The Polyol Pathway and Glucose 6-Phosphate in Human Endothelial Cells Cultured in High Glucose Concentrations

Diabetologia (1987) 30:222-227 Diabetologia Springer-Verlag 1987

The polyol pathway and glucose 6-phosphate in human endothelial cells cultured in high glucose concentrations

1M. Lorenzi, IS.Toledo, 2G. R. Boss, 1M.J. Lane and tD. F. Montisano Department of Medicine, 1Divisions of Endocrinology and Metabolism and 2General Medicine, University of California San Diego, La Jolla, California, USA

Summary. In an attempt to identify the mechanisms underly- high glucose and Sorbinil for 1-3 days versus 14% inhibition ing the ill effects of high glucose previously described in cul- in cells exposed for 14days, p<0.01). Sorbinil treatment tured human endothelial cells, we have investigated in these failed to improve even slightly the abnormalities in cellular cells the activity of the polyol pathway and accumulation of replication induced by high glucose. The cellular content of glucose 6-phosphate, a powerful agent of non-enzymatic gly- glucose 6-phosphate was augmented 3-fold by exposure to cosylation. Sorbitol accumulation varied among different 20mmol/l glucose (p<0.001). In conjunction with other batches of cells (primary cultures). In presence of 5 mmol/1 studies these results indicate that in this model the polyol glucose the cellular sorbitol content ranged from 0.04 to pathway is not an osmotically or metabolically important 0.12 nmol/106 cells. When cells were exposed to 20 mmol/1 mechanism of glucotoxicity, and that the inhibitory activity glucose the sorbitol content increased by 2- to 3-fold to con- of Sorbinil on the polyol pathway of human tissues may be a centrations of 0.08-0.38 nmol/106 cells (p< 0.01). Addition to function of their length of exposure to hyperglycaemia. The the culture medium of 100 ~tmol/1 Sorbinil, an inhibitor of consequences of intracellular accumulation of glucose aldose reductase, resulted in a substantial inhibition of sorbi- 6-phosphate await investigation. tol accumulation throughout the 14 days in culture, but the degree of inhibition varied inversely with the duration of cell Key words: Diabetes, angiopathy, endothelial cells, glucose, exposure to high glucose (70% inhibition in cells exposed to sorbitol, glucose 6-phosphate, aldose reductase, Sorbinil.

While it is well established that the morphology [1, 21, the present study we have investigated whether the functional integrity [1, 3, 4] and even survival [5, 6] of polyol pathway plays a role in mediating the replica- vascular endothelium are jeopardized in diabetes, the tive abnormalities of human endothelial cells cultured mechanisms responsible for such degenerative changes in high glucose and whether these cells accumulate are still poorly defined. A role for toxic effects of sus- glucose 6-phosphate, which is a most potent effector of tained hyperglycaemia is suggested by the recent dem- nonenzymatic glycosylation [14, 15]. onstration that in vitro exposure of endothelial cells to high glucose levels results in accelerated death, decreased replication and signs of DNA damage [7-9]. Materials and methods There are at least three recognized mechanisms whereby the chronically elevated ambient glucose Cell culture characteristic of diabetes may affect tissue function and structure: enhanced activity of the polyol pathway Primary cultures of endothelial cells were prepared from human um- [10, 11], accelerated nonenzymatic glycosylation [12] bilical veins as previously described [7, 16]. For proliferation experiments, cells were seeded at a density of 1 x 105 in 35 mm dishes (Fal- and protracted hypertonicity [13]. Although the replica- con, Oxnard, Calif, USA); for metabolic measurements cells were five delay induced in human endothelial cells by high seeded at 4 x 105 in 60 mm dishes (Falcon). The growth medium was glucose can to some extent be mimicked by increasing Medium 199 (Gibco, Grand Island, NY, USA), supplemented with medium osmolarity with mannitol, the magnitude of glutamine (2 mmol/1), Hepes buffer (17.5 mmol/1), 10 ng/ml epider- mal growth factor (EGF culture grade, Collaborative Research Inc., the mannitol effect is much less than that observed Lexington, Mass, USA) and 14% heat-inactivated pooled human se- with equimolar glucose [7]. Therefore, effects specific rum. Incubation volumes were 2 ml for 35 mm dishes and 4 ml for to the glucose molecule itself must be advocated. In 60 mm dishes. M. Lorenzi et al.: The polyol pathway in human endothelial cells 223

Cultures were incubated at 37 ~ in a humidified 5% CO2-95% erythrocytes were sedimented by centrifugation, washed twice with air atmosphere and all media were filtered and supplemented with PBS and aliquots precipitated with 3 volumes of chilled 6% per- penicillin (50U/ml), streptomycin (50btg/ml) and fungizone chloric acid. The remainder of the extraction procedure was as de- (0.25 l*g/ml) purchased from Irvine Scientific, Santa Ana, Calif, scribed above for endothelial cells. USA. Media were changed every third day. At regular intervals, the We had chosen to perform sorbitol measurements with the enzy- endothelial cell monolayers were tested with FITC-conjugated goat matic method based on the activity of sorbitol dehydrogenase [18]. anti-factor VIII related antigen antibodies (Atlantic Antibodies, The specificity of sorbitol dehydrogenase (L-iditol: NAD 5-oxidore- Scarborough, Me, USA) and always found to be brightly and uni- ductase EC 1.1.1.14 from sheep liver; Sigma) was tested against glu- formly stained. The pH of all incubated cultures varied between 7.2 cose, galactitol, mannitol, fructose, myoinositol and xylitol (all pur- and 7.35. chased from Sigma). Reactivity of the enzyme with glycerol is known To study the effects of high glucose concentrations the growth to be very low [19] and was not checked. As expected [19], all the medium was supplemented with D-glucose (Mallinckrodt, Inc., Pa- compounds tested yielded only background readings even at 20 ~tg/ ris, Ky, USA) to achieve a final concentration of 20 mmol/1. Expo- ml concentration, with the exception of xylitol, which reacted in the sure to high glucose was started the day after plating or at the indi- assay exactly as sorbitol. We thus tested endothelial cells for the cated times before cell harvest. Glucose concentration in control presence of measurable amounts of xylitol by enzymatic and chro- medium was 5 mmol/1. matographic methods. The enzymatic measurement of xylitol was In experiments addressing the effect of aldose-reductase inhibi- carried out as previously described [20] employing xylitol: NADP tion on polyol accumulation and endothelial cell proliferation, Sorb- 4-oxidoreductase L-xylulose forming (EC 1.1.1.10 from pigeon liver, inil (kindly provided by Pfizer Research, Groton, Corm, USA) was Sigma). GLC separation, was performed in a Varian Model 3700 Gas added to the growth medium in concentrations of 2, 4, 10 and Chromatograph on a 3% SE30 column (Supelco Inc., Bellefonte, Pa, 100 ~tmol/1. Additions were performed either the day after plating USA) at a flow rate of 50 ml/min and temperature increments of and renewed at every medium change, or at the indicated times prior 2 ~ (140-+215 ~ HPLC separation was performed in a Spec- to cell harvest. Sorbinil was suspended in normal saline and solubi- tra-Physics 8700 instrument equipped with a U.V. detector, employ- lized by sonication after titrating to pH 9.5 with 0.1 N NaOH. The ing a 300 x 7.8 mm Aminex HPX-87 H organic acid column (Biorad, solution was then back titrated to pH 7.4 with 0.1 N HC1. The low Richmond, Calif, USA) which was maintained at 40 ~ equilibrated concentrations of Sorbinil (2-10 p~mol/1) were chosen on the basis of in 30% buffer A (10 mmol/1 H2SO4) and 70% buffer B (purified the ICs0 of this compound on human placenta aldose reductase [17]; HPLC water) and eluted in the isocratic mode at a flow rate of the 100 ~tmol/1 concentration was employed after witnessing the in- 0.5 ml/min. The HPLC effluent was monitored at 195 nm [21]. effectiveness of the lower concentrations. The stability of Sorbinil in The glucose 6-phosphate (G6P) content of cellular extracts was the tissue culture medium was checked by testing the effect of measured spectrophotometrically at 320 nm by following the reduc- 100 ~tmol/1 Sorbinil, incubated with endothelial cells for 3 days in tion of NADP to NADPH in the presence of 50 mmol/1 Tris, presence of 20 mmol/1 glucose, on the accumulation of sorbitol by pH 7.35 and 1 U of glucose 6-phosphate dehydrogenase (EC human erythrocytes. 1.1.1.49, Sigma) in a final volume of 0.2 ml [22]. An enzyme blank, all components except the sample, and a sample blank, all components except the enzyme, were included and had an absorbance change Measurements that was less than 5% of the absorbance change of the full mixture. In proliferation experiments addressing whether treatment with Sorbinil would prevent or reduce the proliferative delay induced by Statistical analys& 20 mmol/1 glucose, cell counts were performed every other day from day 4 to day 12-14 in culture. The procedures employed have been Data are presented as mean + SD in the indicated number of experi- previously described [7]. ments. In each experiment determinations were obtained in dupli- Metabolic intermediates were measured in neutralized perchloric cate-triplicate. Statistical analysis was performed by analysis of vari- acid extracts of endothelial cells and, in selected experiments, of hu- ance and by linear or polynomial regression (time-course of man erythrocytes. For preparation of extracts, cell culture dishes inhibition of sorbitol accumulation by Sorbinil). were cooled on ice, the monolayers were washed twice with phosphate-buffered saline (PBS, 0.15 mol/l, pH 7.4), the wash fluid was carefully and completely aspirated and 100 pJ of chilled 6% perchloric acid was added to each dish in order to precipitate cellular Results material, which was harvested with a rubber policeman. On the basis of cell counts obtained from companion dishes, the appropriate number of dishes from each treatment were pooled to yield the de- Since sorbitol dehydrogenase, the enzyme employed to sired cell number (at least 4 • 106 endothelial cells for each polyol as- measure cellular sorbitol content, showed substrate say and 20 x 106 cells for each glucose 6-phosphate assay). The har- cross-reactivity with xylitol, we tested human endothe- vested material was sonicated and centrifuged. The supernatant was lial cells for the presence of measurable amounts of neutralized with K2CO3 (2 mol/1), the excess salt removed by centrif- xylitol. The enzymatic assay based on xylitol:NADP ugation and the supernatant filtered through a 0.45 ~tm membrane. When subjected to high-performance liquid chromatography 4-oxidoreductase yielded concentrations of xylitol of (HPLC) or gas-liquid chromatography (GLC) the cellular extracts 0.20+0.06 nmol/106 cells. When, however, the same were further purified by application to a column of mixed acidic cat- cellular extracts were tested by GLC or HPLC no xyli- ion and basic anion exchange resin (Dowex MR-3, Sigma Chemical tol peak was observed. A graphic demonstration of the Co., St. Louis, Mo, USA) and then evaporated under vacuum. Before application to the HPLC column the sample was resuspended in absence of a xylitol peak in HPLC tracings clearly 30 pJ of HPLC grade water (Fisher Scientific Co., Fair Lawn, NJ, showing a sorbitol peak is provided in Figure 1. Since USA). For GLC analysis the sample was resuspended in 200 ~tl of the same pattern was evident in GLC separation, we distilled water, dried under nitrogen and derivatized with 50 ~tl of concluded that the enzymatic assay for xylitol was trimethylsilyl reagent (Sigma). When erythrocytes were employed, measuring some compound other than xylitol and that these were obtained from heparinized blood collected from normal volunteers, washed three times with PBS and incubated in 3 volumes in fact, under our culture conditions, human endotheli- of the same culture medium employed for endothelial cells and con- al cells do not accumulate detectable amounts of xyli- taining 5 or 20 mmol/1 glucose. After 4 h of incubation at 37 ~ tol. On the basis of the additional specificity studies re- 224 M. Lorenzi et al.: The polyol pathway in human endothelial cells

165

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Fig. 1. HPLC tracings of (a) standards for glucose, ,% sorbitol and xylitol; (b) extracts of cells cultured in 5 mmol/1 glucose; (c) extracts of cells cultured in 5 mmol/1 glucose + Sorbinil; (d) extracts of cells cultured in 20 mmol/1 glucose; (e) extracts of cells cultured in 20 retool/1 glucose + Sorbinil. Duration of all treat- t/3 ments was 14 days. Abscissa:time; ordinate: intensity of signal detected at 195 nm; arrows: Sorbitol peak (12.5-12.7 rain) ported in the Methods section, it could thus be Addition to the culture medium of 100 ~tmol/1 assumed that the readings obtained with sorbitol dehy- Sorbinil, an inhibitor of aldose reductase, resulted in a drogenase quantitatively represented true sorbitol. substantial inhibition of sorbitol accumulation by en- Sorbitol accumulation by human endothelial cells dothelial cells. It was, however, noted (Table 1) that the in primary culture was quite variable among different degree of inhibition achieved varied inversely with the batches of cells. In presence of physiologic glucose lev- duration of cell exposure to high glucose (r= -0.957, els cellular sorbitol content ranged from 0.04 to p= 0.01), While in cells exposed to high glucose and 0.12 nmol/t06 cells. When cells were exposed to glu- Sorbinil for 24 h sorbitol accumulation was inhibited cose levels mimicking diabetic hyperglycaemia by 71%, in cells exposed for 14 days inhibition was on- (20 nmol/1) the sorbitol content increased by 2- to ly 14% (p<0.01). That the declining efficacy of Sorbi- 3-fold to concentrations of 0.08-0.38 nmol/106 cells nil was a function of length of cellular exposure to (p < 0.01). high glucose and not of loss of inhibitory activity of M. Lorenzi et al.: The polyol pathway in human endothelial cells 225

Table 1. Sorbitol accumulation in human endothelial cells exposed the drug was verified in two ways. First, we compared to high glucose and effect of an aldose reductase inhibitor (Sorbinil the effect of freshly prepared Sorbinil versus Sorbinil 100 ~tmol/1) incubated with endothelial cell cultures for three days Glucose in medium/ Sorbinil/ Sorbitola Inhibition (the maximum interval between medium changes) on duration duration nmol/106 cells by Sorbinilb the accumulation of sorbitol by human erythrocytes mean + SD (RBC). Sorbitol content of RBC incubated for 3 h at 5 mmol/l 14 days - 0.053-I-_ 0.006 89% 37~ in presence of 5 mmol/1 glucose was 7.7+ 5mmol/1 14days + 24h 0.006+0.001 2.6 nmol/ml RBC and increased to 14.6__+0.9 nmol/ml 20 retool/1 24 h - 0.100 + 0.006 71% RBC in presence of 20 mmol/1 glucose. The addition 20 mmol/1 24 h + 24 h 0.029 + 0.006 of Sorbinil reduced this amount to 8.8 ___0.8 nmol/ml 20 mmol/1 3 days - 0.090+_ 0.02 70% RBC (fresh Sorbinil) and to 8.4+0.3 nmol/ml RBC 20 mmol/1 3 days + 3 days 0.027_+ 0.01 (incubated Sorbinil). Second, we contrasted the effects 20 mmol/1 8 days - 0.086 + 0.01 59% of chronic Sorbinil treatment (14 days) on sorbitol ac- 20 mmol/1 8 days + 8 days 0.036 + 0.008 cumulation by cultures exposed to 5 or 20 mmol/1 glu- 20 mmol/1 14 days - 0.082 + 0.008 14% cose for 14 days (Fig.l). While the HPLC tracing of 20 retool/1 14 days + 14 days 0.071 +_ 0.002 cells cultured in 5 mmol/1 glucose showed, in the pres- Cells ,were studied for sorbitol content after 12-14 days in culture and ence of Sorbinil, obvious disappearance of the sorbitol after the indicated duration of treatments, aValues represent mean + peak, the latter was practically unmodified in cells cul- SD of determinations obtained in three experiments, bThe correlation tured in 20 mmol/1 glucose in the presence of Sorbinil. coefficient between duration of exposure to high glucose (days) and No attempts at quantitation of sorbitol were made in percent inhibition of sorbitol accumulation by Sorbinil was -0.957 (p=0.01) these experiments, in which the amount of cellular extracts derived from the 5 mmol/1 glucose cultures was maximized in order to ensure a detectable sorbitol sig- Table 2. Intracellular accumulation of glucose 6-phosphate in hu- nal. man endothelial cells cultured in high glucose We had previously documented [7] that in endothe- Culture conditions Glucose 6-phosphate lial cell cultures exposed to 20 nmol/l glucose the (nmol/106 cells) achievement of saturation density is substantially de- 5 mmol/1 glucose 0.23 _+_0.11 a layed, with cell counts throughout the growth curve be- 20 mmol/1 glucose 0.70 _+ 0.08 a p < 0.001 ing 70-80% of control. We thus investigated whether 20 mmol/l glucose 0.21 + 0.09b the reduction in sorbitol content achieved with Sorbinil switched to 5 mmol/l for 5 h would modify the proliferative delay induced by expo- aValues represent the mean of 6 experiments_ 1 SD. bValues repre- sure to high glucose. As illustrated in Figure 2, none of sent the mean of 2 experiments_+ 1 SD the concentrations of Sorbinil tested (2-100 lxmol/1) succeeded in modifying the hindering effects of high glucose on endothelial cell proliferation. Both in the 12 presence or absence of Sorbinil the growth curve of the cultures exposed to high glucose differed significantly 10 !"T from control (p< 0.005). The intracellular accumulation of glucose 6-phos- ~ 8 phate (Table 2) was enhanced 3-fold in endothelial q~ , I .Q cells cultured in high glucose: 0.70 _+ 0.08 nmol/ E 6 10 6 cells versus 0.23 _+ 0.11 in controls (p< 0.001). In or- z der to exclude that the increased G6P found in high to 4 glucose cultures m~ght be contributed by G6P released from cell membranes during the preparation of extracts, we measured G6P in extracts of endothelial cells that - after having been chronically exposed to 20 mmol/1 glucose - were switched to culture medium I-.~- I I I I I I I.--.~,/ I I I I 0 4 6 8 10 12 14 0 4 6 8 10 12 containing 5 mmol/1 glucose for 5 h. The content of Days in Culture G6P in these cells (Table 2) was the same as in cells Fig,2. Sorbinil treatment (2-10 Ixmol/1 left panel, 100 ~mol/1 fight chronically exposed to 5 mmol/1 glucose, thus exclud- panel) failed to prevent the proliferative delay observed in human en- ing that G6P detected in extracts of cells cultured in dothelial cells cultured in high glucose ( glucose 5 mmol/1; high glucose could derive from dissociation of the sug- glucose 5mmol/l+Sorbinil; [] glucose 20mmol/l; glucose ar from adducts possibly formed with cellular con- 20 retool/1 + SorbiniI) Each point represents the mean + SD of the de- stituents. When reported to cellular protein content terminations obtained in five experiments. The proliferation curves of cells exposed to 20 mmol/l glucose in presence or absence of Sorbinil (- 200 ].tg/106 cells) the amount of G6P found in endo- are significantly different (p< 0.005 by ANOVA) from those of cells thelial cells was substantially higher than that reported exposed to 5 retool/1 glucose in the literature for human erythrocytes (~ 30 nmol/ml 226 M. Lorenzi et al.: The polyolpathway in human endothelialceils

RBC, 1 ml RBC = 371 nag protein). We thus tested the It cannot be determined from these studies whether accuracy of our assay procedure by measuring G6P the minimal extent of polyol pathway activation by high content in human RBC. The G6P content of RBC incu- glucose is a function of tissue culture conditions, type of bated for 3h in 5mmol/1 glucose was 39+ endothelial cells studied or human origin of the cells. 5 nmol/ml RBC, and is in good agreement with pub- The latter characteristic is, however, likely to be impor- lished data [23, 24]. tant, since vascular endothelial cells of the human retina do not stain for aldose reductase [29], and aldose reductase from other human tissues (lens) shows a higher km for glucose than the enzyme present in other species Discussion [301. Exposure of endothelial cells to Sorbinil uncovered Abnormalities of function and structure of vascular en- an aspect of Sorbinil action of potential relevance to its dothelium are likely to represent a central pathogenetic clinical applications. This was the progressively lesser event in the development of diabetic angiopathy [1-6], inhibition of sorbitol formation achieved by Sorbinil and clarification of the mechanisms of endothelial cell with increasing time of exposure of endothelial cells to injury should assist in developing rational strategies for higla glucose. Since the', phenomenon could not be clinical intervention. Although the tissue culture envi- ascribed to diminished intrinsic potency of the drug, it ronment is quite remote from the complex and dynamic may represent a consequence of the "activation" of hu- in vivo milieu, it offers the opportunity to study vascular man aldose reductase. Das and Srivastava have shown endothelial cells of human origin not otherwise readily [31] that, in human tissues chronically exposed to high accessible. glucose, aldose reductase changes its kinetic properties We have previously shown [7, 8] that human endo- and becomes less sensitive than in its native form to in- thelial cells in primary culture are susceptible to "glu- hibition by various drugs, including Sorbinil. Together cose toxicity" as manifested by the occurrence of accel- with the low activity of the polyol pathway in human erated death, decreased replication and DNA damage tissues this phenomenon may contribute to the difficul- upon exposure to high glucose concentrations. The ties in establishing a clear-cut effectiveness of aldose re- combined results obtained in this and other studies in- ductase inhibitors in the treatment of diabetic complica- dicate that the polyol pathway is not of major impor- tions in humans. tance in mediating the ill effects of high glucose ob- In spite of the saturation kinetics of hexokinase, ac- served in this model. The amount of intracellular cumulation of glucose 6-phosphate (G6P) appears to be sorbitol accumulated in the presence of high glucose - a common occurrence in tissues obtained from diabetic in good agreement with previous observations [251 - humans or animals; the phenomenon can be ascribed to was two orders of magnitude below that expected to the relief by high ambient glucose of the G6P inhibition possibly increase intracellular osmotic pressure [10]. It of hexokinase [24]. The amount of G6P found in endo- could be argued that measurements of intracellular thelial cells cultured in physiologic glucose was almost sorbitol content might grossly underestimate sorbitol an order of magnitude above that reported for erythro- production if leakage of the metabolite to the medium is cytes [23], brain [13] and kidney [32], but superimposable substantial. From studies in other tissues it is, however, to that found in rat myocardium [33]. The high G6P apparent that, when production of sorbitol is high, this content of endothelial cells could not be attributed to continues to be mirrored in the intracellular sorbitol inaccuracy in the assay nor to release of G6P from pos- content in spite of obvious signs of cellular leakage [26]. sible adducts; it is unclear at this time whether it repre- A more important consideration is that intracellular sents a tissue-related characteristic (as in myocardium) sorbitol content does not in itself reflect other possible or a consequence of the in vitro incubation conditions. ill effects of enhanced polyol pathway activity, the best Nevertheless, the almost 3-fold increase in G6P ob- recognized of which is compromised energy balance served upon exposure to high glucose indicates that en- secondary to myoinositol depletion [11]. In various tis- dothelial cells respond akin other tissues to ambient sues [11, 26, 27] such ill effects have uniformly been pre- glucose levels. Owing to its open ring structure G6P is a vented by Sorbinil treatment. In human endothelial powerful "glycosylator", ten times as potent as glucose cells, however, treatment with Sorbinil failed to improve on a molar basis [14, 15]. Additionally, reducing sugars even slightly the proliferative delay induced by high have been shown to undergo auto-oxidation in aqueous glucose, despite reducing cellular sorbitol content to milieu with consequent generation of free radicals and concentrations below control levels throughout a good intermediates of dioxygen reduction, and, albeit less po- portion of the growth curve. Moreover, a major role for tent than other monosaccharides, G6P is also in this re- myoinositol depletion in the replicative delay of human spect more potent than glucose [34]. Thus, either endothelial cells exposed to high glucose has been spe- through non enzymatic glycosylation or through free cifically excluded by studies involving supplementation radicals production, accumulation of G6P might dam- of the culture medium with supraphysiologic concen- age intracellular constituents and, among them, nucleic trations of myoinositol [28]. acids [35, 36]. We are particularly interested in exploring M. Lorenzi et al.: The polyol pathway in human endothelial cells 227 this possibility to explain the replicative delay [7, 9] and 17. Kinoshita JH, Fukushi S, Kador P, Merola LO (1979) Aldose re- DNA damage [8] observed in vascular endothelial cells ductase in diabetic complications of the eye. Metabolism 28: maintained in a high glucose milieu, since these abnor- 462-469 18. Malone JI, Knox G, Benford S, Tedesco TA (1980) Red cell sorbi- malities could conceivably be prodromes of the de- tol, an indicator of diabetic control. 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