The role of oxidative stress in streptozotocin-induced early and late stage diabetes mellitus in the rat (Rattus norvegicus, Berkenhout 1769)

Abstract

Vascular disease is the main etiology for death and for a great percent of morbidity in patients with diabetes mellitus. Although pronounced macro- and microvascular endothelial dysfunction is a hallmark of late stage diabetes, the early stage of uncomplicated diabetes is characterized by increased blood flow and reduced peripheral resistance. The underlying mechanisms of these divergent phenomena are still obscure and the aim of this study was to elucidate the role of oxidative stress and its effect on the NO/cGMP pathway in early and late stage diabetes, using the well established model of type 1 diabetes, the streptozotocin (STZ)-induced diabetic rat. The data of this study shows that early diabetes is associated with increased acetylcholine (ACh)-induced, endothelium-dependent vasorelaxation that goes hand in hand with an increased sensitivity of smooth muscle cells (SMC) to the endothelium-independent vasodilator nitroglycerin (NTG). Early diabetes had no effect on the receptor phenylephrine (Phe) mediated contraction nor on the KCl-induced contraction in the absence of the endothelium and moreover, cAMP levels were not affected. Although we observed no change in NADPH oxidase activity, reactive oxygen species (ROS) levels, as assessed with chemiluminescence (CL) and dihydroethidium (DHE) staining as well as the expression of the NAD(P)H oxidase subunit p67phox and HO-1, a marker of oxidative stress, were decreased in early diabetes. Serum NO2- , a marker of •NO bioavailability, was increased in the diabetic animals and this was associated with an increased sensitivity of soluble guanylate cyclase to •NO with a concomitant increase in cGMP levels. Furthermore, our results show that in early diabetes the contribution of •NO to vasorelaxation may not occur via the cGMP/cyclic GMP dependent kinase (cGK-I) pathway (as assessed with vasodilatory stimulated phosphoprotein (VASP) phosphorylation on ser239) and further studies are needed to elucidate the exact mechanism by which •NO elicits its vasodilatory effect in these animals. Late stage diabetes, on the other hand, exhibited a marked decrease in ACh-induced, endothelium-dependent vasorelaxation, which was associated with decreased sensitivity of SMC to the endothelium-independent vasodilator NTG. Although we found no change in Phe mediated contraction of aortic rings, the KCl-induced contraction in the absence of the endothelium was markedly attenuated in late stage diabetes. This may be explained by our observed increase in cAMP in these animals, which was shown to inhibit voltage-dependent calcium channels. ROS levels, as assessed with CL and DHE staining, were increased in the aorta of late stage diabetic animals as was the expression of Cu/ZnSOD, the enzyme responsible for O2•- dismutation. This is associated with an increase in the NAD(P)H oxidase subunit nox1 as well as an increases in the NADPH oxidase activity of aorta. Although eNOS expression was significantly increased in late stage diabetes, most likely due to a compensatory mechanism, the ACh-induced •NO bioavailability was markedly reduced, concomitant with reduced ACh-induced cGK-I activity (as assessed by the phosphorylation state of VASP on ser239), leading to endothelial dysfunction. We can thus conclude that in early diabetes the aorta seems to over-compensate for the oxidative stress that is associated with hyperglycemia by decreasing ROS levels, which in turn leads to a concomitant increase in •NO bioavailability and vasodilation. However, in late stage diabetes, these compensatory mechanisms fail, resulting in hyperglycemia-induced increases in ROS, decreased •NO bioavailability and eventually endothelial dysfunction. This study reconciles the divergent data on endothelial dysfunction found in the literature and emphasizes the need to standardize all procedures when using this model of STZ-induced diabetes mellitus.