MOLECULAR AND CELLULAR BIOLOGY, Mar. 2011, p. 1252–1262
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 31, No. 6
Mouse Cardiac Acyl Coenzyme A Synthetase 1 Deficiency Impairs
Fatty Acid Oxidation and Induces Cardiac Hypertrophy?†
Jessica M. Ellis,1Shannon M. Mentock,1Michael A. DePetrillo,1Timothy R. Koves,2Shiraj Sen,3
Steven M. Watkins,4‡ Deborah M. Muoio,2Gary W. Cline,5Heinrich Taegtmeyer,3
Gerald I. Shulman,5,6Monte S. Willis,7and Rosalind A. Coleman1*
Department of Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 275991; Department of
Medicine, Duke University, Durham, North Carolina 277082; Division of Cardiology, Department of Internal Medicine,
University of Texas Medical School at Houston, Houston, Texas 770303; Lipomics Technologies, Inc., 2545 Boatman Ave.,
West Sacramento, California 956914; Departments of Internal Medicine and of Cellular and Molecular Physiology5
and Howard Hughes Medical Institute,6Yale University School of Medicine, New Haven, Connecticut 06520; and
Department of Pathology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 275997
Received 15 September 2010/Returned for modification 26 October 2010/Accepted 14 December 2010
Long-chain acyl coenzyme A (acyl-CoA) synthetase isoform 1 (ACSL1) catalyzes the synthesis of acyl-CoA
from long-chain fatty acids and contributes the majority of cardiac long-chain acyl-CoA synthetase activity. To
understand its functional role in the heart, we studied mice lacking ACSL1 globally (Acsl1T?/?) and mice
lacking ACSL1 in heart ventricles (Acsl1H?/?) at different times. Compared to littermate controls, heart
ventricular ACSL activity in Acsl1T?/?mice was reduced more than 90%, acyl-CoA content was 65% lower, and
long-chain acyl-carnitine content was 80 to 90% lower. The rate of [14C]palmitate oxidation in both heart
homogenate and mitochondria was 90% lower than in the controls, and the maximal rates of [14C]pyruvate and
[14C]glucose oxidation were each 20% higher. The mitochondrial area was 54% greater than in the controls
with twice as much mitochondrial DNA, and the mRNA abundance of Pgc1? and Err? increased by 100% and
41%, respectively. Compared to the controls, Acsl1T?/?and Acsl1H?/?hearts were hypertrophied, and the
phosphorylation of S6 kinase, a target of mammalian target of rapamycin (mTOR) kinase, increased 5-fold.
Our data suggest that ACSL1 is required to synthesize the acyl-CoAs that are oxidized by the heart, and that
without ACSL1, diminished fatty acid (FA) oxidation and compensatory catabolism of glucose and amino acids
lead to mTOR activation and cardiac hypertrophy without lipid accumulation or immediate cardiac
The mitochondrial oxidation of long-chain fatty acids (FAs)
provides 60 to 90% of heart ATP (9, 43, 49). Reduced cardiac
FA oxidation and increased glucose utilization are a proposed
consequence of pathological left ventricular hypertrophy
(LVH) (22, 33). However, when genes that encode enzymes of
FA oxidation are knocked out in mice, LVH develops (11, 20).
Thus, it remains unclear whether the shift in substrate use is a
cause or consequence of cardiac hypertrophy and whether the
increased use of glucose interferes with cardiac function.
Long-chain acyl coenzyme A (acyl-CoA) synthetase (ACSL)
isoenzymes convert FAs to acyl coenzyme A (acyl-CoA) in an
ATP-dependent manner, simultaneously activating and trap-
ping FAs within cells (4). Activation to acyl-CoA is required
before FAs can be either oxidized to provide ATP or esterified
to synthesize triacylglycerol (TAG) or membrane phospholip-
ids (PL). The activation of FA is catalyzed by one of a family
of five long-chain acyl-CoA synthetases (ACSLs), long-chain
acyl-CoA synthetase isoform 1 (ACSL1), ACSL3, ACSL4,
ACSL5, and ACSL6, which differ in substrate preference, en-
zyme kinetics, subcellular location, and tissue-specific expres-
sion (10). Because amphipathic acyl-CoAs can move freely
within a membrane monolayer or be transported to distant
membranes, all acyl-CoAs should, theoretically, be metaboli-
cally equivalent, no matter which ACSL isoenzyme catalyzes
their formation and no matter which subcellular organelle is
the site of their synthesis. Yet, both loss-of-function and gain-
of-function studies suggest that each ACSL isoenzyme has a
distinct function in directing acyl-CoAs to one or more specific
downstream pathways (5, 29, 32). We have reported that mice
lacking ACSL1 specifically in adipose tissue have defects in
adipose FA oxidation (15); however, mice lacking ACSL1 in
the liver have minor defects in both hepatic FA oxidation and
triacylglycerol synthesis (28), suggesting that the ACSL iso-
forms may have roles that differ in different tissues. The role
that ACSL1 plays in cardiac FA metabolism has remained
Although FAs provide the major substrate for oxidation in
the heart, it is unknown whether ACSL activity affects FA
oxidation rates or whether a particular ACSL isoenzyme acti-
vates FAs destined for oxidation. The total ACSL activity of
the mouse embryonic heart (embryonic day 16.5 [E16.5]) in-
creases 14-fold during the week after birth and 90-fold by
adulthood (13). This dramatic increase in ACSL activity par-
* Corresponding author. Mailing address: Department of Nutrition,
CB 7461, University of North Carolina at Chapel Hill, 135 Dauer
Drive, MHRC 2301, Chapel Hill, NC 27599. Phone: (919) 966-7213.
Fax: (919) 843-8555. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://mcb
‡ Present address: Tethys Bioscience, 3410 Industrial Boulevard,
Suite 103, West Sacramento, CA 95691.
?Published ahead of print on 18 January 2011.
allels the transition of the developing heart’s substrate prefer-
ence from glucose prenatally to FAs after birth. In mice, this
transition is accompanied by a 6-fold increase in Acsl1 mRNA
abundance and a ?90% decrease in the mRNA abundance of
Acsl3, the predominant Acsl1 in fetal heart (13). In rats, the
increases in ACSL activity and Acsl1 mRNA expression par-
allel the postnatal heart’s increased workload, rate of ATP
generation, oxidative preference for FAs, and expression of FA
oxidative genes (19). Together, these data suggested that
ACSL1 might be the major activator of the FAs that are oxi-
dized in postnatal cardiac tissue.
In order to understand the relationship between cardiac
substrate use and hypertrophy, we created a mouse model that
lacks acyl-CoA synthetase 1 (ACSL1) in multiple tissues, in-
cluding the heart. We reasoned that the absence of ACSL1
would enable us to learn whether a block in FA activation
prevents potential lipotoxicity and abnormal heart function
and to determine the mechanism by which a shift in substrate
use from FAs to glucose causes cardiac hypertrophy. Herein
we report that mice lacking ACSL1 in a multitissue-specific
manner and in a heart-specific manner have markedly reduced
FA oxidation in the heart and develop cardiac hypertrophy.
MATERIALS AND METHODS
Animal treatment. All protocols were approved by the University of North
Carolina Institutional Animal Care and Use Committee. Mice were housed in a
pathogen-free barrier facility (12-h light/12-h dark cycle) with free access to
water and food (Prolab RMH 3000 SP76 chow). Mice with Loxp sequences
inserted on either side of exon 2 in the Acsl1 gene (9) were backcrossed to the
C57BL/J6 strain six times and then interbred with mice in which Cre expression
is driven either by a ubiquitous promoter enhancer or by an ?-myosin heavy-
chain promoter, both of which are induced by tamoxifen [B6.Cg-Tg(cre/
Esr1)5Amc/J or B6.Cg-Tg(Myh6-cre/Esr1)1Jmk/J; Jackson Labs] to generate
(Acsl1H?/?) Acsl1 knockout mice. When the mice were 6 to 8 weeks old, tamox-
ifen (Sigma, St. Louis, MO), dissolved in corn oil (20 mg/ml), was injected
intraperitoneally (i.p.) for 4 consecutive days (3 mg/40 g of body weight) into
Acsl1T?/?, Acsl1H?/?, and littermate Acsl1flox/floxcontrol mice. Tissues were
removed and snap-frozen in liquid nitrogen. To isolate the mitochondria, the
hearts were removed, rinsed in phosphate-buffered saline (PBS), minced, and
homogenized with 10 up-and-down strokes, using a motor-driven Teflon pestle
and glass mortar in ice-cold buffer (0.2 mM EDTA, 0.25 M sucrose, 10 mM
Tris-HCl [pH 7.8], protease inhibitor [Roche, Florence, SC]). Homogenates
were centrifuged at 1,000 ? g for 10 min at 4°C, the supernatant was spun at
12,000 ? g for 15 min at 4°C, and the resulting pellet was washed once and
resuspended in oxidation buffer. Protein content was determined by using the
bicinchoninic acid (BCA) assay kit (Pierce, Rockford, IL) with bovine serum
albumin (BSA) as the standard. Plasma was collected from mice in 5% 0.5 M
EDTA. Plasma triacylglycerol (TAG), ?-hydroxybutyrate (Stanbio, Boerne, TX),
cholesterol, free FAs (FFAs), glucose (Wako, Richmond, VA), and free and
total glycerol (Sigma) were measured with colorimetric assays. Insulin tolerance
tests were performed by i.p. injection with insulin (0.5 U/kg of body weight), and
the tail blood glucose level was measured at baseline, 15, 30, 60, and 120 min
using a One Touch Ultra glucometer (Lifescan, Inc., Milpitas, CA). Blood
pressure was measured by CODA high-throughput noninvasive tail blood pres-
sure system (Kent Scientific, Torrington, CT). Mouse echocardiograms were
performed on unanesthetized mice with the VisualSonics (Toronto, Ontario,
Canada) Vevo 770 ultrasound biomicroscopy system. M-mode images of the left
ventricle were analyzed using VisualSonics software. Transverse arch banding
was performed in mice using a slipknot technique as previously described (44).
ACSL assay. ACSL initial rates were measured with 50 ?M [1-14C]palmitic
acid (Perkin Elmer, Waltham, MA), 10 mM ATP, and 0.25 mM coenzyme A
(CoA) in total membrane fractions (0.5 to 4.0 ?g) or in ventricular mitochondria
Reverse transcription-PCR (RT-PCR). Total RNA was isolated from ventri-
cles (RNeasy fibrous tissue kit; Qiagen, Alameda, CA), and cDNA was synthe-
sized (Applied Biosystems high-capacity cDNA reverse transcription kit). Total
DNA was isolated using the QIAmp DNA microkit (Qiagen). DNA or cDNA
was amplified by real-time PCR using SYBR green (Applied Biosystems, Foster
City, CA) detection with primers specific to the gene of interest. Results were
normalized to the housekeeping gene Gapdh for mRNA or H19 for DNA and
expressed as arbitrary units of 2???CTrelative to the control group.
Immunoblots. Total protein lysates were isolated in lysis buffer (20 mM Tris
base, 1% Triton X-100, 50 mM NaCl, 250 mM sucrose, 50 mM NaF, 5 mM
Na2P2O7, plus protease inhibitor [catalog no. 11836153; Roche, Florence, SC]).
Total membrane fractions were isolated in medium I (10 mM Tris [pH 7.4], 1
mM EDTA, 0.25 M sucrose, 1 mM dithiothreitol). Equal amounts of protein (40
to 60 ?g) were loaded and resolved on 10% SDS-polyacrylamide gels and
transferred to nitrocellulose membranes. Blots were probed with antibodies
against ACSL1 (catalog no. 4047), phosphorylated AMP-activated protein kinase
(P-AMPK) (catalog no. 2535), and phosphorylated p70 (P-p70) S6 kinase (S6K)
(catalog no. 9234) and were then stripped and reprobed with either total
AMPK? (catalog no. 2532) or p70 S6K (catalog no. 9202) antibody (all antibod-
ies from Cell Signaling, Danvers, MA). The purity of mitochondrial fractions was
verified by immunoblotting with antibodies against the mitochondrial protein
voltage-dependent anion channel protein (VDAC) (ab16816) and the endoplas-
mic reticulum (ER) protein calnexin (ab13504) (both antibodies from Abcam,
Histology. The hearts were gravity perfused and fixed for 24 h in PBS con-
taining 4% paraformaldehyde and transferred to 70% ethanol. The fixed tissue
samples were embedded in paraffin, serial sectioned, and stained with hematox-
ylin and eosin or Masson’s trichrome. For lectin staining, paraformaldehyde-
fixed cardiac tissue samples were deparaffinized, hydrated, and incubated with
Triticum vulgaris lectin tetramethyl rhodamine isothiocyanate (TRITC) conju-
gate (Sigma). The sections were subsequently examined by fluorescence micros-
copy. For electron micrograph analysis, the animals were euthanized, and their
hearts were perfused with a freshly made solution containing 2% paraformalde-
hyde and 2.5% glutaraldehyde in 0.15 M sodium phosphate buffer, pH 7.4.
Cardiac tissue was imaged using a LEO EM910 transmission electron micro-
scope at 80 kV (LEO Electron Microscopy, Thornwood, NY) and photographed
using a Gatan BioScan digital camera (Gatan, Inc., Pleasanton, CA). Mitochon-
drial area and myocyte size were quantified using NIH ImageJ software.
Fatty acid oxidation. Freshly isolated heart ventricles and livers were minced
and homogenized with 10 up-and-down strokes using a motor-driven Teflon
pestle and glass mortar in ice-cold buffer (100 mM KCl, 40 mM Tris-HCl, 10 mM
Tris base, 5 mM MgCl2? 6H2O, 1 mM EDTA, and 1 mM ATP [pH 7.4]) at a
20-fold dilution (wt/vol), or 40 ?g of isolated mitochondria was used for oxida-
tion (37). Oxidation was measured in a 200-?l reaction mixture containing 100
mM sucrose, 10 mM Tris-HCl, 10 mM KPO4, 100 mM KCl, 1 mM
MgCl2? 6H2O, 1 mM L-carnitine, 0.1 mM malate, 2 mM ATP, 0.05 mM coen-
zyme A, and 1 mM dithiothreitol (pH 7.4) with either 8 ?M [1-14C]palmitate (0.1
?Ci/reaction) and 100 ?M sodium palmitate complexed to BSA, 80 ?M
[1-14C]pyruvate (0.1 ?Ci/reaction mixture) and 5 mM pyruvate, 4 ?M
[1-14C]palmitoyl-CoA (0.04 ?Ci/reaction mixture) and 50 ?M palmitoyl-CoA, or
8 ?M [U-14C]glucose (0.1 ?Ci/reaction mixture) and 200 ?M glucose. Oxidation
studies measured the production of14C-labeled carbon dioxide (CO2) and acid-
soluble metabolites (ASM) for 30 min without substrate competition in a two-
well oxidation system: one well contained the reaction mixture with the tissue
homogenate, and the adjoining well contained 1 NaOH. The reaction was ter-
minated by adding 70% perchloric acid to the assay well, and then the plate was
incubated for 1 h to drive the CO2into the NaOH. Radioactivity of ASM in the
supernatant of the reaction mixture and CO2was determined by liquid scintil-
lation. Fatty acid oxidation was quantified using the following formula: [(dpm ?
BL)/SA]/[gram of tissue [wet weight] ? time (in hours) of reaction mixture
incubation]), where dpm is the disintegrations per minute, BL is the dpm of
blank wells, and SA is the FA-specific radioactivity.
Tissue lipid, nucleotide, and glucose-6-phosphate content. Acyl-carnitines
were quantified by liquid chromatography and tandem mass spectrometry (1).
Complex lipid content was analyzed by Lipomics Technologies, Inc. (West Sac-
ramento, CA) (50). Acyl-CoA and diacylglycerol composition and content were
assessed from whole hearts extracted with internal standards (36). After sepa-
ration, purification, and elution, lipid metabolite extracts were separated by
high-performance liquid chromatography (HPLC), and individual and total lipid
species were analyzed by liquid chromatography and tandem mass spectrometry
(36). Frozen hearts were homogenized in 0.4 M perchlorate and neutralized in 4
M K2CO3as described previously (31). Nucleotides were separated by HPLC (7)
using a Varian Prostar solvent delivery system (PS-210; Varian, Palo Alta, CA)
and a Luna 5?m C18100A column (Phenomenex, Torrance, CA). Peaks were
detected using a Gilson 118 UV detector (Middleton, WI). Glucose-6-phosphate
was measured by a spectrophotometric enzymatic analysis using glucose-6-phos-
VOL. 31, 2011CARDIAC FA OXIDATION REQUIRES ACYL-CoA SYNTHETASE 11253
phate dehydrogenase coupled to NADPH production with extinction at 340 nm
PPAR? activity. Nuclear fractions were isolated from fresh hearts (nuclear
extraction kit; Cayman Chemicals, Ann Arbor, MI) and used with the peroxi-
some proliferator-activated receptor ? (PPAR?) complete transcription factor
assay kit (Cayman Chemicals, Ann Arbor, MI) according to the manufacturer’s
2-Deoxyglucose uptake. Anesthetized mice were injected retro-orbitally with
10 ?Ci of 2-[1-14C]deoxyglucose in saline (Moravek Biochemicals, Brea, CA).
Tissues were harvested and flash frozen in liquid N230 min after injection.
Radioactivity was measured in tissue homogenates and normalized to the counts
present in 10 ?l of serum obtained 5 min after injection [(dpm/mg of tissue)/dpm
in 10 ?l serum].
Cardiac ACSL activity and acyl-CoA content are reduced in
mice that lack ACSL1. In mice lacking ACSL1 globally
(Acsl1T?/?) mice, the largest and most persistent reduction in
total ACSL enzyme activity and ACSL1 protein was observed
in hearts, compared to livers, kidneys, and adipose tissue (Fig.
1A to D). Ten weeks after tamoxifen was injected, total ACSL
activity in heart ventricles was 97% lower than in the littermate
controls (Fig. 1A). The ACSL activity that remained in tissues
was probably due to other ACSL isoenzymes and to residual
ACSL1 activity in cells that had not been exposed to tamox-
ifen. The appearance, histology, and weights of the liver and
adipose tissue did not differ for the different genotypes (Table
1; histology not shown). Two weeks after the tamoxifen injec-
tion, heart Acsl1 mRNA was nearly absent, but 40% of ACSL1
protein remained (Fig. 1B and E), suggesting that the half-life
of ACSL1 may be as long as 2 weeks. By 10 weeks after the
tamoxifen injection, virtually no ACSL1 protein was present.
Other differences in the measurements at the two time points
included 37% lower plasma triacylglycerol (TAG) and 2.5-
fold-higher plasma fatty acid (FA) concentrations in the
Acsl1T?/?mice compared to the controls at 2 weeks, but not at
10 weeks after the tamoxifen injection (Table 1). In hearts
from Acsl1T?/?mice, loss of ACSL1 reduced the total pool of
long-chain acyl-CoAs by 65% (Fig. 1F), with 67 to 75% reduc-
tions in 16:0-, 16:1-, 18:1-, 18:2-, and 18:3-CoA species and a
26% reduction in 18:0-CoA (Fig. 1G). Expressed as a percent-
age of total acyl-CoA content, 18:0-CoA was twice as high in
Acsl1T?/?hearts as in the control hearts, and all other species
except 16:0-CoA were reduced ?20%. Thus, aside from the
relative increase in 18:0-CoA, lack of ACSL1 did not markedly
change the FA composition of the acyl-CoA pool. These data
indicate that ACSL1 is the major activator of long-chain FA in
the heart and that the knockdown of Acsl1 was virtually com-
plete and persistent in cardiac tissue for as long as 10 weeks.
Despite the 65% reduction in long-chain acyl-CoA content in
Acsl1T?/?hearts, total cardiac TAG content did not change,
suggesting that ACSL1 does not limit the acyl-CoA pool used
for the synthesis of TAG (Fig. 1H).
ACSL1 is required for heart FA oxidation. To determine
whether ACSL1 provides acyl-CoAs for FA oxidation, we as-
sessed the rates of [1-14C]palmitate incorporation into CO2
and acid-soluble metabolites (ASM), which are measures of
complete and incomplete oxidation, respectively. Compared to
the controls, the rate of maximal FA oxidation in Acsl1T?/?
heart homogenates was 95% lower (Fig. 2A). Thus, the ab-
sence of ACSL1 nearly abolished FA oxidative capacity. In
contrast, in Acsl1T?/?livers, in which ACSL1 protein and
FIG. 1. ACSL activity and acyl-CoA content are reduced in mice that lack ACSL1. (A to D) Total ACSL activity (A and C) and ACSL1 protein
(B and D) in control (Con) and Acsl1T?/?tissues 2 weeks (2wk) and 10 weeks (10wk) after the tamoxifen injection (n ? 5 to 7). Kid, kidney; WAT,
white adipose tissue; BAT, brown adipose tissue. (E) Acsl isoenzyme mRNA abundance in ventricles 2 and 10 weeks after the tamoxifen injection
in control and Acsl1T?/?mice (n ? 6). (F and G) Heart total (F) and individual (G) long-chain acyl-CoA content in control and Acsl1T?/?mice
10 weeks after the tamoxifen injection (n ? 7 or 8). (H) Heart triacylglycerol (TAG) content in control and Acsl1T?/?mice 10 weeks after the
tamoxifen injection (n ? 7 or 8). The values are means plus standard errors of the means (SEMs) (error bars). Values that are significantly different
(P ? 0.05) from the values for the control are indicated by an asterisk. Values that are significantly different 2 weeks versus 10 weeks after the
tamoxifen injection within mice of the same genotype are indicated by a # symbol.
1254 ELLIS ET AL.MOL. CELL. BIOL.
activity remained similar to those of controls, the rate of
[1-14C]palmitate oxidation was unchanged (Fig. 2A). The oxi-
dation rates for the ACSL1 product [1-14C]palmitoyl-CoA
were similar for the two genotypes (Fig. 2B), indicating that
carnitine palmitoyltransferase 1 (CPT1)-mediated transport of
acyl-CoA into the mitochondria and its subsequent oxidation
to CO2were unimpaired and that the severe block in FA
oxidation in Acsl1T?/?hearts was due to the lack of FA acti-
The 95% lower rates of FA oxidative capacity in Acsl1T?/?
heart homogenates strongly suggested that ACSL1 activates
the pool of FAs that is directed toward mitochondrial oxida-
tion. This interpretation is further supported by the presence
of residual ACSL1 in purified heart mitochondria 2 weeks after
tamoxifen was injected (Fig. 2C). At that time, when ?40% of
ACSL1 protein still remained, [1-14C]palmitate oxidation in
Acsl1T?/?heart mitochondria was 40% lower than in the con-
trols; however, 10 weeks after the tamoxifen injection, when
ACSL1 protein was absent, [1-14C]palmitate oxidation was
90% lower than in the controls (Fig. 2D). These data showing
TABLE 1. Body weight, adipose weight, plasma metabolites, and blood pressure in male micea
Value (mean ? SEM) at the following time after the tamoxifen injection in micec:
2 wk 10 wk
Body wt (g)
G-WAT wt (% of BW)
Plasma TAG concn (mg/dl)
Plasma FA concn (mmol/liter)
Glucose concn (mg/dl)
Insulin concn (ng/ml)
25.5 ? 1.5
0.87 ? 0.1
47.2 ? 3
0.08 ? 0.03
136 ? 8
23.3 ? 1.7
0.92 ? 0.1
29.7 ? 2.0*
0.28 ? 0.04*
131 ? 9
31.4 ? 0.8†
1.7 ? 0.2†
36.7 ? 5†
0.08 ? 0.01
164 ? 3.6
2.73 ? 0.8
4,981 ? 298
30.1 ? 0.4†
1.8 ? 0.2†
33.8 ? 2
0.09 ? 0.01†
136 ? 5.3*
1.67 ? 0.2
4,828 ? 357
Systolic (mm Hg)
Diastolic (mm Hg)
155 ? 4.0
120 ? 4.3
158 ? 3.4
125 ? 3.6
aMale control and Acsl1T?/?mice (n ? 8 to 15) were studied 2 and 10 weeks after the tamoxifen injection. The body weight and adipose tissue weight, plasma
metabolites, area under the curve in response to insulin tolerance tests, and blood pressure were determined.
bG-WAT, gonadal white adipose tissue; TAG, triacylglycerol; FA, fatty acid; area under the curve (AUC) in response to the insulin tolerance test (ITT).
cValues that are significantly different (P ? 0.05) from the value for the control mice are indicated by an asterisk. Values that are significantly different, (P ? 0.05)
at 2 weeks versus 10 weeks after the tamoxifen injection within mice of the same genotype are indicated by a † symbol.
FIG. 2. Acsl1T?/?hearts have impaired fatty acid (FA) oxidation. (A and B) [1-14C]palmitate oxidation to CO2and acid-soluble metabolites
(ASM) in heart and liver homogenates (A) and heart [1-14C]palmitoyl-CoA oxidation into CO2from control (Con) and Acsl1T?/?(T?/?) mice (n ?
5 or 6) (B). (C) Representative immunoblots against ACSL1, VDAC, and calnexin in control and Acsl1T?/?mitochondrial fractions 2 and 10 weeks
after the tamoxifen injection. (D) [1-14C]palmitate oxidation to CO2and ASM from control (Con) and Acsl1T?/?(T?/?) ventricular mitochondria
2 and 10 weeks after the tamoxifen injection (n ? 5 or 6). (E to H) Free acyl-carnitines (E), acetyl-carnitine (F), medium-chain acyl-carnitines (G),
and long-chain acyl-carnitines (H) in control and Acsl1T?/?hearts (n ? 5 to 7). The values are means plus SEMs (error bars). Values that are
significantly different (P ? 0.05) from the values for the control are indicated by an asterisk.
VOL. 31, 2011CARDIAC FA OXIDATION REQUIRES ACYL-CoA SYNTHETASE 11255
that the rate of FA oxidative capacity and the amount of
ACSL1 protein decreased in parallel support the idea that
ACSL1 is required to activate cardiac FAs before they can be
Cellular acyl-carnitines are markers of metabolite flux
through degradative and oxidative pathways (30). In
Acsl1T?/?cardiac tissue, total free carnitine was 77% higher
than in control hearts, indicating that carnitine was not
limiting (Fig. 2E), whereas acetyl-carnitine was 51% lower,
consistent with reduced mitochondrial acetyl-CoA levels
(Fig. 2F). Confirming the impaired FA oxidation, nearly all
medium-chain (8- to 12-carbon) acyl-carnitine species in the
Acsl1T?/?hearts were 60 to 80% lower than in the control
hearts (Fig. 2G), and long-chain acyl-carnitines were 80 to
90% lower than in the control hearts (Fig. 2H). Total cho-
lesteryl ester and diacylglycerol were reduced ?20% in
Acsl1T?/?hearts, but total phospholipid (PL) and TAG
were unaffected (see Fig. S1 in the supplemental material).
Because the levels of PL and TAG were normal in Acsl1T?/?
hearts but the abundance of nearly all acyl-carnitine species
was reduced ?90%, it appears that ACSL1 activity does not
affect the synthesis of acyl-CoAs used in the pathways of PL
and TAG synthesis but, instead, specifically provides the
acyl-CoAs required for FA oxidation.
Altered FA composition of Acsl1T?/?cardiac lipids. If all
acyl-CoAs were metabolically equivalent, the composition
of complex lipids would reflect changes in the acyl-CoA
pool. However, despite the fact that the Acsl1T?/?hearts
contained twice as much 18:0-CoA as the control hearts (as
a percentage of total long-chain acyl-CoAs), the amount of
18:0-CoA did not change in the TAG, phosphatidylethano-
lamine (PE), and phosphatidylserine (PS) fractions of
Acsl1T?/?hearts and was significantly lower in the cardio-
lipin (CL) and phosphatidylcholine (PC) fractions (see Fig.
S1 in the supplemental material). Acsl1T?/?18:1-CoA con-
tent was ?60% lower than in the controls and, as a percent-
age of total long-chain acyl-CoAs, was 10 to 20% lower;
however, the total levels of 18:1-CoA were not altered in the
TAG, PE, and PS fractions and were increased in the CL
and PC fractions. As a percentage of total esterified FA,
18:1-CoA was increased in TAG, PE, CL, and PC fractions,
but not in the PS fraction (see Fig. S1 in the supplemental
material). These data indicate that the FA composition of
TAG and PL in Acsl1T?/?hearts did not reflect the changes
in the acyl-CoA pool and suggest that other ACSL isoen-
zymes contributed to the acyl-CoAs that were esterified in
the pathways of glycerolipid synthesis.
Pathological cardiac hypertrophy and mitochondrial bio-
genesis in the Acsl1T?/?mice. The weights of Acsl1T?/?female
and male hearts as a percentage of body weight were 23% and
17% greater than the control hearts, respectively (Fig. 3A).
Cardiomyocyte area was 27% larger than for the controls (Fig.
3B), and ventricular walls appeared thickened (Fig. 3C). Echo-
cardiography confirmed that Acsl1T?/?ventricular walls were
10 to 41% thicker than for the controls (Table 2) with a 34%
heavier left ventricular mass (Fig. 3D and E). Echocardiogra-
phy analysis also showed that the Acsl1T?/?left ventricular
inner diameter was ?30% smaller than for the controls, sug-
gesting concentric ventricular hypertrophy (Table 2). Despite
left ventricular hypertrophy (LVH), the percent fractional
shortening and ejection fraction were similar to the values for
the controls, indicating that cardiac function remained normal
for at least 2 months after the ablation of Acsl1 (Fig. 3F and
Upregulation of fetal genes occurs with pathological hyper-
trophy (18); thus, the 4- to 5-fold-higher mRNA expression of
FIG. 3. Acsl1T?/?mice develop cardiac hypertrophy. (A) Weight (Wt) (wet weight) of control and Acsl1T?/?female and male hearts expressed
as a percentage of body weight (n ? 10 to 20). (B) Quantification of cardiomyocyte area from lectin-stained control and Acsl1T?/?male hearts
(n ? 3). (C) Representative hematoxylin-and-eosin-stained control and Acsl1T?/?hearts. (D) Echocardiography calculation of left ventricular (LV)
mass to the body mass of male control and Acsl1T?/?mice (n ? 8 to 10). (E and F) Representative echocardiogram M-mode images (E) and
percent fractional shortening (%FS) (F) in control and Acsl1T?/?male mice (n ? 8 to 10). (G) mRNA abundance of fetal gene markers, ?-skeletal
actin (?SkAc) and brain natriuretic peptide (Bnp) in control and Acsl1T?/?hearts (n ? 6). (H to J) Heart weight (H), percent fractional shortening
(I), and ACSL1 protein content (in arbitrary units [Au]) (J) in control and Acsl1T?/?mice 4 weeks after transverse arch banding (TAB) (n ? 4
to 6). The values are means plus SEMs (error bars). Values that are significantly different (P ? 0.05) from the values for the control are indicated
by an asterisk.
1256ELLIS ET AL.MOL. CELL. BIOL.
the fetal genes for ?-skeletal actin and brain natriuretic pep-
tide (Fig. 3G), together with the 6-fold upregulation of Acsl3,
the major Acsl mRNA expressed in fetal heart (13) (Fig. 1E),
is compatible with pathological hypertrophy. Fibrosis was not
present (data not shown). Thus, the absence of ACSL1 for 10
weeks severely impaired FA oxidation and induced ventricular
hypertrophy with upregulation of fetal genes but without car-
diac dysfunction or failure.
To evaluate the responses of hearts from Acsl1T?/?mice
under stress conditions, we induced pressure overload by trans-
verse arch banding in control and Acsl1T?/?mice. The banding
procedure resulted in a doubling of heart weight (Fig. 3H) and
a decrease in fractional shortening to ?40% (Fig. 3I) in both
control and Acsl1T?/?hearts. With transverse arch banding-
induced hypertrophy, the ACSL1 protein decreased (Fig. 3).
These data show that ACSL1 is repressed and not required for
pressure overload-induced cardiac hypertrophy.
Evaluation of cardiomyocyte organelles by electron micros-
copy showed that the mitochondrial area in Acsl1T?/?ventri-
cles was 54% greater than in the controls (Fig. 4A and
B). Confirming the enhanced mitochondrial biogenesis in
Acsl1T?/?ventricles, the levels of mitochondrial DNA for
NADH dehydrogenase subunit 1, cytochrome b, and cyto-
chrome c oxidase 1 were at least 2-fold higher than for the
controls (Fig. 4C). Mitochondrial biogenesis is driven by sev-
eral transcription factors, including peroxisome proliferator-
activated receptor (PPAR) coactivator 1? (PGC1?) and estro-
gen-related receptor ? (ERR?) (35). In Acsl1T?/?ventricles,
the mRNA levels for the Pgc1? and Err? genes were 100% and
41% greater, respectively, than in controls 2 weeks after ta-
moxifen was injected (Fig. 4D). PPAR? transcription factor
activity in nuclear extracts from control and Acsl1T?/?hearts
did not differ for the two genotypes (Fig. 4E), but several
Acsl1T?/?hearts, including muscle carnitine palmitoyltrans-
ferase 1, cytosolic thioesterase 1, and Acsl3 (6) (Fig. 4F and G
and 1E). The increased mRNA abundance of mitochondrial
?-oxidation genes could reflect either increased mitochondrial
content in Acsl1T?/?hearts or the activation of PPAR? by
mammalian target of rapamycin (mTOR) (46). Because FA
oxidation was severely impaired, we suspected that an energy
deficit might exist that would activate the energy-sensing en-
TABLE 2. Echocardiogram diameter and functional characteristicsa
Value (mean ? SEM) for micec:
0.98 ? 0.02
2.88 ? 0.06
0.86 ? 0.01
1.29 ? 0.02*
2.47 ? 0.06*
1.23 ? 0.02*
1.67 ? 0.03
1.31 ? 0.04
1.48 ? 0.03
1.89 ? 0.04*
1.08 ? 0.03*
1.80 ? 0.04*
LV mass (mg)
86.6 ? 0.50
86.5 ? 3.57
88.3 ? 0.51
115.4 ? 4.24*
aTransthoracic echocardiogram imaging was performed on conscious control
and Acsl1T?/?male mice (n ? 6 to 8) 10 weeks after the tamoxifen injection.
M-mode images were analyzed.
bIVST, interventricular septum thickness; LVID, left ventricule inner diam-
eter; LVPWT, left ventricule posterior wall thickness; % EF, percent ejection
fraction; LV, left ventricule.
cValues that are significantly different (P ? 0.05) from the values for the
control are indicated by an asterisk.
FIG. 4. Mitochondrial excess in Acsl1T?/?hearts. (A and B) Representative electron microscopy images (bars ? 2 ?m) (A) and quantification
of mitochondrial area from the ventricles of control and Acsl1T?/?male mice (B) 10 weeks after the tamoxifen injection (n ? 3). (C) Quantification
of the mitochondrial DNA genes for cytochrome c oxidase 1 (Co1), cytochrome b (Cytb), and NADH dehydrogenase subunit 1 (Nd1) relative to
nuclear DNA in the ventricles from male and female control and Acsl1T?/?mice 10 weeks after the tamoxifen injection (n ? 6). (D) mRNA
abundance of Pgc1? and Err? genes in control and Acsl1T?/?ventricles 2 weeks after the tamoxifen injection (n ? 6). (E) PPAR? transcription
factor activity (TFA) (in arbitrary units [Au] per microgram of protein) in nuclear extracts from control and Acsl1T?/?hearts (n ? 6 to 8). (F and
G) mRNA abundance of Cte1, mCpt1, Mcad, Cd36, Fas, and Ppar? genes in control and Acsl1T?/?ventricles (n ? 6). (H) Quantification of AMPK
phosphorylation (AMPK-P) at Thr172over total AMPK in control and Acsl1T?/?ventricles 10 weeks after the tamoxifen injection (n ? 5 to 7).
(I) Myocardial ATP and AMP content determined by HPLC in control and Acsl1T?/?hearts. The values are means ? SEMs (error bars). Values
that are significantly different (P ? 0.05) from the values for the control are indicated by an asterisk.
VOL. 31, 2011CARDIAC FA OXIDATION REQUIRES ACYL-CoA SYNTHETASE 11257
zyme, AMP-activated kinase (AMPK), which can upregulate
Pgc1?. However, compared to the controls, AMPK phosphor-
ylation (Thr172) was ?40% lower in Acsl1T?/?ventricles (Fig.
4H). Further, myocardial ATP and AMP content did not differ
for the two genotypes (Fig. 4I), suggesting that glucose oxida-
tion had increased sufficiently to maintain the cellular ATP/
Increased glucose use in Acsl1T?/?hearts. Because the ca-
pacity of Acsl1T?/?hearts to oxidize FAs was markedly im-
paired yet cellular ATP content was preserved, we questioned
whether the oxidative capacity for non-FA substrates had in-
creased to compensate. In Acsl1T?/?heart homogenates, the
maximal rate of [U-14C]glucose incorporation into [14C]CO2
was 20% higher than in the controls (Fig. 5A), and in
[1-14C]pyruvate to [14C]CO2was 39% higher, suggesting in-
creased pyruvate dehydrogenase activity (Fig. 5B). Consistent
with increased glucose use, plasma glucose in Acsl1T?/?mice
was 17% lower than in the control mice (Table 1). Confirming
enhanced glucose metabolism, the uptake of [1-14C]2-deoxy-
glucose into Acsl1T?/?hearts compared to the control hearts
was 9-fold greater (Fig. 5C). 2-Deoxyglucose uptake into
Acsl1T?/?liver and gonadal adipose tissue was similar to that
in control mice, but the uptake of 2-deoxyglucose into brown
adipose tissue (BAT) was 2-fold greater (Fig. 5D). The amount
of glucose-6-phosphate was 3-fold higher in the Acsl1T?/?
hearts than in the control hearts, suggesting an enhanced rate
of glycolysis with accumulation of glycolytic intermediates (Fig.
5E). In addition, propionyl- and methyl-malonyl/succinyl-car-
nitines were 71% and 174% higher in Acsl1T?/?hearts than in
the control hearts (Fig. 5F). These carnitine species are me-
tabolites of glucose and of branched-chain and ketogenic
amino acids, suggesting that oxidation of these alternate sub-
strates had increased in Acsl1T?/?hearts. Consistent with the
idea that protein catabolism provided an alternative fuel
source, the total amino acid content in Acsl1T?/?hearts was
32% higher than in the control hearts (Fig. 5G). This increase
in amino acid content was reflected by increases in nearly every
amino acid species (Fig. 5H and I). Together, these data
strongly support the conclusion that reduced FA oxidation in
Acsl1T?/?hearts leads to compensatory increases in glycolysis
and protein catabolism.
mTOR stimulates hypertrophy in Acsl1T?/?hearts. The
mTOR kinase activates pathways that increase cell growth (3,
12). mTOR kinase activation is required for both thyroid hor-
mone-induced and spontaneous hypertensive rat cardiac hy-
pertrophy (14, 25). To determine whether mTOR was linked to
the hypertrophy observed in Acsl1T?/?hearts, we quantified
the phosphorylation of the mTOR target, p70-S6 kinase (S6K).
Compared to the controls, S6K phosphorylation at Thr389was
5-fold greater in Acsl1T?/?hearts (Fig. 5J). These data suggest
that the cardiac hypertrophy in Acsl1T?/?mice was mediated
by activation of the mTOR pathway. Possible causes of en-
hanced mTOR signaling include elevations in amino acid avail-
ability (2), enhanced glycolytic flux (41), elevated plasma insu-
lin (21), and hypertension (42). mTOR activation in Acsl1T?/?
hearts probably resulted from a combination of several of these
factors, including the 32% higher amino acid content and the
increase in glucose uptake and glycolysis (Fig. 5). In addition,
diminished AMPK activity, as shown by the ?40% reduction in
FIG. 5. Glucose oxidation, amino acid catabolism, and S6 kinase
activation increased in Acsl1T?/?hearts. (A) [U-14C]glucose oxidation
to CO2in heart homogenates from control and Acsl1T?/?mice (n ? 5
or 6). (B) [1-14C]pyruvate oxidation to CO2in mitochondria from the
hearts of control and Acsl1T?/?mice (n ? 3 or 4). (C and D) [1-14C]2-
deoxyglucose ([1-14C]2DG) uptake into the heart (C) and liver, gas-
trocnemius muscle, gonadal white adipose tissue (WAT), and brown
adipose tissue (BAT) (D) (see Materials and Methods) (n ? 3 or 4).
(E) Glucose-6-phosphate content in control and Acsl1T?/?ventricles
(n ? 5 to 7). (F) Short-chain acyl-carnitine content in control and
Acsl1T?/?hearts (n ? 5 to 7). Short-chain acyl-carnitine abbreviations:
3, propionyl-carnitine; 5OH/3DC, 3-hydroxy-isovalerly- or malonyl-
carnitine; 4DC/i4DC, methylmalonyl- or succinyl-carnitine. (G to I)
Total amino acids (AA) (G) and individual amino acids (H and I) in
control and Acsl1T?/?hearts (n ? 5 to 7). (J) Representative immu-
noblot (n ? 4 or 5) and quantification (n ? 10) of p70-S6K phosphor-
ylation (S6K-P) at Thr389and total p70-S6K in control and Acsl1T?/?
ventricles. The values are means plus SEMs (error bars). Values that
are significantly different (P ? 0.05) from the values for the control are
indicated by an asterisk.
1258 ELLIS ET AL.MOL. CELL. BIOL.
Acsl1T?/?heart AMPK phosphorylation (Fig. 4H), would re-
lieve the inhibition of mTOR by AMPK (23). Other activators
of mTOR signaling were not present; neither plasma insulin
nor the area under the curve during an insulin tolerance test
differed for mice with the two genotypes, and hypertension was
not present (Table 1). Because the downstream substrate of
mTOR, S6 kinase, is not hyperphosphorylated in brown adi-
pose or gonadal adipose tissue from Acsl1T?/?mice (data not
shown), it appears that mTOR is activated by the deficiency of
ACSL1 specifically in cardiac tissue. Glucose-6-phosphate is
also an activator of mTOR (41), and compared to the control
hearts, the Acsl1T?/?hearts contained 3-fold-more glucose-6-
phosphate (Fig. 5E). These data implicate enhanced glycolytic
flux, the accumulation of glycolytic intermediates, and de-
creased AMPK activation as the major signals that mediate
mTOR activation and hypertrophy in Acsl1T?/?hearts.
Heart-specific ACSL1 deficiency causes hypertrophy. Be-
cause the Acsl1T?/?mice were deficient in ACSL1 in multiple
tissues (Fig. 1A), it might be argued that the observed cardiac
hypertrophy had resulted from some aspect of the whole-body
ACSL1 deficiency. Thus, we examined mice that were deficient
in ACSL1 only in ventricular cardiomyocytes (Acsl1H?/?). In
these Acsl1H?/?mice, real-time PCR showed nearly absent
expression of Acsl1 mRNA in the ventricles (Fig. 6A) and a
50% lower expression of Acsl5 mRNA (data not shown).
ACSL1 protein was absent in Acsl1H?/?ventricles but was
present in the liver and skeletal muscle and at levels similar to
those of the controls (Fig. 6B). The activity of ACSL in
Acsl1H?/?atria was similar to that of controls, whereas ven-
tricle ACSL activity was 90% lower (Fig. 6C). Confirming that
ACSL1 is required for cardiomyocyte mitochondrial fatty acid
oxidation, the rate of [1-14C]palmitate incorporation into CO2
and ASM in isolated mitochondria from Acsl1H?/?ventricles
was reduced ?90% compared to control ventricles (Fig. 6D).
Supporting the conclusion that cardiac hypertrophy was a di-
rect result of ACSL1 cardiomyocyte deficiency, the weights of
the hearts from female and male Acsl1H?/?mice were 25%
and 20% greater than the hearts from the control mice, re-
spectively (Fig. 6E). The left ventricle masses, calculated from
echocardiograph M-mode images, were 34% heavier (Fig. 6F)
with a 30% larger left ventricular wall diameter in the
Acsl1H?/?hearts than in the control hearts. The percent frac-
tional shortening and ejection fraction were similar in mice
with both genotypes, indicating that cardiac function remained
normal despite ventricular hypertrophy (data not shown). To
determine whether mTOR was linked to the hypertrophy ob-
served in Acsl1H?/?hearts, we quantified the phosphorylation
of S6K. S6K phosphorylation at Thr389was 5-fold greater in
the Acsl1H?/?hearts than in the control hearts (Fig. 6G).
These data strongly suggest that the cardiac hypertrophy in
both Acsl1T?/?and Acsl1H?/?mice was mediated by activation
of the mTOR pathway and that hypertrophy is a direct conse-
quence of ACSL1 deficiency in cardiomyocytes.
The primary finding of this study is that ACSL1 provides the
FAs used for cardiac oxidation, and that in the absence of
ACSL1, the heart compensates by increasing the oxidation of
glucose and amino acids. Normally, FAs provide 60 to 90%
of the heart’s energy requirements, and glucose and lactate
oxidation provide the remaining ATP (43, 49). The use of these
three energy sources is controlled by substrate availability,
physiological conditions, and transcriptional and hormonal
regulation. Our data suggest that the use of FAs as an oxidative
substrate is additionally controlled by ACSL1.
Whether a shift from lipolytic to glycolytic oxidation is a
cause or a consequence of cardiac hypertrophy has remained
unclear. Data from human deficiencies in FA oxidation and
rodent models with genetically or chemically impaired FA ox-
idative capacity support the interpretation that reduced FA
oxidation causes cardiac hypertrophy. For example, in humans,
defects in the Na?-carnitine cotransporter (Na?-driven or-
ganic cation transporter 2 [OCTN2]) cause a cardiomyopathy
characterized by cardiac lipid accumulation and hypertrophy
(34). Similarly, mice with juvenile visceral steatosis (JVS)
mouse mimics human systemic carnitine deficiency because
mice with JVS have a spontaneous deficiency of OCTN2 that
results in cardiac lipid accumulation and hypertrophy, as well
FIG. 6. Impaired oxidation, hypertrophy, and S6 kinase activation
in the cardiomyocyte-specific ACSL1 knockout mice. (A) Acsl1 mRNA
abundance in the heart 10 weeks after the tamoxifen injection in
control and Acsl1H?/?mice (n ? 6). (B) Representative immunoblot
against ACSL1 protein in heart, liver, and gastrocnemious (gastroc)
muscle 10 weeks after the tamoxifen injection. (C) Total ACSL activity
in control and Acsl1H?/?atria and ventricles 10 weeks after tamoxifen
(n ? 5 to 7). (D) [1-14C]palmitate oxidation into carbon dioxide (CO2)
and acid-soluble metabolites (ASM) in heart homogenates from con-
trol and Acsl1H?/?mice (n ? 5 or 6). (E) Weight (wet weight) of
control and Acsl1H?/?male and female hearts expressed as a percent-
age of body weight (n ? 10 to 20). (F) Echocardiography calculation of
left ventricular (LV) mass in control and Acsl1H?/?male mice (n ? 8
to 10). (G) Representative immunoblot of p70-S6K phosphorylation at
Thr389and total p70-S6K in control and Acsl1H?/?ventricles 20 weeks
after tamoxifen (n ? 4 or 5). The values are means plus SEMs (error
bars). Values that are significantly different (P ? 0.05) from the values
for the control are indicated by an asterisk.
VOL. 31, 2011 CARDIAC FA OXIDATION REQUIRES ACYL-CoA SYNTHETASE 1 1259
as a 2-fold increase in cardiac mitochondrial area (45). Inhibi-
tion of CPT1 and acyl-carnitine synthesis in rats leads to
cardiac hypertrophy (39), and mice deficient in long-chain acyl-
CoA dehydrogenase (LCAD) (11) or very-long-chain acyl-
CoA dehydrogenase (VLCAD) (17) also develop cardiac hy-
pertrophy, increased mitochondrial biogenesis, and TAG
accumulation. Our data also strongly support the idea that the
shift to glycolysis promotes cardiac hypertrophy.
When ACSL1 is overexpressed 12-fold in the mouse heart,
left ventricular mass doubles by the time the mouse is 3 weeks
old, and heart failure, characterized by a 50% reduction in
percent fractional shortening, occurs by the time the mouse is
4 weeks old (8). The rates of FA oxidation were not assessed in
these hearts, but the levels of TAG and phosphatidylcholine in
the hearts were 12-fold and 1.5-fold higher, respectively, than
in the control hearts. These data suggest that when ACSL1 is
markedly overexpressed, the acyl-CoAs produced are used to
synthesize TAG and phospholipid (PL). In this model, ACSL1
overexpression probably activated large amounts of acyl-CoA
within cardiomyocytes, without a concomitant increase in oxi-
dative demand. Thus, similarly to VLCAD and LCAD null
mice, in which mitochondrial FA oxidation is partially blocked
(11, 17), excess and potentially toxic acyl-CoAs are esterified to
form triacylglycerol (TAG). In contrast to these models, lack of
ACSL1 prevents FA activation, thereby both blocking FA me-
tabolism and preventing FAs from being trapped as acyl-CoA
within cells. The reduction in ACSL activity allows nonacti-
vated FAs to leave cells, thereby decreasing the apparent rate
of FA uptake and the amount of FAs retained (48). Thus,
Acsl1T?/?and Acsl1H?/?mice have severely impaired cardiac
FA oxidation that results in hypertrophy without TAG accu-
The fact that the levels of PL and TAG were unchanged in
the hearts of Acsl1T?/?mice despite a 90 to 97% reduction in
ACSL activity suggests that ACSL1 does not substantially con-
tribute to the synthesis of acyl-CoAs that are incorporated into
glycerolipids. If the metabolic fate of a cellular acyl-CoA were
nonspecific, then a 65% decrease in long-chain acyl-CoA con-
tent should diminish both oxidation and TAG synthesis
equally. The normal content of TAG, the 80 to 90% reduction
in long-chain acyl-carnitines, and the 90% decrease in palmi-
tate oxidation, all support the conclusions that ACSL1 chan-
nels FAs specifically toward oxidation and that acyl-CoAs syn-
thesized by other ACSL isoenzymes are used for complex lipid
synthesis, but not for oxidation (Fig. 7).
A major regulator of cell size is the mTOR kinase, which
activates S6 kinase (S6K) to initiate transcriptional activity that
induces cell growth (27). Activated mTOR is present in several
models of cardiac hypertrophy and is likely to be an important
signal in the pathway that mediates hypertrophy. For example,
S6K phosphorylation is increased in the hypertrophied hearts
of spontaneous hypertensive rats (SHR), and treatment with
the mTOR inhibitor rapamycin attenuates the hypertrophy
without altering the hypertension (42). Similarly, rapamycin
inhibits thyroid hormone-induced S6K phosphorylation and
cardiac hypertrophy (25). Exercise-induced cardiac hypertro-
phy in mice also increases S6K phosphorylation (24), whereas
mouse hearts lacking acetyl-CoA carboxylase 2 (ACC2) have
reduced S6K phosphorylation and smaller hearts (16). These
data strongly suggest that mTOR activation mediates cell
growth in several models of cardiac hypertrophy and that when
mTOR is inhibited, the hypertrophy reverses or diminishes.
Several features present in Acsl1T?/?hearts could enhance
mTOR activation, including elevated amino acid content, en-
hanced glycolysis, and diminished AMP-activated kinase
(AMPK) activity. AMPK activity decreases in isolated skeletal
muscle after exposure to glucose or to branched-chain amino
acids and results in increased phosphorylation of mTOR and
S6K and increased protein synthesis (40). This induction of the
mTOR pathway in muscle depends on the reduced AMPK
phosphorylation and activation (40), probably because AMPK
phosphorylates the tumor suppressor complex that coverts
GTP-Rheb to GDP-Rheb and inactivates mTOR (23). Thus,
the decrease in AMPK activity relieves the GDP-Rheb inhibi-
tion of mTOR. Although it remains unclear how increased
glucose and amino acid metabolism activates the mTOR path-
way in cardiac muscle, AMPK suppression is likely to play a
key role in this process. It has been proposed that long-chain
acyl-CoAs inhibit AMPK by allosterically interacting with
LKB1/AMPKK; thus, the 65% reduction in long-chain acyl-
CoAs in Acsl1T?/?hearts could lead to reduced AMPK acti-
vation (47). The resulting diminished content of activated
AMPK would relieve LKB1/AMPKK inhibition and allow
greater mTOR activity. In the isolated working heart of a
mouse, the initial phosphorylation of glucose appears to be
critical for glucose-induced S6K phosphorylation; S6K phos-
phorylation occurred after exposing cardiomyocytes to 2-de-
oxyglucose, which can be phosphorylated, but not after expo-
sure to 3-O-methylglucose, which cannot be phosphorylated
(41). The 3-fold increase in glucose-6-phosphate in the
FIG. 7. Overview of metabolic disturbance and pathway activation
in the Acsl1T?/?heart. The loss of ACSL1 prevents uptake and acti-
vation of fatty acids (FAs) for oxidation. Other ACSL isoforms
(ACSLx) activate FAs that are used for triacylglycerol (TAG) and
phospholipid (PL) synthesis. The inability of Acsl1T?/?heart to oxidize
FA is compensated for by increased glucose and amino acid catabo-
lism. The shift in oxidative metabolism leads to reduced AMPK phos-
phorylation and the activation of the mTOR pathway causes cardiac
hypertrophy in Acsl1T?/?and Acsl1H?/?mice. S6 Kinase-P, S6 kinase
phosphorylation; Glucose 6-P, glucose-6-phosphate.
1260 ELLIS ET AL.MOL. CELL. BIOL.
Acsl1T?/?hearts suggests that glycolytic intermediates may
activate the mTOR pathway. Our data suggest that the shift
from FA to glucose oxidation, the consequent increase in gly-
colytic flux, the increase in amino acid availability, and the
reduction in AMPK phosphorylation all contributed to mTOR
activation in Acsl1T?/?hearts (Fig. 7).
In summary, the loss of ACSL1 in the mouse heart results in
severely impaired FA oxidation, a compensatory increase in
glucose oxidation, and cardiac hypertrophy without systolic
dysfunction or lipid accumulation. The 5-fold increase in S6K
phosphorylation suggests that activation of the mTOR pathway
initiates the observed cardiac hypertrophy in Acsl1T?/?and
Acsl1H?/?hearts. In support of the role of ACSL1 in activating
FAs for oxidation, mice with an adipose-tissue-specific knock-
out of ACSL1 had reduced FA oxidation rates in white and
brown adipocytes and were severely cold intolerant (15). Like
ACSL1 in adipose tissue, it appears that ACSL1 functions in
cardiac tissue to activate FAs destined for ?-oxidation. In this
model, the shift in substrate use from FAs to glucose causes
the ensuing cardiac hypertrophy, although cardiac function was
not impaired during the time period studied.
This work was supported by NIH grants DK59935 (R.A.C.) and
DK40936 (G.I.S.), UNC NORC grant DK056350 from the National
Institute of Diabetes and Digestive and Kidney Diseases, a grant from
the American Diabetes Association (D.M.M.), an NIH Predoctoral
Training grant T32-HL069768 (J.M.E.), and a predoctoral (J.M.E.)
fellowship from the American Heart Association Mid-Atlantic Divi-
1. An, J., et al. 2004. Hepatic expression of malonyl-CoA decarboxylase re-
verses muscle, liver and whole-animal insulin resistance. Nat. Med. 10:268–
2. Avruch, J., et al. 2009. Amino acid regulation of TOR complex 1. Am. J.
Physiol. Endocrinol. Metab. 296:E592–E602.
3. Balasubramanian, S., et al. 2009. mTOR in growth and protection of
hypertrophying myocardium. Cardiovasc. Hematol. Agents Med. Chem.
4. Black, P. N., and C. C. DiRusso. 2003. Transmembrane movement of exog-
enous long-chain fatty acids: proteins, enzymes, and vectorial esterification.
Microbiol. Mol. Biol. Rev. 67:454–472.
5. Bu, S. Y., M. T. Mashek, and D. G. Mashek. 2009. Suppression of long chain
acyl-CoA synthetase 3 decreases hepatic de novo fatty acid synthesis through
decreased transcriptional activity. J. Biol. Chem. 284:30474–30483.
6. Cao, A., H. Li, Y. Zhou, M. Wu, and J. Liu. 2010. Long chain acyl-CoA
synthetase-3 is a molecular target for peroxisome proliferator-activated re-
ceptor delta in HepG2 hepatoma cells. J. Biol. Chem. 285:16664–16674.
7. Chen, Y., D. Xing, W. Wang, Y. Ding, and L. Du. 2007. Development of an
ion-pair HPLC method for investigation of energy charge changes in cere-
bral ischemia of mice and hypoxia of Neuro-2a cell line. Biomed. Chro-
8. Chiu, H. C., et al. 2001. A novel mouse model of lipotoxic cardiomyopathy.
J. Clin. Invest. 107:813–822.
9. Clark, H., D. Carling, and D. Saggerson. 2004. Covalent activation of heart
AMP-activated protein kinase in response to physiological concentrations of
long-chain fatty acids. Eur. J. Biochem. 271:2215–2224.
10. Coleman, R. A., T. M. Lewin, C. G. Van Horn, and M. R. Gonzalez-Baro ´.
2002. Do acyl-CoA synthetases regulate fatty acid entry into synthetic versus
degradative pathways? J. Nutr. 132:2123–2126.
11. Cox, K. B., et al. 2009. Cardiac hypertrophy in mice with long-chain acyl-CoA
dehydrogenase or very long-chain acyl-CoA dehydrogenase deficiency. Lab.
12. Cunningham, J. T., et al. 2007. mTOR controls mitochondrial oxidative
function through a YY1-PGC-1alpha transcriptional complex. Nature 450:
13. de Jong, H., A. C. Neal, R. A. Coleman, and T. M. Lewin. 2007. Ontogeny of
mRNA expression and activity of long-chain acyl-CoA synthetase (ACSL)
isoforms in Mus musculus heart. Biochim. Biophys. Acta 1771:75–82.
14. Diniz, G. P., M. S. Carneiro-Ramos, and M. L. Barreto-Chaves. 2009. An-
giotensin type 1 receptor mediates thyroid hormone-induced cardiomyocyte
hypertrophy through the Akt/GSK-3beta/mTOR signaling pathway. Basic
Res. Cardiol. 104:653–667.
15. Ellis, J. M., et al. 2010. Adipose acyl-CoA synthetase-1 (ACSL1) directs fatty
acids towards ?-oxidation and is required for cold thermogenesis. Cell
16. Essop, M. F., et al. 2008. Reduced heart size and increased myocardial fuel
substrate oxidation in ACC2 mutant mice. Am. J. Physiol. Heart Circ.
17. Exil, V. J., et al. 2003. Very-long-chain acyl-coenzyme A dehydrogenase
deficiency in mice. Circ. Res. 93:448–455.
18. Ghatpande, S., S. Goswami, E. Mascareno, and M. A. Siddiqui. 1999. Signal
transduction and transcriptional adaptation in embryonic heart development
and during myocardial hypertrophy. Mol. Cell. Biochem. 196:93–97.
19. Glatz, J. F., and J. H. Veerkamp. 1982. Postnatal development of palmitate
oxidation and mitochondrial enzyme activities in rat cardiac and skeletal
muscle. Biochim. Biophys. Acta 711:327–335.
20. Graham, B. H., et al. 1997. A mouse model for mitochondrial myopathy and
cardiomyopathy resulting from a deficiency in the heart/muscle isoform of
the adenine nucleotide translocator. Nat. Genet. 16:226–234.
21. Huang, J., and B. D. Manning. 2009. A complex interplay between Akt,
TSC2 and the two mTOR complexes. Biochem. Soc. Trans. 37:217–222.
22. Ingwall, J. S. 2009. Energy metabolism in heart failure and remodelling.
Cardiovasc. Res. 81:412–419.
23. Inoki, K., et al. 2006. TSC2 integrates Wnt and energy signals via a coordi-
nated phosphorylation by AMPK and GSK3 to regulate cell growth. Cell
24. Kemi, O. J., et al. 2008. Activation or inactivation of cardiac Akt/mTOR
signaling diverges physiological from pathological hypertrophy. J. Cell.
25. Kuzman, J. A., T. D. O’Connell, and A. M. Gerdes. 2007. Rapamycin pre-
vents thyroid hormone-induced cardiac hypertrophy. Endocrinology 148:
26. Lang, G., and G. Michal. 1974. D-Glucose-6-phosphate and D-fructose-6-
phosphate, p. 1316–1319. In H. U. Bergmeyer (ed.), Methods of enzymatic
analysis. Verlag Chemie International, Deerfield Beach, FL.
27. Lee, C. H., K. Inoki, and K. L. Guan. 2007. mTOR pathway as a target in
tissue hypertrophy. Annu. Rev. Pharmacol. Toxicol. 47:443–467.
28. Li, L. O., et al. 2009. Liver-specific loss of long chain acyl-CoA synthetase-1
decreases triacylglycerol synthesis and beta-oxidation and alters phospho-
lipid fatty acid composition. J. Biol. Chem. 284:27816–27826.
29. Li, L. O., et al. 2006. Overexpression of rat long chain acyl-CoA synthetase
1 alters fatty acid metabolism in rat primary hepatocytes. J. Biol. Chem.
30. Makowski, L., et al. 2009. Metabolic profiling of PPARalpha?/? mice
reveals defects in carnitine and amino acid homeostasis that are partially
reversed by oral carnitine supplementation. FASEB J. 23:586–604.
31. Manfredi, G., L. Yang, C. D. Gajewski, and M. Mattiazzi. 2002. Measure-
ments of ATP in mammalian cells. Methods 26:317–326.
32. Mashek, D. G., M. A. McKenzie, C. G. Van Horn, and R. A. Coleman. 2006.
Rat long chain acyl-CoA synthetase 5 increases fatty acid uptake and parti-
tioning to cellular triacylglycerol in McArdle-RH7777 cells. J. Biol. Chem.
33. McMullen, J. R., and G. L. Jennings. 2007. Differences between pathological
and physiological cardiac hypertrophy: novel therapeutic strategies to treat
heart failure. Clin. Exp. Pharmacol. Physiol. 34:255–262.
34. Melegh, B., et al. 2004. Phenotypic manifestations of the OCTN2 V295X
mutation: sudden infant death and carnitine-responsive cardiomyopathy in
Roma families. Am. J. Med. Genet. A 131:121–126.
35. Mirebeau-Prunier, D., et al. 2010. Estrogen-related receptor alpha and
PGC-1-related coactivator constitute a novel complex mediating the biogen-
esis of functional mitochondria. FEBS J. 277:713–725.
36. Neschen, S., et al. 2005. Prevention of hepatic steatosis and hepatic insulin
resistance in mitochondrial acyl-CoA:glycerol-sn-3-phosphate acyltrans-
ferase 1 knock out mice. Cell Metab. 2:55–65.
37. Noland, R. C., et al. 2007. Peroxisomal-mitochondrial oxidation in a rodent
model of obesity-associated insulin resistance. Am. J. Physiol. Endocrinol.
38. Polokoff, M. A., and R. M. Bell. 1978. Limited palmitoyl-CoA penetration
into microsomal vesicles as evidenced by a highly latent ethanol acyltrans-
ferase activity. J. Biol. Chem. 253:7173–7178.
39. Rupp, H., and R. Jacob. 1992. Metabolically-modulated growth and pheno-
type of the rat heart. Eur. Heart J. 13(Suppl. D):56–61.
40. Saha, A. K., et al. 2010. Downregulation of AMPK accompanies leucine- and
glucose-induced increases in protein synthesis and insulin resistance in rat
skeletal muscle. Diabetes 59:2426–2434.
41. Sharma, S., P. H. Guthrie, S. S. Chan, S. Haq, and H. Taegtmeyer. 2007.
Glucose phosphorylation is required for insulin-dependent mTOR signalling
in the heart. Cardiovasc. Res. 76:71–80.
42. Soesanto, W., et al. 2009. Mammalian target of rapamycin is a critical reg-
ulator of cardiac hypertrophy in spontaneously hypertensive rats. Hyperten-
VOL. 31, 2011CARDIAC FA OXIDATION REQUIRES ACYL-CoA SYNTHETASE 11261
43. Stanley, W. C., F. A. Recchia, and G. D. Lopaschuk. 2005. Myocardial substrate Download full-text
metabolism in the normal and failing heart. Physiol. Rev. 85:1093–1129.
44. Stansfield, W. E., et al. 2007. Characterization of a model to independently
study regression of ventricular hypertrophy. J. Surg. Res. 142:387–393.
45. Suenaga, M., et al. 2004. Identification of the up- and down-regulated genes
in the heart of juvenile visceral steatosis mice. Biol. Pharm. Bull. 27:496–503.
46. Sun, X., et al. 2009. Nicotine stimulates PPARbeta/delta expression in hu-
man lung carcinoma cells through activation of PI3K/mTOR and suppres-
sion of AP-2alpha. Cancer Res. 69:6445–6453.
47. Taylor, E. B., W. J. Ellingson, J. D. Lamb, D. G. Chesser, and W. W. Winder.
2005. Long-chain acyl-CoA esters inhibit phosphorylation of AMP-activated
protein kinase at threonine-172 by LKB1/STRAD/MO25. Am. J. Physiol.
Endocrinol. Metab. 288:E1055–E1061.
48. Tong, F., P. N. Black, R. A. Coleman, and C. C. DiRusso. 2006. Fatty acid
transport by vectorial acylation in mammals: roles played by different isoforms
of rat long-chain acyl-CoA synthetases. Arch. Biochem. Biophys. 447:46–52.
49. van der Vusse, G. J., J. F. Glatz, H. C. Stam, and R. S. Reneman. 1992. Fatty
acid homeostasis in the normoxic and ischemic heart. Physiol. Rev. 72:881–
50. Watkins, S. M., P. R. Reifsnyder, H. J. Pan, J. B. German, and E. H. Leiter.
2002. Lipid metabolome-wide effects of the PPARgamma agonist rosiglita-
zone. J. Lipid Res. 43:1809–1817.
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