Cardiovascular Journal of Africa: Vol 24 No 2 (March 2013)

CARDIOVASCULAR JOURNAL OF AFRICA • Vol 24, No 2, March 2013

AFRICA

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,

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 setting 4. Lysates were cleared from particulate matter by

centrifuging for 15 min at 14 000 rpm in a microfuge (Eppendorf

Mini-spin plus, Hamburg, Germany) and the protein content was

determined by the method of Bradford.

Samples were diluted

in Laemmli sample buffer, boiled for 5 min and stored at –80°C.

Equal amounts of cytosolic proteins were separated on a SDS

poly-acrylamide gel and electro-transferred to Immobilon

-P

PVDF membranes. Transfer and equal loading of proteins was

determined with Ponceau red reversible stain. The membranes

were blocked for two hours in Tris-buffered saline (TBS)

containing 0.1% Tween-20 and 5% non-fat milk powder and

incubated overnight in the primary antibodies (diluted in

TBS–Tween according to the manufacturer’s instructions). The

following antibodies from cell signalling were used: insulin

receptor beta-subunit, phospho-PI3K P85 (Tyr458), total and

phospho-PTEN (Ser380/Thr382/383), total and phospho-PKB/

Akt (Ser473), Glut 1 and Glut 4.

Blots were stripped using a 5-min incubation in 2% NaOH

after washing in distilled water and re-probed with a beta-tubulin

antibody to confirm equal loading. Bands were visualised using

the ECL detection system and quantified by laser-scanning

densitometry with suitable software (Silk Scientific Inc, USA).

For comparison purposes, total pixels of bands were expressed as

a ratio of the mean of the controls on the same blot.

Statistical analyses

Data are presented as mean

SEM and were analysed using

either a one-way or two-way ANOVA followed by a Bonferroni

post-hoc

test for differences between groups. The blood pressure

effects were analysed using a repeated-measures two-way

ANOVA. Statistical significance was set at

0.05.

Results

After the 16-week diet animals from model 1 (DIO) presented

with significantly increased body- and intra-peritoneal fat

weight (Table 1). As summarised in Table 1, these animals had

significantly elevated blood glucose and insulin levels, leading to

an increased homeostatic model assessment of insulin resistance

index (HOMA-IR), indicative of whole-body insulin resistance.

In neither control nor DIO animals did the treatment with

glandulosa

have any effect on the body weight or the intra-

peritoneal fat weight of the animals. After treatment of the DIO

animals with

glandulosa

, the blood glucose levels were no

longer significantly elevated compared to the treated controls but

the HOMA-IR was still significantly higher. However, as shown

in Fig. 1, the two-hour blood glucose values after intra-peritoneal

glucose tolerance analyses were significantly lower in the treated

DIO animals, underscoring a slight effect on blood glucose

handling, as previously reported.

Infarct size

After 16 weeks of the obesity-inducing diet, the

vivo

perfused

hearts of the DIO animals presented with significantly larger

infarct sizes, calculated as percentage of the area at risk, than

the hearts from the control animals (DIO 49.48

3.25 vs control

40.62

2.21%,

0.05,

17 per group). The area at risk did

not differ between the groups and averaged 54.13

2.21%.

An eight-week treatment regime with

glandulosa

conjunction with the diet significantly improved the ability of the

hearts to withstand a period of ischaemia, and smaller infarcts

developed. There was no significant effect in the hearts from

control rats (Fig. 2). Two-way ANOVA indicated a significant

effect of the treatment on infarct size (

0.01).

To confirm these results and rule out any effect of insulin

levels on the cardioprotective role of

glandulosa

, we used a

mouse model with a conditional ablation of the insulin receptor

in cardiomyocytes.

Subjecting these animals and their normal

C57Bl6 littermates to

vivo

perfusion and NICA, followed by

reperfusion, we found that the hearts of both control and CIRKO

mice were protected by the

glandulosa

treatment. This was

Fig. 1. DIO or control chow-fed rats for 16 weeks with

P glandulosa

treatment for the last eight weeks were

subjected to intra-peritoneal glucose-tolerance testing

after an 18-hour fast. Blood was collected by tail prick

and analysed over a 120-min period using a commercial

glucometer. Data given are the 120-min values. *

0.05

vs control and DIO plus treatment,

6 per group.

Control

DIO

Clucose concentration (mmol/l)

minus treatment

plus treatment

TABLE 1. BIOMETRIC DATA – MODEL 1: DIO

Control

Control +

P glandulosa

DIO

DIO +

P glandulosa

Weight

433.7

9.3

438.6

9.3

507.7

22.9***

534.3

11.7***

Intra-peritoneal fat

2.7

1.8

28.0

1.74***

1.4***

Blood glucose (mmol/l)

5.42

0.17

5.4

0.18

6.4

0.17*

5.6

0.19

Serum insulin (

U/ml)

17.12

0.8

14.07

1.50

34.33

9.06*

35.93

10.21*

HOMA-IR

4.73

0.71

3.40

0.40

8.96

2.65*

7.88

3.30*

0.05 vs the respective control; ***

0.001 vs the respective control. Analysis by two-way ANOVA,

6 per group.