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Acute effects of insulin on the endothelium-dependent dilation of rat cremaster muscle arteries

G. Ivanov, S. Senadheera, S.L. Sandow and T.V. Murphy, Department of Physiology, School of Medical Sciences, University of New South Wales, NSW 2052, Australia.

Introduction: Obesity and type II diabetes are hyperinsulinemic conditions that increase the risk of cardiovascular diseases such as atherosclerosis and hypertension.  Endothelial dysfunction characterizes these states. However, the underlying mechanisms for cardiovascular and endothelial dysfunction are not well understood. Recent studies have suggested hyperinsulinemia and insulin resistance alter vascular endothelial cell function, possibly contributing to the vascular complications of obesity. (De Boer et al., 2012). Recent observations in our laboratory (Howitt et al., 2012) demonstrated that dietary obesity increased endothelial nitric oxide synthase (NOS) activity, while decreasing the activity of the large-conductance, Ca2+-dependent K+ channels (BKCa) in the rat cremaster muscle artery.  It was observed that obesity caused an increase in plasma insulin in this model; therefore, it is possible that the obesity-induced changes in the cremaster muscle artery function could have been produced by insulin.

Hypothesis:  Insulin mediates the effects of diet-induced obesity on endothelium-dependent vasodilation in the rat cremaster muscle artery.

Method:  Six-week-old male Sprague-Dawley rats were anesthetized (Sodium pentathol, 100 mg/kg, i.p.) and the cremaster muscles removed.  First-order arteries were dissected free.  In pressure myography studies, arteries were cannulated and maintained at 70mmHg/34°C.  Internal diameter was measured using video microscopy.  Vessels were incubated with or without insulin (100 nM) on the intra-luminal side for 2 h.  For biochemical assays, including immunohistochemistry, whole vessels were incubated with or without insulin (100 nM) for 2 h at 34°C, then snap-frozen in liquid nitrogen.

Results: In isolated, pressurized cremaster muscle arteries with myogenic tone (diameter 52.5 ± 2.9% of maximum, n=14), the endothelium-dependent vasodilator acetylcholine (ACh) caused a concentration-dependent dilation of arteries (98.1 ± 5.1%, n=8).  This dilation was significantly reduced by the combination of the NOS and guanylate cyclase inhibitors L-NAME (100 μM) and ODQ (10 μM) respectively (86.4 ± 2.8%) and further addition of the BKCa blocker TEA (1 mM; 69.9 ± 5.5%).  Pre-incubation of the vessels with insulin (100 nM) did not alter ACh-induced vasodilation, but greatly reduced the ability of L-NAME, ODQ and TEA to inhibit the dilation (86.9 ± 4.9%, n=5). Further addition of the intermediate-conductance KCa (IKCa) inhibitor TRAM-34 (1 μM) blocked ACh-induced vasodilation of the insulin treated vessels.  The effects of insulin were unaltered by PD 98,059 (10 μM), a MAP-kinase inhibitor.  Insulin also inhibited vasodilation caused by the BKCa activator NS1619 and the NO donor sodium nitroprusside. Immunohistochemistry studies showed IKCa and low levels of BKCa-α subunit were present in the smooth muscle of control and insulin-treated cremaster muscle arterioles, with IKCa also being present at a comparatively low level in the endothelium.

Conclusion: Acute 2 h treatment with insulin reduced the contribution of NO and BKCa to endothelium-dependent dilation of the rat cremaster muscle artery, but increased the role of IKCa.  These effects of insulin on BKCa are similar to those that were observed in diet-induced obesity (Howitt et al., 2012)

De Boer MP, Meijer RI, Wijnstok NJ, Jonk AM, Houben AJ, Stehouwer CD, Smulders YM, Eringa EC, Serné EH. (2012) Microcirculation 19: 5-18.

Howitt L, Grayson TH, Morris MJ, Sandow SL, Murphy TV. (2012) American Journal of Physiology Heart and Circulatory Physiology 302: H2464-76.