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Characterization of [2-3H]deoxy-d -glucose uptake in retina and retinal pigment epithelium of normal and diabetic rats
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Pergamon
0197-0186(95)00068-2
Neurochem. Int. Vol. 28, No. 2, pp. 213-219, 1996 Copyright © 1996 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0197-0186/96 $15.00+ 0.00
CHARACTERIZATION OF [2-3H]DEOXY-D-GLUCOSE UPTAKE IN RETINA A N D RETINAL PIGMENT EPITHELIUM OF NORMAL AND DIABETIC RATS C A R M E N V I L C H I S and R O C I O S A L C E D A * Instituto de Fisiologia Celular, UNAM. Mrxico, D.F. 232, Mexico (Received 20 July 1994 ; accepted 17 April 1995)
Abstract--The outer blood-retinal barrier which results from the tight junctions between retinal pigment epithelial cells (RPE) restricts the flow of nutrients reaching the retina. We characterize the transport of [2-3H]deoxy-D-glucose (2-DG) across isolated mammalian neural retina and RPE in terms of their kinetics constants. In addition, the effect of insulin on glucose transport was studied by using streptozotocininduced diabetic rats. RPE accumulates 2-DG by a temperature-sensitive and energy-dependent complex kinetics mechanism. The retina takes up 2-DG by an energy and Na÷-dependent saturable system with an apparent Km of 2 mM. Insulin induced an increase of 2-DG uptake by normal retina. The retina of diabetic rats shows lower levels of 2-DG accumulation. These levels can be returned to the normal ones by exposure to insulin. Although insulin does not affect, significantly, 2-DG accumulation by RPE, 2-DG uptake of RPE from diabetic rats shows a normal saturable kinetics with an apparent K,, of 20 mM. Those findings suggest the presence of different types of glucose transporters in retina and RPE. Insulin-sensitive glucose transport in retina might be involved in the manifestation of diabetic retinopathy.
Two blood systems occur in the retina, the retinal vascular system that nourishes the inner layers, and the choroidal vessels received by the retinal pigment epithelium (RPE), whose cells are attached to each other through tight junctions. These tight junctions constitute the blood-retinal barrier (Cunha-Vaz, 1976). Glucose is the major fuel for energy metabolism in the retina (Berman, 1991). Glucose transport by R P E (Miller and Steinberg, 1976; Zadunaisky and Degnan, 1976; Pascuzzo et al., 1980; Masterson and Chader, 1981; Stramm and Paulter, 1982; Crosson and Paulter, 1982) and in the retinal microvasculature (Betz et al., 1983; Li et al., 1985) has been studied, and there is general agreement that glucose is transported by a facilitated system. Retinopathy is one of the clinical manifestations of long standing diabetes meUitus (Davies, 1988). A major biological action of insulin is to promote glucose metabolism and this effect is thought to be largely due to acceleration of glucose transport (Olefsky, 1978). Insulin-stimulated increase in deoxyglucose transport (Allen and Gerritsen, 1986) and insulin receptors have been detected in endothelial cells from blood vessels (King et al., 1985 ;
Haskell et al., 1984) and neural retina (Waldbilling et al., 1987 ; Rosenzweig et al., 1990). The regulatory mechanisms that maintain glucose homeostasis in retina and RPE may include hormonal action, and it would be interesting to compare the properties of glucose transport operating under basal and insulin-stimulated conditions. Therefore, we undertook experiments to characterize the glucose transport in the normal rat retina and RPE, and compared them with those of diabetic animals.
EXPERIMENTAL PROCEDURES Materials [2-3H]deoxy-D-glucose (2-DG ; 37 Ci/mmol) and ~4C-inulin (0.05 mCi/17 mg) were obtained from New England Nuclear (Boston, MA, U.S.A.). Streptozotocin and insulin were acquired from Sigma Chemical Co. (St Louis, MO, U.S.A.). The kit for glucose determination was obtained from Salubridad (Mexico, D.F.). All other reagents were of analytical grade. Animals Adult Long Evans rats (170-200g) were utilized in this study. Diabetes was induced by a single intraperitoneal injection of streptozotocin (65 mg/kg) in 0.05 M citrate buffer, p H 4.5 (M ackerer e t al., 1971). Rats were allowed free access to food and water. The streptozotocin-injected animals were
*Author to whom all correspondence should be addressed. 213
214
Carmen Vilchis and Rocio Salceda
used a~er 20 days of treatment and considered diabetic if serum glucose exceeded 300mg/dl. Analytical methods The rats were decapitated, the eyes were excised and the sclera was eliminated from the posterior part of the eye. The anterior part of the eye was removed, the retina and the RPE were gently peeled away using fine forceps. Retina and RPE were incubated at 37C with 2 ml of Krebs ringer bicarbonate (118 mM NaCI, 1.2 mM K H,PO4, 4.7 mM KCI, 2.5 mM CaCI_~, 1.17 mM MgSO4, 25 mM NaHCO~, plus 5.6 mM glucose, pH 7.4) containing 0.5 #Ci of 2-DG (13 #M). At the end of the incubation period, the tissue was washed with cold medium, weighed and dissolved in 0.5 ml of 1% sodium dodecyl sulfate (SDS). Radioactivity in the solubilized tissue was measured after the addition of 5 ml Tritosol and counts for radioactivity performed in a liquid scintillation spectrophotometer. For kinetics studies the tissue was incubated for 2 rain in 1 ml Krebs medium containing 0.5 #Ci 2-DG and different concentrations of non-radioactive 2-deoxy4)-glucose, in the absence of glucose. For efflux experiments the tissue was loaded with 2-DG for 20 rain incubation in Krebs medium containing 0.5 l*Ci 2-DG. After the incubation, loaded tissue was transferred to 1 ml of non-radioactive medium for different periods of time. The radioactivity released into the medium and that remaining in the tissue was determined as above. For exchange experiments the tissue was incubated for 20 rain in I ml Krebs medium. After that, 0.5 #Ci 2-DG was added and the uptake was measured as described above. Media depleted of sodium ions was prepared by replacement of NaCI with isotonic quantities of choline chloride. When the effect of inhibitors were tested, they were added together with 2-DG. The extracellular space was estimated as described by Ames et al. (1967) and Frank and Scboffeniels (1972). Retina and RPE were incubated for 30 rain with 1.0 /~Ci/ml of '4C-inulin. After incubation, tissue was weighted and its radioactivity measured as described above. The inulin space was calculated from the ratio of the ~4C-inulin content of tissue to the ~4C-inulin in the incubation medium. The obtained values were around 27% and were substracted from 2-DG uptake values. Glucose concentration in serum was determined by the orthotoluidine reaction by means of a commercial kit. Results were compared by analysis of variance (ANOVA) and multiple comparisons by Tukey's analysis.
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~
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Time (min) Fig. 1. Time course of 2-DG uptake in rat retina and RPE. Tissue was incubated in a Krebs bicarbonate medium, pH 7.4 in the presence of 5.6 mM glucose and 0.5/~Ci of 2-DG. Retina (A), RPE (91). The values are the mean ± S E M of 4 experiments.
where exchange transport was measured. After previous equilibration with Krebs medium for 20 min 2D G was added. U n d e r these conditions uptake o f 2D G reached values o f 8.1 __+0.7 and 1.3 + 0 . 0 9 / l m o l / g lbr retina and RPE, respectively. Consistent with these results, the efflux experiments (Fig. 2) demonstrated a facilitated diffusion type o f glucose transporter. Efftux o f 2 - D G was not affected in the retina nor the R P E o f diabetic rats (Fig. 2). The effect o f various c o m p o u n d s on 2-DG accumu-
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RESULTS
2-DG, an analogue o f glucose that is metabolized to 2-deoxy-D-glucose-6-phosphateand trapped within cells, has been used extensively to study hexose transport in many cell types. Figure I shows a time course o f 2-DG uptake in rat retina and RPE. The accumulation o f 2-DG is linear for the first 10 rain (not shown), reaching plateau levels at approx. 30 rain o f 10.8 and 1.37/~mol/g in retina and RPE, respectively (Fig. 1). We examined the uptake o f 2-DG under conditions
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I 20
I 30
Time (rain) Fig. 2. Efflux of 2-DG from retina and RPE. Tissue was loaded by 20 min incubation in Krebs medium containing 0.5 l~Ci of 2-DG. After this, tissue was incubated consecutively with non-radioactive medium for 5 min periods. Results of three different experiments, in which SE was less than 10%. have been averaged, and the data normalized to the 2-DG loaded after preincubation (100%). Normal retina (0). diabetic retina (A), normal RPE (©), diabetic RPE (A).
215
Uptake of 2-DG in retina and RPE lation is shown in Table 1. The uptake of 2-DG of both retina and RPE, was not significantly affected by 1 mM KCN, 0.1raM ouabain, nor 0.1 mM phloridizin, whereas low temperature abolished it. Iodoacetate (lmM) significantly inhibited 2-DG uptake by about 75 and 50% in retina and RPE, respectively. Accumulation of 2-DG in RPE was not significantly modified in the absence of sodium, whereas in retina it was inhibited 90% (Table 1). On the other hand, phloretin slightly (30%) affected accumulation of 2-DG. Cytochalasin B, an inhibitor of glucose uptake, reduced 2-DG accumulation by 40 50% in both RPE and retina (Table 1). The kinetics characteristics of the 2-DG were studied by measuring the 2-DG accumulation within a concentration range of 0.5-30 mM 2-DG. In the retina, a saturation curve was observed with an apparent K m of 2 mM and a Vmax of 0.75 #mol/g/min (Fig. 3). Uptake of 2-DG by RPE showed a complex kinetics, that could not be adjusted to the MichaelisMenten equation, suggesting allosteric interactions (Segel, 1975) (Fig. 4). Kinetics studies of 2-DG uptake by diabetic retina revealed an apparent Km of 5.0 mM and a Vmaxof 1.5 #mol/g/min (Fig. 3). Interestingly, 2-DG uptake by RPE from diabetic rats showed a normal Michaelis-Menten kinetics, with an apparent Km of 20 mM and a Vmaxof 0.75 ttmol/g/min (Fig. 4). The effect of insulin on 2-DG accumulation was studied in the RPE and retina. Addition of insulin to the incubation medium increased accumulation of 2D G by retina. The effect of insulin is dose-dependent, with maximal response (60%) observed at 5 10 ng/ml
(Fig. 5). On the contrary, insulin did not affect significantly 2-DG accumulation by RPE (Fig. 5). To determine whether the effect of insulin reflects the physiological situation, we studied the 2-DG uptake in the retina of streptozotocin-induced diabetic rats. As show in Fig. 6, the rate of 2-DG accumulation by diabetic retina is decreased by 25-30% compared with that of normal retina ; in agreement with these results, addition of insulin to the incubation medium re-established normal 2-DG uptake rate (Fig. 6).
DISCUSSION
The RPE provides a major transport pathway responsible for exchange of metabolites and ions between the choroidal blood supply and the neural retina (Cunha-Vaz, 1976 ; Miller and Steinberg, 1976). The RPE is believed to play an important role in a number of ocular diseases, including diabetic retinopathy (Bresnick, 1983) and proliferative vitreoretinopathy (Machemer and Laqua, 1975). As the neural retina has a very high rate of glucose metabolism, transport of glucose by RPE is probably one of its functions. Most, if not all, mammalian cells have integral membrane proteins that translocate glucose down its concentration gradient by a process of facilitative diffusion (Elbrink and Bihler, 1975). Recent cloning and sequencing studies suggest the existence of several different glucose transport proteins (Glut 1-5) for carrier mediated diffusion of glucose across cell membranes in various tissues and with different kinetics properties (Gould and Bell, 1990 ; Gould et al., 1991 ; Nishimura et al., 1993). The tissue distribution of the different glucose transporters is not well known and more than one transporter may be present in the same Table 1. Effect of different compounds on 2-DG uptake tissue. Glutl type glucose transport is reported to be conRetina RPE centrated in the cells that form occluding junctions, Addition (/zmol/g) thus constituting blood-tissue barriers, including the RPE (Harik et al., 1990 ; Takata et al., 1990). Human -7.67__+0.18 1.26+0.15 cultured RPE cells predominantly express the Glutl KCN (lmM) 8.03+0.40 1.02+0.03 gene and protein, with minor expression of Glut3 and Ouabain (0.1mM) 6.38+0.43 1.05+0.08 Iodoacetate (1 mM) 1.88+0.08" 0.59+0.04* Glut5 genes (Takagi et al., 1994). Phloridizin (0.1mM) 7.46+0.57 1.28+0.17 In agreement with previous studies (Miller and StePhloretin (0.1mM) 5.24+0.37* 0.81 +0.08* inberg, 1976; Zadunaisky and Degnan, 1976; PasCytochalasin B (10/~M) 3.56+ 1.12" 0.75 +0.04* Low temperature (4=C) 0.19+0.02" 0.16___0.02" cuzzo et al., 1980; Masterson and Chader, 1981; Sodium free medium 0.97+0.07* 1.08+0.01 Stramm and Paulter, 1982; Crosson and Paulter, 1982), our results show that 2-DG accumulation by Tissue was incubated for 20 n-finin a Krebs bicarbonate RPE is a carrier mediated process. We found a commedium with 5 mM glucose in the presence of 2-DG (0.5 #Ci). Each value is the mean _ SEM of at least three exper- plex kinetics for 2-DG uptake, that might be explained by allosteric interactions of the transporter and/or the iments. *P < 0.01.
216
Carmen Vilchis and Rocio Salceda 20
1.0 15
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0.5
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J 2.5
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i 5
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J Io
1/[s] mM
Fig. 3. Accumulation of 2-DG by rat retina as a concentration function. Incubation was carried out for 2 min with different concentrations of 2-DG in a Krebs bicarbonate medium without glucose. Right: double reciprocal plot. The points are the mean of at least three experiments. Normal retina (O), diabetic retina (A). existence of more than one carrier mediated process (Segel, 1975). Accumulation of 2-DG was energy dependent as indicated by the inhibition caused in the presence of iodoacetate and to a lesser extent by KCN. As reported for frog RPE (Zadunaisky and Degnan, 1976), we found 2 - D G uptake to be Na +-independent. The inhibition of 2-DG uptake by phloretin and cytochalasin B also evidence the glucose carrier. Addition of insulin to the incubation medium did not affect significantly 2 - D G uptake by normal RPE, although it was slightly decreased in RPE from streptozotocin-induced diabetic rats. In agreement with this result, RPE of diabetic animals show a normal saturable kinetics for 2-DG uptake suggesting that
insulin might modulate its transport. In this way, it should be noted that cultured human RPE cells synthesize and release insulin like growth factor I (ILGFl) (Waldbilling et al., 1991), and that 2 - D G uptake is stimulated when exposed to different growth factors, including I L G F - | (Takagi et al., 1994). Accumulation of 2 - D G by neural retina is considerably higher than that of RPE. We found that the retina exhibits a saturable temperature-dependent uptake of 2-DG. The inhibition of 2 - D G accumulation observed by iodoacetate but not by K C N , might be related to the high glycolytic activity of the retina (Graymore, 1970). An exceptionally high rate of both anaerobic and aerobic glycolysis and lactate production has been reported in the mammalian retina.
40 ="5 ~E m,
~L
I
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1/Is] mM
Fig. 4. Uptake of 2-DG in rat RPE. The RPE was incubated for 2 min with different concentrations of 2DG in Krebs medium without glucose. Normal rat RPE (O), diabetic rat RPE (A). Right: Michaelis Menten plot of 2-DG uptake in diabetic rat RPE. The points are the mean of 5 experiments in which SE was less than 10%.
Uptake of 2-DG in retina and RPE 200
RPE
RETINA
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Insulin (ng/ml)
Fig. 5. Effect of insulin on 2-DG uptake in normal rat retina and RPE. Tissue was incubated for 40 min in a Krebsbicarbonate medium, pH 7.4 containing 2-DG (0.5/~Ci), in the absence or presence of different insulin concentrations (ng/ml). Results shown are mean -I-SEM of 4-6 experiments. *P < 0.01.
15
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5
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Fig. 6. Uptake of 2-DG by normal and diabetic rat retina. Retina from normal ([]) or streptozotocin-induced diabetic rats (1~) were incubated in a Krebs bicarbonate buffer in the absence ( ~ ) or presence ( 9 ) of 10 ng/ml insulin. Results are mean ±SEM of 3-5 experiments. *P < 0.01. Electric activity of the retina is abolished either by withdrawal of oxygen supply or by addition of iodoacetate to the incubation medium (Berman, 1991 ; Winkler, 1981). Although ouabain should collapse the sodium gradient, the external sodium concentration appears to be sufficient to permit 2-DG accumulation, since it was 90% inhibited in the absence of sodium in the incubation medium. Accumulation of 2-DG occurs by a saturable system with an apparent Km of 2 raM, a value similar to the Km reported for the Na+-
217
glucose cotransport system (Brot Laroche et al., 1986) as well as for the Glutl type glucose transporter in human red blood cells (Nishimura et al., 1993) and photoreceptor outer segments (Hsu and Molday, 1991). Glutl has been reported to be present in Mialler cells, ganglion cells, photoreceptor cell bodies and outer limiting membrane in normal and diabetic human retina (Kumagai et al., 1994) showing high density in photoreceptor outer segments (Hsu and Molday, 1991). It is worth noting that 2-DG accumulation by retina is stimulated by insulin. These results agree with the lower accumulation levels of 2-DG observed in the diabetic retina. Kinetics analyses of the uptake in the retina of diabetic animals, suggest that insulin stimulates 2-DG accumulation by increasing its capacity. This is similar to reports on other cell types where the effect of insulin was mediated by an increase in the Vmax (Olefsky, 1978 ; Whitesell and Abumrad, 1985). Since exposure of streptozotocin-treated rat retina to insulin increases 2-DG accumulation to normal values, without modification of the efflux, insulin could induce incorporation of the glucose transporter to the membranes from an internal pool, as has been reported for muscle and adipose tissues (Suzuki and Kono, 1980). The insulin-sensitive 2-DG accumulation observed is most likely to be associated with the neural retina, since longer periods of incubation with insulin are necessary to demonstrate effects on hexose transport in cultured endothelium cells (Allen and Gerristen, 1986), and insulin receptors have also been detected in non vascular portions of bovine retina (Im et al., 1986; Waldbilling et al., 1987; Rosenzweiz et al., 1990). Down regulation of the glucose transporter in diabetic retina may represent a homeostatic mechanism controlling glucose uptake under high glucose concentrations. It is interesting that retinal glucose concentration was not modified by diabetes, showing values of 10.9__ 1.6 /~g/mg protein, quite similar to that in the normal retina (8.39 +0.2 #g/mg protein). But the retina's capacity to respond to insulin is limited compared with adipocytes (Calderhead et al., 1990). The insulin-sensitive glucose transporter represents less than 50% of the total transport activity in the retina. This limited response capacity to high glucose levels could lead to diabetic retinopathy. Our results indicate that both retina and RPE possess different glucose transporters. The characterization of these transporters, their regulation and interaction with the insulin-sensitive transporter requires further studies.
218
Carmen Vilchis and Rocio Salceda
Acknowledgement- The authors would like lo thank Mr G. S~inchez-Ch~ivez for his technical assistance and Dr P. Bissett Mandeville for statistical analyses. REFERENCES
Ames A., Tsukada Y. and Nesbett F. B. (19673 lntracellular Na +, Ca 2~ , Mg 2+, and P in nervous tissue: response of glutamate to changes in extracellular calcium. J. Neurochem. 14, 145 159. Allen L. A. and Gerristen M. E. (1986) Regulation of hexose transport in cultured bovine retinal microvessel endothelium by insulin. Exp. Eve Res. 43, 679 686. Berman E. R. (1991) Biochemistry of the Eve. pp. 309 467. Plenum Press, New York. Betz A. L., Bowman P. D. and Goldstein G. W. (19833 Hexose transport in microvascular endothelial cells cultured from bovine retina. Exp. Eve Res. 36, 269 277. Bresnick G. H. (1983) Diabetic maculopathy. A critical review highlighting diffuse macular edema. Ophthalmolo,qr 90,1301 1312. Brot-Laroche E., Serrano M. A., Delhomme B. and Aivarado F. (1986) Temperature sensitivity and substrate specificity of the two distinct N a +-activated ~)-glucose transport system in guinea pig jejunal brush border membrane vesicles. J. biol. Chem. 261, 6168-6176. Calderhead D. M., Kitagawa K., Tanner L. l., Holman G. D. and Lienhard G. E. (1990) Insulin regulation of the two glucose transporters in 3T3-LI adipocytes. J. biol. Chem. 265, 13,800-13,808. Crosson C. E. and Pautler E. L. (1982) Glucose transport across isolated bovine pigment epithelium. Exp. F4'e Res. 35, 371 377, Cunha-Vaz J. G. (1976) The blood retinal barriers. Doc. Ophthalmol. 41,287 327. Davies M. E. (19883 Diabetic retinopathy: a clinical overview. Diabetes Metab. Ret. 4, 291-322. Elbrink J. and Bihler I. (1975) Membrane transport: its relation to cellular metabolic rates. Science 188. 1177 1184. Frank G. and Schoffeniels E. (19723 Cationic composition of rat cerebral cortex slices. Comparative study during development. J. Neurochem. 19, 395 402. Graymore C. (19703 Biochemistry of the Ere. pp. 645 735. Academic Press Inc., London. Gould G. W. and Bell G. 1. (19903 Facilitative glucose transporters : an expanding family. Trends Biochem. 15, 18 23. Gould G. W., Thomas H. M., Jess T. J. and Belt G. 1. (1991 ) Expression of human glucose transporters in )~,nopus oocytes: kinetics characterization and substrate specificities of the erythrocyte, liver and brain isoforms. Biochemistry 30, 5139 5145. Harik S. I., Kalaria R. N., Whitney P. M., Anderson L A., Lundahl P., Ledbetter S. L. and Perry G. (19903 Glucose transporter are abundant in cells with "'occluding" junctions at the blood-eye barriers. Proc. natn. Acad. Sci. U.S.A. 87, 4261 4264. Haskell J. F., Meezan E. and Pillion D. J. (1984) Identification and characterization of the insulin receptor of bovine retinal microvessels. Endocrinolocly 115, 698 704. Hsu S. C. and Molday R. S. (19913 Glycolytic enzymes and a Glut-I glucose transporter in the outer segments of rod and cone photoreceptor cells. J. biol. ('hem. 266, 21,745 21.752.
lm J. H., Pillion D. J. and Meezan E. (1986) Comparison of insulin receptors from bovine retinal blood vessels and nonvascular retinal tissue, lm,est. Ophthalmol. Vis. Sci. 27, 1681 1690. King G. L., Goodman A. D., Buzney S., Moses A. and Kahn C. R. (1985) Receptors and growth-promoting effects of insulin and insulin like growth factors on cells from bovine retinal capillaries and aorta. J. clin. Invest. 75, 1028 1036. Kumagai A. K., Glasgow B. J. and Pardridge W. M. (19943 Glutl glucose transporter expression in the diabetic and nondiabetic human eye. lm,est. Ophthalmol. Vis. Sci. 35, 2887 2894. Li W., Chan L. S , Khatami M. and Rockey J. H. (19853 Characterization of glucose transport by bovine capillary pericytes in culture. Exp. Eye Res. 41,191-199. Mackerer C. R., Paquet R. J., Mehlman M. A. and Tobin R. B. ( 1971 ) Oxidation and phosphorylation in liver mitochondria from alloxan and streptozotocin diabetic rats. Proc. Soc. exp. Biol. 137, 992-995. Machemer R. and Laqua H. (1975) Pigment epithelium proliferation in retinal detachment (massive periretinal proliferation). Am. J. Ophthalmol. 80, 1 23. Masterson E. and Chader G. J, (19813 Characterization of glucose transport by cultured chick pigmented epithelium. Exp. Eve Res. 32, 279 289. Miller S. and Steinberg R. H. (1976) Transport of taurine, L-methionine and 3-O-methyl-D-glucose across frog retinal pigment epithelium. Exp. E3e Res. 23, 177 189. Nishimura H., Pallardo F. Y., Seidner G. A., Vanucci S,, Simpson I. A. and Birnbaum M. J. (1993) Kinetics of Glut I and Glut4 glucose transporters expressed in Xenopus oocytes. J. biol. Chem. 268, 8514-8520. Olefsky ,h M. (19783 Mechanisms of the ability of insulin to activate the glucose transport system in rat adipocytes. Biochem. J. 172, 137 145. Pascuzzo G. J.. Johnson J. E. and Pautler E. L. (19803 Glucose transport in isolated mammalian pigment epithelium. Exp. Eye Res. 30, 53 58. Rosenzweig S. A., Zetterstrom C. and Benjamin A. (19903 Identification of retinal insulin receptors using site-specific antibodies to a carboxyl-terminal peptide of the human insulin receptor ~ subunit : up-regulation of neuronal insulin receptors in diabetes. J. biol. Chem. 265, 18,030-- 18,034. Segel I. H. (1975) Enzyme Kinetics. pp. 346-464. John Wiley & Sons, New York. Stramm L. E. and Pautler E. L. (19823 Transport of 3-0methyl-glucose in isolated rat retinal pigment epithelial cells. Exp. Eye Res. 35, 91 97. Suzuki K. and Kono T. (1980) Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc. ham. Acad. Sci. U.S.A. 77, 2542-2545. Takagi H., Tanihara H., Seino Y. and Yoshimura N. (19943 Characterization of glucose transporter in cultured human retinal pigment epithelial cells : gene expression and effect of growth factors. Invest. Ophthalmol. Vis. Sei. 35, 170177. Takata K., Kasahara M., Ezaki O. and Hirano O. (1990). Erythrocyte HEPG2-type glucose transporter is concentrated in cells of blood-tissue barriers. Biochem. hiophys. Res. Commun. 173, 67 73. Waldbilling R. J., Fletcher R. T., Chader G. J., Rajagopalan S., Rodrigues M. and LeRoith D. (1987) Retinal insulin
Uptake of 2-DG in retina and RPE receptors. 1. Structural heterogeneity and functional characterization. Exp. Eve Res. 45, 823 835. Waldbilling R. J., Pfeffer B. A., Schoen T. J., Adler A. A., Shen-Orr Z., Scavo L., LeRoith D. and Chader G. J. (1991) Evidence for an insulin-like growth factor autocrine-paracrine system in the retinal photoreceptor-pigment epithelial cell complex. J. Neurochem. 57, 1522-1533. Whitesell R. R. and Abumrad N. A. (1985) Increased affinity
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Pergamon
0197-0186(95)00068-2
Neurochem. Int. Vol. 28, No. 2, pp. 213-219, 1996 Copyright © 1996 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0197-0186/96 $15.00+ 0.00
CHARACTERIZATION OF [2-3H]DEOXY-D-GLUCOSE UPTAKE IN RETINA A N D RETINAL PIGMENT EPITHELIUM OF NORMAL AND DIABETIC RATS C A R M E N V I L C H I S and R O C I O S A L C E D A * Instituto de Fisiologia Celular, UNAM. Mrxico, D.F. 232, Mexico (Received 20 July 1994 ; accepted 17 April 1995)
Abstract--The outer blood-retinal barrier which results from the tight junctions between retinal pigment epithelial cells (RPE) restricts the flow of nutrients reaching the retina. We characterize the transport of [2-3H]deoxy-D-glucose (2-DG) across isolated mammalian neural retina and RPE in terms of their kinetics constants. In addition, the effect of insulin on glucose transport was studied by using streptozotocininduced diabetic rats. RPE accumulates 2-DG by a temperature-sensitive and energy-dependent complex kinetics mechanism. The retina takes up 2-DG by an energy and Na÷-dependent saturable system with an apparent Km of 2 mM. Insulin induced an increase of 2-DG uptake by normal retina. The retina of diabetic rats shows lower levels of 2-DG accumulation. These levels can be returned to the normal ones by exposure to insulin. Although insulin does not affect, significantly, 2-DG accumulation by RPE, 2-DG uptake of RPE from diabetic rats shows a normal saturable kinetics with an apparent K,, of 20 mM. Those findings suggest the presence of different types of glucose transporters in retina and RPE. Insulin-sensitive glucose transport in retina might be involved in the manifestation of diabetic retinopathy.
Two blood systems occur in the retina, the retinal vascular system that nourishes the inner layers, and the choroidal vessels received by the retinal pigment epithelium (RPE), whose cells are attached to each other through tight junctions. These tight junctions constitute the blood-retinal barrier (Cunha-Vaz, 1976). Glucose is the major fuel for energy metabolism in the retina (Berman, 1991). Glucose transport by R P E (Miller and Steinberg, 1976; Zadunaisky and Degnan, 1976; Pascuzzo et al., 1980; Masterson and Chader, 1981; Stramm and Paulter, 1982; Crosson and Paulter, 1982) and in the retinal microvasculature (Betz et al., 1983; Li et al., 1985) has been studied, and there is general agreement that glucose is transported by a facilitated system. Retinopathy is one of the clinical manifestations of long standing diabetes meUitus (Davies, 1988). A major biological action of insulin is to promote glucose metabolism and this effect is thought to be largely due to acceleration of glucose transport (Olefsky, 1978). Insulin-stimulated increase in deoxyglucose transport (Allen and Gerritsen, 1986) and insulin receptors have been detected in endothelial cells from blood vessels (King et al., 1985 ;
Haskell et al., 1984) and neural retina (Waldbilling et al., 1987 ; Rosenzweig et al., 1990). The regulatory mechanisms that maintain glucose homeostasis in retina and RPE may include hormonal action, and it would be interesting to compare the properties of glucose transport operating under basal and insulin-stimulated conditions. Therefore, we undertook experiments to characterize the glucose transport in the normal rat retina and RPE, and compared them with those of diabetic animals.
EXPERIMENTAL PROCEDURES Materials [2-3H]deoxy-D-glucose (2-DG ; 37 Ci/mmol) and ~4C-inulin (0.05 mCi/17 mg) were obtained from New England Nuclear (Boston, MA, U.S.A.). Streptozotocin and insulin were acquired from Sigma Chemical Co. (St Louis, MO, U.S.A.). The kit for glucose determination was obtained from Salubridad (Mexico, D.F.). All other reagents were of analytical grade. Animals Adult Long Evans rats (170-200g) were utilized in this study. Diabetes was induced by a single intraperitoneal injection of streptozotocin (65 mg/kg) in 0.05 M citrate buffer, p H 4.5 (M ackerer e t al., 1971). Rats were allowed free access to food and water. The streptozotocin-injected animals were
*Author to whom all correspondence should be addressed. 213
214
Carmen Vilchis and Rocio Salceda
used a~er 20 days of treatment and considered diabetic if serum glucose exceeded 300mg/dl. Analytical methods The rats were decapitated, the eyes were excised and the sclera was eliminated from the posterior part of the eye. The anterior part of the eye was removed, the retina and the RPE were gently peeled away using fine forceps. Retina and RPE were incubated at 37C with 2 ml of Krebs ringer bicarbonate (118 mM NaCI, 1.2 mM K H,PO4, 4.7 mM KCI, 2.5 mM CaCI_~, 1.17 mM MgSO4, 25 mM NaHCO~, plus 5.6 mM glucose, pH 7.4) containing 0.5 #Ci of 2-DG (13 #M). At the end of the incubation period, the tissue was washed with cold medium, weighed and dissolved in 0.5 ml of 1% sodium dodecyl sulfate (SDS). Radioactivity in the solubilized tissue was measured after the addition of 5 ml Tritosol and counts for radioactivity performed in a liquid scintillation spectrophotometer. For kinetics studies the tissue was incubated for 2 rain in 1 ml Krebs medium containing 0.5 #Ci 2-DG and different concentrations of non-radioactive 2-deoxy4)-glucose, in the absence of glucose. For efflux experiments the tissue was loaded with 2-DG for 20 rain incubation in Krebs medium containing 0.5 l*Ci 2-DG. After the incubation, loaded tissue was transferred to 1 ml of non-radioactive medium for different periods of time. The radioactivity released into the medium and that remaining in the tissue was determined as above. For exchange experiments the tissue was incubated for 20 rain in I ml Krebs medium. After that, 0.5 #Ci 2-DG was added and the uptake was measured as described above. Media depleted of sodium ions was prepared by replacement of NaCI with isotonic quantities of choline chloride. When the effect of inhibitors were tested, they were added together with 2-DG. The extracellular space was estimated as described by Ames et al. (1967) and Frank and Scboffeniels (1972). Retina and RPE were incubated for 30 rain with 1.0 /~Ci/ml of '4C-inulin. After incubation, tissue was weighted and its radioactivity measured as described above. The inulin space was calculated from the ratio of the ~4C-inulin content of tissue to the ~4C-inulin in the incubation medium. The obtained values were around 27% and were substracted from 2-DG uptake values. Glucose concentration in serum was determined by the orthotoluidine reaction by means of a commercial kit. Results were compared by analysis of variance (ANOVA) and multiple comparisons by Tukey's analysis.
o_
1.5
15
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,o
o~
7 o_o
i E
~
I
I 2O
4O
Time (min) Fig. 1. Time course of 2-DG uptake in rat retina and RPE. Tissue was incubated in a Krebs bicarbonate medium, pH 7.4 in the presence of 5.6 mM glucose and 0.5/~Ci of 2-DG. Retina (A), RPE (91). The values are the mean ± S E M of 4 experiments.
where exchange transport was measured. After previous equilibration with Krebs medium for 20 min 2D G was added. U n d e r these conditions uptake o f 2D G reached values o f 8.1 __+0.7 and 1.3 + 0 . 0 9 / l m o l / g lbr retina and RPE, respectively. Consistent with these results, the efflux experiments (Fig. 2) demonstrated a facilitated diffusion type o f glucose transporter. Efftux o f 2 - D G was not affected in the retina nor the R P E o f diabetic rats (Fig. 2). The effect o f various c o m p o u n d s on 2-DG accumu-
100
._= -~ o_o ~a
5O
O..
RESULTS
2-DG, an analogue o f glucose that is metabolized to 2-deoxy-D-glucose-6-phosphateand trapped within cells, has been used extensively to study hexose transport in many cell types. Figure I shows a time course o f 2-DG uptake in rat retina and RPE. The accumulation o f 2-DG is linear for the first 10 rain (not shown), reaching plateau levels at approx. 30 rain o f 10.8 and 1.37/~mol/g in retina and RPE, respectively (Fig. 1). We examined the uptake o f 2-DG under conditions
I t0
I 20
I 30
Time (rain) Fig. 2. Efflux of 2-DG from retina and RPE. Tissue was loaded by 20 min incubation in Krebs medium containing 0.5 l~Ci of 2-DG. After this, tissue was incubated consecutively with non-radioactive medium for 5 min periods. Results of three different experiments, in which SE was less than 10%. have been averaged, and the data normalized to the 2-DG loaded after preincubation (100%). Normal retina (0). diabetic retina (A), normal RPE (©), diabetic RPE (A).
215
Uptake of 2-DG in retina and RPE lation is shown in Table 1. The uptake of 2-DG of both retina and RPE, was not significantly affected by 1 mM KCN, 0.1raM ouabain, nor 0.1 mM phloridizin, whereas low temperature abolished it. Iodoacetate (lmM) significantly inhibited 2-DG uptake by about 75 and 50% in retina and RPE, respectively. Accumulation of 2-DG in RPE was not significantly modified in the absence of sodium, whereas in retina it was inhibited 90% (Table 1). On the other hand, phloretin slightly (30%) affected accumulation of 2-DG. Cytochalasin B, an inhibitor of glucose uptake, reduced 2-DG accumulation by 40 50% in both RPE and retina (Table 1). The kinetics characteristics of the 2-DG were studied by measuring the 2-DG accumulation within a concentration range of 0.5-30 mM 2-DG. In the retina, a saturation curve was observed with an apparent K m of 2 mM and a Vmax of 0.75 #mol/g/min (Fig. 3). Uptake of 2-DG by RPE showed a complex kinetics, that could not be adjusted to the MichaelisMenten equation, suggesting allosteric interactions (Segel, 1975) (Fig. 4). Kinetics studies of 2-DG uptake by diabetic retina revealed an apparent Km of 5.0 mM and a Vmaxof 1.5 #mol/g/min (Fig. 3). Interestingly, 2-DG uptake by RPE from diabetic rats showed a normal Michaelis-Menten kinetics, with an apparent Km of 20 mM and a Vmaxof 0.75 ttmol/g/min (Fig. 4). The effect of insulin on 2-DG accumulation was studied in the RPE and retina. Addition of insulin to the incubation medium increased accumulation of 2D G by retina. The effect of insulin is dose-dependent, with maximal response (60%) observed at 5 10 ng/ml
(Fig. 5). On the contrary, insulin did not affect significantly 2-DG accumulation by RPE (Fig. 5). To determine whether the effect of insulin reflects the physiological situation, we studied the 2-DG uptake in the retina of streptozotocin-induced diabetic rats. As show in Fig. 6, the rate of 2-DG accumulation by diabetic retina is decreased by 25-30% compared with that of normal retina ; in agreement with these results, addition of insulin to the incubation medium re-established normal 2-DG uptake rate (Fig. 6).
DISCUSSION
The RPE provides a major transport pathway responsible for exchange of metabolites and ions between the choroidal blood supply and the neural retina (Cunha-Vaz, 1976 ; Miller and Steinberg, 1976). The RPE is believed to play an important role in a number of ocular diseases, including diabetic retinopathy (Bresnick, 1983) and proliferative vitreoretinopathy (Machemer and Laqua, 1975). As the neural retina has a very high rate of glucose metabolism, transport of glucose by RPE is probably one of its functions. Most, if not all, mammalian cells have integral membrane proteins that translocate glucose down its concentration gradient by a process of facilitative diffusion (Elbrink and Bihler, 1975). Recent cloning and sequencing studies suggest the existence of several different glucose transport proteins (Glut 1-5) for carrier mediated diffusion of glucose across cell membranes in various tissues and with different kinetics properties (Gould and Bell, 1990 ; Gould et al., 1991 ; Nishimura et al., 1993). The tissue distribution of the different glucose transporters is not well known and more than one transporter may be present in the same Table 1. Effect of different compounds on 2-DG uptake tissue. Glutl type glucose transport is reported to be conRetina RPE centrated in the cells that form occluding junctions, Addition (/zmol/g) thus constituting blood-tissue barriers, including the RPE (Harik et al., 1990 ; Takata et al., 1990). Human -7.67__+0.18 1.26+0.15 cultured RPE cells predominantly express the Glutl KCN (lmM) 8.03+0.40 1.02+0.03 gene and protein, with minor expression of Glut3 and Ouabain (0.1mM) 6.38+0.43 1.05+0.08 Iodoacetate (1 mM) 1.88+0.08" 0.59+0.04* Glut5 genes (Takagi et al., 1994). Phloridizin (0.1mM) 7.46+0.57 1.28+0.17 In agreement with previous studies (Miller and StePhloretin (0.1mM) 5.24+0.37* 0.81 +0.08* inberg, 1976; Zadunaisky and Degnan, 1976; PasCytochalasin B (10/~M) 3.56+ 1.12" 0.75 +0.04* Low temperature (4=C) 0.19+0.02" 0.16___0.02" cuzzo et al., 1980; Masterson and Chader, 1981; Sodium free medium 0.97+0.07* 1.08+0.01 Stramm and Paulter, 1982; Crosson and Paulter, 1982), our results show that 2-DG accumulation by Tissue was incubated for 20 n-finin a Krebs bicarbonate RPE is a carrier mediated process. We found a commedium with 5 mM glucose in the presence of 2-DG (0.5 #Ci). Each value is the mean _ SEM of at least three exper- plex kinetics for 2-DG uptake, that might be explained by allosteric interactions of the transporter and/or the iments. *P < 0.01.
216
Carmen Vilchis and Rocio Salceda 20
1.0 15
"'5 OE o
1o
b
0.5
I
J 2.5
P 5.0
i 5
[2 - DG] mM
J Io
1/[s] mM
Fig. 3. Accumulation of 2-DG by rat retina as a concentration function. Incubation was carried out for 2 min with different concentrations of 2-DG in a Krebs bicarbonate medium without glucose. Right: double reciprocal plot. The points are the mean of at least three experiments. Normal retina (O), diabetic retina (A). existence of more than one carrier mediated process (Segel, 1975). Accumulation of 2-DG was energy dependent as indicated by the inhibition caused in the presence of iodoacetate and to a lesser extent by KCN. As reported for frog RPE (Zadunaisky and Degnan, 1976), we found 2 - D G uptake to be Na +-independent. The inhibition of 2-DG uptake by phloretin and cytochalasin B also evidence the glucose carrier. Addition of insulin to the incubation medium did not affect significantly 2 - D G uptake by normal RPE, although it was slightly decreased in RPE from streptozotocin-induced diabetic rats. In agreement with this result, RPE of diabetic animals show a normal saturable kinetics for 2-DG uptake suggesting that
insulin might modulate its transport. In this way, it should be noted that cultured human RPE cells synthesize and release insulin like growth factor I (ILGFl) (Waldbilling et al., 1991), and that 2 - D G uptake is stimulated when exposed to different growth factors, including I L G F - | (Takagi et al., 1994). Accumulation of 2 - D G by neural retina is considerably higher than that of RPE. We found that the retina exhibits a saturable temperature-dependent uptake of 2-DG. The inhibition of 2 - D G accumulation observed by iodoacetate but not by K C N , might be related to the high glycolytic activity of the retina (Graymore, 1970). An exceptionally high rate of both anaerobic and aerobic glycolysis and lactate production has been reported in the mammalian retina.
40 ="5 ~E m,
~L
I
~0
20
[2 - DG] mM
3o
I 2.5
I 5.0
1/Is] mM
Fig. 4. Uptake of 2-DG in rat RPE. The RPE was incubated for 2 min with different concentrations of 2DG in Krebs medium without glucose. Normal rat RPE (O), diabetic rat RPE (A). Right: Michaelis Menten plot of 2-DG uptake in diabetic rat RPE. The points are the mean of 5 experiments in which SE was less than 10%.
Uptake of 2-DG in retina and RPE 200
RPE
RETINA
~)
:::1 o
I00
~u a"6
I i
i I
0
510100
h 0
510100
Insulin (ng/ml)
Fig. 5. Effect of insulin on 2-DG uptake in normal rat retina and RPE. Tissue was incubated for 40 min in a Krebsbicarbonate medium, pH 7.4 containing 2-DG (0.5/~Ci), in the absence or presence of different insulin concentrations (ng/ml). Results shown are mean -I-SEM of 4-6 experiments. *P < 0.01.
15
-6 E
I0
:=L
(5 a I
5
t%l
50
40
Time (rain)
Fig. 6. Uptake of 2-DG by normal and diabetic rat retina. Retina from normal ([]) or streptozotocin-induced diabetic rats (1~) were incubated in a Krebs bicarbonate buffer in the absence ( ~ ) or presence ( 9 ) of 10 ng/ml insulin. Results are mean ±SEM of 3-5 experiments. *P < 0.01. Electric activity of the retina is abolished either by withdrawal of oxygen supply or by addition of iodoacetate to the incubation medium (Berman, 1991 ; Winkler, 1981). Although ouabain should collapse the sodium gradient, the external sodium concentration appears to be sufficient to permit 2-DG accumulation, since it was 90% inhibited in the absence of sodium in the incubation medium. Accumulation of 2-DG occurs by a saturable system with an apparent Km of 2 raM, a value similar to the Km reported for the Na+-
217
glucose cotransport system (Brot Laroche et al., 1986) as well as for the Glutl type glucose transporter in human red blood cells (Nishimura et al., 1993) and photoreceptor outer segments (Hsu and Molday, 1991). Glutl has been reported to be present in Mialler cells, ganglion cells, photoreceptor cell bodies and outer limiting membrane in normal and diabetic human retina (Kumagai et al., 1994) showing high density in photoreceptor outer segments (Hsu and Molday, 1991). It is worth noting that 2-DG accumulation by retina is stimulated by insulin. These results agree with the lower accumulation levels of 2-DG observed in the diabetic retina. Kinetics analyses of the uptake in the retina of diabetic animals, suggest that insulin stimulates 2-DG accumulation by increasing its capacity. This is similar to reports on other cell types where the effect of insulin was mediated by an increase in the Vmax (Olefsky, 1978 ; Whitesell and Abumrad, 1985). Since exposure of streptozotocin-treated rat retina to insulin increases 2-DG accumulation to normal values, without modification of the efflux, insulin could induce incorporation of the glucose transporter to the membranes from an internal pool, as has been reported for muscle and adipose tissues (Suzuki and Kono, 1980). The insulin-sensitive 2-DG accumulation observed is most likely to be associated with the neural retina, since longer periods of incubation with insulin are necessary to demonstrate effects on hexose transport in cultured endothelium cells (Allen and Gerristen, 1986), and insulin receptors have also been detected in non vascular portions of bovine retina (Im et al., 1986; Waldbilling et al., 1987; Rosenzweiz et al., 1990). Down regulation of the glucose transporter in diabetic retina may represent a homeostatic mechanism controlling glucose uptake under high glucose concentrations. It is interesting that retinal glucose concentration was not modified by diabetes, showing values of 10.9__ 1.6 /~g/mg protein, quite similar to that in the normal retina (8.39 +0.2 #g/mg protein). But the retina's capacity to respond to insulin is limited compared with adipocytes (Calderhead et al., 1990). The insulin-sensitive glucose transporter represents less than 50% of the total transport activity in the retina. This limited response capacity to high glucose levels could lead to diabetic retinopathy. Our results indicate that both retina and RPE possess different glucose transporters. The characterization of these transporters, their regulation and interaction with the insulin-sensitive transporter requires further studies.
218
Carmen Vilchis and Rocio Salceda
Acknowledgement- The authors would like lo thank Mr G. S~inchez-Ch~ivez for his technical assistance and Dr P. Bissett Mandeville for statistical analyses. REFERENCES
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