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RESEARCH ARTICLE

SA JOURNAL OF DIABETES & VASCULAR DISEASE

64

VOLUME 13 NUMBER 2 • DECEMBER 2016

reperfusion and culminating in the formation of an infarct has been

ascribed, among others, to the activity of the phosphatidylinositol-

3-kinase (PI-3K) pathway. In view of the previously reported

improvements in insulin sensitivity of cardiomyocytes, induced by

P glandulosa

treatment,

8

we systematically analysed the proteins

involved in this signalling cascade.

As summarised in Table 2 and shown in Fig. 4, hearts from the DIO

animals presented with a significantly lower phosphorylated:total

ratio of the central protein in this cascade, protein kinase B or Akt.

This ratio was significantly improved by treatment. In addition, the

expression of the p85 regulatory subunit of the PI-3K enzyme was

significantly lower in hearts from the DIO animals, whereas this was

not the case after treatment.

Treatment also resulted in a lower expression of the phosphatase

and tensin homologue deleted on chromosome 10 (PTEN)

with a higher state of phosphorylation of this enzyme (Fig. 5).

Phosphorylation of PTEN further inactivates this enzyme, responsible

also for the dephosphorylation of PKB/Akt.

17,18

Fig. 2.

After the 16-week diet plus P glandulosa treatment, isolated hearts from

DIO rats were perfused ex vivo in the working-heart mode. They were subjected

to regional ischaemia as described in Methods. Infarct size was determined as a

percentage of the area at risk of infarction. *

p

< 0.05, **

p

< 0.01,

n

= 15–17

per group.

Fig. 3.

After the eight weeks of treatment, hearts were removed from the CIRKO

mice and perfused ex vivo in the Langendorff mode and subjected to NICA as

described in Methods. Infarct size was determined throughout the whole heart

and expressed as a percentage of the total surface. **

p

< 0.01, ***

p

< 0.001,

n

= 9 per group.

Fig. 4.

Hearts from the treated and untreated DIO animals were removed without

any intervention and stored in liquid nitrogen. Tissue lysates were prepared and

Western blotting was performed as described in Methods.

A

: bar charts of the

expression of PKB protein as well as the ratio of phosphorylated vs total protein.

*

p

< 0.05 vs control; **

p

< 0.01 vs untreated DIO,

n

= 6 individual hearts

analysed per group.

B

is a representative blot depicting these proteins and beta-

tubulin, used as an indicator of equal loading.

Anti-hypertensive effects

As the DIO diet does not cause high blood pressure, we used a

modification of a high-fat diet to induce hypertension in the

animals.

12

As can be seen in Fig. 5, these animals developed a

significant elevation of their blood pressure within four weeks (HFD

135.88 ± 2.0 vs control 125.85 ± 1.9 mmHg,

p

< 0.05,

n

= 8 per

group).

We either pre-treated the animals with

P glandulosa

, starting

at the onset of the diet, or we allowed the animals to become

severely hypertensive (12 weeks) and then started the treatment.

We included a group of animals treated with the angiotensin

converting enzyme (ACE) inhibitor captopril from the onset of the

diet, as a positive control in this study.