perspectives in diabetes hyperglycemic pseudohypoxia and...

Perspectives in Diabetes Hyperglycemic Pseudohypoxia and Diabetic Complications JOSEPH R. WILLIAMSON, KATHERINE CHANG, MYRTO FRANGOS, KHALID S. HASAN, YASUO IDO, TAKAHIKO KAWAMURA, JENS R. NYENGAARD, MARIA VAN DEN ENDEN, CHARLES KILO, AND RONALD G. TILTON

Vasodilation and increased blood flow are characteristic early vascular responses to acute hyperglycemia and tissue hypoxia. In hypoxic tissues these vascular changes are linked to metabolic imbalances associated with impaired oxidation of NADH to NAD+ and the resulting increased ratio of NADH/NAD+. In hyperglycemic tissues these vascular changes also are linked to an increased ratio of NADHINAW, in this case because of an increased rate of reduction of NAD+ to NADH. Several lines of evidence support the likelihood that the increased cytosolic ratio of free NADH/NAD+ caused by hyperglycemia, referred to as pseudohypoxia because tissue partial pressure oxygen is normal, is a characteristic feature of poorly controlled diabetes that mimics the effects of true hypoxia on vascular and neural function and plays an impohant role in the pathogenesis of diabetic complications. These effects of hypoxia and hyperglycemia-induced pseudohypoxia on vascular and neural function are mediated by a branching cascade of imbalances in lipid metabolism, increased production of superoxide anion, and possibly increased nitric oxide formation. Diabetes 42:801-13, 1993

From the Departments of Pathology, Pediatrics, and Internal Medicine, Washington University School of Medicine, St. Louis, Missouri; the Depart- ment of Internal Medicine, Chubu Rosai Hospital. Nagoya, Japan; the Stereological Research Lab, Aarhus University, Aarhus, Denmark; and the Department of Internal Medicine, M~ddelheim Hospital, Antwerp, Belgium.

Address correspondence and reprint requests to Dr Joseph R. Williamson, Department of Pathology, Box 81 18, 660 South Euclid Avenue, St. Louis, MO 631 10.

Received for publication 2 February 1993 and accepted in revised form 12 March 1993

PO,, part~al pressure of oxygen, SDH, sorbitol dehydrogenase; LDH, lactate dehydrogenase; GAP, glyceraldehyde 3-phosphate: 1.3-DPG, 13- bisphospho- glycerate: AR, aldose reductase; ARI, aldose reductase Inhibitor; snGBP, s n glycerol 3-phosphate; DHAP, dihydroxyacetone phosphate; DAG. 1,2-diacyl- snglycerol; GFR, glomerular filtrat~on rate; PKC, prote~n kinase C, CDP-DAG, cytidine diphosphate-I ,2-diacyl-snglycerol, PG, prostagland~n; Ptdlns, phos- phat~dylnositol; PIP,, phosphatidyl~nositol-bisphosphate; LCA-CoA, long-chain acyl ester of coenzyme A, LCA-C, long-cha~n acyl ester of carn~tine; FFA, free fatty acid; O;, superoxide anion; NO, nitrlc oxide; GSH, glutathione, G-6-P, glucose-6-phosphate; 6-PG, 6-phosphogluconate; 6-PG-DH, 6-phosphogluconate-dehydrogenase; R-5-P, r~bulose-5-phosphate; G-6-P-DH, glucose-6- phosphate-dehydrogenase, DPH, d~phenhydramine.

v asodilation and increased blood flow are among the earliest, if not the earliest, vascular changes associated with diabetes (see APPEN-

DIX, note 1) and with acute hyperglycemia of 4- to 5-h duration induced by infusion of glucose in nondiabetic humans and animals (10-18). Vasodilation and increased blood flow also are characteristic vascular responses to tissue hypoxia (19-22) (see APPENDIX, note 2). In hypoxic tissues these vascular changes are closely linked to an increase in NADH/NAD+ (because of impaired oxidation of NADH to NAD+) and associated metabolic imbalances. An increase in cytosolic-free NADH/NAD+ also is observed in tissues exposed to elevated glucose levels (in vivo or in vitro) at normal tissue PO, (Table 1) (14,23-28). Linkage of this redox imbalance to increased metabolism of glucose via the sorbitol pathway has been documented for the aster- isked tissues in Table 1. In contrast to hypoxia, the redox imbalance induced by elevated glucose levels is largely the result of increased oxidation of sorbitol to fructose coupled to reduction of NAD+ to NADH in the second step of the sorbitol pathway (Fig. 1). The considerable number of cells and tissues listed in Table 1, together with several lines of evidence discussed in this perspective, suggests that this hyperglycemia-induced redox imbalance is a characteristic feature of poorly controlled diabetes that mimics the effects of true hypoxia on vascular and neural function and plays an important role in the pathogenesis of diabetic complications.

A comprehensive discussion of all metabolic and functional consequences of an increased ratio of NADHI NAD+ and of other current hypotheses for the pathogenesis of diabetic complications is beyond the scope and space limitations of this perspective. Attention will be drawn, however, to evidence and hypotheses consistent with the role of hyperglycemia-induced pseudohypoxia in mediating diabetic complications. In

DIABETES, VOL. 42, JUNE 1993 80 1

TABLE 1 Tissues manifesting hyperglycemic pseudohypoxia

Cornea* Endoneurium* Erythrocytes* Glomeruli* Granulation tissue*

Lens' Retina* Islets of Langerhans Liver Skeletal muscle

'Hyperglycemic pseudohypoxia is linked to increased flux of glucose via the sorbitol pathway.

addition, explanations will be suggested for observations discordant with the thesis of this perspective. Recent reviews of other hypotheses as well as supporting data relevant to this perspective have been reported previ- ously (1 4,26,27-42).

Several parallels between functional abnormalities associated with an increased NADHINAD' in diabetic tissues and in hypoxic or ischemic myocardium are depicted in Fig. 2 (albiet the metabolic imbalances that mediate these abnormalities may differ in hypoxic and hyperglycemic tissues). This redox imbalance in tissues of diabetic animals appears to result largely from an increased rate of oxidation of sorbitol to fructose by SDH. In hypoxic and ischemic myocardium, the same redox imbalance results from impaired mitochondrial oxidation of NADH to NAD' because of decreased PO,. Functional consequences associated with this redox imbalance in isolated ~erfused hearts include 1 ) electrophysiological dysfunction (arrhythmias), 2) impaired myocyte contractile function, 3) increased vascular permeability, and 4) increased blood flow during reflow after mild hypoxia or brief ischemia but decreased blood flow after prolonged hypoxia/ischemia. Corresponding functional changes in diabetic tissues include 1 ) electrophysiological dysfunction in many tissues, best characterized in peripheral nerve and retina; 2) impaired contractile function of heart, skeletal muscle, and vascular smooth muscle; 3) increased vascular permeability; and 4) increased blood flow early after the onset of diabetes but decreased blood flow later in the course of diabetes. All of these early changes in tissues of diabetic animals are prevented by inhibitors of AR (13,26,27,43-48) (see APPENDIX, note 3).

An important feature of this glucose-induced redox imbalance is that it provides an explanation for the increased susceptibility of diabetic subjects to hypoxic

AR SDH D-GLUCOSE - SORBITOL - D-FRUCTOSE - n

w w 6PG- G6P LACTATE-PYRUVATE R5P

G6PDH LDH 6PGDH

FIG. 1. Reduction of glucose to sorbitol and oxldatlon of sorbitol to fructose in the sorbitol pathway. Reduction of glucose to sorbltol by AR Is coupled to oxidatlon of NADPH to NADP'. NADP' Is reduced to NADPH by the hexose monophosphate pathway. Oxidation of sorbltol to fructose by SDH is coupled to reduction of NAD' to NADH. The cytosolic ratio of free NADHINAD' is in equlllbrlum wlth lactate and pyruvate.

tSorbitol Oxidation

-1

j Dysfunction Dysfunction I

: 1. Electrical 1. Electrical 1 2. Mechanical 2. Mechanical j 3. t Vascular Permeability 3. t Vascular Permeability j 4. t - 8 Blood Flow 4. t - 4 Blood Flow ! 5. Vascular Sclerosis

Hyperglycemic Pseudohypoxia

FIG. 2. Parallels between functional consequences of an increased cytosollc NADHINAD' llnked to hyperglycemlc pseudohypoxla in diabetic tissues and hypoxla or ischemla In myocardlai tissue.

and ischemic injury (32,35,49,50) (see APPENDIX, note 4). Relatively mild hypoxic or ischemic episodes insufficient to cause dysfunction in nondiabetic subjects, when su- perimposed on preexisting pseudohypoxia induced by hyperglycemia, would result in a higher cytosolic NADHI NADf that would cause tissue dysfunction and injury in diabetic subjects.

IMBALANCES IN GLUCOSE METABOLISM CONTRIBUTING TO PSEUDOHYPOXIA The ratio of free NADH to NAD' modulates the activity of many metabolic pathways (54). Because the cytosolic pool of free NADH and NADf is in equilibrium with cytosolic lactate and pyruvate, and because oxidation of NADH to NADf by LDH is coupled to reduction of pyruvate to lactate (Fig. I ) , an increased ratio of free NADHINAD' will be reflected by an increased lactate1 pyruvate ratio. Indeed, the tissue lactatelpyruvate ratio is a more reliable parameter of the cytosolic ratio of free NADH/NADf than measurements of NADH and NADf in tissue extracts because it is not possible to distinguish between mitochondrial and cytosolic pools and between free and bound nucleotides in tissue extracts (23). Thus, for simplicity, in this perspective, changes in tissue lactatelpyruvate ratios are referred to as changes in cytosolic NADH/NADf. In like manner, an increase in mitochondrial NADHINAD' is reflected by an increase in the ratio of p- hydroxybutyrate/acetoacetate (23). lncreased sorbitol pathway metabolism. lncreased metabolism of glucose via the sorbitol pathway is prob- ably the most important mechanism by which hyperglycemia increases cytosolic NADH/NADf (Fig. 1 ) . Be- cause the K, of AR for glucose is very high (70 mM), the rate of reduction of glucose to sorbitol increases with increasing glucose levels in tissues that do not require insulin for glucose uptake (33). lncreased sorbitol levels, in turn, will increase the rate of oxidation of sorbitol to fructose coupled to a reduction of NADf to NADH. At elevated glucose levels, glucose metabolism via the sorbitol pathway accounts for 33% of glucose consump-

DIABETES, VOL. 42, JUNE 1993

t Glucose

f La.m& t So rb i t o l

f FSP 1 c NAD' 3 * SDHi

1 ' N4DH

Pyruvate f Fructose

t FDP t

t

\ 1 /-- t sew, FA

snG3P t LCA-C and LCA-CoA

FA -3 Phosphatidic ac~d

J t CDP-DAG t o a G

f PKC - 4 Na+K+-ATPase

DPH FIG. 3. Imbalances in glucose, sorbitol, and lipid metabolism linked to hyperglycemic pseudohypoxla and vascular and neural dysfunction. *Pharmacological interventions that prevent glucose-Induced vascular dysfunction include ARls, SDH inhlbltors, acetyl-L-carnltlne, PKC inhlbitors, pyruvate, myelnositol, and the antlhistamine DPH. Substances that mimic glucose effects on vascular function are italicized and underlined: lactate, sorbltol, DAG, and LCA-C.

tion by the lens and 10% by human erythrocytes (24,55). The extent to which tissue sorbitol levels are increased by elevated glucose levels is influenced by several factors including the ratio of AR to SDH and the availability of cofactors for both enzymes (56). Thus, tissue sorbitol levels are roughly proportional to the ratio of AR to SDH in diabetic rats (56). lncreased glycolysis. An increased rate of glycolysis also can alter the cytosolic redox state of NADHINAD+. lncreased glucose metabolism to lactate by human erythrocytes (induced by elevating intracellular inorganic phosphate, which activates phosphofructokinase) and in isolated perfused hearts (by increasing the work load), even under aerobic conditions and at constant glucose levels, is associated with an increased cytosolic NADHI NAD+ (57,58). Under these conditions of markedly accelerated glycolysis, oxidation of GAP to 1,3-DPG by GAP dehydrogenase (Fig. 3) appears to become the rate-limiting step in glycolysis (57,58). This reaction is coupled to reduction of NAD+ to NADH and is inhibited by NADH. The mechanism by which an increased rate of glycolysis increases free cytosolic NADH/NAD+ is not fully understood; it appears to result from a dysequilib- rium between the rate of oxidation of GAP to 1,3-DPG and the rate of reduction of pyruvate to lactate (coupled to oxidation of NADH to NAD+) (57,58). These observations raise the possibility that the increased rate of glycolysis in retina, glomeruli, endoneurium, and granulation tissue (but not lens or erythrocytes) exposed to elevated glucose levels may contribute to an increase in cytosolic NADHINAD+.

The likelihood that increased sorbitol pathway flux is the more important of these two mechanisms for increasing cytosolic NADHINAD+ in diabetes is supported by evidence that inhibitors of AR prevent this glucose- induced redox imbalance in tissues in which glycolysis is increased as well as in those in which it is not (14,24,26,28), and decrease sorbitol levels and NADHI NAD+ in tissues exposed to normal glucose levels. The latter finding implies that flux of glucose via the sorbitol pathway modulates cytosolic NADHINAD+ even at physiological glucose levels. Furthermore, exposure of human erythrocytes to elevated sorbitol levels (at normal glucose levels) in vitro markedly increases cytosolic NADHINAD+ (59). The importance of oxidation of sorbi- to1 to fructose in mediating this diabetes-induced redox imbalance and associated vascular and neural dysfunction also is supported by recent investigations with a new inhibitor of SDH. Inhibition of SDH attenuates diabetes- induced vascular dysfunction in retina, peripheral nerve, and aorta and the increased cytosolic NADHINAD+ in retina (the only tissue in which it has been examined at this time), despite a four- to sixfold increase in tissue sorbitol levels above those in untreated diabetic rats (60, 60a). All of these observations force the conclusion that sorbitol pathway-linked vascular and neural dysfunction are more likely the consequence of an increased rate of oxidation of sorbitol to fructose than to putative osmotic stress associated with increased sorbitol levels Der se or to metabolic imbalances linked to reduction of giucose to sorbitol (33,36).

Many studies in diabetic animals in which ARls have failed to prevent early vascular or neural dysfunction may be attributable to drug underdosing, i.e., failure to completely inhibit the sorbitol pathway, despite normalization of tissue sorbitol levels (13,45,47). The same problem may explain some reports that ARls fail to prevent metabolic imbalances (linked to early vascular and neural dysfunction) in cultured cells and incubated tissues. The importance of documenting normalization of fructose levels (in addition to sorbitol levels) as a parameter of glucose flux via the sorbitol pathway cannot be overem- phasized (45,47) (see APPENDIX, note 5). Both Ao et al. (45) and Cameron and Cotter (47) have reported that doses of ARls that normalized neural fructose as well as sorbitol levels also normalized nerve conduction in diabetic rats, whereas doses that normalized sorbitol but not fructose levels failed to normalize electrophysiological function. In addition, Tilton et al. (13) reported that in diabetic rats treated with two different doses of an ARI, vascular dysfunction was normalized only by the higher dose despite normalization of tissue sorbitol levels by both doses.

It is unclear whether vascular and neural dysfunction in chronically diabetic animals can be prevented by com- plete inhibiton of AR or by other interventions. The disappointing effects of ARls on complications in diabetic humans and in chronic studies in animals may be explained by the fact that the studies in human subjects have all been intervention trials, and it is now evident that even subclinical changes are very poorly reversible even by normalization of glucose levels, and the doses of ARls


used in diabetic humans and animals may not have completely inhibited metabolism of glucose via the sorbitol pathway.

NADH generated in the cytosol by glycolysis (coupled to oxidation of GAP to 1,3-DPG) or by oxidation of sorbitol to fructose must be reoxidized to NADf, or glycolysis will be blocked at the level of GAP dehydrogenase as indicated above. In the cytosol, NADH is oxidized to NADf by LDH (coupled to reduction of pyruvate to lactate) and by snG3P dehydrogenase (coupled to reduction of DHAP to snG3P) (Fig. 3). Cytosolic reducing equivalents also are transported (by the snG3P and/or the malate-aspartate shuttle) into mitochondria for oxidation by the electron transport chain. The limited capacity of many cells and tissues to oxidize cytosolic NADH by the sum of these mechanisms is well documented as discussed above.

This discussion has emphasized hyperglycemia-induced changes in cytosolic NADHINAD' because of evidence linking this redox imbalance to increased production of reducing equivalents by cytosolic enzymes. An increase in cytosolic NADH/NAD+ may or may not be accompanied by corresponding increases in mitochondrial NADH/NAD+, because in the liver of diabetic rats, the ratio is increased in the cytosol but decreased in mitochondria (23). The same discordance between cytosolic and mitochondrial ratios of NADH/NAD+ is observed in isolated perfused hearts subjected to an increased work load at normal PO, and constant glucose levels (58). In hypoxic tissues (and in cyanide-poisoned cells) the redox imbalance originates in mitochondria but also affects the cytosol because reducing equivalents generated in the cytosol can no longer be oxidized in the mitochondria.

If an increase in cytosolic NADH/NAD+ plays an important role in mediating vascular and neural dysfunction associated with diabetes and increased sorbitol pathway metabolism, the linkage between cytosolic ratios of lactatelpyruvate and NADH/NAD+ (depicted in Figs. 1 and 3) suggests that glucose-induced vascular dysfunction might be prevented simply by raising tissue pyruvate levels sufficiently to drive oxidation of NADH to NAD' as rapidly as NAD+ is reduced by oxidation of sorbitol to fructose. Conversely, elevation of lactate levels should mimic the effects of hyperglycemia on vascular function. Both of these predictions have been confirmed experimentally. Elevation of tissue pyruvate levels prevents glucose-induced vascular dysfunction in the tissue chamber model as well as in nondiabetic rats with acute hyperglycemia of -5-h duration induced by glucose infusion (61, 61a). Pyruvate also prevents sorbitol-induced vascular dysfunction in skin chamber granulation tissue (62). In contrast to inhibitors of AR and SDH, pyruvate has little impact on flux of glucose via the sorbitol pathway (63). Although pyruvate can function as a free-radical scavenger and can be oxidized in mitochondria via the citric acid cycle, these reactions would not decrease cytosolic NADH/NAD+ or normalize cytosolic redox-linked metabolites. In the skin chamber granulation tissue model, addition of lactate (at normal glucose levels) increases blood flow and vascular per-

meability. In nondiabetic and diabetic human subjects infusion of lactate increases renal blood flow and GFR independent of glucose levels (64). It is of interest that cyanide and ethanol (both of which increase tissue NADH/NADf at normal glucose levels) also are gluco- mimetic in their effects on vascular function in the skin chamber model.

The implications of these observations are that 1) pseudohypoxia of any cause, i.e., elevated levels of glucose, sorbitol, lactate, cyanide, or ethanol, induces vascular responses, i.e., dysfunction (see APPENDIX, note 6), like those caused by true hypoxia; and 2) an important determinant of the susceptibility of cells/tissues to sorbitol pathway-mediated injury is whether they can reoxidize NADH to NADf rapidly enough to keep pace with the increased rate of reduction of NAD+ to NADH associated with increased flux of glucose via the sorbitol pathway. Although it remains unclear why some cells and tissues (i.e., retina, kidney, and nerve) appear to be more susceptible to injury by the diabetic milieu, their vulner- ability may reflect unusual metabolic, structural, and/or functional characteristics in addition to a possible higher content of sorbitol pathway enzymes (resulting in more pronounced pseudohypoxia in response to elevated glucose levels). In tissues containing fructokinase (in addition to AR and SDH) fructose, produced from oxidation of sorbitol, may be phosphorylated and enter glycolysis directly (bypassing phosphofructokinase, which normally limits metabolism of glucose via glycolysis).

METABOLIC CONSEQUENCES OF AN INCREASE IN CYTOSOLIC NADHINA D' An increase in cytosolic NADH/NAD+ may impact on the activity of numerous cytoplasmic and mitochondrial enzymes that use NADH and NAD+ as cofactors and/or are inhibited or activated by NADH or NAD+. Thus, an important feature of this redox imbalance is that it has the potential to explain many metabolic imbalances associated with the diabetic milieu as well as the increased susceptibility of diabetic subjects to vascular and neural injury by factors independent of the diabetic milieu. Several imbalances in lipid metabolism linked to hyperglycemic pseudohypoxia have been implicated as mediators of diabetes-induced vascular and neural dysfunction. The roles of these imbalances in mediating vascular and neural dysfunction may vary in different cells and tissues depending on differences in their capacity for lipid uptake, synthesis, storage, and/or oxidation. lncreased de novo synthesis of DAG. lncreased tissue levels of DAG and/or evidence of glucose- and diabetes- induced increased de novo synthesis of DAG (and associated activation of PKC) have been reported in retina, isolated glomeruli, heart, cultured retinal capillary endothelial cells, cultured aortic endothelial and smooth muscle cells, and granulation tissue (14,26,27,61,65- 69). On the other hand, DAG levels were unchanged or decreased in peripheral nerve and aorta of diabetic rats (70,71), although PKC activity was increased in cultured aortic smooth muscle cells exposed to elevated glucose levels (72). An increased cytosolic NADH/NAD+ favors

804 DIABETES, VOL. 42, JUNE 1993

increased de novo synthesis of DAG by two mechanisms. The increase in NADH favors reduct~on of DHAP to snG3P, which is the first step in one pathway for de novo synthesis of DAG (Fig. 3) (61). It also inhibits GAP dehydrogenase (Fig. 3), thereby impairing the oxidation of GAP to 1,3-DPG, which increases availability of DHAP (which is in equilibrium with fructose 1,6-bisphosphate and GAP) as substrate for DAG synthesis. Activation of PKC, presumably by DAG and/or by LCA-C (discussed below), has been linked to many metabolic and functional vascular and neural changes in diabetes, i.e., decreased Na+K+-ATPase activity, increased PG synthetic activity, and vascular dysfunction in glomeruli, aorta, peripheral nerve, and granulation tissue (61,65, 68,69).

Although the beneficial effects of myeinositol supple- mentation on glucose- and diabetes-induced vascular and neural dysfunction have been attributed to normalization of Ptdlns metabolism and Na'K'-ATPase activity (34,40), they may also (or instead) accrue from concom- itant decreases in DAG levels and PKC activity, because elevated myeinositol levels favor incorporation of DAG (via CDP-DAG into Ptdlns [Fig. 31). This view is supported by evidence that 1) agonist-induced increases in DAG and CDP-DAG (resulting from hydrolysis of PIP,) (Fig. 3) are prevented by myeinositol (73-76), 2) glucose-induced vascular dysfunction in skin chamber granulation tissue (which is associated with an increase in DAG mass) is attenuated by an inhibitor of PKC (61), and 3) decreased neural Na'K'-ATPase activity in diabetic mice is prevented by inhibitors of PKC (68). The prevention of glucose- and diabetes-induced decreased Na+K+-ATPase activity and imbalances in Ptdlns metabolism in aorta and nerve by ARls also is consistent with the possibility that these changes are linked to sorbitol pathway-mediated pseudohypoxia (34,40). Inhibition of fatty acid oxidation. The second step in @-oxidation of long-chain fatty acids is inhibited by an increase in mitochondrial NADH/NAD+ (31). As discussed earlier, mitochondrial NADH/NAD+ may be increased by impaired oxidation of NADH to NAD+ in the mitochondrdia (hypoxia) or by increased transport into the mitochondria of reducing equivalents generated in the cytosol (i.e., by oxidation of sorbitol to fructose); it also can result from increased oxidation of fatty acids (77,78), In hypoxic and ischemic myocardium, long- chain fatty acids accumulate as esters of CoA and of carnitine (Fig. 3) (1 4,26,31 ,32,49). These long-chain acyl esters, like DAG, are amphipathic molecules and are potent modulators of many enzymes whose activities are altered by hypoxia and by diabetes, i.e., Na+K+-ATPase (inhibition), PKC (activation), and Ca++-ATPase (inhibition) (31,32). Accumulation of these esters in hypoxic and ischemic hearts and in hypoxic cultured myocytes is associated with impaired myocyte contractile function as well as electrophysiological dysfunction (31,49,79,80). These changes are more pronounced in hearts from diabetic animals and are made worse by elevating FFA levels in the perfusate (14,26,32,49). All of these changes are attenuated by addition of L-carnitine or short-chain

esters of L-carnitine (acetyl-L-carnitine, propionyl-L-carnitine).

Exposure of skin chamber granulation tissue vessels to palmitoyl-L-carnitine (an LCA-C) in vivo mimics the effects of elevated glucose levels and of hypoxia on blood flow and vascular permeability (1 4,26,27,81); exposure of cultured myocytes to palmitoyl-L-carnitine also induces electrophysiological dysfunction like that caused by hypoxia (80). These effects of palmitoyl-L-carnitine on vascular and electrophysiological dysfunction are prevented by coadministration of L-carnitine, acetyl-L-carnitine, and/or propionyl-L-carnitine. Acetyl-L-carnitine also prevents vascular and neural (electrophysiological) dysfunction in diabetic rats (82,83). Note that plasma and myocardial levels of L-carnitine are decreased by diabetes (84). At this time, it is unclear how the beneficial effects of L-carnitine, acetyl-L-carnitine, and propionyl- L-carnitine are mediated. Several mechanisms have been proposed that include scavenging of free radicals, increasing the availability of free CoA for energy metabolism, and compensating for detergent effects of long-chain acylcarnitines on membranes. Recent observations in diabetic rats' indicate that acetyl-L-carnitine prevents diabetes-induced decreases in endoneurial Na'K'-ATPase and PKC activity (84a) lncreased PG synthetic activity. PG synthetic activity is increased in hypoxic and ischemic cells and tissues (85-87) as well as in glomeruli and aorta of diabetic and nondiabetic animals exposed to elevated glucose levels in vitro (48,88-92). The particular PGs synthesized (and the balance between vasodilator versus vasoconstrictor prostanoids) varies in different vascular cells and tissues and with the severity and duration of hypoxia and hyperglycemia (85,88). The increased GFR observed early after the onset of poorly controlled diabetes in experimental animals is associated with increased production of vasodilatory PGs (88-90); a decreased ratio of vasodilator/vasoconstrictor PGs is associated with decreased blood flow and GFR later in the course of diabetes (88). Impaired contractile function of aortic rings from diabetic and nondiabetic rabbits exposed to elevated glucose levels in vitro is associated with increased production of vasoconstrictor prostanoids (91,92).

lncreased PG synthetic activity in glomeruli from diabetic rats is associated with increased de novo synthesis of DAG (67), activation of PKC (65), increased activity of some phospholipase A, isozymes (93), and increased GFR (1 3,14,88). All of these phenomena are attenuated to varying degrees by inhibitors of AR (13,14,26,89). A plausible linkage of these observations is depicted in Fig, 4.

EFFECTS OF AN INCREASED NADHINAD' ON FREE-RADICAL PRODUCTION lncreased free-radical production associated with hypoxia and ischemia. The importance of increased production of 0; and oxygen reactive species derived from it, i.e., hydrogen peroxide and hydroxyl radical, in mediating vascular injury during reperfusion after hypoxia and ischemia is well known (30,31,94,95). 0; production also is increased in cultured endothelial cells subjected to

DIABETES. VOL. 42, JUNE 1993 805

1 ( HYPERGLYCEMIA I 4 , . ? ~ ~ ~ ~ $ d ~ f a ' ~ ~ s tsorbitol pathway activity

a~vcarion 1

4 r - *Protein kinase C1 1 lAb ~ P h o s p h ~ i p a s e A 2 +Na+K+-ATPase I

1 VASCULAR AND NEURAL DYSFUNCTION 1 FIG. 4. Tentative scenario tor linkage of metabolic Imbalances that mediate the effects of hyperglycemia and pseudohypoxla (increased cytosolic NADH/NAD+) on vascular and neural dystunctlon.

hypoxia (96,97). On the other hand, increased NO (endothelium-derived relaxing factor) (98,99) production plays an important role in mediating hyperemia during reperfusion after mild hypoxia and brief ischemia (21,22). 0; and NO both cause vasodilation and increased blood flow under conditions of normoxemia and euglycemia (98,100); synthesis of both of these free radicals is oxygen dependent and therefore is markedly inhibited by anoxia and severe hypoxia. lncreased free-radical production associated with elevated glucose levels. Numerous reports of elevated levels of lipid peroxidation and glycoxidation products in diabetic humans and animals attest to diabetes-induced increased oxidative stress (see APPENDIX, note 7). It is not known, however, to what extent these parameters of increased oxidative stress reflect increased production of free radicals and/or impaired free-radical scavenger function (29). Recent studies in animal models of diabetes are consistent with the likelihood that increased production of 0, and relative or absolute increases in NO (and/or related monoxides [99]) formation mediate vascular and neural dysfunction induced by acutely elevated glucose levels (in vitro and in vivo) and by diabetes of short duration (1 7,18,42,101-104). Increases in blood flow and vascular albumin permeation induced by acute (-5- h duration) hyperglycemia in nondiabetic rats and in rats with short duration diabetes (2-5 wk) are largely prevented by free-radical scavengers (probucol and/or superoxide dismutase) and by inhibitors of NO synthase, AR, and PG synthetic activity (1 3,14,26,27, 42,103,104,104a; J.R.W., K.C., R.G.T., unpublished observations). Compounds that release NO independent of NO synthase activity, such as nitroglycerin, sodium nitro- prusside, and 3-(4-morpholinyl)sydnone irnine (Sin I ) , all increase vascular permeability as well as blood flow in the skin chamber model (104).

Impaired contractile function of aortic rings exposed to elevated glucose levels is prevented by free-radical

scavengers, ARls, and inhibitors of cyclooxygenase (48,101) as noted earlier. At this time, it is unclear whether NO production is increased by diabetes or whether the effects of NO synthase inhibitors on vascular dysfunction associated with diabetes reflect correction of an imbalance in vasodilators versus vasoconstrictors (inhibition of NO production could compensate for increased vasodilator prostanoid production and/or decreased responsiveness to endothelin-1 , a potent vasoconstrictor). Because plasma levels of endothelin-1 have been reported to be increased in diabetic humans, note that 1 ) endothelin-1 plasma levels are increased by insulin (105), 2) the biochemical effects of endothelin-1 on cultured pericytes are blunted by exposure to elevated glucose levels (106), and 3) low-affinity endothelin-1 receptors are downregulated in glomeruli from diabetic rats (1 07). This downregulation is prevented by an inhibitor of PKC given in vivo. Mechanisms of increased 0,' production by NADH. lncreased 0; production resulting from an increased rate of reduction of NAD+ to NADH may be mediated by several different reactions. The likelihood that an increased NADH/NAD+ will increase 0,- production via increased PG synthetic activity is supported by evidence discussed earlier linking increased PG synthetic activity in diabetes to pseudohypoxia, together with evidence of 0,- production coupled to PGH synthase activity (1 00,108,109). Vascular dysfunction induced by topical application of arachadonic acid is prevented by superoxide dismutase and cyclooxygenase inhibitors (100). Reduction of PGG, to PGH, by PG hydroperoxidase produces 0,- via a sidechain reaction that uses NADH (and NADPH but not GSH) as a reducing cosubstrate (100). Subsequent autoinactivation of prostacyclin synthase by 0; (produced by PG hydroperoxidase) (1 08,109), would explain diabetes-induced decreased production of prostacyclin and other vasodilatory prostanoids reported by some investigators. This would result in a decreased ratio of vasodilator/vascoconstrictor prostanoids because thromboxane synthase is resistant to inactivation by 0,. (108,109). Higher levels of oxygen free radicals also inactivate PG cyclooxygenase activity. Note that NO has been reported to inhibit lipid oxidation by lipoxygenase and cyclooxygenase (1 10). If increased PG synthetic activity associated with diabetes is indeed triggered by increased availability of arachadonic acid, then metabolism of arachadonic acid and associated formation of 0,. by microsomal cytochrome P,,, also may be increased (30,111).

0,- is a normal byproduct of oxidation of NADH to NAD+ by the electron transport chain in mitochondria (30,31). Reduced electron transport chain components can undergo spontaneous auto-oxidation with 0,. formation (97). If cytosolic-reducing equivalents produced by accelerated sorbitol pathway metabolism are transported into mitochondria rapidly enough, autoxidation of reduced electron transport components and 0,. formation may be increased.

NADH also has been reported to increase 0,. production by cultured human fibroblasts exposed to interleukin-1 (1 12). The nature of the reactions(s) that mediate


this effect of NADH are unclear but may be analogous to production of 0; by plasma membrane-associated NADPH oxidase in leukocytes. These observations raise the possibility that increased availability of NADH may favor increased 0; formation by other peroxidases, i.e., GSH peroxidase (1 00).

Note that increased oxidative stress and 0; formation by the above mechanisms may occur in the absence of an increased cytosolic ratio of NA DH/NADf provided that the increased rate of reduction of NADf to NADH by SDH does not exceed the maximal rate at which the cell can reoxidize NADH to NADf. Mechanisms of increased NO production by NADH. lncreased NO production associated with hypoxia and diabetes may be initiated by an increase in intracellular calcium (i.e., by increased 0,- formation [ I 13,114]), which would activate the constitutive isoform of NO synthase (98), and/or by activation of PKC (by increased levels of DAG and/or LCA-C and LCA-CoA), which has been reported to phosphorylate the constitutive isoform of NO synthase that results in a -40% increase in activity (1 15,116). Note that a marked increase in NO production may increase NADH/NADf by inactivating aconitase and electron transport chain enzymes (98). It also is of interest that NO synthase, like AR, requires NADPH as a cofactor (98).

Many (but not all) investigators have reported that acute hyperglycemia and diabetes impair acetylcholine- induced (NO-mediated) relaxation of vessels in vitro (46,48,91,92,101,117-120). Most reports suggesting impaired NO production are based on experiments in which vessels were incubated for several hours in media lack- ing L-arginine as substrate for NO synthesis. The possibility that impaired NO production by tissues from diabetic animals (and from nondiabetic animals incubated at elevated glucose levels) in L-arginine-deficient media may be an artifact of the in vitro milieu is suggested by several lines of evidence: 1) when the concentration of L-arginine (or H, biopterin, another cofactor required for NO synthesis) is suboptimal, activation of purified brain NO synthase results in production of hydrogen peroxide rather than NO (121); 2) glucose- and diabetes-induced decreases in Naf Kf -ATPase activity in rabbit aortic rings are not observed in tissues incubated in media containing L-arginine (122); and 3) impaired acetylcholine-induced relaxation of arteries from hypercholesterolemic rabbits appears to be an in vitro artifact of increased free-radical production (catalyzed by trace metals in the buffer) (123); trace metal-catalyzed free-radical production would be even greater in hyperglycemic media because of autoxidation of glucose (29). Whereas glycated proteins and 0; react with (and inactivate) NO in vitro (98,124), the pathophysiological significance of such observations is unclear because the rate of NO quenching versus rates of NO production and advanced glycation product formation in vivo (in diabetic subjects) is unknown. Further studies are needed to resolve these discordant observations regarding the nature and time course of changes in NO production as well as changes in other vasodilators and vasoconstrictors induced by diabetes.

Second messenger effects and cell injury linked to increased 0,' production. Increasing evidence attests to important roles for both 0; and NO in modulating normal cellular metabolism and mediating responses to vasoactive agents and injury (94,98,99,114). Therefore, if 0; production is increased in diabetic subjects, it could mediate many (otherwise apparently unrelated) diabetes-induced metabolic imbalances and biochemical changes, including extracellular matrix changes. For example, ascorbic acid-induced increases in collagen production and expression of procollagen a1 (1) mRNA levels in human fibroblasts are mediated by reactive aldehyde products of lrpid peroxidation (caused by 0;) and are prevented by probucol (125) (which also prevents glucose-induced vascular dysfunction [104]). Thus, increased 0; production could potentially mediate glucose-induced increased gene expression (in cultured vascular and mesangial cells) for extracellular matrix constituents, i.e., type IV collagen, fibronectin, and p1 laminin (1 261 27). lncreased 0; production linked to hyperglycemic pseudohypoxia also could mediate DNA damage and impaired cell replication in cultured endothelial cells and tissues exposed to elevated glucose levels (126,127). Oxygen free radicals have been reported to depress sarcolemmal Naf Kf -ATPase and Caf + -ATPase activity (52), increase intracellular Caf +

(1 13,114), release arachadonic acid and stimulate cyclooxygenase activity (1 14,128), and inhibit NO synthase activity (1 29).

GALACTOSE-INDUCED PSEUDOHYPOXIA Rodents fed galactose-enriched diets develop vascular and neural dysfunction and early vascular structural changes identical to those in diabetic animals and which, like those in diabetic animals, are prevented by inhibitors of AR (7,26,27,33). Unlike sorbitol (which is readily oxidized to fructose by SDH, galactitol does not appear to be further metabolized except in the liver. For this reason, many (if not most) investigators have attributed sorbitol pathway-linked diabetic complications to increased intracellular concentration of sorbitol (osmotic stress) and/or to redox changes and metabolic imbalances linked to reduction of glucose to sorbitol (33,36). Redox changes and metabolic imbalances associated with oxidation of sorbitol to fructose were considered unlikely candidate mechanisms for mediating sorbitol pathway-linked complications of diabetes.

In view of these considerations, it was of great interest to find that in human erythrocytes and in rat granulation tissue exposed to elevated galactose levels (in vitro and in vivo, respectively) cytosolic NADH/NADf and associated increases in triose phosphates and snG3P equal or exceed those induced by elevated glucose levels and are prevented by ARls and by pyruvate (26,27,130; J.R.W., unpublished observations). Exposure of human erythrocytes to sorbitol also causes a marked increase in NADH/NADf that is attenuated by pyruvate (59); galactitol has no effect on NADH/NADf. These observations suggest the existence of an as yet unidentified polyol pathway-linked mechanism for increasing cytosolic

DIABETES, VOL. 42, JUNE 1993 807

NADH/NAD+. In any case, these findings are consistent with the likelihood that increased cytosolic NADH/NAD+ mediates polyol pathway-linked vascular and neural functional and early structural changes induced by galactosemia (33) as well as by hyperglycemia. It is of interest in this regard that elevated galactose levels (like elevated glucose levels) stimulate de novo synthesis of DAG in cultured vascular cells (G. King, unpublished observations).

Depletion of GSH levels and increased susceptibility to oxidative stress in selected tissues (i.e., lens and erythrocytes) of diabetic and galactose-fed animals have been widely attributed to competition between AR and GSH reductase for NADPH cofactor. Even though NADPH levels are modestly decreased by accelerated polyol pathway activity, recent studies indicate that the decrease is not rate limiting for reduction of oxidized GSH because of the much lower K, of GSH reductase versus AR for NADPH (131). The likelihood that sorbitol pathway-linked depletion of reduced GSH in these tissues reflects increased oxidative stress (i.e., an increased NADH/NAD+) that results from accelerated oxidation of sorbitol to fructose is consistent with depletion of reduced GSH levels linked to the increased NADHINAD~ in hypoxic tissues of nondiabetic subjects (1 32).

In view of this new evidence of galactose-induced pseudohypoxia and earlier evidence that nonenzymatic glycation of lens crystallins by galactose is prevented by GSH (133), note that ARls prevent galactose-induced 1) depletion of lens GSH, 2) increased fluorescence (indic- ative of nonenzymatic glycation) and crosslinking of lens crystallins (134), and 3) pseudohypoxia. These observations are consistent with the putative importance of polyol pathway-induced oxidative stress mediated by pseudohypoxia (i.e., an increased NADH/NAD+) in the pathogenesis of diabetic cataracts and nonenzymatic glycation as well as in other complications of diabetes.

IMPACT OF INCREASED CYTOSOLIC NADH/NAD+ ON THE SUSCEPTIBILITY OF VESSELS AND NERVES TO INJURY BY FACTORS INDEPENDENT OF DIABETES The nature of the metabolic imbalances and functional consequences linked to hyperglycemic pseudohypoxia suggests new insights for understanding the increased susceptibility of diabetic subjects to vascular and neural injury by risk factors independent of diabetes as well as by other systemic metabolic and biochemical imbalances associated with the diabetic milieu. Included among such factors are hypoxic and ischemic injury, elevated plasma levels of atherogenic lipoproteins and FFAs, hypertension, cigarette smoking, ethanol abuse, and increased oxygen-derived free radicals associated with nonenzymatic glycation and glycoxidation. Potential interactions between hyperglycemic pseudohypoxia and each of these factors are considered below.

First, the mechanism by which an increase in cytosolic NADH/NAD+ could increase the susceptibility of tissues to hypoxic and ischemic injury is depicted in Fig. 2. As discussed earlier, cells with an increased NADH/NAD+ attributable to hyperglycemic pseudohypoxia will require

less severe hypoxia or ischemia to increase NADHI NAD+ to the same level caused by hypoxia or ischemia alone.

Second, because albumin permeation into the aorta is increased two- to threefold by diabetes, it is likely that vascular permeation by other macromolecules such as atherogenic lipoproteins also will be increased. Thus, even in diabetic subjects with normal cholesterol and lipoprotein profiles, permeation of atherogenic lipoproteins may be increased two- to threefold, which corre- sponds to the increased frequency of heart attacks in diabetic subjects (35). Any further increase in plasma levels of atherogenic lipoproteins would result in a corresponding increased flux into the vessel wall.

The likelihood that elevated plasma FFA levels in diabetic subjects may accentuate hyperglycemic pseudohypoxia and contribute to excessive accumulation of LCA-C and LCA-CoA, causing more vascular and neural injury, is supported by evidence that 1) elevation of FFAs leads to increased rates of fatty acid oxidation and reduction of NAD+ to NADH (resulting in an increase in mitochondria1 NADH/NAD+) (77,78), which will accentuate oxidative stress and pseudohypoxia induced by hyperglycemia; 2) elevated FFAs increase hypoxic and ischemic injury to the isolated perfused heart; 3) levels of cytotoxic LCA-C and LCA-CoA are higher in hypoxic and ischemic hearts from diabetic than from nondiabetic subjects; and 4) microalbuminuria in diabetic humans and animals is significantly correlated with elevated plasma triglycerides (which also correlate with plasma nonesterified fatty acids) (1 4).

Third, the finding that blood flow in diabetic rats is preferentially increased in tissues prone to late complications, in the absence of an increase in systemic blood pressure, implies dilation of resistance arterioles and impaired smooth muscle contractile function in the affected tissues. Thus, systemic blood pressure will be transmitted further downstream causing microvascular hypertension (Fig. 5). Hypertension is a well- known stim- ulus for vascular collagen production (and is associated with increased 0; production) (loo), which will result in sclerosis and stiffening of the vessel wall (1 4,26,27). Impaired contractile function of resistance arterioles also will permit any increase in systemic blood pressure to be transmitted further downstream resulting in even more severe microvascular hypertension. These hemody- narnic changes, coupled with increased vascular permeability, may account for the more severe and widespread hyalinization of arterioles in diabetic than nondiabetic subjects. The dilation of resistance arterioles induced by diabetes is in sharp contrast to the increased peripheral resistance (attributable to contraction of resistance arterioles) that contributes to hypertension in large arteries while tending to limit transmission of systemic blood pressure into the microvasculature distal to resistance arterioles. Thus, these observations predict that at any arterial blood pressure, microvascular blood flow will be higher in these (complication-prone) tissues of diabetic subjects than in nondiabetic subjects and much higher than in nondiabetic hypertensive subjects (until blood flow is reduced by vascular sclerosis).


NEG 4

I t 4Sorbitol pathway flux + 4 reduction of NAD* to NADH

t Superoxide 4 Free radicals, t Na*K*-ATPase

'-i +

+Vascular permeability +Ratio of vasodilatorlvasoconstrictor agents

Impaired contractile function of resistance arterioles t

Vasodilation, increased blood flow, microvascular hypertension + 4 Collagen synthesis -w vascular scarring

t +Vascular compliance + + pulsatile blood flow

t Tissue PO, + +oxidation of NADH to NAD*

t Progressive vascular sclerosis -w organ failure

FIG. 5. Translation of early reversible metabolic Imbalances associated with hyperglycemlc pseudohypoxia and vascular dysfunctlon into self-perpetuating vascular structural changes culminating in organ failure.

Fourth, cigarette smoking has been implicated as a significant risk factor for vascular complications of diabetes. lncreased CO levels in smokers could impact on diabetes-induced vascular injury in several ways. CO, like NO, reacts with iron-containing electron transport chain enzymes in the mitochondria, thereby impairing oxidation of NADH; binding of CO to Hb also would impair oxygen delivery to tissues. In addition, CO (like NO) activates guanylate cyclase (129,133, which could contribute to vasodilation and increased blood flow.

Fifth, hyperglycemic pseudohypoxia also may provide an explanation for reports that neuropathy is more common and more severe in diabetic subjects who consume excessive amounts of ethanol (136) (assuming the presence of alcohol dehydrogenase in neural tissue). Oxidation of ethanol to acetaldehyde by alcohol dehydrogenase, like oxidation of sorbitol to fructose, is coupled to reduction of NAD+ to NADH. Thus, the impact of ethanol ingestion on cytosolic NADH/NAD+ is equivalent to higher glucose levels and an increased flux of glucose via the sorbitol pathway.

Sixth, numerous reports attest to increased 0; production by glycated proteins and by autoxidative glycoxidation reactions (29,137-139). Glycated proteins induce vascular dysfunction (during euglycemia) like that caused by elevated glucose levels, which also is prevented by free-radical scavengers such as probucol and superoxide dismutase (104a; R.G.T., K.C., J.R.W., unpublished observations). These observations suggest that elevated glucose levels initiate production of free radicals by two independent mechanisms: 1) hyperglycemic pseudohypoxia, and 2) nonenzymatic glycation and autoxidative glycoxidation. Therefore, increased production of oxygen free radicals appears to be a strong candidate mechanism for effecting nonenzymatic as well as enzymatically mediated glucose effects on vascular and neural dysfunction and the late complications of diabetes (Figs. 4 and 5).

CONCLUSIONS A considerable body of evidence 1) indicates that in many tissues hyperglycemia-induced metabolic imbal-

ances increase the cytosolic ratio of free NADH/NADf (despite normal tissue PO,) that results in pseudohypoxia; 2) attests to the similarity of vascular, neural, and redox changes induced by true hypoxia and hyperglycemia-induced pseudohypoxia; and 3) supports the importance of oxidative stress and a branching cascade of metabolic imbalances linked to pseudohypoxia in mediating vascular and neural dysfunction induced by diabetes. Imbalances in lipid metabolism, increased 0; production, and perhaps increased NO formation appear to play important roles in mediating these functional disorders. Tentative scenarios for translation of these early metabolic imbalances and vascular dysfunction into irreversible progressive vascular sclerosis and organ failure are suggested in Figs. 2-5.

lncreased production of 0; and NO appears to play an important role in mediating early vascular and neural dysfunction linked to true hypoxia, hyperglycemic pseudohypoxia, and nonenzymatic glycation. Clearly, much more work is needed to delineate the mechanisms by which elevated glucose levels cause pseudohypoxia and the role of hyperglycemia-induced pseudohypoxia (and associated oxidative stress and metabolic imbalances) in mediating complications of diabetes.

ACKNOWLEDGMENTS This research was supported by grants from the National Institutes of Health (HL-39934, AM-20579, and EY- 06600) and by Pfizer-Central Research Division, Sigma Tau s.p.a. Rome, and the Kilo Diabetes and Vascular Research Foundation. J.R.N. is supported by the Danish Diabetes Association, Danish Medical Research Council, and Novo Foundation.

We thank Drs. John W. Baynes, Peter 6. Corr, Peter J. Oates, and John W. Turk for critical readings of this perspective and for thoughtful comments and sugges- tions. We thank Wanda Allison, Susan Berhorst, Judi Burgan, Antoinette M. Faller, Joyce Marvel, Eva Ostrow, Nemani Rateri, and Samuel R. Smith for expert technical assistance and Maria Himmelmann for preparation of the manuscript.

APPENDIX First, because of the putative importance of early vascular dysfunction (and of vascular responses to hypoxia and pseudohypoxia) in the pathogenesis of diabetic vascular complications, it is appropriate at the outset to acknowledge and address discordant reports of decreased retinal and endoneurial blood flow in acutely diabetic/hyperglycemic rats (1-3). Such reports are derived largely from indirect assessments of blood flow (transit time of fluorescein-labeled albumin for retina [3], hydrogen clearance or laser Doppler methods for endoneurium [1,2]) and/or invasive procedures (hydrogen clearance and laser Doppler methods for endoneurium). The problem with indirect measures of blood flow is that they can be influenced by many variables independent of blood flow, i.e., changes in tissue blood volume and/or in vascular permeability. Hydrogen clearance values based on oxidation of hydrogen by a platinum electrode also


can be affected by tissue edema and by a number of metabolites, including ascorbic acid and oxygen-reactive species (4); thus, redox changes and increased production of oxygen-reactive species associated with the diabetic milieu may affect blood flow values based on hydrogen clearance. The problem with invasive procedures is that vasoreactivity in many tissues is altered by diabetes (5,6); thus, neural trauma and cooling inherent to surgical exposure required for hydrogen clearance and laser Doppler methods (and insertion of electrodes into endoneurium for the hydrogen clearance method) may cause a relative or absolute decrease in neural blood flow in diabetic versus control subjects. For these reasons, indirect assessments of blood flow obtained by invasive procedures lack credibility if they are discordant with more direct measures of blood flow (i.e., appropri- ately sized microspheres) obtained by noninvasive tech- niques.

Blood flow measurements based on radiolabeled mi- crosphere injection methods are not subject to any of the above limitations. The major considerations in the use of microspheres are that the microspheres must be large enough to be trapped efficiently by the tissue vasculature examined, and enough microspheres must be captured to ensure reliable sampling and counting (this can be a problem in small tissues like retina and sciatic nerve of rats) (7). These potential confounding problems have been excluded in the experimental models we have used by the demonstration that molecular microspheres (i.e., 3H-desmethylimipramine, 266,000 M,) yield blood flow values identical to those obtained with conventional 10- to 15-pm microspheres in retina and endoneurium of diabetic rats (8). These findings, coupled with elevated microvascular hematocrits (a rheological parameter of increased blood flow) in retina and sciatic nerve tissue of diabetic rats (9), strongly support the conclusion that blood flow in these tissues is increased by acute hyperglycemia and by diabetes of short duration.

Second, true hypoxia is defined as decreased tissue p02 levels (in the absence of restricted blood flow) that result from a decrease in 0, content of blood (hypox- emia) delivered to tissue. Ischemia is defined as decreased tissue PO, levels that result from decreased volumetric flow of blood with a normal PO, content.

Third, despite these provocative similarities in neural, vascular, and myocyte contractile dysfunction induced by diabetes and hypoxia, it is clear that chronic hypoxic conditions (i.e., chronic lung disease, cyanotic heart disease, etc.) are not associated with diabetic complications and vice versa (although retinal capillary microan- eurysms and diabetic vascular changes in retina, kidney, and muscle are not unique to diabetes). Clearly, the combined effects of chronically elevated glucose levels and pseudohypoxia are essential for development of the late complications of diabetes. In addition, other risk factors such as hypertension appear to play a critical role in the pathogenesis of late complications. Indeed, we have proposed that the major impact of the diabetic milieu on the vasculature and nerves is to induce metabolic imbalances and dysfunction, which make them more susceptible to injury by risk factors independent of

the diabetic milieu (such as hypertension, hypercholes- terolemia, cigarette smoking, ethanol abuse, etc.) as discussed later.

Fourth, in hearts from diabetic animals, basal myocardial contractile function is impaired (49,50), and global ischemia-induced contractile dysfunction develops more rapidly than in control hearts (49). Paradoxically, diabetes improves recovery of myocyte and vascular smooth muscle contractile function during reperfusion after ischemia (50,51). Whereas endothelial barrrier functional integrity in diabetic hearts does not differ from control hearts before ischemia, albumin leakage in diabetic hearts during reflow exceeds that in control hearts by two- to threefold (50). These paradoxical effects of diabetes may be explained by differential responses of endothelium versus vascular smooth muscle and cardiac myocytes to the combination of preexisting pseudohypoxia and impaired Ca++ transport function in the sar- colemma and endoplasmic reticulum (31,32,51,52). Impaired Ca++ transport function could limit accumulation of Ca++, which is believed to play an important role in mediating hypoxic injury (31,32,51). It is of interest that a similar paradoxical effect of ischemia is observed in peripheral nerve in which resistance to ischemic conduction failure is increased by diabetes (53).

Fifth, even the combined measurements of tissue fructose and sorbitol will tend to underestimate sorbitol pathway activity because fructose can be further metabolized and also readily diffuses out of cells to be carried away by the blood. About 66-75% of the fructose produced by incubated tissues is recovered in the medium.

Sixth, vasodilation and increased blood flow may be viewed as normal vascular responses to hypoxia and pseudohypoxia as well as to various vasoactive substances. The point at which these normal vascular responses should be considered vascular dysfunction is unclear. As long as thevessels are dilated and blood flow is increased in response to sustained pseudohypoxia, they may be considered to be reacting normally. On the other hand, vessels that are dilated in response to hyperglycemic pseudohypoxia may dilate and constrict less in response to vasodilating and vasoconstricting agents than vessels in a euglycemic milieu. Although the dampened responses of such vessels to vasoactive agents is generally viewed as evidence of vascular dysfunction, it may just as well be considered a normal response to the combined effects of pseudohypoxia and the particular vasoactive agent examined. In any case, as discussed later, chronic vasodilation and increased blood flow imply microvascular hypertension, which is an important risk factor for diabetic retinopathy and ne- phropathy.

Seventh, glycoxidation products are formed by reactions between sugar-derived autoxidation products (such as glucosone) and proteins, lipids, etc. Oxidative stress is defined as an increase in steady-state levels of oxygen-reactive species (including O;, hydrogen peroxide, and hydroxyl radical) that result either from increased production of precursors of reactive oxygen species or decreased free-radical scavenger activity (29).


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