United States Department of Agriculture/Agriculture Research Service Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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Infant pigs (8.5 kg) were fasted for 16 h and
infused for 6 h with
[U-13C]glucose. The
fractional abundances of all 13C
mass isotopomers of plasma glucose, lactate, and pyruvate and of
plasma, hepatic, and very low density lipoprotein apolipoprotein B-100
(apoB-100) alanine, glutamate, and aspartate were measured. The ratios
of
[13C3]aspartate,
[13C3]glutamate,
and
[13C3]alanine
in apoB-100 were used to estimate the positional equilibrium of
[13C3]oxaloacetate,
the fractional contribution of pyruvate carboxylase to the hepatic
oxaloacetate flux, and the activity of hepatic pyruvate dehydrogenase.
The values were compared with those based on glucose labeling and
previously published equations. The two methods [Katz and Lee
method (J. Katz, P. A. Wals., and W.-N. P. Lee. J. Biol. Chem. 264: 12994-13001, 1989) and apoB
method] gave similar estimates of the positional equilibrium of
[13C3]oxaloacetate
(0.59, Katz and Lee method; 0.61, apoB method) but slightly different
estimates of the contribution of pyruvate carboxylase to the
oxaloacetate flux (0.36, Katz and Lee; 0.31 apoB). Gluconeogenesis
apparently contributed between 71 (Katz and Lee method) and 80% (apoB
method) of the glucose entry rate (25 µmol · kg1 · min
1),
and pyruvate dehydrogenase contributed 20% of the hepatic acetyl-CoA. We conclude that the labeling of aspartate in apoB-100 provides a good
estimate of the isotopomer distribution in hepatic oxaloacetate but may
underestimate the absolute isotopic enrichment by 50%.
hepatic amino acids; stable isotopes
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INTRODUCTION |
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THE IN VIVO quantitative analysis of the metabolic pathways that underlie glucose homeostasis remains controversial. Published estimates of the metabolic recycling of glucose and the contribution of gluconeogenesis to blood glucose turnover (15-18, 21, 22, 25, 29) differ widely and continue to generate substantial argument (21, 22, 25, 26). Although the development and application of the 2H2O method (21, 22) may lead to the emergence of a consensus over the absolute rate of gluconeogenesis, there is a continuing need for carbon tracer studies of glucose metabolism. Uniformly labeled [U-13C]glucose has been used in a number of studies of glucose turnover and gluconeogenesis in both animals and humans (3, 15-18, 20, 29-31). Apart from offering the potential for the calculation of the gluconeogenic rate, this tracer has the advantage that it allows the investigation of other aspects of glucose metabolism, including the relationships between glucose and amino acid metabolism.
In theory, when [U-13C]glucose is used as a tracer, the calculation of the contribution of gluconeogenesis (or more correctly the contribution of gluconeogenesis derived from pyruvate-lactate) to the plasma glucose entry rate, should be simply a matter of measuring the relative isotopic enrichments of [13C3]glucose (i.e., that derived from [13C3]pyruvate) and [13C6]glucose. However, the relative labeling of [13C3]- and [13C6]glucose underestimates the contribution of gluconeogenesis to the glucose entry rate because of isotopic dilution in the three-carbon (pyruvate-lactate) and tricarboxylic acid (TCA) cycle intermediate pools.
The estimation of the dilution within the TCA cycle ketoacid pools remains problematic. Equations, based on the ratios of the isotopic enrichments of [13C1]glucose to [13C3]glucose and [13C2]glucose to [13C3]glucose, have been devised to estimate the parameters necessary for the calculation of gluconeogenesis with [U-13C]glucose (15-18). However, this approach is indirect and relies on the assumptions that the activity of pyruvate dehydrogenase is negligible and that no phosphoenolpyruvate (PEP) synthesized from oxaloacetate (OAA) recycles via the pyruvate pool. The method is also susceptible to errors associated with 13CO2 fixation at the pyruvate carboxylase and isocitrate dehydrogenase (7) reactions.
There has been, therefore, continuing interest in other approaches to the determination of the isotopic enrichment of TCA cycle intermediates in the liver. The most extensive work in this area has been that of Des Rosiers, Brunengraber, and their colleagues (e.g., 7, 8, 10). These studies are, however, in vitro and involve direct sampling of the liver. In vivo, there are fewer possibilities of determining estimates of intrahepatic ketoacid labeling. To this date, sampling the hepatic ketoglutarate pool has involved the administration of phenylacetate to trap hepatic glutamine as its phenylacetyl-glutamine conjugate (23). Unfortunately, this approach involves the ingestion of gram quantities of phenylacetic acid, and it is not certain whether phenylacetate affects hepatic glutamine metabolism.
In this study, we have investigated the utility of a different approach
to the noninvasive sampling of the hepatic ketoacid pool. Because the
very low density lipoprotein (VLDL) particle is rapidly processed to
particles of higher density, the apolipoprotein B-100 (apoB-100)
contained within the VLDL fraction has a very rapid rate of turnover.
In fact, it is sufficiently rapid that, during the course of a tracer
amino acid infusion of <12 h, apoB-100 reaches isotopic equilibrium
(27). By definition, once this state has been achieved, the isotopic
enrichment of the tracer amino acid in VLDL apoB-100 defines directly
the isotopic enrichment of the hepatic pool from which it derived. We
have shown that, during infusions of either
[U-13C]alanine (27) or
[U-13C]glucose (12),
the plasma lactate, pyruvate, and apoB-100-bound alanine pools reach
near-isotopic equilibrium. Thus it appears that alanine incorporated
into apoB-100 derives from a pool of alanine that is synthesized by
transamination of hepatic pyruvate. In designing the present study, we
reasoned that, by analogy to the results with alanine, the equilibrium
labeling of glutamate and aspartate in apoB-100 might also measure the
labeling of the hepatic -ketoglutarate (
-KG) and OAA pools.
The objectives of the present work were threefold: first, to use the labeling of the 13C3 isotopomers of alanine, aspartate, and glutamate isolated from VLDL apoB-100 to calculate the dilution factors necessary for the calculation of gluconeogenesis; second, to compare these estimates with those obtained from calculations based solely on glucose labeling (15-17, 29, 30); and third, to use apoB-100 amino acid and glucose labeling to calculate in vivo estimates of the activity of hepatic pyruvate dehydrogenase and the relative contributions of glycolysis and OAA to the three-carbon pool of the liver.
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METHODS |
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The protocol was approved by the Animal Protocol Review Committee of Baylor College of Medicine. Care and maintenance of the animals conformed to US Department of Agriculture guidelines.
Animals and Surgery
We studied five female pigs, purchased at 2 wk of age from Bud Adams Ranch (Houston, TX). Surgery was performed after a 2-wk period, during which the animals were offered a milk-replacer diet (Litter Life; Merrick, Middleton, WI). Before surgery, animals were fasted overnight and then anesthetized with 5% isoflurane in 50% oxygen (Aurrane, Anaquest) administered with a face mask. A tracheal tube was then inserted, and anesthesia was maintained with 1-2% isoflurane. Under sterile conditions, 20-gauge polyethylene catheters (Intramedic, Parsippany, NJ) were inserted in the carotid artery and right external jugular vein. The catheters were filled with NaCl (0.154 mol/l containing 30 U/ml heparin) and tunneled subcutaneously to emerge between the scapulae. The catheters were protected with a gauze pad and secured with an elastic bandage. Animals were monitored closely until they became ambulant and then at frequent intervals over the next 12 h. The infusion was carried out after a 3-day period during which the animals recovered their presurgery weight gain. At the time of the infusion they were 31 days old and weighed 6.4 ± 0.5 kg.Isotopic Tracer
D-[U-13C]glucose was purchased from Cambridge Isotopes (Woburn, MA). Mass spectrometric analysis confirmed its chemical and isotopomer purity (91.7% [13C6]glucose, 7.4% [13C5]glucose).Infusion
The animals were fasted for 16 h before the start of the infusion. After blood samples were withdrawn for baseline measurements, animals were given a primed constant infusion of [U-13C]glucose (prime 160 µmol/kg; infusion 2 µmol · kgSample Analysis
The blood samples were collected in tubes containing Na2EDTA and immediately centrifuged at 4°C. The plasma was divided into two aliquots. One was taken for analysis of plasma metabolites, the other for isolation and analysis of VLDL apoB-100.For analysis of plasma glucose, lactate, pyruvate, and amino acids, a 1-ml sample of plasma was mixed with an equal volume of acetic acid (1 mol/l) and applied to a 2-ml bed volume column of Dowex AG50 ×8 200 mesh H+ form. The loading volume and a 2-ml water wash was collected, treated with activated charcoal to remove bile salts, and centrifuged, and the supernatant was dried under vacuum. This was used for the measurements of glucose, pyruvate, and lactate labeling. Amino acids were released from the column with 2 ml of 2 M NH4OH. This solution was also dried.
For analysis of VLDL apoB-100, 1 ml of plasma was layered under a solution (0.1 mol/l NaBr, 1 mmol/l Na2EDTA, 1 mmol/l NaN3) of final specific gravity 1.0063 g/ml. This was centrifuged at 100,000 revolutions/min at 22°C for 4 h in a 100.3 rotor in a Beckman (Palo Alto, CA) TL 100 ultracentrifuge. The VLDL fraction was removed by aspiration, and apoB-100 was precipitated with isopropanol (9). The precipitate was mixed with HCl (5.4 mol/l) and hydrolyzed for 24 h at 110°C. The hydrolysate was dried, dissolved in water, redried, and redissolved in water. It was then applied to a Dowex 50 column, and the amino acids were isolated as described above for plasma amino acids.
Samples of liver (1 g) were homogenized in 10 ml of perchloric acid (0.5 mol/l). The homogenate was centrifuged, and the supernatant was neutralized with KOH (4 mol/l). After removal of the potassium perchlorate, a portion of the supernatant was used to isolate amino acids as described above for plasma samples.
Mass Spectrometry
Labeled glucose was analyzed as its pentaacetate derivative by methane-positive chemical ionization (12). Ions with a mass-to-charge ratio (m/z) of 331-337 were monitored. Plasma pyruvate and lactate were analyzed as their pentafluorobenzyl derivatives (11) by methane-negative chemical ionization. We monitored m/z 87-90 for lactate and 89-92 for pyruvate. The amino acids were analyzed by methane negative chemical ionization of the n-propyl esters of their heptafluorobutyramide derivatives (27). Ions monitored were m/z 307-310 for alanine, 393-397 for aspartate, and 407-412 for glutamate. Glucose was separated with a DB 1701 column and lactate, pyruvate, and amino acids with a DB 5 column. Both columns were 30 m long, ID 0.25 mm, and film thickness 0.25 µm and were purchased from J & W Scientific (Folsom, CA). Gas chromatography-mass spectrometry was carried out in triplicate by injection of 1-µl samples with a 1:30 split into a Hewlett-Packard (Palo Alto, CA) 5988A quadrapole instrument. ![]() |
CALCULATIONS |
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The raw ion abundances of all metabolites were converted to fractional abundances (i.e., mol isotopomer/100 mol analyte) using the matrix approach of Brauman (4) as applied in previous papers (1, 6). The baseline spectrum used for the calculation was that of derivatives prepared from plasma samples taken immediately before the start of the infusion rather than pure unlabeled samples. Throughout the following section, for simplicity of presentation, the nonstandard term [M+X] is used to denote the tracer-to-tracee ratio of an isotopomer containing X 13C atoms.
All the calculations of gluconeogenesis are based on the assumption that, during a [U-13C]glucose infusion, glucose synthesis occurring via pyruvate results in the recycling of 13C to glucose. In the following calculations we have used two ways of quantifying the tracer recycling: 1) measurements of the fractional abundances of the [M+3] and [M+6] isotopomers of glucose (16) and 2) data on 13C recycling (29, 30).
The three basic equations used to analyze the glucose labeling were as follows.
Glucose Entry Rate
Glucose entry rate (Ra glucose) is measured in micromoles per kilogram per hour
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(1) |
Glucose Isotopomer Recycling
Glucose isotopomer recycling (GIR) is a proportion of glucose Ra
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(2) |
13C Recycling
13C recycling (GC) is a proportion of glucose Ra)
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(3) |
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(4) |
The crude recycling terms GIR and GC are underestimates of gluconeogenesis because, once glucose carbon is metabolized via the glycolytic pathway, it becomes diluted, either in the three-carbon (pyruvate-lactate-alanine) pool or by 13C-12C exchange and 12C dilution within the TCA cycle. Of the two equations, Eq. 2, which is based on the recycling of a specific isotopomer, is the more theoretically satisfactory because [M+3]glucose can be synthesized only by the direct metabolism of glucose. However, to use the recycling of the [M+3] isotopomer to calculate gluconeogenesis, account must be taken of a further form of dilution that arises because of the operation of the fumarate-OAA metabolic cycle. This cycle leads to the production of a mixture of [M+3]OAA molecules that are either labeled in C-1 to C-3 (directly formed from [M+3]pyruvate) or in C-2 to C-4 (recycled from fumarate). Of these, only [M+3]C-1-C-3OAA yields [M+3]glucose. Because at complete equilibration [M+3]OAA becomes an equimolar mixture of the two positional isotopomers, even without carbon dilution from amino and fatty acids, the labeling of [M+3]glucose can underestimate gluconeogenesis by as much as twofold.
We adopted two approaches to the derivation of the various dilution factors used in conjunction with isotopomer recycling.
Dilution factor for the three-carbon pool. This is the reciprocal of the fractional contribution of glucose to lactate synthesis. Isotopomer method
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(5) |
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(6) |
Dilution at the level of OAA. For the isotopomer recycling data we adopted two approaches to the determination of dilution at the level of OAA.
METHOD OF KATZ AND LEE. The method of Katz and Lee (15-17) uses the labeling [M+1]-, [M+2]-, and [M+3]glucose to calculate two parameters: 1) A = the fraction of [M+3]OAA labeled in C-1 to C-3; this yields a positional dilution factor of 1/A; 2) a factor Y = an estimate of the contribution of pyruvate carboxylation to the OAA flux. This yields a 12C dilution factor of (1 + Y)/Y. The "TCA cycle" dilution factor (D) is then calculated as
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(7) |
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(8) |
Calculation of the Positional Equilibrium of [M+3]OAA (A)
The first decarboxylation step of the TCA cycle occurs at isocitrate dehydrogenase and leads to the loss of C-1 of OAA. It follows from this that citrate synthesized from [M+3]C-1-C-3OAA leads to the production of [M+2]
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(9) |
Calculating the Contribution of Pyruvate Carboxylase to OAA
During a [U-13C]glucose infusion, the only source of hepatic [M+3]OAA is the carboxylation of [M+3]pyru- vate. Again with the assumption that apoB-100 alanine and apoB-100 aspartate sample the hepatic pools of pyruvate and OAA, respectively, then the contribution of pyruvate carboxylase to the flux of OAA can be estimated from
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(10) |
Note that the estimates of A and Y
from apoB-100 amino acid labeling use no measurements of the
[M+1] and
[M+2] isotopomers of
aspartate, alanine, and glutamate. The calculations should not be
affected either by recycling of the
[M+2] isotopomers in the
TCA cycle or by entry of
[M+2]acetyl-CoA.
Furthermore, the decarboxylation-recarboxylation cycle at isocitrate
dehydrogenase (7) does not affect the abundance of
[M+3]-KG because the carbon involved is C-1 of the OAA portion of isocitrate.
Tayek and Katz Method
The Tayek and Katz (29) method uses the 13C enrichment of glucose and lactate. The base measurements are 13C recycling (Eq. 3) and the three-carbon dilution (Eq. 6). Because this approach is unaffected by the fumarate-OAA cycle, the only additional parameter needed for the calculation is an estimate of carbon dilution in the TCA cycle. In this method this is estimated as
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(11) |
Tayek and Katz Method
This Tayek and Katz (30) method developed from their original reasoning (29). In this report, we used an alternative method of quantifying recycling, which they termed molecular recycling. This term is defined as the fraction of all labeled glucose molecules that have arisen from recycling, irrespective of dilution, and is given by the expression
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(12) |
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(13) |
Contribution of Pyruvate Dehydrogenase to Acetyl-CoA Synthesis
In the model of the TCA cycle used by Katz and Lee (15), it is assumed that there is no label entry into the TCA cycle via pyruvate dehydrogenase. However, there is no assurance that its activity is completely suppressed, and, without the isotopic analysis of different fragments ofContribution of
[M+3]C-1-C-3OAA
to
[M+2]-KG
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(14) |
Contribution of
[M+2]OAA
to
[M+2]-KG
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(15) |
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(16) |
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(17) |
Contribution of Gluconeogenesis, Glycolysis, and Triose Cycling to Glucose and Three-Carbon Pool Labeling
A second assumption in the Katz and Lee method (15-17) is that PEP, synthesized from OAA, does not recycle via pyruvate kinase. Moreover, in theory [M+3]glucose could be formed by cycling between [M+6]glucose and the triose phosphate pool without there necessarily being any synthesis of pyruvate. We also used the isotopomer distribution in apoB-100 alanine and aspartate and in [M+1]-[M+3]glucose and lactate in plasma, and hepatic alanine to examine these assumptions.Isotopomer distribution in PEP derived from OAA. Given information on the positional equilibrium of [M+3]OAA (A), the relative abundances of the [M+1], [M+2], and [M+3] isotopomers of PEP derived from OAA can be calculated from apoB-100 aspartate as follows
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(18a) |
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(18b) |
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(18c) |
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(19) |
Isotopomer distribution in triose phosphate derived from glucose. This can be calculated as follows
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(20a) |
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(20b) |
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(20c) |
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(21) |
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(22) |
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RESULTS |
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The time courses of glucose and apoB-100 alanine, apoB-100 aspartate, and apoB-100 glutamate labeling are shown in Figs. 1 and 2, respectively. [13C6]glucose attained an isotopic steady state early on in the infusion, although the isotopomers that derived from 13C recycling did not achieve a plateau until at least 4 h. The labeling of [13C3]alanine, -aspartate, and -glutamate in apoB-100 had also reached stable values by 4 h of infusion. By this time, plasma lactate and pyruvate and the 13C1 and 13C2 isotopomers of apoB-100 amino acids had also reached isotopic steady state (data not shown).
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The mean steady-state values for the fractional abundances of glucose and its metabolites are shown in Table 1. Plasma lactate and pyruvate had achieved almost complete isotopomeric equilibrium, and there was an approximate twofold dilution of 13C between glucose [51.8 atoms percent excess (APE)] and plasma lactate and pyruvate (12.2 APE). ApoB-100 [13C3]alanine and plasma [13C3]alanine had almost identical isotopomer distributions and 13C enrichments. The isotopomer distributions in plasma and apoB-100 alanine were also very similar to those in plasma pyruvate and lactate, but both apoB-100 and plasma alanine were between 32 and 38% less enriched with 13C than plasma lactate and pyruvate. Although the 13C enrichment of hepatic free alanine was similar to that of plasma and apoB-100 alanine, the isotopomer distribution was different; the 13C3 isotopomer was of lesser abundance and the 13C1 and 13C2 isotopomers of hepatic alanine were of greater abundance than the respective isotopomers in apoB-100 and plasma alanine.
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There was a further 2.2-fold dilution of 13C between apoB-100 alanine (8.4 APE) and apoB-100 aspartate (4.1 APE). There was also marked heterogeneity among the pools of aspartate. First, the 13C enrichment of apoB-100 aspartate was twofold higher than hepatic free aspartate and sevenfold higher than plasma aspartate. Second, the isotopomer distribution in the three sampled aspartate pools was strikingly different. The glutamate pools, on the other hand, were apparently in complete isotopic equilibrium.
The base measurements of glucose metabolism are shown in Table
2. The glucose entry rate was 24.6 ± 3.2 µmol · kg1 · min
1;
11 ± 2% of the
[U-13C]glucose had
recycled as
[13C3]glucose
and 24 ± 3% of the total 13C
in glucose was contained in the lower mass isotopomers. According to
the formulation of Tayek and Katz (30) 50.2 ± 3.8% of
the circulating glucose molecules had been labeled via tracer
recycling. The fractional abundance of
[13C3]lactate
was 40% of that of its combined precursor
[[13C6]glucose + (0.5 × [13C3]glucose)],
leading to a three-carbon dilution factor of 2.14 ± 0.18. The
corresponding dilution factor for total
13C in the lactate pool was 2.14 ± 0.2.
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The ratios of the key isotopomers of apoB-100 alanine, aspartate, and glutamate are shown in Table 3. On average, the fractional abundance of [13C3]glutamate was 40.9 ± 4.2% of that of [13C3]aspartate, and the fractional abundance ratio of [13C3]aspartate was 31.2 ± 1.9% of that of [13C3]alanine. According to the calculations based on Eq. 15, the fractional abundance of [13C2]glutamate derived from acetyl-CoA was 0.524 ± 0.126 mole percent. This was 37 ± 6% of the total [13C2]glutamate. After taking into account the fractional abundance of [13C2] and [13C3] apoB-100 alanine, we calculated that 20.0 ± 1.6% of the hepatic acetyl-CoA pool had derived from three-carbon precursors via the pyruvate dehydrogenase reaction.
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Table 4 presents a comparison of the positional equilibration of OAA and the "TCA cycle dilution" factors as calculated from the glucose and apoB-100 amino acid data. The estimates of the proportion of the [13C3]OAA pool that was labeled in C-1 to C-3 were very similar (Katz estimate 0.59 ± 0.03, apoB-100 estimate 0.61 ± 0.04). The two methods gave estimates of the contribution of pyruvate carboxylase to the OAA flux (Katz method, 0.36 ± 0.03; apoB-100, 0.31 ± 0.01) that differed by 11% (P < 0.05). The final dilution factor derived from the two approaches differed by 14.6%.
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The calculated rates of gluconeogenesis are shown in Table 5. With the use of plasma lactate labeling as a common base for the estimation of the three-carbon dilution factor, the fractional gluconeogenic rate (70.6 ± 8.8%, Katz and Lee method; 80.3 ± 10.4%, apoB-100 method) differed by 14% (P < 0.05 by paired t-test). However, it should be noted that one of the values (animal 3) calculated by the apoB-100 method was greater by 100%. The fractional gluconeogenic rate determined with the Tayek and Katz method (29) using 13C recycling (81.3 ± 3.3%) was similar to that derived from the apoB-100 method. Strikingly though, the values calculated with the most recent Tayek and Katz method (30) gave values that were consistently >100%.
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Table 6 summarizes the calculations of the pathways of synthesis of the 13C1 and 13C3 isotopomers of glucose, lactate, and alanine. The results suggested that the large majority (82-91%) of the isotopomer distribution in [13C1]- and [13C3]glucose could be ascribed to gluconeogenesis via OAA and that, by difference, triose recycling made a minor contribution. Between 92 and 95% of the apoB-100 alanine derived from glycolysis, and as a consequence the recycling of PEP derived from OAA into the pyruvate responsible for the synthesis of apoB-100 alanine was negligible. A slightly (84-90%) but nonsignificantly lower proportion of plasma lactate derived from glycolysis. However, the hepatic free alanine pool was clearly of mixed origin, with 52-65% being derived from gluconeogenesis and 35-48% from glycolysis.
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DISCUSSION |
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Calculating Gluconeogenesis
The starting point for the experiment described in this report was the investigation of four different approaches to the determination of gluconeogenesis using [U-13C]glucose as the tracer. In all cases the calculated fractional gluconeogenic rate was high (>70), higher than the values previously reported after 16 (21) and 24 h (28) of fasting in adult humans. In one respect, given the high metabolic rate of young pigs, it might be expected that the rate of gluconeogenesis might also be high, but one approach to the calculation (30) yielded values that were consistently >100%, which is an irrational result. The question is, of course, whether the somewhat lower values given by the other approaches to the calculation are correct or whether all the methods consistently overestimated the rate of gluconeogenesis.Although the recently developed 2H2O method for the determination of the absolute rate of gluconeogenesis (21, 22) appears to offer promise for giving accurate estimates of the pathway, the source of the carbon used for gluconeogenesis and, in particular, the relative contributions of amino acid and glycerol carbon remain important questions. To answer these questions, carbon tracers are almost obligatory. Both [13C]lactate (2, 3, 18) and [13C]glycerol (25) can be used for the purpose of estimating gluconeogenesis. However, we are of the opinion that [U-13C]glucose is particularly useful, because, in addition to estimating the rate of gluconeogenesis, it allows the simultaneous measurement of the glucose Ra and allows investigations of other aspects of glucose metabolism. Nevertheless, the use of this tracer (or indeed, any tracer of the pyruvate pool) for the determination of gluconeogenesis requires that information on the absolute and positional labeling of OAA be obtained, and this has proved to be very difficult in vivo.
In 1985, Kelleher (19) proposed that one approach to both the targeted
introduction of carbon tracers into the TCA cycle and the determination
of the carbon labeling of the TCA cycle keto acids (OAA and -KG)
might be to use their transamination partners, aspartate and glutamate.
In this respect,
-KG and glutamate are particularly useful for the
purpose of modeling the TCA cycle, because different portions of the
-KG molecule derive from acetyl-CoA and OAA. After fragmentation of
glutamate or glutamine, the labeling and positions of labeling of each
moiety can be measured (7, 8, 10, 23) and used to calculate the
contribution of acetyl-CoA and OAA to
-KG labeling. If this approach
could be combined with a method for noninvasively sampling the hepatic
pool of
-KG, it should be possible to generate useful information
about the organization of the TCA cycle in vivo as well as determining
the necessary dilution factors for the calculation of gluconeogenesis (7, 10). To this point, the main method (23) proposed for this purpose
has been to administer phenylacetate and use measurements of urinary
phenyl acetyl-glutamine to probe the hepatic glutamine pool. This
method undoubtedly samples hepatic glutamine but requires the ingestion
of substantial quantities of phenylacetate, and there is no practicable
way of checking whether the drug has altered hepatic glutamine
metabolism.
The method that we have applied in the present work relies on the
following assumptions. First, the only mechanism whereby [13C3]glutamate
can be formed from
[U-13C]glucose is via
[13C3]C-2-C-4OAA.
Second, in a protein at isotopic steady state, the labeling of any
given amino acid defines, unequivocally, that of the protein synthetic
precursor pool from which it derived. Third, there is equilibrium
between the pools of pyruvate, OAA, and -KG involved in the TCA
cycle and gluconeogenesis and the pools of alanine, aspartate, and
glutamate-glutamine that become incorporated into rapidly turning over
hepatic protein apoB-100.
As far as we can ascertain, when [U-13C]glucose is the tracer, the only mechanism available to mammals to incorporate three labeled carbons into the four-carbon (i.e., the fumarate-malate-OAA-aspartate) pool is via pyruvate carboxylase. Thus we believe that the first assumption is sound. The second assumption, regarding the precursor-product relationship in a protein at isotopic steady state, must, by definition, also hold, although we should point out that it is critical to establish that a steady state exists for all isotopomers. In the present experiment this was easily achieved within a short period of time because the rate of protein turnover in piglets is very high (12). This kinetic requirement, however, poses a practical limitation on the method in humans. In our experience, unless the amino acid pool is deliberately overprimed (as in Ref. 24), at least 8 h are necessary to achieve isotopic steady state in VLDL apoB-100 (27), even in normolipidemic individuals. Thus, for this method to be applicable to studies in humans, prolonged tracer infusions must be used. The third assumption is, of course, crucial. Unfortunately it is also very difficult to test.
When the validity of the assumption regarding keto-amino acid
equilibration is examined, it is important to recognize that there are
two ways of viewing isotopic equilibriums in an experiment based on the
present techniques. The first is isotopomer equilibrium. This is
attained when the distribution of the
13C1,
13C2,
and
13C3
isotopomers in the precursor (e.g., pyruvate) and product (e.g., alanine) pools is the same. The second is isotopic equilibrium. This is
attained when both the relative and absolute isotopic enrichments of
each isotopomer in the precursor and product are the same. On the basis
of the close isotopomer and isotopic equilibrium among plasma, hepatic,
and apoB-100 glutamate, it seems very likely that there is an equally
good equilibrium between the glutamate-glutamine and -KG pools.
However, there is less surety with regard to alanine-pyruvate relationships. In the context of the present study, it is important to recognize that it is possible for there to be isotopomer equilibrium (i.e., the labeled precursor is the sole source of labeled product) but not isotopic equilibrium (i.e., there are other, unlabeled, sources of the product). Such a circumstance appears to apply to the alanine pools. Thus the isotopomer distribution in apoB-100 alanine, apoB-100 pyruvate, and apoB-100 lactate was virtually identical (i.e., the pools had attained isotopomer equilibrium), but all three isotopomers of apoB-100 alanine were less enriched with 13C than either plasma lactate or pyruvate (i.e., they had not attained isotopic equilibrium). This circumstance probably reflects the entry of unlabeled alanine from hepatic proteolysis. However, the free alanine in the hepatic pool was in neither isotopomer nor isotopic equilibrium with apoB-100 alanine pool, in as much as hepatic free [13C3]alanine had a lower fractional abundance than apoB-100 [13C3]alanine and the free alanine was more enriched with 13C1 and 13C2 isotopomers than the apoB-100 alanine. These observations suggest that there are two hepatic pyruvate pools that separately derive from glycolysis and gluconeogenesis and that these pools are metabolically separate. Labeled apoB-100 alanine derives specifically from pyruvate synthesized via glycolysis, rather than from pyruvate synthesized via OAA, whereas free alanine derives from both sources of pyruvate.
Not surprisingly, given previous observations (10), there was neither isotopomer nor isotopic equilibrium among the aspartate pools. However, we believe that the apoB-100 aspartate measures accurately the isotopomer distribution in the pool of OAA used for PEP synthesis. Our reasoning is as follows.
When
[13C3]pyruvate
is the source of
[13C]OAA, then a
precursor-product relationship exists between
[13C3]OAA
(as precursor) and
-[13C3]KG
(as product). Under equilibrium conditions, there should also be
a similar relationship between
[13C3]aspartate
and
[13C3]glutamate.
However,
[13C3]OAA
synthesized from
[13C3]pyruvate
is initially labeled in C-1 to C-3, and the production of
-[13C3]KG
occurs only from
[13C3]C-2-C-4OAA.
As the limit is an equimolar mixture of the two positional isomers of
[13C3]OAA,
then the fractional abundance of
[13C3]glutamate
should never be >50% of that of
[13C3]aspartate.
Thus it is important to note that the fractional abundances of hepatic
free
[13C3]aspartate
(0.29 mol percent) and glutamate (0.24 mol percent) were similar and
plasma
[13C3]glutamate
was threefold more enriched than plasma
[13C3]aspartate.
On the other hand, the fractional abundance apoB-100 [13C3]glutamate
was 41% that of apoB-100
[13C3]aspartate.
This is entirely compatible with incomplete but near equilibrium
between
[13C3]C-1-C-3
and
[13C3]C-2-C-4OAA.
Because the predicted isotopomer distribution in PEP derived from OAA
and the measured isotopomer distribution in
[13C1]-
and
[13C3]glucose
(see below) were also compatible with one another, we believe that
isotopomer equilibrium between the gluconeogenic pool of OAA and the
apoB-100 precursor pool of aspartate had been achieved.
However, despite the isotopomeric equilibrium, we believe that the absolute isotopic enrichment of apoB-100 aspartate underestimates that of the gluconeogenic OAA pool. On the assumptions 1) that the isotopomer distribution in apoB-100 aspartate reflects directly that of the gluconeogenic OAA pool precursor for glucose synthesis and 2) that the calculated positional equilibrium of [13C3]C-1-C-3OAA and [13C3]C-2-C-4OAA is correct, then the absolute and relative fractional abundances of [13C1]- and [13C3]glucose can be estimated. Because the synthesis of a glucose molecule requires the condensation of two OAA molecules, the absolute fractional abundances of the 13C1 and 13C3 isotopomers of glucose should be twice those of the PEP pool from which they derive. On the basis of the labeling of apoB-100 aspartate, the estimated fractional abundances of the PEP used for glucose synthesis were 1.0 ± 0.2, 0.58 ± 0.13, and 0.41 ± 0.05 mole percent for the 13C1 , 13C2, and 13C3 isotopomers, respectively. The fractional abundances of the 13C1, 13C2, and 13C3 isotopomers of glucose synthesized from this PEP would have been 2.0 ± 0.4, 1.2 ± 0.2, and 0.8 ± 0.1. However, the measured values (2.8 ± 0.4, 2.2 ± 0.4, and 1.6 ± 0.2 mol percent, for the 13C1, 13C2, and 13C3 isotopomers of glucose) were 45 ± 5, 52 ± 4, and 47 ± 2% higher than those predicted from apoB-100 aspartate. The similarity in the degree of underestimation of the labeling of each isotopomer suggests that there is isotopomeric equilibrium between the gluconeogenic pool of OAA and apoB-100 aspartate but that apoB-100 aspartate underestimates the absolute labeling of the OAA pool by ~50%. In this context, it is also of interest that the 13C dilution for the OAA pool as calculated by the method of Tayek and Katz (29) (1.6 ± 0.01) is 37% less than that measured from the 13C isotopic enrichment of apoB-100 alanine and aspartate (2.2 ± 0.13). If this conclusion is correct, then fractional gluconeogenesis lay between 45 and 60% when calculated by the methods in Refs. 16 and 29 as well as by the apoB-100 method.
We have no ready explanation as to why the method of Tayek and Katz (30), which appears to yield rational values in adults, led to calculated values of fractional gluconeogenesis in piglets of >100%.
Calculating Other Pathways of Glucose Metabolism
The calculation of gluconeogenesis using the labeling of the [M+2] and [M+3] isotopomers of glucose requires some other limiting assumptions. First, irrespective of whether [U-13C]glucose, -pyruvate, or -lactate is used as tracer, it is necessary to assume that the activity of pyruvate dehydrogenase in the liver is either zero or negligible. This assumption limits the application of the method to the fasted state and probably also to conditions in which insulin stimulation of glucose metabolism is negligible. This is unfortunate, because it hampers the use of the method in the fed state, a condition in which further information on gluconeogenesis would be invaluable, particularly with regard to interactions between protein and glucose metabolism (13), and especially in non-insulin-dependent diabetes (5). Second, although the synthesis of [M+3]glucose from [U-13C]glucose implies that [M+3]pyruvate must have been synthesized at some site in the body, these methods also necessarily assume that there is little or no recycling of [M+3]PEP (synthesized from OAA) via pyruvate kinase in the liver. Third, if the calculation of gluconeogenesis involves measurements of the recycling of the [M+3]isotopomer, it should also be recognized that cycling between labeled [U-13C]glucose via the triose phosphate pool will also lead to the synthesis of [13C3]glucose and appear as "gluconeogenesis." Indeed, the fact that labeled glycerol is an effective way of labeling circulating glucose (25, 26) implies that this pathway is possible. Finally, any method that involves data on the [M+1]isotopomer is vulnerable to the influence of 13CO2 fixation in the pyruvate carboxylase reaction and, possibly, at the isocitrate dehydrogenase step in the TCA cycle (7).One potential advantage of the apoB-100 method is that it relies only on data derived from the amino acid [M+3] isotopomers. Thus 13CO2 fixation (and hence the generation of excess enrichment in the [M+1] isotopomers) does not affect the calculation. Furthermore, because the apoB-100 method uses none of the information on the [M+2] isotopomers, it should be unaffected by the incorporation of [13C2]acetyl-CoA into citrate.
Katz et al. (17, 18) have presented equations that allow the potential effects of pyruvate dehydrogenase and PEP recycling to be estimated. This approach involves examining the impact of specific assumptions regarding the activities of the respective enzymes and does not measure them directly. We would propose, however, that irrespective of whether the absolute fractional abundances of the apoB-100 amino acids are direct reflections of the gluconeogenic precursor pool, data on the relative labeling of the [M+2] and [M+3] isotopomers of apoB-100 alanine, aspartate and glutamate, allow estimates of 1) the degree to which pyruvate contributes to the hepatic TCA cycle acetyl-CoA pool, 2) the degree to which PEP, derived via phosphoenolpyruvate carboxykinase, contributes to the three-carbon pool of the liver, and 3) the degree to which triose recycling contributes to the flux of [M+1]- and [M+3]glucose.
According to the calculations shown in Table 2 it appears that, under the conditions of the experiment (i.e., an 18- to 24-h fast), 20% of the acetyl-CoA in the hepatic TCA cycle had derived from pyruvate. The results of the calculations summarized in Table 6 suggest that plasma pyruvate and lactate, as well as labeled apoB-100 alanine, derived almost exclusively from glycolysis and that recycling of PEP derived from OAA into these pools was negligible. On the other hand, the results suggested that between 50 and 65% of the bulked hepatic free alanine pool derived from pyruvate that had, in its turn, been synthesized from the gluconeogenic pool of PEP. Finally, the relative labeling of [M+1]- and [M+3]glucose suggest that between 82 and 91% of glucose synthesis derived from pyruvate rather than from recycling from the triose phosphate pool.
The present results lead us to conclude that the heterogeneity of the hepatic OAA pool may be of sufficient magnitude that it is difficult to see how direct measurements of the absolute isotopic enrichment of the mitochondrial pool of OAA that acts as the gluconeogenic precursor can be made with confidence in vivo. This compartmentation presumably reflects the existence of three functional pools of OAA involved in the TCA cycle (intramitochondrial), gluconeogenesis (intra- and extramitochondrial), and ureagenesis (extramitochondrial). As a result, and contrary to our starting hypothesis, we conclude that the absolute isotopic enrichment of apoB-100 aspartate underestimates that of the gluconeogenic pool of OAA by ~50%. If so, gluconeogenesis via pyruvate contributed ~50% of total glucose entry. Nevertheless, we believe that the relative steady-state labeling of the 13C1,13C2, and 13C3 isotopomers of apoB-100 aspartate are good reflections of the relative enrichments of the same isotopomers of the gluconeogenic and TCA cycle pools of OAA. That being so, it appears that after a 24-h fast in the piglet, 1) the activity of hepatic pyruvate dehydrogenase is not negligible, 2) the large majority of hepatic gluconeogenesis derives from pyruvate, as opposed to glycerol, and 3) little PEP, derived from OAA, recycles to the hepatic pyruvate pool.
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ACKNOWLEDGEMENTS |
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We are grateful to L. Loddeke for sound editorial advice.
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FOOTNOTES |
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This work is a publication of the United States Department of Agriculture/Agriculture Research Service (USDA/ARS) Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, TX.
Funding has been provided in part from the USDA/ARS under Cooperative Agreement No. 58-6258-6100.
The contents of this publication do not necessarily reflect the views or policies of the USDA. Mention of trade names, commercial products, or organizations does not imply endorsement by the US government.
Present address of L. J. Wykes: School of Diabetics and Human Nutrition, McGill University, St. Anne de Bellevue, QC, Canada H9X 3V9.
Address for reprint requests: P. J. Reeds, USDA/ARS Children's Nutrition Research Center, 1100 Bates St., Houston, TX 77030.
Received 7 March 1997; accepted in final form 8 October 1997.
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