Contribution of liver and skeletal muscle to alanine and lactate metabolism in humans AGOSTINO CONSOLI, NURJAHAN NURJHAN, JAMES J. REILLY, JR., DENNIS M. BIER, AND JOHN E. GERICH Clinical Research Center, Departments of Medicine, Surgery, and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; and Metabolism Division, Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110
CONSOLI, AGOSTINO, NURJAHAN NURJHAN, JAMES J. REILLY, JR., DENNIS M. BIER, AND JOHN E. GERICH. Contribution of liver and skeletal muscle to alanine and lactate metab-
anine and lactate uptake because it measures only their net balance and because the gastrointestinal tract produces these substrates (6, 12, 18). Such limitations could olism in humans. Am. J. Physiol. 259 (Endocrinol. Metab. 22): cause underestimation of the contribution of alanine and E677-E684, 1990.-To quantitate alanine and lactate glucolactate to gluconeogenesis by as much as 50-100% (14, neogenesis in postabsorptive humans and to test the hypothesis 18) that muscle is the principal source of these precursors, we Furthermore, although delivery of precursors t’o the infused normal volunteers with [3- “‘Cllactate, [ 3-“‘Clalanine, liver is considered a major determinant of gluconeogenand [6-“HIglucose and calculated alanine and lactate incorpoesis (9, 17), the key tissues supplying alanine and lactate ration into plasma glucose corrected for tricarboxylic acid cycle in humans remain unclear. On the basis of earlier studies carbon exchange, the systemic appearance of these substrates, and their forearm fractional extraction, uptake, and release. (6, 18), muscle might be expected to be a major source of Forearm alanine and lactate fractional extraction averaged 37 these precursors via proteolysis and the Cori and alanine t 3 and 27 k 2%, respectively; muscle alanine release (2.94 * cycles. However, currently available data suggest that 0.27 prnol. kg body wt-l l rein-‘) accounted for -70% of its muscle makes only a minor contribution. Experiments systemic appearance (4.18 t 0.31 prnol. kg body wt-l. min); employing the limb balance technique indicate a net muscle lactate release (5.51 t 0.42 prnol. kg body wt-l min-‘) output of -0.2 pmol/lOO ml tissue for alanine (2, 19, 34, accounted for -40% of its systemic appearance (12.66 t 0.77 40, 44-47) and -0.4 ~mollO0 ml tissue-’ l rein-1 for prnol. kg body wt-’ l rein-‘); muscle alanine and lactate uptake lactate (2-4, 29, 34, 40, 45-47). This would correspond (1.60 t 0.7 and 3.29 t 0.36 prnol. kg body wt-‘. min-‘, respecto -1.0 prnol kg body wt-l min-’ for alanine and 2.0 tively) accounted for -30% of their overall disappearance from pmol . kg body wt-’ . min-’ for lactate and would account plasma, whereas alanine and lactate incorporation into plasma for ~20% of their overall systemic rates of appearance glucose (1.83 t 0.20 and 4.24 t 0.44 prnol. kg body wt-’ min-‘, respectively) accounted for -50% of their disappearance from (-5 and 14 prnol. kg-‘. min-‘, respectively) (2, 5, 8, 10, plasma. We therefore conclude that muscle is the major source 11, 15, 21, 23, 30, 35-37, 39, 49, 57) and thus ~20% of of plasma alanine and lactate in postabsorptive humans and their potential contribution to gluconeogenesis. that factors regulating their release from muscle may thus exert It is well established, however, that use of the net an important influence on hepatic gluconeogenesis. balance technique underestimates the release of a substrate from a tissue when that tissue utilizes as well as gluconeogenesis; muscle produces a substrate (50, 52). For example, net balance of a substrate across a tissue is calculated as the product of the arteriovenous concentration difference and blood IN POSTABSORPTIVE HUMANS, gluconeogenesis normally flow. Thus, if the arterial and venous concentrations of accounts for -30% of overall hepatic glucose output (14, a substrate were 0.7 and 0.9 pmol/ml, respectively, and 43). Although the factors controlling gluconeogenesis blood flow were 3 ml/min, the net balance of the subhave been extensively studied, several important issues strate would be -0.60 pmol/min, indicating net output. remain unsettled; among these are the contributions of If, however, the tissue utilized 30% of the substrate specific precursors and the tissues supplying these pre- delivered to it (i.e., 30% fractional extraction), the actual cursors to the liver. release of the substrate produced in that tissue [(arterial Both isotope dilution and splanchnic balance expericoncentration x blood flow x fractional extraction) ments indicate that alanine and lactate are major glu- net balance] would be 1.23 pmol/min, i.e., a value twice coneogenic precursors (5, 10, 15, 17, 21, 35-37, 51), but as great as that estimated by the net balance technique. their exact contribution remains to be determined. The Because muscle takes up and consumes alanine and conventional isotopic approach underestimates gluco- lactate while also producing these substrates (2, 50), use neogenesis from these precursors because of carbon ex- of the net balance technique must underestimate the change in the tricarboxylic acid cycle and intrahepatic contribution of muscle as a source of these gluconeogenic dilution of precursor specific activity (14,19, 25,31). The precursors; the magnitude of this underestimation resplanchnic balance technique underestimates hepatic al- mains to be determined, but it could be considerable (50). l
l
l
0193~1849/90
$1.50
Copyright
cc>1990 the American
Physiological
Society
E677
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on August 24, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
E678
HUMAN
ALANINE
AND
To calculate muscle release of these precursors, it is necessary to measure their net balance as well as their fractional extraction. Regarding measurement of hepatic gluconeogenesis from alanine and lactate, Katz and Hetenyi (2531) have proposed an isotopic approach that can theoretically correct for tricarboxylic acid cycle carbon exchange and thus provide a more precise estimate of gluconeogenesis. Despite its assumptions (14, 3l), this method approximates gluconeogenic precursor-product relationships better than previous approaches, and its application to assess gluconeogenesis in humans has yielded results that support its usefulness (14, 15). In the present studies, therefore, we have applied this isotopic approach in conjunction with infusions of [314C] lactate, [ 3-“Cl alanine, and [ 6-“H] glucose in normal volunteers to estimate alanine and lactate incorporation into plasma glucose, as well as the rates of appearance of these substrates in plasma. At the same time, we also used the forearm balance technique in conjunction with measurement of forearm alanine and lactate fractional extraction to estimate muscle uptake and release of these gluconeogenic precursors. Thus, with the use of alanine and lactate labeled with different carbon isotopes in conjunction with the limb balance technique, we have been able to determine simultaneously whole body and forearm metabolism of alanine and lactate for the first time and calculate their contributions to new glucose production at the same time. Our results indicate that, in postabsorptive humans, muscle provides -70% of the alanine and 40% of the lactate released into the systemic circulation and that alanine and lactate incorporation into plasma glucose accounts for -50% of their disappearance from plasma and ~80% of overall gluconeogenesis. We conclude, therefore, that muscle is the major source of plasma alanine and lactate in postabsorptive humans and that factors regulating release of these substrates from muscle may thus exert an important influence on hepatic gluconeogenesis. METHODS
Subjects. Informed, written consent was obtained from eight volunteers (6 men, 2 women) with normal weight (80 t 3 kg, body mass index 26 t 1 kg/m’) aged 52 t 1 yr. All subjects consumed a weight-maintaining diet containing at least 200 g carbohydrate for 3 days before the study. Protocol. Subjects were admitted to the University of Pittsburgh General Clinical Research Center the evening before the experiments between 5:00 and 7:00 P.M., were given a standard meal (10 Cal/kg; 50% carbohydrate, 35% fat, and 15% protein), and were studied the next morning after a l2- to 15-h fast. At 5:00 A.M., primed continuous infusions of [3J4C]lactate (60 &i, 0.60 &i/min), [3-‘“Clalanine (174 mg, 1.74 mg/min), and [6-‘HIglucose (25 ,&i, 0.25 &i/min) were started through an antecubital vein; shortly thereafter the ipsilateral radial artery was cannulated at the wrist, and a contralateral deep antecubital vein was cannulated retrogradely to obtain blood that was drain-
LACTATE
METABOLISM
ing the muscle tissue (4, 34). After allowing 4 h for isotopic equilibration, blood samples were taken simultaneously from the radial artery and the deep antecubital vein at 15-min intervals between 9:00 and 10:00 A.M. After each blood sampling, forearm blood flow was determined using electrocapacitance plethysmography as previously described (34). Between 9:00 and 1O:OO A.M., total CO2 production was continuously measured using a metabolic measurement cart (Sensor Medic, Anaheim, CA) equipped with an infrared CO2 analyzer and an in-line turbo transducer for determination of expired gas volume (34). Before and after CO2 production measurements, breath samples of the subjects were collected through a small plastic tube into 2 ml of a 0.5 M solution of hyamine hydroxide in ethanol to trap expired COZ for later determination of its 14C specific activity and 13C enrichment. Analytical procedures. Plasma glucose was determined with a Yellow Springs Instruments glucose analyzer (Yellow Springs, OH). Plasma lactate and alanine were determined by standard fluorometric methods (34). The specific activities (dpm/pmol) of plasma [ 3H] glucose, [ “C]glucose, and [ 14C] lactate were determined after isolating lactate and glucose using ion-exchange chromatography (14). The radioactivity of the isolated lactate and glucose were corrected for recovery by means of external standards. Distribution of the 14C radioactivity within the plasma glucose molecule was obtained as previously described ( 14). Carbon-13 enrichment in plasma glucose and alanine were measured by chemical ionization and selected ion monitoring gas chromatography-mass spectrometry of the penta-acetate (5) and N-acetyl-n-propyl ester (41) derivatives, respectively. For N-acetyl-n-propyl alanine, the protonated molecular ion region comprising mass per unit charge (m/x) 174 and 175 was monitored. The former ion reflects unlabeled alanine and the latter corresponds to [ 3J3C] alanine when corrected appropriately for natural isotopic abundance. For glucose penta-acetate, the m/z ratios 332/331 and 170-169 were monitored simultaneously. The former is the protonated molecular ion region and the latter is a fragment ion that also contains all six glucose carbons. In each instance, the higher mass reflects glucose singly labeled with “C, when appropriate natural isotopic abundance corrections are applied, and the lower mass reflects unlabeled glucose. Monitoring two sets of ions provided an internal check of analytical accuracy, and these ion current ratios were nearly identical in all instances. The average of the two ion current ratios at each time point was used in subsequent calculations. In addition, the ion current ratios m/z 333/33l and 334/331 were monitored to assess the presence of glucose molecules doubly or triply labeled with “C. In each case, no multiple-labeled glucose molecules were observed at these ion current ratios (limit of detection -0.05-0.1 atom %excess). “COZ enrichment in expired CO2 was determined by dual-inlet, dual-collector isotope ratio mass spectrometry (42). Calculations. At steady state, the rate of appearance (R,) of a substrate in plasma equals its rate of disappearance (Rd) from plasma (53). Plasma glucose R, was obtained by dividing the [6-“HIglucose infusion rate
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HUMAN
ALANINE
AND LACTATE
(dpm. kg body wt-’ .min-l) by the steady-state arterial [6-“H] glucose specific activity (SA; dpm/pmol) (53). Plasma lactate R, was obtained by dividing the [3J4C]lactate infusion rate (dpm. kg body wt-’ l rein-‘) by the steady-state arterial [14C]lactate SA (dpm/pmol) (53). Plasma alanine R, was obtained according to the following equation (53) alanine R, = I(Ei/E,
- 1)
where I is the [l”C]alanine infusion rate (pmol/kg body wt-’ l rein-‘), Ei is the enrichment of the infusate (mole %excess), and E, is the arterial isotopic enrichment of plasma alanine (mole %excess). Assuming that pyruvate dehydrogenase activity would be negligible in the liver in the postabsorptive state, the correction factor (CF) to account for exchange of label within the tricarboxylic acid cycle was calculated with the following equation derived from the Katz model of the tricarboxylic acid cycle (31) CF = P4
+ 2YbO
+ Y)1/[(5
+ 4YbYl
where y is obtained from the ratio (R) of the specific activities of plasma glucose carbons 1, 2, 5, and 6 and that of carbons 3 and 4, according to the formula (31) y = (R - 2)/Z The percent of plasma glucose R, derived from lactate was calculated according to the equation percent plasma glucose from lactate = (arterial [14C]glucose SA)/Z (arterial [ 14C]lactate SA) CF
x
l
The incorporation of lactate into plasma glucose (pm01.kg body wt-’ l rein-‘) was calculated by multiplying overall plasma glucose R, by the percent of the rate of appearance derived from lactate. The percent of plasma lactate R, incorporated into plasma glucose was obtained dividing the rate of incorporation of lactate into plasma glucose by the lactate R,. Lactate oxidation (pm01 kg-‘. min-‘), as represented by the incorporation of 14C into COz, was calculated according to the following equation l
lactate oxidation =
[ko2-
( 14COzSA - Z)]/O.Sl .arterial [“Cl lactate SA
where ho2 is CO2 production, the factor 0.81 is introduced to correct for retention of 14C02in the bicarbonate pool (53), and Z represents the amount of 14C incorporated into CO2 by exchange within the tricarboxylic acid cycle. Z was calculated as incorporation of lactate into glucose . arterial [“Cl lactate SA . [ (CF - l)/CF] where CF is the correction factor used to correct for the tricarboxylic acid cycle carbon exchange when calculating lactate incorporation into glucose and [ (CF - l)/CF] represents the percent of carbons of the gluconeogenic pathway lost to COz in the tricarboxylic acid cycle. The percent of plasma glucose R, derived from alanine
E679
METABOLISM
was calculated according to the equation percent glucose from alanine = (arterial EP,13c,glucoJ2 arterial EPI’:~(.lalilninr) CF l
l
The incorporation of alanine into plasma glucose was calculated by multiplying overall plasma glucose R, by the percent R, derived from alanine. The percent of alanine R, converted to glucose was obtained dividing the rate of alanine incorporation into plasma glucose by alanine R,. Alanine oxidation (pm01 kg body wt-‘. min-‘), as represented by the incorporation of 13Cinto COz, was calculated according to the following equation (16) l
alanine oxidation = (Vcoz EPnCO ’ - Z)/O.81 (arterial EP,l.I(,ii ,anlne - Ei) l
2
where 0.81 corrects for recovery of label in expired COZ, and Z is calculated as in the calculation of lactate oxidation. Net forearm balance of lactate and alanine forearm was calculated by multiplying arteriovenous (AV) differences in substrate concentrations by forearm blood flow (4, 7). Forearm fractional extraction of [14C]lactate was calculated as the AV difference in lactate radioactivity divided by the arterial lactate radioactivity (50). Forearm alanine fractional extraction was calculated as the AV difference in [“Clalanine concentration divided by the arterial [ 13C]alanine concentration (52). Forearm substrate uptake was calculated as arterial concentration x blood flow x fractional extraction (22). Forearm substrate release was calculated according to the equation (22) release = uptake - net balance Forearm data per 100 ml per tissue were converted to kilograms per forearm muscle by multiplying the data by 13.3, assuming that muscle tissue has a density of 1.0, that it represents -60% of the forearm volume, and that 80% of the blood flow to the forearm is directed to the muscle tissue (4, 7). Forearm volume was determined using the equations for a truncated cone (7, 34). Total body muscle mass (kg) was calculated according to the equations by Heymsfield et al. (27). Total body muscle alanine and lactate uptake and release were calculated by multiplying forearm muscle data by total body muscle mass. Data are given as means t SE. RESULTS
Overall systemic appearance of alanine and lactate. Arterial and venous alanine and lactate concentrations, lactate specific activities, and alanine isotopic enrichments are given in Table 1. Systemic rates of appearance of lactate and alanine (Table 2) were 12.7 t 0.8 and 4.2 t 0.3 pmol. kg body wt-‘. min-‘, respectively. Rates of lactate and alanine oxidation. 14C02 specific activities and “COz enrichments in expired air and total COz production are given in Table 3. Lactate and alanine oxidation (Table 2) were 7.7 t 0.5 and 1.2 t 0.1 prnol kg body wt-‘. min-‘, respectively, accounting for 62 t 4 and 31 t 4% of their respective turnovers.
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E680
HUMAN
ALANINE
AND
1. Arterial and venous concentrations and 14C specific activities of lactate and concentrations and ‘
CONSOLI, AGOSTINO, NURJAHAN NURJHAN, JAMES J. REILLY, JR., DENNIS M. BIER, AND JOHN E. GERICH. Contribution of liver and skeletal muscle to alanine and lactate metab-
anine and lactate uptake because it measures only their net balance and because the gastrointestinal tract produces these substrates (6, 12, 18). Such limitations could olism in humans. Am. J. Physiol. 259 (Endocrinol. Metab. 22): cause underestimation of the contribution of alanine and E677-E684, 1990.-To quantitate alanine and lactate glucolactate to gluconeogenesis by as much as 50-100% (14, neogenesis in postabsorptive humans and to test the hypothesis 18) that muscle is the principal source of these precursors, we Furthermore, although delivery of precursors t’o the infused normal volunteers with [3- “‘Cllactate, [ 3-“‘Clalanine, liver is considered a major determinant of gluconeogenand [6-“HIglucose and calculated alanine and lactate incorpoesis (9, 17), the key tissues supplying alanine and lactate ration into plasma glucose corrected for tricarboxylic acid cycle in humans remain unclear. On the basis of earlier studies carbon exchange, the systemic appearance of these substrates, and their forearm fractional extraction, uptake, and release. (6, 18), muscle might be expected to be a major source of Forearm alanine and lactate fractional extraction averaged 37 these precursors via proteolysis and the Cori and alanine t 3 and 27 k 2%, respectively; muscle alanine release (2.94 * cycles. However, currently available data suggest that 0.27 prnol. kg body wt-l l rein-‘) accounted for -70% of its muscle makes only a minor contribution. Experiments systemic appearance (4.18 t 0.31 prnol. kg body wt-l. min); employing the limb balance technique indicate a net muscle lactate release (5.51 t 0.42 prnol. kg body wt-l min-‘) output of -0.2 pmol/lOO ml tissue for alanine (2, 19, 34, accounted for -40% of its systemic appearance (12.66 t 0.77 40, 44-47) and -0.4 ~mollO0 ml tissue-’ l rein-1 for prnol. kg body wt-’ l rein-‘); muscle alanine and lactate uptake lactate (2-4, 29, 34, 40, 45-47). This would correspond (1.60 t 0.7 and 3.29 t 0.36 prnol. kg body wt-‘. min-‘, respecto -1.0 prnol kg body wt-l min-’ for alanine and 2.0 tively) accounted for -30% of their overall disappearance from pmol . kg body wt-’ . min-’ for lactate and would account plasma, whereas alanine and lactate incorporation into plasma for ~20% of their overall systemic rates of appearance glucose (1.83 t 0.20 and 4.24 t 0.44 prnol. kg body wt-’ min-‘, respectively) accounted for -50% of their disappearance from (-5 and 14 prnol. kg-‘. min-‘, respectively) (2, 5, 8, 10, plasma. We therefore conclude that muscle is the major source 11, 15, 21, 23, 30, 35-37, 39, 49, 57) and thus ~20% of of plasma alanine and lactate in postabsorptive humans and their potential contribution to gluconeogenesis. that factors regulating their release from muscle may thus exert It is well established, however, that use of the net an important influence on hepatic gluconeogenesis. balance technique underestimates the release of a substrate from a tissue when that tissue utilizes as well as gluconeogenesis; muscle produces a substrate (50, 52). For example, net balance of a substrate across a tissue is calculated as the product of the arteriovenous concentration difference and blood IN POSTABSORPTIVE HUMANS, gluconeogenesis normally flow. Thus, if the arterial and venous concentrations of accounts for -30% of overall hepatic glucose output (14, a substrate were 0.7 and 0.9 pmol/ml, respectively, and 43). Although the factors controlling gluconeogenesis blood flow were 3 ml/min, the net balance of the subhave been extensively studied, several important issues strate would be -0.60 pmol/min, indicating net output. remain unsettled; among these are the contributions of If, however, the tissue utilized 30% of the substrate specific precursors and the tissues supplying these pre- delivered to it (i.e., 30% fractional extraction), the actual cursors to the liver. release of the substrate produced in that tissue [(arterial Both isotope dilution and splanchnic balance expericoncentration x blood flow x fractional extraction) ments indicate that alanine and lactate are major glu- net balance] would be 1.23 pmol/min, i.e., a value twice coneogenic precursors (5, 10, 15, 17, 21, 35-37, 51), but as great as that estimated by the net balance technique. their exact contribution remains to be determined. The Because muscle takes up and consumes alanine and conventional isotopic approach underestimates gluco- lactate while also producing these substrates (2, 50), use neogenesis from these precursors because of carbon ex- of the net balance technique must underestimate the change in the tricarboxylic acid cycle and intrahepatic contribution of muscle as a source of these gluconeogenic dilution of precursor specific activity (14,19, 25,31). The precursors; the magnitude of this underestimation resplanchnic balance technique underestimates hepatic al- mains to be determined, but it could be considerable (50). l
l
l
0193~1849/90
$1.50
Copyright
cc>1990 the American
Physiological
Society
E677
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on August 24, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
E678
HUMAN
ALANINE
AND
To calculate muscle release of these precursors, it is necessary to measure their net balance as well as their fractional extraction. Regarding measurement of hepatic gluconeogenesis from alanine and lactate, Katz and Hetenyi (2531) have proposed an isotopic approach that can theoretically correct for tricarboxylic acid cycle carbon exchange and thus provide a more precise estimate of gluconeogenesis. Despite its assumptions (14, 3l), this method approximates gluconeogenic precursor-product relationships better than previous approaches, and its application to assess gluconeogenesis in humans has yielded results that support its usefulness (14, 15). In the present studies, therefore, we have applied this isotopic approach in conjunction with infusions of [314C] lactate, [ 3-“Cl alanine, and [ 6-“H] glucose in normal volunteers to estimate alanine and lactate incorporation into plasma glucose, as well as the rates of appearance of these substrates in plasma. At the same time, we also used the forearm balance technique in conjunction with measurement of forearm alanine and lactate fractional extraction to estimate muscle uptake and release of these gluconeogenic precursors. Thus, with the use of alanine and lactate labeled with different carbon isotopes in conjunction with the limb balance technique, we have been able to determine simultaneously whole body and forearm metabolism of alanine and lactate for the first time and calculate their contributions to new glucose production at the same time. Our results indicate that, in postabsorptive humans, muscle provides -70% of the alanine and 40% of the lactate released into the systemic circulation and that alanine and lactate incorporation into plasma glucose accounts for -50% of their disappearance from plasma and ~80% of overall gluconeogenesis. We conclude, therefore, that muscle is the major source of plasma alanine and lactate in postabsorptive humans and that factors regulating release of these substrates from muscle may thus exert an important influence on hepatic gluconeogenesis. METHODS
Subjects. Informed, written consent was obtained from eight volunteers (6 men, 2 women) with normal weight (80 t 3 kg, body mass index 26 t 1 kg/m’) aged 52 t 1 yr. All subjects consumed a weight-maintaining diet containing at least 200 g carbohydrate for 3 days before the study. Protocol. Subjects were admitted to the University of Pittsburgh General Clinical Research Center the evening before the experiments between 5:00 and 7:00 P.M., were given a standard meal (10 Cal/kg; 50% carbohydrate, 35% fat, and 15% protein), and were studied the next morning after a l2- to 15-h fast. At 5:00 A.M., primed continuous infusions of [3J4C]lactate (60 &i, 0.60 &i/min), [3-‘“Clalanine (174 mg, 1.74 mg/min), and [6-‘HIglucose (25 ,&i, 0.25 &i/min) were started through an antecubital vein; shortly thereafter the ipsilateral radial artery was cannulated at the wrist, and a contralateral deep antecubital vein was cannulated retrogradely to obtain blood that was drain-
LACTATE
METABOLISM
ing the muscle tissue (4, 34). After allowing 4 h for isotopic equilibration, blood samples were taken simultaneously from the radial artery and the deep antecubital vein at 15-min intervals between 9:00 and 10:00 A.M. After each blood sampling, forearm blood flow was determined using electrocapacitance plethysmography as previously described (34). Between 9:00 and 1O:OO A.M., total CO2 production was continuously measured using a metabolic measurement cart (Sensor Medic, Anaheim, CA) equipped with an infrared CO2 analyzer and an in-line turbo transducer for determination of expired gas volume (34). Before and after CO2 production measurements, breath samples of the subjects were collected through a small plastic tube into 2 ml of a 0.5 M solution of hyamine hydroxide in ethanol to trap expired COZ for later determination of its 14C specific activity and 13C enrichment. Analytical procedures. Plasma glucose was determined with a Yellow Springs Instruments glucose analyzer (Yellow Springs, OH). Plasma lactate and alanine were determined by standard fluorometric methods (34). The specific activities (dpm/pmol) of plasma [ 3H] glucose, [ “C]glucose, and [ 14C] lactate were determined after isolating lactate and glucose using ion-exchange chromatography (14). The radioactivity of the isolated lactate and glucose were corrected for recovery by means of external standards. Distribution of the 14C radioactivity within the plasma glucose molecule was obtained as previously described ( 14). Carbon-13 enrichment in plasma glucose and alanine were measured by chemical ionization and selected ion monitoring gas chromatography-mass spectrometry of the penta-acetate (5) and N-acetyl-n-propyl ester (41) derivatives, respectively. For N-acetyl-n-propyl alanine, the protonated molecular ion region comprising mass per unit charge (m/x) 174 and 175 was monitored. The former ion reflects unlabeled alanine and the latter corresponds to [ 3J3C] alanine when corrected appropriately for natural isotopic abundance. For glucose penta-acetate, the m/z ratios 332/331 and 170-169 were monitored simultaneously. The former is the protonated molecular ion region and the latter is a fragment ion that also contains all six glucose carbons. In each instance, the higher mass reflects glucose singly labeled with “C, when appropriate natural isotopic abundance corrections are applied, and the lower mass reflects unlabeled glucose. Monitoring two sets of ions provided an internal check of analytical accuracy, and these ion current ratios were nearly identical in all instances. The average of the two ion current ratios at each time point was used in subsequent calculations. In addition, the ion current ratios m/z 333/33l and 334/331 were monitored to assess the presence of glucose molecules doubly or triply labeled with “C. In each case, no multiple-labeled glucose molecules were observed at these ion current ratios (limit of detection -0.05-0.1 atom %excess). “COZ enrichment in expired CO2 was determined by dual-inlet, dual-collector isotope ratio mass spectrometry (42). Calculations. At steady state, the rate of appearance (R,) of a substrate in plasma equals its rate of disappearance (Rd) from plasma (53). Plasma glucose R, was obtained by dividing the [6-“HIglucose infusion rate
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HUMAN
ALANINE
AND LACTATE
(dpm. kg body wt-’ .min-l) by the steady-state arterial [6-“H] glucose specific activity (SA; dpm/pmol) (53). Plasma lactate R, was obtained by dividing the [3J4C]lactate infusion rate (dpm. kg body wt-’ l rein-‘) by the steady-state arterial [14C]lactate SA (dpm/pmol) (53). Plasma alanine R, was obtained according to the following equation (53) alanine R, = I(Ei/E,
- 1)
where I is the [l”C]alanine infusion rate (pmol/kg body wt-’ l rein-‘), Ei is the enrichment of the infusate (mole %excess), and E, is the arterial isotopic enrichment of plasma alanine (mole %excess). Assuming that pyruvate dehydrogenase activity would be negligible in the liver in the postabsorptive state, the correction factor (CF) to account for exchange of label within the tricarboxylic acid cycle was calculated with the following equation derived from the Katz model of the tricarboxylic acid cycle (31) CF = P4
+ 2YbO
+ Y)1/[(5
+ 4YbYl
where y is obtained from the ratio (R) of the specific activities of plasma glucose carbons 1, 2, 5, and 6 and that of carbons 3 and 4, according to the formula (31) y = (R - 2)/Z The percent of plasma glucose R, derived from lactate was calculated according to the equation percent plasma glucose from lactate = (arterial [14C]glucose SA)/Z (arterial [ 14C]lactate SA) CF
x
l
The incorporation of lactate into plasma glucose (pm01.kg body wt-’ l rein-‘) was calculated by multiplying overall plasma glucose R, by the percent of the rate of appearance derived from lactate. The percent of plasma lactate R, incorporated into plasma glucose was obtained dividing the rate of incorporation of lactate into plasma glucose by the lactate R,. Lactate oxidation (pm01 kg-‘. min-‘), as represented by the incorporation of 14C into COz, was calculated according to the following equation l
lactate oxidation =
[ko2-
( 14COzSA - Z)]/O.Sl .arterial [“Cl lactate SA
where ho2 is CO2 production, the factor 0.81 is introduced to correct for retention of 14C02in the bicarbonate pool (53), and Z represents the amount of 14C incorporated into CO2 by exchange within the tricarboxylic acid cycle. Z was calculated as incorporation of lactate into glucose . arterial [“Cl lactate SA . [ (CF - l)/CF] where CF is the correction factor used to correct for the tricarboxylic acid cycle carbon exchange when calculating lactate incorporation into glucose and [ (CF - l)/CF] represents the percent of carbons of the gluconeogenic pathway lost to COz in the tricarboxylic acid cycle. The percent of plasma glucose R, derived from alanine
E679
METABOLISM
was calculated according to the equation percent glucose from alanine = (arterial EP,13c,glucoJ2 arterial EPI’:~(.lalilninr) CF l
l
The incorporation of alanine into plasma glucose was calculated by multiplying overall plasma glucose R, by the percent R, derived from alanine. The percent of alanine R, converted to glucose was obtained dividing the rate of alanine incorporation into plasma glucose by alanine R,. Alanine oxidation (pm01 kg body wt-‘. min-‘), as represented by the incorporation of 13Cinto COz, was calculated according to the following equation (16) l
alanine oxidation = (Vcoz EPnCO ’ - Z)/O.81 (arterial EP,l.I(,ii ,anlne - Ei) l
2
where 0.81 corrects for recovery of label in expired COZ, and Z is calculated as in the calculation of lactate oxidation. Net forearm balance of lactate and alanine forearm was calculated by multiplying arteriovenous (AV) differences in substrate concentrations by forearm blood flow (4, 7). Forearm fractional extraction of [14C]lactate was calculated as the AV difference in lactate radioactivity divided by the arterial lactate radioactivity (50). Forearm alanine fractional extraction was calculated as the AV difference in [“Clalanine concentration divided by the arterial [ 13C]alanine concentration (52). Forearm substrate uptake was calculated as arterial concentration x blood flow x fractional extraction (22). Forearm substrate release was calculated according to the equation (22) release = uptake - net balance Forearm data per 100 ml per tissue were converted to kilograms per forearm muscle by multiplying the data by 13.3, assuming that muscle tissue has a density of 1.0, that it represents -60% of the forearm volume, and that 80% of the blood flow to the forearm is directed to the muscle tissue (4, 7). Forearm volume was determined using the equations for a truncated cone (7, 34). Total body muscle mass (kg) was calculated according to the equations by Heymsfield et al. (27). Total body muscle alanine and lactate uptake and release were calculated by multiplying forearm muscle data by total body muscle mass. Data are given as means t SE. RESULTS
Overall systemic appearance of alanine and lactate. Arterial and venous alanine and lactate concentrations, lactate specific activities, and alanine isotopic enrichments are given in Table 1. Systemic rates of appearance of lactate and alanine (Table 2) were 12.7 t 0.8 and 4.2 t 0.3 pmol. kg body wt-‘. min-‘, respectively. Rates of lactate and alanine oxidation. 14C02 specific activities and “COz enrichments in expired air and total COz production are given in Table 3. Lactate and alanine oxidation (Table 2) were 7.7 t 0.5 and 1.2 t 0.1 prnol kg body wt-‘. min-‘, respectively, accounting for 62 t 4 and 31 t 4% of their respective turnovers.
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E680
HUMAN
ALANINE
AND
1. Arterial and venous concentrations and 14C specific activities of lactate and concentrations and ‘
