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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 28, No 6, November/December 2017
AFRICA
363
short-term melatonin consumption also reduced systemic insulin
resistance and conferred cardioprotection.
33
However, whether
melatonin treatment affects myocardial insulin sensitivity and
glucose uptake remains unknown.
The aim of this study was therefore to investigate the effect
of melatonin treatment on myocardial glucose uptake using
cardiomyocytes isolated from insulin-resistant rats and their
aged-matched controls. To investigate whether melatonin has
a direct effect on myocardial glucose uptake, melatonin was
administered
in vitro
to isolated cardiomyocytes and
in vivo
for
the measurement of glucose uptake. To evaluate the effect of
ageing, cardiomyocytes isolated from normal control rats (seven
to eight weeks old) were also included.
Methods
Sixty male Wistar rats were obtained from the University of
Stellenbosch Central Research Facility. They were housed with
free access to water and food and a 12-hour dark/light cycle
(light from 06:00 to 18:00) with temperature and humidity kept
constant at 22ºC and 40%, respectively.
The experimental procedure was assessed and approved by
the Committee for Ethical Animal Research of the Faculty of
Medicine andHealth Sciences, University of Stellenbosch (ethical
clearance no P08/05/008). Animals were treated according 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 1985) and the revised
South African National
Standard for the Care and Use of Animals for Scientific Purposes
(South African Bureau of Standards, SANS 10386, 2008).
For evaluation of insulin responsiveness and sensitivity,
cardiomyocytes were isolated from (1) normal rats (225−250 g)
(
n
=
12) or (2) diet-induced obese rats (group D) (
n
=
24) and
their age-matched controls (group C) (
n
=
24) fed a high-calorie
diet and standard rat chow, respectively. The high-calorie diet
consisted of 65% carbohydrates, 19% protein and 16% fat, while
the standard rat chow consisted of 60% carbohydrate, 30%
protein and 10% fat.
32
The diet-induced obese and age-matched
control rats were seven to eight weeks old at the onset of the
experimental programme, which was continued for a period of
16 to 23 weeks. To evaluate the progressive changes in insulin
sensitivity, the feeding regime of our existing model of diet-
induced obesity and insulin resistance
32
was varied from 16
to 23 weeks to exacerbate the effects of obesity, as previously
reported.
33
To determine whether short-term melatonin administration
in vitro
had a direct effect on myocardial glucose uptake,
melatonin was administered to the cardiomyocytes after isolation
(see below for cardiomyocyte preparation). Briefly, isolated
cardiomyocytes were incubated with phloretin (glucose-uptake
inhibitor, 400
μ
M), and melatonin (100 nM) with or without
insulin (1–100 nM). Fresh melatonin (Sigma-Aldrich, St Louis,
MO, USA) solution was used; melatonin was dissolved in a small
quantity of ethanol and then in medium buffer to yield a final
concentration of 1 nM, 10 nM, 100 nM, 1
μ
M or 10
μ
M (with
<
0.005% ethanol). Ethanol at that concentration had no effect
on glucose uptake by the cardiomyocytes (results not shown).
Phloretin (Sigma-Aldrich, St Louis, MO, USA) was dissolved
in dimethyl sulfoxide (DMSO), stored at −80°C as stock, and
diluted with medium buffer immediately before use.
To evaluate the effect of
in vivo
melatonin treatment on
myocardial glucose uptake, only rats fed for 20 weeks were
used. While studying the effect of
in vitro
melatonin treatment,
we observed that compared to their age-matched control rats,
only cardiomyocytes isolated from obese rats fed for more than
20 weeks showed a significant decrease in insulin-stimulated
glucose uptake (Fig. 3). Four groups were studied including: (1)
untreated control (C), (2) treated control (CM), (3) untreated
diet (D), and (4) treated diet (DM).
Melatonin was orally administered in the drinking water (4 mg/
kg/day) for six weeks starting from the 14th week of feeding, as
described previously.
32,33
This is the lowest concentration to have a
significant effect in our model of diet-induced obesity.
33
Drinking
water with or without melatonin was replaced every day one hour
before lights off (18:00) and was available throughout the light
and dark cycles.
33
In contrast to humans, rats are active during
the night, when their blood melatonin levels are high. A period of
six weeks has been shown as the shortest to elicit marked effects
of melatonin on the hearts from diet-induced obese rats and to
reverse several of the harmful effects of obesity.
33
Animals were anaesthetised with sodium pentobarbitone
(160 mg/kg, intraperitoneally). The hearts were immediately
removed and perfused for isolation of cardiomyocytes, as
described previously.
34
The body weight and visceral fat mass
were recorded. Adiposity index was calculated as the ratio of
visceral fat mass to body weight, multiplied by 100.
33
Blood glucose levels were determined in the fasting state, as
described previously,
35
at the same time (10:00–12:00). Blood
was obtained via a tail prick and levels were determined using
a conventional glucometer (Cipla MedPro, Bellville, South
Africa). Intraperitoneal glucose tolerance (IPGT) curves were
generated in animals after an overnight fasting period. Animals
were injected with 1 g/kg of a 50% sucrose solution and blood
glucose levels were recorded over a two-hour period.
Calcium-tolerant adult ventricular myocytes were isolated
from the different animal groups, as previously reported.
34
After
isolation, the myocytes were suspended in a medium buffer
containing (in mM): HEPES 10, KCL 6, NaH
2
PO
4
0.2, Na
2
HPO
4
1, MgSO
4
1.4, NaCl 128, pyruvate 2, glucose 5.5, and 2% BSA
(fraction V, fatty acid free) plus calcium 1.25 mM, at pH 7.4. The
cells were left for one to two hours under an oxygen atmosphere
on a gently shaking platform to recover from the trauma of
isolation. After recovery, the cells were allowed to settle into a
loose pellet and the supernatant was removed. This procedure
routinely rendered in excess of 80% viable cells, as measured by
trypan blue exclusion. They were additionally washed twice with
and suspended in a suitable volume of the above medium buffer
but without glucose and pyruvate for subsequent glucose uptake
determinations.
Cardiomyocyte glucose uptake was measured essentially as
described previously
34
in a final assay volume of 750
μ
l. Cells
prepared from the different groups of animals were incubated
with or without one, 10 or 100 nM insulin for 30 minutes. After
a total incubation period of 45 minutes, glucose uptake was
initiated by addition of 2-deoxy-D-[
3
H] glucose (2DG) (1.5
μ
Ci/
ml; final concentration 1.8
μ
M) (Perkin Elmer, Boston, USA).
Glucose uptake was allowed to progress for 30 minutes before
stopping the reaction by adding phloretin (final concentration
400
μ
M). Thereafter, the cells were centrifuged at 1 000 g for one
minute and the supernatant containing radiolabelled 2DG was