Are Diabetic Neuropathy, Retinopathy and Nephropathy Caused by Hyperglycemic Exclusion of Dehydroascorbate Uptake by Glucose Transporters?

J. theor. Biol. (2002) 216, 345–359 doi:10.1006/jtbi.2002.2535, available online at http://www.idealibrary.com on

Are Diabetic Neuropathy, Retinopathy and Nephropathy Caused by Hyperglycemic Exclusion of Dehydroascorbate Uptake by Glucose Transporters? Robert Root-Bernsteinz*, JuliaV. Busikn and Douglas N. Henrynw n

Department of Physiology, Michigan State University, East Lansing, MI 48824, U.S.A. and w Department of Pediatrics and Human Development, Michigan State University, East Lansing, MI 48824, U.S.A. (Received on 4 October 2001, Accepted in revised form on 8 January 2002)

Vitamin C exists in two major forms. The charged form, ascorbic acid (AA), is taken up into cells via sodium-dependent facilitated transport. The uncharged form, dehydroascorbate (DHA), enters cells via glucose transporters (GLUT) and is then converted back to AA within these cells. Cell types such as certain endothelial and epithelial cells as well as neurons that are particularly prone to damage during diabetes tend to be those that appear to be dependent on GLUT transport of DHA rather than sodium-dependent AA uptake. We hypothesize that diabetic neuropathies, nephropathies and retinopathies develop in part by exclusion of DHA uptake by GLUT transporters when blood glucose levels rise above normal. AA plays a central role in the antioxidant defense system. Exclusion of DHA from cells by hyperglycemia would deprive the cells of the central antioxidant, worsening the hyperglycemia-induced oxidative stress level. Moreover, AA participates in many cellular oxidation–reduction reactions including hydroxylation of polypeptide lysine and proline residues and dopamine that are required for collagen production and metabolism and storage of catecholamines in neurons. Increase in the oxidative stress level and metabolic perturbations can be expected in any tissue or cell type that relies exclusively or mainly on GLUT for co-transport of glucose and DHA including neurons, epithelial cells, and vascular tissues. On the other hand, since DHA represents a significant proportion of total serum ascorbate, by increasing total plasma ascorbate concentrations during hyperglycemia, it should be possible to correct the increase in the oxidative stress level and metabolic perturbations, thereby sparing diabetic patients many of their complications. r 2002 Elsevier Science Ltd. All rights reserved.

Introduction: The Problem of Diabetic Complications Hyperglycemia is well known to be a major causative factor for the development of diabetic neuropathies, retinopathies, and nephropathies (DCCT, 1993) but the exact mechanism or zAuthor to whom correspondence should be addressed. 0022-5193/02/$35.00/0

mechanisms are not known. It has been suggested, for example, that hyperglycemia might lead to microvascular complications including capillary fragility resulting in microangiopathies, and these, in turn, might deplete oxygen and nutrient delivery to retinal, neuronal, and other cells (Lorenzi & Gerhardinger, 2001). Indeed the hyperglycemia-induced end organ damage in diabetes has long been associated with the r 2002 Elsevier Science Ltd. All rights reserved.

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activation of the protein kinase C (PKC) pathway (Aiello et al., 1998; Koya & King, 1998), increased flux of glucose through the polyol metabolic pathway (Frank et al., 1997; Henry et al., 2000; Lindsay, et al., 1998; Knott et al., 1993; Vinores et al., 1988; Winkler et al., 1997), and accumulation of advanced glycation end (AGE) products (Brownlee, 1994). A recent report that all hyperglycemia-induced changes, including increases in aldose reductase (AR), PKC and AGE are reversed by inhibition of glucose-induced reactive oxygen species (ROS) production (Nishikawa et al., 2000) provides the exciting possibility that blocking glucose-induced oxidative stress also may prevent damage caused by the other pathways. AA plays a central role in the antioxidant defense system and should help to relieve the oxidative stress associated with diabetic complications. Indeed, high doses of ascorbic acid have been found to ameliorate the production of AGEs (Will & Byers, 1996), to lower polyol production (Bode et al., 1993; Kowluru et al., 1997) and to improve complications associated with diabetic retinopathy in rats (Kowluru et al., 2001). We present a hypothesis that simply and coherently explains these phenomena on the basis of glucose competition for dehydroascorbic acid transport into some specific cell types such as neurons and in renal and retinal epithelial and endothelial tissues associated with diabetic complications. Hypothesis: Glucose Competes with DHA for Transport into Cell Types Most Affected by Diabetic Complications Recent discoveries concerning the uptake and transport of ascorbate compounds by cells suggest a novel hypothesis for explaining the causes of some diabetic complications. It has been known for about 25 years (Mann & Newton, 1975) that two distinct mechanisms exist for transport of vitamin C into cells. One is a sodium-dependent mechanism mediated by a pair of AA transporters that have recently been cloned (Tsukaguchi et al., 1999; Daruwala et al., 1999; Wang et al., 2000). Sodium-dependent transport of AA predominates in blood-brain

barrier, osteoblasts, muscles, placenta, small intestines, kidney brush-border cells and many other tissues and is largely unaffected by blood glucose levels (Mooradian, 1987; Agus et al., 1997; Daruwala et al., 1999; Tsukaguchi et al., 1999; Wang et al., 2000). A second mechanism for ascorbate uptake, however, is extremely sensitive to blood glucose levels. Some members of the GLUT family of glucose transporters (especially GLUT1, GLUT3 and GLUT4, but probably not GLUT2 or GLUT5) (Vera et al., 1993; Rumsey et al., 1997, 2000) also transport DHA into cells. Once the DHA enters cells, it is then converted into and stored as ascorbic acid (Packer & Fuchs, 1997). Tissues vary in terms of how they take up vitamin C. Some cell types, including retinal pigmented epithelial cells [Khatami (1987) and data provided in this paper in Fig. 3] and certain types of cells in kidney and intestines (Goldenberg & Schweinzer, 1994) appear to rely mainly on DHA uptake through GLUT transporters. Other tissues, including liver, brain, muscle and most endocrine and neuroendocrine systems, rely primarily upon a sodium-dependent ascorbate uptake system that is not glucose sensitive (Wang et al., 2000; Goldenberg & Schweinzer, 1994). Some cell types, such as lymphocytes (Ng et al., 1998) and red blood cells (Bianchi & Rose, 1986a) utilize both sodium-dependent and GLUTmediated mechanisms of ascorbate uptake. The co-transport of glucose and DHA by GLUTs in certain cell types suggests a novel mechanism for causing pathologies in these specific cell types and not others. Recent studies provide mounting information on hyperglycemia-induced increases in the production of reactive oxygen species (ROS) in the end organs affected by diabetes. ROS production is suggested to be the major causative pathway leading to the development of diabetic complications (Nishikawa et al., 2000; Kowluru et al., 1997; Obrasova et al., 2001; Cameron et al., 2001; Coppey et al., 2001). AA scavenges watersoluble ROS and regenerates tocopherol from tocopheroxyl radical in membranes and lipoproteins. The ascorbate radical formed in these reactions is relatively unreactive, being neither strongly oxidizing nor strongly reducing (Halliwell & Whiteman, 1997; Wells & Che-Hun,

DIABETIC COMPLICATIONS, DEHYDROASCORBATE AND GLUT

1997). AA exclusion from the cells through competition of glucose and DHA for common transport mechanism will deprive the cells of the central antioxidant and could lead to ROS accumulation followed by activation of PKC, AR pathways and AGE production in diabetes. Also, AA is a required cofactor for several intracellular hydroxylases, including lysine and proline hydroxylase and dopamine-b-hydroxylase (Burri & Jacob, 1997). Dopamine-b-hydroxylase converts dopamine into norepinephrine. Norepinephrine is, in turn, the substrate for epinephrine and other catecholamines (Stone & Townsley, 1973; Nagatsu, 1973). Notably, dopamine-b-hydroxylase is found in key tissues that are specific targets of diabetic complications, including retina (which contains dopamine, norepinephrine and epinephrine) (Versaux-Botteri et al., 1992; Hadjiconstantinou & Neff, 1984); Schwann cells (Unsicker & Chamley, 1976); peripheral sensory nerves (Jonsson & Sachs, 1971) and both renal mesangial cells (Vlahovic & Stefanovic, 1994) and the sympathetic enervation of the kidney (Kline et al., 1986). In addition to the increase in oxidative stress levels, chronic hyperglycemia is therefore a likely cause for tissue- or cell-specific metabolic perturbations characterized by disregulation of catecholamine metabolism mediated by GLUT co-transport of glucose and DHA. These effects will not be systemic, as occurs when all tissues are deprived of ascorbate (scurvy), but limited to specific cell types that perform catecholamine metabolism and/or depend on GLUT co-transport of glucose and DHA. The relationship between these competitive molecules for their common transporter is described by the equation: vDHA ¼ KDHA 1 þ

½DHAVmax ! h i GLUCOSE KGLUCOSE

þ ½DHA

ðSegel; 1976Þ; where v is the rate of entry of DHA into cells and Vmax is the maximum velocity of DHA entry into cells; and KDHA and KGLUCOSE are the measured Km values.

347

The Km for glucose transport via GLUT in the cells from the end organs affected by diabetes has been measured to be between 1 and 7 mM, depending on the transporter and experimental conditions (Heilig et al., 1997; Henry et al., 1999). The Km for DHA transport into lymphoblasts has been measured to be 1047/84 mM1 and the Vmax for DHA is about 30–37 nM1hr1/ 106cells1 (Ngkeekwong & Ng, 1997; Ng et al., 1998). In THP-1 monocytes, the DHA Km is 450 mM and the Vmax is 35 nM1hr1106 cells1(Laggner et al., 1999). The Km values for isolated GLUT isoforms are in the same general range: GLUT1 is 1.1 mM; GLUT3 is 1.7 mM; and GLUT4 is 980 mM (Rumsey et al., 1997, 2000). Normal blood glucose is about 3–5 mM and normal blood vitamin C levels are about 50– 110 mM (Sinclair et al., 1991; Moeslinger, et al., 1995; Seghieri, 1998). DHA is produced by metal-binding proteins such as serum albumin and ceruloplasmin that are found in blood plasma. (Mouithys-Mickalad et al., 1998). There is some disagreement in the field on what normal blood plasma levels of DHA may be. Most studies suggest that the DHA concentration in normal individuals is about 10–60% of the total blood ascorbate concentration, a figure of 10–20% probably being the most accurate (Chatterjee & Banerjee, 1979; Som et al., 1981; Sinclair et al., 1991; Deutsch & Kolhouse, 1993; Moeslinger et al., 1995; Tessier et al., 1996; Seghieri et al., 1998; Bakaev et al., 1999). There are some reports, however, that no measurable DHA is present in blood plasma. (Dhariwal et al., 1991; Wang et al., 1992). Some of the measured differences are clearly due to the use of different methodologies for measuring plasma DHA (e.g. spectroscopic vs. HPLC), but it has also been suggested that the presence of DHA itself may be an artifact of the oxidation of ascorbate during the measurement process. Since there are ascorbate oxidizing enzymes in blood plasma, and since there appear to be DHA-dependent tissues, and since the vast majority of the existing literature reports the presence of significant amounts of DHA in blood plasma, we have assumed that DHA is normally present. Using the conservative range of the plasma DHA data (10–20% of ascorbate), the influx of

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DHA relative to glucose via GLUT transporters will be about one-to-ten, since each compound is present at concentrations approximately equal to its Km for GLUT transporters. The Ki for glucose inhibition of DHA uptake in lymphoblasts is 2.2 mM (Ngkeekwong & Ng, 1997). The vDHA for a normal, healthy individual can be calculated from the data and the equation given above to be about 1.5 – 4.5 nM1hr1106 cells1. Because DHA and glucose compete for GLUT transporters, each can inhibit the transport of the other (Ngkeekwong & Ng, 1997). Baseline blood glucose in poorly regulated diabetes is typically raised to a baseline of about 8–11 mM (Seghieri et al., 1998; DCCT Research Group, 1993) and, during hyperglycemic episodes, rises transiently to 20 mM or higher (DCCT Research Group, 1993). Moreover, ascorbate levels tend to be significantly decreased in poorly regulated diabetes so that typical plasma values are in the range of 10– 40 mM. (Seghieri et al., 1998, 1994; Sinclair et al., 1991; Stankova et al., 1984, Som et al., 1981). These lowered ascorbate levels occur even in hyperglycemic diabetic patients who eat ascorbate-rich diets (Sinclair et al., 1991; Seghieri et al., 1998). The mechanism of ascorbate loss appears to be due to the fact that ascorbate is excreted along with glucose in the kidneys and its reuptake blocked by the increased sugar concentration and decreased reabsorbtion by the renal tubules with osmotic diuresis and glycosuria (Stankova et al., 1984; Segheiri et al., 1994; Klein et al., 1995). On the other hand, the system compensates, perhaps through upregulation of ascorbate oxidase-like activity, by increasing the ratio of DHA to ascorbate in blood plasma from 10 to 20% percent to between 80 and 120%, so that DHA-dependent cells are able to take up a greater proportion of the total serum ascorbate that is available (Chatterjee & Banerjee, 1979; Som et al., 1981; Sinclair et al., 1991; Seghieri et al., 1994, 1998). Inserting the range of data summarized in the previous paragraphs into the equation given above yields vDHA of 0.5–2.5 nM hr1106cells1 for poorly regulated diabetes at baseline glucose and vDHA of 0.1–1.6 nM hr1106 cells1 during hyperglycemic episodes. These figures are, on average, a 30–80% decrease in the normal rates

of DHA entry into cells. Thus, DHA transport into nerves, retina, kidney, and other tissues that are solely or mainly GLUT-dependent for ascorbate will be decreased dramatically and chronically. This decreased influx of vitamin C in cells dependent on GLUT transport of DHA is the equivalent of that which would be expected systemically in a non-diabetic individual suffering from moderate to severe scurvy (Sauberlich, 1975). (To achieve the same decrease in DHA transport in a normal person that is seen in a hyperglycemic individual at their baseline glycemic index, the normal person’s blood ascorbate level would have to be below 25 mM, or 1/3 of normal. To achieve the decrease in DHA transport that occurs during a severe hyperglycemic episode is the equivalent of the normal person having a blood ascorbate level of 15 mM or less, or less than one-sixth of normal.) Thus, hyperglycemia is likely to result in the selective AA deficiency of particular types of cells (such as peripheral neurons, retinal pigmented epithelial cells and retinal vascular endothelial cells) that depend mainly or exclusively upon GLUT transporters for vitamin C uptake (Fig. 1 and Table 1). We reiterate that tissues utilizing sodium-dependent ascorbate uptake mechanisms will not have their ascorbate uptake affected by hyperglycemia and therefore that hyperglycemia does not and cannot produce systemic scurvy. Clinical Consequences of the Hypothesis Our theoretical calculations are consistent with laboratory results. Agus et al. (1997) observed that in the presence of normal concentrations of DHA, doubling blood glucose from 6 to 12 mM halves the transport of DHA. At 24 mM glucose, DHA transport decreased to two-fifths of the normal. These decreases would be proportionally greater in mildly scorbutic diabetic patients. The scorbutic effect of high blood sugar will not be systemic because, as we mentioned above, many types of cells utilize an alternative (sodium-dependent) AA uptake mechanism. It is obvious from the foregoing that if vitamin C was excluded from cells metabolizing catecholamines, these cells would develop serious metabolic problems and neurotransmission would be

349

DIABETIC COMPLICATIONS, DEHYDROASCORBATE AND GLUT

GLUCOSE DHA

GLUT

SORBITOL GLUCOSE

H20 + DHA

AA+ O-

AO

NE

DHA +H2O DβH

AR NADPH

AA +O-

NADP+

AGEs

OCT

TYR DOP

MELANINS

Fig. 1. Simplified schema of the proposed effects of diabetic hyperglycemia on cell metabolism in cell types dependent exclusively on GLUT-mediated uptake of dehydroascorbic acid (DHA). Some major effects mediated through glucose uptake are increased production of sorbitol via aldose reductase (AR), increased protein kinase C activation (not shown) and also advanced glycolsylation end-products (AGEs). Both effects are associated with neuropathological sequelae. Hyperglycemia also decreases DHA uptake through GLUT. Diabetic patients tend to have low serum ascorbic acid (AA) levels to begin with, limiting DHA production via ascorbate oxidases (AO) such as serum albumin and ceruloplasmin (Mouithys-Mickalad et al., 1998). Only DHA can be transported via GLUT. DHA is reconverted into AA within cells, and AA is required for dopamine beta hydroxylase (DBH) activity. DBH activity is, in turn, required for the transformation of dopamine (DOP) and tyramine (TYR) into their respective metabolites norepinephrine (NE) and octopamine (OCT). DOP and TYR build up as DBH activity decreases and oxidize rapidly into melanins in the absence of AA. Some melanins (including lipofuscins) are incorporated into AGEs. Increased melanin production and deposition are cytopathic. See Table 1 for additional metabolic effects of this system and the text for references.

impaired. Hyperglycemia has the potential to decrease greatly the amount of vitamin C available to neurons and epithelial cells producing cellspecific metabolic disruption. (Fig. 1 and Table 1). Clinical evidence suggests the plausibility of our hypothesis. Human hemorrhagic retinopathies, both in diabetes and in other syndromes, are associated with low serum ascorbate and can be produced in guinea pigs by induction of scurvy (Greco et al., 1980). The incidence of microangiopathy, retinopathy and nephropathy in diabetic patients is directly correlated with low serum ascorbate (Ng et al., 1998; Sinclair et al., 1991; Seghieri et al., 1994; Hirsch et al., 1998; Ali

& Chakraborty, 1989; Jennings et al., 1987). Alcoholic patients characterized by low serum ascorbate are also unusually prone to retinopathies, peripheral neuropathies, and nephropathies (Boyd et al., 1981). And finally, scurvy patients do display these pathologies in addition to the bleeding gums, connective tissue disorders and the other symptoms that are more commonly used to characterize the disease (Hurlimann & Salomon, 1994; Hood, 1969). Thus, patients with scurvy show some of the same symptoms as do diabetic patients with complications as would be predicted from our hypothesis. On the other hand, diabetic patients do not

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Table 1 HYPOTHESIZED SPECIFIC AND COMMON METABOLIC CONSEQUENCES OF GLUCOSE COMPETITIVE INHIBITION OF DEHYDROACORBIC ACID UPTAKE ON CELLULAR METABOLISM IN DIABETES Hyperglycemic Inhibition of DHA Uptake

Cellular Metabolism

Common Metabolism

-

activation of protein kinase C

-

lipofuscin

-

oxido-reductive stress

-

growth factor/cytokine release

deposition

-

polyol metabolism

-

“up-regulation” of glucose

adrenergic

-

nitric oxide formation

metabolism

-

advanced glycosylation

-

transport

end-products -

decreased DHA transport

Note: See Fig. 1 for details of the mechanism and the text for references

develop all of the symptoms of scurvy because only GLUT-dependent DHA uptake is antagonized by hyperglycemia in diabetes. Moreover, the consequences of low ascorbate on catecholamine metabolism explain some of the particular symptoms that develop in diabetic complications. Interference with ascorbate uptake by hyperglycemia would result in increased production of melanins such as lipofuscins due to polymerization of catecholamine metabolites (Dillon & Root-Bernstein, 2000; Root-Bernstein & Dillon, 1997; Hegedus, 2000; Kameyam et al., 1996; Hegedus & Nayak, 1994; Pawelek & Lerner, 1978). It is suspected that lipofuscins may be a key component in glycation endproducts (AGEs) (Jobst & Lakatos, 1996), which have been associated with diabetic pathologies (Hammes et al., 1991). Indeed, many oxidative intermediates in the conversion of catcholamines into melanins are known to be cytopathic (Kameyam et al., 1996; Hegedus & Nayak, 1994; Pawelek & Lerner, 1978). Accumulation of melanins is known to occur in retinal cells (Rozaowska et al., 1997; Augsten et al., 1997), Schwann cells (Schmidt et al., 1984), and pancreatic islet cells of diabetic patients (Clark et al., 1989). In addition, there is a build-up of soluble melanins in the blood and urine of diabetic patients and animals, resulting in increased exposure of renal mesangial cells to melanin-associated cytotoxicity (Khattab et al.,

1972; Tsuchida et al., 1985; Hegedus et al., 1988). Thus, the specific end-organ targets damaged as a result of chronic hyperglycemia correspond well with the DHA-mediated mechanism we are proposing. We stress that hyperglycemia does not cause scurvy itself because the mechanism of DHA inhibition by glucose does not extend to glucose inhibition of sodium-dependent AA uptake and therefore does not affect most cells or tissues in the body. Ngkeekwong & Ng (1997) have found that while glucose competitively inhibits DHA uptake with a Ki of 2.2 mM in lymphoblasts, AA uptake is significantly inhibited only above 20 mM glucose. Mooradian (1987) found similarly that the brain uptake index of glucose was not altered by any physiologically reasonable dose of AA, but that DHA inhibited the glucose brain uptake index with a Ki of 13.0 mM. These data are consistent with the data of Agus et al. (1997) described above. Thus, cells that utilize sodiumdependent AA transport mechanisms such as brain, intestines, osteoblasts, muscles, and placenta, and cells such as oocytes and lymphocytes will not have their ascorbate intake affected significantly by hyperglycemia. The effects of the mechanism proposed by our hypothesis will be limited to specific cell types in the same ways that diabetic complications are limited. Diabetic patients should not experience the general systemic complications associated with scurvy.

351

DIABETIC COMPLICATIONS, DEHYDROASCORBATE AND GLUT

Preventing and Treating Diabetic Complications A most important implication of our hypothesis is that some types of complications associated with diabetes, including retinopathies, neuropathies, and nephropathies, may be avoidable or treatable. The kinetics of DHA-glucose inhibition suggest that the cell-specific scorbutic effects of hyperglycemia can be corrected in diabetic patients by increasing their ascorbate intake without interfering with glucose uptake by brain or other critical organ systems (Fig. 2 and Table 2). We can solve the equation given above for the concentration of ascorbate that

will correct the velocity of DHA entry into GLUT-dependent cells in poorly controlled diabetic patients. The plasma concentration of ascorbate required to rectify DHA entry in the presence of 8 mM glucose (a typical baseline glucose level for poorly regulated diabetes) is calculated to be 100–120 mM. Renormalization of DHA entry into cells during severe hyperglycemia should be achievable with plasma ascorbate levels of 150–250 mM. Such concentrations are within the range of clinical possibility: steady-state levels plasma concentrations of 150750 mM have been achieved transiently with supplementation of 1 or more grams of vitamin C

H 20 +

GLUCOSE

DHA AA+ O-

DHA GLUT

GLUCOSE

SORBITOL

AO

DHA +H2O

AR

NADPH

NE Dβ H

AA +O-

NADP+

AGEs

OCT TYR DOP

MELANINS

Fig. 2. Simplified schema of the proposed effects of ascorbic acid (AA) supplementation on cell metabolism in cell types dependent exclusively on GLUT-mediated uptake of dehydroascorbic acid (DHA) in diabetes. Significantly increasing plasma AA concentrations results in increased production of dehydroascorbic acid (DHA) production via ascorbate oxidases (AO) such as serum albumin and ceruloplasmin (Mouithys-Mickalad et al., 1998). DHA competes with glucose for GLUT, lowering the influx of glucose into the cells. Decreased glucose uptake lowers production of sorbitol via aldose reductase (AR), decreases protein kinase C activity (not shown) and also advanced glycolsylation end-products (AGEs), thereby decreasing cytopathological processes. Meanwhile, the increase in DHA transport relative to glucose results in higher levels of conversion into AA within cells, stimulating dopamine beta hydroxylase (DBH) activity. Increased DBH activity, in turn, increases the rate at which dopamine (DOP) and tyramine (TYR) are transformed into their respective metabolites norepinephrine (NE) and octopamine (OCT), optimizing neuronal function. Melanin production and the incorporation of melanins (including lipofuscins) into AGEs are minimized, as are the cytopathological consequences. See Table 2 for additional metabolic effects of this system and the text for references.

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Table 2 HYPOTHESIZED SPECIFIC AND COMMON METABOLIC CONSEQUENCES OF DEHYDROACORBIC ACID COMPETITIVE INHIBITION OF GLUCOSE UPTAKE ON CELLULAR METABOLISM IN DIABETES DHA Inhibition of Glucose Uptake

Cellular Metabolism

Common Metabolism

-

activation of protein kinase C

-

-

oxido-reductive stress

-

growth factor/cytokine release

-

polyol metabolism

-

“up-regulation” of glucose

-

nitric oxide formation

-

advanced glycosylation

deposition -

transport

-

lipofuscin

adrenergic metabolism

end-products

increased DHA uptake

Note: See Fig. 2 for details of the mechanism and the text for references

per day (Sinclair et al., 1991; Blanchard et al., 1997). Higher levels can be achieved by intravenous administration of AA (Kodama et al., 1993). We note that it would probably be unwise to administer high doses of DHA intravenously since such treatments have been shown to cause membrane disruption in erythrocytes and renal brush border membranes (Bianchi & Rose, 1986b) and may be toxic to pancreatic beta cells (Rose & Bode, 1993). Ascorbate supplementation itself has, however, already been shown to decrease retinopathyassociated metabolic alterations in diabetic rats (Kowluru et al., 1997) and improve the clinical signs of diabetic retinopathy (Kowluru, 2001). Vitamin C supplementation, but not vitamin E, significantly improves glomerular size and function in diabetic rats (Craven et al., 1997). Moreover, vitamin C supplementation of human patients has been shown to reduce cellular sorbitol concentrations significantly (Bode et al., 1993; Will & Byers, 1996). We predict that adequate ascorbate supplementation should protect both experimental animals and human patients against many aspects of retinopathies, neuropathies, and nephropathies. Preliminary Experimental Tests of Hypothesis Although DHA-glucose competition has been well-studied in lymphocytes and oocytes, no

studies have yet been performed in cells of tissues associated with diabetic pathologies. In particular, it is necessary to demonstrate that such tissues are, as required by our hypothesis, dependent on GLUT transport of DHA rather than a sodium-dependent AA mechanism of ascorbate uptake. In order to test the plausibility of our hypothesis, we therefore performed preliminary experiments using primary cell cultures of human retinal pigmented epithelial (hRPE) cells and human retinal vascular endothelial (hRVE) cells, both of which are cell types well-known to be adversely affected during diabetic retinopathies (Henry et al., 2000). Both types of cells responded to DHA similarly, so only the hRPE cell data will be reported here and should be taken to be representative of the hRVE cells as well. hRPE cells were established by a modification of the method of Del Monte & Maumenee (1980) as described previously (Henry et al., 1993) and cultured in RPMI-1640 medium supplemented with 15% fetal calf serum, 1% penicillin/streptomycin and 5.5 mM glucose as described previously. Initial rates of glucose transport were determined using [3H]-3-O-methylglucose as a tracer. The non-transported stereoisomer [14C]-lglucose was used simultaneously to determine non-specific association of hexose with the cell layer. hRPE cells were rapidly washed and then

DIABETIC COMPLICATIONS, DEHYDROASCORBATE AND GLUT

6 DHA, 1 mM 5

_

_

pmol DHA mg protein 1 2 1 min

7

4 3 2

AA, 1mM

1 0 bgnd

0

0

5.5

11

22

Glucose concentration (mM)

Fig. 3. Competition of glucose and DHA for transport into human retinal pigment epithelial (hRPE) cells. (a) Ascorbic acid (AA, (&), 1.0 mM) and dehydroascorbic acid (DHA, (N), 1.0 mM) uptake by hRPE cells in the presence of 0-22 mM glucose. hRPE cells are clearly DHA-dependent and hyperglycemia clearly interferes with DHA uptake by hRPE cells. Note that AA and DHA concentrations are above those observed physiologically, so that hyperglycemia can be expected to have an even greater inhibition of DHA uptake in vivo.

0.035

_1

s

0.04

_

nmol glucose mg protein 1 12

incubated at 371C with phosphate-buffered saline (PBS) containing 5.5 mM glucose, 3.26 mCi ml1 of [3H]-3-O-methylglucose and 0.0022 mCi ml1 of [14C]-l-glucose, and 0– 100 mM of AA or freshly prepared DHA for 12 s. The uptake was stopped with ice-cold PBS containing 10 mM cytochalasin B, the cells were solubilized with 0.05 N NaOH. An aliquot was used for beta scintillation counting in 3H and 14C windows, and the remainder was saved for protein determination. Initial rates of DHA and AA uptake were determined as follows. RPE cells were rapidly washed and incubated for 2 min with 1 mM of 14 C-labelled AA or DHA (0.5 mCi ml1) in a buffer containing 0–22 mM glucose. The uptake was stopped with an ice-cold PBS containing 10 mM cytochalasin B. The cells were solubilized in 0.05 N NaOH, an aliquot was used for beta scintillation counting in 14C window, and the remainder was saved for protein determination. DHA was prepared by bromine oxidation method just before each experiment (Washko et al., 1993).

353

0.03 0.025 0.02 0.015 0.01 0.005 0 0

0.01 0.1 1 10 DHA concentration (mM)

100

Fig. 4. Glucose uptake (at 5.5 mM) by hRPE cells in the presence of 0–100 mM dehydroascorbic acid (DHA). DHA clearly competes with glucose for entry into hRPE cells such that physiologically attainable levels of DHA (ca. 100 mM) result in a 40% decrease in glucose entry. Thus, DHA has the potential to decrease hyperglycemia-associated oxidative stress in these cells concomitant with increasing internal ascorbate accumulation.

The results of these experiments are shown in Figs. 3 and 4. Fig. 3 demonstrates that hRPE cells are DHA dependent. AA entry into the cells was not significantly different from background noise. At a DHA concentration of 1 mM, increasing concentrations of glucose competitively inhibited DHA entry into the cells such that the accumulation of DHA was half maximal at about 20 mM glucose. These data (as well as the similar data on hRVE cells that is not shown) demonstrate that hyperglycemia will decrease DHA entry into at least some end-organ target tissues in diabetes, as predicted. Thus, the mechanism we have proposed for injury to end-organ tissues is plausible. Figure 4 demonstrates that increasing DHA concentrations in the presence of a constant concentration of glucose (5.5 mM) results in a dose-dependent decrease in glucose entry into hRPE cells; 100 mM DHA, which is physiologically attainable (see above) produced a 40% decrease in glucose entering cells. These data (and the similar data on hRVE cells that is not shown) again support the prediction that increasing DHA levels in diabetic patients may

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protect end-organ tissues from glucose-associated oxidative stress and concomitant ascorbate deficiency, thereby helping to spare these tissues from pathogenic consequences. Clearly, further kinetics experiments and experiments on other primary cell cultures derived from target tissues of diabetic pathologies are necessary, as well as animal and human clinical studies, before any definite conclusions regarding the potential efficacy of DHA supplementation for diabetics can be considered reasonable, but we view these preliminary results as encouraging. Other Testable Mechanistic Implications of the Hypothesis Our hypothesis has many testable implications. Hyperglycemic conditions should lead to oxidative stress observable as measurable increases in ROS, accumulation of melanins such as lipofuscins, and decreases in catecholamine metabolism within tissues targeted during diabetic pathologies. These hyperglycemia-induced metabolic alterations within cells should be preventable or correctable by exposing the cells to higher levels of DHA. At the cellular level, kinetic studies of glucose and DHA transport can be performed using in vitro cell model systems of the blood retinal barrier (Henry et al., 2000) and the renal mesangial cell (Henry et al., 1999). Cytochalasian B should inhibit facilitative uptake of DHA in these cells as a non-specific inhibitor of GLUTs. Specific competition of glucose and DHA has not been previously demonstrated in cells from end organs affected by diabetes. The predominant GLUTs present in these cells are believed known and direct testing of competition of DHA and glucose can be undertaken. Furthermore, terminal outcome measures of any clinical benefit of AA therapy in the prevention or treatment of end-organ damage should include animal models and eventually human trials of ascorbate therapy in diabetes. Presently, our understanding of isoform-specific GLUTmediated facilitative transporters in end organs affected by diabetes permits testing of our hypothesis in a cell-specific and GLUT-specific approach.

Our hypothesis has other cellular implications as well. One is that cells will differ in their susceptibility to diabetic pathologies, the ones that are most susceptible being those that use GLUT transport of DHA mainly or exclusively as their source of vitamin C. Second, among these GLUT-dependent cells, those most susceptible will be involved in catecholamine metabolism and storage. Thus, various forms of pathology associated with diabetes, such as nephropathy, retinopathy, neuropathy, dementia and immune dysfunction may all owe some measure of their pathology to cell-specific metabolic disruption. A similar hypothesis has been proposed previously for the origins of atherosclerosis and angiopathy in diabetic patients (Mann, 1974; Cunningham, 1988; Fay et al., 1990) and for their increased incidence of periodontal disease (Aleo, 1981). Thus, the considerations that have gone into the calculations made here may have very general applications. Our hypothesis has implications for understanding which diabetic patients are at greatest risk for pathologies and how to lower that risk. It explains why diabetics who rarely have hyperglycemia are observed to be less likely to develop neuropathies, retinopathies and nephropathies than those who frequently experience hyperglycemia (DCCT, 1993). The hypothesis also explains why it has been observed that diabetic patients with low vitamin C intake or borderline scurvy are more likely to develop neuropathies, retinopathies and nephropathies than those with normal or high vitamin C intakes. The hypothesis goes beyond these observations to provide a testable mechanism by which hyperglycemia and low vitamin C levels interact to create pathologies. And most importantly, we predict that protection against neuropathies, retinopathies and nephropathies will be conferred by high dose vitamin C regimens that rebalance glucose and DHA transport by GLUT under hyperglycemic conditions.

Other Implications of the Hypothesis Our model may also apply to other disease processes. For example, glucose uptake is increased in a variety of different cancer cells.

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Many cancers are known to have increased glucose transport and utilization that are dependent on abnormal expression of GLUT1 and other GLUT isoforms and may play a significant role in glucose uptake by these tumors (Spielholz et al., 1997). Furthermore, GLUT1 expression by human carcinomas may indicate an increased glucose uptake, and probably increased utilization of energy, which may correlate with an aggressive behavior. Malignant transformation associated with high levels of GLUT1 expression are considered poor prognosticators of survival in colon, breast, rectal, lung and other cancers. Notably, the administration of ascorbate in the treatment of cancer is presently under investigation by various investigators (e.g. Riordan et al., 1995). Randomized clinical trials of oral vitamin C have failed to demonstrate a beneficial effect in cancer therapy, as might be expected from our model, since it is not possible to raise plasma ascorbate levels high enough to interfere with glucose uptake in most tissues and organs through oral ingestion. Intravenous administration of ascorbate can, however, produce millimolar plasma concentrations, which are toxic to many cancer cell lines. This toxicity of intravenous ascorbate may be due to a variety of mechanisms, including inhibition of facilitative glucose uptake, disruption of glucose utilization, increased oxidation products from the conversion of DHA to ascorbate within the cells, and pH disruption due to these reactions (high dose ascorbate causes urine acidosis, for example [Burns, 1975]). Any or all of these factors might make the cancer more susceptible to chemotherapy. Two final notes. Competitive inhibition has been reported for enzyme-mediated reactions associated with diabetes. Excretion of vitamin C from patients taking megadoses of the vitamin interferes with accurate measurements of urine glucose by test strips (Berg, 1986). Thus, the pathological consequences of competitive transport that we have described here may have broader implications in terms of competitive metabolic processes beyond uptake and transport. Lastly, we suspect that the pathological implications of competitive transport described here will be found to explain other pathological

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