Gluconeogenesis in humans with induced hyperlactatemia during low-intensity exercise

Mark J. Roef1, Kees de Meer1,2, Satish C. Kalhan3, Helma Straver4, Ruud Berger4, and Dirk-Jan Reijngoud5

1 Departments of Pediatric Gastroenterology and 2 Clinical Chemistry and Pediatrics, VU University Medical Center, 1007 MB Amsterdam; 4 Laboratory for Metabolic Diseases, University Children's Hospital, 3508 AB Utrecht; and 5 Laboratory for Metabolic Diseases, Department of Pediatrics, University Hospital Groningen, 9700 RB Groningen, The Netherlands; and 3 Robert Schwartz, M.D., Center for Metabolism and Nutrition, MetroHealth Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio 44109


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We studied the role of lactate in gluconeogenesis (GNG) during exercise in untrained fasting humans. During the final hour of a 4-h cycle exercise at 33-34% maximal O2 uptake, seven subjects received, in random order, either a sodium lactate infusion (60 µmol · kg-1 · min-1) or an isomolar sodium bicarbonate infusion. The contribution of lactate to gluconeogenic glucose was quantified by measuring 2H incorporation into glucose after body water was labeled with deuterium oxide, and glucose rate of appearance (Ra) was measured by [6,6-2H2]glucose dilution. Infusion of lactate increased lactate concentration to 4.4 ± 0.6 mM (mean ± SE). Exercise induced a decrease in blood glucose concentration from 5.0 ± 0.2 to 4.2 ± 0.3 mM (P < 0.05); lactate infusion abolished this decrease (5.0 ± 0.3 mM; P < 0.001) and increased glucose Ra compared with bicarbonate infusion (P < 0.05). Lactate infusion increased both GNG from lactate (29 ± 4 to 46 ± 4% of glucose Ra, P < 0.001) and total GNG. We conclude that lactate infusion during low-intensity exercise in fasting humans 1) increased GNG from lactate and 2) increased glucose production, thus increasing the blood glucose concentration. These results indicate that GNG capacity is available in humans after an overnight fast and can be used to sustain blood glucose levels during low-intensity exercise when lactate, a known precursor of GNG, is available at elevated plasma levels.

lactate; hyperlactatemia; stable isotopes


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GLUCONEOGENESIS (GNG) is regulated by hormones (such as glucagon and insulin) and requires three-carbon substrates, i.e., pyruvate (from lactate and alanine) or glycerol. The relationship among substrate availability, the rate of GNG, and the concentration of the product, glucose, in the blood is not yet fully understood. Increased lactate concentration in blood is known to occur in a number of metabolic disorders, e.g., respiratory chain disorders, pyruvate dehydrogenase deficiency, and diabetes mellitus (13). The role of GNG under the condition of increased substrate availability in these disorders is of pathophysiological interest. In patients with a respiratory chain disorder, plasma lactate concentrations are substantially increased at low-intensity levels of exercise (35, 36); in patients with mitochondrial myopathy due to complex I deficiency, we observed steady-state plasma lactate concentrations of between 4 and 7 mM during prolonged low-intensity cycle exercise at 15% of their maximal workload (Wmax). Their mean lactate rate of appearance (Ra) was 73 ± 12 µmol · kg-1 · min-1 during exercise, which was more than double their resting lactate Ra (35, 36). In another low-intensity exercise study in mitochondrial myopathy patients with complex I deficiency during fasting, plasma lactate concentrations increased from 1 mM at rest to ~4 mM during exercise and were associated with an increased fractional contribution of lactate to glucose production as well as increased rates of glucose production (37).

In healthy human subjects in the resting condition, increased availability of gluconeogenic precursors does not result in an increased rate of glucose Ra (1, 8, 14, 16, 17, 30, 41, 43) but does cause an increase in the fractional contribution of total GNG (fracGNGTOT) to glucose Ra. These observations suggest that, at rest, the increase in the Ra of glucose via GNG in the circulation is matched by a decrease in the contribution of glycogenolysis. However, it is also possible that an increase in GNG from one particular three-carbon precursor suppresses GNG from other three-carbon precursors (14). During exercise, whole body glucose consumption and hepatic glucose production have both been shown to be elevated, suggesting that additional demand is introduced on gluconeogenic precursors (5, 9-11, 42). Glycogen is a major source of glucose after overnight fasting (22), and glycogenolysis increases during exercise and overnight fasting (49). Whether increased availability of other gluconeogenic precursors would increase the fractional and absolute rates of GNG and the glucose Ra in healthy humans during exercise and fasting is not known. From our findings in mitochondrial myopathy patients, we hypothesized that increased lactate availability increases GNG from pyruvate and glucose Ra in healthy humans. To observe this effect of increased precursor availability on GNG from pyruvate during the condition of additional demand posed by exercise, no exogenous carbohydrate, protein, or energy should be provided. We therefore chose to study human subjects after overnight fasting. In healthy humans, the plasma concentration of lactate is not substantially elevated over a wide range of submaximal exercise levels, however, and the plasma lactate level rises to values above 1 mM only at exercise levels exceeding 65% of maximal oxygen consumption (VO2 max) (3, 23). Untrained volunteers are able to endure levels of exercise that induce lactate concentrations of ~4 mM (as observed in patients with respiratory chain disorders at very low levels of exercise) only during short periods of time (6, 34).

In view of these considerations, we studied the effect of lactate on the rate of GNG in fasting healthy humans when lactate concentrations were increased to a similar extent to that observed in exercising patients with a respiratory chain disorder. Preliminary experiments showed that low-intensity exercise decreased the fractional GNG from pyruvate (25) and that continuous intravenous infusion of sodium lactate at 60 µmol · kg-1 · min-1 for 1 h during low-intensity cycle exercise at 15-20% Wmax resulted in the desired steady-state hyperlactatemia (Roef MJ and de Meer K, unpublished observations). Volunteers thus received an external infusion of lactate during the last hour of a 4-h low-intensity exercise protocol. This experimental model enabled us to study the role of substrate use, GNG, and glucose Ra before and during hyperlactatemia in healthy subjects from the context of normal physiology. The aim of this study in exercising healthy subjects during fasting was to answer the following questions: 1) does infusion of lactate during hyperlactatemia induce an increase in the rate of GNG; and 2) does infusion of lactate result in an increased glucose Ra?

In the study design, measurements of whole body glucose production and separate measurements of GNG from pyruvate and total GNG were performed, the difference representing the contribution of glycerol. Calculations were made of glucose Ra from isotope dilution with [6,6-2H2]glucose and of the fractional contributions from both pyruvate and glycerol to total GNG by use of labeling of body water with deuterium oxide and quantifying the appearance of 2H in blood glucose at carbon positions C-5 and C-6 (18, 19, 22).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects. Seven healthy active volunteers (3 male and 4 female), aged 21-28 yr, volunteered for the study. They regularly engaged in sport activities (range: 1-3 h/wk), but none of the subjects was performing or had been performing athletic training. None of the subjects had a family history of diabetes mellitus or was taking any medications. Written informed consent was obtained from each subject. The experimental protocol was approved by the Medical Ethics Committee of the University Children's Hospital in Utrecht.

Preexperiment testing. All participants reported to the laboratory >= 3 days before the experiments to perform an incremental maximal exercise test on an electrically braked cycle ergometer (Lode Instruments, Groningen, The Netherlands). Wmax and VO2 max were assessed as previously described (12). The results of this test were used to calculate a workload of 18% Wmax as was used in the study. The physical characteristics are listed in Table 1.

                              
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Table 1.   Physical characteristics of the study subjects

Experimental protocol. Subjects reported to the laboratory at 0730 on two occasions separated by >= 6 wk. A sodium lactate or sodium bicarbonate infusion study was performed and assigned in random order. All subjects participated in both studies. Female subjects were studied during the second half (luteal phase) of their menstrual cycle. Four days before each study, the subjects were placed on a diet containing >= 200 g of carbohydrate daily. The subjects ate their last meals at 1800 the evening before the day of the study. For measurement of GNG, they were given oral 2H2O (>99% 2H; Isotec, Miamisburg, OH) and 5 g/kg body water divided into four doses (at 2100, 2200, 2300, and 2400). Body water was estimated to be 73% of fat-free mass, which was assessed by skinfold measurements. One indwelling catheter was inserted into a cubital vein for infusion. Another catheter was inserted into a dorsal hand vein of the contralateral arm for blood sampling; this hand was kept warm in a heated box to achieve arterialization of the venous blood (21). After the blood sample was drawn, the catheter was flushed with heparinized saline (2.5 U/ml). Subjects were acclimatized to room conditions for 30 min in the supine position. At 0800, a basal blood sample was obtained (time -60 min), and subsequently a primed constant-rate infusion of [6,6-2H2]glucose (98% enriched; MassTrace, Woburn, MA) was commenced (and continued for 5 h; prime 15 µmol/kg; continuous 0.15 µmol · kg-1 · min-1). Subjects rested in the supine position for 1 h. The study started at 0900 (time 0, i.e., 15 h after the last meal), when they were seated on an electronically braked cycle ergometer (Lode Instruments) and performed stationary cycle exercise at 18% Wmax for 4 h (0-240 min). To measure oxygen consumption, open-circuit indirect calorimetry was performed using a face mask before (0-60 min) and during (180-240 min) the infusion.

Interventions. To mimic changes in osmolar and acid-base balance associated with sodium lactate infusion (17), a bicarbonate infusion was performed in each subject as a control experiment. In lactate experiments, a primed constant-rate infusion of 1 M sodium L(+)-lactate [11.1%, wt/vol, purity 93% L(+)-lactate; ICN Biomedicals, Zoetermeer, The Netherlands] was started after 3 h of cycle exercise (180 min) and continued for 60 min (prime 675 µmol/kg for 5 min; continuous 60 µmol · kg-1 · min-1). In control experiments, 1 M sodium bicarbonate (8.4%, wt/vol; prime 675 µmol bicarbonate/kg for 5 min; continuous 60 µmol · kg-1 · min-1) was infused during the same time interval. In none of the experiments did the infused volume exceed 250 ml.

Blood sampling and urine collection. Blood samples were drawn at regular intervals. Glucose and lactate were measured every 30 min at rest and during the first 3 h of exercise and then at 10-min intervals during the lactate/bicarbonate infusion. Pyruvate and glycerol were measured every 60 min before the infusion and every 20 min during infusion. Alanine, triglycerides, free fatty acids, beta -hydroxybutyrate, acetoacetate, insulin, cortisol, and blood gasses were measured at baseline (time 0), preinfusion (180 min), and at the end of the infusion (240 min). The samples were placed on ice and transferred into sodium fluoride-containing tubes for measurement of plasma glucose, lactate, and triglyceride or into lithium-heparin-containing tubes for measurement of free fatty acid, glycerol, alanine, insulin, and cortisol or into heparin-containing syringes for blood gas analysis. Whole blood was deproteinized for measurement of blood pyruvate, beta -hydroxybutyrate, and acetoacetate. Blood samples for determination of 2H enrichments were immediately deproteinized with Ba(OH)2 and ZnSO4, centrifuged at 4°C (1,000 g, 10 min), and stored at -70°C. Urine was voided at time 0 and at 240 min and was collected for measurement of 2H enrichment in body water.

Sample analysis. Plasma glucose, lactate, and triglyceride concentrations were measured enzymatically with autoanalyzers (Dimension AR and ACA SX, respectively; Dade). Blood gas analysis was performed on a standard blood gas analyzer (Ciba Corning 278, Hanover, Germany). The concentrations of [6,6-2H2]glucose used for the tracer infusions were measured with the autoanalyzer with calibration curves from weighted, water-free glucose (Merck, Darmstadt, Germany). Blood pyruvate, beta -hydroxybutyrate, acetoacetate, plasma free fatty acids, and glycerol were measured with automated enzymatic colorimetric methods (COBAS FARA II; Hoffmann-La Roche, Montpellier, France). Plasma alanine was measured by gas chromatography (5). Plasma insulin and cortisol concentrations were measured with a microparticle enzyme immunoassay method (IMX analyzer; Abbott, Chicago, IL) and a fluorescence polarization immunoassay (TDX analyzer; Abbott), respectively. The lowest detection limit for the insulin essay is 5 U/l.

The 2H enrichments of hydrogen on carbon-5 (C-5) and carbon-6 (C-6) of glucose were measured in blood samples at 0, 180, and 240 min and at -60, 0, 150, 180, 200, 220, and 240 min, respectively. Laboratory analyses were performed as described previously (22). Briefly, glucose was isolated from deproteinized plasma by successive ion exchange chromatography and high-performance liquid chromatography. For determination of 2H enrichment on C-5, glucose was first converted into xylose, and the C-5 of glucose with its hydrogens was converted into formaldehyde by periodate oxidation. Formaldehyde was condensed with ammonium hydroxide to form hexamethylenetetramine. C-6 of glucose was converted directly to formaldehyde by periodate oxidation and then condensed with ammonia to form hexamethylenetetramine. Enrichment of hexamethylenetetramine [coming from 2H enrichment of glucose C-5 or C-6 (M+1)] was analyzed on a gas chromatography-mass spectrometry system (HP 5970 equipped with an HP 5890 gas chromatograph; Hewlett-Packard, Palo Alto, CA). The [6,6-2H2]glucose enrichment was also measured [(M+2) at C-6 of glucose] together with (M+0) and (M+1) enrichments in the same run. Electron impact ionization (70 eV) was used, and ions of mass-to-charge ratio 140 and 141 were monitored using the selected-ion-monitoring software. 2H enrichment in urine water (sampled at 0 and 240 min) was determined in acetylene gas by a mass spectrometry system, according to Previs et al. (33). Briefly, urine samples were reacted with calcium carbide to form acetylene gas, in which enrichment was determined by gas chromatography-electron impact ionization mass spectrometry. All samples were reacted in triplicate, and the measured 2H enrichments showed a coefficient of variation <5%.

Respiratory calorimetry. Continuous gas analysis and volume measurements were conducted (Oxycon Champion, Breda, The Netherlands), and standardizations were performed as previously described (37). Oxygen consumption was expressed as percentage of VO2 max.

Calculations. Steady-state whole body glucose Ra was calculated as
glucose R<SUB>a</SUB> (&mgr;mol · kg<SUP>−1</SUP> · min<SUP>−1</SUP>) = i / TTR
where i is the rate of infusion of [6,6-2H2]glucose tracer in micromoles per kilogram per minute, and TTR is the tracer-to-tracee molar ratio in plasma glucose (46). Isotopic enrichments were considered to be in steady state when TTRs of four consecutive samples had a coefficient of variation <10% and a slope not significantly different from zero. When the isotopic enrichments were not in steady state, turnover rate was calculated with Steele's equations for non-steady-state conditions (36, 40).

The contribution of GNG to glucose Ra was calculated as described previously (22, 37)
fracGNG<SUB>TOT</SUB> = 100 × (<SUP>2</SUP>H enrichment of glucose C-5/ 

<SUP>2</SUP>H enrichment in body water)
and
fracGNG<SUB>PYR</SUB> = 100 × 0.5 (<SUP>2</SUP>H enrichment of glucose C-6/ 

<SUP>2</SUP>H enrichment in body water)
where fracGNGTOT is the fractional contribution of total GNG to glucose Ra (in %), and fracGNGPYR is the fractional contribution of GNG from pyruvate to glucose Ra (in %). The difference between fracGNGTOT and fracGNGPYR provides an estimate of the fractional contribution of GNG from glycerol to glucose Ra. The absolute rates of GNGTOT and GNGPYR were calculated by multiplying the respective fractional rates by glucose Ra.

Data analysis and statistics. Data are presented as means ± SE unless otherwise stated. The paired Student's t-test was used to assess the significance of differences between time points, and the unpaired t-test was used to assess the difference between lactate and control infusions. The Wilcoxon rank sum test was used when the data were not normally distributed or when data for fewer than five subjects were available. Linear regression analysis was performed to test whether the slope of enrichment for [6,6-2H2]glucose was significantly different from zero. Multiple analysis of variance (MANOVA) for repeated measures was used for assessment of differences in blood glucose concentration, GNGPYR, fracGNGPYR, and glucose Ra. Available data between -30 and 180 min were used to test the effect of exercise over time, and available data between 190, 200, ... 230, and 240 min were used to assess the effect of the intervention (lactate infusion vs. control) and time. Statistical analyses were performed with SPSS for Windows (version 7.5; SPSS, Chicago, IL). Statistical significance was set at P < 0.05 (two-tailed).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Oxygen consumption during exercise. Mean oxygen consumption during exercise in the control and lactate experiments was 30 ± 2 and 32 ± 2% VO2 max, respectively, before the infusions. During infusion of bicarbonate and lactate, oxygen consumption rose to 34 ± 2 and 33 ± 2% VO2 max.

Metabolite concentrations. In the control experiments, the mean plasma glucose levels (Table 2 and Fig. 1A) decreased significantly from baseline to 180 min (MANOVA, time effect P < 0.001). The preinfusion and end-of-infusion glucose concentrations (4.5 ± 0.2 and 4.2 ± 0.3 mM, respectively) were significantly lower than the baseline value (5.0 ± 0.2 mM, both P < 0.05). Before the lactate infusions, the glucose levels (Fig. 1A) decreased also from baseline (MANOVA, time effect P < 0.001). Lactate infusion resulted in a significant increase in plasma glucose level from 4.4 ± 0.2 (180 min, preinfusion) to 5.0 ± 0.3 mM (end of infusion, P < 0.05). Plasma glucose levels were significantly higher during lactate infusion than during bicarbonate infusion [MANOVA, intervention effect P < 0.001, time effect not significant (NS)]. Lactate infusion significantly increased plasma lactate levels to 4.4 ± 0.6 mM (Fig. 1B; P < 0.01); significant increases in blood pyruvate (P < 0.05) and lactate-to-pyruvate ratio (P < 0.01) were also present. In the control experiments, lactate levels (and pyruvate levels; data not shown) did not increase significantly during the final hour of exercise (Fig. 1B). Blood pH, bicarbonate, and base excess were significantly increased during both infusions (P < 0.01). Alanine concentrations did not show major changes, but glycerol and free fatty acid concentrations increased (4- to 6-fold) during exercise compared with basal values under both infusion conditions. Plasma beta -hydroxybutyrate and acetoacetate concentrations also increased during exercise and showed significant differences before infusion (lactate vs. bicarbonate at 180 min, P < 0.05) but not at the end of the infusions. Plasma insulin and cortisol concentrations were similar before and after both infusion studies.

                              
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Table 2.   Plasma metabolite and hormone concentrations before and during lactate or bicarbonate (control) infusion



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Fig. 1.   Mean plasma glucose (A) and lactate (B) concentrations during lactate (LAC, ) and bicarbonate infusion experiments (BIC, ). Values are means ± SE in 7 subjects. A: lactate vs. bicarbonate infusion (t-test): *P < 0.05. B: during lactate infusion (180-240 min), plasma lactate concentration was significantly increased at all time points (t-test, P < 0.01).

Fractional GNG. Isotopic enrichments (M+1) on C-6 of glucose over time, resulting from GNG from pyruvate before and during the experiment, are depicted in Fig. 2A (see also Tables 3 and 4). FracGNGPYR decreased significantly during the first 3 h of exercise (preinfusion vs. baseline, P < 0.05). FracGNGPYR did not change during bicarbonate infusion, but lactate infusion significantly increased fracGNGPYR (from 29 ± 4 to 46 ± 4%; MANOVA, intervention effect P < 0.001, time effect NS). Data on total GNG during exercise were available from three control and four lactate experiments. In these experiments, fracGNGTOT remained unchanged during the first 3 h of exercise (preinfusion 39%) and during bicarbonate infusion (46%; range 42-52%). Lactate infusion significantly increased fracGNGTOT [from 45 (range: 36-63%) to 64% (63-64%), preinfusion vs. end of infusion, respectively: P < 0.05, lactate vs. control infusion at 240 min: P < 0.05]. The difference between fracGNGTOT and fracGNGPYR, i.e., the fractional contribution of glycerol to glucose Ra, did not change during lactate infusion.


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Fig. 2.   Isotopic enrichments of (M+1) and (M+2) at C-6 of glucose over time during lactate () and bicarbonate infusion () experiments. Values are means ± SE calculated from available data (minimum n = 6). Exercise was performed at 18% maximal workload (Wmax; 0-240 min), and lactate or bicarbonate was infused between 180 and 240 min. Isotopic enrichments (M+1) of hydrogens on C-6 of glucose and body water are combined to calculate (GNGPYR), and isotopic enrichment (M+2) of hydrogens on C-6 is used to calculate glucose rate of appearance (Ra) from [6,6-2H2]glucose dilution. Statistical comparisons of GNGPYR and glucose Ra during the time series and between the interventions are presented in RESULTS and Tables 3 and 5.


                              
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Table 3.   2H enrichment in body water and C-6 of glucose and estimates of GNG from pyruvate before and during lactate or bicarbonate (control) infusion


                              
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Table 4.   2H enrichment in body water and at C-5 of glucose and estimates of total GNG before and during lactate or bicarbonate (control) infusion

Glucose kinetics and absolute rate of GNG from pyruvate. Isotopic enrichments (M+2) on C-6 of glucose over time, resulting from [6,6-2H2]glucose dilution at baseline and during the exercise and interventions, are depicted in Fig. 2B (see also Table 5). Exercise increased glucose Ra substantially. In the control experiments, glucose Ra reached a plateau during the final hour of exercise, and GNGPYR remained unchanged (5.1 µmol · kg-1 · min-1 both at 240 min). During lactate infusion, glucose Ra increased significantly compared with the control infusion (MANOVA, intervention effect P < 0.05, time effect NS), and GNGPYR increased from 5.1 ± 0.4 to 8.8 ± 0.4 µmol · kg-1 · min-1 (preinfusion vs. end of infusion, respectively, P < 0.001; lactate vs. control infusion: MANOVA, P < 0.001).

                              
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Table 5.   Rates of glucose production and GNG from pyruvate before and during lactate or bicarbonate (control) infusion


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study shows that, in humans performing prolonged exercise at low intensity after an overnight fast, blood glucose concentration decreased and that lactate infusion at a rate that induced hyperlactatemia ("lactate clamp") 1) abolished the decrease in blood glucose concentration, 2) increased the whole body glucose Ra, and 3) increased the fractional contribution of GNG from pyruvate to whole body glucose production.

The deuterium oxide method was used for the quantitative assessment of GNG in this study. This method uses the incorporation of 2H into glucose at carbon positions C-5 and C-6 to measure the relative contribution from pyruvate to GNGTOT (18, 19, 22). The absolute value of the contribution of GNGPYR to GNGTOT is underestimated to some extent, because exchange of 2H in labeled body water and methyl-carbon atom of pyruvate (and C-6 of glucose) is ~80% complete after fasting (24). This is due to incomplete exchange of protons at this methyl-carbon atom in the oxaloacetate pool, which is not in full equilibrium with fumarate. The effect of the exchange between fumarate and oxaloacetate depends to some degree on the relative rates of oxaloacetate-fumarate interconversion and tricarboxylic acid (TCA) cycle activity in the liver during exercise. Whether oxygen consumption and TCA cycle activity in the liver are changed during low-intensity exercise or lactate infusion is not known. During intense exercise, hepatosplanchnic uptake of oxygen has been reported to be 66% increased over resting values (31). In our study subjects, the conversion of pyruvate to phosphoenolpyruvate via oxaloacetate (i.e., GNGPYR) was 20% increased over baseline values during exercise under both study conditions and increased with 72% over preinfusion values during lactate infusion. With these considerations, we do not expect the underestimation of fracGNGPYR and GNGPYR under the conditions of our study protocol to be much different from ~20%. Other isotope methods, such as studies using carbon-labeled lactate or other carbon-labeled gluconeogenic precursors that enter via pyruvate, also result in an underestimation of fracGNGPYR caused by additional dilution of the labeled carbon atom by hepatic oxaloacetate and require a number of assumptions to correct for this additional dilution (21, 22).

In our study, plasma ketone body concentrations at 180 min were higher in experiments in which lactate was subsequently infused than in those in which bicarbonate was infused, but at rest and at the end of the infusion period no difference was found. From a study by Kalhan et al. (18), one can deduce that a pronounced increase in GNG from pyruvate could be expected in view of the preinfusion increase of plasma ketone bodies. The preinfusion difference in ketone body concentration in our study subjects was not associated with a significant difference in fractional and absolute GNG from pyruvate at the preinfusion time point. Only a minor, nonsignificant increase was observed in fractional and absolute GNG from pyruvate. Accordingly, we do not think that this preinfusion difference in plasma ketone body concentration will affect our interpretation of the data.

We observed a significant decrease of blood glucose concentration during exercise from 5.0 to 4.2 mM after 4 h. A decrease of similar extent has been reported by others, albeit in humans performing exercise at higher-intensity levels, up to 65% VO2 max (15, 28, 29, 44, 45). In those studies, subjects were studied 7.5 h after their last meal, and exercise commenced 12 h following completion of their evening meal. In our study, exercise started 15 h after the last meal, thus after an overnight fast. The observed decrease from baseline glucose levels in our study subjects occurred despite an increase in glucose Ra (60% increase over resting values) and indicates that the rate of disappearance of glucose during exercise at 30-35% VO2 max is even higher than the Ra.

Hyperlactatemia induced by lactate infusion increased the absolute rate of GNG from pyruvate and glucose Ra in our study subjects. The latter finding is in contrast to several studies in humans and other mammalian species studied at rest (1, 8, 14, 16, 17, 41, 43), which showed that infusion of gluconeogenic precursors did not result in an increased glucose Ra. Infusion rates in those studies were much smaller (e.g., for glycerol or alanine between 6 and 15 µmol · kg-1 · min-1) compared with our study protocol (60 µmol lactate · kg-1 · min-1). The increase in GNGPYR in our study, from 5 to 8.8 µmol glucose · kg-1 · min-1, is equivalent to a disposal of 7.6 (i.e., 2 × 3.8) µmol lactate · kg-1 · min-1, representing ~13% of the disposal of the infused lactate. It is possible that a potential increase in GNG from precursor infusion was not measured with sufficient power in previous studies, which used infusion rates 4-10 times lower than the infusion rate that we chose, assuming an equivalent proportional increase in GNG at such lower rates. Differences in physiological state could also explain the difference between our findings (obtained during prolonged low-intensity exercise) and those of previous studies (obtained in subjects under resting conditions). Recently, Miller and colleagues (28, 29) studied the effect of hyperlactatemia due to lactate infusion (lactate clamp) in healthy volunteers at rest and under exercise at moderate and high intensity. In contrast to our observations, they found a decrease in glucose Ra during lactate infusion under exercise. According to those authors, lactate inhibited peripheral glucose utilization and prevented a decrease in blood glucose concentration and thereby the necessity for the liver to increase glucose production. The contrasting findings in the rate of GNGPYR and glucose Ra of human volunteers during similar levels of induced hyperlactatemia but performing different levels of exercise (low-intensity exercise in our study and moderate-to-intense exercise in the studies of Miller and colleagues) might be explained by differences in preferential use of lactate over glucose by working muscle and associated differential sparing of blood glucose and autoregulation by the liver to increase glucose output (see below). However, other explanations are also possible. It might be that exercise at higher levels than those used in our study reapportioned cardiac output preferentially to working muscles and thereby limited substrate supply to the liver. Differences in hormonal responses to exercise at different levels could also be involved. In our study, insulin levels remained low, and cortisol concentrations were unchanged during the lactate and control infusions. This suggests that the increased availability of lactate resulted in an increase in glucose output in the lower range of euglycemia without much change in the hormonal regulation in our fasting human volunteers.

The increased rate of hepatic glucose production during lactate infusion was accompanied by an increased fracGNGTOT of 19% (from 45 to 64% in 4 subjects) and by an increased fracGNGPYR of 17% (from 29 to 46% in 7 subjects). In control experiments, no such changes were observed in the same subjects. Previous studies did not quantify the contribution of GNGPYR to GNGTOT. Our findings suggest for the first time that increased availability of lactate as a precursor, although it increased GNGPYR under the condition of decreased blood glucose concentration during low-intensity exercise, did not change the GNG from glycerol in fasting exercising adult volunteers. The mean value for GNGTOT during hyperlactatemia may be representative of the change in the rate of GNGTOT during exercise and hyperlactatemia in the human population, but caution should be used due to the limited number of subjects in our study.

Our data indicate that GNG represents a minor (~13% of the load; see above) contribution to lactate disposal under the conditions studied here. Lactate oxidation and muscle glyconeogenesis are also metabolic fates of excess lactate in healthy humans. Glyconeogenesis can remove excess circulating lactate and has been demonstrated in resting skeletal muscle in rodents (25, 27, 32). In perfused, noncontracting rat hindlimb, McLane and Holloszy (27) found a proportional increase in muscle glycogen synthesis during induced hyperlactatemia between 4 and 25 mM. However, estimations of the relative contribution of lactate to muscle glyconeogenesis in the literature vary widely, and data in humans performing submaximal exercise are not available (25). Oxidation is an important mechanism for disposal of lactate during exercise and has been demonstrated in skeletal muscle in humans during and after work (6, 9, 24, 48). Our study focused on quantification of GNG to remove lactate and on glucose Ra, and the contributions of glyconeogenesis and oxidation to lactate removal were not measured separately.

Regulation of the gluconeogenic contribution to glucose synthesis resides in part in (re)partitioning of hepatic glucose 6-phosphate between hepatic glycogen synthesis and hepatic glucose production (2, 47). Excess gluconeogenic glucose 6-phosphate (e.g., during GNG precursor infusion in resting humans) will most likely be repartitioned into glycogen synthesis to avoid hyperglycemia. This combination, of a continuous de novo synthesis of glucose 6-phosphate from an infused substrate with subsequent partitioning of newly synthesized glucose 6-phosphate depending on blood glucose concentrations, offers a way of acute regulation of hepatic glucose production independent of hormonal changes. This provides the mechanism for the liver to respond directly to changes in blood glucose with changes in hepatic glucose production, a phenomenon known as hepatic autoregulation (30). Recently, Coker et al. (7) demonstrated in dogs that moderate excercise in the absence of changes in circulating hormone concentrations resulted in a moderate decrease of blood glucose concentrations accompanied by increased hepatic glucose production. It appears that this autoregulation of glucose production in the liver is a way to keep blood glucose at low physiological concentrations during exercise and thereby prevent hypoglycemia from occurring. Our data showing increased GNGPYR and glucose Ra in fasting human volunteers during low-intensity exercise and hyperlactatemia are in agreement with this explanation. Jenkins et al. (15) have previously shown in fasting humans that glucose production during exercise at 55% VO2 max was subject to feedback control by circulating glucose within the range of 5.1-5.4 mM, with insulin levels (studied during control and insulin infusions) inversely related to glucose Ra. Although our data were obtained in humans at lower exercise conditions (30-35% VO2 max) than those of Jenkins et al., the glucose concentrations (range 4.2-5.0 mM) and low insulin levels in our subjects suggest that sensitivity of glucose production to circulating glucose levels was present. There are no data on the partitioning of hepatic glucose 6-phosphate between blood glucose and glycogen synthesis under the conditions of our experimental protocol, and the net effect of hepatic glycogen synthesis and glycogenolysis during lactate infusion thus cannot be estimated with our present data.

Our study results have potential implications for the understanding of human health and disease. We used the same experimental protocol to measure GNG during prolonged low-intensity exercise in patients with a respiratory chain disorder (mitochondrial myopathy due to complex I deficiency). During fasting, the mean resting plasma lactate concentration in these patients was 1.1 ± 0.1 mM, and the rate of GNGPYR was 4.5 ± 1.4 µmol · kg-1 · min-1. During cycle exercise at 15% of their Wmax, their lactate levels rose to 4.0 ± 1.0 mM, and GNGPYR increased to 7.4 ± 1.9 µmol · kg-1 · min-1 (37). These results compare favorably with our data in healthy volunteers at rest and exercising at 18% of Wmax during induced hyperlactatemia (4.6 ± 0.6 mM). The combined data suggest that patients with a respiratory chain defect in the mitochondria of their skeletal muscles exhibit a normal gluconeogenic response to increases in circulating lactate concentrations during low-intensity exercise. The present study demonstrates that this response is also present in healthy humans exercising at similar relative levels of low-intensity exercise during induced hyperlactatemia and underpins the central role of lactate as an intermediate linking glycolysis, GNG, and the TCA cycle.

We conclude that healthy humans under conditions of induced hyperlactatemia during prolonged low-intensity exercise increase lactate disposal via GNG, increase whole body glucose production, and sustain the blood glucose concentration. Our results indicate that GNG capacity is available in untrained active humans after an overnight fast and can be used to sustain blood glucose levels during low-intensity exercise when lactate, a known precursor of GNG, is available at elevated plasma levels.


    ACKNOWLEDGEMENTS

This work was supported by grants from the Netherlands Organization for Scientific Research and the Foundation "De Drie Lichten" in the Netherlands, and a grant from the National Institutes of Health (RR-00080) Core Mass Spectrometry Laboratory.


    FOOTNOTES

Address for reprint requests and other correspondence: K. de Meer, Dept. of Clinical Chemistry, VU University Medical Center, Reception K, PO Box 7507, 1007 MB Amsterdam, The Netherlands (E-mail: novosilski{at}hotmail.com).

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.

First published February 25, 2003;10.1152/ajpendo.00425.2002

Received 2 October 2002; accepted in final form 13 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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