SummaryThis review summarises the results and discussions of an UNESCO-MCBN supported symposium on oxidative stress and its role in the onset and progression of diabetes. There is convincing experimental and clinical evidence that the generation of reactive oxygen species (ROI) is increased in both types of diabetes and that the onset of diabetes is closely associated with oxidative stress. Nevertheless there is controversy about which markers of oxidative stress are most reliable and suitable for clinical practice. There are various mechanisms that contribute to the formation of ROI. It is generally accepted that vascular cells and especially the endothelium become one major source of ROI. An important role of oxidative stress for the development of vascular and neurological complications is suggested by experimental and clinical studies. The precise mechanisms by which oxidative stress may accelerate the development of complications in diabetes are only partly known. There is however evidence for a role of protein kinase C, advanced glycation end products (AGE) and activation of transcription factors such as NFkB, but the exact signalling pathways and the interactions with ROI remain a matter of discussion. Additionally, results of very recent studies suggest a role for ROI in the development of insulin resistance. ROI interfere with insulin signalling at various levels and are able to inhibit the translocation of GLUT4 in the plasma membrane. Evidence for a protective effect of antioxidants has been presented in experimental studies, but conclusive evidence from patient studies is missing. Large-scale clinical trials such as the DCCT Study or the UKPDS Study are needed to evaluate the long-term effects of antioxidants in diabetic patients and their potential to reduce the medical and socio-economic burden of diabetes and its complications.
Due to the high permeability of endothelial cell layers derived from macrovascular vessels, precise determination of their barrier function towards ion movement requires refined experimental techniques. We thus cultured bovine aortic endothelial cells (BAEC) directly on thin gold-film electrodes and measured the electrochemical impedance to study their passive electrical properties in general and during beta-adrenergic stimulation. Impedance spectra (10-2.10(6) Hz) of confluent cell monolayers revealed that the electrical characteristics of the cells can be modelled by a simple resistor-capacitor parallel network. Under control conditions the overall resistance of confluent BAEC monolayers was 3.6+/-0.6 Omega.cm2 (n=30) and the capacitance was 0. 6+/-0.1 microF/cm2. Both quantities are discussed with respect to morphological characteristics of these cells. Stimulation of BAECs with the synthetic beta-adrenoceptor agonist isoproterenol leads to a concentration-dependent, highly specific increase of the cell layer resistance characterized by a concentration for half-maximal response (EC50) of 0.3+/-0.1 microM. The cell layer capacitance, however, remained unaffected. Using impedance measurements at a single frequency, we analysed the response of BAECs to treatment with isoproterenol in comparison with several chemically unrelated compounds known to stimulate the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent signal transduction cascade. These studies confirmed that the enhancement of the cell layer resistance after beta-adrenergic stimulation is mediated by an increase in intracellular cAMP.
These observations provide in vivo evidence that the generation of ROS plays an important role in the onset of diabetes and the development of vascular dysfunction in GK rats with type 2 diabetes.
Several lines of evidence suggest that endothelial dysfunction and damage present early steps in the pathophysiology of vascular complications in diabetes mellitus [1±3]. Several studies using cultured endothelial cells clearly show that incubation of these cells with high concentrations of glucose leads to severe changes in the proliferation, the adhesive and synthetic properties [1]. Consequently, regulation of vascular relaxation by endothelium becomes disturbed in diabetes [2,3]. In addition to glucose, high concentrations of proinsulin have been shown to promote the synthesis of the plasminogen activator inhibitor type-1 (PAI-1) [4]. This observation indicates that endothelial function can be directly influenced not only by glucose, but also by proinsulin. Thus, high proinsulin levels may contribute to the development of vascular complications in diabetes, if secreted excessively. In line with this assumption proinsulin is increased in the late pre-insulin-dependent diabetes mellitus [5] and especially in non-insulin-dependent diabetic patients with abnormal prohormone convertase PC2 and PC3 activity [6] or with a point mutation in the Diabetologia (1998)
There is evidence that reactive oxygen intermediates (ROI) play an important role in the pathogenesis of vascular complications in diabetes. On the other hand, metformin, one of the most often used antidiabetic compounds has not only been shown to reduce the risk for vascular complications, but in addition these protective effects are largely independent of its well-known antihyperglycemic action. Therefore, to explain the vasculoprotective effects of metformin, a direct antioxidative action of this compound has been suggested. We show here that human endothelial cells (HUVEC) generate ROI not only in response to high glucose (30 mmol/l glucose), but also in response to palmitic acid, and advanced glycation end-products (carboxymethyllysine and S100 proteins). Metformin inhibited the production of ROI in response to all these stimuli. By double staining-dichlorofluorescein as marker of ROI and Mitotracker CMH-Ros for mitochondria-the mechanism of ROI generation was analyzed in more detail in smooth muscle cells. Our data suggest that ROI are generated by uncoupling of the mitochondrial respiratory chain as well as by activation of the cytosolic NADPH-oxidase. A complete inhibition of ROI generation is only achieved by simultaneous inhibition of the mitochondrial electron flux (theonyltrifluoroacetone) and NADPH-oxidase (apocynin). Our data suggest that the various processes contributing to generation of ROI are closely linked. Activation of AMP kinase may represent an important mechanism to understand the antioxidative effects of metformin on the mitochondrial and cytosolic generation of ROI.
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