Tài liệu Báo cáo khoa học: Metabolic gene switching in the murine female heart parallels enhanced mitochondrial respiratory function in response to oxidative stress pdf

Metabolic gene switching in the murine female heart
parallels enhanced mitochondrial respiratory function in
response to oxidative stress
M. Faadiel Essop
1,2
, W. Y. A. Chan
2
and Heinrich Taegtmeyer
3
1 Department of Physiological Sciences, Stellenbosch University, South Africa
2 Hatter Heart Research Institute, Faculty of Health Sciences, University of Cape Town, South Africa
3 Department of Internal Medicine, Division of Cardiology, University of Texas, Houston Medical School, TX, USA
Premenopausal women have a lower risk for develop-
ing cardiovascular disease as compared to age-matched
males [1]. Moreover, experimental studies show increa-
sed resistance to ischemia⁄ reperfusion injury in female
versus male hearts [2,3]. The molecular regulatory
mechanisms underlying such gender-based differences
are unclear. However, estrogen may play a key role in
this process [4], and is thought to signal its cardio-
protective effects via the prosurvival serine-threonine
protein kinase, Akt (also known as protein kinase B)
[2]. In agreement with this, elevated levels of activated
Akt in female hearts are linked to improved cardiac
cell survival [5], and a recent study implicated the
PI3-K ⁄ Akt signaling pathway in estrogen-mediated
cardioprotection [6].
Adaptive metabolic remodeling is considered to be
an important component of cardioprotective mecha-
nisms in response to decreased oxygen supply. For
example, enhanced glucose utilization is proposed to

confer cardioprotective effects in response to ische-
mia ⁄ reperfusion [7]. Conversely, higher rates of fatty
acid oxidation during ischemia may uncouple mito-
chondrial oxidative phosphorylation and ⁄ or increase
proton production, contributing to impaired contractile
Keywords
bioenergetics; cardiovascular disease;
gender differences; gene expression;
mitochondrial respiration
Correspondence
M. F. Essop, Department of Physiological
Sciences, Stellenbosch University,
Room 2009, Mike De Vries Building,
Merriman Avenue, Stellenbosch 7600,
South Africa
Fax: +27 21 808 3145
Tel: +27 21 808 4507
E-mail:
(Received 14 December 2006, revised
14 August 2007, accepted 17 August 2007)
doi:10.1111/j.1742-4658.2007.06051.x
The mechanisms underlying increased cardioprotection in younger female
mice are unclear. We hypothesized that serine-threonine protein kinase
(protein kinase B; Akt) triggers a metabolic gene switch (decreased fatty
acids, increased glucose) in female hearts to enhance mitochondrial bio-
energetic capacity, conferring protection against oxidative stress. Here, we
employed male and female control (db ⁄ +) and obese (db ⁄ db) mice. We
found diminished transcript levels of peroxisome proliferator-activated
receptor-alpha, muscle-type carnitine palmitoyltransferase 1 and pyruvate
dehydrogenase kinase 4 in female control hearts versus male hearts. More-

over, females displayed improved recovery of cardiac mitochondrial respi-
ratory function and higher ATP levels versus males in response to acute
oxygen deprivation. All these changes were reversed in female db ⁄ db
hearts. However, we found no significant gender-based differences in levels
of Akt, suggesting that Akt-independent signaling mechanisms are respon-
sible for the resilient mitochondrial phenotype observed in female mouse
hearts. As glucose is a more energetically efficient fuel substrate when oxy-
gen is limiting, this gene program may be a crucial component that
enhances tolerance to oxygen deprivation in female hearts.
Abbreviations
Akt, serine-threonine protein kinase (protein kinase B); GLUT, glucose transporter; MCAD, medium-chain acyl-CoA dehydrogenase; mCPT1,
muscle-type carnitine palmitoyltransferase 1; PDK-4, pyruvate dehydrogenase kinase 4; PGC-1, peroxisome proliferator-activated receptor-
gamma coactivator-1; PPARa, peroxisome proliferator-activated receptor-alpha; UCP3, uncoupling protein 3.
5278 FEBS Journal 274 (2007) 5278–5284 ª 2007 The Authors Journal compilation ª 2007 FEBS
function [8]. Likewise, high fatty acid oxidation rates
in the diabetic heart results in reduced cardiac effi-
ciency [9].
In addition to its cytoplasmic role, Akt can also
translocate to the nucleus, where it has transcriptional
effects. For example, constitutively activated Akt
resulted in reduced myocardial gene expression
of peroxisome proliferator-activated receptor-alpha
(PPARa) and peroxisome proliferator-activated recep-
tor-gamma coactivator-1 (PGC-1), pivotal nuclear
regulators of numerous fatty acid metabolic genes
[10]. In light of this, we hypothesized that Akt trig-
gers a metabolic gene switch from fatty acids to
increased glucose metabolism in female hearts,
thereby conferring protection against acute oxygen
deprivation. Moreover, we propose that this selective

advantage would be lost with the onset of obesity
and type 2 diabetes.
Results and Discussion
The main finding of this study is the identification of a
metabolic gene switch from fatty acids to glucose in
the murine female heart at baseline linked to enhanced
mitochondrial respiratory capacity in response to oxi-
dative stress. Moreover, we found that this ‘female
advantage’ is lost with the onset of obesity ⁄ type 2 dia-
betes. However, our data show that these changes
occurred in an Akt-independent manner.
Laboratory-based studies show that female hearts
exhibit increased resilience to ischemia ⁄ reperfusion
injury and cell death as compared to males [2,3].
Estrogen is thought to play an important role in this
process, and is proposed to signal its cardioprotection
via the prosurvival kinase Akt [2,4]. For example,
higher levels of activated Akt were reported in pre-
menopausal women as compared to men [11]. More-
over, estrogen stimulation resulted in elevated nuclear
levels of activated Akt [11]. We tested our hypothesis
in male and female hearts collected from db ⁄ db mice,
a well-characterized model of obesity-induced type 2
diabetes (Table 1). However, we did not find any sig-
nificant changes in Akt activation in female hearts at
baseline as compared to males (Fig. 1).
To further investigate our hypothesis, we measured
steady-state transcript levels of various cardiac meta-
bolic genes. Here, myocardial PPARa gene expression
was reduced in females at baseline (P<0.001 versus

male controls) (Fig. 2A). In parallel, expression of the
genes encoding muscle-type carnitine palmitoyltransfer-
ase 1 (mCPT1) and medium-chain acyl-CoA dehydro-
genase (MCAD) (both PPARa target genes) was
Table 1. Baseline characterization of male and female obese mice.
Values are expressed for 18–20-week-old male and female db ⁄ +
versus db ⁄ db mice (mean ± SEM, n ¼ 10 animals). ** P < 0.001
compared with age-matched db ⁄ +mice.
Body weight (g)
Heart ⁄ body
weight ratio
(· 1000)
Fasting blood
glucose
(mmolÆL
)1
)
Male
db ⁄ + 28.0 ± 0.7 4.0 ± 0.1 5.5 ± 0.4
db ⁄ db 47.2 ± 1.7** 2.1 ± 0.1** 26.8 ± 0.9**
Female
db ⁄ + 20.8 ± 0.5 3.6 ± 0.1 4.2 ± 0.2
db ⁄ db 44.7 ± 1.2** 2.2 ± 0.1** 25.9 ± 1.2**
A B
C D
Fig. 1. Immunohistochemical analysis of
phospho-Akt in male and female control
(db ⁄ +) versus obese (db ⁄ db) mice. (A) Male
control, (B) male obese, (C) female control
and (D) female obese mice. Phospho-Akt is

stained brown and the nucleus is counter-
stained blue. Magnification · 200 (n ¼ 6).
M. F. Essop et al. Metabolic gene switching in murine female heart
FEBS Journal 274 (2007) 5278–5284 ª 2007 The Authors Journal compilation ª 2007 FEBS 5279
diminished in female hearts (Fig. 2B,C). However,
uncoupling protein 3 (UCP3) expression remained
unaltered versus males (Fig. 2D). Pyruvate dehydroge-
nase kinase 4 (PDK-4) transcript levels were markedly
attenuated in female hearts at baseline (P<0.001 ver-
sus male controls) (Fig. 2E), whereas glucose trans-
porter (GLUT) 4 expression was not significantly
different as compared to males (Fig. 2F). Interestingly,
we found that gene changes in the female heart were
abolished with the onset of obesity ⁄ type 2 diabetes.
Together, these data suggest that murine female hearts,
unlike male hearts, may display a reduced reliance on
fatty acids as fuel substrate. In agreement, lower
PDK-4 gene expression in female hearts indicates that
myocardial glucose metabolism may be increased in
parallel. As optimization of glucose metabolism is
increasingly highlighted as a therapeutic intervention
for ischemia-induced and ischemia–reperfusion-induced
cardiac damage [7], our data provide a novel mecha-
nism for how signaling cascades induce transcriptional
pathways to augment glucose-mediated cardioprotec-
tion in younger females. In agreement with this con-
cept, we propose that this advantage is eliminated in
obesity ⁄ type 2 diabetes, due to higher fatty acid utili-
zation by the diabetic heart [9].
To functionally assess the significance of these

findings, we evaluated mitochondrial respiratory
function at baseline and in response to acute oxygen
AB
CD
EF
Fig. 2. Cardiac metabolic gene expression in male and female control versus obese mice. (A) PPARa, (B) mCPT1, (C) MCAD, (D) UCP3,
(E) GLUT4 and (F) PDK-4 in male and female obese versus control mice. Data are expressed as mean ± SEM. *P < 0.001 versus male
control mice; **P < 0.01 versus male obese mice;
#
P < 0.01 versus female control mice;
##
P < 0.001 versus female control mice;
§
P < 0.001 versus male control mice.
Metabolic gene switching in murine female heart M. F. Essop et al.
5280 FEBS Journal 274 (2007) 5278–5284 ª 2007 The Authors Journal compilation ª 2007 FEBS
deprivation. Here, females displayed increased mito-
chondrial respiratory function at baseline as compared
to males (Table 2). The efficiency of respiration
(ADP ⁄ O) and ADP phosphorylation rate were similar
between male and female mitochondria at baseline. In
contrast to what was expected, our data suggest that
male obese mice coped well when challenged by oxi-
dant stress; that is, respiratory function and myo-
cardial ATP levels were not significantly altered
(Fig. 3A,B). We are unsure why this occurred, and
propose that the stress applied was not severe
enough or that some adaptive mechanisms were initi-
ated in the male obese mice. However, female controls
exhibited enhanced recovery of state 3 mitochondrial

respiration versus males in response to oxygen lack
(23.6 ± 1.6 versus 16.4 ± 2.6 nmolÆmin
)1
Æmg
)1
pro-
tein) (P < 0.05) (Fig. 3A). Moreover, this was associ-
ated with higher postanoxic myocardial mitochondrial
ATP levels in females as compared to male controls
(39.0 ± 2.7 versus 26.2 ± 2.6 lmolÆmg
)1
protein)
(P<0.05) (Fig. 3B). Again, these changes were abol-
ished in female db ⁄ db heart mitochondria. Together,
these data suggest that the greater reliance of female
murine cardiac mitochondria on glucose than on fatty
acids as compared to their male counterparts may
result in enhanced cardioprotection when they are
challenged by a biological stress, e.g. oxygen lack.
Enhanced glucose utilization in response to oxygen
deprivation may occur because glucose is a more oxy-
gen-efficient fuel substrate for the generation of ATP
as compared to fatty acids. Also, fatty acid-mediated
uncoupling of mitochondrial oxidative phosphoryla-
tion may result in diminished mitochondrial ATP pro-
duction for a given rate of oxygen consumption [12].
However, it is likely that additional mechanisms play a
role, as increased myocardial ATP levels were observed
in female controls in response to oxygen stress. We
propose that estrogen-mediated mitochondrial biogene-

sis may also be implicated in this process. In agree-
ment, recent studies reported that the orphan nuclear
receptor estrogen receptor-alpha, proposed to mediate
estrogen signaling, may play a transcriptional role in
mitochondrial biogenesis [13,14]. Further studies are,
however, required to investigate this possibility.
Limitations
Although the gene data in this study support a fuel
substrate switch away from fatty acids, further studies
measuring actual cardiac fuel substrate utilization are
Table 2. Cardiac mitochondrial respiration for male and female obese mice. Heart mitochondria were isolated from 18–20-week-old mice as
described. Values are expressed as mean ± SEM (n ¼ 7 animals). * P < 0.05 compared with male db ⁄ +mice. ** P < 0.05 compared with
male obese db ⁄ db mice.
Male Female
db ⁄ +db⁄ db db ⁄ +db⁄ db
State 2 respiration (nmolÆmin
)1
Æmg
)1
protein) 29.4 ± 1.6 32.6 ± 1.9 36.6 ± 1.7* 35.0 ± 2.0
State 3 respiration (nmolÆmin
)1
Æmg
)1
protein) 145.0 ± 9.6 165.8 ± 9.8 175.6 ± 7.1 177.8 ± 12.9
State 4 respiration (nmolÆmin
)1
Æmg
)1
protein) 33.5 ± 2.0 30.7 ± 2.4 38.8 ± 1.03 36.8 ± 1.6

ADP ⁄ O 2.5 ± 0.1 2.6 ± 0.1 2.2 ± 0.04 2.3 ± 0.1**
Phosphorylation rate (nmolÆmin
)1
Æmg
)1
protein) 363.4 ± 28.3 436.0 ± 32.3 381.0 ± 20.4 411.6 ± 40.0
AB
Fig. 3. Improved mitochondrial respiratory function in female control mouse hearts in response to oxygen lack. (A) Percentage recovery of
state 3 respiration after 20 min of oxygen deprivation. (B) Total postanoxic mitochondrial ATP levels in male and female cardiac mitochondria.
Data are presented as mean ± SEM. *P < 0.05 versus male control mice;
#
P < 0.05 versus female control mice;
##
P < 0.001 versus female
control mice (n ¼ 7).
M. F. Essop et al. Metabolic gene switching in murine female heart
FEBS Journal 274 (2007) 5278–5284 ª 2007 The Authors Journal compilation ª 2007 FEBS 5281
required to confirm these findings. Moreover, mito-
chondrial respiratory functional analyses were only
performed using fatty acids as substrate. Additional
studies measuring mitochondrial respiratory function
using a more representative substrate for glucose oxi-
dation should provide additional insights into this
interesting question.
Conclusions
In summary, we have identified a novel metabolic
gene switch (decrease in fatty acid utilization,
increase in glucose utilization) in the murine female
heart. Moreover, our data suggest that these changes
occur in an Akt-independent manner. Further studies

are therefore required to identify the precise signaling
mechanisms that control the metabolic remodeling
that we observed in the female murine heart at base-
line. As glucose is a more energetically efficient fuel
substrate than fatty acids when oxygen is limiting,
we believe that this mechanism may represent a
crucial component underlying enhanced recovery
in younger female hearts in response to oxygen
deprivation.
Experimental procedures
Animals
To investigate our hypothesis, we employed 18–20-week-
old male and female leptin-receptor deficient (db ⁄ db)
(BKS.Cg-m+ ⁄ +Lepr
db
⁄ J strain) and heterozygous (db ⁄ +)
mice. Mice were obtained from Jackson Laboratory (Bar
Harbor, ME) and exposed to a reverse 12 h light ⁄ 12 h
dark cycle with free access to standard mouse chow and
water. Two weeks before mice were killed, blood glucose
levels were measured using a glucose meter (ACCU-
CHECK Active Meter; Roche, Basel, Switzerland) after a
6 h fast. All animal experiments were approved by the
University of Cape Town’s Animal Research Ethics
Committee, and the investigation conforms to the Guide
for the Care and Use of Laboratory Animals published by
the US National Institutes of Health (NIH Publication
no. 85-23, revised 1996).
Immunohistochemistry
Heart tissues were fixed with paraformaldehyde and embed-

ded in paraffin wax. Paraffin sections (2–3 lm thick) were
dewaxed in xylene and rehydrated through graded ethanol.
Expression of phospho-Akt was detected using mouse
monoclonal anti-phospho-Akt (Ser473) IgG (Cell Signaling,
Danvers, MA).
Gene analysis
RNA extraction and real-time quantitative RT-PCR of
samples were performed using previously described methods
[15]. Primers for gene analysis in this study have been
described previously [15–18]. We determined transcript
levels of: mCPT1, the rate-limiting mitochondrial fatty
acid-transferring enzyme; MCAD, a representative fatty
acid b-oxidation enzyme; PPARa, a key transcriptional
regulator of fatty acid genes; UCP3; the cardiac-enriched
GLUT4; and PDK-4, an indirect inhibitor of glucose oxida-
tion. Gene expression was normalized to 18S rRNA.
Mitochondrial isolation and respiration studies
Animals were anesthetized using intraperitoneal sodium
pentobarbital (50 mgÆkg
)1
) and heparinized to prevent
blood clotting. Mouse hearts were dissected and mitochon-
dria isolated as described previously [19], with modifica-
tions. Ventricular tissue was homogenized in ice-cold
potassium ⁄ EDTA (KE) buffer (0.18 m KCl, 10 m m EDTA,
pH 7.4), and the homogenate was centrifuged at 660 g for
5 min at 4 °C in a fixed angle rotor (Sigma 202 MK,
Heraues, Germany). The supernatant was again centrifuged
at 960 g for 5 min, and the mitochondrial pellet was resus-
pended in 50 lL of KE buffer. Mitochondrial respiratory

rates were polarographically measured at 25 °Cas
described previously [20], with modifications. Isolated mito-
chondria were added to the electrode chamber containing
incubation medium (25 mm Tris ⁄ HCl, 250 mm sucrose,
8.5 mm KH
2
PO
4
, pH 7.4). We employed a mixture of
5mm malate and 25 lm palmitoyl-l-carnitine as oxidative
substrates. State 2 respiration (resting) was measured after
addition of oxidative substrates, and state 3 respiration
after the addition of 300 lm ADP to the electrode
chamber.
To test the ability of mitochondria to withstand oxidative
stress, 3 mm ADP was added after state 4 respiration, and
the chamber was closed and sealed for a 20 min period. As
a result, oxygen in the closed chamber would be used to
convert ADP to ATP, and an anaerobic condition estab-
lished. After 20 min of oxygen lack, the chamber was
reoxygenated for 6 min, and the percentage recovery of
state 3 respiration was calculated as the ratio of oxygen
consumption before and after oxygen lack. All mitochon-
drial polarographic studies were normalized to total mito-
chondrial protein content [21].
Mitochondrial ATP levels
Postanoxic mitochondrial ATP concentration was assayed
using a luciferin ⁄ luciferase luminometry luminescence
method [22] with modifications. Freshly isolated mitochon-
dria were placed in boiling water (3 · sample volume) for

Metabolic gene switching in murine female heart M. F. Essop et al.
5282 FEBS Journal 274 (2007) 5278–5284 ª 2007 The Authors Journal compilation ª 2007 FEBS
10 min, and thereafter on ice for an additional 10 min to
disrupt mitochondrial membranes. The mixture was sub-
sequently centrifuged at 960 g for 5 min at 4 °C in a fixed
angle rotor (Sigma 202 MK). Ten microliters of supernatant
was added to 175 lL of distilled water and 25 lL of ATP
assay mix [Bioluminescent Somatic Cell Assay Kit (FL-AA);
Sigma, St Louis, MO] containing luciferin and luciferase.
The reaction of the luciferin ⁄ luciferase mixture, energy
donor (ATP) and oxygen results in the emission of light.
The intensity of the bioluminescence was detected using a
luminometer, and the mitochondrial ATP concentration was
determined. Mitochondrial protein concentration was deter-
mined [21], and a standard curve was constructed using
1 · 10
)5
m to 1 · 10
)9
m ATP. The concentration of mito-
chondrial ATP was expressed as micromoles of ATP per
milligram of mitochondrial protein.
Statistical analysis
Data are presented as the mean ± SEM. Statistical differ-
ences between groups were calculated using the unpaired
Student’s t-test. P < 0.05 was considered to indicate statis-
tical significance.
Acknowledgements
The authors are grateful to Mei Gong for expert
technical assistance. MFE thanks the South African

Medical Research Council and National Research
Foundation for financial support. The work of HT
was supported in part by grants from the NHLBI
(RO1-HL073162-01 and T32-HL07591).
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