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ORIGINAL PAPER
Metabolism, Compartmentation, Transport and Productionof Acetate in the Cortical Brain Tissue Slice
Caroline Rae • Aurelie D. Fekete •
Mohammed A. Kashem • Fatima A. Nasrallah •
Stefan Broer
Received: 30 January 2012 / Revised: 12 July 2012 / Accepted: 13 July 2012 / Published online: 1 August 2012
� Springer Science+Business Media, LLC 2012
Abstract Acetate is a two carbon intermediate in metab-
olism. It is an accepted marker of astrocytic metabolism, and
a substrate for production of metabolites such as glutamine,
glutamate and GABA. However, anomalies exist in the
current explanations of compartmentation and metabolism
of acetate. Here, we investigated these anomalies by exam-
ining transport, production and metabolism of acetate.
Acetate is a good substrate for the neuronal monocarboxylate
transporter MCT2 (KM = 2.58 ± 0.8) and the glial MCT1
but a poor substrate for the glial MCT4. Acetate is accu-
mulated by brain cortical tissue slices to concentrations in
excess of those in the media, suggesting active transport,
possibly via the sodium dependent SMCT. [2-13C]Acetate is
produced from [3-13C]pyruvate, [3-13C]lactate and
[1-13C]glucose with the rate of production related to acetyl-
CoA levels, which is likely generated in a ubiquitous cyto-
solic compartment via acetyl-CoA hydrolase. Citrate
breakdown occurs in response to demand for acetyl-CoA
units; this citrate is not derived from acetate carbon but its
fate is influenced by acetate levels. Finally, use of acetate is
altered by levels of nicotinamide or NAD?. This suggests
that metabolism of acetate is controlled rigorously at the
enzyme level, via changes in the acetylation status of acetyl-
CoA synthetase and is not regulated by restriction of uptake.
Keywords Acetate � 13C NMR spectroscopy �Neuron-glia interactions
Introduction
Glucose is the mandatory substrate for energy supply in the
brain. Brain can also use the glucose catabolites lactate,
pyruvate and acetate. Both pyruvate [1] and lactate have
been shown to be adequate substrates for brain metabolism
(Fig. 1) although there is some debate about the full extent
to which they can subserve brain activity [2, 3]. Acetate has
long been considered a marker for glial metabolism where
it is incorporated mostly into glutamine, with label also
reported from a small glutamate pool [4].
Acetate must be metabolized by conversion to acetyl-
CoA in a reaction catalysed by acetyl-CoA synthetase (E.C.
6.2.1.1); this reaction requires ATP and releases pyrophos-
phate. Two isoforms are known in mammalian cells [5].
AceCS2 is a mitochondrial isoform channeling acetate into
the TCA cycle, while AceCS1 is a cytosolic isoform, which
provides acetyl-CoA for fatty acid biosynthesis. Acetate
units (Fig. 1) are also used in the brain for lipid synthesis [6],
for acetylation of histone proteins in the nucleus [7], for
synthesis of the neurotransmitter acetylcholine [8] and for
many other acetylated compounds such as N-acetylaspartate,
N-acetylaspartylglutamate and acetylcarnitine.
Both AceCS isoforms are subject to complex layers of
regulation; the cytosolic form is increased by the presence
of acetate which may come from a variety of sources, such
as via blood from oxidation of ingested ethanol, from
histone deacetylases, or by the activity of the ubiquitous
acetyl-CoA hydrolase. AceCS2 levels are induced by
ketogenic conditions but the mechanisms of this induction
Special Issue: In Honor of Leif Hertz.
C. Rae (&) � A. D. Fekete � M. A. Kashem � F. A. Nasrallah
Neuroscience Research Australia, and Brain Sciences,
University of New South Wales, Barker St, Randwick,
NSW 2031, Australia
e-mail: [email protected]
S. Broer
Research School of Biology, The Australian National University,
Canberra, ACT 0200, Australia
123
Neurochem Res (2012) 37:2541–2553
DOI 10.1007/s11064-012-0847-5
remain unclear. In the brain AceCS activity is found in
both neurons and glia with a mostly mitochondrial distri-
bution, although a small but significant fraction of it is also
likely to be found in the cytosol [9, 10].
AceCS isoforms are also subjected to post-translational
regulation via acetylation under control of the deacetylat-
ing silent information regulator protein family, the Sirtuins.
Specifically, AceCS1 is regulated by SIRT1 and AceCS2
by SIRT3 [11]. Sirtuins are under cellular control via
NAD?; nicotinamide is an inhibitor of the deacetylase
reaction such that low-energy status may turn off energy
consuming reactions such as AceCS. In the brain, SIRT1 is
located mostly, if not exclusively, in neurons [12].
Acetate is taken up and metabolized by neurons in
culture [13] as well as by hippocampal nerve terminals [8]
and in whole rat brain [14]. It is a poor substrate for cortical
tissue slices and needs to be used in conjunction with
substrates such as glucose for adequate slice respiration
[15, 16]. In vivo, acetate is rapidly incorporated into glu-
tamine, GABA, glutamate and aspartate, with the highest
specific activity in glutamine.
Acetate is a substrate for the monocarboxylate trans-
porters (MCTs). These are members of the SLC16 family
and are proton symporters with a wide range of monocarb-
oxylate substrates. MCT1 is a low affinity transporter found
mainly in the blood–brain barrier. Lower levels are found in
astrocytes throughout the brain. MCT2 on the other hand is
generally a higher affinity transporter found in neurons in the
post-synaptic density [17, 18]. MCT4 is a low affinity
transporter and is selectively expressed in astrocytes [19].
Recently another lactate transporter family has been
described which is sodium dependent. SMCT1 (sodium-
dependent monocarboxylate transporter 1; SLC5A8) is a
high-affinity transporter found in neurons, while SMCT2
(SLC5A12) has been reported in glial cells [20].
Waniewski and Martin [21] suggested that the reason
acetate is a relatively poor substrate for neurons was due to
transporter affinity, based on poor uptake of acetate by syn-
aptosomal fractions compared to astrocytes. The uptake of
acetate by neurons was not measured in this work, and their
findings disagreed with the earlier results of Clarke et al. who
showed that acetate is taken up avidly by synaptosomes,
where, rather than being metabolized, it is used to acetylate
synaptosomal proteins. This acetate is released upon activa-
tion of sodium channels, suggesting that acetylation reactions
are involved in dynamic regulation of synaptic activity [22].
Here, we investigated the basis of the compartmentation of
acetate metabolism by studying its metabolic fates in Guinea
pig brain cortical tissue slices along with a range of competing
substrates, and its transport in Xenopus oocytes to clarify
which MCT isoforms are capable of transporting acetate.
Methods
Materials
Guinea pigs (Dunkin-Hartley), weighing 400–800 g, were fed
ad libitum on standard Guinea pig/rabbit pellets, with fresh
cabbage leaves and lucerne hay roughage. Animals were main-
tained on a 12 h light/dark cycle. All experiments were con-
ducted in accordance with the guidelines of the National Health
and Medical Research Council of Australia and were approved
by the institutional (UNSW) Animal Care Ethics Committee.
Sodium [3-13C]pyruvate, sodium [3-13C]L-lactate, sodium
[2-13C]acetate and [1-13C]D-glucose and sodium [13C]formate
Fig. 1 Cartoon showing metabolism of acetate in the brain
2542 Neurochem Res (2012) 37:2541–2553
123
were purchased from Cambridge Isotope Laboratories Inc
(Andover, MA, USA). Sodium pyruvate, sodium lactate,
sodium acetate, nicotinamide, NAD? and ethylenediamine-
tetraacetic acid (EDTA) were purchased from Sigma-Aldrich
(St Louis, MO). All other reagents were of Analytical Reagent
grade.
Other data were taken from experiments that have
already been published, including those with AR-C122982,
a potent, specific MCT inhibitor [23], those with ligands
active at GABA(B) receptors [24] and those examining the
effect of blocking transport of alanine [25]. 1H MRS
spectra obtained from these experiments were used to
measure the amount of 13C-labelled acetate present using
the resolved 13C-satellite resonance at d = 1.78 ppm. The
data from a previously published experiment examining
metabolism of 5 mM [1-13C]D-glucose in the presence of
the inhibitor 4-hydroxycyanocinnamate (7 mM) [23] was
also examined for acetate production.
Acetate Uptake in Xenopus Oocytes
Uptake of [14C]acetate in Xenopus laevis oocytes was
carried out as described in detail previously [26–28]. A
series of concentrations were used and the uptake rate
calculated using [14C]acetate standards to obtain KM. In
flux experiments the activity of non-injected oocytes was
subtracted at all substrate concentrations to correct for
endogenous uptake of monocarboxylates. Endogenous
uptake of monocarboxylates in oocytes is Na?-dependent
and therefore is not observed when monocarboxylate
uptake is monitored by pH-sensitive microelectrodes [29].
Preparation of Brain Cortical Tissue Slices
Following cervical dislocation, guinea pig brains were
removed from the cranial vault and 350 lm paraxial cortical
slices were obtained using a McIlwain tissue chopper. The
slices were then washed three times in a modified Krebs-
Henseleit buffer [15], resuspended for 1 h in fresh buffer
containing 10 mmol/L unlabelled glucose and gassed with
95 % O2/5 % CO2 in a shaking water bath, maintained at
37 �C, to allow metabolic recovery [30]. Slices were then
washed three times in glucose-free buffer and resuspended in
fresh buffer with the substrate of choice.
Synthesis of Acetate from Various Substrates in Brain
Cortical Tissue Slices
To determine labeling patterns from each substrate a
number of experiments were made where label fate and
pool sizes were monitored in the presence and absence of
competing monocarboxylates. The scope of these experi-
ments was limited by the requirement to maintain brain
slice respiration at maximal rates so that changes in
metabolism did not reflect simple limitations on mito-
chondrial respiration rates due to substrate unavailability.
The following experiments were performed:
To determine the effect of competing acetate with
pyruvate:
1. 2 mM sodium [3-13C]pyruvate
2. 2 mM sodium [3-13C]pyruvate plus 5 mM sodium
acetate
3. 2 mM sodium pyruvate plus 5 mM sodium
[2-13C]acetate
Five mM acetate was chosen to fully saturate both MCT1
and MCT2. Historically, this concentration of acetate has
been shown to be metabolized mostly in an astrocyte pool if
metabolism is limited to a short time frame [15].
To determine the effect of competing acetate with
lactate:
1. 0.74 mM sodium [3-13C]lactate and 2 mM D-glucose
2. 0.74 mM sodium [3-13C]lactate and 2 mM D-glucose
plus 5 mM sodium acetate
3. 0.74 mM sodium lactate and 2 mM D-glucose plus
5 mM sodium [2-13C]acetate.
This concentration of lactate was chosen to compete
mainly at MCT2 (KM = 0.74 mM, Table 1) and less so at
MCT1 (KM = 5.6 mM, Table 1). We added 2 mM glucose
to allow the slices to maintain their respiratory rate at
adequate levels as 0.74 mM lactate is an insufficient
amount of substrate to maintain respiration [30].
To determine the effect of competing lactate with
pyruvate:
1. 2 mM sodium [3-13C]pyruvate
2. 2 mM sodium [3-13C]pyruvate plus 0.74 mM sodium
lactate
3. 2 mM sodium pyruvate plus 0.74 mM sodium
[3-13C]lactate
To determine the effect of modulating levels of NAD?
or nicotinamide on acetate metabolism:
1. 5 mM D-glucose and 5 mM sodium [2-13C]acetate
2. 5 mM D-glucose and 5 mM sodium [2-13C]acetate plus
250 lM NAD?
3. 5 mM D-glucose and 5 mM sodium [2-13C]acetate plus
250 lM nicotinamide.
All experiments with lactate and pyruvate as substrates
were incubated for 60 min, while the experiment with
glucose was incubated for 90 min.
To measure citrate in the 13C spectrum, 4 mM EDTA was
added to the samples to chelate paramagnetic species and
sharpen the resonance [15] and 13C NMR spectra of these
samples were reacquired. This was not successful with the
Neurochem Res (2012) 37:2541–2553 2543
123
experiment with lactate and pyruvate as the samples had
deteriorated due to repeated removal from the NMR tubes
and signal to noise was poor. Instead the citrate concentration
was estimated from the broadened metal-bound resonance.
The number of samples was N = 4 in all cases.
Preparation of Samples and NMR Analysis
On completion of the incubation period, slices were removed
from the incubation buffer by rapid filtration and extracted in
methanol/chloroform according to the method of Le Belle
[31]. Extracts were lyophilized, and the pellet retained for
protein estimation by the Lowry technique. Lyophilized
supernatants were stored at -20 �C until required for NMR
analysis. Samples were resuspended in 0.60 mL D2O con-
taining 2 mM sodium [13C]formate as an internal intensity
and chemical shift reference (13C d 171.8). Fully relaxed 1H
and 1H[13C-decoupled] spectra (total cycle of 30 s, compris-
ing 24 s relaxation delay, 4 s water suppression and *1 s
acquisition time), WURST-40 [32] with a 112-step phase
cycle [33], decoupling during acquisition) were obtained at
800.15 MHz on a Bruker Avance-III 800 MHz spectrometer
equipped with a TCI cryoprobe probe, followed by 13C
[1H-decoupled] spectra (800 transients, cycle of 4 s com-
prising 2 s relaxation delay and *2 s acquisition time, con-
tinuous WALTZ-65 decoupling, 185180 data points).
Assignments were made as described previously [34].13C [1H-Decoupled] spectra were Fourier transformed
using 1 Hz exponential line-broadening and peak areas
were determined by integration using standard Bruker
software (TOPSPIN, Version 1.3, or 2.5) following base-
line correction. Peak areas were adjusted for nuclear
Overhauser effect, saturation and natural abundance effects
and quantified by reference to the area of the internal
standard resonance of [13C]formate. Glu C3 was not
quantified due to resonance overlap with pyruvate signal.
Metabolite pool sizes (lactate, alanine, GABA, glutamate,
glutamine and aspartate) were determined by integration of
resonances in fully relaxed 800 MHz 1H[13C-decoupled]
spectra using [13C]formate as the internal intensity refer-
ence. The amount of acetate labeled with 13C at C2 was
also determined from the 13C satellite at d = 1.776 ppm in
the fully-relaxed 1H NMR spectrum.
Experimental data (N = 4) are given as means (standard
deviation). Statistical analysis was done using ANOVA for
comparing experimental interventions with control (N = 4,
followed, only where statistical significance was indicated by
Scheffe F test, by a nonparametric (Mann–Whitney U) test
(Statview Student)). Significance was assumed at a = 0.05.
Principal Components Analysis
In order to discover what metabolic factors influence the
presence of labeled acetate in the cortical tissue slice we
constructed a statistical model using data from previously
published experiments [35, 36], but also including integrals
from the CH313C satellites of acetate C2. Fifteen variables
from 163 experiments were used as input for a principal
components model using Simca P? (v11.5, Umetrics,
Umea, Sweden). Each dataset for a particular manipulation
was imported as the relative change from the average value
obtained from the control group for that particular experi-
ment. Data were univariance scaled to standardize variance
between the high and low concentration metabolites [37],
to ensure that the 13C labelling and steady state pool size
concentrations equally contributed to the model.
Table 1 Substrate affinities for the various monocarboxylate
transporters
Transporter Substrates KM (mM)
MCT1 Acetate 1.6 ± 0.4 [68]
D, L-b-hydroxybutyrate 12.5 ± 0.26 [69]
Lactate 5.6 [70]
D-lactate [60 [71]
16 ± 6 [68]
L-lactate 4.38 ± 0.74 [71]
3.5 ± 0.4 [28]
5 ± 1 [68]
Pyruvate 1.01 ± 0.06 [28]
2.09 ± 0.37 [71]
MCT2 Acetate 2.58 ± 0.8 Fig. 2
D,L-b-Hydroxybutyrate 1.2 ± 0.2 [27]
L-lactate 0.74 ± 0.07 [27]
1.0 ± 0.2 [72]
Pyruvate 0.08 ± 0.01 [27, 71]
0.025 [73]
MCT4 Acetate Non-saturable Fig. 2
L-b-Hydroxybutyrate 824 ± 64 [71]
D-b-Hydroxybutyrate 130 ± 9.6 [71]
Lactate 17–34 [72]
D-lactate 519 ± 35 [71]
L-lactate 28 ± 4 [71]
33.7 ± 4.9 [74]
Pyruvate 153 ± 6 [71]
SMCT1 Acetate 1.6 ± 0.1 [75]
2.46 ± 0.89 [76]
L-b-Hydroxybutyrate 2.33 ± 0.17 [40]
D-b-Hydroxybutyrate 1.44 ± 0.12 [40]
L-lactate 0.184 ± 0.008 [40]
0.235 ± 0.24 [76]
D-lactate 1.088 ± 0.068 [40]
0.742 ± 0.33 [76]
Pyruvate 0.387 ± 0.043 [40]
SMCT2 L-lactate 16.9 ± 3.7 [77]
2544 Neurochem Res (2012) 37:2541–2553
123
Results
Acetate Uptake by MCT2 and MCT4
Acetate was found to be a good substrate for MCT2 with a
Michaelis–Menten constant comparable to that of MCT1
(Fig. 2a). Acetate is a very poor substrate for MCT4 (Fig. 2b).
The potential use of acetate by any cell type in the brain
will depend on the availability of transport proteins and
metabolic enzymes that can convert acetate to acetyl-CoA.
In situ hybridization data from the Allen Brain Atlas
(http://mouse.brain-map.org) suggest that expression of the
mitochondrial acetyl-CoA synthetase 2 (AceCS2) in the
brain is extremely low. The cytosolic AceCS1, by contrast,
is found in significant amounts in all brain areas. In terms
of transport, the brain expresses MCT1, MCT2, MCT4 and
SMCT1. It appears that MCT2 and SMCT1 are largely
neuronal transporters, while MCT1 and MCT4 are
expressed in astrocytes. Apart from MCT4, all of these are
capable of transporting acetate with similar KM-values
(Fig. 2, Table 1). There is an argument that although the
KM for acetate is low enough, capacity is limited by low
levels of MCT2 expression in neurons. However, it is
evident from the residual 13C acetate signal that acetate
was accumulated by at least one brain compartment to
levels higher than in the medium. If this accumulation were
limited to just the astrocytic pool which is thought to me-
tabolise acetate (estimated at around 30 % of brain vol-
ume) this would mean that the concentration of acetate in
this compartment would be in the order of [30 mM.
Thus, it can be reasonably expected that acetate could
penetrate both astrocytes and neurons, but that this may
involve transporters other than MCTs.
Acetate Synthesis and Presence in NMR Spectra
Acetate is reliably present in 1H NMR spectra of cortical
tissue slices, appearing as a singlet resonance which is co-
resonant with the bCH2 of GABA at 1.8 ppm (Fig. 3). It is
difficult to know whether this acetate is endogenous or
whether it might arise due to a putative breakdown of N-
acetylaspartate in the extraction process. There is a doublet
(1JCH = 127.1 ± 0.03 Hz) which is collapsed by broad-
band 13C decoupling of the 1H spectrum, indicating that a
portion of the acetate present is also labeled with 13C at C2
(i.e. in excess of natural abundance at 1.1 %). This doublet
is present in 1H NMR spectra of brain slice extracts fol-
lowing incubation with [3-13C]pyruvate, [3-13C]lactate and
[1-13C]glucose and indicates that acetate labeled at C2 is
synthesized from these substrates (Fig. 3).
Acetate Compartmentation—Pyruvate and Lactate
The effects of adding 0.74 mM L-lactate to 2 mM pyruvate
are shown in Fig. 4. The amount of label incorporated into
acetate C2 was reduced by addition of unlabelled lactate
(control acetate C2 = 1.04 (0.21) cf. 0.76 (0.12) lmol/h/
100 mg protein.). When the label is transferred to lactate
by use of 0.74 mM [3-13C]lactate and 2 mM pyruvate label
was also incorporated into acetate C2. Acetate is therefore
also made in a compartment accessible to 0.74 mM lactate.
Scrutiny of the predicted vs actual labeling of the other
isotopomers revealed that the labeling of Gln C4 from
[3-13C]pyruvate was not significantly different in the
A
B
Fig. 2 Transport of acetate by monocarboxylate transporters.
Oocytes were injected with cRNA encoding MCT2 (20 ng) or
MCT4 (10 ng). After 3–5 days, uptake of [14C]acetate was measured
over a period of 20 min at the indicated concentrations. Each datapoint represents the mean ± SD uptake activity of 8–10 oocytes
Neurochem Res (2012) 37:2541–2553 2545
123
presence (0.11 (0.03) lmol/h/100 mg protein) or absence
of lactate (0.11 (0.02) lmol/h/100 mg protein). It was not
possible to detect labeling of Gln C4 from 0.74 mM
[3-13C]lactate. This suggests that the astrocyte compart-
ment where glutamine is labeled is not accessible to lactate
under these conditions. Labelling of citrate was not sig-
nificantly affected by addition of lactate, and only a small
amount of citrate was labeled from [3-13C]lactate.
There was also little significant change in total metab-
olite pool sizes due to addition of lactate (Fig. 4b).
Acetate Compartmentation—Pyruvate and Acetate
Incubation of 2 mM [3-13C]pyruvate with 5 mM unlabelled
acetate resulted generally in increased labelling from
[3-13C]pyruvate into all isotopomers measured apart from Gln
C4 and Lac C3, which were unchanged (Fig. 5). Labelling of
acetate C2 increased significantly (0.72 (0.1) cf (1.16 (0.14)
lmol/h/100 mg protein as did labeling of citrate (1.47 (0.38)
cf 2.77 (0.5) lmol/h/100 mg protein) as well as Glu C2 and
GABA C2. When the label source was exchanged, incorpo-
ration into measured isotopomers dropped significantly. In
comparison with the control (2 mM [3-13C]pyruvate and no
acetate) experiment, little label was incorporated. Gln C4 was
the most highly labeled isotopomer and was labeled to
*30 % of the levels of the control experiment. Interestingly,
citrate was not labeled from acetate at all; it was not detected in
any spectra from the experiment with 2 mM pyruvate and
5 mM [2-13C]acetate, showing that citrate was not made from
acetyl-CoA which had been synthesized from acetate under
these circumstances.
The total pool size of glutamate and aspartate were sig-
nificantly increased by addition of [2-13C]acetate (Fig. 5b).
Acetate Compartmentation—Lactate and Acetate
Incubation of 0.74 mM [3-13C]L-lactate with 2 mM glu-
cose and unlabelled acetate (5 mM) had no significant
effect on the incorporation of label from [3-13C]L-lactate
into isotopomers of Glu, GABA or Asp (Fig. 6a). Incor-
poration of label into Gln C4 and Ala C3 was significantly
lower and there was no significant effect of acetate on label
incorporation into citrate. When the label source was
exchanged to [2-13C]acetate, label incorporation dropped
significantly. Label was incorporated above natural abun-
dance in Gln C4, Glu C4 and Ala C3.
Addition of acetate, either labeled or unlabelled, resul-
ted in a significant decrease in the total pool sizes of glu-
tamate, lactate, GABA, aspartate and alanine but had no
significant effect on the total pool size of glutamine.
Acetate Metabolism—Effect of NAD?
and Nicotinamide
When slices were incubated with 5 mM D-glucose and 5 mM
[2-13C]acetate, label was incorporated into Glu C4, GABA
C2 and Gln C4 to levels above natural abundance (Fig. 7).
Gln C4 was labeled more from 5 mM [2-13C]acetate in the
Fig. 3 Sections of fully-relaxed 600 MHz 1H NMR spectra of brain
cortical tissue slice extracts following metabolism of added 13C-
labelled substrates showing 13C-labelled acetate. Spectra in the toprow are [13C]-decoupled, while those in the bottom row are not. Left-most spectra are extracts following one hour of incubation with 2 mM
[3-13C]pyruvate [24], the middle spectra are extracts following
30 min of incubation with 10 mM [1-13C]glucose under depolarizing
(40 mM K?) conditions [23] and the right hand spectra are extracts
following 1 h of incubation with 8 mM [3-13C]lactate [16]. Fully
relaxed 1H and 1H[13C-decoupled] spectra (total cycle of 30 s,
comprising 24 s relaxation delay, 4 s water suppression and *2 s
acquisition time), WURST-40 [32] with a 112-step phase cycle [33],
decoupling during acquisition) were obtained at 600.13 MHz on a
Bruker DRX-600 spectrometer with a 5 mm dual 1H/13C probe
2546 Neurochem Res (2012) 37:2541–2553
123
presence of 5 mM glucose (11.7 %) than in the presence of
2 mM pyruvate (8.8 %) or 0.74 mM lactate and 2 mM
glucose (3.9 %).
Addition of 250 lM NAD? resulted in a significant
decrease in labelling from [2-13C]acetate into Gln C4 and
citrate. There was also a significant decrease in the total
pool size of aspartate and glutamine (Fig. 7b).
Addition of 250 lM nicotinamide resulted in significant
decreases in label incorporation into Glu C2 and C4 and
Gln C4. Total lactate was significantly increased (Fig. 7).
Acetate Production in the Presence of 4-
hydroxycyanocinnamate
The inhibitor 4-hydroxycyanocinnamate (4-CIN) is a broad
spectrum inhibitor of monocarboxylate transporters at
7 mM [38] and also of the mitochondria pyruvate carrier
[39]. It therefore has a significant effect on mitochondrial
metabolism. The effect of this inhibitor on production of
[2-13C]acetate from [1-13C]glucose is shown in Fig. 8.
Labelling of [2-13C]acetate was significantly decreased by
4CIN but the degree of reduction was relatively minor
compared to the reduction of label into mitochondrially
produced metabolites such as glutamate. Labeling of
metabolites produced in the cytosol, such as aspartate C2
and C3, was increased.
Pattern Recognition
Principal components analysis of the data from 163
experiments [35, 36] generated a two component model
accounting for 69 % of the variance in the data (45 and
24 %, respectively) with a Q2 of 60 %. Q2 is a measure of
the goodness of fit of the model. The two principal com-
ponents of the model are shown in Fig. 9, along with the
variables in a ‘‘Bi-plot’’. Inspection of this plot shows that
acetate C2 lies closest to Lactate C3 and is at the opposite
side of the plot to variables such as Glu C2 and C4, Asp C2
and C4 and Gln C4.
A
B
Fig. 4 Effects of competing 0.74 mM lactate with 2 mM pyruvate in
brain cortical tissue slices for 60 min. a Incorporation of 13C. b Total
metabolite pool sizes. Clear bars 2 mM [3-13C]pyruvate (control);
hatched bars 2 mM [3-13C]pyruvate plus 0.74 mM sodium L-lactate;
spotted bars 2 mM sodium pyruvate plus 0.74 mM [3-13C]- L-lactate.
* significantly different to control (P \ 0.05); # significantly different
to 2 mM [3-13C]pyruvate plus 0.74 mM sodium L-lactate
A
B
Fig. 5 Effects of competing 5 mM acetate with 2 mM pyruvate in
brain cortical tissue slices for 60 min. a Incorporation of 13C. b Total
metabolite pool sizes. Clear bars 2 mM [3-13C]pyruvate (control);
hatched bars 2 mM [3-13C]pyruvate plus 5.0 mM sodium acetate;
spotted bars 2 mM sodium pyruvate plus 5.0 mM [2-13C]- acetate.
* Significantly different to control (P \ 0.05); # significantly different
to 2 mM [3-13C]pyruvate plus 5.0 mM sodium acetate
Neurochem Res (2012) 37:2541–2553 2547
123
Discussion
Uptake of Acetate
Acetate proved to be a poor substrate for the low affinity
lactate transporter MCT4 with a KM [ 2 M, but a rela-
tively good substrate for the high-affinity (mostly) neuronal
transporter MCT2 (KM = 2.5 mM; Table 1, Fig. 2). This
value compares well with the reported KM for MCT1 of
1.6 mM (Table 1). It is therefore unlikely that failure of the
monocarboxylate transporter to take up acetate into neu-
rons is an adequate explanation for neuronal failure to use
acetate as a substrate with any efficacy [21]. Moreover,
neurons express the sodium dependent monocarboxylate
transporter (SMCT1, [40]), which has an affinity for acetate
comparable to that of the monocarboxylate transporters
MCT1 and MCT2 (Table 1). This transporter may there-
fore also be involved in accumulating acetate. SMCT1 is a
neuronal transporter but we cannot rule out involvement of
SMCT2. The affinity of SMCT2 for acetate as far as we can
determine has not yet been established, nor has the brain
cellular location of this transporter, although the transporter
is described as a low affinity transporter and has been
reported in Muller cells in the retina [20]. When slices are
incubated with 5 mM [2-13C]acetate, acetate is accumu-
lated to high levels, giving a calculated concentration of
8.85 (0.99) lmol/h/100 mg protein. However, only 9 % of
this acetate was actually metabolized into Krebs cycle
intermediates or their metabolites under the conditions
used (i.e. in competition with 2 mM pyruvate). It has
previously been reported that acetate is taken up into brain
after cisternal injection at high specific radioactivity to a
greater extent than lactate, pyruvate or glucose [41] and
also rapidly (within 2 min) incorporated into the lipid
fraction. The fact that acetate is accumulated further
A
B
Fig. 6 Effects of competing 5 mM acetate with 0.74 mM lactate in
brain cortical tissue slices for 60 min. a Incorporation of 13C. b Total
metabolite pool sizes. Clear bars 0.74 mM [3-13C]lactate (control);
hatched bars 0.74 mM [3-13C]lactate plus 5.0 mM sodium acetate;
spotted bars 2 mM sodium lactate plus 5.0 mM [2-13C]-acetate.
* Significantly different to control (P \ 0.05); # significantly different
to 0.74 mM [3-13C]lactate plus 5.0 mM sodium acetate. Slices were
also incubated with 2 mM D-glucose to provide adequate respiratory
support
A
B
Fig. 7 Effects of addition of NAD? and nicotinamide to incorpo-
ration of label supplied from 5 mM [2-13C]acetate. Slices were
incubated with 5 mM D-glucose and 5 mM [2-13C]acetate (clear bars;
control); 250 lM NAD? (hatched bars) or 250 lM nicotinamide
(spotted bars)
2548 Neurochem Res (2012) 37:2541–2553
123
supports a role of SMCT1 in brain acetate transport,
because MCTs only equilibrate intracellular and extracel-
lular pools of monocarboxylates in the absence of sub-
stantial pH gradients [38].
Other workers have shown uptake of acetate by neurons
[8, 13] and also by synaptosomes [42] in contrast to the
conclusions of Waniewski and Martin [21]. Here, we have
shown that the neuronal MCT2 is capable of transporting
acetate, plus provided corroborating evidence that brain
accumulates acetate, suggesting a role for active uptake in
acetate transport.
Production of Acetate
It is apparent from the data that acetate is synthesized in
non-trivial amounts from a range of different substrates
supplied to brain tissue cortical slices. While it might be
expected that acetate be synthesized from 2 mM pyruvate,
since this relatively high substrate concentration is likely to
cause accumulation of acetyl-CoA [43], acetate is also
synthesized from added lactate and also from glucose.
Pyruvate is the most efficacious substrate, producing the
most labeled acetate (mean value *0.35 lmol (h mmol
substrate 100 mg protein)-1, but lactate (used at 5 mM
concentration [23]) also produces non-trivial amounts
(*0.16 lmol (h mmol substrate 100 mg protein)-1,
exceeded when lower amounts of lactate (0.74 mM) are
used *0.54 lmol (h mmol substrate 100 mg protein)-1.
Glucose is also a good source when adjusted for the
amount of label (*0.22 lmol/(90 min mmol substrate
100 mg protein)-1; data taken from the control experiment
with 4-CIN using 5 mM glucose).
Acetate is made by hydrolysis of acety-lCoA by the
ubiquitous enzyme acetylCoA hydrolase [44] an enzyme
which may be product-stimulated by acetate [45, 46].
Acetyl-CoA can come from citrate via the ATP-citrate
lyase an enzyme which is expressed in both neurons and
glia [47].
Free acetate has been detected in brain by NMR in areas
of ischemia and ischemic penumbra [48] but not in normal
brain tissue in vivo. Levels of free acetate have been
measured by enzymatic assay at 1.51 ± 0.34 lmol/g tissue
in mouse brain, significantly higher than levels in liver
[49]. Acetate is reported as a common metabolite across a
Fig. 8 Effects of 4-hydroxycyanocinnamate on incorporation of 13C
in brain cortical tissue slices incubated with [1-13C]glucose for
90 min. Clear bars, control (N = 4); hatched bars, 7 mM 4-CIN
(N = 4). Values shown are means, while error bars show standard
deviations. * Significantly different to control
Fig. 9 Bi-plot showing principal components and variable clustering.
The figure shows the two principal components generated by the
model plus the individual variables and allows us to interpret the
observations in terms of the variables. Data from 163 experiments
(observations) [35, 36] was used as input, with 15 variables as shown
on the plot. Data were univariance scaled to standardize variance
between the high and low concentration metabolites [37], to ensure
that the 13C labelling and steady state pool size concentrations equally
contributed to the model, and were expressed as the relative change
from the average value obtained from the control group for that
particular experiment. Observations are shown as grey squares,
variables as black stars with labels
Neurochem Res (2012) 37:2541–2553 2549
123
range of primary cell cultures, including in neurons,
astrocytes and oligodendrocytes [50].
Acetate production was not significantly impacted by
inhibition of the mitochondrial pyruvate carrier with the
broad spectrum inhibitor 4-CIN (Fig. 8). This is in keeping
with the cytosolic location of acetyl-CoA hydrolase [46].
Inspection of Fig. 9 shows that acetate C2 is on the
opposite side of the principal components plot to variables
such as GluC2, C4, Asp C2 and C3 and Gln C4 indicating
that production of acetate occurs when Krebs cycle flux is
low. Acetate C2 is located near to lactate C3, indicating
that acetate production relates to pyruvate clearance. One
could go a step further and infer that acetate C2 production
relates to clearance of acetyl-CoA; i.e. high levels of
acetyl-CoA due to lowered flux in the Krebs cycle corre-
spond to high rates of acetate C2 production.
Metabolism of Acetate
It is apparent from the data that acetate is used to make
glutamine. This likely occurs in the so-called ‘‘small
compartment’’ and has been the subject of many previous
reports [15, 51–54]. Acetate has previously been reported
to label citrate. Citrate is problematic to study by NMR due
to its role in chelation of paramagnetic ions which results in
broadening of the NMR spectrum. It is possible to reduce
the linewidth of the two citrate CH2 carbons in the 13C
NMR spectrum through addition of the chelating agent
EDTA [55]. Here, we found that addition of lactate to
2 mM pyruvate did not significantly alter incorporation of
label into citrate, while addition of unlabelled acetate
almost doubled incorporation of label from [3-13C]pyru-
vate into citrate. By contrast, very little labelling of citrate
was detected when label was supplied as 5 mM
[2-13C]acetate (less than 4 % of that labelled by pyruvate).
Very little label was incorporated into citrate when
0.74 mM [3-13C]lactate was supplied as labelled substrate
and this incorporation was not altered by addition of ace-
tate. Incorporation of label into citrate from [2-13C]acetate,
although minimal, was reduced further by addition of
NAD? and nicotinamide.
Citrate has been shown to be synthesised in both neu-
rons and astrocytes [56], with astrocytes exporting a much
higher fraction of citrate than neurons [57]. Waagepetersen
has described two separate mitochondrial compartments in
astrocytes that metabolise acetate; one that produces glu-
tamine and one that preferentially produces citrate [58].
This is in agreement with earlier work showing that the
citrate, which enters the small metabolic pool that also
labels glutamine, does not fully interact with glutamine
labelling from acetate [41], and with work that has sug-
gested further subdivision of the ‘‘small’’ glutamate pool
[59]. Here, it is apparent that acetate is influencing the rate
of synthesis of citrate, since incorporation of label into
citrate from [3-13C]pyruvate doubles when unlabelled
5 mM acetate is present. It is also apparent that the added
acetate does not participate fully in this reaction, as very
little label is incorporated from added 5 mM [2-13C]ace-
tate. This is consistent with the subdivision of the small
glutamate pool proposed previously [41, 59]. There was no
significant increase in anaplerotic activity (as measured by
the difference between label incorporation into Asp C3 vs.
C2) in the presence of added acetate [0.23 (0.07) vs. 0.23
(0.01) lmol/h/100 mg protein] suggesting that the synthe-
sis of citrate was not a net cataplerotic process.
We would suggest that, rather than seeing a reduction in
citrate synthesis, we are seeing an alteration in the break-
down of citrate. Citrate is in an equilibrium with acetyl-
CoA, and with acetate [60]. Therefore, in situations where
acetate is plentiful (and by inference, acetyl-CoA) the
activity of ATP-citrate lyase is reduced. This may explain
why adding acetate appears to increase the amount of cit-
rate that is labeled; it does this by inhibiting the breakdown
of citrate.
Alteration of Acetate Metabolism by NAD?
and Nicotinamide
Modulation of enzyme activity by acetylation is emerging
as a major metabolic control mechanism [61]. The major
acetate metabolising enzymes, acetyl-CoA synthetases 1
and 2 are but one set of enzymes whose activity is con-
trolled by reversible acetylation. De-acetylaction of these
enzymes is under the control of the silent information
regulator enzymes, the NAD?-dependent deacetylases
known as sirtuins [62]. Nicotinamide is a potent non-
competitive inhibitor of SIRT1 [63]while NAD? is a sub-
strate although the effect of altering NAD? on SIRT
activity is not well understood[64]. Extracellular NAD?
has been shown to enter cells and influence metabolic
activity [65, 66]. Here, we conducted preliminary experi-
ments to determine whether or not nicotinamide or NAD?
could influence acetate metabolism.
Both molecules decreased the amount of acetate being
used as a substrate for glutamine synthesis along with a
reduction in label incorporation into citrate, which we have
postulated is due to increased breakdown of citrate by
ATP-citrate lyase. This points to a lower availability of
acetyl-CoA.
This is important information to consider when inter-
preting data acquired using the acetate/glucose model of
metabolism. The original papers describing the model all
contained a warning that using this approach from a strictly
compartmentalised viewpoint had limitations [55, 67]. The
possibilities opened up by considering changes in metab-
olism of acetate caused by cellular energy and acetylation
2550 Neurochem Res (2012) 37:2541–2553
123
status offer a potentially more interesting and useful
interpretation of data obtained using this model.
Summary
We have shown that acetate is transported by the neuronal
MCT2, that acetate is actively accumulated by tissue slices
but not catabolised to the same degree as other mono-
carboxylates such as pyruvate or lactate. A likely expla-
nation for the lack of enthusiasm for catabolism of acetate
resides in the control of metabolism via acetylation of
enzymes, in particular acetylation of acetyl-CoA synthetase
[5]. Our data suggest that levels of acetyl-CoA are strictly
controlled and that acetate and citrate are key metabolites
in this control.
AcetylCo-A is a central metabolite in many key reac-
tions (see Fig. 1) and plainly we have much further to go to
reach a full understanding of how these reactions are reg-
ulated in the brain.
Acknowledgments The authors are grateful to Dr Ann Kwan of the
School of Molecular Biosciences at the University of Sydney for
expert technical assistance. This work was supported by the Austra-
lian National Health and Medical Research Council (Grant #568767
to CR and Fellowship to CR #630516).
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