[ H]Inositol Uptake by Glucose and Sorbitol in Cultured Bovine Lens

Modulation of Myo-[3H]inositol Uptake by Glucose and Sorbitol in Cultured Bovine Lens Epithelial Cells

/. Restoration of Myo-inositol Uptake by A/dose Reductase Inhibition

Patrick R. Cammarara,* Hai-Qing Chen,* Jinhua Yang,* and Thomas Yoriot

The association between high-ambient glucose, the polyol pathway, and aldose reductase inhibition on in vitro myo-[3H]inositol uptake was examined in cultured bovine lens epithelial cells (BLECs). Myo- [3H]inositol accumulation in the presence of 5.5 mmol/1 D-glucose was rapid and linear for 8 hr. When Na+ was replaced on an equal molar basis with N-methyl-D-glucamine chloride, myo-[3H]inositol uptake was reduced by more than 95%. The myo-inositol transport system appear to be distinct from glucose transport, based upon three criteria: (1) 2-deoxy-D-[3H]glucose uptake, unlike myo- [3H]inositol uptake, was largely sodium independent; (2) L-glucose was a competitive inhibitor of myo-[3H]inositol uptake but had no effect on 2-deoxy-D-(3H]glucose uptake; and (3) 2-deoxy-D- [3H]glucose uptake appeared independent of myo-inositol concentration. Sodium-dependent myo- [3H]inositol uptake was substantially inhibited after chronic (20 hr) exposure of cultured cells to 40 mmol/1 glucose. Inhibition of aldose reductase activity partially prevented the inhibitory effect of glucose on myo-[3H]inositol accumulation. No significant difference in the rates of passive efflux of myo-|3H]inositol from preloaded high glucose-treated and control cultures was observed. Although the coadministration of sorbinil to the high-glucose medium partially protected against the attendant decrease in transport activity, the failure to normalize myo-[3H]inositol uptake suggested that glucose- sensitive and sorbitol-sensitive processes were involved in the uptake of myo-inositol. Invest Ophthal- mol Vis Sci 33:3561-3571,1992.

Increased aldose reductase activity and reduced tis- PKC substrates, including maintenance of Na+- sue myo-inositol are believed to contribute to early- K+-ATPase activity. Such conditions might explain onset diabetic complications in the lens and other tis- the underlying cause of diabetic complications. sues.1"5 Hyperglycemia promotes polyol accumula- Greene et al postulated that disruption of Na+- tion, which, by unknown mechanisms, causes a K+-ATPase activity is associated with myo-inositol reduction in intracellular myo-inositol content.6"8 depletion and the onset of diabetic neuropathy.9 How- Myo-inositol depletion could lead to a deficit in myo- ever, the correlation that diabetes mellitus causes in- inositol-containing phospholipids, which could se- creased polyol pathway activity associated with de- verely affect diacylglycerol mass and inositol trisphos- creased tissue-free myo-inositol has been inconsis- phate release. Alterations in diacylglycerol formation tent.10 or dysfunction in the release of normal second messen- To identify the cellular mechanisms by which exper- gers ultimately could lead to a decrease in protein ki- imental diabetes, coincident with polyol accumula- nase C (PKC) activation and a reduction in activated tion and myo-inositol depletion, might impair normal lens function, a reliable in vitro parameter should be identified. Recently, we reported that galactose in- From the Departments of *Anatomy and Cell Biology and fPhar- hibits the ouabain-sensitive uptake of myo-inositol." macology, Texas College of Osteopathic Medicine/University of The coadministration of the aldose reductase inhibi- North Texas, Fort Worth, Texas. tor sorbinil to the high-ambient galactose medium ap- Supported by National Public Health Service Award EY05570- 05 (PRC). parently corrected the attenuated myo-inositol up- This work represents partial fulfillment of the requirements for take. The intracellular concentration of galactitol was the degree of Master of Science for Hai-Qing Chen. relatively low under those experimental conditions, Submitted for publication: March 25, 1992; accepted July 16, suggesting that myo-inositol uptake was sensitive to 1992. low levels of polyols. That is, the myo-inositol trans- Reprint requests: Patrick R. Cammarata, Texas College of Osteo- pathic Medicine, Department of Anatomy and Cell Biology, Fort port system must be extremely sensitive to the con- Worth, TX 76107. centration of intracellular polyol or indirectly sub-

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jected to modulation by the aldose reductase reaction. Myo-inositol Accumulation Interpretation of the results necessarily were limited because those preliminary experiments were per- Extracellular myo-inositol uptake was determined formed in a growth medium that contained a consis- as follows. The cultured cells were divided into groups tent myo-inositol concentration of approximately 15 and the medium was replaced with one of the follow- /umol/1. Additional experiments, employing a concen- ing: physiological medium (5.5 mmol/1 glucose, tration range of myo-inositol, was needed to uncover MEM); physiologic medium containing 5.5 mmol/1 obscure relationships between elevated intracellular glucose further supplemented with 34.5 mmol/1 fruc- polyol content and myo-inositol transport, which is tose; 40 mmol/1 glucose; or 40 mmol/1 galactose not readily observed using a single dose of extracellu- (Sigma, St. Louis, MO). All culture media contained lar myo-inositol. approximately 15 jimol/1 myo-inositol. The cultures were maintained under these conditions for 20 hr The studies reported here examined the effect of 3 high-ambient glucose on myo-[3H]inositol uptake us- before myo-[ H]inositol was added to the serum- supplemented media. The accumulation of myo- ing a myo-inositol concentration range of 1.5-400 3 jumol/1. The effect of sorbinil, an aldose reductase in- [ H]inositol was achieved by incubating the cultured cells in the presence of 0.25 /iCi/ml medium myo- hibitor, on myo-inositol uptake in cultured bovine 3 lens epithelial cells (BLECs) exposed to high-ambient [ H]inositol (94 Ci/mmol; Amersham, Arlington glucose was investigated under these conditions. We Heights, IL) for up to 8 hr. After isotope incubation, the medium was removed and the culture flasks were show that myo-inositol uptake was inhibited by glu- 2+ cose, but also was associated with intracellular sorbi- rinsed three times with ice-cold Ca -added phos- tol accumulation, suggesting that at least two mecha- phate-buffered saline (137 mmol/1 NaCl, 8 mmol/1 nisms were involved in the glucose-induced inhibi- dibasic sodium phosphate, 0.7 mmol/1 calcium chlo- tion of myo-inositol transport in cultured BLECs. ride, pH 7.2) and drained overnight at 4°C. Five milli- liters of 2% sodium carbonate in 0.1 N sodium hydroxide was added to each flask and left overnight at Materials and Methods room temperature to ensure cell lysis. Replicate 1.0 ml aliquots were taken for liquid-scintillation count- Cell Culture ing (Packard TriCarb 4640, Laguna Hills, CA). Tripli- cate 25 ix\ aliquots were taken for protein determina- BLECs were isolated and cultured, as previously 13 described by Cammarata et al.12 Cells were main- tion by the method of Bradford et al. with bovine serum albumin (Sigma) as standard. Accumulation of tained in a water-humidified atmosphere of 5% CO2/ 3 95% air at 37°C in Eagle's minimal essential medium myo-[ H]inositol was expressed as counts per minute (MEM), which contained 5.5 mmol/1 glucose supple- per milligram of protein in individual culture flasks. mented with 10% bovine calf serum, 20 mg/L genta- To measure passive efflux, BLECs were preloaded mycin sulfate, 5 mg/L ascorbic acid, nonessential for 4 hr with the appropriate serum-supplemented me- amino acids, and basal medium Eagle vitamin solu- dium in the presence of 0.25 /iCi/ml of myo- tion. This growth medium contained approximately [3H]inositol. After the load-up period, the culture 10-15 Aimol/1 myo-inositol, the primary contribution flasks were rinsed 2x with the appropriate medium, deriving from the vitamin solution. Cell outgrowth and 5 ml of fresh medium was added. The efflux ex- from the capsule to the Petri dish after 7-10 days was periments were performed over 5 hr. The efflux was dispersed in Ca2+-Mg2+-free MEM containing 0.125% 2 expressed as the percentage of radioactivity initially trypsin/0.05% EDTA and transferred to a 75 cm cul- present in the cultured lens epithelium. ture flask.Th e cells originating from two to three cap- Sodium dependency on myo-inositol accumula- sules were placed in each culture flask with 40 ml of tion was determined in a manner similar to that just growth medium. Upon reaching confluence, the cells described, except that for these studies, a more simple, again were dispersed and subcultured in a split ratio of 2 serum-free medium (medium A) was employed. Me- 1:10 in 25 cm culture flasks that contained 5 ml of dium A consisted of the following: 5.5 mmol/1 glu- growth medium. The majority of studies were per- 2 cose, 135 mmol/1 NaCl, 5.4 mmol/1 KC1, 1.8 mmol/1 formed with confluent monolayers in 25 cm culture CaCl , 34.5 mmol/1 fructose, and 10 mmol/1 N-2-hy- flasks (representing 2nd-passage cells). To determine 2 2 droxyethylpiperazine-N-2-ethanesulfonic acid, pH intracellular sorbitol, confluent 150 cm culture flasks 7.4. All cells previously cultured in MEM were were employed, again using cells of the 2nd pass. switched to medium A for a 90 min equilibration pe- Where indicated for experimental purposes, further riod. Thereafter, cells were switched to fresh medium supplementations to the medium are described in the A with the addition of myo-[3H]inositol (1.0 jiCi/ml), text of this report. and the uptake of the isotope was determined for an 8

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hr incubation (collecting samples at the end of the D[2,6-3H]glucose (34 Ci/mmol; Amersham). The cul- first and second hour and every 2 hr thereafter) at 15 tured BLECs were acutely exposed to the isotope for 3 jumol/1 myo-inositol (sodium-dependent time course) hrat37°C. or for 3 hr over a concentration range of 0-100 jumol/1 of supplemented myo-inositol (Sigma). To determine Intracellular Sorbitol the sodium-dependency of myo-inositol uptake, studies were carried out in medium A, replacing NaCl on Intracellular sorbitol was identified by anion ex- an equal molar basis with N-methyl-D-glucamine change chromatography and pulsed electrochemical chloride (Sigma), pH 7.4. The osmolarity of NaCl- detection using a Dionex BioLC chromatographic supplemented and N-methyl-D-glucamine chloride- system that consisted of a high pressure liquid chroma- substituted medium A was 320 ± 5 mosm and 314 tography (HPLC) pump, a pulsed electrochemical de- ± 5 mosm, respectively, as determined with a vapor tector, an eluant degassing module, two CarboPac pressure osmometer (model 5500; Wescor, Salt Lake PA1 anion exchange columns (4 X 250 mm) con- City, UT). Sodium-dependent myo-inositol uptake nected in tandem, and a CarboPac PA Guard column was calculated by subtracting the uptake of myo-ino- (Dionex Corp., Sunnyvale, CA). The mobile phase sitol by cells incubated in N-methyl-D-glucamine was delivered by the HPLC pump at 1.0 ml/min at a chloride from the total uptake obtained in the me- pressure that did not exceed 2400 pounds per square dium that contained NaCl. inch. The elution program consisted of column regen- The effect of acute exposure (hereafter opera- eration for 10 min with 250 mmol/1 NaOH followed tionally defined as a 3 hr incubation period in serum- by column equilibration for 20 min in 3.75 mmol/1 free medium A) of cells to 40 mmol/1 D-glucose or 40 NaOH before sample injection. The mobile phase mmol/1 L-glucose (Sigma) on myo-[3H]inositol up- (3.75 mmol/1 NaOH) was delivered for 7 min; then a take was conducted essentially as already described. gradient from 3.75-25 mmol/1 NaOH was introduced For these experiments, cells previously maintained in over the next 33 min. Thereafter, the mobile phase MEM were switched to medium A for a 90 min equili- was held at 25 mmol/1 for an additional 15 min. Sor- bration period before they were divided into three bitol was identified by comparing sample retention groups: (1) medium A containing 5.5 mmol/1 glucose time with the retention time of standard sorbitol. and 34.5 mmol/1 fructose; (2) medium A containing Quantitation was achieved by comparing the sample 40 mmol/1 D-glucose; and (3) medium A containing peak area with that of a known amount of standard 40 mmol/1 L-glucose (neither (2) nor (3) had fructose sorbitol. Myo-inositol, xylitol, galactitol, sorbitol, supplementation). After myo-inositol accumulation, mannitol, galactose, glucose, xylose, mannose, and a trace amount of myo-[3H]inositol (1.0 juCi/ml) was fructose were obtained from Sigma Chemical Co. Re- added over a concentration range of 1.5-400 jumol/1 agent-grade sodium hydroxide solution (50%, weight/ myo-inositol for a 3 hr uptake period at 37°C. weight) was obtained from Baker Chemical Co. (Phil- The effect of chronically exposing (hereafter opera- lipsburg, NJ). HPLC-grade water was produced by a tionally defined as a 20 hr incubation period in Milli-Q water purification system (Millipore, Bed- serum-supplemented physiologic medium) cells to 40 ford, MA). mmol/1 D-glucose on myo-[3H]inositol accumulation Confluent monolayers of bovine lens epithelial cell was carried out in a manner similar to that just de- were maintained in MEM or 40 mmol/1 glucose in the scribed, except that cultured BLECs were maintained presence or absence of 0.1 mmol/1 sorbinil or 0.1 in serum-containing physiologic medium (MEM) or mmol/1 zopolrestat (Pfizer, Groton, CT) for 20 hr be- physiologic medium containing 40 mmol/1 glucose in fore they were dispersed with trypsin, suspended in the presence or absence of 0.1 mmol/1 sorbinil (Pfizer, Ca2+-Mg2+-free MEM, and centrifuged at 2500 X g at Groton, CT) for 20 hr before they were divided into 4°C for 8 min. The cells were resuspended in 1.5 ml of the following three groups for a 90 min equilibration 0.1 mol/1 potassium phosphate, pH 7.6, and cell period: (1) medium A with 5.5 mmol/1 glucose and disruption was accomplished by rapid freezing in liq- 34.5 mmol/1 fructose; (2) medium A with 40 mmol/1 uid nitrogen and thawing at 37°C; the process was glucose; and (3) medium A with 40 mmol/1 glucose repeated three times. Thereafter, the samples were and 0.1 mmol/1 sorbinil. At the end of 90 min, the transferred to a 5.0 ml Dounce homogenizer cultures were switched to the appropriate medium A (Thomas, Philadelphia, PA) and subjected to five containing a trace amount of myo-[3H]inositol (1.0 strokes while being maintained in an ice bath. The AiCi/ml) over a concentration range of 1.5-400 /xmol/l homogenate was centrifuged at 18,000 X g at 4°C for myo-inositol for a 3 hr uptake period at 37°C. 20 min and the cell pellet was saved for protein deter- Analysis of glucose uptake was conducted with mination. The supernatant was adjusted to 2.0 ml by serum-free medium A and 1 /xCi/ml of 2-deoxy- adding 0.1 mol/1 potassium phosphate, and an ali-

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quot was removed for protein determination. The re- throughout the 8 hr exposure (P < 0.01 for difference maining supernatant was deproteinated by centrifuga- with MEM, with a linear regression comparison and a tion at 5000 X g for 90 min using Centricon concen- test for parallelism). In addition, supplementing the trators with a 10,000 MW cutoff (Amicon, Danvers, control physiologic medium (MEM, 5.5 mmol/1 glu- MA). Twenty five microliters of the nitrate was di- cose) with fructose (34.5 mmol/1) to equalize the tonic- rectly applied to the columns without further modifi- ity of the 40 mmol/1 glucose medium resulted in a cation. For the quantitation of myo-inositol, the filter myo-inositol uptake that was virtually indistinguish- concentrator must be excessively pre-rinsed to re- able from the one generated with the cells maintained move impregnated glycerol. in physiologic medium without fructose adjustment (Fig. 1). As a result, all subsequent experiments were Statistical Analysis performed with control medium appropriately ad- Statistical analyses were performed with the statisti- justed for tonicity by fructose supplementation. cal programs from Tallarido and Murray,14 adapted To establish whether the apparent decrease in myo- for the IBM PC-XT. Appropriate statistical analyses inositol uptake was attributable to enhanced efflux of were applied to each group of data as indicated. myo-inositol from the cells to the medium, BLECs were preloaded with myo-[3H]inositol followed by the Results efflux of the radiolabel out of the cell. The efflux of myo-[3H]inositol (Fig. 2) essentially was the same for Figure 1 shows that chronic exposure (20 hr) of the cultured lens cells exposed to 40 mmol/1 glucose or 40 cultured cells to 40 mmol/1 glucose or 40 mmol/1 ga- mmol/1 galactose for 20 hr and physiologic medium lactose resulted in a reduced capacity to accumulate (not significant by the least significant difference test). radiolabeled myo-inositol (as determined by the cyto- 3 solic counts per minute/milligram protein), which re- Likewise, the efflux of myo-[ H]inositol was not af- mained significantly below that of the control fected by the fructose-adjusted control medium, indicating that the medium osmolarity did not account for the lack of difference in efflux of myo-[3H]inositol Myo-[ Hjinositol Uptake from Bovine Lens under these experimental conditions. Epithelial Cells (1 dy incubation) 120000 Sodium-Dependent Myo-inositol Uptake For these experiments, the physiologic medium MEM (5.5 mM glucose) + 100000 - Fructose (34.5 mM) normally used to maintain the cell cultures was re- D Glucose (40 mM) placed with a simpler, serum-free medium (medium V Galactose (40 mM) A) that contained a trace concentration of myo- 80000 - [3H]inositol (1 juCi/ml of medium), and incubation continued for up to 8 hr. The time course of myo- 3 60000 - [ H]inositol uptake in BLECs in medium A was linear for 8 hr and sodium-dependent (Fig. 3). Therefore, a 3 hr incubation period was selected for subsequent 40000 - (acute exposure) experiments. Myo-inositol uptake was sodium-dependent as observed over a myo-inosi- 20000 - tol concentration range of 1.5-100 nl (Fig. 4). Replace- ment of sodium on an equal molar basis with N- methyl-D-glucamine chloride demonstrated that the sodium-dependent myo-inositol uptake accounted for more than 95% of the total uptake.

Fig. I. Effect of glucose and galactose on myo-inositol uptake in BLECs. Confluent BLECs were maintained in physiologic medium Myo-inositol Transport is Distinct from Glucose (Eagle's minimal essential medium [MEM], 5.5 mmol/1 glucose, Transport MEM supplemented with 34.5 mmol/1 fructose, 40 mmol/1 glucose (Glu), or 40 mmol/1 galactose (Gal) for a 20 hr exposure period. At To determine the acute effect of D-glucose and its the end of the incubation period, corresponding fresh medium plus enantiomer, L-glucose, on myo-inositol uptake, cells myo-[3H]inositol (0.25 jtCi/ml) was introduced to cultures, and maintained in physiologic medium (having never time course of radiolabel uptake was followed for 8 hr. Replicate been exposed to high-ambient glucose) were switched cultures were collected for each of the designated times. Data points to serum-free medium A that contained 40 mmol/1 are means ± standard errors. Data points were plotted by linear regression, and the correlation coefficient was 0.99 for all treat- D-glucose or L-glucose and a trace amount of myo- 3 ments. [ H]inositol during a 3 hr uptake period over 1.5-400

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Myo-[ H]inositol Efflux from Preloaded Bovine Lens Epithelial Cells

(1 dy incubation)

125 125

100 100i

O - O MEM (5.5 mM glucose) • MEM (5.5 mM glucose + g 50 50 V - V Galactose (40 mM) Fructose (34.5 mM) D-D Glucose (40 mM) V Galactose (40 mM) D Glucose (40 mM)

25 25

2 3 4 2 3 4 Time (hours) Time (hours) Fig. 2. Effect of glucose and galactose on myo-inositol efflux in cultured BLECs. Lens cultures were maintained in physiologic medium (Eagle's minimal essential medium [MEM], MEM supplemented with 34.5 mmol/1 fructose, 40 mmol/1 glucose (Glu), 40 mmol/1 galactose (Gal) for 1 day before incubation in 0.25 /iCi/ml myo[3H]inositol for 4 hr. Pre-loaded cells were switched to fresh medium without isotope, and triplicate cultures were collected after 1 hr and every hour thereafter up to 5 hr. Efflux of myo-inositol was expressed as a percentage of radioactivity initially present in lens cells. Data points are means ± standard errors.

lxmo\/\ myo-inositol (Fig. 5). Control cells were main- Chronic Effect of 40 mmol/1 Glucose and Aldose tained in 5.5 mmol/1 D-glucose, serum-free medium Reductase Inhibition on Myo-inositol Uptake A. The uptake of myo-[3H]inositol was inhibited by acute exposure of cultured cells to D-glucose- and L- To determine the chronic effect of high-ambient glucose-containing medium A. Lineweaver-Burk glucose on myo-inositol uptake, cultured cells were transformations (inset) were indicative of competitive exposed to 40 mmol/1 glucose for 20 hr and then were inhibition for D-glucose and L-glucose. switched to 40 mmol/1 glucose-containing, serum- free medium A containing a trace amount of myo- Glucose transport by BLECs was examined by 3 monitoring 2-deoxy-D-[3H]glucose accumulation. As [ H]inositol during a 3 hr incubation period. Control shown in Figure 6, specific 2-deoxy-D-glucose uptake cells were maintained in 5.5 mmol/1 glucose-containing, serum-free medium. The uptake of myo- by cultured BLECs primarily was sodium-dependent. 3 However, unlike myo-inositol, a substantial sodium- [ H]inositol was reduced after chronic exposure of independent process also was observed when sodium cultured cells in 40 mmol/1 glucose, as observed for was replaced by N-methyl-D-glucamine chloride. Not 1.5-400 /xmol/1 myo-inositol (Fig. 8). The coadminis- unexpectedly, 2-deoxy-glucose accumulation was tration of sorbinil 0.1 mmol/1 with 40 mmol/1 glucose partially prevented the inhibitory effect of glucose on markedly inhibited by 40 mmol/1 D-glucose in 3 serum-free medium that contained 15 or 150 jumol/1 myo-[ H]inositol uptake. myo-inositol (Fig. 7). However, in contrast to myo-inositol uptake (Fig. 5), 40 mmol/1 L-glucose signifi- Dionex BioLC Chromatography cantly stimulated 2-deoxy-D-[3H]glucose uptake, irre- spective of the myo-inositol concentration. More- To verify that polyol accumulation attenuates 3 over, 2-deoxy-D-[ H]glucose uptake itself appeared to myo-inositol uptake in cultured BLECs, it was neces- be independent of the myo-inositol concentration in sary to demonstrate that the simultaneous administra- the medium. tion of sorbinil with 40 mmol/1 glucose for 20 hours

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Sodium-Dependent [ H]-Myo-inositol Uptake Sodium-Dependent Myo-inositol Uptake

50000 1800

• + sodium (A) 1500 r 40000 • - sodium (B) O A - B o 1200 -

'£o 30000 900 L

600 -

300 L-

25 50 75 100 125 10 Myo-inositol (uM)

Fig. 4. Sodium-dependent myo-inositol uptake. BLECs were in- Fig. 3. Time course of myo-inositol uptake. BLECs were incu- + + cubated in serum-free Na -HEPES buffer (medium A) or this bated in serum-free, Na -HEPES buffer (medium A) or this buffer buffer containing N-methyl-D-glucamine chloride, substituting containing N-methyl-D-glucamine chloride, replacing NaCl on an NaCl on an equal molar basis. Myo-inositol uptake was determined equal molar basis. Both buffers contained 15 ^mol/1 myo- 3 by collecting triplicate flasks after a 3 hr incubation period over a [ H]inositol. Myo-inositol uptake was determined by collecting trip- myo-inositol concentration range of 1.5-100 /zmol/1. The data replicate flasks for each of the designated times. The data represent the resent the means ± standard errors. mean ± standard errors. (A), NaCl-supplemented medium A. (B), N-methyl-D-glucamine chloride-substituted medium A. (C), Na+- dependent myo-inositol uptake. Data points were plotted by linear regression, and the correlation coefficients for (A), (B), and (C) were 0.99, 0.81, and 0.99, respectively. Effect of D- and L-Glucose on Myo-inositol Uptake

4000 prevented sorbitol from forming in the lens cells. A

typical chromatogram of mixed polyol standards by O - O MEM anion exchange chromatography using the Dionex • - • D-Glu BioLC chromatographic system is shown in Figure 3000 V - V L-Glu 9A. The elution order of the polyols and retention time of each component (in minutes) was as follows: myo-inositol, 3.75; xylitol, 4.45; galactitol, 5.77; sor- 2000 bitol, 6.02; and mannitol, 7.10. Aldose sugars were significantly retained relative to their sugar alcohol counterparts with this elution scheme. Galactose and 1000 glucose eluted at 36.4 and 40.2 min, respectively. Under the conditions of the elution scheme, mannose and xylose coelute at 44.3 min (data not shown). Fifty five minutes was required for the entire operation (not including column regeneration and equilibration time). Figure 9A also shows an overlay of a typical -1000 chromatogram taken from a lens cell culture incu- -100 100 200 300 400 500 bated in 40 mmol/1 glucose for 20 hr. The accumula- Myo—inositol (uM) tion of intracellular sorbitol is evident. Figure 9B is an Fig. 5. Effect of D-glucose and L-glucose on myo-inositol uptake. overlay of several elution profiles taken from cells ex- BLECs were incubated in medium A over a concentration range of posed to 40 mmol/1 glucose, or 40 mmol/1 glucose 1.5-400 ^mol/1 myo-inositol with the inclusion or omission of 40 plus the aldose reductase inhibitors sorbinil or zo- mmol/1 D-glucose or 40 mmol/1 L-glucose. After a 3 hr exposure period, myo-inositol uptake was determined by collecting triplicate polrestat. The synthesis and accumulation of intracel- flasks. Data points are means ± standard errors.

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Sodium-Dependent [ H]-2-Deoxy-Glucose Uptake lular sorbitol normally associated with lens cells con- tinuously exposed to high-ambient glucose for 20 hr 60000 was completely inhibited with either aldose reductase + sodium (A) inhibitor. - sodium (B) 50000 Discussion Decreased tissue free myo-inositol is a common 40000 identifiable complication associated with hyperglyce- mja 7,15-17 however, the association between diabetes mellitus and reduced tissue free myo-inositol has not been consistent. In cerebral micro vessels, the myo-inositol content of endothelial cells did not decrease with hyperglycemia, whereas microvascular pericytes exposed to high-ambient glucose showed a loss of intracellular myo-inositol.18 High-ambient glucose also reduced the myo-inositol content of cultured rat glomerular mesangial cells19 and cultured neuroblastoma cells.20 In the streptozocin-induced diabetic rat, uncontrolled diabetes was accompanied by signifi- Time (hours) cant decreases in the levels of myo-inositol in the in- 21 Fig. 6. Time course of 2-deoxyglucose uptake. BLECs were incu- tact lens. High-ambient galactose has been shown to bated in serum-free Na+-HEPES buffer (medium A) or this buffer attenuate the myo-inositol concentrating capability of containing N-methyl-D-glucamine chloride, replacing NaCl on an cultured BLECs.1" In the latter study, we concluded equal basis. Both buffers contained 5.5 mmol/1 D-glucose, a trace 3 this effect was a result of the aldose reductase reaction, amount of 2-deoxy-[ H]glucose, and 15 /umol/1 supplemented myo- as sorbinil, an aldose reductase inhibitor, prevented inositol. Two-deoxyglucose uptake was determined by collecting the detrimental effects of galactose exposure on myo- triplicate flasks for each of the designated times. Data presented are 11 means ± standard errors. inositol uptake.

Effect of D—glucose and L—glucose on [ H]—2—Deoxy—Glucose Uptake

15 uM myo—inositol 150 uM myo—inositol 60000 60000 RX>3 MEM (5.5 mM glucose + KXH MEM (5.5 mM glucose + 34.5 mM fructose) 50000 34.5 mM fructose) 50000 V77\ D-glucose (40 mM) F771 D-glucose (40 mM) L—glucose (40 mM) L—glucose (40 mM) •~ 40000 40000 o u a 30000 to 30000 6

20000 20000

10000 10000

0 Fig. 7. Effect of D-glucose and L-glucose on 2-deoxyglucose uptake. BLECs were incubated in medium A containing 15 jtmol/1 myo-inositol or 150 ^mol/1 myo-inositol in the presence or absence of 40 mmol/1 D-glucose or 40 mmol/1 L-glucose and a trace amount of 2-deoxy- [3H]glucose. After a 3 hr incubation period, 2-deoxyglucose uptake was determined by collecting triplicate flasks. Data presented are means ± standard errors.

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Effect of Glucose and Sorbinil on Myo-inositol Uptake ated by the control cells not supplemented with fruc-

2500 tose (Fig. 1). We cannot rule out the possibility that intracellular osmotic changes, attributable to polyol accumula- 2000 - tion, play a role in the observed reduction in myo-inositol uptake. Alternatively, the decrease in myo-inositol uptake resulting from the incubation of BLECs in o 1500 - 40 mmol/l glucose or 40 mmol/l galactose may be the result of an enhanced cellular efflux of myo-inositol,

1000 - as has been observed in the whole lens after cataracto- genesis.22 However, the rate of efflux of myo- [3H]inositol essentially was identical for cultured lens 500 - cells exposed to 40 mmol/l glucose or 40 mmol/l galactose, as with control cells. Likewise, the rate of efflux of myo-[3H]inositol was unaffected by adjusting 0 - the control cell medium osmoticity with fructose (Fig. 2). Similar observations with cultured rat glomerular -500 mesangial cells incubated in high-glucose concentra- -100 100 200 300 400 500 tions have been recorded.19 Therefore, the early onset Myo—inositol (uM) loss of capability by the 40 mmol/l glucose- and 40 Fig. 8. Effect of chronic glucose exposure and sorbinil on myo-in- mmol/l galactose-exposed cells to concentrate myo- ositol uptake. BLECs were preincubated in 40 mmol/l glucose inositol does not appear to be caused by a disruption (Glu), 40 mmol/l glucose plus 0.1 mmol/l sorbinil (Glu/Sor), or of structural membrane integrity, which otherwise control (5.5 mmol/l glucose) for 20 hr. After the chronic glucose probably would have led to increased membrane per- exposure, BLECs were divided into three groups—medium A (5.5 meability and the rapid efflux of myo-inositol, as de- mmol/l glucose and 34.5 mmol/l fructose), medium A containing 23 40 mmol/l glucose, or medium A containing 40 mmol/l glucose scribed in other studies with whole lenses. Rather, and 0.1 mmol/l sorbinil—for a 3 hr myo-inositol uptake period the attenuation of myo-inositol uptake in cultured over a concentration range of 1.5-400 ^mol/l. Data represent tripli- BLECs by incubation in high-ambient glucose or ga- cate determinations from individual flasks. Data points are means lactose most likely reflects direct impact on the myo- ± standard errors. inositol transporters rather than a generalized change in membrane permeability. This is supported by our previous observation that demonstrated removing The direct impairment of the myo-inositol trans- high-ambient galactose from the incubation medium port system that results from lens cell exposure to normalized myo-inositol uptake. This suggested that high-ambient galactose represents a plausible mecha- the impairment of the myo-inositol transporters was nism that could account for the loss of intracellular reversible." myo-inositol associated with hyperglycemia. In the present study, the relationship between high-ambient Myo-inositol uptake was sodium dependent over a glucose, the polyol pathway, and aldose reductase in- myo-inositol concentration range of 1.5-100 jumol/1. hibition on myo-inositol transport in cultured BLECs Sodium-dependent myo-inositol transport exceeded was further characterized. 95% of the total myo-inositol uptake, as determined Myo-inositol uptake in cultured BLECs was signifi- by the replacement of sodium on an equal molar basis cantly diminished after 20 hr of exposure to 40 mmol/ with N-methyl-D-glucamine chloride (Fig. 4). This 1 glucose or 40 mmol/l galactose. The capability of the observation agreed with previous results from our lab- 40 mmol/l glucose- or 40 mmol/l galactose-exposed oratory that demonstrated myo-inositol uptake in cells to accumulate myo-[3H]inositol remained signifi- cultured BLECs occurred primarily through an oua- cantly below that of the control throughout the 8 hr bain-sensitive transport process.11 In the intact lens, uptake period. The decrease in myo-inositol transport sodium-dependent myo-inositol uptake also has been could not be attributed to the osmotic effect of 40 observed.24 mmol/l glucose or 40 mmol/l galactose, because con- Myo-inositol transport can be characterized as facil- trol (physiologic medium) cells adjusted on an equal itated diffusion or active transport. The myo-inositol osmolar basis with fructose, equal to the osmolarity of concentration of aqueous humor has been reported at 40 mmol/l glucose- or 40 mmol/l galactose-treated 0.22 mmol/l;10 in the human lens, it has been reported cells, displayed a rate of myo-[3H]inositol uptake that as greater than 20 mmol/l.25 These observations sug- was virtually indistinguishable from the curve gener- gest that myo-inositol must accumulate in the lens by

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Myo-inositol . Myo-lnosltol

Xylitol

Sorbitol Glucose/Zopolrestat

Glucose/Sorblnil

Glucose

Fig. 9. (A) Chromatograms of (A) lens cell lysate from BLECs exposed to 40 mmol/1 glucose for 20 hr and (B) 40 nmol each of myo-inositol, xylitol, galactitol, sorbitol, and mannitol. (B) Chromatograms of lens cell lysate from BLECs exposed to 40 mmol/1 glucose, 40 mmol/1 glucose plus 0.1 mmol/1 sorbinil, or 40 mmol/1 glucose plus 0.1 mmol/1 zopolrestat for 20 hr.

an active process. The present findings with N- tol transport was not inhibited by physiologic glucose methyl-D-glucamine chloride substitution of sodium concentrations in pancreatic islets.31 The apparent in- chloride and our previous observations with ouabain consistency regarding whether glucose acts as a com- suggest that myo-inositol transport in BLECs is an petitive or noncompetitive inhibitor, or does not in- energy-dependent process driven by the active trans- hibit myo-inositol transport at all, may depend upon port of sodium. In retinal pericytes, a myo-inositol the characteristics of the particular myo-inositol trans- cotransport process has been described that involves a porter. Myo-inositol transport has been described for ternary complex between the cotransporter, myo-ino- a number of systems as: (1) an active high-affinity, sitol, and sodium.16 The energy required for the myo- low-capacity mechanism; (2) an active low-affinity, inositol transporter is derived from the sodium gra- high-capacity mechanism; (3) exhibiting kinetic pa- dient and the energy transduction of active sodium rameters consistent with active high-affinity and low- transport.26 It was suggested that the binding of so- affinity mechanisms; or (4) as nonactive facilitated dium to the transporter increased its mobility but not diffusion. In our other report in this issue, active, so- its affinity for myo-inositol. Therefore, changes in sodium-dependent, high-affinity, glucose-sensitive and dium concentration may act to regulate the myo-ino- active, sodium-dependent, low-affinity, sorbitol-sen- sitol carrier proteins. sitive myo-inositol transporters were identified in cul- 32 To determine whether glucose-induced inhibition tured BLECs. of myo-inositol concentrating capability was stereo- Although myo-inositol is a structural isomer of glu- specific, we examined the effects of D- and L-glucose cose, in a number of different tissues it appears to on myo-inositol uptake in cultured BLECs. Both D- have transport properties that are distinct from those and L-glucose competitively inhibited sodium-depen- of glucose. In preparations of small intestine33 and dent myo-inositol uptake, as demonstrated by Line- renal brush border vesicles,34 myo-inositol and glu- weaver-Burk plots (Fig. 5). Similar results have been cose both are actively transported. However, in liver reported for cultured mesangial cells,19 the intact cells parenchymal cells,35 exocrine pancreatic acini,36 and of isolated glomeruli,27 and peripheral nerve tissue brain synaptosomes,37 transport of myo-inositol and preparation.28 L-glucose was reported to be more ef- glucose occurs by a nonactive process. In the lens and fective than D-glucose in inhibiting myo-inositol up- endoneurial preparations, uptake of myo-inositol and take in mouse cerebral microvessel endothelial cells29 glucose appear to occur through different processes, and in microvessel endothelial cells isolated from the as myo-inositol is active in lens2438 and endoneurial bovine retina.30 In contrast, D-glucose inhibition of preparation.2839 However, glucose uptake, as in pan- myo-inositol transport in retinal pericytes16 has been creatic islets,31 is likely to be of the facilitated diffusion reported to be noncompetitive, and active myo-inosi- type in lens40 and endoneurial preparation.41 Glucose

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uptake in BLECs, as measured by 2-deoxy- toma, human Y79 retinoblastoma, human HL60 [3H]glucose, occurred by sodium-dependent and so- cells, bovine pulmonary artery endothelial cells,42 and dium-independent processes (Fig. 6). Unlike myo-in- human retinal pigmented epithelial cells.43 In our ositol uptake (Figs. 3 and 4), sodium-independent 2- other report in this issue, we characterize the kinetic deoxy-[3H]glucose uptake represented a substantial parameters of myo-inositol uptake in cultured BLECs part of the total uptake (Fig. 6). This suggested that and describe high- and low-affinity myo-inositol trans- the myo-inositol transport system was distinct from port sites that are differentially affected by glucose that of the glucose transport system. This distinction and the sugar alcohol of glucose.32 was further supported by the observation that 2- In summary, myo-inositol transport in BLECs in deoxy-[3H]glucose uptake appeared to be unaffected culture occurs via a sodium-dependent, ouabain-sen- by the myo-inositol concentration in physiologic me- sitive, saturable, active transport process that appears dium (Fig. 7). Furthermore, D-glucose, but not L-glu- to be independent of the glucose transport system. cose, inhibited 2-deoxy-[3H]glucose uptake, whereas High-ambient glucose attenuates myo-inositol con- D-glucose and L-glucose competitively inhibited centrating capability via competitive inhibition. Inhi- myo-[3H]inositol uptake (Fig. 5). These results are bition of aldose reductase displayed a trend toward similar to that observed in mouse cerebral microves- near-normalization of myo-inositol accumulation in sel endothelial cells.29 Collectively, these observations cultured BLECs, indicating that sorbitol affects the suggest that the myo-inositol transport system and the myo-inositol transport system. In our other report in glucose transport system are distinct and independent this issue, we describe kinetic parameters that indicate in BLECs. myo-inositol uptake in cultured BLECs is maintained We previously reported that the galactose-induced by at least two mechanisms: a sodium-dependent, attenuation in myo-inositol uptake could be pre- glucose-sensitive transport site and a sodium-depen- vented by concomitantly administering the aldose re- dent, sorbitol-sensitive transport site. ductase inhibitor sorbinil to the incubation medium. '' This observation led to the suggestion that the Key words: aldose reductase, bovine, lens cells, hyperglycemia, myo-inositol transport aldose reductase reaction or formation of the product of the aldose reductase reaction was responsible for the impairment of myo-inositol accumulation. In the References present study, a significant sorbitol accumulation was 1. Cammarata PR, Tse D, and Yorio T: Sorbinil prevents the demonstrated after 20 hr of incubation with 40 hypergalactosemic-induced reduction in [3H]-myo-inositol up- 3 mmol/1 glucose, as demonstrated by ion exchange take and decreased [ H]-myo-inositol incorporation into the phosphoinositide cycle in bovine lens epithelial cells in vitro. chromatography and integrated amperometry (Fig. Curr Eye Res 9:561, 1990. 9B). The accumulation of polyol coincided with the 2. Winegrad AL: Banting Lecture 1986: Does a common mecha- attenuation in myo-inositol uptake observed with 40 nism induce the complications of diabetes? Diabetes 36:396, mmol/1 glucose-treated BLECs. Cells exposed to 40 1987. mmol/1 glucose plus the aldose reductase (AR) inhibi- 3. Kawaba T, Cheng H-M, and Kinoshita JH: The accumulation of myoinositol and rubidium ions in galactose-exposed rat lens. tors sorbinil or zopolrestat showed that the accumula- Invest Ophthalmol Vis Sci 27:1522, 1986. tion of sorbitol was conspicuously absent from the 4. MacGregor LC and Matschinsky FM: Treatment with aldose chromatograms (Fig. 9B), suggesting that the synthe- reductase inhibitor or with myo-inositol arrest deterioration of sis of sorbitol was dramatically inhibited in these cells the electroretinogram of diabetic rats. J Clin Invest 76:887, at the concentration of AR inhibitors used. 1985. 5. Finegold D, Lattimer SA, Nolle S, Bernstein M, and Greene In addition, myo-inositol uptake in the presence of DA: Polyol pathway and myo-inositol metabolism: A sug- 40 mmol/1 glucose plus sorbinil was near-normalized. gested relationship in the pathogenesis of diabetic neuropathy. However, a significant component of myo-inositol Diabetes 32:988, 1983. uptake in the range of 1.5-25 /xmol/1 myo-inositol 6. Okuda Y, Bannai C, Nagahama M, Isaka M, and Yamashita remained sorbinil-insensitive. This observation led to K: Restoration of myo- inositol uptake by aldose reductase inhibitor in human skin fibroblasts cultured in high-glucose the suggestion that more than one myo-inositol trans- medium. Horm Metab Res 23:42, 1990. porter might be functioning in cultured BLECs, or 7. Yorek MA, Dunlap JA, and Ginsberg BH: Effect of sorbinil on that a single myo-inositol transporter might display myo-inositol metabolism in cultured neuroblastoma cells ex- two affinity states, each exhibiting different physical posed to increased glucose levels. J Neurochem 51:331, 1988. characteristics, influenced by the myo-inositol con- 8. Greene DA: A sodium-pump defect in diabetic peripheral nerve corrected by sorbinil administration: Relationship to centration. High- and low-affinity myo-inositol trans- myo-inositol metabolism and nerve conduction slowing. Me- port sites also have been described for mouse cerebral tabolism 35:60, 1986. microvessel endothelial cells,29 mouse neuroblas- 9. Greene DA, Lattimer SA, and Sima AAF: Sorbitol, phosphoin-

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ositides, and sodium- potassium-ATPase in the pathogenesis of 26. Turner RJ: Quantitative studies of cotransport systems: Mod- diabetic complication. N Engl J Med 316:599, 1987. els and vesicles. J Membr Biol 76:1, 1983. 10. Loy A, Lurie KG, Ghosh A, Wilson JM, MacGregor LC, and 27. Whiteside CI, Thompson JC, and Ohayon J: Myo-inositol and Matschinsky FM: Diabetes and the myo-inositol paradox. Dia- D-glucose transport in rat glomerular and cultured mesangial betes 39:1305, 1990. cells. Am J Physiol 260:F138, 1991. 11. Cammarata PR, Tse D, and Yorio T: Uncoupling of atten- 28. Greene DA and Lattimer SA: Sodium- and energy-dependent uated myo-[3H]inositol uptake and dysfunction in Na+-K+- uptake of myo-inositol by rabbit peripheral nerve. J Clin Invest ATPase pumping activity in hypergalactosemic cultured bo- 70:1009, 1982. vine lens epithelial cells. Diabetes 40:731, 1991. 29. Yorek MA, Stefani MR, and Moore SA: Acute and chronic 12. Cammarata PR, Jackson T, and Yorio T: Sorbinil prevents the exposure of mouse cerebral microvessel endothelial cells to in- galactose-induced inhibition of prostaglandin synthesis in lens creased concentrations of glucose and galactose: Effect on + + cells. Invest Ophthalmol Vis Sci 29:1452, 1988. myo-inositol metabolism, PGE2 synthesis, and Na /K -ATP- 13. Bradford MM: A rapid and sensitive method for the quantita- ase transport activity. Metabolism 40:347, 1991. tion of microgram quantities of protein utilizing the principle 30. Kollros PE, Goldstein GW, and Betz AL: Myo-inositol trans- of protein-dye binding. Anal Biochem 72:248, 1976. port into endothelial cells derived from nervous system micro- 14. Tallarido RJ and Murray RB: Manual of Pharmacological Cal- vessels. Brain Res 511:259, 1990. culations, 2nd ed. New York, Springer-Verlag, 1987. 31. Biden TJ and Wollheim CB: Active transport of myo-inositol 15. Yue DK, Hanwell MA, Satchell PM, Handelsman DJ, and in rat pancreatic islets. Biochem J 236:889, 1986. Turtle JR: The effects of aldose reductase inhibition on nerve 32. Cammarata PR, Chen H-Q, Yand J, and Yorio T: Modulation sorbitol and myo-inositol concentrations in diabetic and galac- of myo-[3H]inositol uptake by glucose and sorbitol in cultured tosemic rats. Metabolism 33:1119, 1984. bovine lens epithelial cells. II. Characterization of high- and 16. Li W, Chan LS, Khatami M, and Rockey JH: Non-competitive low-affinity myo-inositol transport sites. Invest Ophthalmol inhibition of myo-inositol transport in cultured bovine retinal Vis Sci 33:3572, 1992. capillary pericytes by glucose and reversal by sorbinil. Biochim 33. Caspary WF and Crane RK: Active transport of myo-inositol BiophysActa 857:198, 1986. and its relation to the sugar transport system in hamster small 17. Llewelyn JG, Simpson CMF, Thomas PK, King RHM, and intestine. Biochim Biophys Acta 203:308, 1970. Hawthorne JN: Changes in sorbitol, myo-inositol and lipid ino- 34. Hammerman MR, Sacktor B, and Daughday WH: Myo-inosi- sitol in dorsal root and sympathetic ganglia from streptozoto- tol transport in renal brush border vesicles and its inhibition by cin-diabetic rats. Diabetologia 29:876, 1986. D-glucose. Am J Physiol 239:F113, 1980. 18. Sussman I, Carson MP, Schultz V, Wu XP, McCall AL, Ruder- 35. Prpic V, Blackmore PF, and Exton JH: Myo-inositol uptake man NB, and Tornheim K: Chronic exposure to high glucose and metabolism in isolated rat liver cells. J Biol Chem decreases myo-inositol in cultured cerebral microvascular peri- 257:11315, 1982. cytes but not in endothelium. Diabetologia 31:771, 1988. 36. Bazin R and Lavau M: Effects of high-fat diet on glucose metab- 19. Haneda M, Kikkawa R, Arimura T, Ebata K, Togawa M, olism in isolated pancreatic acini of rats. Am J Physiol Maeda S, Sawada T, Horide N, and Shigeta Y: Glucose inhibits 243:G448, 1982. myo-inositol uptake and reduces myo-inositol content in cul- 37. Warfield A, Hwang SM, and Segal S: On the uptake of inositol tured rat glomerular mesangial cells. Metabolism 9:40, 1990. by rat brain synaptosomes. J Neurochem 31:957, 1978. 20. Yorek MA, Dunlap JA, and Leeney EM: Effect of galactose 38. Varma SD, Chakrapani B, and Reddy VN: Intraocular trans- and glucose levels and sorbinil treatment on myo-inositol me- port of myoinositol. Invest Ophthalmol 9:794, 1970. tabolism and Na+-K+ pump activity in cultured neuroblas- 39. Gillon KRW and Hawthorne JN: Transport of myo-inositol toma cells. Diabetes 38:996, 1989. into endoneurial preparations of sciatic nerve from normal and 21. Yeh L-A, Rafford CE, Goddu KJ, Ashton MA, Beyer TA, and streptozotocin-diabetic rats. Biochem J 210:775, 1983. Hutson NJ: Na+-K+-ATPase pumping activity is not directly 40. Elbrink J and Bihler I: Characteristics of the membrane trans- linked to myo-inositol levels after sorbinil treatment in lenses port of sugars in the lens of the eye. Biochim Biophys Acta of diabetic rats. Diabetes 36:1414, 1987. 282:337, 1972. 22. Broekhuyse RM: Changes in myoinositol permeability in the 41. Greene DA, Winegrad AI, Carpentier J-L, Brown MJ, Fukuma lens due to cataractous conditions. Biochim Biophys Acta M, and Orci L: Rabbit sciatic nerve fascicle and endoneurial 163:269, 1968. preparations for in vitro studies of peripheral nerve glucose 23. Kawaba T, Cheng H-M, and Kinoshita JH: The accumulation metabolism. J Neurochem 33:1007, 1979. of myoinositol and rubidium ions in galactose-exposed rat lens. 42. Yorek MA, Dunlap JA, and Ginsberg BH: Myoinositol uptake Invest Ophthalmol Vis Sci 27:1522, 1986. by four cultured mammalian cell lines. Arch Biochem Biophys 24. Cotlier E: Myo-inositol: Active transport by the crystalline 246:801, 1986. lens. Invest Ophthalmol 9:681, 1970. 43. Del Monte MA, Rabbani R, Diaz TC, Lattimer SA, Nakamura 25. Dickerson JE and Lou MF: Micro-quantitation of lens myo- J, Brennan MC, and Greene DA: Sorbitol, myo-inositol, and inositol by anion exchange chromatography. Curr Eye Res rod outer segment phagocytosis in cultured hRPE cells exposed 9:201, 1990. to glucose. Diabetes 40:1335, 1991.

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