EPILOGUE
Noninvasive tracing of human liver metabolism: comparison of phenylacetate and apoB-100 to sample glutamine

Frédérique Diraison1, Valérie Large1, Cyrille Maugeais2, Michel Krempf2, and Michel Beylot1

1 Institut National de la Santé et de la Recherche Médicale U499, Faculté Laennec, 69008 Lyon; and 2 Centre de Recherche en Nutrition Humaine, 44000 Nantes, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The labeling pattern of hepatic glutamine during infusion of [3-13C]lactate provides information on liver intermediary metabolism and allows us to correct apparent gluconeogenic rates for isotopic dilution in the oxaloacetate (OAA) pool. Liver glutamine can be sampled by its conjugation with phenylacetate to form phenylacetylglutamine (PAGN) but also by purifying the glutamine of the apolipoproteinB-100 of very low-density lipoprotein (apoB-100-VLDL). We compared these methods in normal and non-insulin dependent diabetes subjects. We tested also whether apoB-100-VLDL alanine enrichment could solve the problem of dilution of gluconeogenic precursor enrichments between peripheral blood and liver (prehepatic dilution). In both normal and diabetic subjects, the labeling patterns of glutamine obtained from PAGN or apoB-100-VLDL were comparable. Therefore, metabolic fluxes and correction factors for dilution in the OAA pool were also comparable. With both methods, gluconeogenic rates were not increased in diabetic patients. Use of the enrichment of apoB-100-VLDL alanine to correct for prehepatic dilution led to high estimates of gluconeogenesis; it remains uncertain whether this enrichment provides a correct estimate of liver pyruvate enrichment.

mass spectrometry; stable isotope; gluconeogenesis; chemical biopsy; diabetes


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE QUANTITATIVE contribution of gluconeogenesis (GNG) to the production of glucose in vivo, in both physiological and pathological situations, remains controversial, mainly because of methodological difficulties in this measurement. The recently developed method with deuterated water ingestion associated with deuterated glucose infusion could solve most of these problems (6, 20). However, methods that use tracers labeled with radioactive or stable carbon isotopes are also needed because they give, in addition to measurement of GNG, information on other intrahepatic metabolic pathways and on the turnover rates of gluconeogenic precursors. The quantitative measurement of GNG in vivo with such tracers is complicated by two main problems: isotopic exchanges in the hepatic oxaloacetate (OAA) pool (15, 18, 32) and dilution of the gluconeogenic precursor enrichment between peripheral plasma and liver (23, 29). For example, when lactate labeled on C-3 is infused, labeled carbons entering the OAA pool through pyruvate carboxylase (PC) can, instead of continuing the gluconeogenic pathway through phosphoenolpyruvate carboxykinase (PEPCK), follow the oxydative pathway in the citric acid cycle (CAC) and be lost as labeled CO2 and replaced by unlabeled carbons that can follow the gluconeogenic pathway. If not accounted for, this dilution of labeling leads to an underestimation of the production of glucose from the labeled precursors (15, 18, 32). This problem can be solved if one has access to the distribution of the label in one intermediate of the CAC during the infusion of labeled lactate (or pyruvate or alanine). One approach to obtain this information is the noninvasive chemical biopsy of liver glutamine developed by Magnusson et al. (25). During infusion of lactate labeled on C-3, this labeled carbon entering the CAC is distributed among the five carbon atoms of alpha -ketoglutarate (alpha -KG). This distribution depends on the relative activities of the main enzymes of the CAC and of the gluconeogenic pathway, particularly on the PC-over-pyruvate dehydrogenase activities ratio. Therefore, this labeling pattern can be used to calculate the relative fluxes through these enzymes and the dilution factor (F) of the label at the OAA crossroad with the equations of the model developed by Beylot et al. (5), Large et al. (22), and Magnusson et al. (25). Liver alpha -KG is not accessible in humans, but glutamine is synthesized in the liver from alpha -KG through glutamate without carbon rearrangements. Therefore, hepatic glutamine (and glutamate) carbons reflect those of alpha -KG. Liver glutamine can be noninvasively sampled by its conjugation with phenylacetate-forming phenylacetylglutamine (PAGN), which is excreted in urine (17, 36, 38). Phenylacetate can be safely ingested; its concentration can also be raised by the ingestion of Aspartam (N-L-alpha -aspartyl-phenylalanine-1-methyl ester; Searle, Boulogne, France), which contains phenylalanine, which has a metabolism that produces phenylacetate (38). Phenylacetate or Aspartam has been used to trace liver CAC activity and GNG in normal subjects (25) and in insulin-dependent (19) and non-insulin-dependent diabetic (NIDD) patients (9). Another possibility to noninvasively sample liver glutamine is to purify the apolipoprotein B-100 (apoB-100) of very low-density lipoproteins (VLDL) secreted by the liver (7, 16, 37). After apoB-100 is hydrolyzed and the amino acids are purified, the labeling pattern of hepatic glutamine can be determined. More exactly, because glutamine is converted during these procedures into glutamate, the labeling pattern obtained is that of a composite of hepatic glutamine and glutamate. We previously used Aspartam to sample liver glutamine in control and NIDD subjects (9). The first aim of the present study was to compare the results obtained with the Aspartam and the apoB-100 sampling methods used simultaneously in normal and diabetic subjects. Theoretically, similar estimates of CAC activity and GNG should be obtained with these two methods.

To solve the second problem, dilution of the enrichment of the gluconeogenic precursor between peripheral blood and liver (23, 29), we previously proposed to use plasma alanine isotopic enrichment (IE) as an estimate of intrahepatic pyruvate IE during the infusion of labeled lactate (9). This was based on the fact that pyruvate-alanine interconversion occurs in tissues, and therefore plasma alanine could be representative of liver pyruvate enrichment during infusion of labeled pyruvate or lactate. However, because pyruvate-alanine interconversion occurs in all tissues, this holds only if there is no significant heterogeneity in labeling between tissues. We found that this assumption of an homogenous intratissular enrichment was not verified in rats infused with labeled lactate (23). Although we had evidence that this tissue-labeling heterogeneity is less important in bigger species (23), this makes questionable the use of plasma alanine IE to estimate the liver pyruvate one. The second aim of the present study was to measure during infusion of labeled lactate the IE of apoB-100-VLDL alanine, which should specifically reflect that of intrahepatic alanine, and to test whether it can serve as a better estimate of liver pyruvate enrichment.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects. The study was approved by the local ethical committee and the Institut National de la Santé et de la Recherche Médicale. Informed written consent was obtained from six healthy volunteers (2 men, 4 women, aged 20-47 yr, body mass index: 21-25 kg/m2), and four NIDD patients (4 men, aged 34-50 yr, body mass index: 27-30 kg/m2). The duration of diabetes was 4-8 yr; hemoglobin A1c levels ranged from 9.5 to 11.0% (normal values <6.0%). No control subject had a personal or familial history of diabetes or obesity or was taking any medication. All consumed a weight-maintaining diet with at least 200 g carbohydrate and had abstained from alcohol and heavy physical activity the week before the studies. The diabetic patients were treated by diet alone (3 subjects) or diet and metformin (1 subject). The last 3 days before the tests metformin was interrupted and the diabetic subjects consumed a weight-maintaining diet. The last meal the day before the tests was ingested between 1900 and 2000.

Materials. Tracers were from Eurisotop (Saint Aubin, France; [6,6-2H2]glucose, NaH13CO3, [5,5,5-2H3]leucine), or Mass Trace (Woburn, MA; L-[3-13C]lactate). Reagents and enzymes were from Sigma (St. Louis, MO) or Boehringer Mannheim (Mannheim, Germany).

Protocols. All tests were performed in the Centre de Recherche de Nutrition Humaine of Lyon. Subjects were overnight fasted. At 0730, indwelling catheters were threaded into a forearm vein for tracer infusion and into a dorsal vein of the opposite hand kept at 55°C to collect arterialized blood. Two studies were performed. In the first study (study 1), four healthy subjects and four NIDD patients were studied twice, once with infusions of L-[3-13C]lactate and D-[6,6-2H2]glucose (test 1) and once with an infusion of NaH13CO3 (test 2), with at least 1 wk between the tests. During test 1, after initial blood sampling, primed-continuous infusions of D-[6,6-2H2]glucose (3 mg/kg, 0.02-0.03 mg · kg-1 · min-1 during 150 min) and L-[3-13C]lactate (10 µmol/kg, 0.60-0.77 µmol · kg-1 · min-1 during 360 min) were initiated. Blood was collected at 60, 120, 130, 140, 150, 240, 300, 320, 340, and 360 min. At 60 min, the subjects consumed Aspartam (1 mg/kg). Urine was collected at the end of the test. During test 2, NaH13CO3 (0.75 µmol · kg-1 · min-1) was infused for 360 min. Blood and expired gas samples were collected before tracer infusion and at 240, 300, 320, 340, and 360 min. The subjects also consumed Aspartam at 60 min, and urine was collected at the end of the test.

In the second study (study 2), five healthy subjects (including 3 from the first study) were infused with [5,5,5-2H3]leucine (0.5 µmol/kg, followed by 0.13 µmol · kg-1 · min-1 during 600 min). Blood was sampled before tracer infusion and each hour until the end of the test.

Analytical methods. Concentrations of glucose and lactate were assayed enzymatically (4) on neutralized perchloric acid extracts of blood. Plasma insulin and glucagon levels were measured by radioimmunoassay (13, 14). The t-butyldimethylsilyl and the quinoxalinol t-butyldimethylsilyl derivatives of plasma lactate and pyruvate, respectively, were prepared, and the 13C enrichments [IE, expressed as molar percent excess (MPE)] of lactate [mass-to-charge ratio (m/z) 261] and pyruvate (m/z 217) were measured by gas chromatography-mass spectrometry (GC-MS) as described (23). Plasma alanine was purified by cation-exchange chromatography (AG50 WX4 H+ form; Bio-Rad, Richmond, CA), and the t-butyldimethylsilyl derivative was prepared for IE measurements (m/z 260) (31). Plasma urea was purified by sequential anion-cation exchange chromatography, and its IE was determined as previously described (2). Plasma glucose was purified by ion-exchange chromatography. Its 13C IE was determined for test 2 by gas chromatography-isotope ratio mass spectrometry (34) (Sira12, Vg ISOGAS, Middlewitch, UK). For test 1, deuterium enrichment during the 120- to 150-min period was determined with either the ions of m/z 217 and 219 (containing C-4 to C-6 of glucose) of the aldonitrile pentaacetate derivative or the ions m/z 117 and 119 and m/z 205 and 207 (C-5 and C-6) of the methyloxime trimethylsilyl derivative (3). Corrections for the additional increases in m + 2 induced by 13C incorporation into glucose from the infused labeled lactate were performed with appropriate standard curves (28). Total 13C enrichment of glucose at the end of the test (300-360 min) was measured as described (3) with the bisbutylboronate-acetate or the pentaacetate derivative of glucose. Urinary PAGN was purified and hydrolyzed (38); the labeling pattern of its glutamine moiety, converted into glutamate during the preparation of the sample (38), was determined by GC-MS as previously described (1). 13C enrichment of CO2 in expired gas was measured by gas chromatography-isotope ratio mass spectrometry (12). VLDL were separated by ultracentrifugation from plasma collected at the end (time: 360 min) of the two tests of study 1 and at each sampling time of study 2. A 2.8-ml vol of plasma was mixed with 1.9 ml of 1.006 g/l solution of NaCl in EDTA. VLDL were isolated by a 4-h centrifugation at 100,000 g at 4°C (TLA 100.4 rotor and Optima TL, Beckman ultracentrifuge). The upper fraction containing VLDL was collected by aspiration and stored at -20°C until further analysis. Apolipoproteins were concentrated, and apoB-100 was isolated from other apolipoproteins by SDS-PAGE with a 4:5:10% discontinuous gradient. ApoB-100 bands were excised from polyacrylamide gels and dried under N2. The dessicated gel slices were hydrolyzed with 1 ml of 12 N HCl at 110°C for 24 h. This procedure converts all glutamine into glutamate. Hydrolysates were evaporated to dryness and then diluted in 1 ml of acetic acid. For samples collected during study 2 (deuterated leucine infusion), amino acids were purified by cation-exchange chromatography (AG50 WX4 H+ form; Bio-Rad). The IE of leucine from apoB-100 was determined with the t-butyldimethylsilyl derivative monitoring the ions with the m/z ratios 200 and 203 (31). For samples collected at the end of the tests of study 1, amino acids were first loaded on a anion-exchange column (AG1X8 formate; Bio-Rad) and eluted with water, except for aspartate and glutamate. Glutamate was then eluted with 40 mM HCl. After concentration, the neutral eluate, which contains the other amino acids, was run on a cation-exchange chromatography column (AG50 WX4 H+ form) and amino acids were eluted with 20% NH4OH. After the t-butyldimethylsilyl was dried, derivatives (31) were prepared for determination of the IE of alanine from apoB. The eluate containing glutamate was dried before preparation of the dimethylaminomethylene methyl derivative for determination of glutamate-labeling pattern (1). All GC-MS analysis were performed with a gas chromatograph (HP5890, Hewlett-Packard, Palo Alto, CA) equipped with a 25-m fused silica capillary column (OV1701, Chrompack, Bridgewater, NJ) and interfaced with a HP5971A mass spectrometer (Hewlett-Packard) working in the electron impact mode. Carrier gas was helium. Standard curves prepared by mixing weighted amounts of natural and 2H- or 13C-labeled metabolites were run before and after the corresponding biological samples (all in triplicate). Special care was taken to have comparable peak areas (i.e., <20% difference) between the standard and biological samples, adjusting when necessary the split ratio or the volume injected (26).

Calculations. In study 1, glucose turnover rate (Rt) was calculated from its deuterium IE during the 120- to 150-min period with equations for steady state. Lactate Rt was calculated with either [13C]lactate or [13C]pyruvate enrichments during the 300- to 360-min period with steady state equations. The labeling patterns of glutamate isolated from urinary PAGN and apoB-100-VLDL during test 1 were corrected for the reincorporation of labeled carbon in position 1 from the fixation of 13CO2 by PC as described by Magnusson et al. (25). CAC parameters and the F at the OAA crossroad were calculated from the corrected labeling patterns of glutamate isolated from urinary PAGN and apoB-100-VLDL with the equations of Magnusson et al. (25) and the model shown in Fig. 1. The equations of the model yield rates expressed relative to citrate synthesis [or CAC activity (V3)]. GNG was first calculated from the ratio of one-half of glucose IE to either plasma lactate, pyruvate, plasma alanine, or apoB-100-VLDL alanine IE. These contributions, calculated as percentages of GNG to endogenous glucose production (EGP) (GNG%), were corrected by F, and the corresponding absolute gluconeogenic rates were calculated as GNG = GNG% × F × glucose Rt. Twice this rate corresponds to the rate of phosphoenolpyruvate to glucose (V9) in the model of Magnusson. Once absolute values for V9 were calculated, all the relatives fluxes could then be converted into absolute values.


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Fig. 1.   Model of tricarboxylic acid cycle and gluconeogenesis. PEP, phosphoenolpyruvate; K1-K5 refer to 5 carbon atoms of glutamine; V1-V9 refer to rate of flux in direction of arrow.

We measured the IE of plasma and apoB-100-VLDL alanine to determine whether both of them could be used to estimate its intrahepatic enrichment or whether plasma alanine was inappropriate. However, a 6-h infusion of labeled lactate could be not sufficient to obtain the plateau enrichment in apoB-100-VLDL, because in most studies with labeled amino acids this plateau is obtained only after 8-10 h of infusion (7, 24). The comparison of the IE of apoB-100-VLDL leucine at 6 h with the enrichment at plateau during study 2 was used to correct for this underestimate of intrahepatic alanine enrichment. GNG and CAC rates were then recalculated with this corrected value of apoB-100-VLDL alanine enrichment.

Statistical analysis. Results are shown as average ± SE. Within and between-group comparisons were performed with Student's t-test for paired or nonpaired values.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hormones and metabolites concentrations. The concentrations of plasma insulin, glucagon, glucose, and lactate during study 1 are shown in Table 1. Diabetic patients had significantly higher glucose and lactate concentrations than normal subjects. There were no significant differences in insulin and glucagon levels between the two groups despite a trend for raised insulinemia in diabetic subjects.

                              
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Table 1.   Hormone and metabolite concentrations

Glucose and lactate turnover rates. Glucose Rt was increased in diabetic patients compared with control subjects (15.57 ± 1.1 and 12.57 ± 0.37 µmol · kg-1 · min-1, respectively, P < 0.05). Lactate Rt was calculated with either lactate or pyruvate IE. Whatever the IE chosen, lactate Rt was significantly higher in diabetic compared with normal subjects (15.5 ± 0.68 and 9.86 ± 0.95 µmol · kg-1 · min-1, respectively, calculated with lactate IE, P < 0.01; and 16.83 ± 1.02 and 12.4 ± 0.81 µmol · kg-1 · min-1, respectively, with pyruvate IE, P < 0.05). Plasma lactate, pyruvate, glucose, alanine, and apoB-100-VLDL alanine IE are shown in Table 2. Only apoB-100-VLDL alanine IE were not significantly different between the two groups.

                              
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Table 2.   13C-isotopic enrichments (MPE) of plasma lactate, pyruvate, glucose, alanine, and apoB-100-VLDL alanine

Labeling patterns and metabolic fluxes. In Fig. 2, we compared (Student's t test for paired values for each individual carbon), in the two groups of subjects, the corrected 13C-labeling patterns of glutamine from urinary PAGN with those of glutamine-glutamate obtained from hydrolyzed apoB-100-VLDL. For both groups, there were no differences in the labeling patterns obtained with either PAGN or apoB-100-VLDL. However, for both control and diabetic subjects the total labeling was lower in apoB-100-VLDL glutamine-glutamate than in glutamine from PAGN (controls: 1.64 ± 0.04 vs. 1.88 ± 0.08%; diabetics: 1.06 ± 0.02 vs. 1.16 ± 0.07%; P < 0.05 for both). With the use of the labeling patterns obtained with the two methods, we calculated the corresponding metabolic fluxes, expressed relative to citrate synthesis, which is arbitrary fixed to 10 (Table 3). As expected, because the labeling patterns obtained with these two methods were not different, the relatives fluxes were comparable. Nevertheless, with the labeling patterns obtained from apoB, diabetic patients had, compared with control subjects, a significant decrease in rates from fatty acids to acetyl-CoA (V2), the flux from lactate to pyruvate (V8), and V9 and an increase in the rate of pyruvate to acetyl-CoA (V1). With the use of PAGN method only V8 and V9 were decreased in diabetic subjects. All the other rates were nearly identical. The PC-over-pyruvate dehydrogenase activity ratios were not different either between the two groups, or when the two methods were compared in each group. The intrahepatic F were also comparable between the two groups and between the two methods.


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Fig. 2.   Labeling patterns of glutamine obtained in control subjects (A) and in diabetic patients (B) with phenylacetylglutamine (hatched bars) and apolipoproteinB-100 (apoB-100)-very low-density lipoprotein (VLDL; solid bars). K1-K5 refer to 5 carbon atoms of glutamine. Enrichment for each carbon is expressed in atom percent excess (APE).


                              
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Table 3.   Metabolic fluxes expressed relative to citrate synthesis

The apparent gluconeogenic rates calculated from one-half of the 13C enrichment of glucose and the enrichment of lactate, pyruvate, and plasma alanine are shown on Table 4. Whatever the precursor used, the uncorrected fractional gluconeogenic rate was lower in diabetic patients (P < 0.05). The corrected fractional contribution of GNG to EGP was calculated with for each group the correction factors F calculated from the labeling patterns obtained with the two different biopsy methods. These contributions were always significantly lower in diabetic subjects whatever the precursor enrichment and the correction factor used. We also calculated the uncorrected fractional gluconeogenic rates, using as enrichment of the precursor, the IE of apoB-100-VLDL alanine (Table 4). The values obtained were much higher than those obtained with the other precursors IE and were twice as high in controls than diabetic subjects (P < 0.01). To verify whether apoB-100-VLDL alanine enrichment in study 1 reached the plateau level or not at the end of the protocol (6 h), we measured in study 2 the kinetic of the incorporation of deuterated leucine in apoB-100-VLDL during a 10-h infusion in control subjects of [5,5,5,-2H3]leucine. Figure 3 shows that IE plateau level in apoB-100-VLDL leucine was achieved only after 8 h. Therefore, in study 1, a 6-h infusion was not sufficient to reach a plateau level in apoB-100-VLDL alanine IE, and the fractional gluconeogenic rates calculated during this study with these enrichments were overestimated. To determine the correct plateau level of IE in apoB-100-VLDL alanine, we used an additional correction factor. This factor was calculated by comparing, during study 2, the IE in apoB-100-VLDL leucine at 6 h with the plateau level attained from 8 to 10 h and was roughly 1.32. Fractional gluconeogenic rates were then calculated, using as precursor IE, the apoB-100-VLDL alanine enrichment corrected with this factor (last line of Table 4).

                              
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Table 4.   Contribution of gluconeogenesis to EGP



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Fig. 3.   Evolution in normal subjects of the enrichment (expressed as molar percent excess, MPE) of apoB-100-VLDL leucine during 10-h infusion of deuterated leucine.

We calculated then, from the measured EGP rates and these last corrected fractional gluconeogenic rates, the absolute gluconeogenic rates. Then all the relative rates of Table 3 were converted into absolute fluxes (Table 5). In both control and diabetic groups, the values obtained with the two methods were nearly identical. When expressed in moles per kilogram per minute, whatever the method chosen to sample hepatic glutamine (i.e., PAGN or apoB-100-VLDL), in addition to V9, V8 was lower in diabetic subjects. All the other rates were similar in the two groups. When absolute values of metabolic fluxes were expressed as moles per minute, the difference in EGP between diabetic and control subjects was magnified (1,191 ± 35 vs. 813 ± 34 mol/min, P < 0.01), but the gluconeogenic rates (with PAGN, 479 ± 69 vs. 623 ± 34 mol/min; with apoB-100-VLDL, 466 ± 75 vs. 633 ± 36 mol/min), as well as the rates V8 and V9, were no longer significantly different.

                              
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Table 5.   Absolute values of metabolic fluxes and gluconeogenesis calculated with corrected alanine-apoB IE


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The first aim of this study was to compare two different methods for the in vivo noninvasive sampling of hepatic glutamine: conjugation of phenylacetate with liver glutamine and purification of glutamine and glutamate from apoB-100-VLDL. This was performed in normal and NIDD subjects infused in the postabsorptive state with [3-13C]lactate. Phenylacetate, or its precursor Aspartam, has been already used in humans (9, 19, 25). A potential problem with phenylacetate is that the conjugation of phenylacetyl-CoA with glutamine takes place in perivenous hepatocytes, which have glutamine synthase activity, whereas GNG occurs in periportal hepatocytes, which lack glutamine synthase. Therefore, the glutamine pool sampled could be not representative of the alpha -KG and OAA pool used for GNG. However, the direct comparison of the labeling pattern of the glutamine moiety of urinary PAGN with that of glutamine, glutamate, and alpha -KG in livers of monkeys infused with various tracers supported the validity of this approach (39). In addition, the comparison in humans infused with [3-14C]lactate of the labeling of glucose and of the glutamine part of PAGN showed that they were apparently formed from a common pool of OAA (25). It is conceivable that labeled glutamate formed in periportal hepatocytes was released and taken up by perivenous cells and converted into glutamine. In both control and diabetic subjects, the total labeling of the glutamine sampled was slightly lower when apoB-100-VLDL was used than when phenylacetate was used, but the labeling patterns obtained with the two methods were comparable, showing that they sampled a common, or identical, pool of glutamate and glutamine. Although there were some slight differences when the metabolic fluxes calculated with the two methods were compared, these two methods gave overall comparable results for both liver CAC activity and GNG in both groups of subjects. These results validate the use of the apoB-100-VLDL method to realize the noninvasive biopsy of hepatic glutamine in vivo in humans. They agree with the recent demonstration of a close identity in the labeling of hepatic and apoB-100-VLDL glutamate in piglets infused with [U-13C]glucose (37). Therefore, we think that both methods can be used in human beings. Compared with the PAGN method, the use of apoB-100-VLDL has the advantage that no administration of any xenobiotic is required. However, theoretically, the endogenous production of PAGN should be sufficient to allow its use without ingestion of exogenous phenylacetate or Aspartam (38). The preparation of the samples is more tedious and time consuming with the apoB-100-VLDL approach because it requires ultracentrifugation to separate VLDL from other plasma lipoproteins and gel electrophoresis to purify apoB-100 before its hydrolysis and the purification of amino acids. In the present study, the lower total labeling in apoB-100-VLDL than in PAGN made the determination of the labeling pattern somewhat more difficult. This difference in total labeling is probably related to the small size of the circulating pool of PAGN (38), which was quickly replaced after Aspartam ingestion by PAGN synthesized in the presence of labeled hepatic glutamine. The circulating pool of apoB-100-VLDL is more important and, as discussed below, was probably not fully replaced during the 6-h infusion of [3-13C]lactate by newly synthesized and secreted apolipoprotein. Lastly, whatever the sampling method used, some potential limitations of the model used (25) should be kept in mind. In particular it is assumed that the fluxes through PEPCK and PC are equal and that there are no significant carbon outputs from the CAC other than CO2 production and GNG nor other significant carbon inputs than through PC and acetyl-CoA. An intake of unlabeled glutamate or aspartate would not change the relative distribution of label between the different carbon atoms but could result in a decrease of the total labeling not accounted for by the model used for the calculation of F.

An advantage of the apoB-100-VLDL method is that it allows to sample other hepatic amino acid pools. Wykes et al. (37) used this possibility to sample liver aspartate and alanine, in addition to glutamate-glutamine, during infusion of [U-13C]glucose in piglets. We reasoned that apoB-100-VLDL alanine IE could give a close estimate of the enrichment of liver pyruvate used for GNG and therefore could better solve the problem of the dilution of the precursor IE between peripheral circulation and liver (23, 29) than plasma alanine IE (9). The use of apoB-100-VLDL alanine IE relies on the following assumptions: first, there is, at the sampling time, an isotopic equilibrium between alanine in apoB-100-VLDL and the hepatic amino acids pool from which it derives; second, there is an equilibrium between liver alanine and the pyruvate pool from which it derives; and third, the hepatic pyruvate pool is homogenous or at least apoB-100-VLDL alanine derives from the same pyruvate pool than the one used for GNG. During the infusion of labeled leucine in normal subjects, a plateau level of apoB-100-VLDL leucine was obtained in 6 h in some studies (10, 35) but not in all of them (7, 24). These results, and the comparison of the total labeling in glutamine from PAGN and in the apoB-100-VLDL glutamine-glutamate, made the first assumption uncertain. Actually, when we infused normal subjects, studied in the same conditions, with deuterated leucine, the apoB-100-VLDL leucine at 6 h was only ~75% of the plateau value, therefore confirming that a more prolonged infusion of labeled lactate would have been necessary to satisfy the first assumption. Therefore, we corrected the measured apoB-100-VLDL alanine IE for the difference between the plateau and the 6-h enrichments determined during the labeled leucine infusion test. We used the same correction in control and diabetic subjects. However, studies of apoB-100-VLDL kinetics in NIDDM patients showed that the plateau level in apoB-100-VLDL is obtained later than in control subjects (10). Thus the correction for apoB-100-VLDL alanine IE was probably underestimated in diabetic patients. The second assumption is supported by our previous finding in isolated rat liver perfused with [3-13C]lactate of an identity in the IE of alanine in the effluent and of liver pyruvate (22). However, we cannot exclude during the present in vivo studies the possibility that liver alanine IE was diluted by unlabeled alanine produced by hepatic proteolysis. The third assumption appears questionable because Wykes et al. (37) found, in piglets infused with [U-13C]glucose, that although apoB-100-VLDL alanine and hepatic alanine had identical total 13C enrichment, their isotopomer distributions were quite different. They interpreted their data as showing that labeled apoB-100-VLDL alanine derives specifically from pyruvate synthesized from glycolysis rather than from pyruvate synthesized via OAA and PEP. Thus apoB-100-VLDL alanine could in our present experiments not be truly representative of the hepatic pyruvate pool used for GNG. Further experiments allowing a direct comparison of liver alanine and pyruvate enrichments and label distributions with those of apoB-100-alanine in animals infused with [3-13C]lactate are necessary to solve this point. Before these experiments are performed, we think that plasma and apoB-100-VLDL alanine IE should be considered as giving the upper and lower estimates, respectively, of liver pyruvate IE and therefore the lower and upper estimates of GNG.

With the use of these estimates, we calculated that GNG represents 44-77% of EGP in control subjects studied after 14-18 h of fasting. These limits correspond to the range of GNG previously reported in the literature by authors with nuclear magnetic resonance (30), other tracer methods (6, 20, 21, 33), or a combination of tracer and indirect calorimetry (11). The most accurate estimates are probably those obtained with deuterated water ingestion and the ratio of deuterium enrichments on C-5 and C-2 of glucose (6, 20). These estimates are 48-50% after 14-16 h of fasting, thus much closer to our low limit (44%) than to the high one (77%). They further suggest that apoB-100-VLDL alanine may not be a good estimate of the liver pyruvate pool used for GNG. The contribution of GNG to EGP was only 27 to 40% in NIDD patients. As discussed previously (correction of the apoB-100-alanine IE), it is quite possible that this upper limit was overestimated in diabetic patients. Some (8, 27) but not all (33, 40) previous studies found an increase of the fractional contribution of GNG to EGP in postabsorptive NIDD patients. The increased contribution of GNG found by Consoli et al. (8) is questionable because of the various methodological problems previously discussed (9). The reason for the discrepancy between the results of Periello et al. (27) and the present ones remains unclear. It should be kept in mind that neither phenylacetate nor apoB-100-VLDL allow sampling of glutamine in human kidneys and to assess their intermediary metabolism. Kidneys contribute to GNG in normal subjects, and this contribution could be higher in diabetic patients. Therefore, we may have underestimated GNG somewhat in the present study, and this underestimation could be more important in diabetic subjects. We found that the absolute gluconeogenic flux was also decreased in diabetic patients when expressed relative to body weight. However, this difference disappeared when the gluconeogenic flux was expressed per minute. We think that this last way for expressing GNG is more appropriate because the increase in body weight of diabetic patients is mainly due to an increase in glucose utilizing tissues (i.e., muscles and adipose tissue). There is no evidence for an increase in the mass of glucose producing tissues in non-insulin-dependent diabetic patients.

In conclusion, we found that the phenylacetate and the apoB-100-VLDL methods for the noninvasive sampling of liver glutamine during in vivo studies of hepatic CAC activity and GNG provide comparable results. This was observed in both normal and diabetic subjects. Both methods can be used dependent on the available equipment or individual choice, although we would recommend a longer period of labeled lactate infusion if one wants to use the apoB-100-VLDL approach. More experimental studies, particularly in animals, are necessary to determine whether plasma or apoB-100-VLDL alanine gives a correct estimate of hepatic pyruvate IE and could be used to solve for the prehepatic dilution of the tracer.


    ACKNOWLEDGEMENTS

We wish to thank the nurses for help in performing the tests and all the subjects who volunteered for the study.


    FOOTNOTES

This work was supported in part by grants from the Association Franaise des Diabetiques, the Association de Langue Française pour l'Etude du diabète et des maladies métaboliques, and the Fondation de France.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. Beylot, Institut National de la Santé et de la Recherche Médicale U499, Faculté Laennec, Rue G. Paradin, 69008, Lyon, France (E-mail: beylot{at}laennec.univ-lyon1.fr).

Received 24 November 1998; accepted in final form 29 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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