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1、Banting Lecture 2004The Pathobiology of Diabetic Complications A Unifying MechanismMichael Brownleetsa great honor to join the exceptional club of Banting Award winners, many of whom were my role models and mentors. In addition, giving the Banting Lecture also has a very personal meaning to me, beca
2、use without Frederick Banting, I would have died from type 1diabetes when I was 8years old. However, it was already apparent at the time I was diagnosed that for too many people like me, Bantingsdiscovery of insulin only allowed them to live just long enough to develop blindness, renal failure, and
3、coronary disease. For example, when I started college, the American Diabetes AssociationsDiabe-tes Textbook had this to say to my parents:“Theperson with type 1diabetes can be reassured that it is highly likely that he will live at least into his 30s.”Not surprisingly, my parents did not ndthis part
4、icularly reassuring.At the same time we were reading this in 1967, however, the rstbasic research discovery about the pathobiology of diabetic complications had just been published in Science the previous year. In my Banting Lecture today, I am thus going to tell you a scienticstory that is also pro
5、foundly personal. Ivedivided my talk into three parts. The rstpart is called “piecesof the puzzle,”and in it I describe what was learned about the pathobiology of diabetic complications starting with that 1966Science paper and continuing through the end of the 1990s. In the second part, I present a
6、uniedmecha-nism that links together all of the seemingly unconnected pieces of the puzzle. Finally, in the third part, I focus on three examples of novel therapeutic approaches for the prevention and treatment of diabetic complications, which are all based on the new paradigm of a unifying mechanism
7、 for the pathogenesis of diabetic complications.PIECES OF THE PUZZLEWhen I refer to the tissue-damaging effects of hypergly-cemia, of course, I mean damage to a particular subset of cell types:capillary endothelial cells in the retina, mesan-gial cells in the renal glomerulus, and neurons and Schwan
8、n cells in peripheral nerves. What is distinct about these cells that makes them so vulnerable to hyperglyce-mia? 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 complica-tions? The answer is t
9、hat most cells are able to reduce the transport of glucose inside the cell when they are exposed to hyperglycemia, so that their internal glucose concentra-tion stays constant. In contrast, the cells damaged byFrom the Departments of Medicine and Pathology, Albert Einstein College of Medicine, Bronx
10、, New York.AGE, advanced glycation end product; eNOS, endothelial nitric oxide synthase; FFA, free fatty acid; GAPDH, glyceraldehyde-3phosphate dehydro-genase; MnSOD, manganese superoxide dismutase; NF B, nuclear factor B; PARP, poly(ADP-ribosepolymerase; PKC, protein kinase C; ROS, reactive oxygen
11、species; SOD, superoxide dismutase; TCA, tricarboxylic acid; UCP, uncoupling protein.2005by the American DiabetesAssociation. FIG. 1. General features of hyperglycemia-induced tissue damage.DIABETES, VOL. 54, JUNE 20051615hyperglycemia are those that cannot do this efciently(3,4.Thus, diabetes selec
12、tively damages cells, like endo-thelial 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 mecha
13、nisms going on inside these cells, rather than outside.The rstsuch mechanism that was discovered was the polyol pathway and increased polyol pathway ux,de-scribed in peripheral nerve in the 1966Science paper I refer to above (5.This was the rstpiece of the puzzle. Then, 10years later, in the late 19
14、70s, a second piece of the puzzle emerged:increased formation of advanced glycation end products (AGEs.In the late 1980s and early 1990s, a third piece of the puzzle was discovered:hyper-glycemia-induced activation of protein kinase C (PKCiso-forms. And in the late 1990s, a fourth piece of the puzzl
15、e was discovered:increased hexosamine pathway uxand conse-quent overmodicationof proteins by N -acetylglucosamine. Imgoing to very brieyreview each of these, because I think each one is an important piece of the puzzle.Increased uxthrough the polyol pathway. The polyol pathway, shown schematically i
16、n 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
17、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 reduc
18、ed 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 Enger-man and Tim Kern (7,in which diabetic dogs were treated for 5years with an aldose reductase inh
19、ibitor. Nerve conduc-tion 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 sec-ond
20、 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 rstmechanism, shown at the top of the endothelial cell, is the modicationof intracellular proteins including, m
21、ost importantly, proteins involved in the regulation of gene transcription (8,9and M.B., unpub-lished 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 t
22、he matrix and the cell and causes cellular dysfunction (11.The third mech-anism, 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 modiedcirculating proteins can then bind to AGE receptors and activa
23、te them, thereby causing the production of inam-matory cytokines and growth factors, which in turn cause vascular pathology (1221.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 pharmacologi
24、c inhibition of AGEs prevents late structural changes of experimental diabetic retinopathy.PKC activation. The third mechanism on my “piecesof 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 call
25、ed diacyl-glycerol, which is a critical activating cofactor for the classic isoforms of protein kinase-C, -, -, and -(2326.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 cas
26、e, 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 (NOsynthase (eNOSis decreased, while the vasoconstrictor endothelin-1is increased. Transformi
27、ng growth factor-and plasminogen activator inhibitor-1are also increased. At the bottom of the gure,the row of black boxes lists the pathological effects that may result from the abnormalities in the open boxes (2630.Again, how do we know that this piece ofFIG. 2. Hyperglycemia increases uxthrough t
28、he polyol pathway. From Brownlee M:Biochemistry and molecular cell biology of diabetic complications. Na-ture 414:813820,2001.PATHOBIOLOGY OF DIABETIC COMPLICATIONS1616DIABETES, VOL. 54, JUNE 2005the puzzle is really important? We know that this is important from many animal studies such as several
29、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 “piecesof the puzzle”list was increased uxthrough the hexosamine pathway. As shown schemat-ically in Fig. 5, w
30、hen glucose is high inside a cell, most of that glucose is metabolized through glycolysis, going rstto glucose-6phosphate, then fructose-6phosphate, and then on through the rest of the glycolytic pathway. How-ever, some of that fructose-6-phosphate gets diverted into a signaling pathway in which an
31、enzyme called GFAT (glutamine:fructose-6phosphate amidotransferase con-verts the fructose-6phosphate to glucosamine-6phos-phate and nallyto UDP (uridinediphosphate N -acetyl glucosamine.What happens after that is the N -acetyl glucosamine gets put onto serine and threonine residues of transcription
32、factors, just like the more familiar process of phosphory-lation, and overmodicationby this glucosamine often results in pathologic changes in gene expression (3335.For example, in Fig. 5, increased modicationof theFIG. 3. Increased production of AGE precursors and its pathologic consequences. From
33、Brownlee M:Biochemistry and molecular cell biology of diabetic complications. Nature 414:813820, 2001.FIG. 4. Consequences of hyperglycemia-induced activation of PKC. M. BROWNLEEDIABETES, VOL. 54, JUNE 20051617transcription factor Sp1results in increased expression of transforming growth factor-1and
34、 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 be
35、en shown to play a role both in hyperglycemia-induced abnormalities of glomerular cell gene expression (33and in hypergly-cemia-induced cardiomyocyte dysfunction in cell culture (37.In carotid artery plaques from type 2diabetic sub-jects, modicationof endothelial cell proteins by the hexosamine path
36、way is also signicantlyincreased (38.A UNIFIED MECHANISMOver 13,000articles published since 1966seemed to show that all of these pieces of the puzzle were important in the pathogenesis of diabetic complications, yet two things suggested that something major was missing. First, there was no apparent
37、common element linking these mecha-nisms to each other. Second, clinical trials of inhibitors of these pathways in patients were all disappointing. Trying to make sense of all this, we hypothesized that all of these mechanisms were in fact linked to a common upstream event and that the failure to bl
38、ock all of the downstream pathways could explain the disappointing clinical trials with single-pathway inhibitors. What we discovered is that all of these different pathogenic mechanisms do reecta single hyperglycemia-induced process and that this single unifying process is the overproduction of sup
39、eroxide by the mitochondrial electron transport chain.We began by asking the following question:What pro-cesses 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 downregu
40、lated by hyperglycemia?We discovered that a consistent differentiating feature common to all cell types that are damaged by hyperglyce-mia is an increased production of reactive oxygen species(ROS(36,39.Although hyperglycemia had been associ-ated with oxidative stress in the early 1960s (40,neither
41、the underlying mechanism that produced it nor its conse-quences for pathways of hyperglycemic damage were known.How does hyperglycemia increase superoxide pro-duction by the mitochondria? There are four protein complexes in the mitochondrial electron transport chain, called complex I, II, III, and I
42、V (Fig.6. When glucose is metabolized through the tricarboxylic acid (TCAcycle, 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 FADH 2, formed by succinate dehydrogenase, which donates elec-tron
43、s 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 nallyto molecular oxygen, which they reduce to water.The electron transport system is organized in this way so that the level of
44、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 mitochon-drial membrane. The energy from thi
45、s 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.Thatswhat happens in normal cells. In contrast, in diabetic cells with hi
46、gh glucose inside, there is more glucose being oxidized in the TCA cycle, which in effect pushes more electron donors (NADHand FADH 2 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 thi
47、s point, electron transfer inside complex III is blocked (43,causing the electrons to back up to coenzyme Q, which donates theFIG. 5. Hyperglycemia increases uxthrough the hexosamine pathway. From Brownlee M:Biochem-istry and molecular cell biology of diabetic com-plications. Nature 414:813820,2001.
48、PATHOBIOLOGY OF DIABETIC COMPLICATIONS1618DIABETES, VOL. 54, JUNE 2005electrons 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 H 2
49、O and O 2by 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 intracellu-lar hyperglycemia did indeed increase the volt
50、age 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-1or manganese superoxide
51、dismutase (MnSODon hyperlglycemia-in-duced 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, hy
52、perglycemia does not increase ROS at all when we degrade superoxide by overexpressing the enzyme MnSOD. These data demon-strate 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 show
53、s that the initial ROS formed is indeed super-oxide.To conrmthese ndingsby an independent experimen-tal approach, we depleted mitochondrial DNA from nor-mal endothelial cells to form so-called 0endothelial cells, which lack a functional mitochondrial electron transport chain (Fig.7B . When the mitoc
54、hondrial electron transport chain is removed, the effect of hyperglycemia on ROS production is completely lost (M.B.,unpublished observa-tions. Similarly, in 0endothelial cells, hyperglycemia completely fails to activate the polyol pathway, AGEFIG. 6. Hyperglycemia-induced production of superoxide b
55、y the mitochondrial electron transport chain.FIG. 7. Hyperglycemia-induced ROS production in wild type and 0endothelial cells. A :From ref. 39. B :From M.B., M.H. Zou, X. Du, D. Edelstein, unpublished data.M. BROWNLEEDIABETES, VOL. 54, JUNE 20051619formation, PKC, or the hexosamine pathway (M.B.,unp
56、ub-lished observations.We also looked at the effect of either UCP-1overexpres-sion 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 col-lapsed by U
57、CP-1or when the superoxide produced was degraded by MnSOD (39.We have veriedall of these endothelial cell culture experiments in transgenic mice that overexpress MnSOD (M.B.,unpublished observa-tions. When wild-type animals are made diabetic, all four of the pathways are activated in tissues where d
58、iabetic 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 B (NFB, a transcription factor that itself activates many proinammatorygenes in the vasculature. As ex-pe
59、cted, hyperglycemia-induced PKC activation is pre-vented by either UCP-1or MnSOD, both in cells and in animals.Importantly, inhibition of hyperglycemia-induced super-oxide overproduction using a transgenic approach (su-peroxide dismutase SODalso prevents long-term experimental diabetic nephropathy in the best animal model of this complication:the db/dbdiabetic mouse (45.Hyperglycemia-induced mitochondrial su
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