On this basis it is not surprising that oxidative stress has been implicated in involved in diabetic complications entails the intracellular formation of AGEs. The augmented presence of glucose inside the cell originates reactive dicarbonyl molecules such as glyoxal, methylglyoxal, and 3- deoxyglucosone, which react with the aminic groups of proteins to form AGEs. The modification process does not require the presence of an enzyme and the two-step reaction is not reversible.
On this basis it is not surprising that oxidative stress has been implicated in involved in diabetic complications entails the intracellular formation of AGEs. The augmented presence of glucose inside the cell originates reactive dicarbonyl molecules such as glyoxal, methylglyoxal, and 3- deoxyglucosone, which react with the aminic groups of proteins to form AGEs. The modification process does not require the presence of an enzyme and the two-step reaction is not reversible. Proteins with a very slow turnover rate, such as collagen and hemoglobin, exist in the body partly modified by glucose. These kind of modifications usually imply a loss of functionality and the denaturation of the target protein; moreover, the binding of the modified protein to AGE receptors on endothelial cells, mesangial cells, and macrophages induces the production of reactive oxygen species. AGEs are proved to be responsible for several aspects of diabetic complications involving vessels and kidneys.7,8
PKC is a family of at least 11 serin/treonin protein kinase isoenzymes involved in several cellular responses, such as growth, differentiation, genic expression, angiogenesis, and sorting of proteins inside the cell’s district. Based on their activating substances, the isoenzymes are classified in different families. The conventional PKCs require calcium and diacylglycerol (DAG) for activation, while the new PKCs require DAG but are calciumindependent.9 In diabetic patients, the augmented availability of glucose causes an augmented availability of DAG and a consequent activation of PKCs. Effects of this extra-activation are different and detrimental for vascular functionality with increased vascular permeability,10 deregulated nitric oxide generation via NADPH oxidase activation,11 stabilized vascular endothelial growth factor (VEGF) messenger ribonucleic acid (mRNA) expression through post-transcriptional mechanisms, increased leukocyte-endothelium interaction, and activating nuclear factor kappa Beta (NF-kB). 12
Last but not least, in diabetes there is an augmented flux through the hexosamine pathway (HP). In the healthy metabolism a relatively low amount of fructose-6P is diverted from glycolysis and directed to a cascade of reactions whose end-product is a series of amino-sugars, the building blocks of the glycosyl-side chains of proteins and lipids.13 Unlike AGE formation, the modification of proteins and lipids requires specific enzymes. Once again, the augmented availability of glucose in the diabetic patient causes an accumulation of end-products of this pathway. This was associated with the development of the complications of diabetes, basically due to the modification on serine/threonine residues of selected proteins.14-16 HP was involved in transforming growth factor beta-1 (TGF-β1) and fibronectin expression,17,18 and stimulates plasminogen activator inhibitor 1 (PAI-1) via modification of the transcription factor stimulatory protein 1 (Sp1).19 Augmented flux through the HP was also linked to insulin resistance and several authors proposed that flux through the hexosamine pathway may represent a cellular ‘sensor’ to monitor the flux of a nutrient, glucose, that can became toxic when in excess.20 This position ascribes to insulin resistance a sort of protective action to minimize glucose entrance.
So, there are four apparently distinct pathways whose end-points are diabetic complications, apparently because the unifying theory proposes a unique activating event for all of them: an upstream event able to start all the damaging pathways. This event is the production of ROS at the mitochondrial electron transport chain in high-glucose condition. It has been proved that there exists a threshold protonic potential, above which electron transfer between the complexes of the transport chain is inhibited and a very strong increase in ROS production takes place.21 The electrons, being forbidden the natural transfer, are given up to the molecular oxygen, originating superoxide anion (see Figure 1).
In an elegant series of experiments, complex two of the mitochondrial electron transport chain was proved to be the site of superoxide generation, and it was established that the inhibition of the production of superoxide anion from this site is enough to block the activation of all four damaging pathways discussed above.22 In fact, the same substances effective in preventing mitochondrial ROS formation also inhibited PKC activation, intracellular AGE formation, and polyol pathway flux, preventing sorbitol accumulation. Superoxide production and oxidative stress generation is only the first step; in the following years, the mechanism by which the superoxide produced at the electron transport chain was able to activate the damage pathways was pinpointed.
Recently, Du and colleagues demonstrated that in aortic endothelial cells cultured in 5mM glucose, inhibition of the glyceraldehydes-3-phosphate dehydrogenase (GAPDH) enzyme using antisense oligonucleotides activated the same damaging pathways that were activated in cells cultured in 30mM glucose. Afterwards, it was corroborated that the cascade of events leading to GAPDH inactivation in high-glucose-treated cells started with ROS production at the mitochondrial level, and it was suggested that the oxidative stress so generated determined DNA strand breaks and the activation of the poly (ADP-ribose) polymerase enzyme that modified GAPDH inhibiting it.23
It is not difficult to imagine the effect of inhibiting this key enzyme of glycolysis. In a cell environment stressed by the presence of high glucose concentration, the main pathway of utilization of this molecule becomes impeded. The substrate is then addressed to all the other pathways of utilization, which are the damaged pathways considered above. The low affinity for its substrate of aldose reductase was overtaken by the high glucose presence; in the same way, both the enzymatic and the non-enzymatic glycosylation process were boosted and the augmented availability of DAG causes the activation of the DAG-sensitive isoforms of PKC (see Figure 2).
As we have previously considered, activation of these pathways is reflected in an augmented oxidative stress inside the cell. The presence of sorbitol, a molecule that can hardly pass through membranes, influences the osmotic pressure in the cell, but mainly shoots down cell antioxidant defense subsiding its GSH pool. Once produced, AGEs can react with specific receptors for advanced glycation end-products (RAGEs) on endothelial cells, mesanglial cells, and macrophages in a process that has been proved to mediate signal transduction through generation of reactive oxygen species, and that lead to activation of both the transcription factor NF-kB and farnesyl-protein transferase (p21ras).24,25
Not all the passages have been clearly elucidated, but evidence is amassing and the role of oxidative stress is changing from that of a walker-on to a main character. Understanding the entire cascade of events would give us the capacity to fight diabetic complications at their early beginning, developing new molecules that are more specific and effective. At the moment this is still not possible, even if some pleasant secret is hidden by some drugs already in use. It is now well recognized that thiazolinedones,26 statins,27,28 ACE inhibitors, and angiotensin-1 (AT-1) inhibitors29 have shown beneficial ‘unpredicted’ effects exceeding expectations. Different authors are oriented to ascribe these effects to the antioxidant activity that is now recognized in these molecules. So, a lot of work has still to be done, but the road seems fair and the first steps of this long trip were very promising.