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Department of Nutrition, Case Western Reserve University, Cleveland, OH 44106
3To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT Liver glutamate and...Modeling of the 14C-...Determination of the 14C-...Noninvasive sampling of liver...New knowledge derived from...Future directionsREFERENCES |
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-ketoglutarate, whose labeling pattern
is influenced by the following: 1) the contributions of
glucose and fatty acids to acetyl-CoA, 2) the
relative contributions of pyruvate carboxylase and pyruvate
dehydrogenase to the entry of pyruvate carbon into the citric acid
cycle, and 3) the rate of gluconeogenesis in relation to
citric acid cycle activity. In humans and primates, hepatic glutamate
can be sampled noninvasively via urinary phenylacetylglutamine, which
is formed in liver from phenylacetate (a side product of phenylalanine
catabolism) and glutamine (which equilibrates with liver glutamate and
-ketoglutarate). The 14C- or 13C-labeling
pattern of the glutamate moiety of phenylacetylglutamine can be
measured by sequential degradations to 14CO2,
gas chromatography-mass spectrometry or nuclear magnetic resonance
(NMR). When phenylacetylglutamine is labeled from singly labeled
[14C]- or [13C]substrates, relative
metabolic rates can be computed from the labeling pattern using
Landau’s model. In diabetic patients infused with
[3-13C]pyruvate, the noninvasive sampling of hepatic
glutamate via phenylacetylglutamine allows one to test the degree of
liver insulinization via the (pyruvate carboxylase)/(pyruvate
dehydrogenase) activity ratio. This ratio regulates gluconeogenesis in
part. Its measurement may allow the identification of patients who
might benefit from the intraperitoneal administration of insulin, or
from recently developed antidiabetic drugs.
KEY WORDS: • phenylacetylglutamine • gluconeogenesis • tricarboxylic acid cycle • isotopic methods • diabetes
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Liver glutamate and gluconeogenesis |
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TOPABSTRACTLiver glutamate and...Modeling of the 14C-...Determination of the 14C-...Noninvasive sampling of liver...New knowledge derived from...Future directionsREFERENCES |
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lactate glucose), and alanine, derived from
proteolysis, are converted to glucose via pyruvate by the same
reactions.
A number of isotopic techniques have been developed to study
gluconeogenesis in vivo and ex vivo. This review concentrates on
isotopic techniques developed to assess the regulation of hepatic
gluconeogenesis and its relationship to other pathways of intermediary
metabolism, particularly the citric acid cycle and fatty acid
oxidation. Such investigations are complicated by the fact that
oxaloacetate is an intermediate common to gluconeogenesis and the
citric acid cycle (Landau 1993
). This results in
numerous isotopic exchanges between gluconeogenic and citric acid cycle
intermediates. These isotopic exchanges result in variable losses of
carbon label between gluconeogenic precursors (pyruvate, lactate,
alanine) and glucose. In addition, the substrate cycle pyruvate
oxaloacetate phosphoenolpyruvate pyruvate results in additional
loss of label in phosphoenolpyruvate before it is converted to glucose.
Last, pyruvate enters the citric acid cycle via two reactions, one
catalyzed by pyruvate carboxylase to form oxaloacetate, and the other
by pyruvate dehydrogenase to form acetyl-CoA. The two routes of
entry of pyruvate carbon into the citric acid cycle result in a complex
labeling pattern of cycle intermediates. In addition to affecting the
labeling pattern of citric acid cycle intermediates, the (pyruvate
carboxylase)/(pyruvate dehydrogenase) activity ratio is a key regulator
of gluconeogenesis. It is increased in starvation and diabetes, and
decreased by insulin treatment. Because of the importance of this
activity ratio, much effort has been devoted to setting up techniques
for its estimation, using both invasive and noninvasive procedures.
Some of these techniques take advantage of the rapid interconversion
that occurs in liver mitochondria between -ketoglutarate and
glutamate (Fig. 1
). This interconversion is catalyzed by glutamate dehydrogenase,
glutamate-oxaloacetate aminotransferase and glutamate-pyruvate
aminotransferase. Thus the labeling pattern of liver glutamate,
equivalent to that of -ketoglutarate, yields useful information on
the regulation of hepatic gluconeogenesis.
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Modeling of the 14C- or 13C-labeling pattern of glutamate |
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TOPABSTRACTLiver glutamate and...Modeling of the 14C-...Determination of the 14C-...Noninvasive sampling of liver...New knowledge derived from...Future directionsREFERENCES |
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)
developed a mathematical model of the
14C-labeling pattern of glutamate that is
applicable to experiments in which glutamate is labeled from lactate,
or pyruvate labeled with 14C on carbon 3. The
data introduced into the model are the specific activities of the five
glutamate carbons. Computation yields rates of citric acid cycle
reactions and of gluconeogenesis expressed relative to the net flux
through the citric acid cycle (setting the rate of the citrate synthase
reaction to 1.0). This model is also applicable to experiments
conducted with [3-13C]lactate or
[3-13C]pyruvate, in which the computation uses
the 13C-enrichments of the five glutamate carbons
(Yang et al. 1996
). Magnusson et al. (1991)
and Di Donato et al. (1993)
have
discussed some limitations of the model, particularly the sensitivity
of some parameters to errors in measurements of the small amount of
label on carbon 5 of glutamate. |
Determination of the 14C- or 13C-labeling pattern of glutamate |
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TOPABSTRACTLiver glutamate and...Modeling of the 14C-...Determination of the 14C-...Noninvasive sampling of liver...New knowledge derived from...Future directionsREFERENCES |
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). The 13C-enrichment of the
glutamate carbons can be determined by gas chromatography-mass
spectrometry of various glutamate derivatives. Only ~50 nmol of
glutamate is required for the analyses. When the total
13C-labeling of glutamate is sufficiently low so
that, on average, most molecules contain no more than one
13C atom, analysis of a single derivative of
glutamate yields the complete labeling pattern (Beylot et al. 1993
). When glutamate is more heavily labeled, the analysis
requires running up to five glutamate derivatives on the mass
spectrometer (Di Donato et al. 1993
). Unfortunately, no
present mass spectrometric technique allows the direct measurement of
the 13C-enrichment of carbon 5 of glutamate. This
enrichment is calculated as the difference between the enrichments of
two fragments of the glutamate molecule. When the enrichment of carbon
5 is low compared with that of carbon 4, the value for carbon 5
enrichment is imprecise.
The problem of the low 13C-enrichment of carbon 5
of glutamate can be solved, at a price, by assaying the labeling
pattern of glutamate by high-power 13C or
1H nuclear magnetic resonance (NMR). However, to
be able to assay enrichments on carbon 5 of the order of 0.1–0.5%,
~50 mg of semipurified phenylacetylglutamine is required. Such
amounts cannot be obtained without administering phenylacetate or
phenylbutyrate to the subjects (typically 2.5 g) (Cline et al. 1994
, Jones et al. 1998
). An advantage of
the NMR assay is that it can identify adjacent
13C atoms in the glutamate molecule (Jones et al. 1998
).
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Noninvasive sampling of liver glutamate via urinary phenylacetylglutamine |
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TOPABSTRACTLiver glutamate and...Modeling of the 14C-...Determination of the 14C-...Noninvasive sampling of liver...New knowledge derived from...Future directionsREFERENCES |
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), which is excreted in urine (James et al. 1972
). This
process involves the activation of phenylacetate to
phenylacetyl-CoA, followed by conjugation with glutamine catalyzed
by an acyl-CoA:L-glutamine N-acyltransferase
(Webster et al. 1976
). Exogenous phenylacetate can be
ingested in gram amounts without toxicity. The main fate of this
ingested phenylacetate is the formation and excretion of
phenylacetylglutamine.
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). Because of the
objectionable smell of phenylacetate, it is often administered as a
less volatile precursor, phenylbutyrate. In the liver, phenylbutyrate
undergoes ß-oxidation to phenylacetyl-CoA, the immediate
precursor of phenylacetylglutamine (Piscitelli et al. 1995
). Note that prolonged administration of phenylbutyrate can
lead to glutamine depletion (Darmaun et al. 1998
).
Magnusson et al. (1991)
were the first to use the
labeling pattern of urinary phenylacetylglutamine as a noninvasive
probe of the liver citric acid cycle intermediates and of
gluconeogenesis. This was the first of a number of studies, some of
which are ongoing, that have capitalized on this elegant technique.
Some of these studies used 14C-substrates
(Esenmo et al. 1992
, Landau et al. 1995
, Schumann et al. 1991
); other studies used
13C-substrates, assaying the concentration and/or labeling
pattern of phenylacetylglutamine by gas chromatography-mass
spectrometry (Diraison et al. 1998
, Yang et al. 1993
and 1996
) or NMR (Cline et al. 1994
,
Dugelay et al. 1994
, Jones et al. 1998
).
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New knowledge derived from the labeling pattern of glutamate |
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TOPABSTRACTLiver glutamate and...Modeling of the 14C-...Determination of the 14C-...Noninvasive sampling of liver...New knowledge derived from...Future directionsREFERENCES |
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Katz et al. (1993)
infused food-deprived rats with
[U-13C3]lactate or
[2,3-13C2]lactate and
assayed the mass isotopomer distribution of liver glutamate. From the
mass isotopomer distribution of glutamate and glucose, they deduced the
absolute rates of gluconeogenesis, recycling of phosphoenolpyruvate,
Cori cycling and citric acid cycle flux.
Beylot et al. (1995)
determined the
13C-labeling pattern of rat liver glutamate
(Beylot et al. 1993
) under two sets of conditions as
follows: 1) in isolated livers perfused with
[3-13C]lactate,
[2-13C]acetate or
-keto[3-13C]isocaproate, and 2)
in liver taken from rats that had been infused with the same tracers.
With [3-13C]lactate, the labeling pattern of
liver glutamate was identical in the livers perfused with the tracer
and in livers removed after infusion of the tracer in vivo. In
contrast, with [2-13C]acetate and
-keto[3-13C]isocaproate, the labeling
pattern of glutamate was very different in the livers perfused with the
tracer and in livers removed after infusion of the tracer in vivo. This
discrepancy results from the intense metabolism of
[2-13C]acetate and
-keto[3-13C]isocaproate in muscle, which
releases 13CO2 and
[13C]glutamine; these are carried by blood to
the liver where they modify the labeling pattern of glutamate made
locally. With the latter tracers, the labeling patterns of muscle and
liver glutamine are very different, as shown by direct analysis. This
study confirmed the conclusion of Schumann et al. (1991)
that the bulk of the metabolism of
[2-14C]acetate is extrahepatic (see below).
Also, Large et al. (1997) applied the model of
Magnusson et al. (1991)
to studies of gluconeogenesis in
perfused rat livers with an emphasis on the effect of modulators of
pyruvate metabolism.
Glutamate isolated from urinary phenylacetylglutamine.
Landau and co-workers used the labeling pattern of
phenylacetylglutamine to investigate the suitability of various
14C-tracers to gain insight into the regulation of
gluconeogenesis and the citric acid cycle in human liver. One of the
issues to be resolved was whether, with some tracers, the labeling
pattern of liver glutamate could be altered by circulating labeled
products of the tracer’s metabolism in extrahepatic tissues,
particularly muscle. They compared the labeling patterns of
phenylacetylglutamine and of plasma glucose. They developed a technique
to correct the labeling pattern of phenylacetylglutamine and glucose
for reincorporation of 14CO2, which labels
carbon 1 of the glutamate moiety of phenylacetylglutamine, as well as
carbons 3 and 4 of glucose. This was achieved by using the specific
activity of urea measured in the experiments with various tracers as
well as in control experiments in which the production of
14CO2 was simulated by an infusion of
NaH14CO3. These studies showed that
[14C]lactate, but not [14C]acetate, is
suitable for tracing gluconeogenesis in liver (Magnusson et al. 1991
, Schumann et al. 1991
). They stimulated
Beylot et al. (1995)
to compare the labeling patterns of
liver glutamate in perfused rat livers and in livers from rats infused
with various [13C]substrates (see above).
To determine directly whether the labeling pattern of urinary
phenylacetylglutamine reflects that of liver glutamate and
-ketoglutarate, Yang et al. (1996)
infused
anesthetized monkeys with phenylacetate and various
13C-substrates, which label the citric acid cycle
intermediates either via pyruvate carboxylase and pyruvate
dehydrogenase ([3-13C]lactate,
[U-13C3]lactate), or via other reactions
([1,2-13C2]acetate,
-keto[3-13C]isocaproate). Figure 3
shows the comparison of the labeled mass isotopomer distributions of
the glutamate moiety of phenylacetylglutamine in monkeys infused either
with [U-13C3]lactate (upper
panel) or with [1,2-13C2]acetate.
Mass isotopomers are molecules containing one to four 13C
atoms (M1, M2, M3, M4). In the presence of
[U-13C3]lactate, the mass isotopomer
distributions of liver glutamate and urinary phenylacetylglutamine are
practically identical. The isotopomer distributions of muscle and
kidney glutamate do not match those of liver glutamate and
phenylacetylglutamine. In the presence of
[1,2-13C2]acetate, the mass isotopomer
distribution of phenylacetylglutamine does not match that of liver
glutamate, but is closer to those of muscle and kidney glutamate. This
study demonstrated that the labeling pattern of urinary
phenylacetylglutamine is identical to that of liver -ketoglutarate
and glutamate only when carbon label is administered as lactate or
pyruvate. For a review on the applications of mass isotopomer analysis
to nutrition research, see Brunengraber et al. (1997)
.
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assayed the labeling pattern of
phenylacetylglutamine in control and type II diabetic patients
infused with [1-13C]glucose. They concluded that patients
with poorly controlled insulin-dependent diabetes mellitus have
augmented hepatic gluconeogenesis and relatively decreased rates of
hepatic pyruvate oxidation. Diraison et al. (1998)
infused [3-13C]lactate in control and stable type II
diabetic patients and, using the labeling pattern of
phenylacetylglutamine, did not find evidence of increased
gluconeogenesis. Landau et al. (1995)
studied control
humans and type I diabetics infused with [3-14C]lactate
and NaH13CO3 after 60 h of food
deprivation. From the labeling patterns of glucose and
phenylacetylglutamine, they calculated rates of pyruvate cycling
and contributions of gluconeogenesis to total glucose production (80
and 45% in control and diabetic subjects, respectively).
Recently, Jones et al. (1998)
reported the labeling
pattern of phenylacetylglutamine in humans who had ingested
[U-13C3]propionate. This pattern, determined
by NMR, was used with a model of the citric acid cycle in which label
enters via an anaplerotic process.
Note that computation of the labeling pattern of phenylacetylglutamine or of glutamate does not yield absolute rates of gluconeogenesis, but rather relative rates of cellular reactions that participate in the control of gluconeogenesis. The information, gathered from the labeling pattern of phenylacetylglutamine, is thus complementary to techniques used to measure carbon flux through gluconeogenesis and glycogenolysis.
The precision of the measurements of the distribution of 13C between the glutamate and phenylacetylglutamine carbons depends very much on the following: 1) the nature of the tracer, 2) whether the tracer is singly or uniformly labeled, 3) the dose or administration rate of (expensive) tracers, 4) the sensitivity of the analytical equipment, and 5) the analytical skills of the investigators. Readers are referred to the original references for further evaluation of the technologies.
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Future directions |
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TOPABSTRACTLiver glutamate and...Modeling of the 14C-...Determination of the 14C-...Noninvasive sampling of liver...New knowledge derived from...Future directionsREFERENCES |
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FOOTNOTES |
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2 Supported by grants from the National Institutes of Health (DK35543) and the Cleveland Mt. Sinai Medical Center.
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REFERENCES |
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TOPABSTRACTLiver glutamate and...Modeling of the 14C-...Determination of the 14C-...Noninvasive sampling of liver...New knowledge derived from...Future directionsREFERENCES |
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Beylot M., Soloviev M. V., David F., Landau B. R., Brunengraber H. Tracing hepatic gluconeogenesis relative to citric acid cycle activity in vitro and in vivo.
Comparisons in the use of [3-13C]lactate,
[2-13C]acetate and -keto[3-13C]isocaproate. J. Biol. Chem. 1995;270:1509-1514
3. Brunengraber H., Kelleher J. K., Des Rosiers C. Applications of mass isotopomer analysis to nutrition research. Annu. Rev. Nutr. 1997;17:559-596[Medline]
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Darmaun D., Welch S., Rini A., Sager B. K., Altomare A., Haymond M. W. Phenylbutyrate-induced glutamine depletion in humans: effect on leucine metabolism. Am. J. Physiol. 1998;274:E801-E807
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Di Donato L., Des Rosiers C., Montgomery J. A., David F., Garneau M., Brunengraber H. Rates of gluconeogenesis and citric acid cycle in perfused livers, assessed from the mass spectrometric assay of the [13C]labeling pattern of glutamate. J. Biol. Chem. 1993;268:4170-4180
8. Diraison F., Large V., Brunengraber H., Beylot M. Non-invasive tracing of liver intermediary metabolism in normal subjects and in moderately hyperglycaemic NIDDM subjects. Evidence against increased gluconeogenesis and hepatic fatty acid oxidation in NIDDM. Diabetologia 1998;41:212-220[Medline]
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Esenmo E., Chandramouli V., Schumann W. C., Kumaran K., Wahren J., Landau B. R. Use of 14CO2 in estimating rates of hepatic gluconeogenesis. Am. J. Physiol. 1992;263:E36-E41
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Jones J. G., Solomon M. A., Sherry A. D., Jeffrey F. M., Malloy C. R. 13C NMR measurements of human gluconeogenic fluxes after ingestion of [U, 13C3]propionate, phenylacetate, and acetaminophen. Am. J. Physiol. 1998;275:E843-E852
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Katz J., Wals P., Lee W.-N.P. Isotopomer studies of gluconeogenesis and the Krebs cycle with 13C-labeled lactate. J. Biol. Chem. 1993;268:25509-25521
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