The impact of activation of the renin-angiotensin system in the development of insulin resistance in experimental models of obesity
Date
2009-03
Authors
Perel, Shireen J. C.
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : University of Stellenbosch
Abstract
Insulin stimulates the production of nitric oxide (NO) in endothelial cells and cardiac
myocytes by a signalling pathway that involves the insulin receptor substrate (IRS)-1,
phosphatidylinositol-3-kinase and protein kinase B (PKB/Akt). Physiological
concentrations of NO play an important part in maintaining normal vascular function.
It has been suggested that nitric oxide synthase (NOS) activity and NO production
are chronically impaired in diabetes mellitus by an unknown mechanism. The reninangiotensin
system and subsequent production of angiotensin II (Ang II) are elevated
in obesity and diabetes while antagonism of the AT1 receptor with Losartan has
beneficial effects in patients with insulin resistance and type II diabetes. Aims: We
therefore aimed to investigate (i) the effect of Ang II on myocardial insulin signalling
with regards to key proteins (IRS-1, PKB/Akt, eNOS and p38 MAPK) in correlation
with NO production, (ii) the effect of Losartan on these parameters. Methods:
Hyperphagia-induced obese, insulin resistant rats (DIO=diet supplemented with
sucrose and condensed milk) were compared to age-matched controls. Half the
animals were treated with 10mg/kg Losartan per day for 1 week. Isolated hearts were
perfused with or without 0.03 μIU/mL insulin for 15 min. Blood glucose, bodyweight,
intraperitoneal fat and plasma insulin and Ang II were recorded. Proteins of interest
and their phosphorylation were determined by Western blotting. NO production was
flow cytometrically analyzed. ANOVA followed by the Bonferroni correction was used
with a p< 0.05 considered significant. Results: DIO animals had significant elevated
bodyweight, blood glucose, plasma insulin and Ang II levels. Our data showed that
the hearts from the DIO animals are insulin resistant, ultimately reflected by the
attenuated activation of the key proteins (IRS-1, PKB/Akt and eNOS) involved in
insulin signalling as well as NO production. AT1 receptor antagonism improved NO
production in isolated adult ventricular myocytes from DIO animals while concurrently
enhancing expression of eNOS, PKB/Akt and p38 MAPK. In contrast, NO production
as well as expression of eNOS and PKB/Akt was attenuated in control animals after
Losartan treatment. Conclusion: These results suggested that Ang II via AT1 or
AT2 receptors, modulates protein expression of both PKB/Akt and eNOS. This
encouraged us to investigate the involvement of AT2 receptors in the observed
changes.
To investigate this we needed to establish a culture of neonatal rat cardiac myocytes
treated with raised fatty acids and Ang II. If similar changes were induced as
observed in the hearts of DIO animals, the involvement of the AT1 and AT2 receptors
could be investigated using specific antagonists against these receptors. Primary
cultured ventricular myocytes were isolated from 1-3 day old Wistar rat pups. They
were cultured for 48 hours before the addition of palmitate and oleate at a
concentration of 0.25 mM each and were treated with or without the fatty acids for a
period of 4 days. After 18 hours of serum starvation, cells were stimulated with or
without 10 nM insulin for 15 minutes. The effect of fatty acid treatment on cell viability
and glucose uptake were assessed by trypan blue and propidium iodide staining and
2-deoxy-D-3[H] glucose uptake respectively. Protein levels and phosphorylation of
key proteins (PKB/Akt, PTEN and p38 MAPK) in insulin signalling was determined by
Western blotting. 0.25 mM Fatty acids did not result in the loss of cell viability.
Contrary to expectation, fatty acid treatment led to enhanced basal glucose uptake
but lower Glut 1 protein expression. Basal protein expression of PPARα was,
however, upregulated as was the expression of the phosphatase, PTEN. The latter
could explain the lower PKB/Akt phosphorylation also documented.
From these results we conclude that neonatal cardiac myocytes, cultured in the
presence of elevated fatty acids, did not respond in a similar manner as the intact
hearts of our animals and further modifications of the system might be needed before
it can be utilized as initially planned.
Description
Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2009.
Keywords
Insulin resistance -- Nitric oxide, Theses -- Medicine, Dissertations -- Medicine