Cells infected with Ad.CA-AMPK demonstrated a fourfold increase in Ad.CA-AMPK activity as compared with control cells expressing green fluorescent protein Ad.GFP.. Endogenous MCD activity
Trang 1Malonyl-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 MCDhas 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
MCDis regulated by AMPK Cells infected with
Ad.CA-AMPK demonstrated a fourfold increase in Ad.CA-AMPK activity
as compared with control cells expressing green fluorescent
protein (Ad.GFP) MCDactivity increased 40- to 50-fold
in Ad.MCD+ Ad.GFP cells when compared with
Ad.GFP control Co-expressing AMPK with
MCDfur-ther augmented MCDexpression 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 MCDactivities in cytosolic fractions were not different between the two groups Interestingly, in the mitochondrial fractions, MCDactivity 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 MCDby AMPK was observed The increase in MCDactivity 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 MCDalso has an important role in lowering malonyl-CoA levels [10]
MCDwas originally identified in the uropygial gland of the goose [11] We also showed MCDto be highly expressed
in mammalian cardiac muscle [12], and provided evidence
to suggest that cardiac MCDplays an important role in regulating fatty acid metabolism in the heart [10,13] Regulation of MCDoccurs both at the level of transcription and post-translation [14,15] MCDhas 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: gary.lopaschuk@ualberta.ca
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)
Trang 2that MCDcan either be inhibited or activated by
phos-phorylation [16,17] One potential kinase that could control
MCDactivity is 5¢AMP-activated protein kinase (AMPK)
AMPK is a cellular fuel gauge, 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 MCDto facilitate
malonyl-CoA degradation [12] However, the existing literature on
MCDregulation by AMPK is inconsistent in this regard
Although we [12] and others [25] have demonstrated that
MCDis not a direct substrate for AMPK in vitro, other
studies suggest that MCDis activated by phosphorylation
by AMPK [16,17] The inconsistencies in the literature
regarding AMPK’s role on MCDregulation 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 vitroconditions that do not mimic conditions seen in
the intact cell [25]
Two alternate translational start sites on MCDappear 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 MCDis the 50.7 kDa isoform,
which is primarily expressed in the mitochondria [1,28]
How compartmentalization regulates cardiac MCDactivity
is not clearly understood In the present study we examined
whether cardiac MCDis regulated by AMPK, by
co-overexpressing a constitutively active mutated form of the
catalytic subunit of AMPK and the full length human
MCDin H9c2 cells (a rat cardiac ventricular cell line) using
an adenoviral gene delivery technique As MCDis localized
in various subcellular compartments, we also examined
whether AMPK differentially regulates MCDin
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 mM L-carnitine (Sigma) Dishes
were incubated in a water-jacketed CO2 incubator
main-tained at 37C with 95% O2and 5% CO2 (v/v/v) Cells
were replenished with fresh media every 48 h Cells were
seeded at approximately 4000–5000 cells per cm2 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 mM L-carnitine In the presence of 0.25 mM L-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 mM 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 mM NaCl, 3.5 mM
KCl, 1.3 mMCaCl2, 1.2 mMMgSO4, 1.2 mMKH2PO4) for
20 min at 37C At the end of 20 min, fresh KH solution with or without AICAR (2.0 mMfinal 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 lM) for 2 h, either with or without 2.0 mMAICAR 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 MCDcDNA 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 hMCDwith 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 MCDprotein 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 l1subunit (1–312 amino acid residues) of AMPK (CA-AMPKa1(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-AMPKa1(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 MCDacti-vity (Ad.MCD+ Ad.CA-AMPK) Cells infected with
Trang 3Ad.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 (Sonifier,
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 mM Tris-base, 250 mM mannitol, 1 mM
EDTA, 1 mMEGTA, 50 mMNaF, 5.0 mMNaPPi, 1 mM
dithiothreitol, mammalian protease inhibitor cocktail
(Sigma) and 1% (v/v) Triton X-100 For MCDassays,
lysis buffer containing 75 mMKCl, 20 mMsucrose, 10 mM
Hepes, 1 mM EGTA, 50 mM NaF, 5 mM NaPPi, 1 mM
dithiothreitol, and a protease inhibitor cocktail was used
Samples were subjected to ultrasonication on ice for 5 s
and whole cell lysates were used for MCDassay 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 lM) treatment for 20 min at 37C [34] Each
60 mm dish was treated with buffer containing 30 lM
digitonin, 0.15 mM MgCl2, 10 mMKCl, 10 mM 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-MCTM ultrafiltration
(30 kDa molecular mass cut-off) units, centrifuged at
5500 g for 1 h in 4C
Mitochondrial fraction was prepared from the above
digitonin permeabilized cells, as described previously [35]
Cells were quickly washed with ice-cold NaCl/Pi and
scraped into ice cold NaCl/Pi 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 mM MgCl2, 10 mM KCl,
10 mM 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 0.25M 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
Tris-acetate (pH 6.7) buffer containing 0.15 mM MgCl2,
250 mMsucrose and re-centrifuged at 5000 g for 10 min The pellet was then suspended in 10 mM Tris-acetate (pH 7.0) buffer containing 250 mMsucrose 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 MCDin 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-AMPKa1(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, BDBiosciences 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-Ka1(312) activities were measured as previously described [8] Samples were diluted to a concentration of 1 mgÆmL)1in re-suspension buffer containing 100 mM Tris-base, 1 mM
EDTA, 1 mMEGTA, 50 mMNaF, 5 mMNaPPi, 10% (v/v) glycerol, 1 mMdithiothreitol, 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 lM AMARA (AMARAASAAALARRR) peptide, 200 lM [32P]ATP[c-P], 0.8 mM dithiothreitol,
5 mMMgCl2, 200 lMAMP in buffer (pH 7.0) containing
40 mMHepes/NaOH, 80 mMNaCl, 8% (w/v) glycerol for
5 min at 30C (total volume 25 lL) This incubation leads to incorporation of32P into the AMARA peptide At the end of
5 min, 15 lL of the incubation mixture was blotted onto a
1 cm2phosphocellulose paper The paper was then washed three times for 10 min in 150 mMphosphoric acid followed
by a 5 min final wash in acetone The papers were then dried and counted in 4 mL of scintillation fluid (EcoLiteTM, ICN,
CA, USA) AMPK activity was expressed as picomoles of
32P incorporated into AMARA peptide per minute per milligram protein
Trang 4MCD assay
MCDactivity 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 [14C]citrate by incubation with
[14C]oxaloacetate in the presence of citrate synthase
(0.73 lUÆlL)1) [14C]Oxaloactetate in turn was produced
from [U-14C]aspartate (5 lCiÆmL)1) and a-ketoglutarate
(2 mM) 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 MCDin
nonoverexpressing cells (2.0–3.0 mgÆmL)1protein
concen-tration) or 20–40 times diluted samples for cells
overex-pressing MCDwere incubated with 90 lL incubation buffer
containing phosphatase inhibitors 50 mM NaF, 5 mM
NaPPi, 1 mM dithiothreitol and 100 mM Tris-base
(pH 8.0) The timed reaction was started by adding
1.0 mM malonyl-CoA to the incubation mixture and
incubated at 37C for 20 min to allow formation of
acetyl-CoA The reaction was stopped with 40 lL of 0.5M
perchloric acid, neutralized with 10 lL of 2.2M KHCO3
(pH 10) and centrifuged at 1500 g at 4C for 5 min to
remove precipitated proteins Supernatants containing
formed acetyl-CoA were incubated with 22 lL of a mixture
of 0.01 mM dithiothreitol, 1.0 mM CuSO4, and 400 mM
potassium acetate solution, 20 lL of 60 mM EDTA and
30 lL of 30 mMN-ethylmaleimide to remove excess CoA
remaining in the later stages of the reaction so that the
citrate present could not generate non-MCDderived
acetyl-CoA The unreacted [14C]oxaloacetate was converted back
to aspartate by the addition of glutamic oxaloacetate
transaminase (0.533 lUÆlL)1) in the presence of 6.8 mM
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 (EcoLiteTM, ICN, CA, USA) and counted in a liquid
scintillation counter The radioactivity was converted to
nanomoles of acetyl-CoA formed in the reaction using a
standard curve generated from 0 to 20 nMrange 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 MCDenzyme activity
In vitro phosphorylation of MCD by AMPK using lysates
of cells overexpressing either MCD or CA-AMPKa1(312)
H9c2 cells overexpressing MCDwere lysed with buffer
containing 75 mM KCl, 20 mM Sucrose, 10 mM Hepes,
1 mMEGTA, 1 mMdithiothreitol, and a protease inhibitor
cocktail on ice by ultrasonication for 5 s and whole cell
lysates were used Cells overexpressing CA-AMPKa1(312)
were lysed with buffer containing 50 mMTris-base, 250 mM
mannitol, 1 mM EDTA, 1 mM EGTA, 50 mM NaF,
5.0 mM NaPPi, 1 mMdithiothreitol, mammalian protease
inhibitor cocktail (Sigma) and 1% (v/v) Triton X-100 by
ultrasonication as mentioned above Whole cell lysates of
cells overexpressing MCDwere incubated with the lysates
of cells overexpressing CA-AMPKa1(312)for 20, 30, 60, 120 and 180 min At the end of the indicated time points samples were immunoprecipitated for MCDwith rabbit polyclonal anti-MCDIgG 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 [32P]ATP[c-P] for the above-indicated duration followed by immunoprecipitation as above and autoradiographed for
1 week in)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 MCDactivity only modestly (to 126% of untreated controls, 1.3 ± 0.3 nmolÆmin)1Æmg)1, one tailed P < 0.05) Itu,
an inhibitor of AMPK, inhibited AMPK activity (381 ± 66 pmolÆmin)1Æmg)1) but did not affect MCD activity (1.5 ± 0.6 nmolÆmin)1Æmg)1) However, Itu did inhibit AICAR-stimulatable AMPK activity significantly (394 ± 46 pmolÆmin)1Æmg)1, P < 0.05), as well as the small increase in MCDactivity (1.9 ± 0.2 nmolÆ min)1Æmg)1 in AICAR-treated cells vs 1.3 ± 0.03 nmolÆ min)1Æmg)1in Itu + AICAR-treated cells, P < 0.05)
Endogenous MCD activity by overexpressed CA-AMPKa1(312)
Overexpression of CA-AMPKa1(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-AMPKa1(312) did not increase endogenous cytoplasmic MCDactivity 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-AMPKa1(312) overexpression, which was not statistically significant when compared with Ad.GFP cells (Fig 1Bii, P < 0.07) As shown, the endogenous MCDactivities were very low and difficult to obtain a reproducible result in subcellular fractions There-fore, due to low endogenous MCDactivities in H9c2 cell fractions, we decided to increase the expression of MCDin
Trang 5these cells along with CA-AMPKa1(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.MCDresulted in a
significant increase in MCDprotein 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.MCDand 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.MCDand 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 MCDshowed a trend towards increase
which was not statistically significant (from 80.7 ± 7.3
nmolÆmin)1Æmg)1 in Ad.MCD+ Ad.GFP cells to
108.5 ± 14.2 nmolÆmin)1Æmg)1 in Ad.MCD+ Ad.CA-AMPK cells, P¼ 0.058; Fig 2Biv)
MCD expression and activity in subcellular fractions
of H9c2 cells overexpressing both MCD and AMPK Previous studies have shown that MCDexists 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 MCDdistribution 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 MCDwas present in both cytoplasmic and mitochondrial rich fractions While the majority of over expressed MCDactivity was present in mitochondria, about 30–40% of total MCDactivity was measured in cytoplasmic fractions (33 ± 18 nmolÆmin)1Æ
mg)1in cytoplasmic fractions vs 81 ± 7 nmolÆmin)1Æmg)1
in whole cell lysates) This distribution is consistent with previously published studies [1]
Cytoplasmic MCD Figure 3Bi–iv shows the effect of CA-AMPKa1(312) overexpression on cytoplasmic MCDprotein 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 MCDprotein 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 MCDprotein was not statistically significant Despite the increased expression of MCDprotein, MCDactivity in cytoplasmic fraction (normalized to milligrams of total protein) was not different between two groups (Fig 3iv)
Mitochondrial MCD MCDactivity 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 MCDwas the shorter form (50.7 kDa) The increase in MCD activity in the mitochondrial rich fractions was accompanied by a corres-ponding increase in MCDprotein 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)1of
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 MCDactivity 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 5 experiments.
Trang 6associated proteins like VDAC1 and
ubiquinone-cyto-chrome C-core 2 subunit of complex III are not affected
by increased CA-AMPKa1(312)
Discussion
Regulation of MCDby AMPK remains controversial
[16,17,25] In this study, we demonstrate that AMPK
regulates MCDby increasing levels of mitochondrial MCD
protein and activity whereas, cytoplasmic MCDprotein
levels increased without a change in enzyme activity In vitro
incubation of purified enzymes confirms that MCDmay 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 MCDactivity 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 MCDactivity was very low in H9c2 cells to see a significant change in activity
We therefore, overexpressed CA-AMPKa1(312)in H9c2 cells and examined the regulation of MCDactivity by AMPK However, low levels of endogenous MCDin 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) MCDexpression (i) and activity (ii) in H9c2 cells infected with Ad.MCDor 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.
Trang 7overexpressed MCD Overexpression of CA-AMPKa1(312)
resulted in a three- to fourfold increase in AMPK protein
and activity Similarly, MCDoverexpression yielded a
several-fold increase in MCDexpression and activity When
AMPK was co-overexpressed, there was an increase in
MCDactivity in the mitochondrial fraction, which was due
to an increase in the amount of MCDlocalized to the
mitochondria On the other hand, cytoplasmic fractions
exhibited increases only in MCDprotein 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 MCDprotein 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 MCDis 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 MCDin H9c2 cells both the short and the long forms of MCDwere expressed While the majority of the overexpressed MCDwas 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 MCDprotein 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) MCDexpression (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 6 experiments Lanes 1 and 3 for Ad.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.
Trang 8the 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 MCDprotein, 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 MCDtranscription 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.MCDvirus
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 MCDisoform in mitochondria, with a parallel increase in MCDactivity Although we did not screen for all the mitochondrial proteins, the increased CA-AMPKa1(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 MCDprotein and activity in the mitochondria may be selective to MCDwhen compared with the other proteins tested above
Contrary to our findings, Habinowski et al observed
no differences in MCDactivities between cytoplasmic and mitochondrial fractions [25] In their study, an islet cell line was used, where a greater expression of the longer isoform of MCDis observed Previous studies have shown that pancreatic MCDis 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) MCDexpression (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-Ad.CA-AMPK cells Results represent n ¼ 4 different passages from each group.
Trang 9[12,38] Pancreatic MCDappears in both longer and
shorter forms while heart and muscle show mainly the
shorter form of MCD[12,38] This greater distribution of
MCDin the cytoplasmic compartment may explain the
lack of AMPK regulation of MCDin pancreatic islets in
the above study
MCDprotein 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 MCDusing alkaline
phospha-tase increased MCDactivity suggesting that MCDis down
regulated by phosphorylation [12] However, recent studies
in skeletal muscle demonstrated that phosphorylation of
MCDincreases its activity and that dephosphorylation by
PP2A decreases or prevents the raise in MCD activity in
response to activation of AMPK [16] On the other hand
in vitro incubation of purified MCDwith 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 MCDwith those overexpressing
CA-AMPKa1(312), we did not observe any phosphorylation
of MCD(data not shown) Also, when immunoprecipitated
MCDwas 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 MCDvia
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 MCDas
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 MCDin 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 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 MCDexpression 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 MCDexpression and activity Whereas cytoplasmic
MCDlevels rise without any change in activity, both
mitochondrial MCDlevels and activity increase Whether
this differential regulation of MCDby 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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