The Effects of Gender and Obesity on Myocardial Tolerance to Ischemia

Abstract

Obesity is increasing at an alarming rate globally. Several studies have shown that premenopausal women have a reduced risk of CV disease and a reduced myocardial susceptibility to ischemia/reperfusion injury. The effect of obesity on myocardial tolerance to ischemia in women has not been established. To determine how obesity affects myocardial susceptibility to ischemia/reperfusion injury in both males and females, we fed male and female Wistar rats a high caloric diet (HCD) or a control rat chow diet (CD) for 18 weeks. Rats were subsequently fasted overnight, anesthetized and blood was collected. In separate experiments, 18-week-fed (HCD and CD) rats underwent 45 min in vivo coronary artery ligation (CAL) followed by 2 hours reperfusion. Hearts were stained with TTC and infarct size determined. Both male and female HCD fed rats had increased body and visceral fat weights. Homeostasis model assessment (HOMA) index values were 13.95+/-3.04 for CD and 33.58+/-9.39 for HCD male rats (p<0.01) and 2.98+/-0.64 for CD and 2.99+/-0.72 for HCD fed female rats. Male HCD fed rats had larger infarct sizes than CD fed littermates (43.2+/-9.3 % vs. 24.4+/-7.6 %, p<0.05). Female HCD and CD diet fed rats had comparable infarct sizes (31.8+/-4.3 % vs. 23.9+/-3.3 %). We conclude that male rats on the HCD became viscerally obese, dyslipidemic and insulin-resistant, while female HCD fed rats became viscerally obese without developing dyslipidemia or insulin resistance. Obesity increased myocardial infarct size in males but not the females.

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PHYSIOLOGICAL RESEARCH • ISSN 0862-8408 (print) • ISSN 1802-9973 (online)
© 2011 Institute of Physiology v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic
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Physiol. Res. 60: 291-301, 2011
The Effects of Gender and Obesity on Myocardial Tolerance to
Ischemia
C. CLARK1, W. SMITH1, A. LOCHNER1, E. F. DU TOIT2
1Division of Medical Physiology, Department of Biomedical Sciences, Faculty of Health Sciences,
University of Stellenbosch, Tygerberg, Cape Town, South Africa, 2School of Medical Science,
Griffith University, Gold Coast Campus, QLD, Australia
Received March 30, 2010
Accepted August 5, 2010
On-line November 29, 2010
Summary
Obesity is increasing at an alarming rate globally. Several studies
have shown that premenopausal women have a reduced risk of CV
disease and a reduced myocardial susceptibility to
ischemia/reperfusion injury. The effect of obesity on myocardial
tolerance to ischemia in women has not been established. To
determine how obesity affects myocardial susceptibility to
ischemia/reperfusion injury in both males and females, we fed
male and female Wistar rats a high caloric diet (HCD) or a control
rat chow diet (CD) for 18 weeks. Rats were subsequently fasted
overnight, anesthetized and blood was collected. In separate
experiments, 18-week-fed (HCD and CD) rats underwent 45 min
in
vivo
coronary artery ligation (CAL) followed by 2 hours reperfusion.
Hearts were stained with TTC and infarct size determined. Both
male and female HCD fed rats had increased body and visceral fat
weights. Homeostasis model assessment (HOMA) index values
were 13.95±3.04 for CD and 33.58±9.39 for HCD male rats
(p<0.01) and 2.98±0.64 for CD and 2.99±0.72 for HCD fed female
rats. Male HCD fed rats had larger infarct sizes than CD fed
littermates (43.2±9.3 % vs. 24.4±7.6 %, p<0.05). Female HCD
and CD diet fed rats had comparable infarct sizes (31.8±4.3 % vs.
23.9±3.3 %). We conclude that male rats on the HCD became
viscerally obese, dyslipidemic and insulin-resistant, while female
HCD fed rats became viscerally obese without developing
dyslipidemia or insulin resistance. Obesity increased myocardial
infarct size in males but not the females.
Key words
Obesity • Gender • Myocardial infarction
Corresponding author
E. F. du Toit, School of Medical Science, Griffith University Gold
Coast, Parklands Drive, Southport, QLD, 4217, Australia. Fax:
07 555 28908. E-mail: j.dutoit@griffith.edu.au
Introduction
The incidence of obesity is increasing at an
alarming rate globally. According to a 2005 World Health
Organization (WHO) survey 1.6 billion of the world’s
adults were overweight and another 400 million obese. It
is predicted that by 2015, 2.3 billion adults will be
overweight and more than 700 million will be obese.
Obesity has increased exponentially in women and has
lead to the publication of guidelines for the prevention of
cardiovascular disease in women (Mosca et al. 2007).
Obesity predisposes individuals to cardiovascular disease
and more specifically myocardial infarction and heart
failure (Kenchaiah et al. 2002, Sowers et al. 2003, Yusuf
et al. 2005).
Several studies have shown that premenopausal
women have a reduced risk of cardiovascular disease
when compared with men (Barrett-Connor et al. 1997,
Crabbe et al. 2003) but that cardiovascular disease
increases after menopause (Hayward et al. 2000). A
phenomenon thought to be due to the presence of female
sex hormones before menopause. The relevance of these
observations were however questioned when it became
evident that hormone replacement therapy was unable to
protect postmenopausal women against cardiovascular
disease (Rossouw et al. 2002). Although some studies
suggest that the female myocardium is more resistant to
ischemia/reperfusion injury than the male myocardium
(Mehelli et al. 2002, Bae and Zhang 2005, Gabel et al.
2005, Wang et al. 2006), the effect of obesity on
myocardial tolerance to ischemia in both males and
females remains unresolved.

292 Clark et al. Vol. 60
Data from recent studies suggest that obesity
may alter myocardial metabolism leading to
compromised cardiac function and tolerance to ischemia
(Poirier et al. 2006, Lopaschuk et al. 2007). Elevated
circulating lipids may promote increased myocardial fatty
acid uptake and utilization which could decrease both
myocardial 1) efficiency under normoxic conditions,
(Tuunanen et al. 2006, Lopaschuk et al. 2007) and, 2)
tolerance to ischemia (Opie 1998). Obesity may also
promote cardiac lipid accumulation which has been
implicated in the etiology of insulin resistance
(Lopaschuk et al. 2007). Excessive myocardial free fatty
acid utilization during ischemia is also potentially
detrimental as it not only inhibits glucose oxidation
(Tuunanen et al. 2006), but fatty acids also require more
oxygen for ATP production when compared with glucose
(Wallhaus et al. 2001).
Obesity is also a risk factor for insulin resistance
(Bjorntrop 1993). Several mechanisms have been
proposed to explain how increased adiposity interferes
with insulin signalling and glucose uptake and utilization.
Increased fat accumulation in insulin sensitive tissues
causes dysfunction of the insulin signalling cascade by
activating PKCθ which inhibits IRS-1 (insulin receptor
substrate 1) and PI3-K (phosphoinositide 3-kinase)
activity and causes decreased GLUT-4 translocation and
glucose uptake (Boden and Schulman 2002). Ischemic
hearts rely on glucose as the primary fuel to produce ATP
through glycolysis with insulin resistance compromising
the heart’s ability to tolerate an ischemic event
(Lopaschuk et al. 2002). The effect of gender on obesity
induced insulin resistance is controversial. Although
observations from several laboratory (Horton et al. 1997,
Gomez-Perez et al. 2008) and clinical studies (Ferrara et
al. 2008, Vistisen et al. 2008) have suggested that obesity
does not affect insulin sensitivity in females, others have
shown that both genders are prone to obesity induced
insulin resistance and subsequent diabetes (Coatmellec-
Taglioni et al. 2003, Cordero et al. 2009).
Adiponectin is an adipocytokine that is elevated
in the serum of women when compared with men (Ryo et
al. 2004, Costacou et al. 2005). It improves insulin
sensitivity of insulin sensitive organs (Yamauchi et al.
2001, Kubota et al. 2002, Stefan et al. 2002), decreases
hepatic glucose production (Batterham et al. 2002) and
may consequently protect the heart against
ischemic/reperfusion injury. Similarly, estrogen is
purported to improve lipid profiles (Pedersen et al. 2004),
protect against hyperglycemia (Rincon et al. 1996, Louet
et al. 2004) and be cardioprotective during ischemia
(Wang et al. 2006). The latter effects of estrogen may be
by: 1) inducing nitric oxide synthase (NOS) production
which in turn inhibits the L-type Ca2+ channels (Groné et
al. 1998) or, 2) by differentially activating MAPK to
mediate this protection (Wang et al. 2006).
Effects of obesity on this apparent increased
tolerance to ischemia in women is however unknown.
Previous studies in our laboratory have shown that diet
induced obesity in male rats increased heart susceptibility
to ischemia/reperfusion injury (Du Toit et al. 2005,
2008). We had however not tested the effects of this high
caloric diet (HCD) in female rats. Preliminary
observations by our group had suggested that body
weight gain in female rats on this diet was lower than in
their male littermates. In addition, no other studies had
carefully documented body weight gain and visceral fat
accumulation in female rats on this HCD. We therefore
wished to determine whether: 1) the female rats would
become obese when subjected to a high caloric diet
(HCD), 2) the obesity induced systemic insulin resistance
in the female rats and, 3) obesity impacts on myocardial
tolerance to ischemia in the female rats. Finally, we
wished to determine how obesity influenced circulating
adiponectin and estrogen levels in these animals and
whether there was an association between the levels of
these peptides and insulin sensitivity and myocardial
tolerance to ischemia.
Methods
Feeding program
Age matched (8 week old) male and female
Wistar rats were put on a high caloric diet (HCD) or a
control diet (CD) for 18 weeks (Pickavance et al. 1999,
Du Toit et al. 2008). The CD fed rats had a total energy
intake of 371±18 kJ/day (60 % carbohydrates, 30 %
protein, and 10 % fat) and the HCD fed rats had a total
energy intake of 570±23 kJ/day (65 % carbohydrates,
19 % protein and 16 % fat). The increased caloric intakes
in the HCD fed rats were achieved due to voluntary
hyperphagia. The choice of the dietary composition for
our study was motivated by the fact that global and
particularly African urbanisation is associated with an
increase in the incidence of obesity. The increase in the
incidence of obesity in Africa has been attributed to an
increase in the dietary fat (Khan and Bowman 1999,
MacIntyre et al. 2002) and refined sugar (Khan and
Bowman 1999) content in the diet. The typical diet of

2011 Obesity, Gender and Myocardial Tolerance to Ischemia 293
urban Africans now more closely resembles the Western
diet with a high fat and carbohydrate content (Kruger et
al. 2002, Bourne et al. 2002). This change in the rat diet
would therefore mimic the change in diet often
experienced with urbanisation where both carbohydrate
and fat content of the diet may increase (Khan and
Bowman 1999).
The rats were supplied by, and housed in the
Central Research Facility of the Faculty of Health Sciences
at the University of Stellenbosch (AAALAC accredited).
Animals were provided with fresh food daily and had ad
libitum access to food and water and were housed in
facilities with a 12-hour day-night cycle at 23 oC.
The study was approved by the Committee for
Experimental Animal Research of the Faculty of Health
Sciences, University of Stellenbosch. All animals
received humane care in accordance with the Principles
of Laboratory Animal Care of the National Society for
Medical Research and the Guide for the Care and use of
Laboratory Animals of the National Academy of Sciences
(NIH publication no 80-23, revised 1985).
Experimental design
Sixty four Wistar rats (32 male and 32 female)
were randomly divided in two groups that were placed on
either the CD or the HCD for 18 weeks. Within each
group, 10 rats were randomly selected for in vivo infarct
size quantification and the remaining 6 were used for
biochemical analysis.
In vivo infarct induction and infarct size quantification
Rats were anesthetized with an intraperitoneal
injection of ketamine (7.5 mg/kg) and medetomidine
(0.5 mg/kg), intubated, and placed on a rodent ventilator
(Harvard Instruments, Model 683) before being placed in
a pediatric incubator maintained at a temperature of
34.5 °C. Rat core temperature was monitored using a
rectal temperature probe and was maintained at 36-37 oC
throughout the experiment.
Rats were placed on their right side, the thorax
shaved and a left thoracotomy performed. The ribs were
separated using a rodent retractor (Aesculap, Melsungen,
Germany) and the pericardium removed. The left anterior
descending (LAD) coronary artery was ligated using an
Ethicon silk suture. Hearts were subjected to 45 min
regional ischemia before hearts were reperfused for
120 min (Thim et al. 2006). Myocardial reperfusion was
initiated by release of the silk suture. During CAL and
reperfusion the thoracic cavity was covered with a sterile
saline solution saturated swab to prevent excess fluid loss
and dehydration. Rats were also injected with 2ml of
sterile saline intraperitonealy every 30 minutes to
compensate for any fluid loss which may have occurred
due to the ventilation.
After 120 min reperfusion, hearts were excised,
mounted on an isolated Langendorff perfusion system
and perfused with a Krebs-Henseleit bicarbonate buffer
within 30 sec of excision from the rat. This isolated heart
perfusion lasted 60 sec to allow for staining with Evans
blue dye. The coronary artery was reoccluded and the
heart was stained with 600 μl of Evan’s Blue Dye
(Sigma, Saint Louis, Missouri, USA) administered
through the aortic cannula. Hearts were frozen at –20 °C
overnight after which triphenyl tetrazolium chloride
(TTC) was used to delineate the viable and necrotic
myocardium. Infarct size was expressed as a percentage
of the area at risk.
The sham-operated animals underwent the same
surgical procedure described above except that the suture
that was passed under the LAD coronary artery was not
fastened. The same TTC staining procedure was
followed. Necrotic tissue was present where the suture
passed through the myocardium under the LAD coronary
artery but this necrotic tissue represented less than 2 % of
the left ventricular area at risk. One animal was lost
during the initial surgical procedure during induction of
coronary artery ligation due to excessive bleeding.
TTC staining
After freezing, hearts were cut into 5-7
transverse slices, each approximately 2 mm in size. Heart
slices were immersed in a buffered triphenyltetrazolium
chloride (TTC) solution at room temperature (protected
from light) for 15-20 minutes. They were subsequently
immersed in 5 ml of formaldehyde for 3 hours to enhance
any color differences.
Biochemical analysis
The animals which were set aside for
biochemical analysis were fasted for 10 hours,
anesthetized and blood was collected for biochemical
analysis. Blood was collected by cardio-puncture after
performing a thoracotomy to access the heart. The hearts
from these animals were not used for the infarct size
determinations as fasting and consequent glycogen
depletion of the heart compromises myocardial tolerance
to ischemia/reperfusion. The peritoneal and
retroperitoneal fat was removed and weighed (Sartorius

294 Clark et al. Vol. 60
Pty. Ltd, Johannesburg, South Africa).
For serum collection, blood samples were placed
in serum separation tubes (BD Vacutainer tubes) and
stored on ice for 20 minutes before centrifugation
(Eppendorf Centrifuge 5403, Hamburg, Germany) at
2000 g at 4 °C for 10 minutes. Serum was stored at
-80 °C until assays could be performed. The assays were
all done within 1-2 weeks of collection of the serum
samples. Insulin (Coat-A-Count® Insulin, Siemens
Medical Solutions Diagnostics, California, USA),
estrogen (Assay Designs’ Correlate-EIA™, Michigan,
USA) and adiponectin (AdipoGen, Seoul, Korea) were
determined according to the manufacturers’ instructions.
The respective serum concentrations were analyzed using
a radioimmunoassay (RIA), an immunoassay and two
enzyme-linked immunosorbant assay’s (ELISA).
Serum triglycerides, high density lipoproteins
(HDL) cholesterol, low density lipoproteins (LDL) and
cholesterol levels and glucose levels were determined in
fresh blood using a Cardiochek® lipid analyzer
(Cardiocheck, Indianapolis, USA) and a glucometer
(GlucoPlus™ Inc, Québec, Canada) respectively.
In order to assess insulin resistance in these
animals the homeostasis model assessment (HOMA)
index was determined. Fasting blood glucose and insulin
levels were used to determine the HOMA index using the
standard formula: [fasting insulin (μIU/ml) x fasting
glucose (mmol/l)]/22.5.
Statistical analysis
All results were expressed as the mean ±
standard error of the mean (S.E.M.). For multiple
comparisons, a Two-way ANOVAs was used followed
by a Bonferroni post hoc test. A p-value of less than 0.05
was considered to be significantly different.
Results
Body weights
Rats were age matched and male and female rats
therefore had different body weights at the start of the
study. Female Wistar rats are known to be 100-110 grams
lighter than their male littermates at 8 weeks (Table 1).
Male and female rats on the HCD had increased body
weights compared with their CD fed littermates
(Fig. 1A). The percentage weight gain was higher in both
male and female rats on the HCD (Table 1). Visceral fat
content was also higher in HCD fed male and female rats
compared with CD fed rats (Fig. 1B).
Blood and serum lipid, glucose and insulin levels
Serum triglyceride levels were elevated in the
male HCD fed rats but not in the HCD fed female rats
(Fig. 2A). Triglyceride levels were however significantly
lower in HCD fed females than HCD fed males
(0.78±0.11 mmol/l for females vs. 1.31±0.08 mmol/l for
males, p<0.001). Serum total cholesterol levels were
below the assay detection limit (2.5 mmol/l) for the
females (Fig. 2B) and similar between male CD and HCD
fed groups (Fig. 2B). Serum HDL cholesterol levels were
similar in all four groups (Fig. 2C).
Table 1. Initial body weights and percentage body weight gain
for male and female rats after 18 weeks on the respective diets.
Male Female
Control Diet (CD) 303.6 ± 4.9 g 198.4 ± 4.6 g
High Caloric Diet (HCD) 308.2 ± 5.4 g 206.5 ± 4.8 g
Control Diet (CD) 45 ± 4 % 23 ± 3 %
High Caloric Diet (HCD) 58 ± 5 % # 36 ± 6 % #
# p<0.05 vs. control diet, n=16 for each experimental group.
A
Fig. 1. (A) Final body weight of male and female, CD and HCD
fed rats. (B) Visceral fat weight of male and female, CD and HCD
fed rats. All values are expressed as mean ± S.E.M. n=16 for
each experimental group.

2011 Obesity, Gender and Myocardial Tolerance to Ischemia 295
Fig. 2. (A) Fasting serum triglyceride levels of male and female,
CD and HCD fed rats. (B) Fasting serum total cholesterol levels
of male and female, CD and HCD fed rats. (C) Fasting serum
HDL cholesterol levels of male and female, CD and HCD fed rats.
All values are expressed as mean ± S.E.M. n=6 for each
experimental group.
Fasting blood glucose levels were elevated in
HCD fed compared with CD fed male rats (Fig. 3A).
There were no differences in fasting blood glucose levels
in the female rats. Male obese rats also had higher fasting
blood glucose levels than their female obese littermates
(Fig. 3A).
Male HCD fed rats had elevated insulin levels
compared with CD fed rats (Fig. 3B). No differences
were observed between the two female groups. Male CD
fed rats however also had higher insulin levels than CD
fed females and HCD fed male rats had higher insulin
levels than HCD fed female rats (Fig. 3B).
HOMA values were increased in HCD fed males
compared with the CD fed males (Fig. 3C). HOMA
values were also increased in the HCD fed male rats
compared with the HCD fed females (Fig. 3C).
Fig. 3. (A) Fasting blood glucose levels of male and female, CD
and HCD fed rats. (B) Fasting serum insulin levels of male and
female, CD and HCD fed rats. (C) HOMA values for male and
female, CD and HCD fed rats. All values are expressed as mean ±
S.E.M. n=6 for each experimental group.
Effect of obesity on myocardial infarct size
Infarct was expressed as a % of the area at risk.
The area of the left ventricle at risk was similar for all four
experimental groups (male CD – 45.2±8.2 %, male HCD –
43.6±10.4 %, female CD – 46.8±10.5 % and female
HCD – 40.7±7.0 %). Infarct size was increased in HCD fed
compared with CD fed male rats (Fig. 4). There were no
differences in infarct size between the female groups but
myocardial infarct size was reduced in female rats fed
HCD compared with males on the same diet. Infarct size
was similar for male and female rats on the CD (Fig. 4).
Circulating adiponectin and estrogen levels
The HCD had no effect on serum adiponectin
levels in either the male or the female rats. Female obese
(on HCD) rats however had elevated adiponectin levels
compared to the obese male littermates (23.48±
0.94 μg/ml vs. 19.92±0.74 μg/ml, p<0.001). Male and

296 Clark et al. Vol. 60
female CD fed rats had similar adiponectin levels (male
CD – 19.00±0.73 μg/ml and female CD – 21.46±
0.74 μg/ml)
The fasting serum estrogen levels were similar
for all groups at the time of blood collection and
experimentation (male CD – 971.3±22.4 pg/ml, male
HCD – 984.9±14.1 pg/ml, female CD – 961.4±24.4 pg/ml
and female HCD – 981.0±3.9 pg/ml).
Discussion
We found that both male and female rats were
prone to visceral obesity when subjected to a HCD. This
visceral obesity was associated with dyslipidemia,
elevated insulin and glucose levels and insulin resistance
(as assessed using the HOMA) in the male but not in the
female rats. This obesity and decreased insulin sensitivity
was also associated with increased myocardial infarct
sizes in male but not female rats. The maintained insulin
sensitivity in viscerally obese female rats may be due to
the normal circulating lipids and increased circulating
adiponectin levels in females when compared with their
obese male littermates. The normal tolerance to ischemia
observed in the female rats may be due to their normal
insulin sensitivity but cannot be attributed to elevated
circulating estrogen levels in these animals at the time of
experimentation.
Effect of obesity on serum lipids and insulin resistance in
male and female rats
The effects of obesity and the metabolic
syndrome on cardiovascular risk factors in women are
controversial (Coatmellec-Taglioni et al. 2003, Regitz-
Zagrosek et al. 2007, Cordero et al. 2009). Cordero et al.
(2009), and others (Coatmellec-Taglioni et al. 2003) have
proposed that women may be as prone, if not more prone
to obesity induced insulin resistance than men. Conversely,
data from animal studies suggest that females may be
protected against obesity (Gomez-Perez et al. 2008,
Ferrara et al. 2008, Vistisen et al. 2008) or high sucrose
diet (Horton et al. 1997) induced insulin resistance.
We found that basal insulin levels were lower in
female lean rats than in their lean male littermates
(Fig. 3). This is in agreement with the findings of another
study comparing the effects of a high fat diet on fasting
insulin levels in lean rats (Gomez-Perez et al. 2008). This
group found that fasting serum insulin levels were lower
in female than male rats and that the high fat diet
exacerbated insulin resistance in male rats.
We found that both male and female rats put on a
high caloric diet (containing increased carbohydrates and
fats) had significantly elevated body and visceral fat
weights. The increased body weight was associated with
elevated serum triglyceride levels and insulin resistance in
the male rats while there was no change in lipid levels or
insulin sensitivity in the female rats. These observations
are in agreement with the data showing that high fat diet
induces obesity, dyslipidemia and insulin resistance in
male rats (Gomez-Perez et al. 2008), but not in females
(Gomez-Perez et al. 2008, Aubin et al. 2008). A similar
study by Thakker and co-workers (2006) found that a high
fat diet induced obesity and dyslipidemia in both male and
female mice without causing insulin resistance in the
female animals. Male mouse fasting insulin levels were
four-fold higher in obese male than obese female mice and
the male mice were insulin resistant as determined using
the HOMA-IR (insulin resistance) index. Similarly, in
another study high sucrose diets increased body weight in
female rats without causing insulin resistance (Horton et al.
1997). There is also strong evidence to suggest that
estrogens decrease noradrenalin induced lipolysis in
women by up-regulating the α-2 adrenergic anti-lypolytic
receptors in adipose tissue (Pedersen et al. 2004). The
absence of an effect of the HCD on total cholesterol levels
in the rats in this study is consistent with our observations
(Du Toit et al. 2008) and those of others (Roach et al.
1993, Ferdinandy et al. 1997) and is believed to be due to
down-regulation of hepatic cholesterol synthesis in
response to increased dietary cholesterol consumption in
the rat (Roach et al. 1993).
Potential mechanisms for obesity induced insulin
resistance
A role for elevated serum lipids in the genesis of
Fig. 4. Infarct size (as a percentage of the area at risk) of male
and female, CD and HCD fed rats. All values are expressed as
mean ± S.E.M. n=9-10 for each experimental group.

2011 Obesity, Gender and Myocardial Tolerance to Ischemia 297
peripheral insulin resistance was proposed several years
ago (Kraegen et al. 1991). We now believe that elevated
plasma triglycerides associated with obesity cause
intramuscular triglyceride accumulation despite the
concomitant increased fatty acid oxidation rates in the
muscle (Kraegen et al. 1991, Turner et al. 2007). This may
explain the increased insulin sensitivity observed in female
rats in our study where lipid profiles remained normal
despite the increased body and visceral fat weights of the
HCD fed female rats. Our findings are consistent with
those of Gomez-Perez and co-workers (2008) who found
that a high fat diet induced more severe insulin resistance
(as assessed using HOMA) in the male than the female
rats. This was despite the fact that the body weight gain in
the female rats was 38 % compared with the moderate
weight gain of 16 % observed in the male rats in that study.
The proposed insulin sensitizing effects of
adiponectin are well documented (Berg et al. 2001,
Combs et al. 2001, Fruebis et al. 2001, Ryo et al. 2004).
Yamauchi and co-workers (2001) demonstrated a very
strong correlation between elevated adiponectin levels
and improved insulin sensitivity. They also demonstrated
how chronic exogenous adiponectin administration
improved insulin sensitivity in mice. We could not
demonstrate differences in the levels of circulating
adiponectin between CD fed and HCD fed male rats or
between lean and obese female rats but found that
circulating adiponectin levels were higher in female
obese than in male obese rats. The elevated adiponectin
levels in female obese rats may improve insulin
sensitivity in these animals and therefore protect them
against obesity induced insulin resistance.
Estrogen also plays a role in glucose
homeostasis and metabolism. Estrogen protects animal
models of diabetes against hyperglycemia by increasing
glucose uptake into muscle and decreasing hepatic
glucose synthesis (Rincon et al. 1996, Louet et al. 2004).
Although we did not see any differences in estrogen
levels in the serum of our rats at the time of blood
collection and experimentation, we believe that these rats
were pre-menopausal and would have been affected by
the additional estrogen present in females at certain
stages of the estrus-cycle.
The effects of obesity and consequent insulin resistance
on myocardial susceptibility to ischemia/reperfusion
injury in male and female rats
Our group has previously shown that male rats
on the high caloric diet became obese, dyslipidemic and
insulin resistant and were more prone to
ischemic/reperfusion injury (Du Toit et al. 2005, 2008).
The effects of gender on myocardial susceptibility to
ischemia is however controversial. Studies using
isolated perfused hearts (Wang et al. 2006) or isolated
cardiomyocytes (Ranki et al. 2001) have demonstrated
that the hearts of female rats are more resistant to
ischemia/reperfusion damage than their male
counterpart. Similarly Cross and co-workers (1998,
2002, 2003) have demonstrated increased resistance to
ischemia/reperfusion injury in conditions where
intracellular calcium was elevated prior to ischemia.
They also demonstrated that estrogen may play a
cardioprotective role in females as ovarectomised rats
had levels of ischemic injury that resembled those of
males (Cross et al. 2002). Despite these positive
findings, a large scale clinical trial has failed to show
any cardioprotective benefits from hormone
replacement therapy in postmenopausal women
(Rossouw et al. 2002). It was proposed that the adverse
effects observed in the latter study may be related to
prothrombotic effects of progestins (Rossouw et al.
2002). We were unable to demonstrate an increased
tolerance to ischemia in CD fed female rats when
compared with males when using this in vivo model of
ischemia/reperfusion. This may be due to the fact that
we performed all the experiments at a stage in the estrus
cycle when estrogen levels were low and comparable
with males. This was achieved by performing all
experiments on the same day of the estrus cycle of the
rat. The absence of differences in estrogen levels in the
male and female rats during the time of experimentation
in this in vivo model would eliminate the possible
receptor mediated protective effects of estrogen. The
long term beneficial effects of estrogen that include its
lipid (Pedersen et al. 2004) and glucose (Rincon et al.
1996, Louet et al. 2004) modulating effects cannot
however be discounted.
Conclusions
We conclude that visceral obesity causes
dyslipidemia and insulin resistance in male but not
female rats. The obese insulin resistant male rats were
more prone to ischemic/reperfusion injury than their lean
littermates. Obese female rats on the HCD were not
dyslipidemic or insulin resistant and were more resistant
to ischemic/reperfusion injury than their male obese
littermates.

298 Clark et al. Vol. 60
Conflict of Interest
There is no conflict of interest.
Acknowledgements
This study was supported by funding from the South
African National Research Foundation and the Harry and
Doris Crossley Foundation.
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