Atlantic Endocrinology New York City

Antioxidant Therapy in Diabetic Complications

In diabetes oxidative stress plays a key role in the pathogenesis of vascular complications, and an early step of such damage is considered the development of an endothelial dysfunction. Hyperglycemia directly promotes an endothelial dysfunction inducing process of overproduction of superoxide and consequently peroxynitrite that damages DNA and activates the nuclear enzyme (ADP-ribose) polymerase. 

This process, depleting NAD+, slowing glycolysis, ATP formation and electron transport, results in acute endothelial dysfunction in diabetic blood vessels and contributes to the development of diabetic complications. Classic antioxidants, like vitamin E, failed to show beneficial effects on diabetic complications probably due to their only “symptomatic” action. It is now evident that statins, ACE inhibitors, AT-1 blockers, calcium channel blockers and thiazolidinediones have a strong intracellular antioxidant activity, and it has been suggested that many of their beneficial ancillary effects are due to this property. 

Statins increase NO bioavailability and decrease superoxide production, probably interfering with NAD(P)H activity and modulating eNOS expression. ACE inhibitors and AT-1 blockers prevent hyperglycemia-derived oxidative stress modulating angiotensin action and production. This effect is of particular interest because hyperglycemia is able to directly modulate cellular angiotensin generation. Calcium channel blockers inhibit the peroxidation of cell membrane lipids and their subsequent intracellular translocation. Thiazolinediones bind and activate the nuclear peroxisome proliferator-activated receptor gamma, a nuclear receptor of ligand-dependent transcription factors. The inhibition of these receptors lead to inhibition of the inducible nitric oxide synthase and consequently reduction of peroxynitrite generation. This preventive activity against oxidative stress generation can justify a large utilization and association of this compound for preventing complications in diabetic patients, where antioxidant defenses have been shown to be defective.

What is oxidative stress?

Oxidative stress is defined in general as excess formation and/or insufficient removal of highly reactive molecules such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) ROS include free radicals such as superoxide (•O2-), hydroxyl (•OH), peroxyl (•RO2), hydroperoxyl (•HRO2-) as well as nonradical species such as hydrogen peroxide (H2O2) and hydrochloric acid (HOCl) . RNS include free radicals like nitric oxide (•NO) and nitrogen dioxide (•NO2-), as well as nonradicals such as peroxynitrite (ONOO-), nitrous oxide (HNO2) and alkyl peroxy nitrates (RONOO) . Of these reactive molecules, •O2-, •NO and ONOO- are the most widely studied species and play important roles in the diabetic cardiovascular complications. Thus, these species will be discussed in more detail.

  • NO is normally produced from L-arginine by endothelial nitric oxide synthase (eNOS) in the vasculature. •NO mediates endothelium-dependent vasorelaxation by its action on guanylate cyclase in vascular smooth muscle cells (VSMC), initiating a cascade that leads to vasorelaxation. •NO also displays antiproliferative properties and inhibits platelet and leukocyte adhesion to vascular endothelium. Therefore, •NO is considered a vasculoprotective molecule. However, •NO easily reacts with superoxide, generating the highly reactive molecule ONOO-, and triggering a cascade of harmful events as discussed below. Therefore its chemical environment, i.e. presence of •O2-, determines whether •NO exerts protective or harmful effects.

Hyperglycemia, Oxidative Stress, And Endothelial Dysfunction

At Atlantic Endocrinology & Diabetes Center we know that vascular function in diabetes has been studied extensively in both animal models and humans. Impaired endothelium-dependent vasodilation has been a consistent finding in animal models of diabetes induced by alloxan or streptozotocin. Similarly, studies in humans with type 1 or type 2 diabetes have found endothelial dysfunction when compared with vascular function in nondiabetic subjects.

In vitro, the direct role of hyperglycemia has been suggested by evidence that arteries isolated from normal animals, which are subsequently exposed to exogenous hyperglycemia, also exhibit attenuated endothelium-dependent relaxation. Consistently, in vivo studies have also demonstrated that hyperglycemia directly induces, both in diabetic and normal subjects, an endothelial dysfunction.

The role of free radicals generation in producing the hyperglycemia-dependent endothelial dysfunction is suggested by studies showing that both in vitro and in vivo, the acute effects of hyperglycemia is counterbalanced by antioxidants.

Increased superoxide production in endothelial cells during hyperglycemia: the unifying hypothesis for the development of diabetic complications

Brownlee recently pointed out the key role of superoxide production in endothelial cells at the mitochondrial level during hyperglycemia in the pathogenesis of diabetic complications. This new insight is consistent with the four pathways suggested to be involved in the development of diabetic complications (increased polyol pathway flux, increased advanced glycosylation end product formation, activation of protein kinase C, and increased hexosamine pathway flux) and with a unifying hypothesis regarding the effects of hyperglycemia on cellular dysfunction. The authors used endothelial cells subjected to physiologically relevant glucose concentrations as a model system for analyzing the vascular response to hyperglycemia because the non-insulin-dependent glucose transporter GLUT1 facilitated diffusion of high levels of glucose into the endothelium. In the presence of increased glucose, endothelial generation of reactive oxygen species, particularly superoxide anion, was shown to be enhanced. Several pathways can be considered as likely candidates for oxygen free radical formation in cells. These include NAD(P)H oxidase, the mitochondrial respiratory chain, xanthine oxidase, the arachidonic cascade (lipoxygenase and cyclooxygenase), and microsomal enzymes. Brownlee et al. have determined that the source of free radicals in endothelial cells incubated in high glucose is the transport of glycolysis-derived pyruvate in mitochondria at the level of complex II (succinate:ubiquinone oxidoreductase), one of the four inner membrane–associated complexes central to oxidative phosphorylation. The data in the papers  indicate that, at least in the cell culture, endothelium in an environment mimicking physiological hyperglycemia cannot control its appetite for glucose. Accelerated flux of glucose through glycolysis and feeding of pyruvate (thus formed) to the tricarboxylic acid cycle overloads mitochondria, causing excessive generation of free radicals. Although oxygen free radicals have been shown to have a physiological role in signal transduction, their sustained generation at the levels shown in endothelial cells exposed to high glucose can be expected to have substantial effects on cellular properties. Each of the pathways implicated in secondary complications of diabetes has been shown to arise by a single unifying mechanism. A central contribution of the works of Brownlee et al.  is to demonstrate that suppression of intracellular free radicals, using low molecular inhibitors or by expression of the antioxidant enzyme manganese-superoxide dismutase, prevents each of these events (i.e., glucose-induced formation of oxidants is a proximal step in cell perturbation).