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 |
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 |
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
-ketoglutarate (
-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
-KG is not accessible in humans, but glutamine is
synthesized in the liver from
-KG through glutamate without carbon
rearrangements. Therefore, hepatic glutamine (and glutamate) carbons
reflect those of
-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-
-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 |
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 |
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.
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.
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).
|
|
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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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.
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|
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.
 |
DISCUSSION |
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
-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
-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.
 |
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