Chronic acetyl-L-carnitine alters brain energy metabolism and increases noradrenaline and serotonin content in healthy mice
Acetyl-L-carnitine (ALCAR), the short-chain ester of carnitine, is a common dietary supplement readily available in health food stores, claimed to improve energy levels and muscle strength. ALCAR has numerous effects on brain and muscle metabolism, protects against neurotoxic insults and may be an effective treatment for certain forms of depression. However, little is known about the effect of chronic ALCAR supplementation on the brain metabolism of healthy mice. Here, we investigated ALCAR's effect on cerebral energy and neurotransmitter metabolism after supplementing the drinking water of mice with ALCAR for 25 days, providing a daily dose of about 0.5 g/kg. Thereafter the animals were injected with [1-(13)C]glucose, and (13)C incorporation into and levels of various metabolites were quantified in extracts of the hippocampal formation (HF) and cortex using (1)H- and (13)C-nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography (HPLC). Increased glucose levels were detected in both regions together with a decreased amount of [3-(13)C]lactate, but no alterations in incorporation of (13)C derived from [1-(13)C]glucose into the amino acids glutamate, GABA and glutamine. These findings are consistent with decreased metabolism of glucose to lactate but not via the TCA cycle. Higher amounts of the sum of adenosine nucleotides, phosphocreatine and the phosphocreatine/creatine ratio found in the cortex of ALCAR-treated mice are indicative of increased energy levels. Furthermore, ALCAR supplementation increased the levels of the neurotransmitters noradrenaline in the HF and serotonin in cortex, consistent with ALCAR's potential efficacy for depressive symptoms. Other ALCAR-induced changes observed included reduced amounts of GABA in the HF and increased myo-inositol. In conclusion, chronic ALCAR supplementation decreased glucose metabolism to lactate, resulted in increased energy metabolite and altered monoamine neurotransmitter levels in the mouse brain.
Chronic acetyl-L-carnitine alters brain energy metabolism and increases
noradrenaline and serotonin content in healthy mice
Olav B. Smeland
, Tore W. Meisingset
, Karin Borges
, Ursula Sonnewald
Dept. of Neuroscience, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway
Dept. of Pharmacology, School of Biomedical Sciences, The University of Queensland, St. Lucia QLD 4072, Australia
Received 11 January 2012
Received in revised form 10 April 2012
Accepted 10 April 2012
Available online 23 April 2012
-carnitine (ALCAR), the short-chain ester of carnitine, is a common dietary supplement readily
available in health food stores, claimed to improve energy levels and muscle strength. ALCAR has numer-
ous effects on brain and muscle metabolism, protects against neurotoxic insults and may be an effective
treatment for certain forms of depression. However, little is known about the effect of chronic ALCAR sup-
plementation on the brain metabolism of healthy mice. Here, we investigated ALCAR’s effect on cerebral
energy and neurotransmitter metabolism after supplementing the drinking water of mice with ALCAR for
25 days, providing a daily dose of about 0.5 g/kg. Thereafter the animals were injected with [1-
C incorporation into and levels of various metabolites were quantiﬁed in extracts of the hip-
pocampal formation (HF) and cortex using
C-nuclear magnetic resonance (NMR) spectroscopy
and high performance liquid chromatography (HPLC). Increased glucose levels were detected in both
regions together with a decreased amount of [3-
C]lactate, but no alterations in incorporation of
derived from [1-
C]glucose into the amino acids glutamate, GABA and glutamine. These ﬁndings are con-
sistent with decreased metabolism of glucose to lactate but not via the TCA cycle. Higher amounts of the
sum of adenosine nucleotides, phosphocreatine and the phosphocreatine/creatine ratio found in the cor-
tex of ALCAR-treated mice are indicative of increased energy levels. Furthermore, ALCAR supplementa-
tion increased the levels of the neurotransmitters noradrenaline in the HF and serotonin in cortex,
consistent with ALCAR’s potential efﬁcacy for depressive symptoms. Other ALCAR-induced changes
observed included reduced amounts of GABA in the HF and increased myo-inositol. In conclusion, chronic
ALCAR supplementation decreased glucose metabolism to lactate, resulted in increased energy metabo-
lite and altered monoamine neurotransmitter levels in the mouse brain.
! 2012 Elsevier Ltd. All rights reserved.
-carnitine (ALCAR), the short-chain ester of carnitine, is
endogenously produced within mitochondria and peroxisomes and
is involved in the transport of acetyl-moieties across the mem-
branes of these organelles. Therefore, ALCAR can affect lipid, carbo-
hydrate and amino acid, as well as energy metabolism. Since the
early 1990s, much attention has been directed towards the possi-
ble role of ALCAR as a therapeutic agent in aging, disorders of the
brain and its mechanism of action (reviewed by Jones et al.
(2010)). Plasma and CSF concentrations of ALCAR increase after
oral administration, and the compound is transported across the
blood brain barrier by the organic cation/carnitine transporter
OCTN2 (Kido et al., 2001; Parnetti et al., 1992). Carnitine transport-
ers from the OCTN family are present on both neurons and astro-
cytes (Januszewicz et al., 2010, 2009). In rat brain cells, the
acetyl moiety of ALCAR may be used for the biosynthesis of acetyl-
choline (Dolezal and Tucek, 1981), fatty acids (Ricciolini et al.,
1998), and amino acids (Scaﬁdi et al., 2010). Acute ALCAR admin-
istration altered rat brain energy homeostasis by increasing phos-
phocreatine and decreasing lactate and inorganic phosphate levels
(Aureli et al., 1990), stimulating glycogen synthesis (Aureli et al.,
1998), and regionally increasing [
measured by autoradiography in certain brain regions (Ori et al.,
2002). Also, chronic ALCAR increased [
in similar regions of the rat brain (Freo et al., 2009), as well as the
0197-0186/$ - see front matter ! 2012 Elsevier Ltd. All rights reserved.
Abbreviations: ALCAR, acetyl-
-carnitine; HF, hippocampal formation; DA,
dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; GABA,
5-HIAA, 5-hydroxyindoleacetic; 5-HT, serotonin; HVA, homovanillic acid; MDMA,
3,4-methylenedioxymethamphetamine; NA, noradrenaline; NAA, N-acetylaspar-
tate; NMR, nuclear magnetic resonance; PDH, pyruvate dehydrogenase; TCA,
Corresponding author. Address: Dept. Neuroscience, Faculty of Medicine, Olav
Kyrresgt. 9, N-7030 Trondheim, Norway. Tel.: +47 73 590492; fax: +47 73 598655.
E-mail addresses: email@example.com (O.B. Smeland), firstname.lastname@example.org
(T.W. Meisingset), email@example.com (K. Borges), firstname.lastname@example.org (U.
Joint ﬁrst authors.
Neurochemistry International 61 (2012) 100–107
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/nci
enzyme activities of the sodium potassium and the calcium mag-
nesium ATP-ases by about 30% (Villa et al., 2011).
ALCAR has both antioxidant and anti-apoptotic properties (Ishii
et al., 2000; Liu et al., 1993) and can protect against various neuro-
toxic insults such as excessive glutamate (Forloni et al., 1994 ) and
amyloid-ß exposure (Virmani et al., 2001). Treatment with ALCAR
improved neurological outcome and energy metabolism in various
animal models of ischemia (Aureli et al., 1994; Rosenthal et al.,
1992) and aging (reviewed by Ames and Liu (2004), Jones et al.
(2010)). In light of such promising ﬁndings in animal models, AL-
CAR has been tested in several clinical trials for various disorders.
Small beneﬁcial clinical effects have been reported for Alzheimer’s
disease in a meta-analysis of double blind randomized controlled
clinical trials (Montgomery et al., 2003). However, a Cochrane re-
view found no convincing effects (Hudson and Tabet, 2003). There
is some evidence from small clinical trials with 24–67 patients that
ALCAR can decrease symptoms of depression in the elderly (Garzya
et al., 1990; Tempesta et al., 1987), ﬁbromyalgia patients (Rossini
et al., 2007) and patients with minimal hepatic encephalopathy
(Malaguarnera et al., 2011).
Most psychiatric treatment is centered on altering monoamine
neurotransmitter homeostasis. There are few reports on the effect
of ALCAR on monoamine metabolism in the brain and they are re-
stricted to rats: acute ALCAR pretreatment before 3,4-methylen-
edioxymethamphetamine (MDMA, ecstasy) injection prevented
mitochondrial damage and also loss of serotonin (5-HT) (Alves
et al., 2009). In the control group acute ALCAR only increased the
level of the serotonin metabolite 5-hydroxyindoleacetic acid (5-
HIAA), but not 5-HT, in one of six investigated brain regions,
namely the prefrontal cortex. In a model of attention deﬁcit hyper-
active disorder, chronic ALCAR treatment restored the noradrena-
line (NA) level and the serotonin turnover ratio (5-HIAA/5-HT) in
speciﬁc brain regions towards the values of normal young rats. In
normal young rats the only change was an increased serotonin
turnover ratio in the cingulate cortex (Adriani et al., 2004).
In summary, there is a lack of studies on the effect of chronic
ALCAR administration on brain metabolism in healthy non-elderly
animals other than rats. Speciﬁcally, this is the case for monoamine
neurotransmitters; information undoubtedly important for under-
standing, evaluating and exploiting chronic ALCAR as a therapeutic
agent. Here, we give a comprehensive metabolomic analysis which
includes; glucose, energy, amino acid and monoamine neurotrans-
mitter metabolism after chronic ALCAR supplementation in mice,
showing for the ﬁrst time increases of noradrenaline and serotonin
levels in the healthy brain.
2. Materials and methods
C) and D
O (99.9%) were purchased from
Cambridge Isotope Laboratories (Woburn, MA, USA), ethylene gly-
col from Merck (Darmstadt, Germany), and acetyl-
Sigma–Aldrich (St. Louis, MO, USA). All other chemicals were of the
purest grade available from local commercial sources.
The Norwegian National Animal Research Authority and the lo-
cal ethics committee approved the experimental procedures. Four-
teen male four week old NMRI mice with an average weight of
25 ± 0.7 g (n = 14; Taconic, Ejby, Denmark) were used in the exper-
iment. Nine mice were used in the intervention group and ﬁve as
controls. All animals were maintained under standard laboratory
conditions on a 12/12 h light/dark cycle (lights on at 7AM), at a
constant temperature of 22 "C and a humidity of 60%. Animals
were housed in individual cages with free access to food and water.
The mice were acclimatized to the above conditions for 1 week be-
fore the start of the experiments.
In accordance with previous studies, mice received drinking
water containing ALCAR (1.5 g/L, pH adjusted to 6) ad libitum
(Hagen et al., 2002; Mollica et al., 2001) drinking an average of
9.9 ± 0.6 ml per day (calculated by weighing the water bottles),
which provides a daily ALCAR dose of 496 ± 21 mg/kg body weight.
Control mice receiving ﬁltered tap water drank 7.8 ± 1.2 ml per
day. There was no difference in the weight gain between the
groups. Both groups received saline injections (10 ml/kg i.p.) three
times a week until the last day, due to the fact that they were part
of a bigger study examining the kindling epilepsy model (data not
shown). After 25 days, mice were injected with 543 mg/kg
C]glucose (i.p., using a 0.3 M solution) and 15 min later were
subjected to microwave ﬁxation of the head at 4 kW for 1.7 s
(Model GA5013, Gerling Applied Engineering, Modesto, CA, USA),
a time point that ensures substantial label incorporation without
washout (Hassel et al., 1995). The brains were removed and the
cortices and the HFs were dissected and stored at !80 "C till
extraction. The tissue samples were homogenized in 0.7% per-
chloric acid using a Vibra Cell sonicator (Model VCX 750, Sonics
& Materials, Newtown, CT, USA), potassium perchlorate precipi-
tated by neutralization with 1 M KOH, and supernatants containing
2.3. High performance liquid chromatography (HPLC)
To determine the total amount of glutathione, the samples were
analyzed using a Hewlett Packard 1100 System (Agilent Technolo-
gies, Palo Alto, CA, USA) with ﬂuorescence detection, after deriva-
tization with o-phtaldialdehyde and a standard curve derived
from a standard solution of glutathione (Geddes and Wood,
1984). The components were separated with a ZORBAX SB-C18 col-
umn (4.6 " 150 mm, 3.55 micron, Agilent) using 50 mM sodium
phosphate buffer (pH 5.9) with 2.5% tetrahydrofurane and metha-
nol/tetrahydrofurane (98.75%/1.25%) as eluent.
To quantitate the levels of monoamines NA, 5-HT and dopamine
(DA) and acid metabolites 3,4-dihydroxyphenylacetic acid (DO-
PAC), 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid
(HVA), an Agilent 1200 system with an electrochemical detector
(Coulochem III, ESA, Sunnyvale, CA, USA) was used. Components
were separated with an Eclipse XDB-C18 column (4.6 " 150 mm,
5 micron, Agilent) with an aqueous mobile phase (0.90 mL/min)
containing 90 mM NaH
, 50 mM citric acid, 0.1 mM EDTA,
0.5 mM octanesulfonic acid and 7% methanol. A standard curve de-
rived from standard solutions of monoamines was run repeatedly
at 15 samples intervals for quantitation.
C-nuclear magnetic resonance (NMR) spectroscopy
Lyophilized samples were dissolved in 200
0.1% ethylene glycol as an internal standard for quantitation and
re-adjusted to pH 6.5–7.5 Spectra were recorded at 25 "C using a
BRUKER DRX-600 for HF and a BRUKER DRX-500 spectrometer
for cortex samples (both BRUKER Analytik GmbH, Rheinstetten,
H-NMR spectra were acquired on the same instru-
ments with the following parameters: pulse angle of 90", spectral
width of 32 K data points and the number of scans was 128 and
1024 for cortical and HF extracts respectively. Acquisition time
was 1.36 s and relaxation delay 10 s. Water suppression was
achieved by applying a low-power pre-saturation pulse at the
water frequency. Proton decoupled
C-NMR spectra were ob-
tained using a 30" pulse angle and 30 kHz spectral width with
64 K data points employing an acquisition time of 1.08 s and a
O.B. Smeland et al. / Neurochemistry International 61 (2012) 100–107
relaxation delay of 0.5 s. The number of scans needed to obtain an
appropriate signal to noise ratio was typically 25,000 for cortex
and 35,000 for HF samples.
Relevant peaks in the spectra were identiﬁed and integrated
using XWIN NMR software (BRUKER BioSpin GmbH, Rheinstetten,
Germany). The total amounts and
C labeling of metabolites were
quantiﬁed from the areas under the peaks using ethylene glycol as
an internal standard. Correction for natural abundance, nuclear
Overhauser enhancement and relaxation effects relative to the
internal standard were applied to all relevant resonances. For a
C-NMR spectrum see Fig. 1.
2.5. Labeling patterns from metabolism of [1-
The metabolism of [1-
C]glucose is described in Fig. 2. Via gly-
C]glucose is converted to [3-
C]pyruvate, which can
be further converted to [3-
C]acetyl-CoA may enter the TCA cycle
through condensation with oxaloacetate to form citrate. Eventually
the TCA cycle intermediate
C]ketoglutarate is formed,
which is the precursor of [4-
C]glutamate. Thereafter, [4-
tamate may be transformed to [4-
C]glutamine in astrocytes due
to the astrocyte speciﬁc localization of glutamine synthetase
(Norenberg and Martinez-Hernandez, 1979) and to [2-
in GABAergic neurons. If
C]ketoglutarate is further con-
verted in the TCA cycle it gives rise to different labeling patterns
in these amino acids (Alvestad et al., 2008). In astrocytes
C]pyruvate can be converted to [3-
C]oxaloacetate via pyru-
vate carboxylase (Patel, 1974) which can lead to the formation of
C]glutamine and [4-
2.6. Data analysis
Statistics were performed using the 2-tailed unpaired Student’s
t-test, and p 6 0.05 was regarded as signiﬁcant. Data are presented
as mean ± SD. Due to technical problems, the number of analyses
varied between analytical methods.
3.1. Glucose-derived and related metabolites
Injection of [1-
C]glucose led to labeling of many metabolites
as shown in a typical
C-NMR spectrum from cerebral cortex
Fig. 1. Typical
C-NMR-spectrum of a tissue extract of cerebral cortex from a ALCAR-treated mouse. Peak assignment: 1: creatine C-3; 2: aspartate C-3; 3: taurine C-3; 4:
GABA C-2; 5: glutamate C-4 (peak is truncated); 6: glutamine C-4; 7: glutamate C-3; 8: glutamine C-3.
Fig. 2. Schematic presentation of isotopomers derived from [1-
C]glucose via [2-
C]acetyl-CoA. Black circles indicate
C labeling from pyruvate dehydrogenase (PDH) and
C labeling from pyruvate carboxylase (PC). Only the ﬁrst turn of the TCA cycle is illustrated.
102 O.B. Smeland et al. / Neurochemistry International 61 (2012) 100–107
extract of an ALCAR-treated mouse (Fig. 1). Only the part of the
spectrum containing the C-4 and C-3 glutamate and glutamine,
C-2 aspartate and C-2 and C-3 GABA peaks is shown. The labeling
patterns of metabolites labeled from [1-
C]acetyl-CoA from the ﬁrst turn of the TCA cycle are depicted
in Fig. 2. The C-2 and C-3 positions are labeled in the second and
subsequent turns of the TCA cycle (not shown).
The total and labeled amounts of glucose, lactate, alanine and
amino acids were quantiﬁed using
In the HF the total amounts of glucose and alanine were increased
by 43% (p = 0.05) and decreased by 31% (p = 0.004), respectively
after 25 days of ALCAR treatment (Fig. 3). Furthermore, lactate con-
tent in the ALCAR treated group compared to control was decreased
by 38% (p = 0.08). In the cortex the glucose content was increased
by 55 % (p = 0.01), but amounts of lactate and alanine remained un-
changed. The levels of [3-
C]lactate were lower in both brain re-
gions investigated by 63% (p = 0.04) in the HF and 37% (p = 0.04)
in the cortex. Moreover, [1-
C]glucose content was 38% higher in
the HF of mice treated with ALCAR (p = 0.01) and a similar trend
was seen in cortex (22 % increase, p = 0.06). The amounts of
C]alanine were not signiﬁcantly changed in either brain region.
Following ALCAR administration the amounts of GABA were sig-
niﬁcantly decreased in the HF by 32 % (p = 0.01), but were un-
changed in the cortex (Fig. 4), consistent with regional speciﬁc
metabolic alterations by ALCAR as previously described (Freo
et al., 2009; Ori et al., 2002). The amounts of glutamate and gluta-
mine were unaffected in both brain regions. The same was true for
the levels of [4-
C]glutamine and [2-
C]glucose-derived metabolites labeled in the ﬁrst turn of
the TCA cycle via [2-
C]acetyl-CoA. Consequently, the percent
C enrichment of glutamate and glutamine was unaltered. The
same was the case for GABA since the individual values for
C]GABA content in the ALCAR treated animals were smaller
than those in the control group, even though this difference was
not signiﬁcant (p = 0.30). No alterations in the pyruvate carboxyla-
tion to dehydrogenation ratios were observed between control and
ALCAR treated animals (results not shown).
3.2. Other metabolites
ALCAR treatment signiﬁcantly increased the levels of several
energy metabolites in the cortex, such as the sum of adenosine
nucleotides AMP, ADP, and ATP by 23% (p = 0.03), phosphocreatine
by 66% (p = 0.04), and myo-inositol by 30% (p = 0.02, Table 1). The
ratio of phosphocreatine/creatine (PCr/ratio) was about twofold
higher in the cortex of mice treated with ALCAR (p = 0.03). The
amounts of creatine, succinate, glutathione, choline-containing
compounds and N-acetylaspartate (NAA) were unchanged in both
brain regions investigated. The levels of nicotinamide adenine
dinucleotide (NAD) were not quantiﬁable in the HF and were unaf-
fected in the cortex.
3.3. Monoamine neurotransmitters
ALCAR administration resulted in a signiﬁcant 25% increase in
the level of NA in the HF (p = 0.03), but not in cortex (Fig. 5).
Fig. 3. Glucose metabolism in brain extracts of hippocampal formation and cortex from control (white bars) and ALCAR-treated mice (black bars). Levels of glucose, lactate
and alanine quantiﬁed by
H-NMR spectroscopy (A and B) and [1-
C]lactate and [3-
C]alanine obtained by
C-NMR spectroscopy are shown (C and D). Data
represent mean ± SD of four control mice and nine ALCAR-treated mice for hippocampal formation, and ﬁve control mice and nine ALCAR-treated mice for cortex.
p 6 0.05,
6 0.005, statistically signiﬁcantly different from control group, analyzed by Student’s t-test.
O.B. Smeland et al. / Neurochemistry International 61 (2012) 100–107
Moreover, 5-HT concentrations were signiﬁcantly increased in the
cortex by 20% (p = 0.005), with a similar trend in the HF, although
without statistical signiﬁcance (22%, p = 0.09). The serotonin turn-
over rate (5-HIAA/5-HT) was decreased in the cortex from 0.49 to
0.37 (p = 0.04), a potential reason for the increase in serotonin lev-
els (Table 2). The following parameters measured were not affected
by ALCAR treatment; (1) DA and 5-HIAA amounts in both brain re-
gions and (2) neither DOPAC and HVA levels nor the ratios of DA
metabolites to DA, HVA/DA and DOPAC/DA, measures of DA
turnover, in cerebral cortex. The amounts of DOPAC and HVA in
the HF were below our detection level for quantitation.
The most important ﬁndings of this work are (1) alterations in
glucose and lactate metabolism, (2) increases in high energy phos-
phates and (3) myo-inositol, as well as (4) increases in the levels of
Fig. 4. Amounts of amino acids glutamate, GABA and glutamine (A and B) and
C-labeled amino acids derived from [1-
C]glucose (C and D) in brain extracts of hippocampal
formation and cortex from control (white bars) and ALCAR-treated mice (black bars). Data represent mean ± SD of ﬁve control mice and nine ALCAR-treated mice,
p 6 0.01,
statistically signiﬁcantly different from control group.
mol/g tissue) of metabolites in brain extracts of hippocampal formation and cortex from control and ALCAR-treated mice.
Hippocampal formation Cortex
Control ALCAR Control ALCAR
AMP + ADP + ATP 1.64 ± 0.97 1.83 ± 1.14 2.48 ± 0.50 3.07 ± 0.35
Creatine 8.64 ± 0.93 8.02 ± 0.96 9.57 ± 2.06 8.52 ± 1.08
Phosphocreatine 1.62 ± 1.20 2.02 ± 0.72 1.15 ± 0.61 1.91 ± 0.59
Creatine + phosphocreatine 10.26 ± 1.18 10.05 ± 0.97 10.72 ± 2.45 10.43 ± 0.74
PCr/Cr ratio 0.19 ± 0.15 0.26 ± 0.11 0.12 ± 0.06 0.23 ± 0.09
NADH n.d. n.d. 0.29 ± 0.07 0.36 ± 0.08
Succinate 0.55 ± 0.28 0.63 ± 0.12 0.41 ± 0.10 0.49 ± 0.18
Glutathione 1.30 ± 0.24 1.35 ± 0.30 1.16 ± 0.15 1.32 ± 0.15
1.72 ± 0.06 1.57 ± 0.18 1.78 ± 0.13 1.82 ± 0.14
Myo-inositol 10.12 ± 2.32 11.04 ± 2.13 5.86 ± 0.80 7.60 ± 1.40
N-acetylaspartate 5.51 ± 0.34 5.50 ± 0.67 7.36 ± 0.50 7.79 ± 0.97
All metabolite levels were quantiﬁed using
H-NMR spectroscopy, with the exception of glutathione (HPLC) and myo-inositol (
C-NMR spectroscopy). Abbreviations:
: choline-containing compounds; NADH: nicotinamide adenine dinucleotide; n.d: not determined; PCr/Cr ratio: phosphocreatine/creatine ratio. Data represent
mean ± SD of ﬁve control mice and nine ALCAR-treated mice, and were analyzed with the Student’s t-test.
p 6 0.05, statistically signiﬁcant difference from control mice.
104 O.B. Smeland et al. / Neurochemistry International 61 (2012) 100–107
some monoamine neurotransmitters in hippocampus and/or cor-
tex after chronic ALCAR supplementation in mice. The implications
of our results are discussed below in relation to previous ﬁndings.
4.1. Glucose metabolism
In this study we found that chronic dietary supplementation
with ALCAR increased the amount of glucose in both cerebral cor-
tex and HF. Similarly, Ori et al. (2002) and Freo et al. (2009) re-
ported that acute and chronic ALCAR administration increased
-glucose labeling in various brain regions. Also, AL-
CAR treatment did not alter serum concentrations of glucose
(Aureli et al., 1998; Freo et al., 2009; Ori et al., 2002) or the uptake
-glucose into rat brain slices (Tanaka et al.,
2003). This indicates that ALCAR does not alter brain glucose up-
take. In agreement with the reported reduced amounts of lactate
in the normal adult and aged rat brain after acute ALCAR treatment
(Aureli et al., 1990), we also found decreased [3-
in mice, but no changes in the concentrations of TCA cycle-derived
C-labeled glutamate, glutamine or GABA. Increased glucose
concentration and unchanged TCA cycle activity have also been ob-
served by Nilsen et al. (2011) in mice with reduced
dehydrogenase complex activity. Taken together our results indi-
cate that ALCAR administration reduced glucose metabolism to
lactate without changing glucose metabolism via the TCA cycle,
which lead to higher brain glucose concentrations. Similarly, acute
ALCAR treatment counteracted production of lactate in rat and dog
models of ischemia (Aureli et al., 1994; Rosenthal et al., 1992).
4.2. Energy metabolites
The levels of the sum of AMP + ADP + ATP, phosphocreatine and
the PCr/Cr ratio were signiﬁcantly increased in the cortex in our
mice supplemented with ALCAR compared to control. The fact that
the sum of creatine + phosphocreatine was unaltered indicates that
the increase in phosphocreatine reﬂects a larger reservoir of high-
energy phosphates. Furthermore, the increased amount of the sum
of AMP + ADP + ATP may reﬂect increased concentration of ATP
(see studies below). We could not distinguish the phosphorylation
state of adenosine due to resonance overlap in the
Our ﬁndings are in line with several studies demonstrating that AL-
CAR treatment increased the levels of phosphocreatine and re-
duced the amount of free organic phosphate in the adult and old
rat brain (Aureli et al., 1990), elevated the amounts of phosphocre-
atine and ATP in a rat model of ischemia (Aureli et al., 1994), pre-
vented ATP depletion in neuroblastoma cells exposed to beta-
amyloid (Dhitavat et al., 2002), and ameliorated the decrease of
ATP in rat hippocampus after ischemia (Al-Majed et al., 2006).
Taken together, the data indicate that ALCAR treatment improves
the capacity of the brain to produce high-energy phosphates and
reduces anaerobic glucose metabolism. This potential neuroprotec-
tive ability may prove to be beneﬁcial in conditions with disturbed
The level of myo-inositol was increased in the cortex of mice
supplemented with ALCAR in the present study. Likewise, ALCAR
has been shown to prevent myo-inositol depletion in a streptomy-
cin induced rat model of diabetic neuropathy (Nakamura et al.,
1998; Stevens et al., 1996). Myo-inositol is important for the syn-
thesis of PIP2, IP3, DAG and complex signaling phospholipids. If
myo-inositol is increased due to a decreased synthesis of these
molecules, it would have widespread functional consequences. It
is also an important osmolyte in the brain and is the most abun-
dant form of inositol (Fisher et al., 2002). Myo-inositol has been
found to be particularly enriched in astrocytes (Brand et al.,
1993). Thus, the increased content of this metabolite appears to re-
ﬂect an effect of ALCAR on this cell type. The effect on myo-inositol
suggests that ALCAR can have a positive inﬂuence in disorders
where water homeostasis is altered.
4.4. Monoamine neurotransmitters
ALCAR supplementation enhanced the amounts of NA and 5-HT
in healthy mice. An increased level of NA was detected in the HF,
whereas the amount of 5-HT was increased in the cortex, accompa-
nied by a decreased 5-HIAA/5-HT ratio, the latter reﬂecting re-
duced serotonin turnover. It is of interest in this context that NA
increases oxidative metabolism in cultured astrocytes and freshly
dissociated astrocytes express the relevant receptor subtypes
(Hertz et al., 2010). In several small trials, ALCAR was found to
Fig. 5. Amounts (nmol/g tissue) of monoamines and their metabolites in brain
extracts of hippocampal formation and cortex from control (white bars) and ALCAR-
treated mice (black bars) quantiﬁed using HPLC. Abbreviations: Noradrenaline (NA),
3,4-dihydroxyphenylacetic acid (DOPAC), dopamine (DA), 5-hydroxyindoleacetic
acid (5-HIAA), homovanillic acid (HVA), serotonin (5-HT). Data represent mean ± SD
of ﬁve control mice and seven ALCAR-treated mice for hippocampal formation, and
ﬁve control mice and nine ALCAR-treated mice for cortex.
p 6 0.005 statistically
signiﬁcantly different from control group, analyzed with the Student’s t-test.
‘‘Turnover’’ of dopamine and serotonin in brain extracts of hippocampal formation
and cortex from control and ALCAR-treated mice.
Hippocampal formation Cortex
(n = 5)
(n = 7)
(n = 5)
(n = 9)
DOPAC/DA n.d. n.d. 0.16 ± 0.04 0.15 ± 0.02
HVA/DA n.d. n.d. 0.15 ± 0.06 0.20 ± 0.04
5-HIAA/5-HT 1.31 ± 0.43 0.98 ± 0.16 0.49 ± 0.09 0.37 ± 0.05
The ratios of monoamine neurotransmitters to their metabolites were calculated as
a measure for their turnover rates calculated using data from HPLC. Abbreviations:
n.d. not determined. Data represent mean ± SD, and were analyzed with the Stu-
p 6 0.05, statistically signiﬁcant difference from control mice.
O.B. Smeland et al. / Neurochemistry International 61 (2012) 100–107
be beneﬁcial for patients with depressive disorders and related
conditions (Martinotti et al., 2011; Pettegrew et al., 2002;
Soczynska et al., 2008; Zanardi and Smeraldi, 2006). The reported
increase of 5-HT and NA levels presented here validate further
study of these neurotransmitters, their metabolism and receptors
in relation to ALCAR and depressive disorders, and may provide
rationale for an antidepressant effect of ALCAR.
In conclusion, we report that ALCAR supplementation in healthy
mice resulted in improved energy metabolism and sparing of glu-
cose in both HF and cortex. The amounts of the monoamines NA
and 5-HT were increased in the HF and cortex respectively. These
new insights warrant further studies of ALCAR in clinical settings
especially for diseases known to involve energy deﬁcits or mono-
amine neurotransmitter derangements.
6. Author disclosure
OB. Smeland, TW. Meisingset, K Borges and U. Sonnewald have
no conﬂicts of interest.
The technical assistance of Lars Evje is gratefully appreciated.
The authors wish to thank Elvar M. Eyjolfsson, Sivert H. Sandvik
and Mats L. Skillingstad for their help with the experiments. This
collaboration was supported by a travel scholarship to US and KB
by The University of Queensland. KB is currently funded by the
Australian National Health and Research Council Grant No.
Adriani, W., Rea, M., Baviera, M., Invernizzi, W., Carli, M., Ghirardi, O., Caprioli, A.,
Laviola, G., 2004. Acetyl-
-carnitine reduces impulsive behaviour in adolescent
rats. Psychopharmacology 176, 296–304.
Al-Majed, A.A., Sayed-Ahmed, M.M., Al-Omar, F.A., Al-Yahya, A.A., Aleisa, A.M., Al-
Shabanah, O.A., 2006. Carnitine esters prevent oxidative stress damage and
energy depletion following transient forebrain ischaemia in the rat
hippocampus. Clin. Exp. Pharmacol. Physiol. 33, 725–733.
Alves, E., Binienda, Z., Carvalho, F., Alves, C.J., Fernandes, E., de Lourdes Bastos, M.,
Tavares, M.A., Summavielle, T., 2009. Acetyl-
-carnitine provides effective
in vivo neuroprotection over 3,4-methylenedioximethamphetamine-induced
mitochondrial neurotoxicity in the adolescent rat brain. Neuroscience 158,
Alvestad, S., Hammer, J., Eyjolfsson, E., Qu, H., Ottersen, O.P., Sonnewald, U., 2008.
Limbic structures show altered glial-neuronal metabolism in the chronic phase
of kainate induced epilepsy. Neurochem. Res. 33, 257–266.
Ames, B.N., Liu, J., 2004. Delaying the mitochondrial decay of aging with
acetylcarnitine. Ann. N.Y. Acad. Sci. 1033, 108–116.
Aureli, T., Di Cocco, M.E., Puccetti, C., Ricciolini, R., Scalibastri, M., Miccheli, A.,
Manetti, C., Conti, F., 1998. Acetyl-
-carnitine modulates glucose metabolism
and stimulates glycogen synthesis in rat brain. Brain Res. 796, 75–81.
Aureli, T., Miccheli, A., Di Cocco, M.E., Ghirardi, O., Giuliani, A., Ramacci, M.T., Conti,
F., 1994. Effect of acetyl-
-carnitine on recovery of brain phosphorus
metabolites and lactic acid level during reperfusion after cerebral ischemia in
the rat-study by
H-NMR spectroscopy. Brain Res. 643, 92–99.
Aureli, T., Miccheli, A., Ricciolini, R., Di Cocco, M.E., Ramacci, M.T., Angelucci, L.,
Ghirardi, O., Conti, F., 1990. Aging brain: effect of acetyl-
on rat brain energy and phospholipid metabolism. A study by
spectroscopy. Brain Res. 526, 108–112.
Brand, A., Richter-Landsberg, C., Leibfritz, D., 1993. Multinuclear NMR studies on the
energy metabolism of glial and neuronal cells. Dev. Neurosci. 15, 289–298.
Dhitavat, S., Ortiz, D., Shea, T.B., Rivera, E.R., 2002. Acetyl-
against amyloid-beta neurotoxicity: roles of oxidative buffering and ATP levels.
Neurochem. Res. 27, 501–505.
Dolezal, V., Tucek, S., 1981. Utilization of citrate, acetylcarnitine, acetate, pyruvate
and glucose for the synthesis of acetylcholine in rat brain slices. J. Neurochem.
Fisher, S.K., Novak, J.E., Agranoff, B.W., 2002. Inositol and higher inositol phosphates
in neural tissues: homeostasis, metabolism and functional signiﬁcance. J.
Neurochem. 82, 736–754.
Forloni, G., Angeretti, N., Smiroldo, S., 1994. Neuroprotective activity of acetyl-
carnitine: studies in vitro. J. Neurosci. Res. 37, 92–96.
Freo, U., Dam, M., Ori, C., 2009. Cerebral metabolic effects of acetyl-
rats during aging. Brain Res. 1259, 32–39.
Garzya, G., Corallo, D., Fiore, A., Lecciso, G., Petrelli, G., Zotti, C., 1990. Evaluation of
the effects of
-acetylcarnitine on senile patients suffering from depression.
Drugs Exp. Clin. Res. 16, 101–106.
Geddes, J.W., Wood, J.D., 1984. Changes in the amino acid content of nerve endings
(synaptosomes) induced by drugs that alter the metabolism of glutamate and
gamma-aminobutyric acid. J. Neurochem. 42, 16–24.
Hagen, T.M., Liu, J., Lykkesfeldt, J., Wehr, C.M., Ingersoll, R.T., Vinarsky, V.,
Bartholomew, J.C., Ames, B.N., 2002. Feeding acetyl-
-carnitine and lipoic acid
to old rats signiﬁcantly improves metabolic function while decreasing oxidative
stress. Proc. Natl. Acad. Sci. U. S. A. 99, 1870–1875.
Hassel, B., Sonnewald, U., Fonnum, F., 1995. Glial-neuronal interactions as studied
by cerebral metabolism of [2-
C]acetate and [1-
C]glucose: an ex vivo
NMR spectroscopic study. J. Neurochem. 64, 2773–2782.
Hertz, L., Lovatt, D., Goldman, S.A., Nedergaard, M., 2010. Adrenoceptors in brain:
cellular gene expression and effects on astrocytic metabolism and [Ca(2+)]I.
Neurochem. Int. 57, 411–420.
Hudson, S., Tabet, N., 2003. Acetyl-
-carnitine for dementia. Cochrane Database Syst.
Ishii, T., Shimpo, Y., Matsuoka, Y., Kinoshita, K., 2000. Anti-apoptotic effect of acetyl-
-carnitine and I-carnitine in primary cultured neurons. Jpn. J. Pharmacol. 83,
Januszewicz, E., Bekisz, M., Mozrzymas, J.W., Nalecz, K.A., 2010. High afﬁnity
carnitine transporters from OCTN family in neural cells. Neurochem. Res. 35,
Januszewicz, E., Pajak, B., Gajkowska, B., Samluk, L., Djavadian, R.L., Hinton, B.T.,
Nalecz, K.A., 2009. Organic cation/carnitine transporter OCTN3 is present in
astrocytes and is up-regulated by peroxisome proliferators-activator receptor
agonist. Int. J. Biochem. Cell Biol. 41, 2599–2609.
Jones, L.L., McDonald, D.A., Borum, P.R., 2010. Acylcarnitines: role in brain. Prog.
Lipid Res. 49, 61–75.
Kido, Y., Tamai, I., Ohnari, A., Sai, Y., Kagami, T., Nezu, J., Nikaido, H., Hashimoto, N.,
Asano, M., Tsuji, A., 2001. Functional relevance of carnitine transporter OCTN2
to brain distribution of
-carnitine and acetyl-
-carnitine across the blood-brain
barrier. J. Neurochem. 79, 959–969.
Liu, Y., Rosenthal, R.E., Starke-Reed, P., Fiskum, G., 1993. Inhibition of postcardiac
arrest brain protein oxidation by acetyl-
-carnitine. Free Radical Biol. Med. 15,
Malaguarnera, M., Bella, R., Vacante, M., Giordano, M., Malaguarnera, G., Gargante,
M.P., Motta, M., Mistretta, A., Rampello, L., Pennisi, G., 2011. Acetyl-
reduces depression and improves quality of life in patients with minimal
hepatic encephalopathy. Scand. J. Gastroenterol. 46, 750–759.
Martinotti, G., Andreoli, S., Reina, D., Di Nicola, M., Ortolani, I., Tedeschi, D., Fanella, F.,
Pozzi, G., Iannoni, E., D’Iddio, S., Prof, L.J., 2011. Acetyl-
-carnitine in the treatment
of anhedonia, melancholic and negative symptoms in alcohol dependent
subjects. Prog. Neuropsychopharmacol. Biol. Psychiatry 35, 953–958.
Mollica, M.P., Iossa, S., Soboll, S., Liverini, G., 2001. Acetyl-
stimulates oxygen consumption and biosynthetic function in perfused liver of
young and old rats. Cell. Mol. Life Sci. 58, 477–484.
Montgomery, S.A., Thal, L.J., Amrein, R., 2003. Meta-analysis of double blind
randomized controlled clinical trials of acetyl-
-carnitine versus placebo in the
treatment of mild cognitive impairment and mild Alzheimer’s disease. Int. Clin.
Psychopharmacol. 18, 61–71.
Nakamura, J., Koh, N., Sakakibara, F., Hamada, Y., Hara, T., Sasaki, H., Chaya, S.,
Komori, T., Nakashima, E., Naruse, K., Kato, K., Takeuchi, N., Kasuya, Y., Hotta, N.,
1998. Polyol pathway hyperactivity is closely related to carnitine deﬁciency in
the pathogenesis of diabetic neuropathy of streptozotocin-diabetic rats. J.
Pharmacol. Exp. Ther. 287, 897–902.
Nilsen, L.H., Shi, Q., Gibson, G.E., Sonnewald, U., 2011. Brain [U-
metabolism in mice with decreased
-ketoglutarate dehydrogenase complex
activity. J. Neurosci. Res. 89, 1997–2007.
Norenberg, M.D., Martinez-Hernandez, A., 1979. Fine structural localization of
glutamine synthetase in astrocytes of rat brain. Brain Res. 161, 303–310.
Ori, C., Freo, U., Pizzolato, G., Dam, M., 2002. Effects of acetyl-
-carnitine on regional
cerebral glucose metabolism in awake rats. Brain Res. 951, 330–335.
Parnetti, L., Gaiti, A., Mecocci, P., Cadini, D., Senin, U., 1992. Pharmacokinetics of IV
and oral acetyl-
-carnitine in a multiple dose regimen in patients with senile
dementia of Alzheimer type. Eur. J. Clin. Pharm. 42, 89–93.
Patel, M.S., 1974. The relative signiﬁcance of CO2-ﬁxing enzymes in the metabolism
of rat brain. J. Neurochem. 22, 717–724.
Pettegrew, J.W., Levine, J., Gershon, S., Stanley, J.A., Servan-Schreiber, D.,
Panchalingam, K., McClure, R.J., 2002.
P-MRS study of acetyl-
treatment in geriatric depression: preliminary results. Bipolar Disord. 4, 61–66.
Ricciolini, R., Scalibastri, M., Kelleher, J.K., Carminati, P., Calvani, M., Arduini, A.,
1998. Role of acetyl-
-carnitine in rat brain lipogenesis: implications for
polyunsaturated fatty acid biosynthesis. J. Neurochem. 71, 2510–2517.
Rosenthal, R.E., Williams, R., Bogaert, Y.E., Getson, P.R., Fiskum, G., 1992. Prevention
of postischemic canine neurological injury through potentiation of brain energy
metabolism by acetyl-
-carnitine. Stroke 23, 1312–1318.
Rossini, M., Di Munno, O., Valentini, G., Bianchi, G., Biasi, G., Cacace, E., Malesci, D.,
La Montagna, G., Viapiana, O., Adami, S., 2007. Double-blind, multicenter trial
-carnitine with placebo in the treatment of ﬁbromyalgia
patients. Clin. Exp. Rheumatol. 25, 182–188.
106 O.B. Smeland et al. / Neurochemistry International 61 (2012) 100–107
Scaﬁdi, S., Fiskum, G., Lindauer, S.L., Bamford, P., Shi, D., Hopkins, I., McKenna, M.C.,
2010. Metabolism of acetyl-
-carnitine for energy and neurotransmitter
synthesis in the immature rat brain. J. Neurochem. 114, 820–831.
Soczynska, J.K., Kennedy, S.H., Chow, C.S., Woldeyohannes, H.O., Konarski, J.Z.,
McIntyre, R.S., 2008. Acetyl-
-carnitine and alpha-lipoic acid: possible
neurotherapeutic agents for mood disorders? Exp. Opin. Investig. Drugs 17,
Stevens, M.J., Lattimer, S.A., Feldman, E.L., Helton, E.D., Millington, D.S., Sima, A.A.,
Greene, D.A., 1996. Acetyl-
-carnitine deﬁciency as a cause of altered nerve
myo-inositol content, Na, K–ATPase activity, and motor conduction velocity in
the streptozotocin–diabetic rat. Metab. Clin. Exp. 45, 865–872.
Tanaka, M., Nakamura, F., Mizokawa, S., Matsumura, A., Matsumura, K., Watanabe,
Y., 2003. Role of acetyl-
-carnitine in the brain: revealed by bioradiography.
Biochem. Biophys. Res. Commun. 306, 1064–1069.
Tempesta, E., Casella, L., Pirrongelli, C., Janiri, L., Calvani, M., Ancona, L., 1987.
acetylcarnitine in depressed elderly subjects. A cross-over study vs. placebo.
Drugs Exp. Clin. Res. 13, 417–423.
Villa, R.F., Ferrari, F., Gorini, A., 2011. Effect of in vivo
administration on ATP-ases enzyme systems of synaptic plasma membranes
from rat cerebral cortex. Neurochem. Res. 36 (8), 1372–1382.
Virmani, M.A., Caso, V., Spadoni, A., Rossi, S., Russo, F., Gaetani, F., 2001. The action
-carnitine on the neurotoxicity evoked by amyloid fragments and
peroxide on primary rat cortical neurones. Ann. N.Y. Acad. Sci. 939, 162–178.
Zanardi, R., Smeraldi, E., 2006. A double-blind, randomised, controlled clinical trial
-carnitine vs. amisulpride in the treatment of dysthymia. Eur.
Neuropsychopharmacol. 16, 281–287.
O.B. Smeland et al. / Neurochemistry International 61 (2012) 100–107