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RESEARCH ARTICLE
SA JOURNAL OF DIABETES & VASCULAR DISEASE
62
VOLUME 13 NUMBER 2 • DECEMBER 2016
the Committee for the use of animals in research of the University
of Stellenbosch – numbers P05/11/013 and P07/11/020.
The
P glandulosa
plant material was originally obtained from
naturally growing plants. The material was handled according to a
patented and standardised procedure
8
and pre-packed in capsules
for human consumption, which we emptied and weighed. The
voucher specimen was reported previously.
8
Plasma glucose levels were determined in the fasting state. Blood
was obtained via a tail prick and glucose levels were determined
using a conventional glucometer (Cipla MedPro). Plasma was stored
at –80°C in a Snijders Scientific Ultracool (Tilburg, the Netherlands)
and insulin levels were determined using a coat-a-count assay
(Diagnostic Products).
Intra-peritoneal glucose tolerance curves (IPGTTs) were
generated in the animals after an 18-hour fast. Animals were
injected intra-peritoneally with 1 g/kg of a 50% sucrose solution
and blood glucose levels were monitored over a 120-min period.
After removal, the hearts were arrested in ice-cold Krebs Henseleit
(KH) medium (in mM: NaCl 119, NaHCO
3
25, KCl 4.75, KH
2
PO
4
1.2, MgSO
4
.7H
2
O 0.6, Na
2
SO
4
0.6, CaCl
2
.2H
2
O 1.25, glucose 10)
and immediately (within 30 sec) mounted onto the aortic cannula
of a perfusion rig. The pulmonary vein was connected to a second
cannula in order to perform perfusions in the working-heart mode
with a preload of 15 cm H
2
O and an afterload of 100 cm H
2
O, as
described previously.15 The perfusion medium was continuously
gassed with 95%O
2
/5%CO
2
. Hearts were fitted with a temperature
probe and the temperature was kept constant at 36.5–37°C.
After a stabilisation period of 30 min, rat hearts were subjected
to 35-min regional ischaemia by coronary artery ligation, followed
by reperfusion for one hour, as described previously.
15
Infarct size
was determined according to a well-established protocol,
15
followed
by planimetry, and expressed as a percentage of the area at risk.
Planimetry was performed blind by a third party.
Mouse hearts were perfused retrogradely, meaning via the
aorta without a connection to the pulmonary vein. After the
30-min stabilisation period, the hearts were subjected to 20-min
normothermic ischaemic cardiac arrest (NICA) by stopping all
perfusion. This was followed by one hour of reperfusion, after
which the infarct development through the whole heart was
determined as described above.
To measure blood pressure, rats were placed in restraining
holders with a dark nose cone to calm them. The restrainers were
placed on a heating pad (32 ± 2°C) to warm the rat and maintain
blood flow to the tail. Animals were placed in the restrainers for
at least five minutes before monitoring the blood pressure using
a computerised tail-cuff blood pressure monitor (Kent Scientific
Corporation, Connecticut, USA). Prior to commencement of the
experiment, rats were subjected to the above procedure daily for
at least a week to train the animals for the procedure and to avoid
stress in the rats during experimental determinations.
Animals were placed individually in metabolic cages and the
volume of urine was determined over a period of 24 hours.
Western blotting
Frozen tissues were pulverised with a liquid nitrogen pre-cooled
mortar and pestle and then extracted in lysis buffer containing
in mM: Tris-HCl 20 (pH 7.5), EGTA 1, EDTA 1, NaCl 150, Na
2
VO
3
1, beta-glycerophosphate 1, sodium-pyrophosphate 2.5, PMSF
0.3, Triton X-100 1% (v/v) plus 10 μg/ml leupeptin and aprotinin,
respectively, using a Polytron PT10 homogeniser, 2 × 4 sec, at
anti-infective and anti-parasitic compounds have also been isolated
from this plant.
9
In view of the hypoglycaemic effects of
P glandulosa
, as well as
its ability to partially restore the function of pancreatic tissue and
increase cardiomyocyte insulin sensitivity,
7
we set out to determine
the cardiovascular effects of treatment, using a wellcharacterised
rat model of obesity and pre-diabetes with known cardiovascular
insufficiency and endothelial dysfunction.
10,11
In addition, using a
rat model of high-fat feeding known to develop hypertension,
12
we determined whether
P glandulosa
had any effects on the
development of high blood pressure.
Models
Diet-induced obesity (DIO)
As described previously,
10
Wistar rats (180–200 g) were randomly
divided into a control and diet group. The DIO group was fed
a diet of normal rat chow supplemented with sucrose and
condensed milk for a basic period of eight weeks. From weeks
eight to 16 the rats were treated with
P glandulosa
(100 mg/kg/
day) set in jelly/gelatine blocks and given to each one individually
according to the weight of the animal.
8
This was done to ensure
absolute compliance and dose control. The dose of
P glandulosa
was calculated as previously described.
8
The diet to induce pre-diabetes in the animals was
based on hyperphagia.
13
Animals were anaesthetised with
sodium pentobarbital (160 mg/kg, intra-peritoneally) before
experimentation. At the time of sacrifice, their body weight and
the weight of the intra-peritoneal fat were noted and trunk blood
was collected for biochemical analyses. For Western blot analyses,
the hearts were removed, immediately snap-frozen in
liquid nitrogen and stored at –80°C.
High-fat diet (HFD)
To induce high blood pressure, the rats were fed a diet containing
the following per kg of food: cooking fat 400 g, fructose 100 g,
casein 100 g, cholesterol 10 g, and rat chow pellets 390 g. Blood
pressure was monitored on a weekly basis over 16 weeks. Treatment
with
P glandulosa
(100 mg/kg/day) given in jelly blocks was either
started at the onset of the diet to study the effect on prevention of
the development of hypertension, or after a period of 12 weeks of
the HF diet to study its anti-hypertensive effects. Rats treated with
captopril (50 mg/kg/day) from the onset of the diet were included
as a positive control. All animals were also placed individually in
metabolic cages in order to collect urine samples.
CIRKO mice
A mouse model of animals with a cardiac conditional ablation of
the insulin receptor was used in conjunction with their C57Bl6
littermates.
14
Mice were fed normal chow and treated with
P
glandulosa
at a similar dose to that of the rats for a period of eight
weeks before experimentation.
Methods
Animals had free access to food and water and were kept on a
12-hour day/night cycle in the Central Research Facility of the
Faculty of Health Sciences of the University of Stellenbosch. The
study conformed to the revised South African National Standard for
the Care and Use of Animals for Scientific Purposes (South African
Bureau of Standards, SANS 10386, 2008) and was registered with