Autoregulation of glucose production in men with a glycerol load during rest and exercise

Jeff K. Trimmer, Gretchen A. Casazza, Michael A. Horning, and George A. Brooks

Exercise Physiology Laboratory, Department of Integrative Biology, University of California, Berkeley, Berkeley, California 94720


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Related to hepatic autoregulation we evaluated hypotheses that 1) glucose production would be altered as a result of a glycerol load, 2) decreased glucose recycling rate (Rr) would result from increased glycerol uptake, and 3) the absolute rate of gluconeogenesis (GNG) from glycerol would be positively correlated to glycerol rate of disappearance (Rd) during a glycerol load. For these purposes, glucose and glycerol kinetics were determined in eight men during rest and during 90 min of leg cycle ergometry at 45 and 65% of peak O2 consumption (VO2 peak). Trials were conducted after an overnight fast, with exercise commencing 12 h after the last meal. Subjects received a continuous infusion of [6,6-2H2]glucose, [1-13C]glucose, and [1,1,2,3,3-2H5]glycerol without (CON) or with an additional 1,000 mg (rest: 20 mg/min; exercise: 40 mg/min) of [2-13C]- or unlabeled glycerol added to the infusate (GLY). Infusion of glycerol dampened glucose Rr, calculated as the difference between [6,6-2H2]- and [1-13C]glucose rates of appearance (Ra), at rest [0.35 ± 0.12 (CON) vs. 0.12 ± 0.10 mg · kg-1 · min-1 (GLY), P < 0.05] and during exercise at both intensities [45%: 0.63 ± 0.14 (CON) vs. 0.04 ± 0.12 (GLY); 65%: 0.73 ± 0.14 (CON) vs. 0.04 ± 0.17 mg · kg-1 · min-1 (GLY), P < 0.05]. Glucose Ra and oxidation were not affected by glycerol infusion at rest or during exercise. Throughout rest and both exercise intensities, glycerol Rd was greater in GLY vs. CON conditions (rest: 0.30 ± 0.04 vs. 0.58 ± 0.04; 45%: 0.57 ± 0.07 vs. 1.19 ± 0.04; 65%: 0.73 ± 0.06 vs. 1.27 ± 0.05 mg · kg-1 · min-1, CON vs. GLY, respectively). Differences in glycerol Rd (Delta Rd) between protocols equaled the unlabeled glycerol infusion rate and correlated with plasma glycerol concentration (r = 0.97). We conclude that infusion of a glycerol load during rest and exercise at 45 and 65% of VO2 peak 1) does not affect glucose Ra or Rd, 2) blocks glucose Rr, 3) increases whole body glycerol Rd in a dose-dependent manner, and 4) results in gluconeogenic rates from glycerol equivalent to CON glucose recycling rates.

gluconeogenesis; glycerol kinetics; exertion; stable isotopes


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

UNDER POSTPRANDIAL CONDITIONS, hepatic autoregulation maintains euglycemia by coordinated changes in glycogenolysis and gluconeogenesis (GNG). In resting humans, GNG provides 20-40% of hepatic glucose production (HGP) after an overnight fast (5, 13, 17, 27, 32, 34). The contribution of GNG to HGP increases to 90% after 40-60 h of fasting rest (13, 22, 27). During steady-state exercise, GNG and glycogenolysis increase to match glucose production and utilization. The absolute and relative contributions of GNG to HGP have been reported to increase with exercise duration (1), intensity (5, 9), and training (5).

Investigators have attempted to quantify GNG in humans during rest and exercise by several methods, including splanchnic catheterization (1, 34), labeling the gluconeogenic pathway with tracer CO2 (15, 28), and incorporation of precursor carbon isotope into glucose (5, 17, 18, 20, 30). The most common techniques for the estimation of GNG in vivo are based on precursor-to-product (p/p) relationships. Unfortunately, estimation of GNG by the p/p approach is confounded by several factors, including inaccessibility of the triose phosphate pool (TPP, the "true" gluconeogenic precursor pool) for measurement, and dilution of infused carbon label in the tricarboxylic acid (TCA) cycle (21). In an attempt to account for isotopic dilution, correction factors determined from the recovery of infused tracer acetate (14) have been developed. However, correction factors are species and condition specific and are potentially affected by extrahepatic metabolism of acetate (30).

Recently, Hellerstein and colleagues (12, 26) have developed a technique, termed mass isotopomer distribution analysis (MIDA), to measure fractional GNG without the use of correction factors for determination of GNG in vivo. Unlike the p/p approach, estimation of GNG by MIDA is theoretically not confounded by dilution or isotopic exchange of carbon label in the TCA cycle. Furthermore, MIDA is purported to account for GNG from all, not just 3-carbon gluconeogenic substrates. However, the isotopic glycerol load required for the reliable application of MIDA could elicit a significant increase in blood glycerol concentration, thereby affecting HGP or GNG.

In the past, elevated gluconeogenic precursor supply has been reported to increase both absolute GNG (20) and the relative contributions of the infused substrates to HGP in dogs (31, 36) and humans (18, 38). However, results of splanchnic catheterization (2) and isotopic dilution studies (18, 20, 38) on humans indicate that elevated gluconeogenic precursor supply does not drive HGP. Thus it appears that hepatic autoregulatory mechanisms maintain normal HGP despite elevated gluconeogenic precursor supply in resting humans. However, the effects of increasing GNG precursor supply during exercise, a condition in which HGP is elevated, remain unknown.

The present investigation was performed to evaluate effects of a glycerol load sufficient to perform MIDA on glucose turnover, oxidation, and recycling at rest and during moderate exercise after an overnight fast. Specifically, we evaluated the hypotheses that 1) glucose production would be altered as a result of a glycerol load; 2) decreased glucose recycling would result from increased glycerol uptake; and 3) the absolute rate of GNG from glycerol would be positively correlated to glycerol rate of disappearance (Rd) during a glycerol load. Results indicate tight control of hepatic autoregulation in resting and exercising men.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects. Eight endurance-trained male subjects were recruited from the University of California, Berkeley campus, by posted notice and electronic mailing. Trained subjects 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 the US Cycling Federation or collegiate mountain or road cycling competitions for >3 yr and had a peak oxygen consumption (VO2 peak) >60 ml · kg-1 · min-1 during leg cycling exercise. Subjects were nonsmokers, diet and weight stable with a body fat percentage <10%, had a 1-s forced expiratory volume >= 70% 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 98-4-83), and subjects gave informed written consent.

Screening tests. VO2 peak was determined on three occasions by means of a progressive leg cycle ergometer protocol (Monark Ergometric 829E), beginning at 100 W and increasing 25 or 50 W every 3 min until voluntary cessation. Two VO2 peak tests were performed before the isotope trials to assure a true maximum effort, and blood was collected from an antecubital vein during the second test for determination of lactate threshold. A third VO2 peak test was conducted 1 wk after the last isotope test to ensure that VO2 peak was unchanged over the 6-wk experimental period. 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 were determined every minute by a real-time, online PC-based system (5, 9). Three-day dietary records were collected before the first and fourth isotope trial to assess dietary habits and monitor individual caloric intake and macronutrient composition during the 6-wk testing period. 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 (9).

Experimental design. After screening, five stable-isotope infusion trials were performed on each subject (see Tracer protocol). 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 laboratory staff. The dietary protocol included a final snack (566 kcal: 52% carbohydrate, 33% fat, and 15% protein), consumed 12 h before the onset of exercise. Subjects reported to the laboratory at 0700 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 (easy) or 65% (moderate intensity exercise) VO2 peak. Trials were performed in a randomized order, with no fewer than 5 days between. Subjects were instructed to maintain their initial dietary and training regimens throughout the 6-wk testing period.

Tracer protocol. All trials were conducted at the same time of day. On the morning of the isotope trials, a catheter was inserted into a dorsal hand vein that was subsequently warmed for collection of arterialized blood. A second catheter was placed into the antecubital vein of the contralateral arm for continuous infusion of the isotope and glycerol solutions. After collection of background blood and breath samples, [6,6-2H2]glucose, [1-13C]glucose, and [1,1,2,3,3-2H5]glycerol were continuously infused (Baxter Travenol 6200 infusion pump) without (CON) or with (GLY) the addition of 13C-labeled or unlabeled glycerol (J. T. Baker, Phillipsburg, NJ). Glucose and [1,1,2,3,3-2H5]glycerol isotopes were infused at 4.0 and 0.4 mg/min, respectively, at rest. To maintain isotopic enrichments of blood metabolites, tracer infusion rates were doubled during exercise. The tracer infusion rates employed have been previously demonstrated by our laboratory (9, 10) to maintain stable plasma isotopic enrichments for the measurement of substrate kinetics throughout rest and the two exercise intensities studied. In GLY trials, [2-13C]- or unlabeled glycerol was infused at the rate of 20 mg/min during rest and was increased to 40 mg/min during exercise to replicate conditions required for MIDA. Isotopes (Cambridge Isotope Laboratories, Woburn, MA) and unlabeled glycerol were diluted in 0.9% sterile saline, pyrogenicity and sterility tested (University of California, San Francisco, School of Pharmacy), and, on the day of the experiment, passed through a 0.2-µm Millipore filter (Nalgen, Rochester, NY) before infusion.

Sampling and analyses. Blood was sampled at minutes 0, 180, 195, 210, and 225 of the 3.75-h rest period and at minutes 30, 45, 60, 75 and 90 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 acids (FFA) 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 FFA and glycerol concentrations were determined using commercially available kits (NEFA-C, WAKO, Richmond, VA, and GPO-Trinder, Sigma). Hematocrit was measured at each sampling point, and subjects were instructed to drink tap water to ensure that metabolite and hormone concentrations were not affected by changes in plasma volume.

VO2 and CO2 production (VCO2) were determined via an open-circuit, indirect calorimetry system (Ametek S-3A1 O2 and Ametek CD-3A CO2 analyzers), and respiratory parameters were determined every minute by a real-time, online PC-based system (5, 9). Subjects' respiratory gases were collected for 10 min at each sample time, and values reported represent the average of the final 5 min of each collection period. Respiratory exchange ratios (RER, = VCO2/VO2), and substrate oxidation rates were calculated as previously described (5, 9).

Isotopic enrichment analyses. Glucose and glycerol isotopic enrichments were measured using gas chromatography-mass spectrometry (GC-MS; GC model 5890, series II, and MS model 5989A, Hewlett-Packard, Palo Alto, CA) analyses of the glucose pentaacetate and glycerol triacetate derivatives. Before GC-MS, analysis samples were neutralized with 2 N KOH, transferred to cation (AG 50W-X8, 50- to 100-mesh H+ resin) and anion (AG 1-X8, 100- to 200-mesh formate resin) exchange columns, and eluted with deionized water. Samples were then lyophilized, resuspended in methanol, and transferred to a 2-ml microreaction vial. One hundred microliters of an acetic anhydride-pyridine solution (2:1) were added to each vial and heated for 10 min at 60°C. Samples were subsequently dried under nitrogen, resuspended in 200 µl of ethyl acetate, and transferred to GC-MS vials for analysis. For GC-MS analyses of glucose and glycerol isotopomers, injector temperature was set at 200°C and initial oven temperatures were set at 110°C for glucose and 80°C for glycerol. Oven temperature was increased 35°C/min until final temperatures of 255 and 225°C were reached for glucose and glycerol, respectively. Helium was used as the carrier gas for all analyses with a 35-to-1 ml/min splitless injection ratio; transfer line temperature was set at 250°C, source temperature at 200°C, and quadruple temperature at 116°C, and positive chemical ionization was performed.

At each of the 10 sampling times, duplicate 10-ml aliquots of expired air were collected in 10-ml evacuated containers for the determination of 13CO2 isotopic enrichment. Breath samples were analyzed in duplicate by use of isotope ratio mass spectrometry (IRMS) by Metabolic Solutions (Acton, Nashua, NH).

Calculations. Glucose and glycerol rates of appearance (Ra), Rd, metabolic clearance rate (MCR), glucose Rr, and the fractional incorporation of carbon label into glucose from labeled 3-carbon precursor (GNG) were calculated using equations defined by Steele and modified for use with stable isotopes, as previously described (9). Metabolite kinetics were calculated as
R<SUB>a</SUB>(mg<IT>·</IT>kg<SUP><IT>−1</IT></SUP><IT>·</IT>min<SUP><IT>−1</IT></SUP>) (1)

<IT>=</IT><FR><NU>F<IT>−</IT>V[(C<SUB><IT>1</IT></SUB><IT>+</IT>C<SUB><IT>2</IT></SUB>)<IT>/2</IT>][(IE<SUB><IT>2</IT></SUB><IT>−</IT>IE<SUB><IT>1</IT></SUB>)<IT>/</IT>(<IT>t<SUB>2</SUB>−t<SUB>1</SUB></IT>)]</NU><DE>[(IE<SUB><IT>2</IT></SUB><IT>+</IT>IE<SUB><IT>1</IT></SUB><IT>/2</IT>)]</DE></FR>

R<SUB>d</SUB>(mg<IT>·</IT>kg<SUP><IT>−1</IT></SUP><IT>·</IT>min<SUP><IT>−1</IT></SUP>)<IT>=</IT>R<SUB>a</SUB><IT>−</IT>[V(C<SUB><IT>2</IT></SUB><IT>−</IT>C<SUB><IT>1</IT></SUB>)<IT>/</IT>(<IT>t<SUB>2</SUB>−t<SUB>1</SUB></IT>)] (2)

MCR (ml<IT>·</IT>kg<SUP><IT>−1</IT></SUP><IT>·</IT>min<SUP><IT>−1</IT></SUP>)<IT>=</IT>R<SUB>d</SUB><IT>/</IT>[glucose] (3)

Rr (mg<IT>·</IT>kg<SUP><IT>−1</IT></SUP><IT>·</IT>min<SUP><IT>−1</IT></SUP>)<IT>=</IT>R<SUB>a</SUB><SUP><IT>2</IT></SUP>H<SUB>G</SUB><IT>−</IT>R<SUB>a</SUB><SUP><IT>13</IT></SUP>C<SUB>G</SUB> (4)

GNG from glycerol<IT>=</IT>IE<SUB><IT>1</IT></SUB> glucose<IT>/</IT>IE<SUB><IT>1</IT></SUB> glycerol (5)
where F represents the specific isotope infusion rate (mg · kg-1 · min-1); volume distribution (V) of glucose and glycerol were set at 180 and 270 ml/kg, respectively; C1 and C2 are concentrations at sampling times t1 and t2; IE1 and IE2 are the excess isotopic enrichments of either M1 or M2 glucose or M1 or M5 glycerol at times t1 and t2; and Ra2HG and Ra13CG are glucose Ra measured using [6,6-2H2]glucose and [1-13C]glucose, respectively.

Measured isotopic enrichments were corrected for background from blood samples taken before isotope infusion. For the comparison of glycerol infusion rates, glycerol Rd and glucose Rr units are reported as milligrams per kilogram per minute. The use of milligrams per kilogram per minute allows for the direct comparison of glycerol flux and glucose recycling rates despite the different molecular weights. The rate of glucose oxidation (Glcox) was calculated using the IRMS-measured enrichment of expired CO2 by means of standard equations previously reported by our laboratory, and the fractional and absolute contributions of glucose, carbohydrate, and fat to the total energy expenditure (TEE) were derived using standard equations previously reported by our laboratory (4, 5, 9).

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. When no significant differences were observed between CON and GLY protocols, data were pooled. Significance of mean differences among infusion protocols and exercise intensities was determined with two-factorial ANOVA with repeated measures. Significance differences among groups and changes over time were determined using repeated-measures factorial ANOVA and post hoc analysis. Post hoc comparisons were made with Fisher's protected least significant difference test. Pearson product-moment correlations were conducted as indicated. Statistical significance was set at P = 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Subject characteristics, dietary records, and physiological responses to exercise. Anthropometric data on subjects are reported in Table 1. During the course of the 6-wk study, body composition and VO2 peak were unchanged (Table 1). As determined from 72-h dietary records, macronutrient and energy contents of individual diets were consistent during the experimental period (week 1: 61.1 ± 2.7% carbohydrate, 24.2 ± 2.3% fat, 15.1 ± 0.7% protein, 3,349.9 ± 282.14 kcal vs. week 6: 57.8 ± 2.4% carbohydrate, 25.4 ± 3.3% fat, 16.8 ± 1.4% protein, 3,126.0 ± 182.81 kcal). Infusion of glycerol did not affect VO2, heart rate, or RER during rest or exercise (Table 2). In the transition from rest to exercise, VO2 and heart rate increased in an intensity-dependent manner (Table 2). RER was also increased at 65% VO2 peak compared with rest. The cycle ergometer workloads required for the specific exercise intensities are also reported in Table 2.

                              
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Table 1.   Subject characteristics during 6-wk experimental period


                              
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Table 2.   Ergometric and physiological characteristics during rest and exercise

Metabolite concentrations. Plasma glycerol concentrations were significantly elevated at rest and during both exercise intensities in GLY compared with CON trials (Fig. 1A). In all four trials, glycerol concentration increased in the transition from rest to exercise in an intensity-dependent manner. Resting plasma FFA concentrations were not different between GLY and CON trials (0.28 ± 0.04 vs. 0.27 ± 0.03 mM, GLY vs. CON). Similarly, FFA concentrations increased (P < 0.05) in the transition from rest to exercise in CON and GLY trials (45% VO2 peak: 0.39 ± 0.05 vs. 0.41 ± 0.08; 65% VO2 peak: 0.37 ± 0.05 vs. 0.43 ± 0.06, GLY vs. CON). Furthermore, infusion of glycerol had no effect on glucose concentrations during rest or exercise (Fig. 1B). However, during exercise at 65% VO2 peak, glucose concentration decreased over time in 12-h-fasted men despite the infusion of glycerol. During exercise at 65% VO2 peak, lactate concentrations were increased compared with both rest and 45% VO2 peak. Blood lactate concentrations were unaffected by glycerol infusion during rest or exercise (Fig. 1C).


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Fig. 1.   Plasma concentrations of glycerol (A), glucose (B), and lactate (C) over time during rest and exercise. Values are means ± SE; n = 8. Circles, 45% VO2 peak; squares, 65% VO2 peak; CON, control; GLY, glycerol infusion. *Significantly different from rest; Delta significantly different from 45%; dagger significantly different over time of exercise; ¥significantly different from CON at corresponding sample times (P < 0.05).

Isotopic enrichments. Infusion of unlabeled glycerol significantly reduced [1,1,2,3,3-2H5]glycerol enrichments in GLY compared with CON (Fig. 2A). However, stable isotopic enrichments were obtained during the final 30 min of rest and exercise under both protocols. Similarly, [6,6-2H2]glucose, and [1-13C]glucose enrichments, shown in Fig. 2, B and C, respectively, were stable throughout rest and the final 30 min of exercise in all trials. No differences in [6,6-2H2]glucose enrichments were observed between protocols (Fig. 2B). However, [1-13C]glucose enrichments were lower during 45% VO2 peak GLY compared with CON trials (Fig. 2C). In addition, [6,6-2H2]- and [1-13C]glucose enrichments demonstrated an intensity effect during exercise (Fig. 2, B and C).


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Fig. 2.   Isotopic enrichment (IE) for [1,1,2,3,3-2H5]- (D5)glycerol (A), [6,6-2H2]- (D2)glucose (B), and [13C]glucose (C) over time during rest and exercise. Values are means ± SE; n = 8. Circles, 45% VO2 peak; squares, 65% VO2 peak; CON, control; GLY, glycerol infusion. Delta Significantly different from 45%; dagger significantly different over time of exercise; ¥significantly different from CON at corresponding sample times (P < 0.05).

Glycerol kinetics. Tracer-measured glycerol Ra was significantly increased at rest and at both exercise intensities in GLY compared with CON trials (Fig. 3A). However, differences in glycerol Ra between protocols were abolished when the infusion rate of unlabeled glycerol was subtracted from total glycerol Ra (Fig. 3A). Compared with rest, glycerol Ra increased during exercise in an intensity-dependent manner under both GLY and CON conditions. Glycerol Rd was increased in GLY compared with CON at rest and during both exercise intensities (Fig. 3B). The absolute difference in glycerol Rd (Delta Rd) between protocols during the final 30 min of rest and exercise was equal to the minute infusion rate (If) of unlabeled glycerol in the GLY trials (Fig. 3C).


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Fig. 3.   [1,1,2,3,3-2H5]glycerol rate of appearance (Ra; A), disappearance (Rd; B), and correlation between function of the change in glycerol Rd (Delta Rd) and glycerol infusion (GLY-If; C). Values are means ± SE; n = 8. Circles, 45%VO2 peak; squares, 65%VO2 peak; CON, control; GLY, glycerol infusion; GLY-If, GLY flux minus exogenous infusion. *Significantly different from rest; Delta significantly different from 45%; dagger significantly different over time of exercise; ¥Significantly different from CON at corresponding sample times (P < 0.05).

Glucose kinetics. No effect of glycerol infusion was observed on [6,6-2H2]glucose-measured Ra and Rd at rest and during exercise (Fig. 4, A and B). Compared with rest, glucose Ra increased two- and threefold (P < 0.05) during exercise at 45 and 65% VO2 peak, respectively (Fig. 4A). Mean glucose Ra and Rd were similar in resting men (Fig. 4B). Glucose Ra and Rd both demonstrated a significant intensity effect during exercise. The exercise intensity effect was 17% greater on glucose Rd compared with Ra, resulting in lower blood glucose concentrations during exercise at 65% VO2 peak. Because no effect of glycerol infusion was observed on glucose concentration and Rd, glucose metabolic clearance rate (MCR) was not different between protocols and was scaled to exercise intensity (Fig. 4C).


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Fig. 4.   [6,6-2H2]glucose Ra (A), Rd (B), and metabolic clearance rate (MCR; C) over time during rest and exercise. Values are means ± SE; n = 8. Circles, 45% VO2 peak; squares, 65% VO2 peak; CON, control; GLY, glycerol infusion. *Significantly different from rest; Delta significantly different from 45%; dagger significantly different over time of exercise (P < 0.05).

In CON trials, glucose Rr increased 79 and 108% (P < 0.05) in the transition from rest to exercises at 45 and 65% VO2 peak, respectively (Fig. 5A). However, an exercise intensity effect was not observed when Rr was expressed as a percentage of glucose Ra (Fig. 5B). Absolute differences in glucose Rr and glycerol Rd were highly correlated (r = 0.96) (Fig. 5C).


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Fig. 5.   Glucose recycling rate (Rr), glucose appearance from glycerol, and sum of glucose recycling and glycerol gluconeogenesis (GNG) expressed in absolute units (A), percent glucose Ra (B), and Delta Rd (C). Values are means ± SE; n = 8. *Significantly different from rest; ¥significantly different from all CON values (P < 0.05).

At rest, the percentage of glucose Ra from glycerol during the GLY trials was 13.6 ± 3.2%. During the transition from rest to exercise, the fraction of glucose Ra from glycerol was unchanged (45% VO2 peak, 11.4 ± 4.1%; 65% VO2 peak, 9.8 ± 3.8%). However, the absolute rate of GNG from glycerol was increased during exercise at 65% VO2 peak compared with rest and 45% VO2 peak (Fig. 5A). The rates of GNG from glycerol during GLY trials were positively correlated (r = 0.85) to the absolute increase in glycerol disposal (glycerol Delta Rd).

GLCox and RER. GLCox, determined from expired 13CO2 enrichment, was not affected by glycerol infusion at rest or exercise (Table 2). During exercise, GLCox increased in a time- and intensity-dependent manner. In addition, the contribution of glucose oxidation to the TEE increased from rest to exercise in an intensity-dependent manner. Because glucose Ra, Rd, and oxidation were highly correlated in exercising men, the increase in GLCox during exercise reflected changes in glucose Ra and Rd. The oxidation of other carbohydrate-derived fuels (glycogen and lactate), determined from indirect calorimetry, increased with exercise intensity and was unaffected by the infusion of glycerol.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present investigation is the first to examine the effects of an intravenous glycerol load sufficient to perform MIDA on glucose kinetics in men during sustained exercise. Our results indicate that glucose turnover (Ra and Rd), GLCox, and MCR were not affected by glycerol infusion in resting men. Moreover, glucose Ra and Rd increased in an intensity-dependent manner during light and moderate exercise independently of glycerol infusion. Glucose production was maintained during rest and both exercise intensities despite a twofold increase in glycerol concentration and removal. During infusion of glycerol, GNG from glycerol was correlated to glycerol Rd and was equal to glucose Rr during CON trials. However, provision of exogenous glycerol did not prevent a fall in blood glucose concentration during prolonged moderate (65% VO2 peak) exercise. Our data extend the scope of studies on hepatic autoregulatory control (33) to include the regulation of glucose production in the presence of increased gluconeogenic substrate supply during exercise.

Glycerol kinetics. Tracer-measured glycerol Ras were higher under GLY than under CON conditions, but when corrected for exogenous glycerol infusion, no differences in endogenous glycerol Ras were observed between protocols (Fig. 3A). The glycerol Ra results are interpreted to mean that whole body lipolysis was not affected by glycerol infusion. The lack of effect of increased glycerol concentrations on lipolysis is consistent with our observation that plasma insulin concentrations were not affected by exogenous glycerol infusion (J. K. Trimmer, J. M. Schwarz, G. A. Casazza, M. A. Horning, N. Rodriguez, and G. A. Brooks, unpublished observations). Glycerol Ra was greater during both exercise intensities than it was during rest. In addition, glycerol Ra was significantly increased during the final 15 min of exercise at 65 compared with 45% VO2 peak. In this respect, our results are consistent with those of others showing an exercise intensity effect on glycerol Ra (10, 29). Glycerol Ra also increased over time during exercise at 65% VO2 peak, a result that is in agreement with results of previous studies on men engaged in moderate- to hard-intensity exercise (10, 29). The glycerol Ra values that we determined tended to be higher than values previously reported from our laboratory (10). The distinction likely reflects the difference in dietary controls and duration of exercise. Previously our subjects had been studied 4-6 h postprandially. However, in the present investigation, subjects were studied after an overnight fast, with exercise commencing 12 h after the last meal. Baba et al. (3) have also reported that glycerol Ra increases with time since the last meal.

Glycerol Rd was significantly greater in all GLY compared with CON experiments. The Delta Rd between protocols was correlated to the exogenous glycerol infusion rate and the resulting increases in glycerol concentrations in GLY trials. The differences in glycerol Rd after exogenous infusion indicate that glycerol is removed in a concentration-dependent manner. This effect of exogenous glycerol infusion on glycerol Rd was consistent with effects in previous reports (1, 36, 37).

Considerable controversy surrounds the identity of disposal sites for circulating glycerol. Previously, it was reported that glycerol is removed predominantly by the liver and kidneys (16), although recent evidence may be interpreted to suggest that significant glycerol removal occurs in peripheral tissues (8, 23). However, according to our estimations, during GLY trials, 75-100% of the increase in glycerol Rd during GLY trials could be accounted for by conversion to glucose. Thus our data indicate that the majority of plasma glycerol is metabolized in gluconeogenic organs.

Glucose kinetics. Glucose Ra was unaffected by the infusion of glycerol and increased in an intensity-dependent manner during exercise (Fig. 4A). As well, we observed a secondary rise in glucose Ra (Fig. 4A) after 60 min of exercise after a 12-h fast. Glucose Rd was not affected by glycerol infusion during rest or exercise. Glucose turnover rates observed under CON and GLY conditions were similar to values previously reported for trained men by our laboratory (4, 9) and others (6, 24). Similarly, others have observed a lack of effect of increased gluconeogenic substrate availability on glucose turnover in resting postabsorptive dogs (36) and humans (2, 18, 20, 38). The substrates studied include lactate (2, 20), alanine (18, 38), and glycerol (18, 36). We now report that increasing gluconeogenic precursor availability by glycerol infusion does not increase HGP in exercising men.

Total oxidation rates of glucose and other carbohydrates determined by indirect calorimetry were not different between infusion protocols, despite elevated glycerol concentrations in GLY trials. In this regard, our results are in agreement with the findings of Miller et al. (25), who reported that RER was unchanged after an oral glycerol load that elicited a 100-fold increase in plasma glycerol concentrations. Moreover, because absolute and relative glucose oxidation rates were unaffected by glycerol infusion, we conclude that elevation of blood glycerol does not spare blood glucose or redirect glucose disposal during rest or exercise.

Glucose recycling and GNG from glycerol. Glucose recycling rates observed during the control trials indicate that flux through phosphoenolpyruvate carboxykinase (PEPCK) accounted for ~18% of total glucose appearance during rest. Although we recognize that glucose Rr underestimates total GNG, the recycling rates that we observed are similar to values previously reported by our laboratory for resting men (9). As well, the recycling rates that we observed in CON trials are similar to gluconeogenic rates estimated from the secondary labeling of glucose carbon after [13C]lactate infusions (5, 17). Assuming the conversion of glycerol to glucose accounts for ~6% of glucose Ra in overnight-fasted men (3), we estimate that GNG accounted for 24% of the net glucose production at rest in CON experiments, a value similar to results obtained using MIDA (Refs. 13 and 32 and J. K. Trimmer, J. M. Schwarz, G. A. Casazza, M. A. Horning, N. Rodriguez, and G. A. Brooks, unpublished observations) and [13C]bicarbonate (Ref. 7 and J. K. Trimmer, G. A. Casazza, M. A. Horning, and G. A. Brooks, unpublished observations). In CON trials, glucose recycling accounted for 16 and 12% of glucose Ra during exercise at 45 and 65% VO2 peak, respectively. However, the absolute glucose recycling rates were increased during both exercise intensities compared with rest. Thus the glucose recycling results are interpreted to mean that the absolute, but not relative, gluconeogenic carbon flux through pyruvate increased during exercise in CON trials.

The effects of increasing exercise intensity on GNG measured by incorporation of 13C label during GLY trials are similar to results observed using glucose Rr as an index of GNG. Our calculations of GNG were not corrected for loss of carbon label in the TCA cycle (i.e., Hetenyi number). However, if glycerol is removed and oxidized in extrahepatic tissue, as has been recently suggested (8, 19, 23), incorporation of labeled glycerol into glucose as an index of gluconeogenic flux results in an underestimation of GNG. Despite the potential underestimation of GNG, comparison of gluconeogenic rates estimated by glucose Rr (CON) and incorporation of 13C label into glucose (GLY) suggests that the reduction in glucose Rr during GLY trials could be compensated for by GNG from glycerol (Fig. 5A).

Hepatic autoregulation. Although our results are consistent with the idea that high concentrations of a gluconeogenic precursor such as glycerol can influence its relative contribution to GNG, we do not believe that a gluconeogenic precursor load can increase the absolute gluconeogenic rate; i.e., increases in the contribution of any precursor are likely compensated for by decreased contributions of other precursors. This view influences the way in which we interpret the previous contributions of others. In the present investigation, infusion of glycerol elicited a 66% reduction in glucose recycling at rest and a nearly complete cessation of glucose recycling during light and moderate exercise. These results are consistent with those of Wapnir and Stiel (35), who reported that high levels of dihydroxyacetone phosphate reduced PEPCK activity in rat liver preparations. An abundance of glycerol could make it the predominant gluconeogenic precursor by directly influencing the TPP and by indirectly inhibiting PEPCK by causing a backup of gluconeogenic intermediates.

With regard to the effects of exogenous glycerol on GNG and glycerol disposal sites, the decline in glucose Rr, and the positive correlation between glycerol Delta Rd and the rate of glycerol incorporation into glucose, the data can be interpreted to mean that disposal of exogenous glycerol occurred at gluconeogenic sites. Consistent with this conclusion, studies on rats (26) indicate that 75% of infused glycerol passes through the intrahepatic TPP after a 6-h fast and that the contribution increases to >90% after 11 h of fasting.

Our conclusion, that absolute GNG was unaffected by an intravenous glycerol load reaching 200 µM at rest and 700 µM during exercise, is not entirely consistent with that of Jenssen et al. (20), who concluded that GNG could be affected by precursor load. They observed an increase in tracer alanine incorporation into glucose when accompanied by an exogenous lactate load sufficient to raise blood lactate concentration to 5 mM. It may be that, in our experiments, in which exogenous glycerol did not increase blood lactate concentration, the accumulated gluconeogenic precursors could be cleared by alternative mechanisms. However, in resting subjects with low overall metabolic rates, the lactate load imposed by Jenssen et al. caused GNG to increase by mass action. In contrast, in our studies in which the total precursor load was much less, any increases in GNG from glycerol could have been compensated for by decreases in the contributions from other precursors. Those fluxes could have been redirected to disposal by oxidation and other nongluconeogenic pathways.

In summary, results of the current investigation support the concept of hepatic autoregulation in the production and utilization of glucose in 12-h-fasted men. Furthermore, our results showing a lack of effect of increasing gluconeogenic precursor supply on hepatic glucose production in men engaged in prolonged light- and moderate-intensity exercises extend the concept to include exercising conditions. Our data indicate that, during the infusion of glycerol at rates sufficient to perform MIDA, appropriate glucose production is maintained by dampening gluconeogenic flux through PEPCK to compensate for the increase in incorporation of glycerol into glucose, at rates proportional to changes in glycerol Rd.


    ACKNOWLEDGEMENTS

We thank the subjects for their participation in and compliance with the dietary and exercise protocols.


    FOOTNOTES

This work was supported by National Institutes of Health Grants AR-42906 and DK-19577.

Address for reprint requests and other correspondence: G. A. Brooks, Exercise Physiology Laboratory, Dept. of Integrative Biology, 5101 Valley Life Sciences Bldg., Univ. of California, Berkeley, Berkeley, CA 94720-3140 (E-mail: gbrooks{at}socrates.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.

Received 13 March 2000; accepted in final form 15 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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