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*
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157,
Department of Neuroscience and Anatomy and
**
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033
3To whom correspondence should be addressed at Wake Forest University School of Medicine, Department of Biochemistry, Medical Center Boulevard, Winston-Salem, NC 27157. E-mail: shutson{at}wfubmc.edu.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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-keto acids (BCKA). BCKA, which are poorly
oxidized in astrocytes, exit and are taken up by neurons. Neuronal
BCATc catalyzes transamination of the BCKA with glutamate. The
products, BCAA, exit the neuron and return to the astrocyte. The
-ketoglutarate product in the neurons may undergo reductive
amination to glutamate via neuronal glutamate dehydrogenase. Operation
of the shuttle in the proposed direction provides a mechanism for
efficient nitrogen transfer between astrocytes and neurons and
synthesis of glutamate from astrocyte -ketoglutarate. Evidence in
favor of the hypothesis is: 1) The two BCAT isoenzymes
appear to be localized separately in the neurons (BCATc) or in the
astroglia (BCATm). 2) Inhibition of the shuttle in the
direction of glutamate synthesis can be achieved by inhibiting BCATc
using the neuroactive drug gabapentin. Although gabapentin does not
inhibit BCATm, it does block de novo glutamate synthesis from
-ketoglutarate. 3) Conversely, gabapentin stimulates
oxidation of glutamate. Inhibition of BCATc may allow BCKA to
accumulate in the astroglia, thus facilitating conversion of glutamate
to -ketoglutarate.
KEY WORDS: • branched-chain amino acids • transamination • glutamate • neurotransmitter • brain
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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-keto acids (BCKA). BCATm is found in most
tissues (Hutson et al. 1992
), whereas BCATc is expressed
predominantly in the central nervous system (Hall et al. 1993
). Additionally, BCATc, but not BCATm, is a target of the
anticonvulsant and neuroprotective drug, and leucine analog, gabapentin
(Hutson et al. 1998
). The second catabolic step,
irreversible oxidative decarboxylation of the transamination products
catalyzed by the mitochondrial BCKA dehydrogenase enzyme complex,
commits the BCAA to oxidative degradation, resulting in net transfer of
nitrogen from the BCAA to dispensable amino acids such as glutamate.
The BCAA, particularly leucine, are known to cross the blood–brain
barrier rapidly (Oldendorf 1971
). In brain slices, BCAA
are metabolized more rapidly than they are incorporated into protein
(Chaplin et al. 1976
), and the ratio of BCAT/BCKA
dehydrogenase activity in brain tissue is high (Hutson et al. 1998
), suggesting that in the brain BCAA must serve a function
distinct from one as an energy source. Published results (Bixel et al. 1997
, Hutson et al. 1998
, Yudkoff 1997
, Yudkoff et al. 1996
, Zielke et al. 1997
, Huang et al. 1996
, McKenna et al. 1998
) support the hypothesis that BCKA and BCAA have an
important role in the oxidation and synthesis of excitatory
neurotransmitter glutamate in brain. Both in cultured astrocytes
(Hutson et al. 1998
, Yudkoff et al. 1994
)
and in intact brain (Zielke et al. 1997
), one or more
BCKA but not BCAA, have been shown to stimulate the oxidation of
glutamate.
The importance of maintaining normal brain BCAA homeostasis is also
clear from the inherited neurologic disorders in man that are
associated with defects in the oxidation of BCAA (Chuang and Shih 1995
). Patients with branched-chain keto acidurias
have markedly elevated blood concentrations of BCAA and BCKA. If
untreated by dietary restriction of BCAA, these patients suffer from
seizures and severe mental and physical retardation (Chuang and Shih 1995
). This latter observation is important, because it
indicates a net transfer of nitrogen from BCAA (oxidation) is required
for normal brain function.
Because glutamate is the most widely utilized excitatory
neurotransmitter in the nervous system, it is critical to understand
how neurons replenish the glutamate they release during
neurotransmission. The brain glutamate/glutamine cycle (Glu/Gln Cycle
in Fig. 1
) is the metabolic pathway that involves the synaptic release of
glutamate from neurons, rapid and efficient glutamate uptake by
astroglia, conversion of glutamate to glutamine by astrocytic glutamine
synthetase, followed by release of glutamine to the interstitium, and
uptake by the neurons for conversion back to glutamate (Shank and Aprison 1981
). This process efficiently prevents excessive
accumulation of glutamate in the interstitium that would induce
excitotoxic neurodegeneration.
|
-Ketoglutarate
synthesized in the glia is transaminated to glutamate and then amidated
to glutamine for release and transport back to the neurons. Recent
studies suggest BCAA nitrogen is actually required for the optimal
formation of glutamate in the astroglia (Yudkoff et al. 1996
, Bixel et al. 1997
), because nitrogen
sources such as aspartate are limited in astroglia (Gamberino et al. 1997
, Hutson et al. 1998
). Recently, we
proposed that BCAA participate in a nitrogen shuttle that is obligatory
for optimal rates of de novo glutamate synthesis in brain
(Hutson et al. 1998
). In this report we present evidence
in support of the BCAA shuttle hypothesis. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Male Sprague–Dawley rats (200–400 g) were anesthetized with Nembutal, eyes enucleated, and retinas dissected in ice-cold buffer. The protocol complies with the Guide for the Care and Use of Laboratory Animals.
Retinal incubations.
Half-retinas were preincubated at 37°C for 3 min in Krebs buffer (pH
7.4) also containing 20 mmol/L HEPES, 5 mmol/L glucose, 0.05 mmol/L
NH4Cl, 25 mmol/L NaH14CO3, 0.2
mmol/L pyruvate. Bicarbonate (H14CO3-) was
used to measure CO2 fixation (Gamberino et al. 1997
), and vessels were closed to the atmosphere. At 20 min the
half-retinas were removed and placed in 2% perchloric acid. The
buffer was then acidified with perchloric acid and unreacted
14CO2 was allowed to diffuse out of all the
samples. [1-14C]Leucine transamination and
[U-14C]glutamate oxidation were determined as described
(Hutson et al. 1998
). Metabolites were separated and
quantified by published procedures (Gamberino et al. 1997
).
Immunohistochemistry.
Coronal sections (30 µm) of 4% paraformaldehyde fixed rat brain were used. Free-floating sections were stained after treatment with 10% methanol, 0.3% H2O2 in 0.1 M phosphate-buffered saline. Sections were rinsed in phosphate-buffered saline, blocked in 3% normal goat serum (Vector Laboratories, Burlingame, CA), and incubated overnight at 4°C with immunoaffinity-purified rabbit anti-rat BCATc peptide antibody (1.2 mg/L). The secondary antibody was goat anti-rabbit IgG 1:250 (Jackson ImmunoResearch, West Grove, PA), and samples were developed using a standard diaminobenzidine reaction. With the immunoaffinity-purified rabbit anti-human BCATm antibody, fresh frozen sections were used. For the control, antibody was absorbed overnight with a 50-fold excess of purified BCATc peptide, and tissue slices were incubated with the peptide for 30 min prior to incubation with preabsorbed antibody.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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, Sonnewald et al. 1993
, Gamberino et al. 1997
) indicate that up to
30% of the glutamate taken up by the astrocytes is not converted
directly to glutamine but is actually metabolized to lactate via
conversion of glutamate to -ketoglutarate and subsequent citric acid
cycle decarboxylation (Pyr/Mal Cycle in Fig. 1
). If glial citric acid
cycle decarboxylation occurs, equivalent rates of anaplerosis must take
place to recover the lost carbon of glutamate, i.e., de novo glutamate
synthesis. The anaplerotic enzyme pyruvate carboxylase is expressed
abundantly in the brain, and localized to glia (Shank et al. 1985
). Our previous studies of the rate of anaplerosis in
neonatal cultured astrocytes and the resultant accumulation of citric
acid cycle intermediates showed that pyruvate carboxylase was very
active and dependent on levels of added pyruvate (Gamberino et al. 1997
, Hutson et al. 1998
). This study also
indicated that the conversion of the excess tricarboxylic acid cycle
intermediates, formed by pyruvate carboxylase, to glutamate and
glutamine could be severely limited by lack of a source of nitrogen.
Because NH3 could not supply the needed nitrogen
(Gamberino et al. 1997
) and others previously reported
that BCAA were important donors of nitrogen for glutamate synthesis in
vivo (Kanamori et al. 1998
) and in brain cell culture
(Yudkoff et al. 1996
, Bixel et al. 1997
),
we proposed that BCAA might be the physiological source for the
-amino nitrogen of glutamate (Hutson et al. 1998
).
Equation 1
represents a linear pathway for net de novo glutamate
synthesis:
 | (1) |
Recent results showing a lack of effect of the BCATc-specific
drug gabapentin on glutamate metabolism in astrocyte cultures suggested
that the role of BCAA in glutamate synthesis is more complex than
depicted in eq. 1
. Immunocytochemical analysis of the localization of
BCATm and BCATc in primary cultures of rat brain cells showed that in
neuron-rich primary cultures BCATc is the sole BCAT isoenzyme
expressed in rat cortical neurons (Bixel et al. 1996
),
whereas in astroglia-rich primary cultures, BCATm is the
predominant isoenzyme (Bixel et al. 1997
). Gabapentin
was an effective inhibitor only of leucine transamination in neuronal
cultures (Hutson et al. 1998
). Similarly, immunoblots
revealed that BCATm is the predominant BCAT in astroglial cultures,
whereas BCATc is the predominant isoenzyme found in early neuron
cultures (Hutson et al. 1998
).
These results led to development of the BCAA shuttle hypothesis
depicted in Figure 1
(BCAA Shuttle, Fig. 1
). BCAA enter the astrocyte
where transamination catalyzed by BCATm produces glutamate and BCKA.
Since BCKA are poorly oxidized in astrocytes (Bixel and Hamprecht 1995
, Hutson et al. 1998
), they exit
the astrocyte and can be taken up by the neuron. Neuronal BCATc
catalyzes transamination of the BCKA with glutamate. The products,
BCAA, exit from the neuron and return to the astrocyte. The
-ketoglutarate product in neurons may undergo reductive amination to
glutamate via neuronal glutamate dehydrogenase. Although reversible,
the cycle will be driven in the direction shown in Figure 1
by
anaplerotic synthesis of -ketoglutarate in astrocytes and by the
higher activity of neuronal as opposed to astrocytic glutamate
dehydrogenase under physiological ADP concentrations (Gamberino et al. 1997
, Shashidharan et al. 1997
).
In the intact brain BCATc is found in neurons.
The model predicts cell-specific localization of BCATm and BCATc in
the central nervous system. Therefore, we used immunohistochemistry
with immunoaffinity-purified BCATc peptide antibodies to determine
the distribution of BCATc in paraformaldehyde-fixed rat brain. The
pattern of BCAT isoenzyme expression is consistent with the shuttle
hypothesis. In the brain regions examined, the pattern of staining for
BCATc is neuronal (Fig. 2A–D
). Immunostaining for BCATm, using
immunoaffinity-purified human BCATm antibodies, was found in
astroglia in fresh frozen rat brain cortex (Hutson, S. M.,
unpublished observations). BCATc immunostaining of neurons is observed
in all layers of the somatosensory cortex (Fig. 2A
) with
staining found primarily in the pyramidal neurons (Fig. 2B,
C
) which are glutamatergic. BCATc immunostaining in the cerebellar
cortex showed intense staining in GABAergic Purkinje neurons (Fig. 2D
). Other GABAergic neurons also stained positive for BCATc
(Hutson, S. M., unpublished observations). The intense staining
for BCATc in the Purkinje neuron cell bodies and processes contrasts
sharply with the reported near background immunolabeling for phosphate
activated glutaminase in Purkinje cell bodies (Laake et al. 1999
). These results are intriguing because they suggest that
the full BCAA shuttle that requires both BCAT isoenzymes, glutamine
synthetase and glutaminase, may be more active in glutamatergic rather
than GABAergic pathways. Furthermore, understanding the pattern of
expression of the cycle enzymes may allow for better interpretation of
the effect of drugs, such as gabapentin, that target BCATc.
|
The ex vivo rat retina provides a model system to test the shuttle
hypothesis. The isolated retina is a well-defined model of
glutamatergic neurons, does not require cell culture from immature
cells and can still provide a way of examining the process in the
presence of interacting astroglia (Müller cells in the retina)
and neurons. Immunoblotting of retinal homogenates revealed comparable
levels of BCATc and BCATm compared to whole brain (Hutson, S. M.,
unpublished data). The effect of the competitive BCATc inhibitor,
gabapentin (Hutson et al. 1998
), on metabolism of added
leucine as well as rates of de novo glutamate and glutamine synthesis
(no added leucine) was examined. Addition of 1 mM
gabapentin to the excised retinas resulted in a 50–60% inhibition of
added leucine transamination (Fig. 3
). In a separate experiment, addition of gabapentin to the retina (no
added BCAA) resulted in 
30% inhibition of the de novo synthesis of
glutamine and glutamate from
14HCO3- (2.26 ± 0.05
versus 3.18 ± 0.12 nmol 14C-glutamine +
14C-glutamate/mg protein, P < 0.001) (Lieth E. et al., unpublished observations). These results were
the first demonstration that inhibition of BCATc can affect de novo
glutamate synthesis in an intact neural preparation.
|
Glutamate added to the media surrounding excised rat retinas enters the
tissue through the Müller cells (Pow and Crook 1996
, Poitry et al. 2000
) so that the glutamate
does not have direct access to neurons. Therefore Müller cell
oxidation and amidation of glutamate can be monitored by adding
[U-14C]glutamate to ex vivo excised retinas. It
was previously shown that glutamate oxidation in cultured cortical
astrocytes occurs by an initial transamination step (McKenna et al. 1996
). In cultured astrocytes at physiological levels of
glutamate, oxidation is almost completely blocked by nonspecific
transaminase inhibitors such as aminooxyacetic acid. Our previous
studies (Lieth et al. 2000
) of glutamate oxidation by
excised retinas indicates that addition of 1 mM
aminooxyacetic acid inhibits glutamate oxidation by 40%. The
proposed BCAA shuttle predicts that glutamate oxidation to
-ketoglutarate in the Müller cells will be partially dependent
on a supply of BCKA. We previously showed in studies of cultured rat
cortical astrocytes that addition of BCKA stimulates oxidation of
[U-14C]glutamate (Hutson et al. 1998
). If the hypothesis is correct in predicting that BCATc
normally operates in the direction of synthesis of BCAA at the expense
of BCKA, then inhibiting BCATc should increase the pool of BCKA in the
retina and therefore stimulate [U-14C]glutamate
oxidation. We incubated excised rat retinas for 40 min at 37°C with
0.2 mM BCKA, with 1 mM gabapentin, or with
both. The results, illustrated in Figure 4
, demonstrate that the oxidation of glutamate in the retinas is
increased by both BCKA and gabapentin. The data also show that
production of 14CO2,
14C-lactate and 14C-citric
acid cycle intermediates increases in the presence of both BCKA and
gabapentin and that, when the two are combined in the same incubation,
stimulation is somewhat greater than with either BCKA or gabapentin
alone.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported in part by grants from the National
Institutes of Health Grant DK34738 (to S.M.H.) and Juvenile Diabetes
Foundation International (to E.L.).
4 Abbreviations used: BCAA, branched-chain
amino acid(s), BCATm, mitochondrial branched-chain
aminotransferase; BCATc, cytosolic branched-chain aminotransferase;
BCKA, branched-chain -keto acid(s).
|
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