INTRODUCTION

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IN HUMANS, AS HEPATIC GLYCOGEN stores are reduced during fasting, glucose production (GP) is maintained by an increased contribution of gluconeogenesis (GNG) (28, 29, 38, 39). During exercise, GNG is important also because GP is significantly increased and greater demands are placed on liver glycogen stores. Liver glycogen stores (~72 g in the overnight-fasted condition; Refs. 38, 39, 41) would be completely depleted in <3 h of exercise at ~65% maximal O₂ consumption if hepatic glycogen was the only source of GP (4, 11, 12, 47). Elevated rates of GNG and the fraction of GP from GNG under postabsorptive (PA) conditions are accomplished by alterations in the hormonal milieu (33) and increased delivery and extraction of gluconeogenic precursors across the splanchnic bed (38). During exercise, mobilizations of lactate (6, 35, 44) and glycerol (13, 47) increase in an intensity-dependent manner, thus increasing GNG precursor supply. Results from splanchnic catheterization (1-3, 49, 50) and stable-isotope infusion studies (5, 24-26) suggest that extraction and conversion of gluconeogenic precursors to glucose increases in a concentration-dependent manner during rest and exercise. Consequently, GNG has been estimated to provide as much as 60% of GP after 4 h of moderate exercise (1, 2), and GNG would also be expected to increase with increasing exercise intensity. However, because of the difficulties associated with the measurement of GNG in vivo (27, 46, 51), the effects of exercise intensity on absolute (GNG_ab) and fractional (f_GNG) gluconeogenic rates in humans remains unclear.

Previous investigations employing carbon tracers and the measurements of precursor-to-product ratios have reported variable effects of exercise intensity on GNG (5, 7, 11, 45, 47). The discrepancies are likely the result of methodological limitations because the estimation of GNG by use of carbon tracers and the precursor-to-product ratio is made uncertain by dilution of precursor carbon label in the TCA cycle (27, 46, 48, 51). In an attempt to account for isotopic dilution, our laboratory (48) and others (20-22, 51) have derived correction factors calculated from the recovery of infused carbon-labeled acetate. However, correction factors are species and treatment specific and are affected by extrahepatic metabolism of acetate (22).

A recent technique developed by Hellerstein and colleagues (15, 17, 37), termed mass isotopomer distribution analysis (MIDA), estimates true precursor pool enrichment (p) and subsequently f_GNG without the necessity of correction factors. Previously, we showed that the infusion of unlabeled glycerol at rates required to perform MIDA did not affect GP during rest and exercise (47). In the present investigation, we used MIDA to evaluate the effects of exercise intensity on the apparent rates of GNG and hepatic glycogenolysis (Gly) during 90 min of leg cycling exercise. We hypothesized that coordinated adjustments in GNG and Gly would permit GP to match glucose utilization during moderate (45% O_2 peak) and hard (65% O_2 peak) exercises. Previously, we showed that these exercise intensities raise GP two- to threefold (4, 11, 12, 47).

	METHODS

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Subjects. Eight endurance-trained male subjects were recruited from the University of California, Berkeley campus by posted notice and electronic mailing. Trained cyclists were used to extend the period of time that constant metabolic flux rates could be sustained during exercise. Subjects were considered endurance trained if they had been competing in United States Cycling Federation or collegiate mountain or road cycling competitions for more than 3 yr and had a peak O₂ consumption (O_2 peak) >60 ml · kg¹ · min¹ during leg cycling exercise. Subjects were nonsmokers, were diet and weight stable, had a percent body fat <10%, had a 1-s forced expiratory volume of 70% or more of vital capacity, and were injury and disease free as determined by medical questionnaire and physical examination. The protocol was approved by the University of California Committee for the Protection of Human Subjects (CPHS 97-5-79), and subjects gave informed, written consent to participate.

Screening tests. O_2 peak was determined on three occasions by means of a progressive leg cycle ergometer protocol (Monark Ergometric 839E) beginning at 100 W and increasing 25 or 50 W every 3 min until voluntary cessation. Two O_2 peak tests were performed before the isotope trials to ensure a true maximum effort, and blood was collected from a forearm vein during the second test for determination of lactate threshold. A third evaluation of O_2 peak was conducted 1 wk after the last tracer trial to confirm that O_2 peak was unchanged over the 6-wk experimental period. For indirect calorimetry, respiratory gases were continuously collected and analyzed via an open-circuit indirect calorimetry system (Ametek S-3A1 O₂ and Ametek CD-3A CO₂ analyzers) and respiratory parameters, including respiratory exchange ratio (CO₂ production/O₂ consumption), were determined every minute by a real-time, on-line personal computer-based system (11). Heart rate was monitored throughout the experimental protocols by using a modified 12-lead electrocardiogram. Three-day dietary records were collected before and after completion of the testing period to assess dietary habits and monitor individual caloric intake and macronutrient composition. Analysis of dietary records was performed using the Nutritionist III program (N-Squared Computing, Salem, OR). Body composition was determined by skinfold measurements, as previously reported (11).

Experimental design. After screening, two stable-isotope infusion trials were performed on each subject under each exercise condition. Previously, our laboratory (47, 48) reported on trials using [6,6-²H₂]glucose (D₂-glucose) and [1-¹³C]glucose tracers, with and without exogenous glycerol infusion. Now we report results of separate trials on the same subjects, but infused with [3-¹³C]glycerol and D₂-glucose. During the 24 h preceding each isotope trial, subjects refrained from exercise and consumed a standardized diet (3,240 kcal; 66% carbohydrate, 19% fat, and 14% protein) prepared by the laboratory staff. The dietary protocol included a final snack (609 kcal; 54% carbohydrate, 29% fat, and 17% protein) consumed exactly 12 h before the onset of exercise. Subjects reported to the laboratory at 7:00 AM on the morning of the isotope trial, 7.5 h after their last meal. After collection of background samples, tracer infusion began and subjects rested for 3.75 h followed by 90 min of leg ergometer cycling at either 45% (moderate) or 65% O_2 peak (hard) intensity exercise. Trials were performed in a randomized order with no fewer than 5 days between experiments. Subjects were instructed to maintain their habitual dietary and training regimes throughout the testing period.

Tracer protocol. All trials were conducted at the same time of day. On the morning of isotope trials, a catheter was inserted into a dorsal hand vein that was subsequently warmed by a heating pad for collection of "arterialized" blood. A second catheter was placed into the antecubital or forearm vein of the contralateral arm for continuous infusion of the isotope solutions. After collection of background blood and breath samples, [6,6-²H₂]glucose and [2-¹³C]glycerol were continuously infused (Baxter Travenol 6200 infusion pump). The [6,6-²H₂]glucose was infused at 4.0 and 8.0 mg/min during rest and exercise, respectively; these rates were previously demonstrated by our laboratory to maintain stable plasma isotopic enrichments under the conditions studied (47). The glycerol isotope infusion rates were selected after pilot studies indicated that stable and adequate precursor pool enrichment (p) values were achieved during rest and exercise at the specified infusion rates. Previous studies (18, 30, 37) have shown that measurement of GNG by MIDA requires p in excess of 12% for accurate measurement of fractional GNG. In the present investigation, [2-¹³C]glycerol was infused at 20.0 and 40.0 mg/min during rest and exercise, respectively. Glucose and glycerol isotope tracers (Cambridge Isotope Laboratories, Andover, MA) were diluted in 0.9% sterile saline and were pyrogenicity and sterility tested (University of California, San Francisco, School of Pharmacy). Furthermore, on the day of the experiment, the solutions were passed through a 0.2-µm Millipore filter (Nalgene, Rochester, NY) before infusion.

Blood sampling and analysis. Blood was sampled at minutes 0, 180, 195, 210, and 225 of the 230-min rest period and at 30, 45, 60, 75 and 90 min of exercise. Samples were immediately chilled on ice and centrifuged at 3,000 g for 18 min, and the supernatant was collected and frozen until analysis. Blood samples for determination of glucose and glycerol isotope enrichments and glucose and lactate concentrations were collected in 8% perchloric acid. Samples for free fatty acid and glycerol concentrations were transferred to vacutainers containing EDTA, thoroughly mixed, and chilled on ice before centrifugation. Glucose and lactate concentrations were measured enzymatically in duplicate or triplicate using hexokinase (Sigma Chemical, St. Louis, MO) and lactate dehydrogenase (11), respectively. Plasma free fatty acid and glycerol concentrations were determined as previously reported (11) (NEFA-C, Wako, Richmond, VA, and GP O-Trinder, Sigma Chemical). Hematocrit was measured at each sampling point, and subjects were instructed to drink tap water to prevent changes in plasma volume that would affect metabolite and hormone concentrations.

Isolation of metabolites and preparation for mass spectrometry. Glucose and glycerol samples for isotopic analyses were prepared by using ion-exchange chromatography as previously described (47). After lyophilization and resuspension in methanol, three aliquots were removed from each sample for glucose-pentaacetate, glucose-saccharic acid tetraacetate, and glycerol-triacetate derivatizations. The glucose-pentaacetate and glycerol-triacetate derivatizations have been previously described (47). Glucose samples to be converted to saccharic acid derivatives were lyophilized in a 2-ml microreaction vial, resuspended in 35 µl of concentrated nitric acid and 25 µl of sodium nitrate (0.5 g/ml), and heated at 60°C for 1 h. Samples were then lyophilized, and the two carboxyl groups of saccharic acid were methylated by adding 500 µl of MeOH/HCl, heated to 80°C for 1 h, and then dried under a stream of nitrogen. Acetylation of the hydroxyl groups was performed by adding 300 µl of a 2:1 acetic-anhydride pyridine solution to each vial, which were then heated for 20 min at 60°C. Samples were dried under nitrogen, resuspended in 200 µl of ethylacetate, and filtered through glass wool (18).

Plasma glucose R_a. To calculate glucose rate of appearance (R_a) by dilution of D₂-glucose, the contribution of ¹³C from the gluconeogenic conversion of infused [¹³C]glycerol was "subtracted" from the excess M₂ glucose enrichment before use in the Steele equation. For this purpose, a calculation algorithm was used as described elsewhere (8).

Triose phosphate pool enrichment and fractional GNG. Triose phosphate pool enrichments were estimated by using MIDA and combinatorial probability calculations described briefly herein and in detail previously (15, 37). Combinatorial probabilities were used to predict the expected mass excess (EM)₁- and EM₂-glucose (saccharic) isotopomer enrichments for a theoretical precursor pool enrichment (p) with the following equation

(1)

where M represents ¹³C glucose enrichment above background; n is the number of monomers in the glucose polymer; x is the number of labeled monomers in the glucose polymer, and p is the theoretical precursor pool enrichment. Because the EM₂/EM₁ ratio is uniquely determined by p, endogenous p was calculated by substitution of observed mass excess (M)₂/M₁ ratios into a regression equation derived from theoretical M₂/M₁ values calculated using Eq. 1 (15).

(2)

(3)

(4)

Statistical analyses. Data are presented as means ± SE. Representative values for metabolite concentration and substrate kinetics were obtained by averaging values from the final 30 min of rest and exercise. Significance of mean differences between exercise intensities was determined with one-factorial ANOVA measures. Significance of changes over time was determined by using repeated-measures factorial ANOVA and post hoc analysis. Post hoc comparisons were made with Fisher's protected least significant difference test. Statistical significance was set at  = 0.05.

	RESULTS

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Subject characteristics, dietary records, and physiological responses to exercise. During the course of the 8-wk experimental period, body weight and composition and O_2 peak were unchanged, as previously reported (47, 48). As well, 3-day dietary records indicated that macronutrient and energy contents of individual diets were unchanged during the experimental period. Heart rate, respiratory exchange ratio, and O₂ consumption increased as a function of exercise intensity in a similar manner as reported previously (47, 48).

Metabolite and hormone concentrations. Plasma glucose concentration was not different between rest and 90 min of exercise at 45% O_2 peak but decreased over time during exercise at 65% O_2 peak and was reduced ~10% after 75 min of continuous exercise compared with rest and 45% O_2 peak (Fig. 1). Plasma glycerol concentration increased in the transition from rest to exercise in a time- and intensity-dependent manner (Ref. 47; Table 1). The increase in glycerol was not the result of the increase in glycerol infusion alone. Our laboratory has previously reported that glycerol concentration increases in the transition from rest to exercise of the same intensity (13, 47). Lactate concentrations remained constant during rest and 45% O_2 peak. During exercise at 65% O_2 peak, lactate concentrations were increased approximately twofold compared with rest and 45% O_2 peak (Table 1). Compared with rest, plasma insulin concentration decreased in an intensity-dependent manner during exercise (Table 1). Conversely, plasma glucagon concentration increased in the transition from rest to exercise at 65% O_2 peak (Table 1). As a result of the exercise-induced changes in the glucoregulatory hormones, compared with rest, the mean insulin-to-glucagon ratio was reduced 23 and 55% at 45 and 65% O_2 peak, respectively (Table 1, P < 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Plasma concentrations of glucose (A), glycerol (B), and lactate (C) over time during rest and exercise. Values are means ± SE; n = 8. *Significantly different from rest; significantly different from 45% peak O2 consumption (O2 peak); significantly different over time of exercise (P < 0.05).

Glucose enrichments and kinetics. Stable D₂-glucose isotopic enrichments, corrected for incorporation of [¹³C]glycerol (see above), were obtained during the final 30 min of rest and exercise (Fig. 2A). During exercise, plasma glucose enrichments were significantly different (Fig. 2A). Glucose R_a increased approximately two- and threefold (P < 0.05) during the transition from rest to exercise at 45 and 65% O_2 peak, respectively (Fig. 2B). Mean glucose rate of disappearance (R_d) also scaled to exercise intensity (Fig. 2C). However, during exercise at 65% O_2 peak, the increase in glucose R_d was 12% greater than the gain in R_a, resulting in lower blood glucose concentrations (Fig. 1A). Because of the intensity effect on glucose disposal, glucose metabolic clearance rate increased in an intensity-dependent manner during the transition from rest to exercise (rest, 2.28 ± 0.21; 45%, 4.81 ± 0.53; 65%, 7.78 ± 1.13, ml · kg¹ · min¹, P < 0.05).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Isotopic enrichment of D2 glucose (A), glucose rate of appearance (B), and glucose rate of disappearance (C) over time during rest and exercise. Values are means ± SE; n = 8. *Significantly different from rest; significantly different from 45% O2 peak (P < 0.05).

Triose phosphate pool. Estimated p was stable throughout rest and both exercise intensities (Fig. 3) and in excess of the minimum required enrichment of 12% for the accurate measurement of fractional GNG at all time points (29). There were no observed differences in p between rest (16.98 ± 0.33) and either exercise intensity (45% O_2 peak, 17.85 ± 0.62; 65% O_2 peak, 16.49 ± 0.57). However, p was higher during the final 30 min of exercise at 45% O_2 peak compared with 65% O_2 peak (45%, 17.92 ± 0.62 vs. 65%, 16.29 ± 0.60, P < 0.05).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Triose phosphate pool (TPP) enrichment over time during rest and exercise. Values are means ± SE; n = 8. Significantly different from 45% O2 peak (P < 0.05).

GNG and glycogenolysis. The fraction of GP from GNG (f_GNG) increased (P < 0.05) over time during rest, reaching 25.6 ± 0.9% during the final minutes of rest; 12 h after the last meal (Fig. 4A). Despite the effect of fasting duration, compared with the last rest sample, f_GNG remained constant and was reduced throughout exercise at 45 and 65% O_2 peak, respectively. In addition, an intensity effect was observed during the first 60 min of exercise (Fig. 4A).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Percent of glucose production from gluconeogenesis (GNG; A) and absolute gluconeogenesis (B) over time during rest and exercise. Values are means ± SE; n = 8. *Significantly different from rest; significantly different from 45% O2 peak; significantly different over time (P < 0.05).

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	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We report the first attempt using MIDA to differentiate between the contributions of GNG and Gly to GP in men exercising after an overnight fast. Our results are consistent with those of previous studies indicating that the f_GNG contribution to GP gradually increases during fasting. However, we did not observe a time-dependent increase in f_GNG during 90 min of exercise. Rather, the f_GNG to GP remained constant during moderate-intensity (45% O_2 peak) exercise and decreased compared with rest during hard exercise at 65% O_2 peak. Because of augmentations in absolute GNG and Gly rates, GP increased during both exercise intensities compared with rest. Despite these compensations, peripheral glucose utilization exceeded GP, and blood glucose concentration decreased in the 12-h fasted men during the final 60 min of hard exercise (65% O_2 peak). Thus our data extend the scope of knowledge to include the effects of exercise intensity on absolute and fractional GNG and Gly rates measured by use of the MIDA technique. In this regard we note that, in our laboratory's previous investigations (4, 5, 14, 52), blood glucose concentration was well maintained over 60 min of hard (65% of O_2 peak) exercise when subjects were given a standardized breakfast several hours before exercise. Again, Gly appears to be more important than GNG for maintaining euglycemia in men during high-intensity exercise.

GNG during rest. Our results indicate that euglycemia after an overnight fast is maintained by a time-dependent increase in f_GNG such that GNG contributed 25% (0.56 mg · kg¹ · min¹) of GP after a 12-h fast. Previous studies employing a variety of measurement techniques reported that GNG provided 25-40% of GP after a 10-14 h fast (21, 31, 32, 41, 44, 47), and as the fast progressed GNG increased to account for 60-70% of GP by 22 h of fasting (13, 34, 41, 44, 45) (Fig. 4A). Linear extrapolation of the time-dependent increase in f_GNG in the present study [%GNG = (0.157 + 0.0239 h) × 100] predicted that GNG would account for 67% of GP after a 22-h fast.

GNG during exercise. Glucose production (R_a) scales exponentially to relative exercise intensity (4, 11, 12, 47). Our results on exercising men with MIDA indicate that f_GNG was constant during exercise and inversely related to relative exercise intensity. Despite the effect of exercise intensity on f_GNG, because of the positive effect of exercise intensity on glucose R_a, GNG_ab increased 54 and 103% (P < 0.05) over that shown at rest during exercises at 45 and 65% O_2 peak, respectively.

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Comparison of gluconeogenic rates previously reported during exercise. The GNG rates reported for exercising men in the present study are increased compared with values previously published by our laboratory (5, 11, 23, 45) and others (1-3, 7, 33, 49) at similar exercise intensities. However, MIDA was not used to measure GNG in the previous studies. Rather, GNG was estimated from the incorporation of tracer carbon from "surrogate" precursors, including alanine (33) and lactate (5, 23, 45). However, dilution of surrogate ¹³C label in the mitochondrial oxaloacetate pool results in the underestimation of GNG (27, 51). Comparing the present results obtained with the use of MIDA with results obtained on similar subjects in which GNG was estimated from the appearance of ¹³C in glucose after infusion of [3-¹³C]lactate (5, 23, 45) suggests that dilution factors of 1.30 and 1.45 should be applied during exercise eliciting 45 and 65% of O_2 peak, respectively. The dilution factors for exercise compare with that for resting men (1.86, see above).

Mass isotopomer distribution analysis. Although we and others (18, 19, 37, 42, 43) have employed MIDA to estimate enrichment of the "true" gluconeogenic precursor pool, several groups have challenged the accuracy of MIDA. Concerns are that heterogeneity among gluconeogenic triose phosphate pools results in the inaccurate measures of p and nonphysiological calculations of GNG (10, 30). Those challenges have been addressed by Hellerstein and colleagues (18, 36, 37), who maintain that MIDA accurately estimates GNG when [2-¹³C]glycerol is infused at rates sufficient to raise p above 12%. As noted previously, p was stable and in excess of 15% throughout rest and exercise in the present study (Fig. 3).

Summary and conclusions. The rates of GNG we observed in resting, PA men by using MIDA are in agreement with values previously obtained by others using similar methodology. Furthermore, results obtained by MIDA and other methods support the conclusion that the f_GNG increases in resting individuals over time after eating. Results obtained by using MIDA to estimate GNG in exercising men yielded results 30-45% higher than obtained on similarly treated subjects in whom GNG was estimated by using carbon-labeled precursor product methods. Although the GNG_ab increases during exercise compared with those at rest, as a fraction of GP, the relative role of GNG to GP remains the same during moderate-intensity exercise and decreases during hard exercise. In the exercise conditions we studied, GNG provided only a minor portion (20-25%) of glucose production; hence, Gly was more important than GNG in maintaining blood glucose homeostasis. The combined increases in GNG and Gly in 12-h PA men during 90 min of hard (65% O_2 peak) exercise were insufficient to prevent a decline in blood glucose concentration despite elevations in precursor (lactate and glycerol) supply. Our results indicate that combined Gly and GNG cannot continuously compensate for high rates of peripheral glucose uptake (R_d > 6.0 mg · kg¹ · min¹) in exercising PA men whose liver glycogen content has been compromised by fasting and exercise.