International Journal of Obesity (2000) 24, 639±646
ß 2000 Macmillan Publishers Ltd All rights reserved 0307±0565/00 $15.00
www.nature.com/ijo
Development of high fat diet-induced obesity
and leptin resistance in C57Bl=6J mice
S Lin1, TC Thomas1, LH Storlien1 and XF Huang1*
1
Metabolic Research Center, Department of Biomedical Science, University of Wollongong, NSW 2522, Australia
OBJECTIVE: To investigate the development of high fat diet-induced obesity and leptin resistance.
DESIGN: Two experiments were carried out in this study. Firstly, we fed the mice with a high- or low-fat diet for up to
19 weeks to examine a progressive development of high fat diet-induced obesity. Secondly, we examined peripheral
and central exogenous leptin sensitivity in mice fed high- or low-fat diets for 1, 8 or 19 weeks.
SUBJECTS: A total of 168 C57BL=6J mice (3 weeks old) were used in this study.
MEASUREMENTS: In the ®rst experiment, we measured the body weight, energy intake, adipose tissue mass, tibia
bone length, and plasma leptin in mice fed either a high- or low-fat diet for 1, 8, 15 and 19 weeks. In the second
experiment, body weight change and cumulative energy intake were measured at 6 h intervals for 72 h after leptin
injection in mice fed a high- or low-fat diet for 1, 8 or 19 weeks.
RESULTS: The results from the ®rst experiment suggested that the development of high fat diet-induced obesity in
mice could be divided into early, middle and late stages. Compared with the mice fed a low-fat diet, the mice fed a
high-fat diet showed a gradually increased body weight ( ‡ 5.2%), fat storage (epididymal plus perirenal; ‡ 6.7%) and
plasma leptin ( ‡ 18%) at 1 week; ‡ 11.4%, ‡ 68.1%, and ‡ 223%, respectively, at 8 weeks; and ‡ 30.5%, ‡ 141%, and
‡ 458%, respectively, at 19 weeks. Energy intake of high fat diet-fed mice was equal to that of low fat diet-fed controls
for the ®rst 3 weeks; it fell below control levels over the next 5 week period, but began to increase gradually after 8
weeks of high-fat diet feeding and then increased dramatically from 15 weeks to be 14% higher than that of controls
after 19 weeks. The results from our second experiment showed that: (1) after 1 week of feeding, the mice fed a highfat diet were sensitive to a 2 mg=g (body weight) intraperitoneal (i.p.) injection of leptin, with no differences in body
weight change or cumulative energy intake post-injection; (2) after 8 weeks of feeding, the mice fed a high-fat diet
were insensitive to 2 mg=g (body weight) i.p. leptin, but were sensitive to a 0.1 mg intracerebroventricular (i.c.v.)
injection of leptin; (3) after 19 weeks of feeding, the mice fed a high-fat diet were insensitive to 0.1 mg i.c.v. leptin, but
were sensitive to a high dose of 2 mg i.c.v. leptin.
CONCLUSIONS: The present study demonstrated that the development of high fat diet-induced obesity (19 weeks) in
C57 B1=6J mice could be divided into three stages: (1) an early stage in response to high-fat diet that mice were
sensitive to exogenous leptin; (2) a reduced food intake stage when mice had an increase in leptin production and still
retained central leptin sensitivity; and (3) an increased food intake stage, accompanied by a reduction of central leptin
sensitivity.
International Journal of Obesity (2000) 24, 639±646
Keywords: obesity; dietary fats; hyperleptinaemia; leptin; mice
Introduction
Obesity is de®ned as increased adipose mass resulting
from a chronic imbalance between energy intake and
expenditure.1 ± 3 In rodents, a number of autosomal
recessive gene mutations that result in the obese
phenotype have been described, including ob=ob and
db=db mice.4,5 The products of the ob and db genes
represent a hormone ± receptor pair (leptin, produced
and released into the plasma by adipocytes, and its
receptor, found in hypothalamic nuclei, respectively)
that appears to be a component of a central negative
feedback system involved in the regulation of fat
mass, such that a change in the amount of lipid
*Correspondence: XF Huang, Department of Biomedical
Science, University of Wollongong, NSW 2522, Australia.
E-mail: [email protected]
Received 18 September 1998; revised 23 August 1999 and
8 November 1999; accepted 16 December 1999
stored triggers a response that opposes the change,
so as to maintain adiposity within a narrow range.6 ± 9
Mice unable to produce leptin (ob=ob) or to respond to
it (db=db) are characterized by hyperphagia and
decreased energy expenditure, as well as other metabolic abnormalities such as hyperinsulinaemia, hyperlipidaemia, insulin resistance, glucose intolerance and
diabetes.9,10 Further, treatment with leptin inhibits
feeding, increases energy expenditure, and subsequently reduces body fat in a dose-dependent fashion
in both ob=ob and normal mice, but not in db=db
mice.11,12
The ef®cacy of such murine monogenic models of
obesity in determining the mechanisms underlying
human obesity remains to be seen. There is no convincing evidence to indicate that mutations in the
genes encoding either leptin or its receptor are a
part of the aetiology of normal human obesity.13,14
Indeed, plasma leptin concentrations are closely correlated to body mass index in humans, and ob mRNA
has been shown to be increased in obese humans
High fat diet-induced obese mice
S Lin et al
640
relative to nonobese humans.6,15 ± 17 As such it has
been suggested that obese humans may be insensitive,
or `resistant' to the leptin signal.
In support of this hypothesis, leptin resistance has
recently been described in a rodent model of obesity
that may more closely re¯ect the human condition,
namely, diet-induced obese mice.18 Further, the evidence indicated that the mice and rats fed a high-fat
diet for weeks ( < 10 weeks) develop obesity and
resistance peripherally.18,19 Our study was therefore
undertaken to investigate whether or not high fat dietinduced obesity is associated with a progressive
development of leptin insensitivity peripherally and
centrally, and whether the duration of high fat feeding
may be a factor in the development of such leptin
insensitivity.
Materials and methods
Animals
One-hundred and sixty-eight 3-week-old C57BL=6J
male mice were used in this study. The study contains
two major experiments. In the ®rst experiment, 48
mice were used to compare body weight, food intake,
fat storage and serum leptin concentration between the
mice fed a high and low-fat diet. In the second
experiment, 120 mice were used to examine peripheral and central effects of exogenous leptin following
a high-fat diet for 1, 8 and 19 weeks. Animals were
randomized and housed individually in environmentally controlled conditions (temperature 22 C, light
cycle from 06:00 to 18:00 and dark cycle from 18:00
to 06:00), and were given ad libitum access to tap
water throughout the study.
Diet and experimental procedure
Mice were obtained from the Animal Resource Centre
(Western Australia) and were fed standard laboratory
chow for the ®rst week to allow them to adjust to the
new environment. Mice were subsequently randomly
assigned to one of two groups: a high-fat diet fed
group or a low-fat diet fed group. The composition of
the respective diets is the same as used previously.20
Diets were freshly made every week and stored at
4 C. Mice were fed using small plastic dishes just
before the beginning of the dark cycle.
Experiment 1. Forty-eight mice were divided into
two groups. The mice in the ®rst group were fed with
high-fat diet (HFF) and the mice in the second group
were fed with low-fat diet (LFF). Six mice for each
group were killed after being fed either high or low-fat
diet for 1, 8, 15 and 19 weeks. The body weight and
energy intake were measured every week for the 19week feeding period. After the mice were killed at
week 1, 8, 15 and 19, adipose tissues (epididymal,
International Journal of Obesity
perirenal and inguinal fat masses) and tibial length
were measured. Blood samples were obtained by
puncturing its right ventricle of the heart and plasma
leptin was measured using an RIA kit for mouse leptin
(Linco Inc., St Louis, MO).
Experiment 2. One-hundred and twenty mice were
divided into HFF or LFF groups. In accordance with
the observed variability in energy intake of HFF mice
in the ®rst experiment, three time points were selected
for the administration of leptin as representative of the
three time periods during which rates of consumption
differed, namely, weeks 1, 8 and 19 of the feeding
protocol.
After 1 week of high or low-fat diet, six mice from
each group received an i.p. injection of 2.0 mg=g (body
weight) recombinant murine leptin (R&D Systems
Inc., USA) or saline as controls. Body weight
change and cumulative energy intake were then measured every 6 h until 72 h following the injection.
After 8 weeks of high or low-fat diet, six mice from
each group received an intraperitoneal (i.p.) injection
of 2.0 mg=g (body weight) leptin or saline as controls,
while another six mice from each group received an
intracerebroventricular (i.c.v.) injection of leptin
(0.1 mg) or vehicle (5 ml of 10 mM sodium acetate,
pH 7.4) as controls. Needle placement was con®rmed
by visual examination of the lateral ventricle in the
tissue sections. Body weight change and cumulative
energy intake were then measured at every 6 h until
72 h following the injection.
After 19 weeks of high or low-fat diet, two dosages
of i.c.v. leptin were used for this part of the study. Six
mice from each group received an i.c.v. injection of
0.1 mg leptin or vehicle, while another six mice from
each group received a higher dosage (2 mg) of i.c.v.
leptin or vehicle as controls. Body weight change and
cumulative energy intake were then measured at 6 h
interval until 72 h following the injection.
Food consumption measurement
Food consumption was estimated by subtracting the
amount of food left in the plastic dishes and the
amount of food spilled from the initial food weight.
Food spillage was determined as follows: any food
that spilled was collected on absorbent paper placed
beneath the cages in which the mice were kept. After
removing faeces and woodshavings from the paper,
food spillage was collected and weighed.
Statistical analysis
All data are shown as mean s.e. for groups based on
six mice in each group. The effect of leptin dose,
differences in body weights and cumulative energy
intake over time between HFF and LFF mice were
analysed using a two-way analysis of variance
(ANOVA). Analyses were performed using the JMP
statistical package (SAS institute Inc., NC, USA,
High fat diet-induced obese mice
S Lin et al
1997). Differences in variables between HFF mice
and LFF mice were analysed using Student's t-test.
Results
Experiment 1: measurements of body weight, calorie
intake, fat storage and plasma leptin in mice fed a
high- or low-fat diet for 1, 8, 15 or 19 weeks
A signi®cant increase in body weight gain in mice fed
the high-fat diet was evident after only 2 weeks of
high-fat feeding, and this trend continued throughout
the dietary protocol (Figure 1A). After 8, 15 and 19
weeks feeding, body weight gain of the HFF group
was 11.4%, 23.1% and 30.5%, respectively, higher
than that of the LFF group (all P < 0.0001).
In conjunction with the changes in body weight, the
increases in weight of the epididymal and perirenal fat
masses of the HFF group were signi®cantly greater
than those of the LFF controls after 8, 15 and 19
weeks of feeding (Figure 2A, B). The ®nal gains in
epididymal and perirenal fat masses of the HFF group
were 144% and 130%, respectively. The gain in
inguinal fat mass of the HFF group was not signi®cantly greater than that of the LFF controls at 1 and 8
weeks feeding, and was 33.6% greater in the HFF
group at 19 weeks (Figure 2C). There was no signi®cant difference in tibia bone length between the
two groups (Figure 2E).
In the HFF group, energy intake paralleled that of
the LFF controls for 4 weeks. However, at this point,
the energy intake of HFF mice began to decrease, and
by the ®fth week of feeding, it had fallen signi®cantly
below that of the LFF mice. From 8 weeks, however,
the energy intake of the HFF group began a gradual
increase; then, at approximately 15 weeks, it increased
dramatically, surpassing that of the LFF mice, and
after 19 weeks of feeding, was 14.6% (P < 0.0001)
greater than the energy intake of the LFF controls
(Figure 1B). As it had been noted that there was a
decreased energy intake in HFF group between week
5 and 8, the HFF mice still gained signi®cantly more
epididymal ( ‡ 54%) and perirenal ( ‡ 123%) fats
compared with the LFF mice at 8 week.
Plasma leptin concentration were signi®cantly elevated in the HFF mice compared with LFF mice when
measured at 8, 15 and 19 weeks of feeding (Figure
2D). Compared with the LFF mice, plasma leptin
concentrations were increased 5-fold (P < 0.0001)
after 19 weeks in the HFF mice.
641
Experiment 2.1: peripheral effects of exogenous leptin
in mice after feeding a high-fat diet for 1 week
2 mg=g body weight i.p. leptin. After an i.p. injection
of leptin, HFF mice had a signi®cant 9.3%, 9.1%, and
7.8% reduction in body weight at 24, 48 and 72 h,
compared with saline treated controls. Similarly, after
an i.p. injection of leptin, LFF mice had a signi®cant
10.2%, 10.4%, and 9.7% reduction in body weight at
24, 48 and 72 h, compared with saline treatment
controls (Figure 3A). Cumulative energy intake after
72 h leptin treatment was reduced by 14.7%
(P < 0.0001) in HFF mice and 18.4% (P < 0.0001)
in LFF mice, compared with saline treatment controls
(Figure 4A). However, comparing the mice fed a
high-fat diet with the mice fed a low-fat diet, there
were no signi®cant differences in body weight change
at 24 h (F(1,20) ˆ 0.146, P ˆ 0.706), 48 h (F(1,20) ˆ
0.29, P ˆ 0.596) and 72 h (F(1,20) ˆ 0.796, P ˆ 0.383).
No signi®cant differences in cumulative energy intake
at 72 h (F(1,20) ˆ 4.157, P ˆ 0.055), were found after
i.p. injection of leptin, that is, there was no signi®cant
interaction between diet and injected treatment for
body weight change at 24, 48 and 72 h, nor for
cumulative energy intake at 72 h.
Experiment 2.2: peripheral and central effects of
exogenous leptin in mice after feeding a high-fat diet for
8 weeks
Figure 1 (A) Body weight in mice fed either a high-fat or low-fat
diet for 19 weeks; (B) Energy intake in mice fed either a high-fat
or low-fat diet for 19 weeks. results are mean s.e., *P < 0.05;
comparisons between the mice fed a high and low-fat diet, n ˆ 6
per group.
(A) 2 mg=g body weight i.p. leptin. After an i.p.
injection of leptin until 72 h, HFF mice did not
show leptin effects on body weight (P ˆ 0.9772) and
cumulative energy intake (P ˆ 0.7842; Figures, 3B
and 4B), compared with saline-treated controls. However, after an i.p. injection of leptin, compared with
International Journal of Obesity
High fat diet-induced obese mice
S Lin et al
642
Figure 2 Amount of fat storage in mice fed either a high-fat or low-fat diet for 1, 8, 15 and 19 weeks. (A) Epididymal fat mass; (B)
perirenal fat mass; (C) inguinal fat mass; (D) plasma leptin concentration in mice fed a high-fat or low-fat diet for 1, 8, 15 and 19 weeks;
(E) the length of tibia in mice fed either a high-fat or low-fat diet for 1, 8, 15 and 19 weeks. Results are mean s.e. *P < 0.05;
comparisons between the mice fed a high and low-fat diet, n ˆ 6 per group.
saline treated controls, LFF mice showed signi®cant
4.5% (P < 0.0001), 3.5% (P ˆ 0.012) and 0.6%
(P ˆ 0.045) reduction of body weight at 24, 48 and
72 h, respectively (Figure 3B). Cumulative energy
intake after 72 h leptin treatment was also reduced
by 7.0% (P < 0.0001; Figure 4B).
(B) 0.1 mg i.c.v. leptin. After an i.c.v. injection of
0.1 mg leptin, HFF mice had a signi®cant 3.1%, 0.9%
International Journal of Obesity
and 1.1% reduction in body weight at 24, 48 and 72 h,
compared with saline treated controls (Figure 3C).
Similarly, after an i.c.v. injection of 0.1 mg leptin, LFF
mice had 4.1%, 1.3% and 0.9% reduction in body
weight at 24, 48 and 72 h, compared with salinetreated controls (Figure 3C). Cumulative energy
intake after 72 h leptin treatment was reduced by
17.7% (P ˆ 0.0003) in HFF mice and 18.3%
(P < 0.0001) in LFF mice, compared with salinetreated controls (Figure 4C). However, comparing
High fat diet-induced obese mice
S Lin et al
the mice fed a high-fat with the mice fed a low-fat
diet, no signi®cant differences in body weight change
at 24 h (F(1,20) ˆ 1.264, P ˆ 0.2743), 48 h (F(1,20) ˆ
0.014, P ˆ 0.905) and 72 h (F(1,20) ˆ 0.922, P ˆ 0.348),
and no signi®cant differences in cumulative energy
intake at 72 h (F(1,20) ˆ 0.848, P ˆ 0.368), were found
after i.c.v. injection of leptin.
Experiment 2.3: central effects of exogenous leptin in
mice after feeding a high fat diet for 19 weeks
643
(A) 0.1 mg i.c.v. leptin. This study showed that 0.1 mg
i.c.v. leptin had no signi®cant effects on body weight
and cumulative energy intake in the HFF mice at 24,
48 and 72 h, compared with saline-treated controls
Figure 3 Peripheral and central effects of exogenous leptin on the body weight change of mice fed either a high-fat or low-fat diet for
1, 8, 19 weeks. Although body weight changes were measured at every 6 h, the data is presented at three time points (24, 48 and 72 h).
(A) Effects of intraperitoneal injection (i.p.) of leptin (2 mg=g body weight) in mice, fed a high-fat or low-fat diet for 1 week, on body
weight change at 24, 48 and 72 h. (B) Effects of i.p. injection of leptin (2 mg=g body weight) in mice, fed a high-fat or low-fat diet for 8
weeks, on body weight change at 24, 48 and 72 h. (C) Effects of intracerebroventricular injection (i.c.v.) of leptin (0.1 mg) in mice, fed a
high-fat or low-fat diet for 8 weeks, on body weight change at 24, 48 and 72 h. (D) Effects of i.c.v. injection of leptin (0.1 mg) in mice, fed a
high-fat or low-fat diet for 19 weeks, on body weight change at 24, 48 and 72 h. (E) Effects of i.c.v. injection of leptin (2.0 mg) in mice, fed
a high-fat or low-fat diet for 19 weeks, on body weight change at 24, 48 and 72 h. Results are mean s.e., *P < 0.05, comparing the
effects between the injections of leptin and saline within the same dietary group. HFFL, the mice fed a high-fat diet following leptin
injection; HFFS, the mice fed a high-fat diet following saline injection; LFFL, the mice fed a low-fat following leptin injection; LFFS, the
mice fed a low-fat diet following saline injection; n ˆ 6 per group.
International Journal of Obesity
High fat diet-induced obese mice
S Lin et al
644
(Figure 3D). However, LFF mice, after i.c.v. injection
of 0.1 mg leptin, showed a signi®cant reduction in
body weight of 2.2% (P < 0.0001) and 1.0%
(P ˆ 0.005) at 24 and 48 h, respectively, compared
with saline-treated controls (Figure 3D). Cumulative
energy intake after 72 h leptin treatment was reduced
by 7.0% (P ˆ 0.0013) compared with saline treatment
controls (Figure 4D).
(B) 2 mg i.c.v. leptin. HFF mice, after an i.c.v.
injection of 2 mg leptin, had a signi®cant reduction
Figure 4 Peripheral and central effects of exogenous leptin on the cumulative energy intake of mice fed either a high-fat or low-fat diet
for 1, 8 and 19 weeks. (A) Effects of intraperitoneal injection (i.p.) of leptin (2 mg=g body weight) in mice, fed a high-fat or low-fat diet for
1 week, on cumulative energy intake at 6, 12, 18, 24, 48 and 72 h. (B) Effects of i.p. injection of leptin (2 mg=g body weight) in mice, fed a
high-fat or low-fat diet for 8 weeks, on cumulative energy intake at 6, 12, 18, 24, 48 and 72 h. (C) Effects of intracerebroventricular
injection (i.c.v.) of leptin (0.1 mg) in mice, fed a high-fat or low-fat diet for 8 weeks, on cumulative energy intake at 6, 12, 18, 24, 48 and
72 h. (D) Effects of i.c.v. injection of leptin (0.1 mg) in mice, fed a high-fat or low-fat diet for 19 weeks, on cumulative energy intake at 6,
12, 18, 24, 48 and 72 h. (E) Effects of i.c.v. injection of leptin (2.0 mg) in mice, fed a high-fat or low-fat diet for 19 weeks, on cumulative
energy intake at 6, 12, 18, 24, 48 and 72 h. Results are mean s.e. *P < 0.05, comparing the effects between the injections of leptin and
saline within the same dietary group. HFFL, the mice fed a high-fat following leptin injection; HFFS, the mice fed a high-fat diet
following saline injection; LFFL, the mice fed a low-fat diet following leptin injection; LFFS, the mice fed a low-fat diet following saline
injection; n ˆ 6 per group.
International Journal of Obesity
High fat diet-induced obese mice
S Lin et al
in body weight for 2.5% (P < 0.0001) and 0.7%
(P ˆ 0.007) at 24 and 48 h, respectively, compared
with saline-treated controls (Figure 3E). LFF mice,
after an i.c.v. injection of 2 mg leptin, had 5.4%
(P < 0.0001), 2.7% (P < 0.0001) and 1.1%
(P < 0.0001) reduction in body weight at 24, 48 and
72 h, compared with saline-treated controls (Figure
3E). Compared with saline-treated controls, cumulative energy intake after 72 h leptin treatment was
reduced by 6.7% (P ˆ 0.0019) in HFF mice and
16.6% (P < 0.0001) in LFF mice (Figure 4E). Compared with the LFF mice, the HFF mice, after an i.c.v.
injection of 2 mg leptin, had a signi®cant 2.9%
(F(1,20) ˆ 32.47, P < 0.0001), 2.0% (F(1,20) ˆ 55.35,
P < 0.0001) and 1.0% (F(1,20) ˆ 52.99 P < 0.0001)
lower reduction in body weight at 24, 48 and 72 h,
respectively. After an i.c.v. injection of 2.0 mg leptin,
HFF mice had a signi®cant 9.9% (F(1,20) ˆ 15.11,
P ˆ 0.0009) lower cumulative energy intake than
that of LFF mice at 72 h.
Discussion
The results presented here show that mice exposed to
a high-fat diet for 19 weeks develop obesity and,
progressively, peripheral and then central leptin insensitivity.
The early stage of high fat diet-induced obesity in
mice is characterized by relatively normal food intake
but elevated body weight and fat gain. As shown
previously, in a direct comparison between a high
carbohydrate and a high-fat diet fed isoenergetically, a
high-fat diet appeared to be handled more ef®ciently,
resulting in a greater increase in adipose mass in
rats.21,22 The present study shows, however, that the
mice fed a high-fat diet for 1 week had similar
response in body weight change and cumulative
energy intake following i.p. leptin injection, indicating that no peripheral leptin resistance had occurred.
The middle phase is perhaps the most fascinating.
Here there seems to be an active attempt, possibly
driven by increased plasma leptin concentration, to
control the rate of excess fat gain by signi®cantly
reducing food intake. However, despite the hypophagia, excess fat still accumulates with apparent gains in
energetic ef®ciency at least partially thwarting this
regulatory endeavour. Peripheral, but not central,
leptin insensitivity is evident by 8 weeks of HFF.
This is consistent with other work showing that
central leptin sensitivity was still intact after 10
weeks of HFF diet.18 The hypophagia could then be
driven via activation of hypothalamic leptin receptors.
By some 4 months of high-fat feeding, the energy
intake of the HFF mice increased dramatically, and by
19 weeks their energy intake was 14.6% greater than
that of LFF controls. Furthermore, the inhibitory
effects of central leptin administration on energy
intake and body weight were attenuated in HFF
mice but not in LFF mice; a 0.1 mg injection of
leptin had virtually no effects on the energy intake
and body weight change, and there was signi®cantly
less of an effect from a 2 mg injection. Several theories
have been proposed to account for the apparent leptin
insensitivity. Although the mechanism causing central
leptin insensitivity is not fully understood, some
suggest that the presence of high levels of leptin
may induce desensitization of the hypothalamic
leptin receptor.19,23 ± 25 Alternatively, it has been suggested that the reduction in leptin action may be due
to down-regulation of leptin receptor density or even
saturation of the receptors with endogenous leptin as
a result of the elevated leptin output.19 In addition,
another explanation may be that a high-fat diet affects
hypothalamic neural networks downstream from the
leptin receptor, such as the a-melanocyte-stimulating hormone (a-MSH) pathway. We have recently
observed a reduction of the arcuate hypothalamic aMSH immunoreactivity in long-term (19 weeks) high
fat diet-induced obese mice (unpublished observations). Evidence is accumulating to suggest that this
pathway is involved in mediating leptin effects on
energy intake and body weight.26
645
Conclusions
This study shows that a high fat diet-induced obesity
in C57 B1=6J mice is a progressive course of development and can be divided into three stages over 19
weeks feeding, distinguished in body weight gain,
energy intake, fat storage, and peripheral and central
leptin sensitivity.
Acknowledgements
We wish to thank Dr Ken Russell (Department of
Applied Statistics, University of Wollongong) for
suggestions regarding the statistical analysis. This
study was supported by grants from the National
Health and Medical Research Council of Australia.
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