When I refer to the tissue-damaging effects of hyperglycemia, of course, I mean damage to a particular subset of cell types: capillary endothelial cells in the retina, mesangial cells in the renal glomerulus, and neurons and Schwann cells in peripheral nerves. What is distinct about these cells that makes them so vulnerable to hyperglycemia? We know that in diabetes, hyperglycemia is bathing all the cells of every tissue. So why does damage occur only in the few cell types involved in diabetic complications? The answer is that most cells are able to reduce the transport of glucose inside the cell when they are exposed to hyperglycemia, so that their internal glucose concentration stays constant. In contrast, the cells damaged by hyperglycemia are those that cannot do this efficiently (3,4). Thus, diabetes selectively damages cells, like endothelial cells and mesangial cells, whose glucose transport rate does not decline rapidly as a result of hyperglycemia, leading to high glucose inside the cell. This is important, because it tells us that the explanation for what causes complications must involve mechanisms going on inside these cells, rather than outside.
Increased flux through the polyol pathway.
The polyol pathway, shown schematically in Fig. 2, focuses on the enzyme aldose reductase. Aldose reductase normally has the function of reducing toxic aldehydes in the cell to inactive alcohols, but when the glucose concentration in the cell becomes too high, aldose reductase also reduces that glucose to sorbitol, which is later oxidized to fructose. In the process of reducing high intracellular glucose to sorbitol, the aldose reductase consumes the cofactor NADPH (6). But as shown in Fig. 2, NADPH is also the essential cofactor for regenerating a critical intracellular antioxidant, reduced glutathione. By reducing the amount of reduced glutathione, the polyol pathway increases susceptibility to intracellular oxidative stress.
How do we know that this piece of the puzzle is really important? From studies like one conducted by Ron Engerman and Tim Kern (7), in which diabetic dogs were treated for 5 years with an aldose reductase inhibitor. Nerve conduction velocity in the diabetic dogs decreased over time as it does in patients. In contrast, in diabetic dogs treated with an aldose reductase inhibitor, the diabetes-induced defect in nerve conduction velocity was prevented.
Intracellular production of AGE precursors.
The second discovery listed on my pieces of the puzzle list is the intracellular production of AGE precursors. As shown schematically in Fig. 3, these appear to damage cells by three mechanisms. The first mechanism, shown at the top of the endothelial cell, is the modification of intracellular proteins including, most importantly, proteins involved in the regulation of gene transcription (8,9 and M.B., unpublished observations). The second mechanism, shown on the left, is that these AGE precursors can diffuse out of the cell and modify extracellular matrix molecules nearby (10), which changes signaling between the matrix and the cell and causes cellular dysfunction (11). The third mechanism, shown on the right of Fig. 3, is that these AGE precursors diffuse out of the cell and modify circulating proteins in the blood such as albumin. These modified circulating proteins can then bind to AGE receptors and activate them, thereby causing the production of inflammatory cytokines and growth factors, which in turn cause vascular pathology (12–21). Again, how do we know that this piece of the puzzle is really important? From many animal studies such as one done by Hans-Peter Hammes (22), showing that that pharmacologic inhibition of AGEs prevents late structural changes of experimental diabetic retinopathy.
PKC activation.
The third mechanism on my "pieces of the puzzle" list was the PKC pathway. In this pathway, shown schematically in Fig. 4, hyperglycemia inside the cell increases the synthesis of a molecule called diacylglycerol, which is a critical activating cofactor for the classic isoforms of protein kinase-C, -ß, -{delta}, and -{alpha} (23–26). When PKC is activated by intracellular hyperglycemia, it has a variety of effects on gene expression, examples of which are shown in the row of open boxes in Fig. 4. In each case, the things that are good for normal function are decreased and the things that are bad are increased. For example, starting from the far left of Fig. 4, the vasodilator producing endothelial nitric oxide (NO) synthase (eNOS) is decreased, while the vasoconstrictor endothelin-1 is increased. Transforming growth factor-ß and plasminogen activator inhibitor-1 are also increased. At the bottom of the figure, the row of black boxes lists the pathological effects that may result from the abnormalities in the open boxes (26–30). Again, how do we know that this piece of the puzzle is really important? We know that this is important from many animal studies such as several published by George King, showing that inhibition of PKC prevented early changes in the diabetic retina and kidney (27,31,32).
Increased hexosamine pathway activity.
The last mechanism on the "pieces of the puzzle" list was increased flux through the hexosamine pathway. As shown schematically in Fig. 5, when glucose is high inside a cell, most of that glucose is metabolized through glycolysis, going first to glucose-6 phosphate, then fructose-6 phosphate, and then on through the rest of the glycolytic pathway. However, some of that fructose-6-phosphate gets diverted into a signaling pathway in which an enzyme called GFAT (glutamine:fructose-6 phosphate amidotransferase) converts the fructose-6 phosphate to glucosamine-6 phosphate and finally to UDP (uridine diphosphate) N-acetyl glucosamine.
What happens after that is the N-acetyl glucosamine gets put onto serine and threonine residues of transcription factors, just like the more familiar process of phosphorylation, and overmodification by this glucosamine often results in pathologic changes in gene expression (33–35). For example, in Fig. 5, increased modification of the transcription factor Sp1 results in increased expression of transforming growth factor-ß1 and plasminogen activator inhibitor-1, both of which are bad for diabetic blood vessels (36). Again, how do we know that this piece of the puzzle is really important? Although this piece of the puzzle is the most recent to be recognized as a factor in the pathogenesis of diabetic complications, it has been shown to play a role both in hyperglycemia-induced abnormalities of glomerular cell gene expression (33) and in hyperglycemia-induced cardiomyocyte dysfunction in cell culture (37). In carotid artery plaques from type 2 diabetic subjects, modification of endothelial cell proteins by the hexosamine pathway is also significantly increased (38).
A UNIFIED MECHANISM
We began by asking the following question: What processes are increased by intracellular hyperglycemia in cells whose glucose transport rate is not downregulated by hyperglycemia but not increased in cells whose glucose transport rate is downregulated by hyperglycemia?
We discovered that a consistent differentiating feature common to all cell types that are damaged by hyperglycemia is an increased production of reactive oxygen species (ROS) (36,39). Although hyperglycemia had been associated with oxidative stress in the early 1960s (40), neither the underlying mechanism that produced it nor its consequences for pathways of hyperglycemic damage were known.
How does hyperglycemia increase superoxide production by the mitochondria?
There are four protein complexes in the mitochondrial electron transport chain, called complex I, II, III, and IV (Fig. 6). When glucose is metabolized through the tricarboxylic acid (TCA) cycle, it generates electron donors. The main electron donor is NADH, which gives electrons to complex I. The other electron donor generated by the TCA cycle is FADH2, formed by succinate dehydrogenase, which donates electrons to complex II. Electrons from both these complexes are passed to coenzyme Q, and then from coenzyme Q they are transferred to complex III, cytochrome-C, complex IV, and finally to molecular oxygen, which they reduce to water.
The electron transport system is organized in this way so that the level of ATP can be precisely regulated. As electrons are transported from left to right in Fig. 6, some of the energy of those electrons is used to pump protons across the membrane at complexes I, III, and IV. This generates what is in effect a voltage across the mitochondrial membrane. The energy from this voltage gradient drives the synthesis of ATP by ATP synthase (41,42). Alternatively, uncoupling proteins (UCPs; Fig. 6) can bleed down the voltage gradient to generate heat as a way of keeping the rate of ATP generation constant.
That’s what happens in normal cells. In contrast, in diabetic cells with high glucose inside, there is more glucose being oxidized in the TCA cycle, which in effect pushes more electron donors (NADH and FADH2) into the electron transport chain. As a result of this, the voltage gradient across the mitochondrial membrane increases until a critical threshold is reached. At this point, electron transfer inside complex III is blocked (43), causing the electrons to back up to coenzyme Q, which donates the electrons one at a time to molecular oxygen, thereby generating superoxide (Fig. 6). The mitochondrial isoform of the enzyme superoxide dismutase degrades this oxygen free radical to hydrogen peroxide, which is then converted to H2O and O2 by other enzymes.
How do we know that this really happens in cells known to be damaged by hyperglycemia? First, we looked at such cells with a dye that changes color with increasing voltage of the mitochondrial membrane and found that intracellular hyperglycemia did indeed increase the voltage across the mitochondrial membrane above the critical threshold necessary to increase superoxide formation (44). In order to prove that the electron transport chain indeed produces superoxide by the mechanism we proposed, we examined the effect of overexpressing either UCP-1 or manganese superoxide dismutase (MnSOD) on hyperlglycemia-induced ROS generation (Fig. 7A). Hyperglycemia caused a big increase in production of ROS. In contrast, an identical level of hyperglycemia does not increase ROS at all when we also collapse the mitochondrial voltage gradient by overexpressing UCP (39). Similarly, hyperglycemia does not increase ROS at all when we degrade superoxide by overexpressing the enzyme MnSOD. These data demonstrate two things. First, the UCP effect shows that the mitochondrial electron transport chain is the source of the hyperglycemia-induced superoxide. Second, the MnSOD effect shows that the initial ROS formed is indeed superoxide.
To confirm these findings by an independent experimental approach, we depleted mitochondrial DNA from normal endothelial cells to form so-called {rho}0 endothelial cells, which lack a functional mitochondrial electron transport chain (Fig. 7B). When the mitochondrial electron transport chain is removed, the effect of hyperglycemia on ROS production is completely lost (M.B., unpublished observations). Similarly, in {rho}0 endothelial cells, hyperglycemia completely fails to activate the polyol pathway, AGE formation, PKC, or the hexosamine pathway (M.B., unpublished observations).
We also looked at the effect of either UCP-1 overexpression or MnSOD overexpression on each of these four hyperglycemia-activated pathways. Hyperglycemia did not activate any of the pathways when either the voltage gradient across the mitochondrial membrane was collapsed by UCP-1 or when the superoxide produced was degraded by MnSOD (39). We have verified all of these endothelial cell culture experiments in transgenic mice that overexpress MnSOD (M.B., unpublished observations). When wild-type animals are made diabetic, all four of the pathways are activated in tissues where diabetic complications occur. In contrast, when MnSOD transgenic mice are made diabetic, there is no activation of any of the four pathways.
In endothelial cells, PKC also activates nuclear factor {kappa}B (NF{kappa}B), a transcription factor that itself activates many proinflammatory genes in the vasculature. As expected, hyperglycemia-induced PKC activation is prevented by either UCP-1 or MnSOD, both in cells and in animals.
Importantly, inhibition of hyperglycemia-induced superoxide overproduction using a transgenic approach (superoxide dismutase [SOD]) also prevents long-term experimental diabetic nephropathy in the best animal model of this complication: the db/db diabetic mouse (45).
Referência :
(1) Diabetes. 2005 Jun;54(6):1615-25.
