modulation of myo-[3h]inositol uptake by glucose and sorbitol in

9
Investigative Ophthalmology & Visual Science, Vol. 33, No. 13, December 1992 Copyright © Association for Research in Vision and Ophthalmology Modulation of Myo-[ 3 H]inositol Uptake by Glucose and Sorbitol in Cultured Bovine Lens Epithelial Cells //. Characterization of High- and Low-Affinity Myo-inositol Transport Sites Patrick W. Cammarara,* Hai-Qing Chen,* Jinhua Yang,* and Thomas Yoriof Myo-( 3 H]inositol accumulation in cultured bovine lens epithelial cells (BLECs) occurred by high- and low-affinity, Na + -dependent transport sites. The high- and low-affinity transport systems had a Km of 27 ± 4 and 157 ± 22 fimol/l, respectively, and V max of 652 ± 35 and 2952 ± 308 pmol/mg protein/hr, respectively. The uptake of myo-[ 3 H]inositol was lowered after chronic (20 hr) incubation of cultured cells in 40 mmol/1 glucose throughout the concentration range for 1.5-400 ixmo\/l myo-inositol. The coadministration of sorbinil (0.1 mmol/1) to 40 mmol/1 glucose partially prevented the inhibitory effect of glucose on myo-[ 3 H]inositol uptake. Although the aldose reductase inhibitor prevented the inhibitory effect of glucose on the low-affinity transport site, a glucose-sensitive process for myo-[ 3 H]inositol uptake on the high-affinity transport site was uncovered by Lineweaver-Burk analysis. Acute exposure (3 hr) of cultured BLECs maintained in physiologic medium (Eagle's minimal essential medium, 5.5 mmol/1 glucose) to a range of 5.5-44 mmol/1 glucose plus sorbinil also caused a decrease in myo- ( 3 H]inositol uptake. Oixon plot analysis confirmed that the acute effect of glucose was the result of competitive inhibition of the high-affinity myo-inositol transport site. Acute exposure of cultured cells to 10-40 mmol/1 sorbitol also diminished the accumulation of myo-[ 3 H]inositol. Dixon plot analysis established that the acute effect of exogenous sorbitol was the result of competitive inhibition of the low-affinity myo-inositol transport site. These data support the contention that myo-inositol transport in BLECs is maintained by at least two processes: a glucose-sensitive, relative sorbitol insensitive, sodium-dependent, high-affinity myo-inositol transport system; and a sorbitol (aldose reductase) sensi- tive, sodium-dependent, low-affinity myo-inositol transport system. Invest Ophthalmol Vis Sci 33:3572-3580,1992 The underlying biochemical deficits that contribute to the pathogenesis of diabetic complications in the lens and other tissues remain controversial. 1 " 5 Despite the identification of attendant pathologic manifesta- tions in cell culture and animal models of the disease, explanations of the mechanisms that lead to cellular damage primarily are speculative. Among the preva- lent theories that seek to explain the aberrant bio- chemical alterations that lead to diabetic complica- tions, the "osmotic stress" hypothesis has been one of the most widely disseminated. Activation of the al- dose reductase reaction and formation and accumula- From the Departments of *Anatomy and Cell Biology and fPhar- macology, Texas College of Osteopathic Medicine/University of North Texas, Fort Worth, Texas. Supported by National Public Health Service Award EYO557O- 05 (PRC). This work represents partial fulfillment of the requirements for the degree of Master of Science for Hai-Qing Chen. Submitted for publication: March 25, 1992; accepted July 16, 1992. Reprint requests: Patrick R. Cammarata, Texas College of Osteo- pathic Medicine, Department of Anatomy and Cell Biology, Fort Worth, TX 76107. tion of polyols directly promote osmotic swelling, leading to diabetic complications. 6 - 7 The best evi- dence that aldose reductase and polyols are involved in such complications of diabetes was the demonstra- tion that aldose reductase inhibitors can prevent or delay the onset of complications. 8 ' 9 However, any model that attributes the onset of diabetic complica- tions to polyol accumulation also must consider that many tissues may not accumulate polyols to a con- centration high enough to exert a direct osmotic ef- fect. 10 Alternative models, linking hyperglycemia and dysfunctional myo-inositol metabolism, have been suggested. One model postulated that hyperglycemia may deplete a discrete, small, rapidly turning over, glucose-sensitive, intracellular myo-inositol pool that was essential for maintaining phosphatidylinositol- mediated processes, including Na + -K + -ATPase activ- ity as put forth by Winegrad. 11 A variation of this is the "myo-inositol depletion" hypothesis by Greene et al,' 2 which considers the possibility that chronic hyper- glycemia results in the substantial reduction of intra- cellular myo-inositol content, eventually causing de- creased Na + -K + -ATPase activity. The mechanism by 3572 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933384/ on 02/14/2018

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Page 1: Modulation of myo-[3H]inositol uptake by glucose and sorbitol in

Investigative Ophthalmology & Visual Science, Vol. 33, No. 13, December 1992Copyright © Association for Research in Vision and Ophthalmology

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

//. Characterization of High- and Low-Affinity Myo-inositol Transport Sites

Patrick W. Cammarara,* Hai-Qing Chen,* Jinhua Yang,* and Thomas Yoriof

Myo-(3H]inositol accumulation in cultured bovine lens epithelial cells (BLECs) occurred by high- andlow-affinity, Na+-dependent transport sites. The high- and low-affinity transport systems had a Km of27 ± 4 and 157 ± 22 fimol/l, respectively, and Vmax of 652 ± 35 and 2952 ± 308 pmol/mg protein/hr,respectively. The uptake of myo-[3H]inositol was lowered after chronic (20 hr) incubation of culturedcells in 40 mmol/1 glucose throughout the concentration range for 1.5-400 ixmo\/l myo-inositol. Thecoadministration of sorbinil (0.1 mmol/1) to 40 mmol/1 glucose partially prevented the inhibitory effectof glucose on myo-[3H]inositol uptake. Although the aldose reductase inhibitor prevented the inhibitoryeffect of glucose on the low-affinity transport site, a glucose-sensitive process for myo-[3H]inositoluptake on the high-affinity transport site was uncovered by Lineweaver-Burk analysis. Acute exposure(3 hr) of cultured BLECs maintained in physiologic medium (Eagle's minimal essential medium, 5.5mmol/1 glucose) to a range of 5.5-44 mmol/1 glucose plus sorbinil also caused a decrease in myo-(3H]inositol uptake. Oixon plot analysis confirmed that the acute effect of glucose was the result ofcompetitive inhibition of the high-affinity myo-inositol transport site. Acute exposure of cultured cellsto 10-40 mmol/1 sorbitol also diminished the accumulation of myo-[3H]inositol. Dixon plot analysisestablished that the acute effect of exogenous sorbitol was the result of competitive inhibition of thelow-affinity myo-inositol transport site. These data support the contention that myo-inositol transportin BLECs is maintained by at least two processes: a glucose-sensitive, relative sorbitol insensitive,sodium-dependent, high-affinity myo-inositol transport system; and a sorbitol (aldose reductase) sensi-tive, sodium-dependent, low-affinity myo-inositol transport system. Invest Ophthalmol Vis Sci33:3572-3580,1992

The underlying biochemical deficits that contributeto the pathogenesis of diabetic complications in thelens and other tissues remain controversial.1"5 Despitethe identification of attendant pathologic manifesta-tions in cell culture and animal models of the disease,explanations of the mechanisms that lead to cellulardamage primarily are speculative. Among the preva-lent theories that seek to explain the aberrant bio-chemical alterations that lead to diabetic complica-tions, the "osmotic stress" hypothesis has been one ofthe most widely disseminated. Activation of the al-dose reductase reaction and formation and accumula-

From the Departments of *Anatomy and Cell Biology and fPhar-macology, Texas College of Osteopathic Medicine/University ofNorth Texas, Fort Worth, Texas.

Supported by National Public Health Service Award EYO557O-05 (PRC).

This work represents partial fulfillment of the requirements forthe degree of Master of Science for Hai-Qing Chen.

Submitted for publication: March 25, 1992; accepted July 16,1992.

Reprint requests: Patrick R. Cammarata, Texas College of Osteo-pathic Medicine, Department of Anatomy and Cell Biology, FortWorth, TX 76107.

tion of polyols directly promote osmotic swelling,leading to diabetic complications.6-7 The best evi-dence that aldose reductase and polyols are involvedin such complications of diabetes was the demonstra-tion that aldose reductase inhibitors can prevent ordelay the onset of complications.8'9 However, anymodel that attributes the onset of diabetic complica-tions to polyol accumulation also must consider thatmany tissues may not accumulate polyols to a con-centration high enough to exert a direct osmotic ef-fect.10

Alternative models, linking hyperglycemia anddysfunctional myo-inositol metabolism, have beensuggested. One model postulated that hyperglycemiamay deplete a discrete, small, rapidly turning over,glucose-sensitive, intracellular myo-inositol pool thatwas essential for maintaining phosphatidylinositol-mediated processes, including Na+-K+-ATPase activ-ity as put forth by Winegrad.11 A variation of this isthe "myo-inositol depletion" hypothesis by Greene etal,'2 which considers the possibility that chronic hyper-glycemia results in the substantial reduction of intra-cellular myo-inositol content, eventually causing de-creased Na+-K+-ATPase activity. The mechanism by

3572

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No. 13 MYO-INO5ITOL TRANSPORT SITES IN LENS CELLS / Commororo er ol 3573

which hyperglycemia promotes a reduction in intra-cellular myo-inositol content remains unexplained.

In our other report in this issue, we investigated theeffect of sorbinil on sodium-dependent myo-inositoluptake in a dose-dependent manner, using culturedbovine lens epithelial cells (BLECs) maintained inhigh-ambient glucose or physiologic medium. Thepartial prevention of the inhibitory effect of glucoseon myo-[3H]inositol uptake by inhibition of aldosereductase activity suggested that glucose-sensitive andsorbitol-sensitive processes were involved in the up-take of myo-inositol. The present study describes ananalysis of kinetic parameters that extends our initialfindings and supports the notion that cultured BLECstake up myo-inositol via two sodium-dependent trans-port systems, one of which is competitively inhibitedby glucose, the other being sorbitol-sensitive.

Materials and Methods

Cell Culture

Bovine (Bos taurus) eyes obtained from a localslaughterhouse were brought on ice to the laboratory,where the lenses were removed aseptically. After inci-sions were made on each side of the equator, the ante-rior capsule of each lens, with its epithelium attached,was peeled away from the cortex and placed in a 60mm Petri dish in 5 ml of a growth medium composedof Eagle's minimal essential medium (MEM) supple-mented with 10% calf serum, nonessential aminoacids, 5 mg/L ascorbic acid, 20 mg/L gentamycin sul-fate, and basal medium Eagle vitamin solution andmaintained in a water-humidified atmosphere of 5%CO2-95% air at 37°C, as previously described byCammarata et al.13 Cell outgrowth from the capsuleto the Petri dish after 7-10 days was dispersed in Ca2+-Mg2+ free MEM that contained 0.125% trypsin-0.05%EDTA and transferred to a 75 cm2 culture flask. Thecells, originating from two to three capsules, wereplaced in each culture flask with 40 ml of growth me-dium. When they reached confluence, the cells againwere dispersed and subcultured in a split ratio of 1:10in 25 cm2 culture flasks containing 5 ml of growthmedium. All studies were performed with confluentmonolayers in 25 cm2 culture flasks that representedsecond passage cells.

Kinetic Parameters

The kinetic parameters for myo-inositol uptakewere determined by chronically exposing cells (hereaf-ter operationally defined as a 20 hr incubation periodin serum-supplemented physiologic medium) to 40mmol/1 D-glucose on myo-[3H]inositol accumula-tion. BLECs were maintained in serum-containingphysiologic medium (MEM) or physiologic mediumcontaining 40 mmol/1 glucose in the presence or ab-

sence of 0.1 mmol/1 sorbinil for 20 hr before they weredivided into three groups for a 90 min equilibrationperiod. The three groups were: (1) medium A with 5.5mmol/1 glucose and 34.5 mmol/1 fructose; (2) me-dium A with 40 mmol/1 glucose; or (3) medium Awith 40 mmol/1 glucose and 0.1 mmol/1 sorbinil. Atthe end of 90 min, the cultures were switched to theappropriate medium A containing 1.0 /uCi/ml myo-[3H]inositol (94 Ci/mmol; Amersham, ArlingtonHeights, IL) over a concentration range of 1.5-400/xmol/1 myo-inositol for a 3 hr uptake period at 37 °C.After the incubation, the cells were washed in ice-coldCa2+ added phosphate-buffered saline; radioactivityand protein determinations were performed as de-scribed in our other report in this issue. Medium Aconsisted of: 5.5 mmol/1 glucose, 135 mmol/1 NaCl,5.4 mmol/1 KC1, 1.8 mmol/1 CaCl2, 34.5 mmol/1 fruc-tose, and 10 mmol/1 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, pH 7.4.

Experiments for Dixon plot analysis were per-formed as follows. Cells previously maintained inMEM were switched to medium A for a 90 min equili-bration period before they were divided into fourgroups: medium A containing (1) 5.5 mmol/1, (2) 11mmol/1, (3) 22 mmol/1, and (4) 44 mmol/1 D-glucose.The experiment was performed at a fixed osmolarityof medium A as the fructose concentration was re-duced accordingly and as glucose concentration wasraised from 5.5 mmol/1 to 44 mmol/1. All experimen-tal sets contained 0.1 mmol/1 sorbinil. Myo-inositolaccumulation was followed by the addition of 1.0/uCi/ml myo-[3H]inositol and selected myo-inositolconcentrations for a 3 hr uptake period at 37°C. Es-sentially the same experiment was performed with 10,20, 30, and 40 mmol/1 sorbitol, except that in thissituation, the medium osmolarity was not fixed byfructose readjustment and sorbinil was not presentbefore or during the uptake period.

Statistical Analysis

Statistical analyses were performed with the statisti-cal programs from Tallarido and Murray14 adaptedfor the IBM PC-XT. Appropriate statistical analyseswere applied to each group of data as indicated. Lin-ear regression analysis and correlation coefficientswere generated where appropriate. In some instances,the correlation coefficient suggested that the data didnot conform to a linear transformation.

ResultsKinetic Parameters

Shown in Figure 1 are the Eadie-Hofstee plots ofmyo-[3H]inositol uptake by BLECs, based upon thevelocity curves presented in the preceding manuscript(refer to accompanying article; Fig. 8). Myo-inositol

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3574 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / December 1992 Vol. 33

Eadie-Hofstee Plot of Myo-inositol Uptake

iJUU

2000

1500

1000

500

0

O

" \

- \

°\

-

-

i

MEM(5.5 mM

o

glu)

-- o

1

2000

1500

1000

500

10 20 30 40 50

-500

Glucose(40 mM glu)

10 12 16

V/S

Fig. 1. Eadie-Hofstee plot of myo-inositol uptake. BLECs werepreincubated in physiologic medium containing 40 mmol/1 glucose(Glu) or control (5.5 mmol/1 glucose, minimal essential medium[MEM] for 20 hr. BLECs were switched to medium A containing5.5 mmol/1 glucose and 34.5 mmol/1 fructose or medium A con-taining 40 mmol/1 glucose for a 3 hr myo-inositol uptake periodover a myo-inositol dose range of 1.5-400 /imol/l. Data points aretriplicate determinations from individual flasks and representmeans ± standard errors. The unit of velocity is picomoles/milli-gram protein/hr and S is micromolar. Two myo-inositol transportsites were apparent over the concentration ranges of 1.5-25 ^mol/1and 50-400 Mmol/1. Data points were plotted by linear regression,and the correlation coefficients for the low-affinity transport siteand high-affinity transport site were 0.95 and 0.67 (MEM-treatedcells) and 0.96 and 0.98 (40 mmol/1 glucose-treated cells), respec-tively.

uptake proceeds via two transport sites. The high-af-finity, low-capacity transport system predominates at1.5-25 imol/1 myo-inositol, whereas the low-affinity,high-capacity myo-inositol transport system is promi-nent above 50 /xmol/l myo-inositol. Kinetic constantsfor the high- and low-affinity transport systems areKm of 27 ± 4 (n = 4) and 157 ± 22 (n = 4) ^mol/1,respectively, and Vmax of 652 ± 35 (n = 4) and 2952± 308 (n = 4) pmol/mg protein/hr, respectively.

Chronic exposure (20 continuous hours) of BLECsto 40 mmol/1 glucose revealed an increase in the Kmfor the high- and low-affinity myo-inositol transportsites without a significant change of Vmax, indicatingcompetitive inhibition (Table 1). The concentrationdependence of high- and low-affinity myo-[3H]-inositol uptake was graphically distinguished as twoseparate components by Lineweaver-Burk plots pre-sented in the concentration ranges of 1.5-25 ^mol/1and 50-400 /umol/1 myo-inositol, respectively (Fig. 2).A trend toward normalization of the Km of the low-affinity myo-inositol transport site was apparentwhen sorbinil was concomitantly administered withthe 40 mmol/1 glucose medium. The Km of the high-affinity myo-inositol transport site, although some-what decreased because of the sorbinil accompani-ment in 40 mmol/1 glucose, remained significantlyelevated compared to cells maintained in physiologicmedium. Thus, although the concomitant addition ofan aldose reductase inhibitor with 40 mmol/1 glucoselargely prevented the inhibitory effect of glucose onthe low-affinity transport site, a glucose-sensitive pro-cess for myo-inositol uptake on the high-affinity trans-port site was uncovered by Lineweaver-Burk transfor-mation of the Michaelis-Menten equation (Fig. 2).

Acute Effects of Glucose and Aldose ReductaseInhibition on the High-Affinity Myo-inositol Carrier

The experiment just described indicated that glu-cose competitively inhibited myo-inositol uptake viathe high-affinity transport site, whereas sorbitol ap-peared to primarily inhibit myo-inositol uptake viathe low-affinity transport site. To confirm these re-sults and more conclusively establish the nature of theassociation between high-ambient glucose and myo-inositol transport, lens cells were briefly exposed to 40mmol/1 glucose that contained the inhibitor of sorbi-tol biosynthesis, sorbinil. Thus, the acute (3 hr expo-sure) effects of glucose on myo-inositol transport

Table 1. Kinetic parameters of the low- and high-affinity myo-inositol transporter takenfrom Eadie-Hofstee plots

Transporter MEM (5.5 mmol/l glucose) Glucose (40 mmol/l)

High affinity

v m a x

Low affinityKm

max

27652

1572952

± 4± 35

± 22±308

51 ±577 ±

235 ±2185 ±

12*82

25*262

Values are the means ± standard errors of four separate experiments run intriplicate. * Significant from minimal essential medium (MEM) using theleast significance difference test (P < .01).

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No. 13 MYO-INOSITOL TRANSPORT SITES IN LENS CELLS / Cammororo er ol 3575

Lineweaver-Burk Plot of Low Affinity Myo-inositol Transporter

o•V

!

-0.03

-*• '

MEMGluGlu/Sor

- 0 0 2 ^ :

0.004

0.003

0.002

0.001

s^er o.-0.001

-0.002

-0.003

-0.004

-

-

- 1

)0

-

-

-

0.01

^ § = ^1

0.02

i

0.03

1/S

Lineweaver-Burk Plot of High Affinity Myo-inositol Transporter

O MEM• GluV Glu/Sor

i i i

-0.8 -0.6 -0.4_^.

0.050

0.038

0.025

0.013

/

-0.025

-0.038

&r^ i i i i

0 0.2 0.4 0.6 0.8

-

-

1/S

Fig. 2. Effect of chronic glucose exposure and sorbinil on myo-in-ositol uptake plotted by the Lineweaver-Burk transformation equa-tion. BLECs were preincubated in 40 mmol/1 glucose (Glu), 40mmol/1 glucose plus 0.1 mmol/1 sorbinil (Glu/Sor), or control (5.5mmol/1 glucose, minimal essential medium) for 20 hr. Afterchronic glucose exposure, BLECs were divided into threegroups—medium A (5.5 mmol/1 glucose and 34.5 mmol/1 fructose,medium A containing 40 mmol/1 glucose, or medium A containing40 mmol/1 glucose and 0.1 mmol/1 sorbinil for a 3 hr myo-inositoluptake period over a concentration range of 1.5-400 Mmol/1. Eachdata point represents the mean ± standard error from triplicateindividual flasks. The relationship between glucose and the high-af-finity myo-inositol transport site and sorbitol and the low-affinitymyo-inositol transport site was apparent over the myo-inositol con-centration ranges of 1.5-25 Mmol/1 and 50-400 Mmol/1, respec-tively, when plotted in double reciprocal fashion. Data points wereplotted by linear regression, and the correlation coefficient was 0.99for all treatments.

could be examined in the absence of accumulated sor-bitol. With this approach, it was possible to uncoverany relationship between glucose and the active trans-port of myo-inositol. Figure 3A is a Dixon plot thatresulted from the acute incubation of BLECs with D-glucose and sorbinil. It shows that D-glucose had aninhibitory effect on myo-inositol uptake. The myo-inositol concentrations used for the experiment were

1.57,3.73, and 6.25 /u.mol/1, selected because they rep-resent the range of concentrations of the high-affinitytransport site. The concentrations of glucose used toexamine the inhibition of myo-inositol accumulationwere 5.5, 11, 22, and 44 mmol/1. These concentra-tions covered the typical range of our experimentalconditions.

In Figure 3A, the intersections of the Dixon plotsfor all values of substrate above the inhibitor axis indi-cate that glucose competitively inhibits myo-inositoluptake. The Ki of D-glucose on myo-inositol accu-mulation ranged from 29 to 35 mmol/1 (n = 6). Theresulting apparent Ki of glucose of 35 mmol/1 impliesthat the high-affinity myo-inositol carrier has an af-finity for myo-inositol that is at least 1000 timesstronger than for glucose. The data shown in Figure3B was generated as in Figure 3A, except that themyo-inositol concentrations used for the experimentwere 50, 100, and 200 /umol/l, which represented therange of concentrations of the low-affinity transportsite as defined under these experimental conditions.The failure of the Dixon plots to intersect for all val-ues of substrate provided the evidence that D-glucosedoes not interact with the low-affinity myo-[3H]inositol transport system (correlation coefficientsindicate nonlinearity).

Acute Effects of Sorbitol on Myo-inositol Uptake

To determine the acute effect of exogenous sorbitolon myo-inositol uptake, BLECs maintained in physio-logic medium were switched to serum-free medium Athat contained 10 mmol/1 sorbitol and a trace amountof myo-[3H]inositol over a myo-inositol concentra-tion range of 1.5-800 jumol/1 for a 3 hr incubationperiod (Fig. 4). Control cells were maintained inserum-free medium A that contained 10 mmol/1 fruc-tose. The uptake of myo-inositol with the sorbitol-containing medium was indistinguishable from thecontrol in the range of 1.5-100 /imol/1 myo-inositol,as determined by picomoles/milligram protein/hr ofaccumulated myo-inositol. However, myo-inositoluptake was markedly depressed by the sorbitol treat-ment in the myo-inositol concentration range of 200-800 ^mol/1.

The experiment described above indicates that ex-ogenous sorbitol had little to no effect on myo-inosi-tol uptake via the high-affinity transport system, butcaused significant attenuation of myo-inositol uptakevia the low-affinity myo-inositol transport system. Toconfirm and expand upon these results, the relation-ship between high-ambient sorbitol and the activetransport of myo-inositol was examined by Dixonplot analysis. Figure 5A is a Dixon plot of myo-[3H]inositol uptake resulting from the acute (3 hr) in-

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3576 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / December 1992 Vol. 33

Dixon plot of D-glucose effect on myo-inositol uptake

(high affinity transporter)

0.060 r

Dixon plot of D-glucose effect on myo-inositol uptake

(low affinity transporter)

0.003 r

O 1.57 uM MI• 3.23 uM MIV 6.25 uM MI

1/V

01000-50 -40 -30 -20 -10 0 10 20 30 40 50

Glucose (mil)

0!000-50 -40 -30 - 2 0 - 1 0 0 10 20 30 40 50

Glucose (mil)

Fig. 3. Dixon plot of acute D-glucose and sorbinil exposure on myo-inositol uptake. (A) (High-affinity transporter). BLECs were incubatedin medium A containing 1.57, 3.23, and 6.25 ^mol/1 myo-inositol and a trace amount of myo-[3H]inositol. Data points were plotted by linearregression, and the correlation coefficients for the Dixon plot resulting from 1.57, 3.23, and 6.25 Mmol/1 myo-inositol were 0.85, 0.82, and0.72, respectively. (B) (Low-affinity transporter). BLECs were incubated in medium A containing 50, 100, and 200 Mmol/1 myo-inositol and atrace amount of myo-[3H]inositol. Data points were plotted by linear regression, and the correlation coefficients for the Dixon plot resultingfrom 50, 100, and 200 ^mol/l myo-inositol were 0.22,0.43, and 0.80, respectively. The incubation mixtures for A and B also contained 5.5, 11,22, or 44 mmol/1 D-glucose and 0.1 mmol/1 sorbinil. Myo-inositol uptake was determined after a 3 hr uptake period. Data are means± standard errors of triplicate determinations from individual flasks.

cubation of BLECs to high-ambient sorbitol. Theconcentrations of sorbitol were 10, 20, 30 and 40mmol/1, and the myo-inositol concentrations used forthe experiment were 1.57, 3.23, and 6.25 ^mol/1.When the concentration dependence of myo-[3H]inositol uptake was presented in this way, it wasapparent that exogenous sorbitol had no effect onhigh-affinity myo-inositol transport, because myo-inositol uptake was independent of sorbitol concen-tration (correlation coefficients indicate nonlinear-ity). Figure 5B is a Dixon plot of the acute inhibitoryeffect of sorbitol on low-affinity myo-inositol trans-port. The myo-inositol concentrations used for thisstudy were 100, 200, and 400 pmol/1. Plotted in thisform, this study further indicated that exogenous sor-bitol competitively inhibits low-affinity myo-inositoltransport by cultured BLECs. The Ki of exogenoussorbitol on myo-inositol uptake was approximately19 mmol/1 (n = 6).

Discussion

The mechanism by which intracellular myo-inosi-tol is transported from the surrounding environmentinto cells appears tissue-specific. Indeed, glucose-me-

diated inhibition of myo-inositol uptake has been de-scribed as competitive or non-competitive, dependingupon the system being investigated. Myo-inositoltransport has been described in a number of differentsystems as: (1) an active high-affinity, low-capacitymechanism; (2) an active low-affinity, high-capacitymechanism; (3) sharing physical characteristics of anactive high-affinity and active low-affinity uptakemechanism; or (4) as a nonactive facilitated diffusionmechanism. Myo-inositol uptake in the cultured bo-vine lens epithelial cell model system exhibited char-acteristics of active transport. Support for this is basedupon our observations that myo-inositol is accumu-lated against a concentration gradient, is energy-de-pendent (ouabain-sensitive), is sodium-dependent,and that it exhibited saturation kinetics.15-16 Exposureof the cultured BLECs to high-ambient glucose or ga-lactose resulted in the attenuation of myo-inositol up-take.15'16 Reports of similarly depressed myo-inositoluptake have been described in the intact lens exposedto hyperglycemic conditions17"19 and with mouse cere-bral microvessel endothelial cells20 and cultured neu-roblastoma cells.21

In the present study, we examined the kinetic pa-rameters of myo-inositol uptake in cultured BLECs

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No. 13 MYO-INOSITOL TRANSPORT SITES IN LENS CELLS / Cammorora er ol 3577

Effect of Exogenous Sorbitol on Myo-inositol Uptake

3500

3000

2500

-S 2000

E 1500

1000

500

• FructoseO Sorbitol

200 400 600 800 1000

Myo-inositol (uM)

Fig. 4. Effect of acute exogenous sorbitol exposure on myo-inosi-tol uptake. BLECs maintained in physiologic medium (5.5 mmol/1glucose) were divided into two groups—medium A containing 5.5mmol/1 glucose and 44.5 mmol/1 fructose (control) or medium Acontaining 5.5 mmol/1 glucose, 34.5 mmol/1 fructose, and 10mmol/1 sorbitol, plus a trace amount of myo-[3H]inositol (1 nC\/ml), for a 3 hr myo-inositol uptake period over a myo-inositol con-centration range of 1.5-800 uM. Data represent triplicate determi-nations from individual flasks. Data points are means ± standarderrors.

and determined whether the data was best repre-sented by a one- or two-site transport mechanism.Eadie-Hofstee plots of myo-[3H]inositol accumula-tion by cultured BLECs indicated that myo-inositoluptake proceeded via two transport sites (Fig. 1). Tosupport this observation, we further employed non-linear regression analysis and mass action equationsderived for one and two independent sites and a pro-gram to determine "goodness" of fit by iteration. Asshown in Figure 6, the data were best represented bytwo transport sites, and thus confirmed the Eadie-Hofstee plots. At this time, the data do not permit usto determine whether there are two physically inde-pendent myo-inositol carrier proteins, or, alterna-tively, whether there are two affinity states of the samemyo-inositol transport protein.

Eadie-Hofstee plots indicated that myo-inositol up-take in cultured BLECs occurred by a high- and low-affinity transport system. Both the high- and low-af-finity transports were sodium-dependent. Kineticanalysis of the high-affinity transport site revealed aKm of 27 ^mol/1 compared to 157 /umol/1 for thelow-affinity site. This is similar to that described formyo-inositol uptake in mouse cerebral micro vessel en-dothelial cells,20 where the high- and low-affinity

myo-inositol transport system had a Km of 11 /umol/1and 198 /u.mol/1, respectively. This also is similar tothat described for myo-inositol uptake in human reti-nal pigmented epithelium22 and several other cul-tured mammalian cells.23

Chronic exposure (20 continuous hours) of cul-tured BLECs to 40 mmol/1 glucose produced an atten-uation of myo-inositol uptake that was observed withthe high- and low-affinity transport sites. The increasein Km after incubation in high-ambient glucose, withlittle change in Vmax, suggested that the glucose-me-diated inhibition was competitive (Table 1). Line-weaver-Burk plot analysis also indicated competitiveinhibition (Fig. 2). The aldose reductase inhibitor,sorbinil, near-normalized the myo-inositol uptake ofthe low-affinity transport site, but had little effect onthe glucose-mediated attenuation of myo-inositol up-take of the high-affinity transport site. This suggestedthat glucose was directly affecting the high-affinitytransport site, whereas sorbitol accumulation (result-ing from product formation of the aldose reductasereaction) was impeding myo-inositol uptake via thelow-affinity transport site. Dixon plot analysis con-firmed that glucose competitively inhibited the high-affinity myo-inositol transport site, with an apparentKi of 35 mmol/1 (Fig. 3 A). The latter observation sug-gested that the myo-inositol transporter had a 1000times greater affinity for myo-inositol than for D-glu-cose.

These experiments were performed in the presenceof sorbinil (thereby eliminating the possibility of sor-bitol accumulation), so they were designed to exam-ine the direct action of glucose on myo-inositol up-take. Dixon plot analysis further revealed that glucosedid not interact with the low-affinity myo-inositoltransport site (Fig. 3B). Competitive inhibition ofmyo-inositol uptake by glucose has been reported forcultured rat glomerular mesangial cells24 and mousecerebral microvessel endothelial cells,20 which, likethe lens, appeared to have glucose-sensitive and sorbi-tol-sensitive components of myo-inositol transport.In the latter study, the Ki for glucose was 21 mmol/1,similar to that reported in the present study with lensepithelial cells in culture. Not all tissues respond tohigh-ambient glucose in a similar manner. For exam-ple, cultured neuroblastoma cells21'25 and retinal capil-lary pericytes26 revealed a decrease in myo-inositolaccumulation because of noncompetitive glucose inhi-bition of high-affinity myo-inositol uptake. Such di-versity in the mode of glucose-mediated inhibition ofmyo-inositol uptake in various tissues probably re-flects the physical characteristics of the myo-inositoltransporters present and their predilection to be inhib-ited by the products of the polyol pathway. It is highlylikely that more than one type of myo-inositol carrier

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3578 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / December 1992 Vol. 33

Dixon plot of sorbitol effect on myo-inositol uptake(high affinity transporter)

A

Dixon plot of sorbitol effect on myo-inositol uptake

(low affinity transporter)

0.0020-rO 100 uM MI

B

0.060 r

O 1.57 uM MI

• 3.23 uM MI

V 6.25 uM MI

• 200 uM MIV 400 uM MI

1/V

0!000-50 -40 -30 - 2 0 -10 0 10 20 30 40 50

Sorbitol (mM)

-50 - 4 0 -30 -20 -10 10 20 30 40 50

Sorbitol (mM)

Fig. 5. Dixon plot of acute sorbitol exposure on myo-inositol uptake. (A) (High-affinity transporter). BLECs were incubated in medium Acontaining 1.57, 3.23, and 6.25 ^mol/1 myo-inositol and a trace amount of myo-[3H]inositol. Data points were plotted by linear regression, andthe correlation coefficients for the Dixon plot resulting from 1.57, 3.23, and 6.25 ^mol/l myo-inositol were 0.34,0.72, and 0.70, respectively.(B) (Low-affinity transporter). BLECs were incubated in medium A containing 100, 200, and 400 ^mol/1 myo-inositol and a trace amount ofmyo-[3H]inositol. Data points were plotted by linear regression, and the correlation coefficients for the Dixon plot resulting from 100,200, and400 jtmol/1 myo-inositol were 0.93,0.97, and 0.92, respectively. The incubation medium of A and B also contained 10, 20, 30, or 40 mmol/1sorbitol. Myo-inositol uptake was determined after a 3 hr uptake period. Data points represent means ± standard errors taken from triplicatedeterminations of individual flasks.

protein exists, as has been generally demonstrated forthe glucose transporter, where at least four sodium-in-dependent and two sodium-dependent proteins havebeen identified.2728 Indeed, Nikawa et al29 recentlyreported the isolation and characterization of two dis-tinct myo-inositol transporter genes of Saccharo-myces cerevisiae, suggesting that myo-inositol trans-porters could be classified into the sugar transportersuperfamily.

Although it was demonstrated (Fig. 3B) that glu-cose did not directly impede the low-affinity myo-ino-sitol transport system, results shown here and in ourother report in this issue16 indicated that a sorbitol-sensitive process was involved in the uptake of myo-inositol by the low-affinity transport site in culturedlens epithelial cells. This interaction was investigatedby determining the effects of exogenous sorbitol (10mmol/1) on myo-inositol uptake over a range of myo-inositol concentrations (Fig. 4). Although adding sor-bitol had no effect on the high-affinity transport site,it significantly reduced myo-inositol uptake in themyo-inositol concentration range associated with thelow-affinity transport site. This result was consistentwith the observation that sorbinil prevented the glu-

cose-mediated attenuation of myo-inositol uptake viathe low-affinity transport site as demonstrated by Lin-eweaver-Burk plot analysis (Fig. 2) and indicated thatthe glucose effect on the low-affinity transporter wasassociated with sorbitol accumulation.

Agreeing with this finding was the Dixon plot analy-sis of the concentration-dependent competitive inhibi-tion of sorbitol on myo-inositol uptake of the low-af-finity transporter (Fig. 5B). Varying the concentrationof exogenous sorbitol had no effect on the high-affin-ity myo-inositol transport system (Fig. 5A). The Kifor sorbitol inhibition of myo-inositol uptake on thelow-affinity transport site was 19 mmol/1. Because ex-ogenous sorbitol only was presented to the culturedBLECs for an acute (3 hr) duration, it was highly un-likely that sorbitol accumulated intracellularly to alevel appreciable enough to affect the low-affinitymyo-inositol transport site from the cytoplasmic sideof the cell. Therefore, under these experimental con-ditions, the effect of exogenous sorbitol on myo-inosi-tol uptake probably reflected the capability of this po-lyol to compete for the external, plasma membranelow-affinity myo-inositol transport site. It is temptingto speculate that the low-affinity myo-inositol trans-

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No. 13 AAYO-INOSITOL TRANSPORT SITES IN LENS CELLS / Commororo er ol 3579

Eadie-Hofstee PlotsOne Site vs Two Site Fits

2500

2000 -

1500 -

> 1000 -

500 -

0 -

-500

o

0

-

1 1

o

o

Raw DataPredicted

-Predicted

cP

OneTwo

. o "^CO

1 1

SiteSite

i

10 15 20

V/[S]

25 30 35

Fig. 6. Nonlinear regression analysis for one and two indepen-dent myo-inositol transport sites determined on Eadie-Hofsteeplots of myo-[3H]inositol uptake. The dashed line represents thepredicted one site, and the continuous line represents the predictedtwo sites. The data were derived from the experiment described inFigure 1.

port site is a generalized polyol carrier that regulatesintracellular polyol concentrations during periods ofsevere osmoregulatory stress. However, the experi-ments that used exogenous sorbitol probably did notreveal the true mechanism by which intracellular sor-bitol influences myo-inositol uptake. On the otherhand, such experiments do somewhat indicate the rel-ative affinity of the high- and low-affinity transportsites for sorbitol.

Cultured BLECs appear to have (based on kineticanalysis) two functional myo-inositol carrier proteins(or a single myo-inositol transporter where ligand in-teraction with the transporter varies with the confor-mation of the system) that appear to be distinct fromglucose transport. The high-affinity myo-inositoltransport site predominates at low concentrations ofexternal myo-inositol, and, using the energy of thesodium gradient (external to internal), accumulatesmyo-inositol intracellularly. Given the operationallimitations of this model system, the high-affinitymyo-inositol transport site functions normally in thepresence of physiologic glucose concentrations (5.5mmol/1). However, when extracellular glucose rises—as with diabetes, animal models of hyperglycemia, orwith the introduction of high-ambient glucose to phys-iologic surrogates like the BLEC culture system—myo-inositol uptake via the high-affinity transport

site is reduced by direct competitive inhibition. As aresult, myo-inositol uptake proceeds normally via thelow-affinity transport system only when an adequateextracellular concentration of myo-inositol is avail-able. Thus, a state of hyperglycemia could reducemyo-inositol accumulation and deplete intracellularmyo-inositol, important for the inositol-phospholipidcycle and cellular signal transduction mechanisms.

The situation may be further exacerbated when ele-vated glucose is converted to sorbitol (which we haveshown) and negatively affects the myo-inositol uptakecapability of the low-affinity myo-inositol transportsystem. The depletion of intracellular myo-inositolcontent could have far-reaching repercussions for lenscellular membrane integrity and for the capability ofthe lens epithelium to maintain normal cellular func-tion. Further studies are in progress to identify theendogenous regulators/antagonists of myo-inositoluptake. Such studies are particularly pertinent to thelens, which has limited access to nutrients and reliesgreatly on its microenvironment. Moreover, our re-sults are potentially clinically important in the man-agement of diabetes because of the implication thatinhibition of aldose reductase alone does not fullyprevent the deleterious effect of glucose on myo-inosi-tol accumulation. This implies that this class of com-pounds may not completely protect against the com-plications of diabetes if hyperglycemia is not tightlycontrolled.

Key words: high-affinity site, lens cells, low-affinity site,myo-inositol transport

Acknowledgments

The authors express their gratitude to Dr. Michael Martinfor helpful discussions.

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