Neurochemistry International 36 (2000) 451±459

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Mitochondrial malic enzyme activity is much higher in

mitochondria from cortical synaptic terminals compared with
mitochondria from primary cultures of cortical neurons or
cerebellar granule cells
Mary C. McKenna a,*, Joseph H. Stevenson a, Xueli Huang a, J. Tyson Tildon a, b, Carol
L. Zielke a, Irene B. Hopkins a
a
Department of Pediatrics, University of Maryland School of Medicine, 655 W. Baltimore Street, Baltimore, MD 21201, USA
b
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, MD 21201, USA
Received 2 August 1999; received in revised form 22 September 1999; accepted 23 September 1999

Abstract

Most of the malic enzyme activity in the brain is found in the mitochondria. This isozyme may have a key role in the
pyruvate recycling pathway which utilizes dicarboxylic acids and substrates such as glutamine to provide pyruvate to maintain
TCA cycle activity when glucose and lactate are low. In the present study we determined the activity and kinetics of malic
enzyme in two subfractions of mitochondria isolated from cortical synaptic terminals, as well as the activity and kinetics in
mitochondria isolated from primary cultures of cortical neurons and cerebellar granule cells. The synaptic mitochondrial
fractions had very high mitochondrial malic enzyme (mME) activity with a Km and a Vmax of 0.37 mM and 32.6 nmol/min/mg
protein and 0.29 mM and 22.4 nmol/min mg protein, for the SM2 and SM1 fractions, respectively. The Km and Vmax for malic
enzyme activity in mitochondria isolated from cortical neurons was 0.10 mM and 1.4 nmol/min/mg protein and from cerebellar
granule cells was 0.16 mM and 5.2 nmol/min/mg protein. These data show that mME activity is highly enriched in cortical
synaptic mitochondria compared to mitochondria from cultured cortical neurons. The activity of mME in cerebellar granule
cells is of the same magnitude as astrocyte mitochondria. The extremely high activity of mME in synaptic mitochondria is
consistent with a role for mME in the pyruvate recycling pathway, and a function in maintaining the intramitochondrial reduced
glutathione in synaptic terminals. # 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Malic enzyme; Pyruvate recycling pathway; Synaptic terminals; Mitochondria; Cortical neurons; Cerebellar granule cells; Energy
metabolism

1. Introduction the predominance of this isozyme, its role in the brain

The mitochondrial isozyme of malic enzyme (mME)
is found in highest activity in mammalian and avian
brain (Kochan et al., 1995a), and accounts for 75% of
the activity in human and rat brain (Bukato et al.,
1992, 1994; Salganico€ and Koeppe, 1968). Despite
* Corresponding author. Tel.: +1-410-706-1990; fax: +1-410-706-
0020.
E-mail address: mmckenna@umaryland.edu (M.C. McKenna).
is not well de®ned. The current understanding of the
function of mME relates to a role in providing
NADPH for the reduction of the small, but critical
amount of intramitochondrial glutathione which pre-
vents oxidative damage of mitochondria (Bukato et
al., 1995b; Kochan et al., 1995b; Loeber et al., 1994;
Vogel et al., 1998a,b), and/or a role in the pyruvate
recycling pathway in the brain (Cerdan et al., 1990;
Cruz et al., 1998). This pathway, ®rst reported in the
brain by Cerdan and coworkers (Cerdan et al., 1990;
Kunnecke et al., 1993), uses either malic enzyme or the

0197-0186/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 1 9 7 - 0 1 8 6 ( 9 9 ) 0 0 1 4 8 - 5
452 M.C. McKenna et al. / Neurochemistry International 36 (2000) 451±459
combined activity of pyruvate kinase and PEPCK to
convert TCA cycle intermediates such as malate or
oxaloacetate to pyruvate and thus provides acetyl CoA
to maintain TCA cycle activity (Friedman et al., 1971).
The presence of a pyruvate recycling pathway gives tis-
sues the ability to continuously maintain sucient
energy metabolism to preserve functional activity when
substrates such as glucose and ketone bodies are low
(Cerdan et al., 1990); a protective mechanism that
could be crucial to the survival of neurons during
pathological conditions.
Sonnewald et al. (1996) demonstrated evidence of
pyruvate recycling activity in astrocytes, but not in
neurons. However, that report is in marked contrast
to work from Cerdan's group, including in vivo stu-
dies, documenting pyruvate recycling activity which
appeared to be localized in neurons (Cerdan et al.,
1990; Cruz et al., 1998; Kunnecke et al., 1993).
Indeed, Cruz et al. (1998) recently reported that the
pyruvate recycling system develops after the second
week of life concurrent with the main period of
synaptogenesis in the brain. They also showed that
the increase in pyruvate recycling occurred at a
time when the speci®c activity of malic enzyme
increased, and that of PEPCK decreased, strength-
ening the evidence that malic enzyme may be the
primary enzyme associated with pyruvate recycling
in the brain.
Cruz et al. (1998) used western blots to identify
the mRNAs for only the cytosolic isoforms of malic
enzyme and determined activity in low speed super-
natants from brain and cell homogenates. Cytosolic
malic enzyme, which is almost exclusively a glial
enzyme (Kurz et al., 1993; Vogel et al., 1998b),
could only have a role in a pathway localized in
synaptic terminals if metabolic tracking were
involved in the pathway. Although it is conceivable
that tracking could be involved in such a pathway,
a more likely possibility would be the involvement
of mME in the pyruvate recycling pathway.
McKenna et al. (1995) reported a relatively low, but
distinctly regulated activity for mME activity in
astrocytes. However, a recent report showed the ma-
jority of the immunocytochemical staining for mME
activity was localized primarily in neurons (Vogel et
al., 1998a,b).
The activity of mME in synaptic terminals has not
been studied in detail. The presence of a high activity
of mME in synaptic terminals or neurons would be
consistent with a role for this isozyme in the pyruvate
recycling pathway. The present study was undertaken
to determine the activity and kinetics of mME in mito-
chondrial subfractions isolated from cortical synaptic
terminals, and in mitochondria isolated from primary
cultures of cortical neurons and cerebellar granule
cells.
2. Experimental
2.1. Materials
Culture media (DMEM and EMEM with Earle's
salts without L-glutamine) and Dulbecco's phosphate
bu€ered saline (PBS) were purchased from Biowhit-
taker, Walkersville, MD, USA. Fetal bovine serum,
horse serum, and insulin were purchased from
HyClone Laboratories, Inc., Logan, UT, USA. The
Falcon cell strainers were obtained from Fisher Scien-
ti®c, Pittsburgh, PA, USA, and culture dishes
(NUNC) were obtained from VWR, San Francisco,
CA, USA. L-Glutamine, L-malate, benzethonium hy-
droxide, culture medium supplements, bu€ers and
enzymes utilized for the cerebellar granule cell prep-
aration were purchased from Sigma Chemical, St.
Louis, MO, USA. Clonal peroxidase-antiperoxidase
(PAP) and monoclonal antibodies to synaptic ``t-
snare'' protein (SNAP-25), anti-microtubule-associated
protein 2 (anti-MAP-2), and anti-glial ®brillary acidic
protein (anti-GFAP) were obtained from Sternberger
Monoclonals, Inc., Lutherville, MD, USA. The ¯uor
used for liquid scintillation counting (Ready Solve EP)
was purchased from Beckman Instruments, Inc., Full-
erton, CA, USA. Solutions for the Pierce BCA Micro-
reagent Protein Assay were purchased from Pierce,
Rockford, IL, USA. All other reagents and chemicals
were of the highest analytical grade. The radioactive
compound L-[U-14C]malate was purchased from Amer-
sham Corporation, Arlington Heights, IL, USA.
2.2. Animals
Time-pregnant Sprague±Dawley rats were purchased
from Zivic±Miller, Zelienople, PA, USA for the neur-
onal cultures. Age speci®ed rats were ordered for the
synaptosomal studies. Rats were housed in the animal
colony at the University of Maryland School of Medi-
cine until use. Animals were maintained at 228 2 18C,
relative humidity 65%, with a 14 h light/10 h dark
cycle with minimum noise and handling. Animals had
unlimited access to food and water until sacri®ced. All
protocols in this study were approved by the Univer-
sity of Maryland School of Medicine Institutional Ani-
mal Care and Use Committee.
2.3. Preparation of cortical neurons from rat brain
Primary cultures of cortical neurons were prepared
from the cerebral hemispheres of 16 days gestation rat
embryo using a modi®cation of the methods of Yavin
and Yavin (1980) and Hertz et al. (1985, 1989). The
dissected neopallia were minced, incubated in 0.25%
trypsin at 378C for 2 min and the activity stopped by
addition of an equal volume of medium (EMEM sup-
 M.C. McKenna et al. / Neurochemistry International 36 (2000) 451±459
plemented with 10% heat inactivated horse serum,
10% fetal bovine serum (FBS), 27 mM D-glucose and
2 mM freshly prepared L-glutamine). The cells were
triturated, centrifuged at low speed, resuspended in
fresh media and ®ltered through a sterile Falcon cell
strainer (70 mm pore size), diluted to 3 ml/brain and
seeded on polylydsine coated 60 mm NUNC plastic
tissue culture dishes. Cultures were maintained at 378C
in an atmosphere of 95% air/5% CO2 (v/v) at 90%
relative humidity. After 72 h in vitro the cultures were
treated for 24 h with cytosine arabinocide (®nal con-
centration 10 mm). Cultures were refed with freshly
prepared maintenance media (plating media minus
FBS), and were used 7 days after preparation.
Additional neuronal cultures prepared at the same
time in our laboratory were immunocytochemically
stained by the peroxidase-antiperoxidase (PAP)
method of Sternberger (Sternberger, 1976; Sternberger
and Sternberger, 1987) at 7 days in vitro and examined
for morphology, neuronal markers and the presence of
contaminating cells. Neuronal cultures were stained
with antibodies to microtubule-associated protein 2
(MAP 2) to identify developing dendritic processes
(Alaimo-Beuret and Matus, 1985; Caceres et al., 1984;
DeCamilli et al., 1984; Kosik and Finch, 1987), and
antibodies to synaptic ``t-snare'' protein (SNAP-25) to
identify axons (Osen-Sand et al., 1993; Oyler et al.,
1989, 1991, 1992). Cultures were evaluated for the pre-
sence of astrocytes using antibodies to glial ®brillary
acidic protein (GFAP) (Antanitus et al., 1975; Bignami
et al., 1972; Ra€ et al., 1979).
The cortical neurons cultured from fetal brain con-
tained healthy, well developing neurons with abundant
processes that stained with antibody to SNAP-25 (data
not shown). Examination by inverted phase contrast
microscopy revealed that the cortical neurons consisted
of mostly small refractile cells, either associated in
small clusters or as individual cells. Anti-SNAP-25
stained a ®ne network of neurites as well as thick fasci-
cles of neurites that frequently extended long distances
over the culture dish. Anti-MAP 2 stained both the
cell perikarya and developing neurites, but not the
neurites stained by anti-SNAP-25. The anti-SNAP-25
was di€usely localized in developing axonal processes
indicating that the cells may not have completely dif-
ferentiated. The immunocytochemical staining patterns
for the cultured cells with anti-MAP 2, anti-SNAP-25
and anti-GFAP were consistent with a neuronal mor-
phology with some contamination by glial cells. How-
ever, the high total cell number in the cultures utilized
for metabolic studies precluded an accurate count of
the di€erent cell types. Hertz and coworkers (Hertz et
al., 1989; Walz and Hertz, 1982) have reported that
cultures prepared by this method contain >90% neur-
ons and exhibit the morphology and K+ induced
GABA release characteristics of mature neurons.
453
2.4. Preparation of primary cultures of rat cerebellar
granule cells
Primary cultures of rat cerebellar granule cells were
prepared by the method of Schousboe et al. (1989)
from the cerebella of 7-day-old rat pups. The cells
were isolated as described by Schousboe et al. (1989),
seeded on poly-D-lysine coated NUNC tissue culture
dishes and incubated under the same conditions as cor-
tical neurons. Cells were treated with 20 mM cytosine
arabinoside 48 h in culture to arrest the proliferation
of glial cells. Cerebellar granule cells were used at 10
days in vitro. The cerebellar granule cultures consisted
of small refractile cells with processes, with many of
the cells associated in small clusters. These cells
showed healthy, well developing neurons with many
processes that stained with antibody to SNAP-25. The
immunocytochemical staining patterns for anti-MAP 2
and anti-SNAP-25 were similar to those for the corti-
cal neurons. According to Schousboe et al. (1989) cul-
tures prepared by this method contain >90% granule
cells.
2.5. Isolation of subcellular fractions
Cultured neurons (cortical neurons and cerebellar
granule cells) were rapidly washed four times with iso-
lation media (IM) containing 0.25 M sucrose, 10 mM
Tris, 0.5 mM K+-EDTA, pH 7.4. The cells were
scraped into a glass hand held homogenizer with a
plastic cell lifter using 0.5 ml of isolation media per
®ve plates. The cells were homogenized by hand using
14 up and down strokes and decanted into a centrifuge
tube. The homogenate was centrifuged at 600  g for
6 min and the pellet containing nuclear debris dis-
carded. The supernatant was recentrifuged at 34,000 
g for 10 min to obtain the crude mitochondrial pellet
(P2 fraction) and the soluble/cytosolic fraction (i.e. the
post-mitochondrial supernatant). The pellet was
washed, resuspended in isolation media and sonicated
for 10 s three times at 30 s intervals in a Branson Soni-
®er 200.
2.6. Preparation of rat brain synaptosomes
Cortical synaptosomes were prepared as described
by Lai and Clark (1979), with minor modi®cations
previously described by McKenna et al. (1995), from
brain tissue from 7±8-week-old male Sprague±Dawley
rats. The tissue from four brains was minced, washed
in IM and centrifuged to obtain the crude mitochon-
drial pellet (P2). Synaptosomes were isolated from the
P2 fraction by ultracentrifugation through a discon-
tinuous Ficoll gradient (Lai and Clark, 1979) of 7.5%
(w/w) Ficoll layered on 10% (w/w) Ficoll medium and
centrifuged at 99,000  g for 30 min in a SW28 swing-
454 M.C. McKenna et al. / Neurochemistry International 36 (2000) 451±459
ing bucket rotor in a Beckman L8-70 refrigerated
ultracentrifuge. After ultracentrifugation the synapto-
somal layer was washed with isolation media to
remove the Ficoll, and the heavy (SM2) and light
(SM1) synaptic mitochondrial fractions were isolated
as described by Lai and Clark (1979).
2.7. Malic enzyme assay
The activity of malic enzyme [malate-NADP+-
oxidoreductase decarboxylating (EC 1.1.1.40)] was
measured as described by McKenna et al. (1995) using
a sensitive decarboxylation assay developed in our lab-
oratories. Aliquots of the sonicated mitochondrial tis-
sue (in IM) were incubated in Tris/HCl bu€er
(containing 75 mM Tris/HCl, pH 7.4) in the presence
of 5 mM MnCl2, 4 mM NADP+ and increasing con-
centrations of malate from 0.1±20 mM, containing L-
[U-14C]malate, as described below. The reaction was
started by adding the tissue to the incubation tubes.
Samples were incubated at 378C for 30 min in a shak-
ing water bath, and the reaction stopped by the ad-
dition of 0.3 ml of 10% (v/v) trichloroacetic acid, and
the 14CO2 released was trapped in center wells contain-
ing benzethonium hydroxide. The trapped 14CO2 was
counted in a liquid scintillation spectrometer. Protein
was determined by the Pierce BCA Protein Assay
microreagent system (Smith et al., 1985). In all deter-
minations, portions of the reaction mixtures for each
concentration of malate used were incubated without
tissue for 30 min and the values subtracted from the
tissue values to correct for any non-speci®c degra-
dation of labeled malate.
Previous studies in our laboratory veri®ed that this
assay is speci®c for malic enzyme, and that the 14CO2
released was due only to the activity of malic enzyme
since only 1.47% of the label was released as 14CO2 in
the absence of NADP+ (McKenna et al., 1995). The
activity of malic enzyme reported represents the dpms
of 14CO2 released from the 1-position of the U-14C-
labeled malate divided by one quarter of the speci®c
activity of the substrate in the medium/min/mg pro-
tein. These rates represent minimal values since the
speci®c activity of the intracellular substrate pools may
be lower than that of the medium as pointed out by
Fitzpatrick et al. (1983).
2.8. Statistical analysis
Kinetic parameters were determined using linear re-
gression analysis of the data in the Lineweaver±Burk
plots of 1/velocity vs 1/substrate (malate) concen-
tration (Snedecor and Cochran, 1967; Ze€ren and
Hall, 1973).
3. Results
The activity of malic enzyme determined using
0.5 mM malate, the physiological concentration of
malate in the brain (Hawkins and Mans, 1982), was
more than 10-fold higher in freshly isolated cortical
synaptic mitochondria than in mitochondria from pri-
mary cultures of cortical neurons (Fig. 1). The activity
in synaptic mitochondria was also considerably higher
than the activity in mitochondria from cerebellar gran-
ule cells (Fig. 1), and the activity previously reported
in mitochondria from astrocytes (McKenna et al.,
1995). At physiological concentrations of malate, the
mME activity in light (SM1) and heavy (SM2) synap-
tic mitochondrial fractions was 13.8 and 20.6 nmol/
min/mg protein, respectively. The malic enzyme ac-
tivity, using the same concentration of malate, was
1.23 and 4.62 nmol/min/mg protein in mitochondria
from primary cultures of cortical neurons and cerebel-
lar granule cells, respectively.
The rates of malic enzyme activity in the SM1 and
SM2 mitochondrial fractions isolated from cortical
synaptic terminals were measured using a 100-fold
range of malate concentrations (0.05±5 mM) so that
kinetic parameters could be determined. With both
synaptic mitochondrial preparations, mME activity
increased with increasing malate concentration, and
appeared to reach saturation between 1 and 2 mM
malate as shown in Fig. 1. The activity of mME in the
SM2 mitochondrial fraction was always higher than
the activity in SM1 mitochondria, regardless of malate
concentration. It is important to note that the majority
of mitochondria in cortical synaptic terminals from
adult rat brain are the heavier SM2 mitochondria; the
protein content of this mitochondrial fraction is gener-
ally about 4- to 5-fold higher than the protein content
Fig. 1. The activity of malic enzyme at various concentrations of
malate in SM2 (closed circles) and SM1 (open circles) subfractions
of synaptic mitochondria from cortex from 7±8-week-old rat brain,
and in mitochondria from primary cultures of cerebellar granule
cells (triangles) and cortical neurons (squares). The mitochondria
were isolated and the experiments were carried out as described in
section 2.
 M.C. McKenna et al. / Neurochemistry International 36 (2000) 451±459 455

Fig. 3. An enlarged view of the data for malic enzyme activity in

Fig. 2. Double-reciprocal plot of data in Fig. 1, showing 1/rate vs 1/
mitochondria from primary cultures of cortical neurons (squares)
concentration of malate for malic enzyme in SM1 (open circles) and
and cerebellar granule cells (triangles) shown in Fig. 1 (note the
SM2 (closed circles) synaptic mitochondrial subfractions. The lines
di€erence in scale). Cultures were prepared and experiments were
were calculated by linear regression.
carried out as described in section 2.

of the SM1 fraction. Lineweaver±Burk plots of 1/ac-

4. Discussion
tivity vs 1/malate concentration for the data from SM1
and SM2 mitochondrial fractions gave straight lines
Data from the present study demonstrate that the
demonstrating that the enzyme exhibited Michaelis±
activity of mME is highly enriched in cortical synaptic
Menten kinetics (Fig. 2). The Km and Vmax values,
terminals, consistent with the high mME in cortex
determined by linear regression analysis of the data in
reported by Bukato et al. (1994), and with the im-
the Lineweaver±Burk plots are summarized in Table 1.
munocytochemical localization in cells morphologically
The malic enzyme activity in mitochondria isolated
identi®ed as neurons reported by Vogel et al. (1998a).
from primary cultures of cortical neurons and cerebel-
Although the much lower activity in cortical neurons
lar granule cells was much lower than in synaptic
di€ers somewhat from the immunohistochemical stain-
mitochondria, but also exhibited saturation with
ing seen around the nuclei and beginning of dendritic
increasing malate concentration (Fig. 1). This is more
processes in some neuronal cell bodies reported by
apparent when the data are replotted with an
that group (Vogel et al., 1998a), this di€erence may
expanded scale (Fig. 3). The mME activity in both
re¯ect the relative immaturity of neurons grown in pri-
types of cultured neurons reached saturation at 0.5 mM
mary culture compared to those in mature brain.
malate, a lower concentration than in the synaptic
Alternatively, the activity of mME may simply be
mitochondria. Lineweaver±Burk plots of the mME
higher in the mitochondria from synaptic terminals
enzyme activity data from the primary cultures of
than in the mitochondria localized in neuronal cell
neurons also gave straight lines consistent with
bodies. Indeed, the heterogeneity of mitochondria in
Michaelis±Menten kinetics (Fig. 4). The lower Km and
the brain and synaptic terminals is well established
Vmax values in Table 1 show that the mME in both
(Lai and Clark, 1979; McKenna et al., 1990; Sonne-
the cultured cortical neurons and cerebellar granule
cells had a higher anity for malate, but considerably
lower maximal activity than mME in synaptic mito-
chondria.

Table 1
Kinetic parameters of mitochondrial malic enzyme in brain cells and
synaptic mitochondria

Fraction or cell type Km Vmax

(mM) (nmol/min/mg protein)

SM1 synaptic mitochondria (cortical) 0.30 22.4

SM2 synaptic mitochondria (cortical) 0.37 32.6
Cortical neurons 0.10 1.4
Cerebellar granule cells 0.16 5.2 Fig. 4. Double-reciprocal plot of data in Fig. 3, showing 1/rate vs 1/
Astrocyte mitochondria (total) 0.21 6.7 concentration of malate for malic enzyme in mitochondria from cor-
tical neurons (squares) and cerebellar granule cells (triangles). The
 lines were calculated using linear regression.
Values from McKenna, unpublished data.
456 M.C. McKenna et al. / Neurochemistry International 36 (2000) 451±459
wald et al., 1993b). The relatively low activity of mME
in cerebellum in the present study is consistent with
the lower activity reported by Bukato et al. (1994) and
weaker immunoreactivity in this area seen by Vogel et
al. (1998a).
Earlier studies demonstrated that mME in brain tis-
sue preferentially acts in the direction of oxidative dec-
arboxylation of malate (Bukato et al., 1995a,b;
Frenkel, 1972; Simpson and Eastabrook, 1969).
Bukato et al. (1995b) have proposed a role for mME
in the degradation of GABA which enters the TCA
cycle as succinate. Cortical synaptic terminals are
known to be the primary site of GABA reuptake and
oxidative degradation (Bukato et al., 1995b). Thus the
extremely higher activity of mME in synaptic mito-
chondria would be consistent with such a role.
The results presented above demonstrate that the
speci®c activity and Vmax for mME in synaptic mito-
chondria are 4- to 5-fold higher than the activity pre-
viously reported by our group for astrocytes (Table 1
and McKenna et al., 1995). Although the presence of
multiple forms of mME have been reported in some
tissues (Taroni and Didonato, 1988), the present study
does not address this question. The di€erences in ac-
tivity of mME in astrocytes, neuronal cell bodies and
synaptic terminals are likely to re¯ect the in¯uence of
the lipid composition of the mitochondrial membranes
within each type of brain cell or microenvironment,
which are known to di€er considerably (Ruggiero et
al., 1992). The kinetic parameters determined in the
present study are consistent with earlier reports that
cytosolic and mitochondrial malic enzymes are distinct
isozymes which do not crossreact (Kurz et al., 1993;
Vogel et al., 1998a), and di€er in kinetic parameters,
chromatographic and electrophoretic mobility (Fren-
kel, 1972).
Numerous reports demonstrate the presence of
either a partial (Bachelard et al., 1993; McKenna et
al., 1996; Sonnewald et al., 1993a), or complete (Bak-
ken et al., 1997; Cerdan et al., 1990; Cruz et al., 1998;
Kunnecke et al., 1993; Sonnewald et al., 1996) pyru-
vate recycling pathway in the brain. There is contro-
versy about the site of this pathway which has been
reported to be localized in astrocytes (Sonnewald et
al., 1996), neurons (Cerdan et al., 1990; Kunnecke et
al., 1993), and synaptic terminals (Cruz et al., 1998).
The genes for both malic enzyme and PEPCK have
been identi®ed in primary cultures of astrocytes, neur-
ons and also in synaptosomes from adult rat brain
(Cruz et al., 1998) demonstrating that all of these frac-
tions are capable of pyruvate recycling. However, the
demonstration of Cruz et al. (1998) that both the pyru-
vate recycling activity and the mRNA for malic
enzyme increases in conjunction with synaptogenesis,
strengthens the evidence for the neuronal localization
and the potential role of malic enzyme in this pathway.
This concept is strengthened by the ®nding that the ac-
tivity of PEPCK decreases while those of ME and pyr-
uvate recycling increase (Cruz et al., 1998).
Another controversial aspect of the involvement of
malic enzyme in pyruvate recycling is the question of
whether the mitochondrial or cytosolic isozyme of
malic enzyme is involved in the pyruvate recycling
pathway. Bakken et al. (1997) reported pyruvate recy-
cling in mitochondria from rat brain. In contrast, Cruz
et al. (1998) reported an increase in total malic enzyme
activity in conjunction with synaptogenesis and pyru-
vate recycling capability. However, it is widely
accepted that cytosolic malic enzyme (cME) is primar-
ily, if not exclusively, a glial enzyme (Bukato et al.,
1994; Kurz et al., 1993; McKenna et al., 1995; Vogel
et al., 1998b). Both the demonstration of pyruvate
recycling activity in isolated mitochondria and the
extremely high activity of mME in synaptic terminals
found in the present study give support to the concept
that mME is the isoform involved in this pathway in
the brain. In situations such as low glucose, where pyr-
uvate recycling would be necessary, an initial lack of
acetyl CoA would decrease the rate of citrate for-
mation, leading to an increase in mitochondrial oxa-
loacetate (OAA). Any increase in intramitochondrial
OAA would strongly inhibit mitochondrial malate de-
hydrogenase (Malik et al., 1993) and potentiate the
conversion of malate to pyruvate via mitochondrial
malic enzyme. The localization of pyruvate dehydro-
genase in the mitochondrial membrane as well as the
high anity of mME for malate, would make mME
ideally suited to participate in the pyruvate recycling
pathway. Furthermore, since the normal concentration
of malate in the brain is 00.5 mM (Hawkins and
Mans, 1982; McKenna et al., 1990), the enzyme would
be responsive to changes in the concentration of
malate within the physiological range.
This role of malic enzyme may be particularly im-
portant in neurons, since studies by Hassel and Sonne-
wald (1995) reported evidence of 020% of metabolism
via such a pathway. The high activity of mitochondrial
malic enzyme in synaptosomes reported above sup-
ports this concept. Furthermore, the data from Cer-
dan's group (Cerdan et al., 1990; Kunnecke et al.,
1993) suggest that this neuronal pyruvate recycling
pathway may preferentially utilize substrates released
by glial cells since the pathway was more prominent in
brain tissue incubated with labeled acetate or 3-hydro-
xybutyrate as substrates, than with labeled glucose. It
is well documented that neurons require an external
supply of substrates to maintain TCA cycle activity
since they lack the anaplerotic enzyme pyruvate car-
boxylase (Shank et al., 1985; Yu et al., 1983), and that
the need for substrates is supplied by tracking from
astrocytes (Hertz and Schousboe, 1988; Shank and
Campbell, 1984; Westergaard et al., 1995).
 M.C. McKenna et al. / Neurochemistry International 36 (2000) 451±459
Another potentially very important role of mME is
the production of NADPH which could be utilized to
maintain intramitochondrial glutathione in its reduced
state. A de®ciency of glutathione leads to mitochon-
drial damage (Jain et al., 1991; Mizui et al., 1992) and
is thought to have a role in several neurodegenerative
diseases (Bains & Shaw, 1997; Dringen et al., 1999;
Sian et al., 1994; So®c et al., 1992). The fraction of
glutathione in mitochondria is a small proportion of
the total glutathione in the brain (Bolanos et al., 1996;
Mizui et al., 1992). Recent studies underscore the key
role of intramitochondrial glutathione in survival of
neurons during hypoxia, ischemia or other insults
(Bolanos et al., 1996). Dringen et al. (1999) have
demonstrated that neurons are more ecient at using
cysteine and the dipeptide Cys±Gly as glutathione pre-
cursors than astrocytes. However, the rapid turnover
of neuronal glutathione would render these cells more
dependent on continuous biosynthesis and reduction
of glutathione (Dringen et al., 1999). Evidence sup-
porting the involvement of mME in maintaining intra-
mitochondrial reduced glutathione levels has recently
been reported (Vogel et al., 1999).
It should be noted that a role of mME in maintain-
ing reduced glutathione is not totally dissociated from
the potential role of this enzyme in pyruvate recycling
since the need for formation of substrates via this
pathway would be greatest during conditions such as
hypoxia, ischemia, and excitotoxicity when the protec-
tive activity of reduced intramitochondrial glutathione
is also most essential. The ability to continuously
maintain sucient energy metabolism to preserve
membrane potential and other functional activity is
crucial for the survival of brain cells (Heales et al.,
1999). It is well documented that metabolic inhibition
and/or dysfunction is a feature of many pathological
conditions and can directly lead to neurodegeneration.
The enrichment of mME in synaptic mitochondria
would serve to protect synaptic terminals from oxi-
dative damage and maintain neuronal activity.
Acknowledgements
These studies were supported in part by NIH Grant
HD 16596. We thank Craig Stewart for his technical
assistance in isolating some of the synaptic mitochon-
dria.
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