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1Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario N1G 2W1; and 2Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
Submitted 10 October 2003 ; accepted in final form 14 June 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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0.40 ± 0.04 vs. 0.07 ± 0.01 mM). The respiratory exchange ratio (RER) was increased throughout exercise (0.020 + 0.008) after NA ingestion. However, the provision of energy from fat oxidation only decreased from 33% of the total in the control trial to 26% in the NA trial, suggesting increased IMTG oxidation in the NA trial. Mean HSL activity was 2.25 + 0.15 mmol·kg dry mass–1·min–1 at rest and increased (P < 0.05) to 2.94 ± 0.20 mmol·kg dry mass–1·min–1 at 5 min in control. Contrary to the hypothesis, mean HSL was not activated to a greater extent in the NA trial during exercise (2.20 + 0.28 at rest to 2.88 + 0.21 mmol·kg dry mass–1·min–1 at 5 min). No further HSL increases were observed at 20 or 40 min in both trials. There was variability in the response to NA ingestion, as some subjects experienced a large increase in RER and decrease in fat oxidation, whereas other subjects experienced no shift in RER and maintained fat oxidation despite the reduced FFA availability in the NA trial. However, even in these subjects, HSL activity was not further increased during the NA trial. In conclusion, reduced plasma FFA availability accompanied by increased epinephrine concentration did not further activate HSL beyond exercise alone. intramuscular triacylglycerol; fat oxidation; triacylglycerol lipolysis
Nicotinic acid (NA) has previously been used to examine the effects of reduced plasma free fatty acid (FFA) availability on skeletal muscle metabolism. NA inhibits adipose tissue lipolysis by binding to HM74 receptors that are coupled to Gi proteins. Subsequent decreases in cAMP levels prevent the protein kinase A (PKA)-induced activation of HSL, leading to decreased systemic FFA release (22). This effect would not be expected in skeletal muscle because HM74 receptors are not expressed. In lightly trained subjects, oral NA ingestion resulted in elevated carbohydrate (CHO) oxidation and decreased fat oxidation as indicated by an increased respiratory exchange ratio (RER) (3, 17, 21). In these studies, fat oxidation with attenuated plasma FFA availability was reduced by 6 kJ/min (37%), but some of the unavailable plasma FFA may have been compensated for by oxidation of more IMTG. In support of this, Coyle et al. (4) reported no difference in whole body fat oxidation in endurance-trained cyclists during 40 min of exercise at 50% maximal O2 uptake (
O2 max) after the ingestion of Acipimox (an NA analog), despite reduced rates of plasma FFA oxidation. It was inferred that these subjects were able to maintain the same level of fat oxidation by increasing IMTG metabolism. Although the mechanisms of this increase were not examined, the finding of increased plasma epinephrine and an adrenergic effect on HSL activity with NA ingestion (10) may explain how HSL activity and IMTG hydrolysis may be increased after NA ingestion (16, 27).
Hence, the purpose of this study was to use NA to examine the effects of reduced FFA availability on the activation of HSL in human skeletal muscle. We hypothesized that HSL activity would be increased after NA ingestion, perhaps as the result of elevated plasma epinephrine levels.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Eleven active male subjects volunteered to participate in this study. All subjects engaged in at least four 
30-min aerobic workouts per week, which included activities such as running or cycling. Their mean (± SE) age, weight, and O2 max were 22.5 ± 1.4 yr, 72.1 ± 3.0 kg, 54.1 ± 2.2 ml·kg–1·min–1, respectively. Subjects were informed of the experiment protocol, and the possible associated risks of the study were explained to subjects both orally and in writing before written, informed consent was obtained. The ethics committees of the University of Guelph and McMaster University approved the experimental protocol.
Preexperimental Protocol
All subjects reported to the University of Guelph testing laboratory on two separate occasions before the experimental trials. The first visit consisted of a O2 max test, whereby each subject completed an incremental cycling test (Quinton Excalibur, Quinton Instruments, Seattle, WA) until volitional exhaustion was reached. The second visit was a familiarization trial, where subjects cycled for 40 min after NA supplementation to confirm the exercise power output of 55% and ensure that subjects tolerated the NA before any invasive measures were made. The mean power output was 144 ± 8 W.
Dietary analysis for each subject was completed before the experimental trials. On the basis of 1 day of dietary recall a "preexperimental diet" was devised (50% CHO, 30% fat and 20% protein), where each subject would consume the same predetermined diet, including the final meal, the day before each experimental trial. Subjects were also instructed to abstain from intense exercise and caffeine and alcohol ingestion 1 day before each experimental trial.
Experimental Protocol
Each subject completed two experimental trials that were conducted exactly 1 wk apart. Subjects reported to the McMaster University laboratory after an overnight fast (10 h), where an indwelling catheter was inserted into their antecubital vein for blood sampling and a saline drip was attached to maintain a patent line. One leg was prepared for muscle biopsies by making four incisions in the skin and underlying connective tissue over the vastus lateralis muscle under a local anesthetic (2% lidocaine, no epinephrine). During the 60 min before the exercise, subjects ingested either NA or nothing (control) in a randomized fashion. Before exercise commenced, resting blood samples (–60, 0 min) and a resting muscle biopsy (0 min) were taken. Subjects then cycled for 40 min at 55% O2 max. Expired gases were collected (Quinton Q-plex 1, Quinton Instruments) at 10, 20, 30, and 40 min of exercise for the measurement of ventilation and expired fractions of O2 and CO2 and the determination of whole body fat and CHO oxidation rates. Additional blood samples were taken at 5, 10, 20, 30, and 40 min of exercise. Muscle biopsies were taken at 5, 20, and 40 min of exercise. When the biopsies were taken, 30 s elapsed between the cessation of exercise, obtaining of the muscle biopsy, and recommencing of cycling. All muscle samples were immediately frozen in liquid N2 for future analysis.
NA Administration
Each subject ingested a total of 20 mg/kg body mass NA (ICN Canada, Montreal, Canada) in three doses. The first dose of 10 mg/kg body mass was given 60 min before exercise, and the following two doses of 5 mg/kg body mass were administered 30 min before and immediately before exercise. NA is a powerful antilipolytic agent that inhibits the activation of HSL in adipose tissue, thus significantly reducing plasma FFA and glycerol concentrations. This dosing protocol, as outlined by Hawley et al. (10) and Stellingwerff et al. (21), was intended to minimize adverse side effects, which can include gastrointestinal distress and headaches. All subjects experienced redness/flushing of their skin over most of their body, beginning 10–15 min after their first dose. The flushing was often associated with a tingling or itchy sensation. These side effects usually began to subside by the commencement of exercise and in most cases were minimal by the end of the trial. Although it is unlikely that these symptoms affected whole body or skeletal muscle responses to exercise, we cannot completely rule this out.
Analysis
Venous blood samples were placed in a heparinzed tube and partitioned into three fractions. An aliquot of 200 µl of whole blood was added to 1 ml of 0.6% (wt/vol) HClO4 and centrifuged. The deproteinized supernatant was stored at –80°C and later analyzed for glucose, lactate, and glycerol (2). A second aliquot of 1.5 ml of blood was added to 30 µl of EGTA and reduced glutathione and thoroughly mixed and centrifuged. The supernatant was analyzed for plasma epinephrine by radioimmunoassay (Adrenaline RIA, Labor Diagnostika, Nord, Germany). The remaining blood (1.5 ml) was immediately centrifuged and the plasma removed for analysis of plasma FFAs (Wako NEFA C test kit, Wako Chemicals, Richmond VA).
Muscle samples were removed from liquid N2 and freeze-dried, dissected free of blood and connective tissue, powdered and stored at –80°C until analysis. A 6- to 8-mg aliquot of powdered muscle was used for the determination of hormone sensitive lipase activity. The method has been described by Langfort et al. (16) and modified by Watt et al. (26). Briefly, the powdered muscle was homogenized on ice in 20 vol of homogenizing buffer by using a rotating Teflon pestle on glass. After centrifugation, the supernatant was removed and stored on ice for immediate analysis of HSL activity. A 4-ml substrate consisting of 14 x 106 disintegrations/min [9,10-3H]triolein at a final concentration of 5 mM, 0.6 mg phospholipid (phosphatidylcholine-phosphatidylinositol 3:1 wt/wt), 0.1 M potassium phosphate, and 20% BSA was emulsified by sonication. The muscle homogenate supernatant (14 µl) was incubated at 37°C with enzyme dilution buffer (86 µl) and 100 µl of triolein substrate. The reaction was stopped after 20 min by the addition of 3.25 ml of a methanol-chloroform-heptane (10:9:7 vol/vol/vol) solution, and 1.1 ml of 0.1 M potassium carbonate and 0.1 M boric acid were added to facilitate the separation of the organic and aqueous phases. The mixture was mixed on a vortex, centrifuged at 1,100 g for 20 min, and 1 ml of the upper phase containing the released fatty acids was removed for determination of radioactivity on a beta spectrometer (model LS 5000TA, Beckman). All measurements were made in triplicate, and the mean of these values is reported.
A second aliquot of 10–12 mg was extracted with 0.5 M HClO4 (1 mM EDTA) and neutralized with 2.2 M KHCO3. The supernatant was subsequently analyzed for phosphocreatine (PCr), creatine, lactate, glucose-6-phosphate (G-6-P), and ATP by enzymatic spectrophotometric analysis (2, 8) and for pyruvate by fluorometric analysis (18). A third aliquot of muscle (3 mg) from the rest and 40-min biopsies was used to determine muscle glycogen content (8).
Calculations
Free ADP (ADPf) and AMP (AMPf) were calculated by assuming equilibrium constants for the creatine kinase and adenylate kinase reactions. Specifically ADPf was calculated by using the measured contents of PCr, creatine, and ATP; the estimated H+ concentration (20); and an equilibrium constant for creatine kinase of 1.66 x 109. AMPf was calculated using the measured contents of ATP, the estimation of ADPf, and the equilibrium constant for adenylate kinase of 1.05. Free inorganic phosphate (Pif) was calculated by adding the resting Pif content of 10.8 mmol/kg dry mass (6) to the difference in PCr content minus the accumulation of G-6-P between rest and 5, 20, and 40 min of exercise.
CHO and fat oxidation rates (g/min) were calculated according to the following equations (19): CHO oxidation = 4.585 CO2 production (CO2) (l/min) – 3.226 O2 uptake (O2) (l/min), fat oxidation = 1.695 O2 (l/min) – 1.701 CO2 (l/min). To convert oxidation rates to kilojoules per minute, values were multiplied by 16.19 for CHO and by 40.80 for fat.
Statistics
Data are presented as means ± SE. A two-way repeated-measures ANOVA (treatment x time) was used to determine significant differences between treatments. When a significant F-ratio was obtained, post hoc analysis was completed by using a Student-Newman-Keuls test. A single-tailed paired t-test was used to assess net glycogen utilization between trials. Statistical significance was accepted atP < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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O2 was elevated (P < 0.05) at 30 and 40 min compared with 10 min for both trials (Table 1). Throughout the NA trial, O2 was also slightly greater (P < 0.05, main effect) than control. Similarly, ventilation at 40 min was greater (P < 0.05) than at 10 min for both trials, and ventilation was greater in the NA compared with control (P < 0.05, main effect). RER progressively decreased (P < 0.05) after 10 min of exercise during both trials (Fig. 1). Accordingly, CHO and fat oxidation rates during both trials were significantly different at 20, 30, and 40 min compared with 10 min of exercise (Fig. 2). CHO oxidation decreased by 11 and 9% from 10 to 40 min in the control and NA trials (control, 32.4 to 28.9 kJ/min; NA, 37.1 to 33.6 kJ/min). Fat oxidation increased by 38 and 53% from 10 to 40 min in the control and NA trials (control, 12.6 to 17.4 kJ/min: NA, 9.2 to 14.1 kJ/min). RER in the NA trial was also greater (P < 0.05) than control throughout exercise. Consequently, the average CHO oxidation rate throughout exercise was 14% higher in the NA vs. control trial (34.6 vs. 30.4 kJ/min) and the average fat oxidation rate was 19% lower in the NA vs. control trial (12.3 vs. 15.1 kJ/min).
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Blood glucose remained constant throughout the control trial (Table 2). Glucose was increased (P < 0.05) at rest after NA ingestion and remained elevated for the initial 20 min of exercise. Thereafter, blood glucose returned to resting levels. Blood lactate was unaffected by NA supplementation at rest (Table 2). During exercise lactate levels increased (P < 0.05) above resting values in both trials; however, at 5, 10, and 20 min of the NA trial, lactate was greater (P < 0.05) than the control trial. Resting plasma FFA concentrations were not different between trials before NA supplementation (Fig. 3). Plasma FFA was decreased after NA ingestion at rest (P < 0.05). The average plasma FFA concentrations were also lower in the NA vs. control trial (NA, 0.07 + 0.01 mM; control, 0.40 + 0.04 mM) throughout exercise (P < 0.05). Before NA ingestion, plasma glycerol was not different between trials (Table 2). Plasma glycerol was greater (P < 0.05) than rest by 30 min of exercise in control. During NA, plasma glycerol was lower than control (P < 0.05) at all time points. Plasma epinephrine concentrations were not different before NA ingestion (Fig. 4). In both the NA and control trials, plasma epinephrine was elevated from resting levels during exercise; however, this response was augmented (P < 0.05) in NA at 0, 5, 10 and 40 min.
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There were no differences in HSLa between NA and control trials (Fig. 5). NA ingestion had no effect on resting HSLa. Resting HSL activity for the combined trials averaged 2.22 ± 0.15 mmol·kg dry mass–1·min–1 and increased (P < 0.05) by a mean of 49 ± 18% by 5 min. No further increases were observed at 20 or 40 min.
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PCr decreased (P < 0.05) during exercise in both NA and control trials (Table 3). No differences in ATP occurred during either trial (Table 3). ADPf, AMPf, and Pif increased (P < 0.05) at 5 min and remained elevated for the duration of the exercise in both trials (Table 3). Muscle glycogen contents were not different between trials at rest or after 40 min of exercise (Table 4). However, net glycogen breakdown was significantly greater (P < 0.05) during exercise in the NA compared with control (197 ± 26 vs. 154 ± 26). G-6-P, pyruvate, and lactate increased (P < 0.05) during exercise compared with rest in both NA and control, and there were no differences between trials (Table 4).
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Shift in RER vs. degree of HSL activation. The difference in RER between NA and control trials (i.e., increase in RER) across all time points averaged 0.020 ± 0.008 (Fig. 1). However, when individual differences in RER between trials were examined a continuum of responses was apparent (Fig. 6A). The shift in RER ranged from –0.009 to 0.054 among the subjects, indicating that some subjects were able to maintain fat oxidation during the NA trial (subjects 1–3), whereas other subjects compensated for their reduced ability to oxidize plasma FFA by increasing CHO oxidation to varying degrees (subjects 4–11). There was, however, no association between the shift in RER and the differences in HSLa between NA and control trials (r2 = 0.07) (Fig. 6B).
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)] in RER and shift in fat oxidation.
A correlation was also performed between the decrease in plasma FFA concentration ([FFA]) vs. the shift in RER and vs. the shift in fat oxidation that occurred between the control and NA trials: RER = 0.1,239 x [FFA] – 0.0201, r2 = 0.386 (Fig. 7A); and fat oxidation = 17.509 x [FFA] – 3.0165, r2 = 0.308 (Fig. 7B).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Effects of NA on HSL Activity
NA administration had no effect on HSL activity, despite the increased plasma epinephrine concentrations observed in the NA trial. In the present study, plasma epinephrine increased by 45% during NA, which is consistent with that reported in previous studies using similar dosing protocols (10, 13). The precise role of epinephrine in the activation of HSL in skeletal muscle is unclear. Studies that have artificially elevated plasma epinephrine concentrations to high physiological (14, 27) and pharmacological (16) levels have observed increased HSL activity both at rest and during exercise. As in adipose tissue, the stimulation of HSL appears to be mediated by 
-adrenergic activation, resulting in an increase in cAMP and phosphorylation of HSL by PKA (16). Other studies have shown that normal epinephrine concentrations do not necessarily predict HSL activity, with marked increases in plasma epinephrine (5 nM) having no stimulatory effect on HSL activity during prolonged and intense exercise (24, 26). In the present study, we observed HSL activation early in exercise, which is likely due to contraction-related factors, although a permissive effect of epinephrine cannot be discounted. However, as the duration of exercise increased the elevated plasma epinephrine concentrations in NA did not further stimulate HSL activity. Thus it would appear that epinephrine possesses the ability to stimulate HSL at high physiological and supraphysiological concentrations; however, under normal situations other signals, such as contraction-related events, dominate HSL regulation.
NA inhibits adipose tissue lipolysis by interacting with the HM74 receptor and stimulating a Gi protein-coupled cascade that ultimately prevents HSL activation (22). In contrast to the control of adipose tissue lipolysis, the available evidence suggests that NA is not inhibiting HSL in skeletal muscle. First, HM74 mRNA has been identified in white adipose and spleen tissue, but not in skeletal muscle (22). Second, if NA inhibited skeletal muscle HSL, we would have expected decreased HSL activity during exercise in the NA trial, as occurs in adipose tissue (Watt MJ and Febbraio MA, unpublished data). Thus the absence of change in skeletal muscle HSL activity is unlikely to be explained by direct effects of NA.
Variability of the Responses to NA
The calculated energy derived from fat during the control and NA trials was 33 and 26% of the total energy turnover, respectively. Recent work using isotopic tracers estimated that FFAs contributed up to 50% of the total energy derived from fat during moderate-intensity exercise (55–65% O2 max) of 30–60 min (7, 23). Accordingly, in the control trial of this study, we expected a large proportion of the total fat oxidation to be derived from plasma FFA and the majority of the remainder, or the non-plasma fatty acid oxidation, to be derived from IMTG. Previous work had established that the oxidation of FFA from circulating very low-density lipoproteins and plasma triacylglycerol was negligible during exercise under normal dietary conditions (9, 11). Conversely, in the NA trial with reduced FFA availability, we expected that fat oxidation would decrease and CHO oxidation would increase to make up for the unavailable or missing plasma FFA. The other possibility was that some of the unavailable plasma FFA may have been compensated for by oxidation of more IMTG. We did not measure IMTG content in the present study because net changes were unlikely to be observed after 40 min of exercise because some investigators report no net IMTG changes after 2 h of moderate-intensity exercise (25). However, the results of Coyle et al. (4) support this possibility in the present study because they reported no significant difference in RER after NA ingestion, whereas directly measured plasma FFA oxidation was dramatically reduced.
Although there was a significant decrease in fat oxidation and increase in CHO oxidation with NA in the present study, there was a continuum of responses to NA ingestion (Figs. 6 and 7). In subjects 9–11, there was a large increase in RER and decrease in fat oxidation in the NA trial, whereas in subjects 1–3, there was no shift in RER or fat oxidation with NA. From these data, it is tempting to conclude that subjects 9–11 increased only CHO oxidation to compensate for the missing plasma FFA and that subjects 1–3 increased IMTG oxidation to compensate for the reduced plasma FFA availability. It is unclear why some individuals might have the potential to upregulate IMTG oxidation, whereas others cannot. However, an alternate explanation may account for some of this variability. The magnitude of the decrease in plasma [FFA] in the NA trial accounted for aproximately one-third of the variability in the RER and the fat oxidation change (Fig. 7). In other words, the higher the [FFA] during the control trial, and therefore the larger the decrease with the NA trial, the larger the increase in RER and decrease in fat oxidation with the NA trial. The strength of these relationships were modest at r2 = 0.31–0.38, suggesting that other factors were also at work, including the use of IMTG as proposed above. Specifically, it appeared that five of the subjects experienced large decreases in plasma [FFA] with NA yet did not demonstrate substantial increases in RER or decreases in fat oxidation. However, because IMTG use was not measured in this study we are not able to conclusively state that IMTG use was increased during the NA trial in these subjects.
Summary
NA supplementation decreased plasma FFA availability, increased CHO oxidation, and decreased fat oxidation from 33 to 26% of the total energy oxidized in the NA trial. Contrary to our hypothesis, NA supplementation had no effect on HSL activation during moderate aerobic exercise despite elevated plasma epinephrine concentrations. In both trials, HSL activity increased early in exercise and remained elevated for the duration of the exercise. There was a continuum of responses to NA ingestion in the RER response, with some subjects able to maintain fat oxidation despite reduced plasma FFA availability. A portion of the variability could be accounted for by the NA-induced reduction in plasma [FFA]. However, even in the individuals who maintained fat oxidation, HSL activity did not increase beyond the exercise effect alone.
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GRANTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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NA trial significantly different from control, P < 0.05.
Significantly different from corresponding time point for NA, P < 0.05.
