer, T, Hagobian T, Wolfel EE, and Brooks GA, unpublished observation).

The importance of endogenous ovarian hormones in the regulation of human lipid metabolism.   Whole body fatty acid R_ox was not different between menstrual cycle phases at rest or during 60 min of exercise at either 45 or 65% O_{2 peak}. These findings corroborate results of previous investigations that found no effect of menstrual cycle phase on whole body fatty acid R_ox during 20–90 min of exercise at 33–90% O_{2 peak} (7, 37, 40, 56). The present report extends the results of those findings by showing for the first time that fluctuations in ovarian hormones across the normal menstrual cycle do not influence plasma FFA turnover, R_ox, or fatty acid R_s at rest or during moderate-intensity exercise. Heiling and Jensen (34) performed the only other study to date to examine plasma FFA kinetics across the menstrual cycle. Using [9,10-³H]palmitate and [9,10-³H]oleate, they also found that plasma FFA R_a was not different between FP and LP at rest in the overnight fasted state. Additionally, plasma FFA R_a increased similarly in FP and LP during 3 h of somatostatin-induced hypoinsulinemia, indicating that the suppressive effects of insulin on lipolysis are not affected by fluctuations in ovarian hormone concentrations.

The literature is not entirely consistent with respect to the effect of menstrual cycle phase on substrate partitioning. Others (10, 63) have found higher whole body fatty acid R_ox in LP than FP during exercise, and the disparity of these results was initially explained by differences in subjects' nutritional status. Campbell et al. (10) demonstrated that whole body fatty acid R_ox during 120 min of exercise at 70% O_{2 peak} was 17% higher in LP than FP in the overnight-fasted condition, but this difference was abolished when subjects were fed glucose before and during exercise. However, others have found no difference in whole body fatty acid R_ox across the normal menstrual cycle in overnight-fasted subjects (37), indicating that nutritional status does not completely explain the lack of agreement in the literature. Perhaps more important than the methodological differences between studies is the physiological variability of circulating ovarian hormone concentrations. In well-controlled studies that have reported circulating ovarian hormone concentrations (3, 7, 10, 37, 56), average midluteal concentrations of plasma estradiol (range of 82–232 pg/ml) and progesterone (range of 8–40 ng/ml) have differed by as much as three- to fivefold among studies.

Due to the potential antiestrogen effects of progesterone, the ratio of circulating estradiol-to-progesterone concentrations (E/P) rather than absolute concentrations alone should be considered when assessing the potential regulatory roles of ovarian hormones (16, 37). The E/P expressed as a percent increases from 7–8% in the early FP (3, 37, 56) to 14–22% in the mid-FP (7, 37) due to a rise in estradiol and no change in progesterone. The E/P then drops to 0.5–1.5% in the mid-LP due to a much greater increase in progesterone (2- to 25-fold) than estradiol (2- to 3-fold) (3, 7, 10, 37, 56). Interestingly, this approach would lead one to predict that the ability of estradiol to promote fatty acid mobilization and oxidation would be most evident in the mid-FP rather than the mid-LP. The E/P seems to explain the results of D'Eon et al. (17), who employed pharmacological control of serum ovarian hormone concentrations in eumenorrheic women and found that whole body fatty acid R_ox during 60 min of exercise at 60% O_{2 peak} was 50% greater under conditions of high estradiol-low progesterone (E/P = 16.5%) than either low estradiol-low progesterone (E/P = 3.6%) or high estrogen-high progesterone (E/P = 4.9%). However, Horton et al. (37) found no difference in whole body fatty acid R_ox during 90 min of exercise at 50% O_{2 peak} in nonpharmacologically controlled eumenorrheic women tested in the early FP, mid-FP, and mid-LP, indicating that the reason for the lack of complete agreement in the literature is likely multifactorial.

The lack of an effect of endogenous ovarian hormones on fatty acid mobilization or oxidation in the present study also does not agree with results of studies using animal models that have manipulated ovarian hormones by supplementing male or ovariectomized female rats with pharmacological doses of estrogen and progesterone. Those studies have found that, compared with treatments containing placebo or progesterone, estrogen treatment alone stimulates adipose lipolysis (31) and increases plasma FFA availability (41) and R_ox during prolonged exercise (32). These results may be explained in part by the recent finding that estrogen treatment of ovariectomized female rats increases the expression of several proteins critical to the regulation of skeletal muscle lipid oxidation (11, 12). The disagreement between human and animal studies is not surprising, given not only the species differences but also the vast differences in circulating ovarian hormone concentrations. Whereas circulating estradiol and progesterone concentrations may range from 20 to 250 pg/ml and from 0.3 to 40 ng/ml, respectively, between the early FP and mid-LP of the normal human menstrual cycle (3, 7, 10, 37, 56), rats have been exposed to circulating estradiol and progesterone concentrations of 45–1,700 pg/ml and 8–50 ng/ml, respectively (11, 12). Interestingly, studies on laboratory rodents suggesting that estrogen promotes the expression of proteins integral to fatty acid oxidation employed high-estrogen conditions with an E/P of only 0.6–3% (11, 12).

The ability of oral contraceptive use to alter lipid metabolism.   We previously reported that 4 mo of triphasic oral contraceptive use increased whole body lipolytic rate during moderate-intensity exercise (14). The increase in lipolytic rate was not matched by an increase in whole body or plasma FFA R_ox (Figs. 3D and 4A). Whole body fatty acid R_ox at rest and during 60 min of exercise at both 45 and 65% O_{2 peak} was not affected by oral contraceptive use. These findings corroborate results of previous cross-sectional investigations that found no difference in whole body fatty acid R_ox during 30–90 min of exercise at 45–85% O_{2 peak} between women using mono- or multiphasic oral contraceptives for 12–98 mo and those not taking oral contraceptives (4, 6). Hagenfeldt et al. (26) showed that 3 mo of monophasic oral contraceptive use did not affect oleate turnover at rest. The present report extends those findings by showing for the first time that oral contraceptive use does not influence plasma FFA turnover or R_ox at rest or during moderate-intensity exercise.

To date, there are no definitive studies regarding the methodology used to quantify fatty acid reesterification. Most reports provide estimates of fatty acid reesterification based on equations first advanced by Wolfe at al. (62). Here we propose a slight modification of these equations to account for the reesterification of fatty acids within skeletal muscle that originate from the hydrolysis of IMTG and never entered the plasma pool. We feel that this modification is justified given the significant role of nonhepatic tissues, such as skeletal muscle, in the reesterification of fatty acids at rest (59), during a prolonged fast (39), and during exercise (24, 59).

Few reports exist concerning the dynamics of fatty acid reesterification during exercise. Total fatty acid R_s of BOC at rest and during exercise (Table 4, Fig. 4B) agree well with previously reported values in 4- to 6-h postprandial women (22) and are 25–50% lower than those reported in 12-h-fasted men (59, 62). Interestingly, it appears that exercise intensity plays a role in the partitioning of fatty acid reesterification (Fig. 5). The increase in total fatty acid R_s from rest to exercise was primarily due to an increase in plasma-derived fatty acid R_s, and a majority of reesterification was plasma derived at rest and during exercise at 45% O_{2 peak}. However, plasma-derived fatty acid R_s and its proportion of total fatty acid R_s were lower at 65 than 45% O_{2 peak} in BOC, IP, and HP. Local fatty acid R_s and its proportion to total fatty acid R_s, on the other hand, tended to be higher at 65 than 45% O_{2 peak} in all conditions and reached statistical significance in IP. These findings are supported by previous findings that plasma-derived fatty acid R_s increases during 240 min of exercise at 40% O_{2 peak} (62), whereas local fatty acid R_s increases during 60 min of exercise at 60% O_{2 peak} (59) in 12-h-fasted men. It has been suggested that local fatty acid R_s is primarily regulated by the capacity to transport liberated FFA, which is a function of both adipose tissue blood flow and adequate albumin binding sites (62). Therefore, the shift in fatty acid R_s from plasma-derived to local R_s with increasing exercise intensity may be the result of a reduction in adipose tissue blood flow and/or albumin binding site availability.

View larger version (9K): [in this window] [in a new window]   Fig. 5. Model of the effect of exercise intensity on the partitioning of FA reesterification. Total FA rate of reesterification (Rs) was derived from the difference between lipolytic rate (3 times glycerol rate of appearance) and whole body FA Rox. Local FA Rs represents the reesterification of FAs that never left their site of origin. Plasma-derived Rs represents the reesterification of FAs that exited their site of origin and were reesterified in another tissue (adipose, liver, or skeletal muscle).

Acute hypercortisolemia has been shown to increase protein degradation in humans (8, 53) and increase the activity of branched-chain -ketoacid dehydrogenase, the rate-limiting enzyme of branched-chain amino acid oxidation, in rat skeletal muscle (5). While the influence of oral contraceptive-induced hypercortisolemia on protein metabolism during exercise may be small relative to exercise intensity and recent carbohydrate intake, it warrants further investigation.

The two- to threefold higher plasma cortisol concentrations in IP and HP compared with BOC at rest in the present study agrees well with the approximate twofold increases noted in previous longitudinal studies of women on triphasic oral contraceptives for 3 mo (1) and various monophasic oral contraceptives for 6 mo (60). It appears that oral contraceptives may increase plasma cortisol concentration by inducing twofold increases in corticosteroid-binding globulin (CBG) (1), thus reducing the clearance of cortisol, and this effect may be scaled to the estradiol content of oral contraceptives (60). However, similar relative increases in plasma concentrations of both total cortisol and CBG raise the possibility that the concentration of the more biologically active free cortisol is not altered by oral contraceptive use. The determination of plasma free cortisol is technically difficult, and future investigations should examine salivary and urinary free cortisol to gain a better understanding of the effect of oral contraceptive use on cortisol availability. Another possibility is that plasma cortisol concentrations are increased during oral contraceptive use by an increase in the sensitivity of the hypothalamic-pituitary-adrenal axis or a reduction in hepatic metabolism of cortisol in addition to CBG binding of cortisol (51).

Summary and conclusions.   Endogenous ovarian hormones and exogenously administered synthetic hormones found in low-dose oral contraceptives do not exert a measurable influence on whole body fatty acid R_ox or plasma FFA turnover or R_ox at rest or during moderate-intensity exercise in the 3-h postprandial state when carbohydrate use predominates. The increase in whole body lipolytic rate during exercise induced by oral contraceptive use was not matched by an increase in fatty acid oxidation and instead resulted in an increase in reesterification. We conclude that the hierarchy of regulators of fatty acid oxidation is as follows: exercise intensity > recent carbohydrate nutrition > synthetic > endogenous ovarian hormones.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This study was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-42906 and a gift from the Brian and Jennifer Maxwell Foundation.

	ACKNOWLEDGMENTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The authors thank the subjects for participation in the study and Monika Bautista, Joe Vivo, Zinta Zarins, Ryan McMillan, and Andre Rosenberg for providing most of the laboratory support for this study. We also thank Anne L. Friedlander for insightful advice related to the modification of the fatty acid reesterification equations.

	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 ACKNOWLEDGMENTS REFERENCES

 

1. Aden U, Jung-Hoffmann C, and Kuhl H. A randomized cross-over study on various hormonal parameters of two triphasic oral contraceptives. Contraception 58: 75–81, 1998.
2. Anton AH and Sayre DF. A study of the factors affecting the aluminum oxide-trihydroxyindole procedure for the analysis of catecholamines. J Pharmacol Exp Ther 138: 360–375, 1962.
3. Bailey SP, Zacher CM, and Mittleman KD. Effect of menstrual cycle phase on carbohydrate supplementation during prolonged exercise to fatigue. J Appl Physiol 88: 690–697, 2000.
4. Bemben DA, Boileau RA, Bahr JM, Nelson RA, and Misner JE. Effects of oral contraceptives on hormonal and metabolic responses during exercise. Med Sci Sports Exerc 24: 434–441, 1992.
5. Block KP and Buse MG. Glucocorticoid regulation of muscle branched-chain amino acid metabolism. Med Sci Sports Exerc 22: 316–324, 1990.
6. Bonen A, Haynes FW, and Graham TE. Substrate and hormonal responses to exercise in women using oral contraceptives. J Appl Physiol 70: 1917–1927, 1991.
7. Braun B, Mawson JT, Muza SR, Dominick SB, Brooks GA, Horning MA, Rock PB, Moore LG, Mazzeo RS, Ezeji-Okoye SC, and Butterfield GE. Women at altitude: carbohydrate utilization during exercise at 4,300 m. J Appl Physiol 88: 246–256, 2000.
8. Brillon DJ, Zheng B, Campbell RG, and Matthews DE. Effect of cortisol on energy expenditure and amino acid metabolism in humans. Am J Physiol Endocrinol Metab 268: E501–E513, 1995.
9. Brooks GA and Mercier J. Balance of carbohydrate and lipid utilization during exercise: the "crossover" concept. J Appl Physiol 76: 2253–2261, 1994.
10. Campbell SE, Angus DJ, and Febbraio MA. Glucose kinetics and exercise performance during phases of the menstrual cycle: effect of glucose ingestion. Am J Physiol Endocrinol Metab 281: E817–E825, 2001.
11. Campbell SE and Febbraio MA. Effect of ovarian hormones on mitochondrial enzyme activity in the fat oxidation pathway of skeletal muscle. Am J Physiol Endocrinol Metab 281: E803–E808, 2001.
12. Campbell SE, Mehan KA, Tunstall RJ, Febbraio MA, and Cameron-Smith D. 17-Estradiol upregulates the expression of peroxisome proliferator-activated receptor alpha and lipid oxidative genes in skeletal muscle. J Mol Endocrinol 31: 37–45, 2003.
13. Carter S, McKenzie S, Mourtzakis M, Mahoney DJ, and Tarnopolsky MA. Short-term 17-estradiol decreases glucose R_a but not whole body metabolism during endurance exercise. J Appl Physiol 90: 139–146, 2001.
14. Casazza GA, Jacobs KA, Suh SH, Miller BF, Horning MA, and Brooks GA. Menstrual cycle phase and oral contraceptive effects on triglyceride mobilization during exercise. J Appl Physiol 97: 302–309, 2004.
15. Casazza GA, Suh SH, Miller BF, Navazio FM, and Brooks GA. Effects of oral contraceptives on peak exercise capacity. J Appl Physiol 93: 1698–1702, 2002.
16. D'Eon T and Braun B. The roles of estrogen and progesterone in regulating carbohydrate and fat utilization at rest and during exercise. J Womens Health 11: 225–237, 2002.
17. D'Eon TM, Sharoff C, Chipkin SR, Grow D, Ruby BC, and Braun B. Regulation of exercise carbohydrate metabolism by estrogen and progesterone in women. Am J Physiol Endocrinol Metab 283: E1046–E1055, 2002.
18. Divertie GD, Jensen MD, and Miles JM. Stimulation of lipolysis in humans by physiological hypercortisolemia. Diabetes 40: 1228–1232, 1991.
19. Djurhuus CB, Gravholt CH, Nielsen S, Mengel A, Christiansen JS, Schmitz OE, and Moller N. Effects of cortisol on lipolysis and regional interstitial glycerol levels in humans. Am J Physiol Endocrinol Metab 283: E172–E177, 2002.
20. Friedlander AL, Casazza GA, Horning MA, Buddinger TF, and Brooks GA. Effects of exercise intensity and training on lipid metabolism in young women. Am J Physiol Endocrinol Metab 275: E853–E863, 1998.
21. Friedlander AL, Casazza GA, Horning MA, Huie MJ, and Brooks GA. Training-induced alterations of glucose flux in men. J Appl Physiol 82: 1360–1369, 1997.
22. Friedlander AL, Casazza GA, Horning MA, Huie MJ, Piacentini MP, Trimmer JK, and Brooks GA. Training-induced alterations of carbohydrate metabolism in women: women respond differently from men. J Appl Physiol 85: 1175–1186, 1998.
23. Friedlander AL, Casazza GA, Horning MA, Usaj A, and Brooks GA. Endurance training increases fatty acid turnover, but not fat oxidation, in young men. J Appl Physiol 86: 2097–2105, 1999.
24. Guo Z, Burguera B, and Jensen MD. Kinetics of intramuscular triglyceride fatty acids in exercising humans. J Appl Physiol 89: 2057–2064, 2000.
25. Hagenfeldt L. Turnover of individual free fatty acids in man. Fed Proc 34: 2246–2249, 1975.
26. Hagenfeldt L, Hagenfeldt K, and Wahren J. Influence of contraceptive steroids on the turnover of plasma free arachidonic and oleic acids. Horm Metab Res 9: 66–69, 1977.
27. Hagenfeldt L, Hagenfeldt K, and Wennmalm A. Turnover of plasma free arachidonic and oleic acids in men and women. Horm Metab Res 7: 467–471, 1975.
28. Hagenfeldt L and Wahren J. Human forearm muscle metabolism during exercise. II. Uptake, release and oxidation of individual FFA and glycerol. Scand J Clin Lab Invest 21: 263–276, 1968.
29. Hagenfeldt L, Wahren J, Pernow B, and Raf L. Uptake of individual free fatty acids by skeletal muscle and liver in man. J Clin Invest 51: 2324–2330, 1972.
30. Halliwell KJ, Fielding BA, Samra JS, Humphreys SM, and Frayn KN. Release of individual fatty acids from human adipose tissue in vivo after an overnight fast. J Lipid Res 37: 1842–1848, 1996.
31. Hansen FM, Fahmy N, and Nielsen JH. The influence of sexual hormones on lipogenesis and lipolysis in rat fat cells. Acta Endocrinol 95: 566–570, 1980.
32. Hatta H, Atomi Y, Shinohara S, Yamamoto Y, and Yamada S. The effects of ovarian hormones on glucose and fatty acid oxidation during exercise in female ovariectomized rats. Horm Metab Res 20: 609–611, 1988.
33. Havel RJ, Carlson LA, Ekelund LG, and Holmgren A. Turnover rate and oxidation of different free fatty acids in man during exercise. J Appl Physiol 19: 613–618, 1964.
34. Heiling VJ and Jensen MD. Free fatty acid metabolism in the follicular and luteal phases of the menstrual cycle. J Clin Endocrinol Metab 74: 806–810, 1992.
35. Hellstrom L, Blaak E, and Hagstrom-Toft E. Gender differences in adrenergic regulation of lipid mobilization during exercise. Int J Sports Med 17: 439–447, 1996.
36. Horning MA, Friedlander AL, Casazza GA, Huie MA, and Brooks GA. Arterial and "arterialized" sampling sites do not change isotopic enrichment using [6,6-D-gluc