©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tracing Hepatic Gluconeogenesis Relative to Citric Acid Cycle Activity in Vitro and in Vivo
COMPARISONS IN THE USE OF [3-C]LACTATE, [2-C]ACETATE, AND alpha-KETO[3-C]ISOCAPROATE (*)

(Received for publication, September 30, 1994; and in revised form, November 16, 1994)

Michel Beylot (1) Maxim V. Soloviev (2) France David (2) Bernard R. Landau (2) (3) (4) Henri Brunengraber (2) (4)(§)

From the  (1)Institut National de la Santé et de la Recherche Médicale U197, Lyon 69008, France and the Departments of (2)Nutrition, (3)Medicine, and (4)Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The validity of the use of a carbon tracer for investigating liver intermediary metabolism in vivo requires that the labeling pattern of liver metabolites not be influenced by metabolism of the tracer in other tissues. To identify such specific tracer, livers from 48-h starved rats were perfused with recirculating buffer containing [3-C]lactate, [2-C]acetate, or alpha-keto[3-C]isocaproate. Conscious 48-h starved rats were infused with the same tracers for 5 h. The labeling patterns of liver glutamate and extracellular glucose were assayed by gas chromatography-mass spectrometry. In vivo data were corrected for CO(2) reincorporation into C-1 of glutamate and C-3 and C-4 of glucose, using data from control rats infused with NaHCO(3). With [3-C]lactate the labeling pattern of liver glutamate was the same in perfused organs and in vivo. In contrast, with [2-C]acetate and alpha-keto[3-C]isocaproate the labeling pattern of liver glutamate in vivo was clearly influenced by the expected labeling pattern of citric acid cycle intermediates formed in non-gluconeogenic organs, presumably glutamine made in muscle. Indeed, the labeling pattern of plasma glutamine and liver glutamate were similar in experiments with [3-C]lactate but different in experiments with [2-C]acetate and alpha-keto[3-C]isocaproate. Similar conclusions were drawn from the labeling patterns of glucose. Therefore, labeled lactate appears as the best tracer for studies of liver intermediary metabolism in vivo. Our data also show that a substantial fraction of alpha-ketoisocaproate metabolism occurs in peripheral tissues.


INTRODUCTION

Carbon-labeled tracers are used extensively to investigate various aspects of liver intermediary metabolism, in particular gluconeogenesis (GNG). (^1)Measurements of GNG are complicated by isotopic exchanges at the level of oxaloacetate (OAA), leading to underestimations of the rate of GNG calculated from the incorporation of labeled lactate, pyruvate, or alanine into glucose(1, 2, 3) . Various novel techniques, using acetate tracers, have been proposed to correct for this isotopic dilution(4, 5, 6, 7) . These techniques were used in a number of studies(8, 9, 10) , but their limitations were soon recognized for in vivo studies. First, labeled acetate is probably used mostly in peripheral tissues where it is oxidized to labeled CO(2)(11, 12) . The latter is carried by blood to the liver where it is incorporated into C3 and C4 of glucose. Indeed, some investigators calculate an index of GNG from the incorporation of labeled CO(2)(10, 13, 14, 15) . Second, in muscle, acetate metabolism may label glutamine, which is transported to the liver, where it can influence the labeling pattern of citric acid cycle (CAC) intermediates and of glucose(12) . Third, some models use the label of mitochondrial acetyl CoA estimated from the labeling of C-1 and C-2 of plasma acetoacetate or R-beta-hydroxybutyrate(5, 6) . This estimation is inaccurate especially in vivo because of (i) dilution of the labeling of acetoacetate in peripheral tissues via reversal of 3-oxoacid-CoA transferase, a process we called pseudoketogenesis(16, 17) , and (ii) heterogeneity of the labeling of liver mitochondria acetyl CoA, so that with some tracers, the labeling of acetyl CoA going to acetoacetate is different from that going to citrate(18) .

The problems raised with the utilization of labeled acetate could be prevented, at least in part, if one had a selective tracer for liver mitochondrial acetyl CoA. In conceiving the present study, we hypothesized that alpha-keto[3-C]isocaproate ([3-C]KIC) could be such a tracer. This hypothesis was based on the notion that KIC is formed from leucine in peripheral tissues and is degraded in liver(19) . In addition, we wanted to find out whether labeled lactate is metabolized in peripheral tissues to compounds that influence the labeling pattern of liver CAC intermediates. This question is important in view of the recent development of a noninvasive technique for determining the labeling pattern of liver CAC intermediates in humans(12, 20, 21, 22) . One can sample liver glutamine by its conjugation with exogenous or endogenous phenylacetate, forming phenylacetylglutamine (PAGN), which is excreted in urine(23) . It is assumed that the labeling pattern of the glutamine moiety of PAGN reflects that of liver alpha-ketoglutarate (alphaKG). This labeling pattern is used to calculate parameters of the CAC and the degree of isotopic dilution of gluconeogenic carbon at the OAA cross-road(12, 20, 21, 22) .

In the present study we compared the labeling patterns of liver glutamate and extracellular glucose labeled in vivo and in isolated organs from [3-C]lactate, [2-C]acetate, or [3-C]KIC. Our hypothesis was that a liver-specific tracer should yield the same labeling patterns in vivo and in perfused livers.


EXPERIMENTAL PROCEDURES

Materials

Chemicals were obtained from Sigma, and enzymes were from Boehringer Mannheim. Sodium [2-C]acetate, [3-C]lactate, [3-C]pyruvate, and NaHCO(3) (all 99% MPE) were from MSD isotopes. [C]Urea, NaB^2H(4), ^2H(2)O, and NaO^2H were from Isotec. [3-C]KIC (99%) was purchased from Tracer Technologies. 2-Hydroxy-[2,3,3-^2H(3)]isocaproate was prepared from KIC by exchange with ^2H(2)O and NaO^2H, followed by reduction with NaB^2H(4)(24) .

Liver Perfusions

Livers from 48-h starved Sprague-Dawley rats (200-220 g, Charles River Laboratories) were perfused (25) with recirculating Krebs Ringer bicarbonate buffer containing 4% dialyzed bovine serum albumin (fraction V, fatty acid poor, Miles) and 4 mM glucose, 1 mM lactate, 0.2 mM pyruvate, 0.2 mM KIC, and 0.2 mM acetate. Following orientation experiments, lactate, pyruvate, and KIC were infused into the reservoir at 8.0, 1.6, and 2.0 µmolbulletmin, respectively, to compensate for uptake. In perfusions with [2-C]acetate, the latter was infused at 1.25 µmolbulletmin. In some experiments, lactate and pyruvate were replaced by [3-C]lactate/[3-C]pyruvate (15%). In other experiments, acetate was replaced by [2-C]acetate, and KIC was replaced by [3-C]KIC (99%). Samples of perfusate were taken at regular intervals for analyses. After 2 h, the livers were freeze-clamped and kept in liquid N(2) until analysis.

In Vivo Experiments

Rats were housed in metabolic cages and fed chow ad libitum. After 1 week, they were anesthetized with pentobarbital and fitted with permanent catheters in a carotid artery and in the contralateral jugular vein. The catheters were kept patent by constant infusion of non-heparinized saline (0.25 mlbulleth each). The rats were used for experiments 4 days after surgery and after 2 days of fasting. Then they were infused intravenously in the conscious state for 5 h with isotonic solutions of [2-C]acetate (20 µmolbulletminbulletkg), [3-C]lactate (10 µmolbulletminbulletkg), [3-C]KIC (10 µmolbulletminbulletkg), or NaHCO(3) (20 µmolbulletminbulletkg). All infusions were preceded by a loading dose corresponding to 1 h of infusion. This loading dose was delivered over 6 min. Arterial blood was sampled at 3, 4, and 5 h. At 5 h, the rats were killed by a blow to the head. After laparotomy, the livers were freeze-clamped within 10-15 s of killing.

Analytical Procedures

Samples of perfusate and blood were deproteinated with perchloric acid (final concentration, 3%) and freeze-clamped. Concentrations of glucose, lactate, pyruvate, R-beta-hydroxybutyrate, acetoacetate, and acetate were assayed enzymatically on neutralized perchloric acid extracts of perfusate. The MPE of acetate was assayed by gas chromatography-mass spectrometry after conversion to acetyl difluoroaniline. (^2)Other samples of perfusate were treated with NaB^2H(4) to reduce KIC to 2-hydroxyisocaproate, whose concentration and C MPE were assayed by gas chromatography-mass spectrometry using an internal standard of 2-hydroxy-[2,3,3-^2H(3)]isocaproate(24) . The MPE of lactate(26) , pyruvate(26) , R-beta-hydroxybutyrate (27, 28) , CO(2)(29) , and urea (30) were measured by published methods. Neutralized perchloric acid extracts of liver were chromatographed on AG 1-X8-Cl (31) to isolate glutamine, glutamate, alphaKG, and PEP. The C-labeling pattern of glucose and glutamate isolated from liver perfusate and blood were determined as described by Beylot et al.(32, 33) . The total MPE of glutamine, alphaKG, and PEP were measured as trimethylsilyl or tert-butyl dimethylsilyl derivatives.

Calculations

In experiments with [3-C]lactate, citric acid cycle parameters and the ratio of MPE of lactate/PEP were calculated from the absolute labeling pattern of glutamate, using the equations of Magnusson et al.(20) . For in vivo experiments, calculations used the labeling patterns of liver glutamate corrected for CO(2) reincorporation on C-1(20) . Also, the MPE of portal vein lactate was calculated from the MPE of arterial lactate, using an MPE ratio (portal vein)/artery of 0.52. (^4)

In experiments with [2-C]acetate and [3-C]KIC, the ratio of enrichments of PEP/(mitochondrial acetyl CoA) was calculated using the glucose -labeling ratio, R = (average enrichment of C-1, C-2, C-5, and C-6)/(average enrichment of C-3 and C-4) and Katz equations(6) . Then, using the MPE of C-1 and C-2 of R-beta-hydroxybutyrate as reflecting that of mitochondrial acetyl CoA, we calculated the MPE of PEP. The MPE of PEP was also directly measured.


RESULTS

In orientation liver perfusion experiments, we determined the rates of substrate infusion into the recirculating perfusate that are necessary to maintain constant concentrations (see ``Experimental Procedures''). With this protocol, lactate/pyruvate concentrations ranged from 0.6 to 1.2 mM, whereas KIC and acetate concentrations remained between 0.2 and 0.3 mM. Glucose concentration increased from 4 to about 7 mM over 2 h. R-beta-Hydroxybutyrate/acetoacetate concentration increased to 2.8-3.0 mM over 2 h. The uptake of lactate plus pyruvate was 120-150 µmolbulletg, whereas the net accumulation of glucose was 54-78 µmolbulletg, corresponding to 75-100% of the net lactate plus pyruvate uptake. There was no significant difference in the above balance parameters between the three series of perfusions.

During infusion of labeled substrates, their MPE stabilized in the recirculating perfusate. The MPEs of [3-C]lactate/[3-C]pyruvate (infused at 15% MPE) stabilized from 60 to 120 min at 7.7 ± 0.5 and 10.3 ± 0.4%, respectively. Since the rate of [3-C]lactate infusion was 5 times that of [3-C]pyruvate infusion, the apparent rate of production of endogenous lactate + pyruvate was about equal to the rate of labeled substrate infusion, i.e. 9.6 µmolbulletmin. This dilution of infused [3-C]lactate and [3-C]pyruvate could result from glycolysis, isotopic exchanges, or both(34, 35, 36) . We showed previously that livers from 2-day starved rats perfused with 4 mM glucose release very little lactate(37) . Therefore, in the present experiments, we ascribe most of the dilution of infused [3-C]lactate to isotopic exchanges. Still, we cannot exclude some substrate cycling between glucose and pyruvate/lactate.

The MPEs of [2-C]acetate and [3-C]KIC (infused at 99%) stabilized at 50 ± 5 and 97 ± 2%, respectively. Thus, the rate of production of endogenous acetate, presumably from acetyl-CoA hydrolysis(38, 39, 40) , was about 1.25 µmolbulletmin. This confirms the coexistence of acetate uptake and production in perfused rat livers(41, 42) . The negligible dilution of the MPE of infused KIC is consistent with the low activity of branched-chain amino acid transaminase in the liver(19) .

In in vivo experiments with [3-C]lactate (99%) infused at 10 µmolbulletminbulletkg, the MPE of arterial plasma lactate stabilized at 13-18%. Thus, the apparent turnover of plasma lactate was 55-76 µmolbulletminbulletkg. This apparent turnover represents a combination of lactate production and isotopic exchanges (34, 35, 36) .

Fig. 1and Fig. 2show the labeling patterns (^3)of liver glutamate and glucose isolated from liver perfusate and from rat blood, expressed as relative enrichments for glutamate and labeling ratio, R, for glucose. In liver perfusion experiments, there was no detectable C enrichment in perfusate bicarbonate and urea. This results from the extensive gassing of the oxygenator with 95% O(2), 5% CO(2) (1 literbulletmin). Therefore, reincorporation of CO(2) via pyruvate carboxylase did not contribute to the labeling pattern of glucose and glutamate in liver perfusions. In contrast, in in vivo experiments, the MPE of urea was detectable in the final blood sample: 0.63 ± 0.11% (S.D., n = 8) from [3-C]lactate, 1.09 ± 0.27% (n = 6) from [2-C]acetate, and 0.38 ± 0.08% (n = 10) from [3-C]KIC. Therefore, it is likely that in these experiments, reincorporation of CO(2) contributed to the labeling of C-3 and C-4 of glucose and C-1 of glutamate. We therefore infused NaHCO(3) into a fourth series of catheterized rats and measured the MPE of urea (2.06 ± 0.7%, n = 6), C-1 of glutamate (0.50 ± 0.15%), and C-3 and C-4 of glucose (1.07 ± 0.39%, average of C-3 and C-4). Using equations developed by Magnusson et al.(20) , we corrected the labeling patterns of plasma glucose and liver glutamate for reincorporation of CO(2). Corrected values are also included in Fig. 1and Fig. 2.


Figure 1: Labeling pattern of liver glutamate in perfused organs and in vivo. Each panel refers to experiments with the indicated [C]substrate. Each set of three bars corresponds to experiments (i) in perfused livers (open bars), (ii) in vivo, with the labeling pattern as measured (hatched bars), and (iii) in vivo, with the labeling pattern corrected for CO(2) reincorporation on C-1 (solid bars). All labeling patterns are corrected for natural C enrichment. Data are presented as mean ± S.D. The number of experiments with [3-C]lactate, [2-C]acetate, and [3-C]KIC is 10, 6, and 9 in perfused livers and 7, 7, and 8 in in vivo, respectively. *, significantly different from liver perfusions (p < 0.05).




Figure 2: Labeling ratio of glucose. Other data are shown from the same experiments as in Fig. 1. R is defined as (average enrichment of C-1 + C-2 + C-5 + C-6)/(average enrichment of C-3 + C-4). Each set of three bars refers to experiments with the indicated [C]substrate and includes experiments (i) in perfused livers (open bars), (ii) in vivo, with the labeling pattern as measured (hatched bars), and (iii) in vivo, with the labeling pattern corrected for CO(2) reincorporation on C-3 and C-4 (solid bars). Statistical significance is by comparison with perfused liver data *, (p < 0.05);**, (p < 0.01).



In isolated livers perfused with [3-C]lactate/[3-C]pyruvate, most of the glutamate label is on C-2 and C-3, as shown previously(31, 32) . In contrast, with [2-C]acetate and [3-C]KIC, most of the glutamate label is on C-4. This was expected based on previous work with [2-C]acetate (31) and on the notion that [2-C]acetate and [3-C]KIC are both precursors of [2-C]acetyl-CoA, which labels mostly C-4 of glutamate.

In the presence of [3-C]lactate, the labeling patterns of glutamate are very similar in perfused livers and in vivo, except for higher labeling of C-1 in vivo. After correction for CO(2) reincorporation the labeling on C-1 is no longer different between isolated livers and in vivo livers. In contrast, in the presence of [2-C]acetate or [3-C]KIC, the labeling of glutamate is different for all carbons between isolated livers and in in vivo livers, before correction for CO(2) reincorporation. After such correction, labeling of C-2 to C-5 still remains different.

The data on glucose labeling pattern^3 were used to calculate the R labeling ratio in glucose (1) isolated from liver perfusate and rat plasma (Fig. 2). For plasma glucose, R is presented without and with correction for CO(2) reincorporation into C-3 and C-4 of glucose. R values differ between perfused liver and in vivo data uncorrected for CO(2) reincorporation. However, after such correction, R values in [3-C]lactate experiments are not significantly different in perfusion and in vivo experiments. Significant differences remain in [2-C]acetate and [3-C]KIC experiments.

Fig. 3shows comparisons between the total MPE of liver alphaKG, glutamate, and glutamine in isolated organs and in vivo. Fig. 3also includes the MPE of plasma glutamine. In isolated livers, the MPEs of alphaKG and glutamate are the same for all tracers. The MPE of glutamine is about one-fifth less than the MPE of glutamate. In vivo, the MPE of glutamate is less than that of alphaKG. The main difference between tracers is in the relative labeling of liver glutamate and plasma glutamine. With [3-C]lactate, plasma glutamine is significantly less labeled than liver glutamate. With [2-C]acetate and [3-C]KIC, the corresponding differences in MPE are not significant. In addition, in experiments with [2-C]acetate and [3-C]KIC, the plasma glutamine labeling ratios (C-1 to C-3)/(C-1 to C-5) were different from the corresponding liver glutamate ratios (0.66 ± 0.06 versus 0.57 ± 0.06 (n = 7, p < 0.05) for [2-C]acetate and 0.67 ± 0.05 versus 0.59 ± 0.03 (n = 7, p < 0.05) for [3-C]KIC). In contrast, in experiments with [3-C]lactate these ratios were not significantly different (0.87 ± 0.09 versus 0.91 ± 0.03, n = 8).


Figure 3: C Enrichment of liver alpha-ketoglutarate, glutamate, and glutamine in perfused organs (left panels) and in vivo (right panels). The labeled substrates infused were [3-C]lactate (top panels), [2-C]acetate (middle panels), and [3-C]KIC (bottom panels). The right panels (in vivo) also include the enrichment of plasma glutamine. *, significantly different (p < 0.05) from alphaKG (all panels) or from glutamate (upper right panel).



The absolute distributions of C in glutamate labeled in perfused livers and in vivo from [3-C]lactate were introduced into the equations of Magnusson et al.(20) to calculate rates shown in Table 1. These equations yield rates expressed relative to the CAC flux taken as 10. In perfused liver experiments, these relative rates were converted to absolute rates since (i) we measured the net rate of glucose accumulation, and (ii) the other rates of the Magnusson et al. model are linked to the rate of glucose production (V9). Note that the rate of lactate/pyruvate uptake calculated from the model, i.e. 1.26 µmolbulletminbulletg is very close to the actually measured rate of uptake, i.e. 1.20 µmolbulletming. Thus, in these livers perfused with lactate/pyruvate/KIC, the uptake of lactate/pyruvate is practically converted to glucose. Comparing the relative rates of reactions in perfused livers and in livers in vivo, the main differences are in the uptake of lactate/pyruvate and in fluxes related to gluconeogenesis. All other relative rates are almost identical. Note that in perfused livers, the concentration of pyruvate in the perfusate when the livers were freeze-clamped was 0.18 mM. This concentration is 3-4 times that of plasma pyruvate in starved rats. Since PC has a K(m) for pyruvate of about 0.4 mM(43) , GNG in perfused livers was clearly stimulated by substrate supply. This also explains the higher PC/PDH flux ratio in perfused livers compared to livers in vivo.



The calculated and measured MPE ratios, lactate/PEP (Table 1) are very similar in perfused livers and in vivo provided one takes into account the dilution of the MPE of portal vein lactate, compared with arterial lactate (see ``Experimental Procedures'').

Table 2compares the labelings of PEP and C-1, C-2, and C-3 of glutamate with one-half the MPE of glucose, in perfused livers and in vivo. The MPEs of PEP and of C-1, C-2, and C-3 of glutamate are very similar in perfused livers and in vivo, except in the presence of [2-C]acetate. The labeling of glucose in vivo is close to that of liver PEP. Note that in vivo, labeled substrates were infused for 5 h. Under similar conditions, the turnover rate of glucose is about 45 µmolbulletminbulletkg, corresponding to a half-life of extracellular glucose of about 15 min(44) . Thus, after 5 h, the initial pool of unlabeled extracellular glucose in live animals had been replaced by labeled glucose synthesized during the experiment.



In contrast, in isolated livers, the labeling of perfusate glucose was still lower than that of liver PEP. During the 2 h of the experiment, glucose concentration increased from 4 to 7 mM. Thus, the initial pool of unlabeled glucose was not entirely replaced by newly synthesized labeled glucose.

Data from experiments with [2-C]acetate and [3-C]KIC, i.e. the R glucose labeling ratios and the MPE of C-1 and C-2 of R-beta-hydroxybutyrate were used to calculate the MPE of PEP, using Katz equations(6) . In perfused livers, there was good agreement between the calculated and measured MPE (Table 2). In contrast, in in vivo experiments, the calculated and measured MPE of PEP were very different, although R ratios had been corrected for CO(2) reincorporation.


DISCUSSION

The main finding of this report is that the labeling pattern of liver glutamate is identical in the perfused liver and in the in vivo liver, when [3-C]lactate is the labeled substrate (Fig. 1). With [3-C]lactate, the glucose labeling ratio is also similar in perfused liver and in vivo (Fig. 2). These identities require correcting the labeling patterns of glutamate and glucose for CO(2) reincorporation (20) .

In contrast, with [2-C]acetate and [3-C]KIC, the labeling patterns of liver glutamate and extracellular glucose are very different in isolated livers from those in vivo, even after corrections for CO(2) reincorporation ( Fig. 1and Fig. 2).

In the liver, [2-C]acetate and [3-C]KIC yield [2-C]acetyl CoA, which labels C-4 of glutamate more than C-2 and C-3. This is because label on C-2 and C-3 leaves the CAC on the gluconeogenic pathway. In the starved liver, [3-C]lactate yields a labeling pattern of glutamate with enrichments C-2 > C-3 > C-4. This is because (i) [3-C]pyruvate derived from [3-C]lactate is both carboxylated to [3-C]OAA and decarboxylated to [2-C]acetyl CoA, (ii) the flux ratio PC/PDH is greater than unity, and (iii) the randomization of OAA label via reversible reactions catalyzed by malate dehydrogenase and fumarase is incomplete (31, 45, 46, 47) . In non-gluconeogenic tissues such as muscle, the three labeled substrates are converted to [2-C]acetyl CoA, which should, at steady state, label alphaKG, glutamate, and glutamine almost equally on C-2, C-3, and C-4. The randomization of label on C-2 and C-3 is complete because all of the label passes through symmetrical succinate.

Peripheral tissues can influence the labeling pattern of liver CAC intermediates by two mechanisms. First CO(2) formed in peripheral tissues is incorporated in liver into C-1 of alphaKG and glutamate and into C-3 and C-4 of glucose. This results from carboxylations catalyzed by PC and possibly from the reversal of PEP carboxykinase and malic enzyme. This incorporation of CO(2) is corrected using data from control experiments with labeled bicarbonate(20) .

Second, glutamine released by muscle is deamidated in liver to glutamate, which could influence the labeling pattern of CAC intermediates by both isotopic exchange and anaplerosis. Note that glutaminase is located in periportal hepatocytes, which are the main site of gluconeogenesis(48) . The extent to which muscle glutamine influences the labeling pattern of liver CAC intermediates depends on (i) the difference in total labeling of muscle and liver CAC intermediates, (ii) the difference in labeling pattern of muscle and liver CAC intermediates, (iii) the rate of glutamine flux from muscle to liver (this rate depends on the rate of protein catabolism and the nutritional status), (iv) the extent to which glutamine carbon is used as a gluconeogenic substrate in liver, and (v) the relative rates of label entry into liver CAC via glutamine on the one hand and acetyl CoA and/or OAA on the other hand. Based on these observations, a discussion of the observed labeling patterns of liver glutamate follows.

In the presence of [2-C]acetate, the relative labeling of C-2 and C-3 of glutamate is increased, and the relative labeling of C-4 is decreased in vivo compared with the perfused liver. This results from the superposition of liver type glutamate with C-2 = C-3 < C-4 and muscle type glutamate with C-2 = C-3 approx C-4. This is confirmed by the higher labeling ratio (C-1 to C-3)/(C-1 to C-5) of plasma glutamine than liver glutamate.

In the presence of [3-C]KIC, the labeling patterns of glutamate are almost identical to what was observed with [2-C]acetate. Also, the labeling ratio of plasma glutamine is higher than that of liver glutamate. This was surprising given the classical concept that KIC is formed in peripheral tissues and degraded in liver(19) . We were expecting that the relative labeling patterns of liver glutamate would be identical in perfused organs and in vivo. This is why we had [3-C]KIC synthesized as a potential specific tracer of liver intermediary metabolism. In fact, it is clear that a substantial fraction of [3-C]KIC must be metabolized in peripheral tissues, probably in muscle. This is consistent with recent studies of the state of activation of branched-chain ketoacid dehydrogenase (reviewed in (49) ). Although the activity of muscle branched-chain ketoacid dehydrogenase is low in rat muscle compared with liver(19) , the large amount of muscle compared with liver (30 versus 4% of body weight) can probably metabolize a large fraction of infused [3-C]KIC.

In the presence of [3-C]lactate, the identity of the liver glutamate labeling pattern in perfused organ and in vivo results probably from the much higher rate of direct entry of lactate label via OAA/acetyl CoA, compared with indirect entry via glutamine made in muscle. That this must be the case can also be deduced from examination of Fig. 3. When [3-C]lactate or [3-C]KIC is infused in vivo, the MPE of plasma glutamine is two-thirds that of liver alphaKG (Fig. 3, B and F). However, when [3-C]KIC is infused, muscle glutamine appears to have a greater influence on the labeling pattern of liver CAC intermediates than when [3-C]lactate is infused. Therefore, the direct influx of lactate label into liver CAC intermediates is greater than the direct influx of KIC label. Also, the labeling ratio (C-1 to C-3)/(C-1 to C-5) of plasma glutamine is similar to the corresponding ratio in liver glutamate. This is compatible with reports showing that anaplerosis of the CAC via PC occurs in skeletal muscle and heart (50, 51, 52, 53) . Metabolism of [3-C]pyruvate via PC increases the amount of label on C-2 and C-3 of muscle glutamine. Therefore, the relative labeling pattern of muscle glutamine and liver alphaKG should be very similar. Thus, the influence of muscle glutamine on the labeling pattern of liver CAC intermediates must be minimal.

The data on the labeling pattern of liver glutamate were computed using the model of Magnusson et al.(20) , which yields relative rates of CAC reactions. In a previous study (31) conducted in isolated rat livers perfused with non-recirculating buffer containing [3-C]lactate, we converted the relative rates calculated from the model to absolute rates, using the measured uptake of lactate/pyruvate. This yielded a rate of GNG that we could not verify by balance measurements since the difference in glucose concentration across the liver could not be measured with precision. In the present liver perfusion study, conducted with recirculating buffer, we could measure both lactate/pyruvate uptake and glucose accumulation. We converted the relative rates to absolute rates, using the measured glucose accumulation. Then the calculated rate of lactate/pyruvate uptake (1.26 µmolbulletminbulletg) was the same as the measured rate (1.20 µmolbulletminbulletg). Also, the MPE ratio lactate/PEP calculated from the model was practically the same as that measured in liver (Table 1, last two rows). These good agreements support the applicability of the Magnusson et al. model to estimate rates of GNG in liver. However, as we showed previously(31) , some rates are very sensitive to the amount of label on C-5 of glutamate, i.e. pyruvate kinase (V7), pyruvate carboxylase (V6), and PEP carboxykinase (V6). Since the absolute enrichment on C-5 of glutamate was very low (0.1-0.2%), we do not give much credence to these calculated rates (Table 2). However, we also showed (31) that the other rates of the model are not sensitive to variations in the enrichment of C-5 of glutamate. Therefore, we feel confident about them.

Our data have a bearing on the choice of tracer used to investigate liver intermediary metabolism in vivo. Labeled acetate has been used extensively in studies of GNG to quantitate isotopic exchange at the OAA crossroad(4, 5, 6, 7) . Various authors have warned that the bulk of the acetate tracer is used in peripheral tissues, which can influence the labeling pattern of glucose and of liver CAC intermediates(11, 12, 20) . Our data ( Fig. 1and Fig. 2; Table 2) confirm that labeled acetate is suitable for tracing liver intermediary metabolism in isolated organs but not in vivo. In addition, we show that KIC catabolism occurs to a large extent in peripheral tissues. Thus, [3-C]KIC is also unsuitable to trace liver intermediary metabolism in vivo. Last, we show that labeled lactate is the best tracer for studies of liver intermediary metabolism in vivo, at least in the fasting state. Whether or not labeled lactate is a suitable tracer during a short fast or in the fed state needs to be investigated.

The ^14C- and C-labeling patterns of liver CAC intermediates yield kinetics parameters of the CAC and GNG(31, 54, 55) , including rates of isotopic exchanges with other substrates such as aspartate and glutamate(7) . In experiments with ^14C tracers, this labeling pattern has been probed noninvasively in humans by degradation of urinary PAGN(12, 20, 21, 22) . In humans infused with [^14C]lactate(20) , the labeling pattern of the glutamine moiety of PAGN yielded relative rates of liver CAC reactions that are very similar to those we measured in live rats in the present study. This supports the use of labeled lactate and the PAGN probe in humans.

The probing of human and primate liver CAC intermediates with urinary PAGN can be extended to the use of stable isotopic tracers(56, 57) , particularly [C]lactate. This could yield useful information on metabolic disorders, such as inborn errors of pyruvate metabolism and diabetes.


FOOTNOTES

*
This work was supported by Grants DK35543 (to H. B.) and DK14507 (to B. R. L.) from the National Institutes of Health, the Nutrition Fund of the Cleveland Mt. Sinai Medical Center, and the North Atlantic Treaty Organization. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Nutrition, Mt. Sinai Medical Center, Cleveland, OH 44106-4198. Fax: 216-421-6661.

(^1)
The abbreviations used are: GNG, gluconeogenesis; alphaKG, alpha-ketoglutarate; CAC, citric acid cycle; KIC, alpha-ketoisocaproate; MPE, molar percent enrichment; OAA, oxaloacetate; PAGN, phenylacetylglutamine; PEP, phosphoenolpyruvate; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase.

(^2)
L. Powers, M. K. Osborn, C. L. Kien, R. D. Murray, M. Beylot, and H. Brunengraber, submitted for publication.

(^4)
Tables showing the absolute enrichments of each carbon of glucose and glutamate are available from the authors.

(^3)
V. Large, M. V. Soloviev, H. Brunengraber, and M. Beylot, Am. J. Physiol., in press.


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