Báo cáo khoa học: Malonyl-CoA decarboxylase (MCD) is differentially regulated in subcellular compartments by 5¢AMP-activated protein kinase (AMPK) Studies using H9c2 cells overexpressing MCD and AMPK by adenoviral gene transfer technique potx

Malonyl-CoA decarboxylase (MCD) is differentially regulated in
subcellular compartments by 5¢AMP-activated protein kinase (AMPK)
Studies using H9c2 cells overexpressing MCD and AMPK by adenoviral gene transfer
technique
Nandakumar Sambandam, Michael Steinmetz, Angel Chu, Judith Y. Altarejos, Jason R. B. Dyck
and Gary D. Lopaschuk
Department of Pediatrics, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Canada
Malonyl-CoA, a potent inhibitor of carnitine pamitoyl
transferase-I (CPT-I), plays a pivotal role in fuel selection in
cardiac muscle. Malonyl-CoA decarboxylase (MCD) cata-
lyzes the degradation of malonyl-CoA, removes a potent
allosteric inhibition on CPT-I and thereby increases fatty
acid oxidation in the heart. Although MCD has several Ser/
Thr phosphorylation sites, whether it is regulated by AMP-
activated protein kinase (AMPK) has been controversial.
We therefore overexpressed MCD (Ad.MCD) and consti-
tutively active AMPK (Ad.CA-AMPK) in H9c2 cells, using
an adenoviral gene delivery approach in order to examine if
MCD is regulated by AMPK. Cells infected with Ad.CA-
AMPK demonstrated a fourfold increase in AMPK activity
as compared with control cells expressing green fluorescent
protein (Ad.GFP). MCD activity increased 40- to 50-fold
in Ad.MCD + Ad.GFP cells when compared with
Ad.GFP control. Co-expressing AMPK with MCD fur-
ther augmented MCD expression and activity in
Ad.MCD + Ad.CA-AMPK cells compared with the
Ad.MCD + Ad.GFP control. Subcellular fractionation
further revealed that 54.7 kDa isoform of MCD expression
was significantly higher in cytosolic fractions of
Ad.MCD + Ad.CA-AMPK cells than of the Ad.MCD +
Ad.GFP control. However, the MCD activities in cytosolic

fractions were not different between the two groups.
Interestingly, in the mitochondrial fractions, MCD activity
significantly increased in Ad.MCD + Ad.CA-AMPK cells
when compared with Ad.MCD + Ad.GFP cells. Using
phosphoserine and phosphothreonine antibodies, no
phosphorylation of MCD by AMPK was observed. The
increase in MCD activity in mitochondria-rich fractions of
Ad.MCD + Ad.CA-AMPK cells was accompanied by an
increase in the level of the 50.7 kDa isoform of MCD protein
in the mitochondria. This differential regulation of MCD
expression and activity in the mitochondria by AMPK may
potentially regulate malonyl-CoA levels at sites nearby
CPT-I on the mitochondria.
Keywords: malonyl-CoA decarboxylase; AMPK; cardiac
cells.
Malonyl-CoA is a potent inhibitor of carnitine palmitoyl
transferase-I (CPT-I), thereby playing a pivotal role in fuel
selection in cardiac muscle [1]. CPT-I, localized on the outer
mitochondrial membrane, is the rate-limiting enzyme of
fatty acid transport into mitochondria for b-oxidation [2–4].
As b-oxidation of fatty acids contributes the majority of
energy produced by the normal aerobic heart [5,6], malonyl-
CoA has a key role in regulating cardiac energy metabolism.
Tissue levels of malonyl-CoA are determined by its rate of
synthesis by acetyl-CoA carboxylase (ACC) and by its rate
of degradation by malonyl-CoA decarboxylase (MCD) [1].
Various physiological and pathological conditions result
in rapid changes in malonyl-CoA levels [7–9]. For instance,
malonyl-CoA levels drop rapidly and dramatically during
ischemia and reperfusion, which is associated with a

significant increase in fatty acid oxidation [8]. Similarly,
rapid maturation of fatty acid oxidation in the developing
heart is associated with a significant decrease in malonyl-
CoA levels in the myocardium [7]. While decreased synthesis
of malonyl-CoA by ACC is partly responsible for these
changes in malonyl-CoA, a simultaneous degradation by
MCD also has an important role in lowering malonyl-CoA
levels [10].
MCD was originally identified in the uropygial gland of
the goose [11]. We also showed MCD to be highly expressed
in mammalian cardiac muscle [12], and provided evidence
to suggest that cardiac MCD plays an important role in
regulating fatty acid metabolism in the heart [10,13].
Regulation of MCD occurs both at the level of transcription
and post-translation [14,15]. MCD has several serine and
threonine residues that can potentially be phosphorylated.
Previous studies in our lab and other groups have shown
Correspondence to G. Lopaschuk, 423 Heritage Medical Research
Building, University of Alberta, Edmonton, Alberta T6G 2S2,
Canada. Fax: + 1 780 492 9753, Tel.: + 1 780 4922170,
E-mail:
Abbreviations: ACC, acetyl-CoA carboxylase; AICAR, 5-amino-
imidazole-4-carboxamide riboside; AMPK, 5¢AMP-activated protein
kinase; CPT-I, carnitine pamitoyl transferase-I; GFP, green fluores-
cent protein; Itu, 5¢-iodotubercidin; MCD, malonyl-CoA decarboxy-
lase; moi, multiplicity of infection.
(Received 26 February 2004, revised 14 April 2004, accepted 14 May
2004)
Eur. J. Biochem. 271, 2831–2840 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04218.x
that MCD can either be inhibited or activated by phos-

phorylation [16,17]. One potential kinase that could control
MCD activity is 5¢AMP-activated protein kinase (AMPK).
AMPK is a Ôcellular fuel gaugeÕ, and acts to simulta-
neously shut down ATP consuming biosynthetic processes
and facilitate ATP producing catabolic processes during
periods of metabolic stress [18]. One important stress that
can occur in the heart is ischemia. AMPK is rapidly
activated during myocardial ischemia [8,19,20], leading to
rapid changes in the control of glucose and fatty acid
metabolism. AMPK stimulation of fatty acid metabolism
occurs as a result of AMPK phosphorylation and inhibition
of ACC [18,20–24]. This activation of AMPK and inhibi-
tion of ACC results in a dramatic drop in malonyl-CoA
levels during and following ischemia [8,20].
Alterations in myocardial malonyl-CoA levels can not be
solely explained by suppression of ACC activity unless
simultaneous degradation of malonyl-CoA is occurring. It
has therefore been hypothesized that AMPK could also
play a dual role by activating MCD to facilitate malonyl-
CoA degradation [12]. However, the existing literature on
MCD regulation by AMPK is inconsistent in this regard.
Although we [12] and others [25] have demonstrated that
MCD is not a direct substrate for AMPK in vitro,other
studies suggest that MCD is activated by phosphorylation
by AMPK [16,17]. The inconsistencies in the literature
regarding AMPK’s role on MCD regulation may be partly
due to the fact that the above studies have either used
nonspecific means to activate AMPK [16,17] or have used
in vitro conditions that do not mimic conditions seen in
the intact cell [25].

Two alternate translational start sites on MCD appear to
give rise to two isoforms of molecular weight 54.7 kDa and
50.7 kDa, respectively [11,13,26]. MCD could potentially
exist in different subcellular compartments, including cyto-
plasm, peroxisome or mitochondria [27]. In cardiac myo-
cytes, the majority of the MCD is the 50.7 kDa isoform,
which is primarily expressed in the mitochondria [1,28].
How compartmentalization regulates cardiac MCD activity
is not clearly understood. In the present study we examined
whether cardiac MCD is regulated by AMPK, by co-
overexpressing a constitutively active mutated form of the
catalytic subunit of AMPK and the full length human
MCD in H9c2 cells (a rat cardiac ventricular cell line) using
an adenoviral gene delivery technique. As MCD is localized
in various subcellular compartments, we also examined
whether AMPK differentially regulates MCD in mito-
chondrial and cytosolic fractions of these cardiac cells.
Materials and methods
H9c2 cell culture
H9c2 cells (ATCC, Rockville, MD, USA) were grown as
myoblasts to confluency in 60-mm diameter cell culture
dishes in Dulbecco’s modified Eagles’ medium (DMEM;
Sigma) containing 10% (v/v) fetal bovine serum, 1% (w/v)
PenStrep (Sigma) and 0.25 m
ML
-carnitine (Sigma). Dishes
were incubated in a water-jacketed CO
2
incubator main-
tained at 37 °C with 95% O

2
and 5% CO
2
(v/v/v). Cells
were replenished with fresh media every 48 h. Cells were
seeded at approximately 4000–5000 cells per cm
2
.On
reaching approximately 90% confluency, myoblasts were
allowed to differentiate into myotubes in DMEM contain-
ing 1% (v/v) fetal bovine serum, 1% (w/v) penstrep, and
0.25 m
ML
-carnitine. In the presence of 0.25 m
ML
-carnitine,
full differentiation of myoblast to myotubes occurred within
7 days of adding 1% (v/v) fetal bovine serum, using peak
levels of myo-d expression as a marker of muscle cell
differentiation (data not shown). Passages 12–25 were used
for experiments described in this study.
AICAR treatment
H9c2 cells were treated with 2 m
M
5-aminoimidazole-
4-carboxamide riboside (AICAR) for 2 h, as described
previously [29]. Briefly, DMEM containing 1% (v/v) fetal
bovine serum was removed and cells were incubated with
Krebs’ Henseleit (KH) solution (118 m
M

NaCl, 3.5 m
M
KCl, 1.3 m
M
CaCl
2
,1.2m
M
MgSO
4
,1.2m
M
KH
2
PO
4
)for
20 min at 37 °C. At the end of 20 min, fresh KH solution
with or without AICAR (2.0 m
M
final concentration) was
added to each dish, and cells were incubated for 2 h. Some
cells were also treated with the AMPK antagonist
5¢-iodotubercidin (Itu, 50 l
M
) for 2 h, either with or without
2.0 m
M
AICAR. Four groups were included: (a) control,
(b) AICAR treated, (c) Itu treated, and (d) Itu + AICAR

treated cells. At the end of the 2-h incubation, cells were
rapidly lysed as described previously [29]. Cell lysates were
then used for measurement of AMPK activities.
Construction of recombinant adenovirus encoding
MCD, AMPK, and GFP and infection of H9c2 cells
To construct recombinant adenovirus, full length human
MCD cDNA containing the two putative start sites [30] was
subcloned into a pAdTrack-CMV shuttle vector, linearized
with Pme 1 and inserted into adenovirus using pAdEasy-1
system for homologous recombination in Escherichia coli
[31]. The full-length hMCD with two start sites can express
two isoforms of MCD (a 50 kDa and 54.7 kDa isoforms).
The longer form has a putative mitochondrial targeting
sequence, as well as peroxisomal targeting sequence [32].
The pAdTrack-CMV shuttle vector also contained a gene
encoding enhanced green fluorescent protein (GFP). There-
fore, the adenovirus used to express MCD protein also
expressed GFP, which served as a marker of successful viral
infection and protein overexpression.
A similar protocol was used to construct adenoviruses
encoding a myc-tagged constitutively active (T172D)
catalytic l
1
subunit (1–312 amino acid residues) of AMPK
(CA-AMPKa
1(312)
) [33], as well as an adenovirus encoding
GFP alone (used as a control).
Differentiated H9c2 cells cultured in DMEM with 1%
(v/v) fetal bovine serum were infected with either five

multiplicity of infection (moi) per cell of Ad.MCD, 25 moi
per cell of Ad.GFP or 25 moi per cell of Ad.CA-AMPK.
Ad.CA-AMPK (25 moiÆcell
)1
) were determined to yield
optimum CA-AMPKa
1(312)
expression and activity from
series of Ad.CA-AMPK concentrations (5, 10, 25 and 50
moiÆcell
)1
). Some cells were double infected with Ad.MCD
(5 moi) and Ad.CA-AMPK (25 moi) to study the effect
of overexpressed AMPK on overexpressed MCD acti-
vity (Ad.MCD + Ad.CA-AMPK). Cells infected with
2832 N. Sambandam et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Ad.MCD (5 moi) and Ad.GFP (25 moi) served as the
control (Ad.MCD + Ad.GFP) to the above group. Cells
were allowed to express the proteins for 48 h and lysed
rapidly as described below.
Cell lysis and sample preparation for MCD and
AMPK assays
Cells were subjected to a rapid lysis procedure to avoid
activation of endogenous AMPK, as slow lysis of cells has
been shown previously to increase cellular AMP levels [29].
Culture dishes were placed on ice, ice-cold lysis buffer was
added, cells were scraped carefully with a rubber scraper
and transferred to microfuge tubes. Samples were then
immediately homogenized by ultrasonication (SonifierÒ,
Model W185D, Heat Systems-Ultrasonics, Inc., NY, USA)

and centrifuged at 17 000 g for 3 min [29]. Supernatants
were subsequently collected and stored at )80 °C. For
AMPK and ACC assays, cells were lysed in buffer
containing 50 m
M
Tris-base, 250 m
M
mannitol, 1 m
M
EDTA, 1 m
M
EGTA, 50 m
M
NaF, 5.0 m
M
NaPP
i
,1m
M
dithiothreitol, mammalian protease inhibitor cocktail
(Sigma) and 1% (v/v) Triton X-100. For MCD assays,
lysis buffer containing 75 m
M
KCl, 20 m
M
sucrose, 10 m
M
Hepes, 1 m
M
EGTA, 50 m

M
NaF, 5 m
M
NaPPi, 1 m
M
dithiothreitol, and a protease inhibitor cocktail was used.
Samples were subjected to ultrasonication on ice for  5s
and whole cell lysates were used for MCD assay. Protein
concentrations of the cell lysates were determined by a
Bradford protein assay kit.
Subcelluar fractionation to isolate cytosol and
mitochondrial fractions
To prepare mitochondrial and cytoplasmic fractions, three
60 mm dishes were pooled. Cytoplasmic fractions were
obtained by permeabilization of plasma membrane by
digitonin (30 l
M
) treatment for 20 min at 37 °C[34].Each
60 mm dish was treated with buffer containing 30 l
M
digitonin, 0.15 m
M
MgCl
2
,10m
M
KCl, 10 m
M
Tris/HCl,
pH 6.7). Following incubation the buffer was removed, and

centrifuged at 1500 g for 5 min. Supernatant was con-
centrated using Amicon Ultrafree-MC
TM
ultrafiltration
(30 kDa molecular mass cut-off) units, centrifuged at
5500 g for1hin4°C.
Mitochondrial fraction was prepared from the above
digitonin permeabilized cells, as described previously [35].
Cells were quickly washed with ice-cold NaCl/P
i
and
scraped into ice cold NaCl/P
i
in 15 mL centrifuge tubes.
Cells were pelleted by centrifuging at 1000 g for 10 min. The
pellet was then re-suspended in approximately six volumes
of homogenizing buffer (0.15 m
M
MgCl
2
,10m
M
KCl,
10 m
M
Tris/HCl, pH 6.7), transferred to a glass-Teflon
homogenizer (Potter-Elvehjem, between 0.10 and 0.15 mm
clearance), and homogenized by 10–15 up and down strokes
while revolving at 500 r.p.m. Homogenate was then trans-
ferred to a microfuge tube, and sucrose was added to the

homogenate to a final concentration of 0.25
M
and
dissolved. The homogenate was centrifuged at 1500 g for
3 min to remove nuclei and larger fragments. The superna-
tant was then centrifuged at 5000 g for 10 min to pellet
mitochondria. The pellet was resuspended in 10 m
M
Tris-acetate (pH 6.7) buffer containing 0.15 m
M
MgCl
2
,
250 m
M
sucrose and re-centrifuged at 5000 g for 10 min.
The pellet was then suspended in 10 m
M
Tris-acetate
(pH 7.0) buffer containing 250 m
M
sucrose. This procedure
is known to yield a mitochondrial-rich fraction of high
purity and functional integrity [36].
Voltage dependent anion-selective channel protein 1
(VDAC-1), a mitochondrial porin, was used as a marker
to check the mitochondrial fractions [37]. Digitonin perme-
abilization followed by mitochondrial fractionation did not
affect mitochondrial integrity as determined by negligible
amounts of cytochrome C released into cytosol.

Western blot and SDS/PAGE for AMPK, MCD
and mitochondrial markers
To identify AMPK and MCD in the samples, SDS/PAGE
and Western blot analysis was peformed. Thirty micrograms
of either whole cell lysates or subcellular fractions were
loaded in each well of 10% SDS gel. Following electrophor-
esis, proteins were transferred to nitrocellulose membranes
which were then blocked overnight with either 5% (w/v)
bovine serum albumin (for MCD) or in 5% (w/v) skim-
med milk powder (for AMPK) in NaCl/Tris. For CA-
AMPKa
1(312)
which is myc-tagged, polyclonal anti-myc
(Santa Cruz Biotechnology Inc., CA, USA); and for
MCD, rabbit polyclonal anti-MCD IgG [12,13] were used.
Enhanced chemiluminscence detection was carried out to
visualize the protein bands on an autoradiograph.
Western blot analyses for VDAC1, cytochrome C
oxidase and ubiquinone-cytochrome C core 2 subunit of
complex III were performed using respective primary
antibodies (polyclonal goat anti-VDAC1, Santa Cruz
Biotechnology Inc.; monoclonal mouse anti-cytochrome c,
BD Biosciences Pharmingen, San Diego, CA, USA; mono-
clonal mouse anti-core 2 subunit, Molecular Probes,
Eugene, OR, USA).
AMPK assay
Both endogeous AMPK and overexpressed CA-AMP-
Ka
1(312)
activities were measured as previously described

[8]. Samples were diluted to a concentration of 1 mgÆmL
)1
in
re-suspension buffer containing 100 m
M
Tris-base, 1 m
M
EDTA, 1 m
M
EGTA, 50 m
M
NaF, 5 m
M
NaPPi, 10% (v/v)
glycerol, 1 m
M
dithiothreitol, 0.1% (w/v) mammalian pro-
tease inhibitor cocktail and 0.12% (v/v) Triton X-100. Two
microlitres of the above sample was then incubated with the
synthetic 200 l
M
AMARA (AMARAASAAALARRR)
peptide, 200 l
M
[
32
P]ATP[c-P], 0.8 m
M
dithiothreitol,
5m

M
MgCl
2
,200l
M
AMP in buffer (pH 7.0) containing
40 m
M
Hepes/NaOH, 80 m
M
NaCl, 8% (w/v) glycerol for
5 min at 30 °C (total volume 25 lL). This incubation leads to
incorporation of
32
P into the AMARA peptide. At the end of
5min,15lL of the incubation mixture was blotted onto a
1cm
2
phosphocellulose paper. The paper was then washed
three times for 10 min in 150 m
M
phosphoric acid followed
by a 5 min final wash in acetone. The papers were then dried
and counted in 4 mL of scintillation fluid (EcoLite
TM
,ICN,
CA, USA). AMPK activity was expressed as picomoles of
32
P incorporated into AMARA peptide per minute per
milligram protein.

Ó FEBS 2004 Cardiac malonyl-CoA decarboxylase (Eur. J. Biochem. 271) 2833
MCD assay
MCD activity was determined by radiometric assay that
was slightly modified from a previously described method
[10]. Acetyl-CoA, the product of malonyl-CoA degradation
by MCD, was converted to [
14
C]citrate by incubation with
[
14
C]oxaloacetate in the presence of citrate synthase
(0.73 lUÆlL
)1
). [
14
C]Oxaloactetate in turn was produced
from [U-
14
C]aspartate (5 lCiÆmL
)1
)anda-ketoglutarate
(2 m
M
) by transamination in the presence of glutamic
oxaloacetate transaminase. One hundred microliters of
whole cell lysates or cytoplasmic and mitochondrial frac-
tions of either undiluted samples for endogenous MCD in
nonoverexpressing cells (2.0–3.0 mgÆmL
)1
protein concen-

tration) or 20–40 times diluted samples for cells overex-
pressing MCD were incubated with 90 lL incubation buffer
containing phosphatase inhibitors 50 m
M
NaF, 5 m
M
NaPP
i
,1m
M
dithiothreitol and 100 m
M
Tris-base
(pH 8.0). The timed reaction was started by adding
1.0 m
M
malonyl-CoA to the incubation mixture and
incubated at 37 °Cfor20mintoallowformationof
acetyl-CoA. The reaction was stopped with 40 lLof0.5
M
perchloric acid, neutralized with 10 lLof2.2
M
KHCO
3
(pH 10) and centrifuged at 1500 g at 4 °Cfor5minto
remove precipitated proteins. Supernatants containing
formed acetyl-CoA were incubated with 22 lLofamixture
of 0.01 m
M
dithiothreitol, 1.0 m

M
CuSO
4
, and 400 m
M
potassium acetate solution, 20 lLof60m
M
EDTA and
30 lLof30m
M
N-ethylmaleimide to remove excess CoA
remaining in the later stages of the reaction so that the
citrate present could not generate non-MCD derived acetyl-
CoA. The unreacted [
14
C]oxaloacetate was converted back
to aspartate by the addition of glutamic oxaloacetate
transaminase (0.533 lUÆlL
)1
) in the presence of 6.8 m
M
sodium glutamate. The resulting reaction mixture was then
added to 1 mL of a 1 : 1 suspension of Dowex 50 W-X8
(100–200 mesh, hydrogen form) in distilled water. Dowex
binds the aspartate while leaving citrate in the supernatant.
0.5 mL of supernatant was collected after centrifuging the
slurry at 1000 g for 5 min, mixed with 4 mL of scintillation
fluid (EcoLite
TM
,ICN,CA,USA)andcountedinaliquid

scintillation counter. The radioactivity was converted to
nanomoles of acetyl-CoA formed in the reaction using a
standard curve generated from 0 to 20 n
M
range of standard
acetyl-CoA which underwent similar treatment as that of
samples. Preliminary experiments established that 20 min
incubation and the amount of samples used were in the
linear range of MCD enzyme activity.
In vitro
phosphorylation of MCD by AMPK using lysates
of cells overexpressing either MCD or CA-AMPKa
1(312)
H9c2 cells overexpressing MCD were lysed with buffer
containing 75 m
M
KCl, 20 m
M
Sucrose, 10 m
M
Hepes,
1m
M
EGTA, 1 m
M
dithiothreitol, and a protease inhibitor
cocktail on ice by ultrasonication for  5 s and whole cell
lysates were used. Cells overexpressing CA-AMPKa
1(312)
were lysed with buffer containing 50 m

M
Tris-base, 250 m
M
mannitol, 1 m
M
EDTA, 1 m
M
EGTA, 50 m
M
NaF,
5.0 m
M
NaPP
i
,1m
M
dithiothreitol, mammalian protease
inhibitor cocktail (Sigma) and 1% (v/v) Triton X-100 by
ultrasonication as mentioned above. Whole cell lysates of
cells overexpressing MCD were incubated with the lysates
of cells overexpressing CA-AMPKa
1(312)
for 20, 30, 60, 120
and 180 min. At the end of the indicated time points
samples were immunoprecipitated for MCD with rabbit
polyclonal anti-MCD IgG bound to protein-A sepharose
beads. Immunoprecipitates were subjected to SDS/PAGE
and Western blotting and probed with antiphosphoserine or
antiphosphothreonine antibodies. In some experiments cell
extracts were incubated in the presence of 100 lCi of

[
32
P]ATP[c-P] for the above-indicated duration followed by
immunoprecipitation as above and autoradiographed for
1weekin)20 °C.
Statistical analysis
Data are presented as means ± SEM. Statistically signifi-
cant differences between groups of two were assessed using
the paired Students t-test. A two-tailed value of P <0.05
was considered to be significant.
Results
Effect of AICAR on endogenous AMPK and MCD activities
Incubation of H9c2 cells with AICAR increased AMPK
activity significantly (916 ± 130 pmolÆmgÆmin
)1
)when
compared with untreated control cells (588 ± 81 pmolÆmgÆ
min
)1
). AICAR treatment also increased MCD activity
only modestly (to 126% of untreated controls,
1.3 ± 0.3 nmolÆmin
)1
Æmg
)1
, one tailed P < 0.05). Itu,
an inhibitor of AMPK, inhibited AMPK activity
(381 ± 66 pmolÆmin
)1
Æmg

)1
) but did not affect MCD
activity (1.5 ± 0.6 nmolÆmin
)1
Æmg
)1
). However, Itu did
inhibit AICAR-stimulatable AMPK activity significantly
(394 ± 46 pmolÆmin
)1
Æmg
)1
, P < 0.05), as well as the
small increase in MCD activity (1.9 ± 0.2 nmolÆ
min
)1
Æmg
)1
in AICAR-treated cells vs. 1.3 ± 0.03 nmolÆ
min
)1
Æmg
)1
in Itu + AICAR-treated cells, P < 0.05).
Endogenous MCD activity by overexpressed
CA-AMPKa
1(312)
Overexpression of CA-AMPKa
1(312)
using recombinant

adenovirus (Ad.CA-AMPK) resulted in an increase in
AMPK expression and activity in a concentration-depend-
ent manner when compared with control cells expressing
GFP (Fig. 1Ai,ii). As a concentration of 25 moiÆcell
)1
Ad.CA-AMPK yielded maximum activity, we used the
above concentration of Ad.CA-AMPK for all further
studies. Control cells were infected with an equivalent
amount of Ad.GFP virus per cell.
Overexpression of CA-AMPKa
1(312)
did not increase
endogenous cytoplasmic MCD activity measured when
compared with Ad.GFP cells (Fig. 1Bi). In mitochondrial
rich fractions, there was trend towards an increase in MCD
activity in response to CA-AMPKa
1(312)
overexpression,
which was not statistically significant when compared with
Ad.GFP cells (Fig. 1Bii, P < 0.07). As shown, the
endogenous MCD activities were very low and difficult to
obtain a reproducible result in subcellular fractions. There-
fore, due to low endogenous MCD activities in H9c2 cell
fractions, we decided to increase the expression of MCD in
2834 N. Sambandam et al. (Eur. J. Biochem. 271) Ó FEBS 2004
these cells along with CA-AMPKa
1(312)
to examine the role
of AMPK in the regulation of MCD.
MCD activity in H9c2 cells coinfected with Ad.CA-AMPK

and Ad.MCD
Infection of H9c2 cells with Ad.MCD resulted in a
significant increase in MCD protein and activity when
compared with Ad.GFP cells (Fig. 2Ai,ii). In order to study
the effect of AMPK on MCD, we coinfected H9c2 cells with
Ad.MCD and Ad.CA-AMPK viruses (Ad.MCD +
Ad.CA-AMPK) and compared our results to control cells
coinfected with an equivalent number of viral particles/cells
of Ad.MCD and Ad.GFP (Ad.MCD + Ad.GFP). Our
Western blot analysis show that there was a significant
increase in both the 50.7 and 54.7 kDa isoform of MCD
protein levels in Ad.MCD + Ad.CA-AMPK cells com-
pared with Ad.MCD + Ad.GFP cells (Fig. 2Bi–iii). The
enzyme activity of MCD showed a trend towards increase
which was not statistically significant (from 80.7 ± 7.3
nmolÆmin
)1
Æmg
)1
in Ad.MCD + Ad.GFP cells to
108.5 ± 14.2 nmolÆmin
)1
Æmg
)1
in Ad.MCD + Ad.CA-
AMPK cells, P ¼ 0.058; Fig. 2Biv).
MCD expression and activity in subcellular fractions
of H9c2 cells overexpressing both MCD and AMPK
Previous studies have shown that MCD exists in both the
cytoplasmic and mitochondrial compartments [25]. Hence,

we wanted to determine if there is a differential expression
and activation in various subcellular compartments. We
therefore isolated cytoplasmic and mitochondrial rich
fractions to determine MCD distribution and its regulation
in different compartments in response to increased AMPK
activity.
Mitochondrial-rich fractions showed enrichment of a
mitochondrial specific protein VDAC1 that was absent in
cytoplasmic fractions (Fig. 3Ai). Further, most of the
cytochrome C was confined to mitochondrial rich fractions
and very little of cytochrome C was released into the
cytoplasmic fractions (Fig. 3Aii) suggesting that digitonin
permeabilization resulted in a negligible damage to mito-
chondria. Taken together, our data suggest that the
subcellular fractions were relatively pure. Figure 3Aiii
shows that overexpressed MCD was present in both
cytoplasmic and mitochondrial rich fractions. While the
majority of over expressed MCD activity was present in
mitochondria, about 30–40% of total MCD activity was
measured in cytoplasmic fractions (33 ± 18 nmolÆmin
)1
Æ
mg
)1
in cytoplasmic fractions vs. 81 ± 7 nmolÆmin
)1
Æmg
)1
in whole cell lysates). This distribution is consistent with
previously published studies [1].

Cytoplasmic MCD
Figure 3Bi–iv shows the effect of CA-AMPKa
1(312)
overexpression on cytoplasmic MCD protein levels and
activities. As observed in Western blot analysis, cyto-
plasmic fractions show both isoforms of MCD. In
Ad.MCD + Ad.CA-AMPK cells, there was increase in
MCD protein levels (both long and short isoforms;
Fig. 3Bi) when compared with Ad.MCD + Ad.GFP cells.
This increase was more pronounced with the long isoform
in Ad.MCD + Ad.CA-AMPK cells (optical density
1.29 ± 0.11 AU vs. 0.16 ± 0.02 AU in Ad.MCD +
Ad.GFP cells, P < 0.0001; Fig. 3Bi,ii). Interestingly, the
increase in short isoform of MCD protein was not
statistically significant. Despite the increased expression of
MCD protein, MCD activity in cytoplasmic fraction
(normalized to milligrams of total protein) was not different
between two groups (Fig. 3iv).
Mitochondrial MCD
MCD activity was augmented in mitochondrial fractions
obtained in response to co-overexpression of AMPK
in Ad.MCD + Ad.CA-AMPK cells compared with
Ad.MCD + Ad.GFP cells (Fig. 4Aiii). Unlike the cyto-
plasmic fractions, almost all of the MCD was the shorter
form (50.7 kDa). The increase in MCD activity in the
mitochondrial rich fractions was accompanied by a corres-
ponding increase in MCD protein levels (Fig. 4Ai,ii,iii).
Figure 4Bi,ii shows that levels of other mitochondrial-
Fig. 1. AMPK overexpression by adenoviral gene transfer and
endogenous MCD activity. (A) AMPK expression (i) and activity (ii) in

H9c2 cells infected with Ad.CA-AMPK or Ad.GFP. Control H9c2
cells had no viral infection while Ad.GFP cells had 25 moiÆcell
)1
of
Ad.GFP virus. As AMPK is myc tagged, anti-myc antibody was used
to probe overexpressed CA-AMPK
a1(312)
. Western blot is a represen-
tative of n ¼ 2 experiments, AMPK activity values are average of n ¼
2 experiments. (B) Endogenous MCD activity in cytosolic (i) and
mitochondrial (ii) fractions of H9c2 cells infected with Ad.CA-AMPK
or Ad.GFP. Values are mean ± SE of n ¼ 5experiments.
Ó FEBS 2004 Cardiac malonyl-CoA decarboxylase (Eur. J. Biochem. 271) 2835
associated proteins like VDAC1 and ubiquinone-cyto-
chrome C-core 2 subunit of complex III are not affected
by increased CA-AMPKa
1(312)
.
Discussion
Regulation of MCD by AMPK remains controversial
[16,17,25]. In this study, we demonstrate that AMPK
regulates MCD by increasing levels of mitochondrial MCD
protein and activity whereas, cytoplasmic MCD protein
levels increased without a change in enzyme activity. In vitro
incubation of purified enzymes confirms that MCD may not
be a direct substrate for AMPK [12,25]. However, it cannot
be excluded that AMPK could indirectly modulate MCD
activity in intact cells. Stimulation of MCD activity with
AICAR in intact cells was very modest. This is probably due
to the fact that AICAR stimulation of AMPK is only

modest and also the level of endogenous MCD activity was
very low in H9c2 cells to see a significant change in activity.
We therefore, overexpressed CA-AMPKa
1(312)
in H9c2 cells
and examined the regulation of MCD activity by AMPK.
However, low levels of endogenous MCD in H9c2 cells
posed a practical problem to measure either the protein or
the enzyme activity in subcellular fractions. Hence, we also
Fig. 2. MCD overexpression and activity in H9c2 cells co-expressing Ad.CA-AMPK or Ad.GFP. (A) MCD expression (i) and activity (ii) in H9c2
cells infected with Ad.MCD or Ad.GFP. In the Western blot, lanes 1 and 2 ¼ Ad.GFP and lanes 3 and 4 ¼ Ad.MCD. Western blot is a
representative of n ¼ 3 experiments. Activity values are means ± SE of n ¼ 5 experiments. *Significantly different from Ad.GFP control,
P < 0.05. (B) MCD expression (i), optical density of 54.7 kDa isoform (ii), optical density of 50.7 kDa isoform (iii) and activity (iv) in whole cell
lysates of H9c2 cells coinfected with Ad.MCD + Ad.GFP or Ad.MCD + Ad.CA-AMPK virus. In the representative Western blot, lanes 1 and
3 ¼ Ad.MCD + Ad.GFP and lanes 2 and 4 ¼ Ad.MCD + Ad.CA-AMPK. The relative intensity and activity values are means ± SE of n ¼ 5
experiments. *Significantly different from Ad.MCD + Ad.GFP group, P <0.05.
2836 N. Sambandam et al. (Eur. J. Biochem. 271) Ó FEBS 2004
overexpressed MCD. Overexpression of CA-AMPKa
1(312)
resulted in a three- to fourfold increase in AMPK protein
and activity. Similarly, MCD overexpression yielded a
several-fold increase in MCD expression and activity. When
AMPK was co-overexpressed, there was an increase in
MCD activity in the mitochondrial fraction, which was due
to an increase in the amount of MCD localized to the
mitochondria. On the other hand, cytoplasmic fractions
exhibited increases only in MCD protein levels and no
change in activity compared with control conditions.
In the heart, we and others [1,12] have previously
demonstrated that the majority of MCD protein is in the

short form ( 50.7 kDa) associated with mitochondria.
Whether this short form is as a result of alternate splicing at
the level of transcription or as a result of post-translational
modification of full length protein ( 54.7 kDa), is not yet
known. It was proposed that once MCD is targeted to
mitochondria, it may lose the mitochondrial target sequence
by proteolytic cleavage and exists in the short form [38]. Our
data support this concept. When we overexpressed human
recombinant MCD in H9c2 cells both the short and the long
forms of MCD were expressed. While the majority of the
overexpressed MCD was the short form and was localized
to mitochondria, the long form was expressed in Ad.MCD
cells and was observed primarily in cytoplasm. As mito-
chondria are a rich source of MCD, it is possible that the
short isoform could have leached out of mitochondria into
cytoplasm during the fractionation procedures. In spite of
an increased MCD protein in the cytoplasm, the activity did
not increase in response to AMPK overexpression. In fact,
Fig. 3. MCD expression and activity in cytosolic fractions of H9c2 cells. (A) Western blots for VDAC1 (i), cytochrome C (ii) and MCD expression
(iii) in cytoplasmic and mitochondrial fractions from H9c2 cells infected with Ad.GFP or Ad.MCD. Western blots are representative of n ¼ 2
experiments. (B) MCD expression (i), optical density of 54.7 kDa isoform (ii), optical density of 50.7 kDa isoform (iii) and activity (iv) of
cytoplasmic fractions obtained from H9c2 cells coinfected with Ad.MCD + Ad.GFP or Ad.MCD + Ad.CA-AMPK virus. Western blot is
representative of n ¼ 6 experiments and relative intensities are means ± SE of n ¼ 6experiments.Lanes1and3forAd.MCD+Ad.GFP
and lanes 2 and 4 for Ad.MCD + Ad.AMPK cells. Activity values are means ± SE of n ¼ 5 experiments. *Significantly different from
Ad.MCD + Ad.GFP control, P <0.05.
Ó FEBS 2004 Cardiac malonyl-CoA decarboxylase (Eur. J. Biochem. 271) 2837
the specific activity per amount of protein was lower when
compared with control cells suggesting that the long
isoform, which contributes to most of the increases in
cytoplasmic MCD protein, may be less active than the short

form. Our data suggest that AMPK augments levels of both
isoforms of MCD. Whether this increase in MCD expres-
sion by AMPK is a result of post-transcriptional regulation
either affecting mRNA stability or protein stability is not
known. Although evidence suggests that AMPK may
regulate MCD transcription via PGC1 and PPARa
[14,15,39,40], it may not be applicable here as MCD
overexpression per se is driven by the cytomegalo virus
promoter present in the recombinant Ad.MCD virus.
The heart predominantly expresses the 50 kDa isoform
of MCD [12]. In this study, we observed that this short
isoform is mainly associated with mitochondria. In this
study we demonstrated that AMPK overexpression faci-
litated an increase in the short MCD isoform in
mitochondria, with a parallel increase in MCD activity.
Although we did not screen for all the mitochondrial
proteins, the increased CA-AMPKa
1(312)
activity did not
affect the levels of other mitochondria-associated proteins
like VDAC1 and ubiquinone–cytochrome c–core 2 sub-
unit of complex III. This suggests that the role of AMPK
in increasing MCD protein and activity in the
mitochondria may be selective to MCD when compared
with the other proteins tested above.
Contrary to our findings, Habinowski et al.observed
no differences in MCD activities between cytoplasmic and
mitochondrial fractions [25]. In their study, an islet cell
line was used, where a greater expression of the longer
isoform of MCD is observed. Previous studies have shown

that pancreatic MCD is post-translationally processed and
regulated differently than either heart or muscle MCD
Fig. 4. MCD expression and activity in mitochondrial fractions of H9c2 cells. (A) MCD expression (i), optical density of 50.7 kDa isoform (ii) and
activity (iii) of mitochondrial fractions obtained from H9c2 cells coinfected with Ad.MCD + Ad.GFP or Ad.MCD + Ad.CA-AMPK virus. In
the Western blot, lanes 1 and 2 are for Ad.MCD + Ad.GFP cells and lanes 3 and 4 are for Ad.MCD + Ad.CA-AMPK cells. Western blot is a
representative of n ¼ 3 experiments and relative intensity values are means ± SE of n ¼ 3 experiments. Activity values are means ± SE of n ¼ 5
experiments. *Significantly different from Ad.MCD + Ad.GFP control, P < 0.05. (B) Western blots for VDAC
1
protein (i) and cytochrome c
Core 2 subunit of complex III (ii) in mitochondrial fractions obtained from H9c2 cells infected with Ad.MCD + Ad.GFP or Ad.MCD + Ad.CA-
AMPK virus. Lanes 1, 2, 5 and 6 represent Ad.MCD + Ad.GFP cells and lanes 3, 4, 7 and 8 represent Ad.MCD + Ad.CA-AMPK cells. Results
represent n ¼ 4 different passages from each group.
2838 N. Sambandam et al. (Eur. J. Biochem. 271) Ó FEBS 2004
[12,38]. Pancreatic MCD appears in both longer and
shorter forms while heart and muscle show mainly the
shorter form of MCD [12,38]. This greater distribution of
MCD in the cytoplasmic compartment may explain the
lack of AMPK regulation of MCD in pancreatic islets in
the above study.
MCD protein has several potential Ser/Thr sites, phos-
phorylation of which could result in either a decrease or
increase in activity [12,16,17]. Previously we have shown
that dephosphorylation of MCD using alkaline phospha-
tase increased MCD activity suggesting that MCD is down
regulated by phosphorylation [12]. However, recent studies
in skeletal muscle demonstrated that phosphorylation of
MCD increases its activity and that dephosphorylation by
PP2AdecreasesorpreventstheraiseinMCDactivityin
response to activation of AMPK [16]. On the other hand
in vitro incubation of purified MCD with heterotrimeric

AMPK holoenzyme as well as constitutively active a1
subunit found that there was no phosphorylation of MCD
[12,25]. In the present study, when we incubated the lysates
from cells overexpressing MCD with those overexpressing
CA-AMPKa
1(312)
, we did not observe any phosphorylation
of MCD (data not shown). Also, when immunoprecipitated
MCD was probed for the myc-AMPK by Western blot
analysis, we did not observe AMPK suggesting that there
may be no physical interaction between the two proteins
(data not shown). Taken together, this indicates that MCD
may not be a direct substrate for AMPK in vivo. However,
this does not rule out that AMPK can regulate MCD via
other intermediary protein and by other post-translational
modifications. In this regard, previous studies suggested that
a 40 kDa protein that coprecipitated with MCD could be
an MCD-inhibitory protein [41].
Although this study has limitations in that (a) a
nonphysiological model system overexpressing MCD as
well as AMPK was used, and (b) a constitutively active
fragment of catalytic subunit of AMPK rather than
physiological heterotrimeric form was used, the observa-
tions are interesting and support the possibility of differen-
tial regulation of MCD in different subcellular
compartments. Of particular interest, basal malonyl-CoA
levels in tissues are well above the inhibitory concentration
for CPT-1 [42], suggesting a compartmentalization of
cardiac malonyl-CoA. Thus, it is possible that malonyl-
CoA levels in the vicinity of CPT-I (on the outer mito-

chondrial membrane) could undergo changes sufficient
enough to either activate or inhibit CPT-I. In support of
this, a recent study in human skeletal muscle observed a
moderate increase in malonyl-CoA concentrations (20% of
control) led to significant decrease in fatty acid oxidation
(41% of control) [43]. Therefore, it is tempting to speculate
that an AMPK mediated increase in MCD expression and
activity selectively in mitochondria could potentially
decrease malonyl-CoA levels sufficiently in the vicinity of
CPT-I to increase CPT-I activity. This in turn would
increase fatty acid uptake and oxidation. In summary, our
results demonstrate that increasing AMPK activity by
overexpression of constitutively active AMPK increases
both MCD expression and activity. Whereas cytoplasmic
MCD levels rise without any change in activity, both
mitochondrial MCD levels and activity increase. Whether
this differential regulation of MCD by AMPK is at the post-
transcriptional or post-translational level needs further
investigation.
Acknowledgements
This study was funded by a grant from the Canadian Institute for
Health Research. N.S. is a postdoctoral fellow of the Alberta Heritage
Foundation for Medical Research and Heart and Stroke Foundation
of Canada. J.R.B.D. is a Scholar of the Alberta Heritage Foundation
for Medical Research and a Canadian Institutes of Health Research
New Investigator. G.D.L. is a Medical Scientist of the Alberta Heritage
Foundation for Medical Research.
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