29 / 48
SA JOURNAL OF DIABETES & VASCULAR DISEASE
RESEARCH ARTICLE
VOLUME 14 NUMBER 2 • DECEMBER 2017
67
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
and Health 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 dietinduced 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
μM), 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 μM or 10 μM (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, a
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 μl. 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-[3H] glucose (2DG) (1.5 μCi/ ml; final
concentration 1.8 μM) (Perkin Elmer, Boston, USA).
Glucose uptake was allowed to progress for 30 minutes before
stopping the reaction by adding phloretin (final concentration 400
μM). Thereafter, the cells were centrifuged at 1 000 g for one
minute and the supernatant containing radiolabelled 2DG was
aspirated. The subsequent pellet was washed twice with medium
buffer without substrate and then dissolved in 0.5 M NaOH; 50
μl of this solution was used for the determination of the protein
content by the method of Lowry
et al
.,
36
while the rest was counted
for radioactivity using a scintillation counter (Beckman).
TheWesternblottechniquewasperformedaspreviouslyreported,