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Impaired contractile function and mitochondrial respiratory
capacity in response to oxygen deprivation in a rat model of
pre-diabetes
M. F. Essop,
1
W. Y. Anna Chan,
2
A. Valle,
3
F. J. Garcı
´
a-Palmer
3
and E. F. Du Toit
4
1 Department of Physiological Sciences, Stellenbosch University, Stellenbosch, South Africa
2 Hatter Heart Research Institute, Department of Medicine, University of Cape Town’s Health Sciences Faculty, Cape Town,
South Africa
3 Grup de Metabolisme Energe
`
tic i Nutricio
´
, Universitat de les Illes Balears, Palma de Mallorca, and Ciber Fisiopatologı
´
a Obesidad
y Nutricio
´
n (CB06/03) Instituto Salud Carlos III, Palma de Mallorca, Spain
4 Department of Medical Physiology, Stellenbosch University Faculty of Health Sciences, Tygerberg, South Africa
Received 12 December 2008,
revision requested 29 January 2009,
final revision received 18 June 2009,
accepted 16 July 2009
Correspondence: M. F. Essop,
Department of Physiological
Sciences, Stellenbosch University,
Mike De Vries Building, Merriman
Avenue, Stellenbosch 7600, South
Africa. E-mail: mfessop@sun.ac.za
Abstract
Aim: Obesity is a major contributor to the global burden of disease and is
closely associated with the development of type 2 diabetes and cardiovas-
cular diseases. This study tested the hypothesis that mitochondrial respira-
tory capacity of the pre-diabetic heart is decreased leading to impaired
contractile function and tolerance to ischaemia/reperfusion.
Methods: Eight-week-old male Wistar rats were fed a high caloric diet for
16 weeks after which anthropometric, metabolic, cardiac and mitochondrial
parameters were evalu ated vs. age-matched lean controls. Cardiac function
(working heart perfusions) and mitochondrial respiratory capacity were
assessed at baseline and in response to acute oxygen deprivation.
Results: Rats fed the high caloric diet exhibited increased body weight and
visceral fat vs. the control group. Heart weights of obese rats were also
increased. Triglyceride, fasting plasma insulin and free fatty acid levels were
elevated, while high-density lipoprotein cholesterol levels were reduced in the
obese group. Contractile function was attenuated at baseline and further
decreased after subjecting hearts to ischaemia-reperfusion. Myocardial
infarct sizes were increased while ADP phosphorylation rates were dimin-
ished in obese rats. However, no differences were found for mtDNA levels
and the degree of oxidative stress-induced damage.
Conclusions: These data show that decreased mitochondrial bioenergetic
capacity in pre-diabetic rat hearts may impair respiratory capacity and
reduce basal contractile function and tolerance to acute oxygen deprivation.
Keywords metabolic syndrome, mitochondrial respiration, myocardial
infarction, obesity.
The prevalence of obesity is reaching global epidemic
proportions and it is a major contributor to increased
prevalence of the metabolic syndrome and type 2
diabetes (Eckel et al. 2005, Kelly et al. 2008). Insulin
resistance and type 2 diabetes is characterized by
metabolic derangements that may contribute to cardiac
contractile dysfunction in the absence of atherosclerosis
and hypertension, i.e. the diabetic cardiomyopathy
(reviewed by Boudina & Abel 2007). For example, the
diabetic heart displays higher rates of fatty acid b-oxi-
dation and a corresponding reduction in carbohydrate
utilization (Carley & Severson 2005). Furthermore,
previous studies demonstrated that increased myocar-
dial oxygen consumption and reduced cardiac efficiency
Acta Physiol 2009
2009 The Authors
Journal compilation 2009 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2009.02024.x
1
(work/myocardial oxygen consumption) may be impor-
tant factors contributing to contractile dysfunction of
the diabetic heart (Boudina & Abel 2006, How et al.
2006).
The precise mechanisms underlying this process are
unclear, although fatty acid-induced mitochondrial
uncoupling and lowered mitochondrial ATP production
have been implicated (Boudina et al. 2005). Moreover,
increased mitochondrial reactive oxygen species (ROS)
production by diabetic hearts is proposed to contribute
to uncoupling of mitochondrial oxidative phosphoryla-
tion and oxygen wastage (Boudina et al. 2007). How-
ever, most studies investigating metabolic perturbations
and its effects on cardiac function were performed using
experimental models of overt type 2 diabetes. Further-
more, mitochondrial studies were largely performed on
genetic mouse models of obesity and insulin resistance
(Boudina et al. 2005, 2007). As mutations in the leptin
gene or its receptor are rare in humans and leptin may
have direct effects on cardiac function, it would be
useful to assess the effects of modern-day eating habits
(high caloric diet) and obesity on a normal genetic
background on mitochondrial energetics as emphasized
in a recent review article by Abel et al. (2008).
Importantly, it will be of great benefit to evaluate
whether these changes occur at an earlier stage during
disease progression, i.e. in the pre-diabetic state com-
pared to more advanced stages of disease progression as
studied before. In the light of this, we employed a rat
model of obesity-induced pre-diabetes to test the
hypothesis that mitochondrial bioenergetic capacity of
the pre-diabetic heart is decreased leading to impaired
contractile function.
Materials and methods
Animals
Eight-week-old male Wistar rats were housed at
22.0 0.5 C on a 12-h light/dark cycle and divided
into two groups, i.e. fed standard chow (60% carbohy-
drate, 30% protein, 10% fat) vs. a high caloric diet
(65% carbohydrate, 19% protein, 16% fat) as
described before (Du Toit et al. 2008). After a 16-week
feeding period, rats were weighed and thereafter
anaesthetized using intraperitoneal pentobarbitone
sodium (12 mg kg
)1
). Hearts were rapidly excised and
weighed to determine heart/body weight ratios. All
animals were treated in accordance with the Principles
of Laboratory Animal Care of the South African
Medical Research Council and the Guide for the Care
and use of Laboratory Animals of the National Acad-
emy of Sciences (NIH publication no. 85-23, revised
1996). The study was also ethically approved by the
Committee for Experimental Animal Research of the
Faculty of Health Sciences (Tygerberg campus of
Stellenbosch University, South Africa).
Metabolic assays
During excision of the heart, blood samples were
collected in EDTA tubes for glycosylated haemoglobin
(HbA1c) and free fatty acid (FFA) measurements; potas-
sium oxalate/sodium fluoride tubes for glucose determi-
nations; and serum separation tubes for lipid analysis.
Where appropriate, samples were centrifuged at 1500 g
for 10 min at 4 C and plasma/serum stored at )20 C
for later use. However, blood glucose and HbA1c measure-
ments were performed on the day of sample collection.
Serum assays
Serum total cholesterol (TC), high-density lipoprotein
(HDL) cholesterol and triglycerides were measured
using enzymatic and colorimetric methods as described
previously (Esterhuyse et al. 2005). Plasma FFA levels
were determined using an enzymatic colorimetric assay
(Roche, Penzberg, Germany). Serum insulin levels were
measured using a radio-immunoassay-based kit (Diag-
nostic Products, Los Angeles, CA, USA). Blood glucose
levels were measured using the glucose oxidase method
with one-touch test strips (Accu-Check; Roche Diag-
nostics, Mannheim, Germany). Blood HbA1c levels
were determined using the glycosal spectrophotometric
method (National Health Laboratory Services, Tyger-
berg Hospital, Cape Town, South Africa).
Blood pressure measurements
After 16 weeks, systolic blood pressure was determined
using the standard tail-cuff method (Bunag 1973).
Briefly, rats were sedated with thiopentone sodium
(20 mg kg
)1
) while a tail-cuff and a pneumatic pulse
sensor were fitted. The tail-cuff was linked to a
sphygmomanometer and the pulse sensor to a Lectr-
omed Multitrace 2 chart recorder (Rue Fondon, Jersey,
Channel Islands). The tail-cuff was gradually inflated
until the pulse pressure curve on the chart recorder
disappeared. The sphygmomanometer linked to the tail-
cuff was then deflated until the pulse pressure curve
reappeared on the chart recorder. The sphygmoma-
nometer pressure that coincided with the reappearance
of the pulse pressure curve was considered the systolic
blood pressure. The procedure was repeated three times
and the mean systolic pressure was determined.
Isolated heart perfusions
After 16 weeks of feeding, animals were anaesthetized
using intraperitoneal pentobarbitone sodium (12 mg kg
)1
),
2
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Journal compilation 2009 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2009.02024.x
Heart and respiratory function with obesity
Æ
M F Essop et al. Acta Physiol 2009
hearts rapidly excised and placed in ice-cold Krebs-
Henseleit perfusion buffer (118 mmol L
)1
NaCl,
4.7 mmol L
)1
KCl, 1.2 mmol L
)1
MgSO
4
Æ7H
2
O,
1.25 mmol L
)1
CaCl, 25mm NaHCO
3
, 1.2 mmol L
)1
KH
2
PO
4
and 10 mm glucose). Hearts were subse-
quently transferred to a working heart perfusion appa-
ratus and perfused with Krebs-Henseleit buffer
equilibrated with 95% O
2
/5% CO
2
at 37 C and
100 cmH
2
O pressure. Retrograde perfusion was initi-
ated within 45 s of excision of the heart.
Functional recovery and myocardial infarct size
measurements
Hearts were initially perfused in the Langendorff mode
for a 10-min stabilization period, thereafter for 20 min
in the working heart mode. Pre-ischaemic aortic output
(AO), coronary flow and aortic systolic pressure and
aortic diastolic pressure (ADP) was determined before
hearts were subjected to 15 min normothermic global
ischaemia (37 C). Subsequently, hearts were reperfused
in the Langendorff mode for 5 min before switching to
the working heart mode for an additional 20 min.
Reperfusion functional parameters were again docu-
mented at 10, 15 and 20 min. Percentage AO recovery
was determined as follows: Aortic output recov-
ery = (AO at reperfusion [20 min]/AO at pre-ischaemia
[20 min]) · 100.
In a separate series of hearts myocardial infarct size
was determined as described previously (Du Toit et al.
2008). Briefly, hearts were perfused as above before
being subjected to 40 min regional ischaemia at 37 C.
Regional ischaemia was induced by ligation of the left
anterior descending coronary artery using a silk suture.
The suture was then released and the heart reperfused
for 30 min before the coronary artery was re-occluded.
After re-occlusion, a solution of 2.5% Evans blue was
injected into the coronary arteries to delineate the area
at risk. Hearts were then frozen, cut into slices and
incubated in sodium phosphate buffer containing 1%
w/v triphenyltetrazolium chloride for 15 min to visual-
ize the unstained infarcted region. Infarct and risk zone
areas were determined with planimetry and infarct area
was expressed as a percentage of the area at risk (Du
Toit et al. 2008).
Mitochondrial isolation and measurement of respiratory
function
For a separate set of rats (non-perfused), mitochondria
were isolated as described before, with modifications
(Essop et al. 2007). In brief, ventricular tissues were
minced and gently homogenized in 3 mL ice-cold
potassium-EDTA isolation buffer (0.18 m KCL,
10 mm EDTA) using a glass homogenizer (Tenbroek,
Bellco, Vineland, NJ, USA). After differential centrifu-
gation the mitochondrial pellet was gently resuspended
in 100 lL of incubation buffer (10 mm Tris-HCl,
250 mm sucrose, 8.5 mm KH
2
PO
4
). It is likely that
we isolated subsarcolemmal mitochondria as released
by mechanical disruption, unlike interfibrillar mito-
chondrial isolation that requires protease treatment and
additional centrifugation steps (Palmer et al. 1977) that
were not performed in this study. Mitochondrial protein
concentrations were determined and respiratory capac-
ity was measured polarographically at 25 Cas
described (Essop et al. 2007). Two sets of experiments
were performed: (1) 12.5 mm glutamate or (2) 54 lm
palmitoyl-l-carnitine and 3.3 mm malate used as oxi-
dative substrates. Analyses were only performed when
the respiratory control ratio, i.e. the ratio of state 3
respiration : state 4 respiration, was ‡3.
Mitochondrial respiratory function in response to anoxic
stress
To test the ability of mitochondria to withstand
oxidative stress, mitochondria (still incubated with
respective oxidative substrates) were exposed to
20 min of oxygen deficiency followed by 6 min of
reoxygenation as described (Essop et al. 2007). Recov-
ery of respiratory function was evaluated by determin-
ing the ratio of post-anoxia state 3 respiration over state
3 respiration prior to oxygen deficiency. All mitochon-
drial polarographic studies were normalized to total
mitochondrial protein.
Extraction and quantification of mitochondrial DNA
Mitochondrial DNA (mtDNA) was extracted by
digestion with proteinase K (100 lg lL
)1
) in a buffer
containing 50 mm KCl, 10 mm Tris-HCl, 2.5 mm
MgCl
2
and 0.5% Tween 20. Homogenate samples
were incubated overnight at 37 C and then boiled for
5 min in order to inactivate the enzyme. mtDNA was
linearized by digestion with BclI restriction enzyme for
3 h at 50 C and boiled for 5 min. Samples were
centrifuged at 7000 g for 5 min and the resulting
supernatant was used for amplification. A quantitative
PCR assay was adapted for light cycler amplification
(Koekemoer et al. 1998). PCR was performed to
amplify a 162-nts fragment of the mitochondrial
NADH dehydrogenase subunit 4 gene. The primer
sequences were 5¢-TACACGATGAGGCAACCAAA-3¢
and 5¢-GGTAGGGGGTGTGTTGTGAG-3¢. Increasing
amounts of template were amplified in parallel reac-
tions to obtain a standard curve. Amplification was
carried out in a LightCycler rapid thermal cycler
system (Roche, Basel, Switzerland) using a total
volume of 10 lL containing 0.375 lm of each primer,
2009 The Authors
Journal compilation 2009 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2009.02024.x
3
Acta Physiol 2009 M F Essop et al.
Æ
Heart and respiratory function with obesity
3mm MgCl
2
,1lL LightCycler-FastStart DNA Master
SYBR Green I (Roche), and 2.5 lL of sample prepared
as described above. After initial denaturation (95 C,
10 min), PCR reactions were cycled 35 times with the
following parameters: denaturation at 95 C for 10 s,
annealing at 60 C for 12 s, extension at 72 C for
12s. Total DNA was measured using the diaminoben-
zoic acid method for mtDNA correction (Thomas &
Farquhar 1978).
Measurement of thiobarbituric acid-react ive substances
Lipid peroxidation levels or thiobarbituric acid-reactive
substances (TBARS) were determined as malondialde-
hyde-thiobarbituric acid adducts as described (Buege &
Aust 1978). Peroxidation levels were measured spec-
trophotometrically at 532 nm, using a molar extinction
coefficient of 1.56 · 10
5
m
)1
cm
)2
, and expressed as
nanomoles of TBARS per milligram of protein.
Measurement of carbonyl content
Carbonyl groups were quantified using the Oxyblot
protein oxidation detection kit (Chemicon, Chandlers
Ford, UK). Here 2,4-dinitrophenyhydrazine (DNPH)
derivatization was carried out for 15 min on 5 lgof
homogenate protein following the manufacturer’s
instructions. Proteins were transferred to nitrocellulose
filters by means of a slot-blot system (Bio-Rad, Hercu-
les, CA, USA). After incubation with anti-DNP anti-
body, blots were developed using the ECL detection
system on ChemiDoc XRS (Bio-Rad). Bands were
quantified using the Quantity One software package.
To determine specificity, the oxidized proteins provided
by the kit were included as a positive control. Treatment
of samples with a control solution served as a negative
control for the DNPH derivatization.
Statistics
All data are expressed as mean SEM. The unpaired
Student’s t-test was used to determine statistical signif-
icance. For multiple comparisons significance was
determined by anova followed by the Bonferroni test.
P < 0.05 was considered statistically significant.
Results
Body weight (BW) was significantly higher in rats fed
the high caloric diet compared to controls (Table 1).
The obese group also displayed increased heart weight
to body weight (HW : BW) and left ventricle to body
weight ratios (LV : BW). Moreover, triglycerides, fast-
ing plasma insulin, HbA1c and FFA levels were
elevated, while HDL cholesterol levels were reduced in
the obese group vs. controls (Table 1). However, fasting
plasma glucose levels and systolic blood pressure did
not significantly differ between control and obese rats,
while visceral fat was higher after the high caloric
feeding period (P < 0.05 vs. matched controls). Impor-
tantly, HbA1c levels were not above 6.5% in the high
caloric diet fed rats. Values below this limit are
indicative of good blood glucose management in these
animals.
We next assessed ex vivo contractile functional
parameters and myocardial infarct size after ischaemia.
Here the pre-ischaemic baseline AO was reduced in the
obese group (41.0 1.2 vs. 34.0 1.2 mL min
)1
;
P < 0.05 vs. controls; Fig. 1a). Furthermore, the AO
recovery was 25–30% lower in the obese group after
ischaemia-reperfusion (Fig.1b). For infarct size studies,
the area of the left ventricle at risk was similar for both
groups, i.e. 48.7 1.6% of the left ventricular volume
for controls and 50.4 1.7% for obese rats. In
agreement with the functional data, myocardial infarct
size after ischaemia/reperfusion was increased in the
obese group (24.3 2.1 vs. 42.9 1.8% of area at
risk; P < 0.0001 vs. controls; Fig. 1c).
We employed two different mitochondrial oxidative
substrates to gain insight into fuel substrate utilization.
State 2 respiration was decreased (22.2 1.0 vs.
19.2 0.6 nmol min
)1
mg
)2
protein) in the obese
group when malate palmitoyl-l-carnitine (MPC) was
used as oxidative substrate (P < 0.05 vs. controls;
Table 2). Lower state 2 respiration in the obese rats
merely reflects low endogenous ADP levels of which the
origin is unclear. State 3 respiration and ADP/O ratios
were similar for obese and control mitochondria (using
both oxidative substrates). State 4 respiration did not
differ when MPC was employed as oxidative substrate,
but was lower for the obese group when glutamate was
used as substrate. We also found that the ADP phos-
phorylation rate was decreased in the obese group
(278.1 8.3 vs. 245.6 11.3 nmol ADP min
)1
mg
)1
protein; P < 0.05 vs. controls) when MPC was
employed as oxidative substrate.
To further assess the effects of diet-induced obesity on
mitochondrial respiratory function, we exposed mito-
chondria to 15 min of oxygen deficiency followed by
6 min of reoxygenation. We subsequently determined
state 3 respiration (using MPC as substrate) as an index
of mitochondrial function in response to the anoxic
stress. Here, the percentage recovery of state 3 mito-
chondrial respiration was diminished in mitochondria
isolated from the obese group (51.7 8.2 vs.
28.7 5.4 nmol O
2
min
)1
mg
)1
protein; P < 0.05 vs.
controls; Fig. 2).
We also investigated potential mechanisms to explain
decreased contractile and mitochondrial respiratory
function in obese rats. Here we determined mtDNA
4
2009 The Authors
Journal compilation 2009 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2009.02024.x
Heart and respiratory function with obesity
Æ
M F Essop et al. Acta Physiol 2009
levels but found no significant differences between the
obese and control groups, i.e. 100 10.8 vs.
133 29.5 mtDNA ng
)1
tissue. We also evaluated
the effects of oxidative stress and found no significant
differences for the degree of protein carbonylation
(100 16 vs. 133 25 intensity mm
)2
) and lipid
peroxidation (TBARS; 6.6 1 vs. 5.6 0.3 nmol mg
)1
protein) for control vs. obese rats.
Discussion
Obesity is a major contributor to the global burden of
disease and is closely associated with the development
of type 2 diabetes and cardiovascular diseases. In the
light of this, we investigated cardiac contractile function
and mitochondrial respiratory capacity in a rat model of
obesity-induced pre-diabetes. The main finding of this
Table 1 Baseline characterization
of obese and control rats
Control Obese
Body weight (BW; g) 507.7 7.4 588.6 8.1
Heart weight (HW; g) 1.3 0.02 1.5 0.04
HW : BW ratio 2.4 0.1 2.6 0.1*
LV : BW ratio 2.4 0.1 2.6 0.1*
Total cholesterol (mmol L
)1
) 1.4 0.1 1.3 0.1
HDL cholesterol (mmol L
)1
)1 0.02 0.6 0.03***
Triglycerides (mmol L
)1
) 0.7 0.1 1.9 0.2***
FFA (mmol L
)1
) 0.8 0.1 1.7 0.3*
Glucose (fasting) (mmol L
)1
) 4.8 0.4 5.3 0.2
Insulin (fasting) (lIU mL
)1
) 27.8 3.5 43.4 4.2*
HbA1c (%) 3.5 0.1 4 0.1**
SBP (mmHg) 148 3.9 159 3.9
Visceral fat (%) 29.8 1.6 49.5 2.2*
Eight-week-old male Wistar rats were fed a high caloric diet for 16 weeks and com-
pared with matched controls on a standard laboratory chow. Values are expressed as
mean SEM (n ‡ 5 animals). HDL, high-density lipoprotein; FFA, free fatty acid;
HbA1c, glycosylated haemoglobin; SBP, systolic blood pressure.
*P < 0.05; **P < 0.01, ***P < 0.001 and
P < 0.0001 compared with control rats.
Control
Obese
45
(a)
(b)
(c)
35
40
*
25
30
15
20
5
10
0
Control
70
80
90
Obese
40
50
60
**
*
*
10
20
30
0
Aortic output recovery (%) Aortic output (mL min
–1
)
10 min 15 min 20 min
Reperfusion time (min)
#
50
60
30
40
10
20
Infarct size (% of AAR)
0
Figure 1 Decreased cardiac contractile
function in obese rats at baseline and in
response to an ischaemic insult. (a) Base-
line aortic output (mL min
)1
) for obese
and control rats; (b) percentage aortic
output recovery of rat hearts subjected to
15 min of global ischaemia and reper-
fused for 10, 15 and 20 min; and
(c) myocardial infarct size (expressed as
per cent of area at risk) after ischaemia-
reperfusion. The data are presented as
mean SEM. *P < 0.05, **P < 0.005,
#P < 0.0001 vs. matched controls
(n = 7–8).
2009 The Authors
Journal compilation 2009 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2009.02024.x
5
Acta Physiol 2009 M F Essop et al.
Æ
Heart and respiratory function with obesity
study is that pre-diabetic obese rats displayed reduced
baseline cardiac contractile function and mitochondrial
bioenergetic capacity, and impaired contractile function
and decreased mitochondrial respiratory capacity in
response to acute oxygen deficiency.
Our rodent model of diet-induced obesity closely
resembles the pre-diabetic state/metabolic syndrome
that is typically characterized by insulin resistance,
dyslipidaemia (high triglycerides, low HDL cholesterol
levels), hypertension and visceral obesity (Miranda
et al. 2005). Here we found that obese rats had
increased visceral fat, dyslipidaemia and higher plasma
insulin levels. As elevated circulating FFA levels are
causally linked to the onset of insulin resistance
(Delarue & Magnan 2007) and we previously reported
a higher homeostasis model assessment (HOMA) index
for obese rats using the same model (Du Toit et al.
2008), our data strongly support an insulin-resistant
state. In support, when the feeding regimen is extended
to the 30-week time point, we observe that fasting
insulin and glucose levels continue to increase vs.
16-week-old rats characterized in this study (M.F.
Essop & E.F. Du Toit, unpublished data). In parallel,
the HOMA index is further increased at the 30-week time
point, indicating progressive loss of insulin sensitivity.
Obese rats also exhibited a degree of cardiac remod-
elling, i.e. the development of cardiac hypertrophy.
These data are consistent with previous studies report-
ing a hypertrophic response in obese/type 2 diabetic
rodents (Yue et al. 2007, Du Toit et al. 2008). The
initial hypertrophic response is usually characterized by
increased wall thickness followed by progressive dila-
tation and contractile dysfunction. We found that
contractile function of obese rats was lower at baseline
and further decreased after subjecting hearts to ischae-
mia-reperfusion. In parallel, myocardial infarct sizes
were increased. We found that infarct sizes remain more
or less the same when different substrates (e.g. glucose,
palmitate, insulin) are employed for perfusion studies
(M.F. Essop & E.F. Du Toit, unpublished data).
Glutamate and MPC are not substrates commonly used
in isolated heart perfusions and are more suitable for
isolated mitochondrial studies. These findings are in
agreement with epidemiological data showing that the
metabolic syndrome is associated with a significantly
greater risk of cardiovascular disease (Wilson et al.
2005). However, not many laboratory studies have
assessed cardiac damage in response to ischaemia-
reperfusion in the pre-diabetic state. Multiple factors
may play a role in this process, e.g. altered lipoprotein
levels, ectopic cardiac triglyceride lipid deposition,
activation of inflammatory pathways, metabolic
derangements and perturbed mitochondrial respiratory
capacity (reviewed by Boudina & Abel 2006).
Table 2 Cardiac mitochondrial respiratory function of obese and control rats
Glutamate
Malate and palmitoyl-
l-carnitine
Control Obese Control Obese
State 2 respiration (nmol O
2
min
)1
mg
)1
protein) 11.2 1.1 10.8 1.2 22.2 1 19.2 0.6*
State 3 respiration (nmol O
2
min
)1
mg
)1
protein) 123.4 15.9 121.5 8.6 126.2 4.6 117.1 5
State 4 respiration (nmol O
2
min
)1
mg
)1
protein) 10.5 1.4 5.9 1.0* 11.8 1.7 14.1 1.8
ADP/O 2.4 0.1 2.3 0.03 2.2 0.1 2.1 0.04
Phosphorylation rate (nmol ADP min
)1
mg
)1
protein) 298.7 44.6 248 22.4 278.1 8.3 245.6 11.3*
Heart mitochondria were isolated from rats fed a high caloric diet for 16 weeks vs. controls on a standard laboratory chow.
Respiration parameters were determined using glycolytic (glutamate) or fatty acid (malate and palmitoyl-l-carnitine) as substrates.
Values are expressed as mean SEM (n ‡ 5 animals).
*P < 0.05 compared with age-matched controls.
60
Control
Obese
50
40
30
*
20
Percentage recovery of
State 3 respiration
10
0
Figure 2 Recovery of state 3 mitochondrial respiration in
response to acute oxygen deficiency. Mitochondria from obese
and control rats were isolated, supplied with 12.5 mm malate
and 54 lm palmitoyl-l-carnitine, and subjected to 20 min of
oxygen deficiency followed by 6 min of reoxygenation.
Recovery of respiratory function was evaluated by determining
the ratio of post-anoxia state 3 respiration over state 3 respi-
ration prior to oxygen deficiency. The data are presented as
mean SEM. *P < 0.05 vs. controls (n = 6).
6
2009 The Authors
Journal compilation 2009 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2009.02024.x
Heart and respiratory function with obesity
Æ
M F Essop et al. Acta Physiol 2009
In line with our hypothesis, we next evaluated
whether altered mitochondrial respiratory capacity
may provide a potential mechanism for attenuated
cardiac function in obese rats. Overall, we did not find
many differences between control and pre-diabetic rats.
However, ADP phosphorylation rates were diminished
in obese rats when a fatty acid was employed as a fuel
substrate. Moreover, we found that state 4 respiration
(MPC as substrate) was not significantly altered in the
obese group, suggesting no significant degree of mito-
chondrial uncoupling. Although state 4 respiration was
lower for the obese group when glutamate was used as
oxidative substrate, we are of the opinion that MPC is a
more representative substrate of uncoupling in the heart
as fatty acids are the major fuel substrate of the adult
mammalian heart. Moreover, state 4 respiration has
limitations as a marker for proton leak as additional,
‘non-uncoupling’ factors, e.g. substrate oxidation and
efficiency of electron transport, may influence this
parameter.
To gain further insight into possible mitochondrial
respiratory defects, we exposed isolated heart mito-
chondria to an acute anoxic insult followed by reper-
fusion and found that polarographic mitochondrial
oxygen consumption was impaired for obese rats. Our
mitochondrial data therefore suggest that attenuated
mitochondrial bioenergetic capacity may contribute to
the decreased respiratory capacity observed in response
to acute oxygen deficiency.
We also tested the degree of oxidative damage as
increased fatty acid oxidation rates are linked to
elevated levels of ROS production, thereby contributing
to lipid and protein peroxidation (Boudina et al. 2007).
However, we found no significant degree of oxidative
stress-induced damage. Moreover, as mtDNA levels
were also not significantly altered, our data therefore
support a pre-diabetic state, i.e. with a limited degree of
oxidative stress and damage to intracellular structural
components. We are unclear regarding the precise
mechanisms underlying decreased mitochondrial bioen-
ergetic capacity in obese rats. However, we propose that
downregulation of several nuclear-encoded mitochon-
drial genes and/or reduced protein levels of mitochon-
drial respiratory chain complexes with type 2 diabetes
animal models (Boudina et al. 2005) may be contribut-
ing factors to this process. Alternatively, disorganiza-
tion of mitochondrial ultrastructure may also be
implicated (Dong et al. 2006). These possibilities
require further investigation.
In summary, we have established a rodent model of
pre-diabetes resembling the human metabolic syn-
drome. Here obese rats displayed increased myocardial
damage and attenuated respiratory capacity in re-
sponse to acute oxygen deprivation. We propose that
decreased mitochondrial bioenergetic capacity may
play an important role in this process. Investigations
to enhance this process may therefore result in novel
therapeutic interventions to limit the onset of diabetic
cardiomyopathy.
Conflict of interest
None.
The authors thank Mr Wayne Smith for technical assistance.
This work was supported by the South African Medical
Research Council and the South African National Research
Foundation (to M.F. Essop), and the South African National
Research Foundation (to E.F. Du Toit).
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