Neurochem Res (2010) 35:1566–1574 DOI 10.1007/s11064-010-0216-1
ORIGINAL PAPER
Effects of Taurine on Glial Cells Apoptosis and Taurine Transporter Expression in Retina Under Diabetic Conditions Kaihong Zeng • Hongxia Xu • Mantian Mi • Ka Chen • Jundong Zhu • Long Yi • Ting Zhang • Qianyong Zhang • Xiaoping Yu
Accepted: 3 June 2010 / Published online: 9 June 2010 Ó Springer Science+Business Media, LLC 2010
Abstract Taurine, a ß-aminosulfonic acid, has been reported to reduce the risk of a number of diseases, including cardiovascular disease, diabetes, and also perhaps to reduce neurodegeneration in the elderly. The transport of taurine is known to be mediated by taurine transporter (TauT). The purpose of this study is to examine the effects of taurine on glial cells apoptosis and on TauT expression in retina of diabetic rats and retinal glial cells cultured with high glucose. TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining analysis showed that the number of TUNEL-positive cells in taurine treated diabetic rats was significantly lower than those of untreated diabetic rats over the 8-, and 12-week time courses, respectively (all P \ 0.001). No TUNEL-positive cells were observed in retina of control groups and taurine treated control groups. In cultured retinal glial cells, the apoptosis in high glucose-treated cells was significantly increased vs the control. When the cells were incubated with high glucose and taurine at 0.1, 1.0 and 10 mmol/l, the percentage of apoptosis was significantly decreased to 16.4, 5.7 and 7.6% respectively (all P \ 0.05). With supplementation of taurine in diet and culture medium, higher expression of TauT in retina of diabetic rats and cultured retinal glial cells under diabetic conditions were detected by western-blotting (P \ 0.05). Taken together, our data suggest that diabetes or high glucose induced retinal glial cells apoptosis can be inhibited by taurine, and that taurine
K. Zeng H. Xu M. Mi (&) K. Chen J. Zhu L. Yi T. Zhang Q. Zhang X. Yu Department of Nutrition and Food Hygiene, School of Preventive Medicine, The Third Military Medical University, 30 Gaotanyan Street, Shapingba District, Chongqing 400038, People’s Republic of China e-mail:
[email protected]
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reverses the diabetes-induced or high glucose-induced decrease in TauT expression. Keywords Taurine Retina Glial cells Apoptosis Neuroprotection TauT
Introduction Taurine, a ß-aminosulfonic acid, is the most abundant retinal amino acid and is essential for sustain of retinal structure and function [23]. The taurine concentration in retina is about 100 times higher than that in serum (100– 300 lmol/l) [6, 32]. The transport of taurine is known to be mediated by an Na?- and Cl--dependent taurine transporter (TauT) [29]. TauT knockout mice are reported to display a loss of vision due to severe retinal degeneration, in addition to having low taurine levels in a variety of tissues and reduced fertility [15], suggesting that TauT is critical for normal retinal development and function. Our previous experiments showed that dietary taurine supplementation could increase the level of taurine, decrease the level of glutamate, inhibit glial reactivity and glutamate dysmetabolism in diabetic retina, then exhibits effective prevention against diabetic retinopathy (DR) [34, 35]. DR is the leading cause of blindness in working-age adults [31]. This disease is usually considered a vascular disease, but several evidences have demonstrated that there are also a range of abnormalities of neuronal function that occur early in retina of diabetes. This neuronal dysfunction is exhibited as changes in colour vision, dark adaptation and electrophysiology [1, 3, 10, 19, 20]. Histologically, neuronal apoptosis has been observed in ganglion cells before any alteration could be detected in the retinal vascular system. This neuronal apoptosis is associated with
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glial reactions in glial cells, generally examined following the appearance of retinopathy [4]. Because such neuronal dysfunction appear early in the disease process, it has been suggested that they may ultimum be linked with the retinal vascular pathology. The glial cells in the vertebrate retina are grouped into astrocytes, microglia, and Mu¨ller glial cells. Mu¨ller glial cells are the main glial cells in retina and are actively involved in uptake and degradation of the amino acid neurotransmitters, \gamma[ -amino butyric acid (GABA) and glutamate, are involved in shuttling energy metabolites to neurons and are also important for maintenance of the blood-retinal barrier [8, 21]. Studies of diabetic retina and animal models of hyperglycemia have displayed changes in glial cells morphology, protein expression, and physiology well in advance of detectable retinopathy [19, 20]. We have shown that glial cells undergo diabetes-induced glial reactivity and glutamate dysmetabolism in vivo [35]. Nevertheless, the contribution of glial cells to diabetic retinopathy remains unclear. Apoptosis, or programmed cell death, occurs as a physiological phenomenon during normal embryonic development and in the cell turnover throughout life. However, apoptosis has also been involved in several neurodegenerative diseases. Several studies have shown that neuron and ganglion cells undergo hyperglycemiainduced apoptosis in vitro [1, 17], although the mechanism of initiation of the apoptotic cascade is unknown. The aim of this project was to examine the preventive effect of taurine on apoptosis in retina and cultured retinal glial cells of rats induced by diabetes or high glucose and to further examine the regulative effect of taurine on the expression of TauT in retina and cultured retinal glial cells under diabetic conditions.
Materials and Methods All animal experiments were performed following protocols approved by The Third Military Medical University’s Animal Care and Use Committee in compliance with the NIH Guide for the Care and Use of Laboratory Animals. Model of Diabetes Sprague–Dawley rats of age 14 weeks and weight 180 ± 20 g were housed in standard stainless steel cages at 25°C. After consuming a purified diet based on the AIN-93 formulation [27] for 1 week, The rats were fasted for 12 h but freely drank water, and then treated with a single intraperitoneal (i.p.) injection of streptozotocin 60 mg/kg dissolved in 0.1 mol/l citrate buffer (pH 4.2) to establish
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experimental diabetic model. Subsequently, the animals received AIN-93 formulation and water adlibitum. The animals were examined 72 h later and those with plasma glucose levels of greater than 16.7 mmol/l were classified as diabetic. After that, these rats were randomly divided into two groups as follows: STZ-induced untreated diabetic rats (DM, n = 68); STZ-induced taurine treated diabetic rats (5.0 g of taurine in 100 g diet, DM ? Tau, n = 68). Taurine dose was selected based on previous study showing maximum protection of taurine at the level of 5 g taurine/100 g diet [7]. The rest rats fed AIN-93 formulation were randomly divided into two groups as follows: Normal control rats (Con, n = 52); Taurine treated normal control rats (Con ? Tau, n = 52). All rats had free access to food and water during the whole experimental time. The rats were monitored daily and the blood was drawn from a tail vein weekly for plasma glucose determination by standard laboratory methods. A total of seven diabetic rats died during the study. At 2-, 4-, 8-, 12-week after treatment, the rats were weighed and then killed by decapitation under diethyl ether anesthesia between 10 AM and noon to avoid possible circadian fluctuations in the metabolism of amino acids. Glial Cells Culture and Experimental Conditions Glial cells were obtained by a previously described method [16]. Briefly, enucleated eyes from Sprague– Dawley rats at postnatal (PN) day 5 to PN7 under sterile conditions were soaked as intact eyeballs in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mmol/l glutamine and 1/1000 penicillin/streptomycin overnight at room temperature in the dark. They were then incubated in DMEM containing 0.1% trypsin and 70 U/ml collagenase, 0.5 ml per eye at 37°C for 45 min. The retina were isolated, chopped into about 1 mm2 pieces and cultured in DMEM containing 5 mmol/l D-glucose, 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin at 37°C in a humidified chamber with a 5% CO2-95% air mixture. Medium was changed every day. Glial cells were identified by their expression of glutamine synthetase (GS), vimentin and glutamate transporter (GLAST), as judged by immunocytochemical staining. Nuclei were stained with hoechst. When cultures reached 80% to 90% confluence they were split. Cells that had been in culture for three to five passages were used in this study. Cells were cultured in DMEM in the presence of 5 mmol/l D-glucose (normal glucose), 5 mmol/l D-glucose plus 0.1, 1 and 10 mmol/l taurine, 25 mmol/l D-glucose (high glucose), 25 mmol/l D-glucose plus 0.1, 1 and 10 mmol/l taurine, and 5 mmol/l D-glucose (normal glucose), 25 mmol/l D-glucose (high glucose) plus 20 mmol/l mannitol for at least 3 days.
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Immunofluorescence
Annexin V Binding
Cultured cells were blocked by 5% normal goat serum for 10 min at room temperature, and then incubated with a mixture of two of the following primary antibodies: vimentin, GS (sigma, St. Louis, MO, USA) (1:1000 dilution) or incubated with primary antibodies: GLAST (ADI, San Antonio, TX) (1:1000 dilution) overnight, and then followed by a dilution (1:200) of fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (sigma, St. Louis, MO, USA) or rhodamine (TRITC)-conjugated anti-mouse IgG (sigma, St. Louis, MO, USA), for 2 h. After further rinsing the cells were covered with coverslips. Negative controls were performed routinely by incubating the cells in normal buffered serum instead of the primary antibody. Cells were examined in an Olympus confocal laser scanning microscope (FV 1000, Olympus, Tokyo, Japan).
Annexin V binding was used to detect early stages of apoptosis in retinal glial cells of rats cultured with high glucose. Glial cells were incubated with 5 or 25 mmol/l glucose with or without different concentrations of taurine for 3 days and then subcultured on chambered cover glasses (Fisher, Pittsburgh, PA) for 2 more days. Normal medium containing 20 mmol/l mannitol was performed as osmotic control. Annexin V staining was using to determine the cell apoptosis using a commercial available kit (Annexin V- fluorescein isothiocyanate staining kit; Roche). Annexin V staining was performed as previously described. Briefly, cells grown for 96 h were washed in Annexin V binding buffer and incubated in 500 ll Annexin V staining solution (1:50 dilution of Annexin V-phycoerythrin in Annexin V binding buffer) for 10 min in the dark at 37°C in 5% CO2. Then the cells were washed with the calcium binging buffer and 10 ll of Propidium Iodide (PI) was added and incubated for 10 min at 4°C. Flow cytometry was used to analyze 5000 cells. This allowed the discrimination of live cells (unstained with either fluorochrome) from apoptotic cells (stained with Annexin V) and necrotic cells (stained with PI). All data were collected, stored, and analyzed by Multigraph software (Coulter, Miami, FL).
Combined TUNEL and Double Immunofluorescent Labeling Enucleated eyes were immediately fixed in 4% paraformaldehyde (Sigma, St. Louis, MO) for 2 h. The cornea and lens were removed, and the remaining eye cup was placed in the same fixative for 4 h. Tissues were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA), and sagittal sections were cut through the optic nerve. TdT-mediated dUTP-biotin nickend labeling (TUNEL) staining was performed on 7 lm frozen retinal sections with a kit (TdT-FragEL DNA Fragmentation Detection Kit; Oncogene, Boston, MA), with procedures modified from Gavrieli et al. [13]. The sections were incubated with 20 lg/ml proteinase K and then with Tdt/dUTP reaction mixture for 1 h at 37°C for DNA fragment labeling, followed by three rinses in phosphate-buffered saline (PBS). The tissues were blocked for 1 h with blocking buffer containing 5% normal goat serum in PBS. Then the sections were incubated for 1 h in blocking buffer with rabbit anti-rat GS (1:2 000, Sigma, St. Louis, MO, USA). The secondary antibody used was antirabbit IgG conjugated with TRITC (1:200, Chemicon International, Temecula, CA, USA). Incubation in the secondary antibodies was carried out in the dark at room temperature for 40 min. After further rinsing the sections were covered with coverslips. Positive control experiments demonstrating DNA fragmentation were performed by exposing sections to DNAase, whereas negative controls omitted the TdT/dUTP labeling mixture, resulting in no staining. Sections were viewed in an Olympus confocal laser scanning microscope (FV 1000, Olympus, Tokyo, Japan). The number of TUNEL-positive cells in the inner nuclear layer (INL) was counted in a masked fashion. The area of the INL was measured with OpenLab software.
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Protein Expression Analyzed by Western-blotting Total protein was extracted using the method described by Rauen et al. [26]. Protein concentration was determined using the Bradford method (Bio-Rad Laboratories, Hercules, CA). The protein (60 lg) was separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), and then electrophoretically transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Nonspecific sites were blocked by incubating the membrane with 5% nonfat dry milk in triethanolamine-buffered saline solution (TBS) plus 0.1% Tween (TBS-T) for 60 min. The membranes were then incubated, respectively, with the following primary antibodies: rabbit anti-rat TauT (1:1000; sigma, St. Louis, MO, USA) and mouse anti-rat b-actin (1:2000, sigma, St. Louis, MO, USA) overnight at 4°C. After several washes, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 60 min. Relative quantities analyzed by densitometry using the Bandleader 3.0 software (Magnitec Ltd, Israel). The densitometric reading of each protein was then calculated as a ratio of b-actin intensity reading for normalization. Statistical Analysis The results were expressed as mean ± standard deviation. Statistical analysis was performed by a one-way analysis of
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variance (ANOVA) followed by Tukey’s post-hoc test. Difference was considered significant if P \ 0.05. SPSS version 13.0 was used for all statistical analysis.
Results Confirmation of Experimental Diabetes The induction of STZ-induced diabetes and taurine treatment of all rats was consistent with our previous reports [35]. Diabetic rats fed without or with 5% taurine had a 7.0% or a 68.9% gain in weight from 2 to 12 weeks after onset of diabetes, whereas age-matched controls fed
without or with 5% taurine had a 118.5% or a 117.8% gain in weight. By 12 weeks after onset of diabetes, diabetic rats weighed significantly less than control rats (P \ 0.05). Diabetic rats fed without or with 5% taurine were hyperglycemic, whereas age-matched controls fed without or with 5% taurine were orthoglycemic lasting 12 weeks. The body weights of the taurine treated diabetic rats were significantly lower than those of nondiabetic control rats and higher than those of untreated diabetic rats over the 2-, and 12-week time courses, respectively (all P \ 0.05). Treatment with taurine had no statistical difference in the levels of blood glucose in 2–12 weeks diabetic rats compared to their respective age-matched untreated diabetes (P [ 0.05) (Table 1).
Table 1 Weight and blood glucose levels in the four groups of rats studied Duration of diabetes (weeks) 2
12
Treatment group
n
Weight (g)
Blood glucose (mmol/l)
Con
13
199 ± 10
4.3 ± 0.1
Con ? Tau
13
194 ± 8
4.4 ± 0.2
DM
11
198 ± 10
24.1 ± 0.5a
DM ? Tau
12
196 ± 9
22.2 ± 0.3a
Con
13
436 ± 11
4.5 ± 0.3
Con ? Tau
13
423 ± 13
4.4 ± 0.4
DM DM ? Tau
11 12
213 ± 12a 331 ± 13ab
19.4 ± 0.6a 18.9 ± 0.4a
Results were expressed as mean ± standard deviation a
P \ 0.05, compared to the corresponding value of normal control group
b
P \ 0.05, compared to the corresponding value of untreated diabetic group
Fig. 1 Identification of retinal glial cells. Retinal glial cells were identified by their expression of glutamine synthetase (GS) (a), vimentin (b) and glutamate transporter (GLAST) (e), as judged by
immunocytochemical staining. Nuclei were stained with hoechst (c, f). d Merged labeling of GS, vimentin and hoechst. g Merged labeling of GLAST and hoechst
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Glial Cells Culture Characterization After 7 days in culture, fusiform cells were observed to extend radially from the margin of cell aggregates. Another 7 days later, these spindle-like cells reached 80% confluence. After 14 days in vitro, the culture cells were labeled with antibodies GS, vimentin and GLAST, all markers of glial cells. Nuclei were stained with hoechst. As shown in Fig. 1, cells in this culture system showed positive labeling for GS, vimentin and GLAST. By this immunocytochemical labeling, the cultured cells were thought to be glial cells. Protective Effect of Taurine on Diabetes-Induced Apoptosis in Retina of Rats Apoptosis in rats retina was detected by TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining at 2, 4, 8
Fig. 2 Effect of taurine on diabetes-induced apoptosis in retina of rats. a Representative double labeling for TUNEL and GS (cell-type specific markers of glial cells) by retina of normal control rats (Con, n = 7), taurine treated normal control rats (Con ? Tau, n = 7), untreated diabetic rats (DM, n = 8) and taurine treated diabetic rats (DM ? Tau, n = 8) (Scale bar: 20 lm). b Quantification of TUNELpositive cells in untreated diabetic rats (DM, n = 8) and taurine treated diabetic rats (DM ? Tau, n = 8). Bars represent the means ± SD. ### P \ 0.001 versus untreated diabetic group
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and 12 weeks after induction of diabetes. TUNEL-positive nuclei were observed in retina of diabetic rats and taurine treated diabetic rats at any of the four stages studied. No TUNEL-positive nuclei were observed in retina of control groups and taurine treated control groups at any of the four stages studied. The location of the TUNEL-positive nuclei was detected mainly in the INL (Fig. 2a). The number of TUNEL-positive nuclei in taurine treated diabetic retina was significantly lower than those of untreated diabetic rats over the 8-, and 12-week time courses, respectively (all P \ 0.001) (Fig. 2b). Protective Effect of Taurine on High Glucose-Induced Apoptosis in Retinal Glial Cells of Rats We further assessed the apoptosis in retinal glial cells of rats in vitro by Annexin V/PI staining followed by flow cytometric analysis. We did not find significant variation in
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apoptosis after 48 h of incubation with 25 mmol/l high glucose compared with the control (data not show). At the 72 h of 25 mmol/l high glucose incubation, as shown in Fig. 3, the apoptosis was significantly increased vs the control. The percentage of early apoptotic cells in high glucose incubation groups was 20.6%. When the cells were incubated with high glucose and taurine at 0.1, 1.0 and 10 mmol/l, the percentage of early apoptosis significantly decreased to 16.4, 5.7 and 7.6%, respectively (all P \ 0.05). The mannitol did not change the percentage of apoptosis comparing to the control and high glucose (not significant).
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significantly (P \ 0.001) blocked the decreases in TauT protein expression in diabetic retina compared to their respective untreated diabetes at 8-, and 12-week. TauT expression in normal controls was also significantly increased by taurine treatment (P \ 0.001). Regulative Effect of Taurine on the Protein Level of TauT in Retinal Glial Cells of Rats
The protein expression of TauT (Fig. 4) analyzed by western-blotting showed significantly decreases in diabetic retina compared to their respective controls after 8-, and 12-week (at least P \ 0.05). Treatment with taurine
In Fig. 5, western-blotting analysis shows that there was an decrease in expression of TauT protein in cells treated with high glucose when compared with the normal glucose group (P \ 0.01). The expression of TauT protein was not altered by mannitol treatment. Quantitative densitometry from three independent experiments revealed that the protein level of TauT in cells treated with high glucose decreased significantly compared with normal glucose group. The protein expression of TauT was increased obviously in cells treated with high glucose plus 1 mmol/l and 10 mmol/l taurine, when compared with high glucose
Fig. 3 Effect of taurine on high glucose-induced apoptosis in retinal glial cells of rats. Glial cells were cultured in DMEM in the presence of 5 mmol/l D-glucose (normal glucose), 5 mmol/l D-glucose plus 0.1, 1 and 10 mmol/l taurine, 25 mmol/l D-glucose (high glucose), 25 mmol/l D-glucose plus 0.1, 1 and 10 mmol/l taurine, and
5 mmol/l D-glucose, 25 mmol/l D-glucose plus 20 mmol/l mannitol. Three days after cultured, the cells were stained by Annexin V/PI staining followed by flow cytometric analysis. Bars represent the means ± SD. *** P \ 0.001 versus normal glucose; @ P \ 0.05 and @@@ P \ 0.001 versus high glucose
Regulative Effect of Taurine on the Protein Level of TauT in Retina of Rats
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Fig. 4 Effect of taurine on the protein expression of TauT in retina of diabetic rats. Representative western-blotting of TauT and b-actin and the quantitation of signals from western-blotting by retina of 2-, 4-, 8-, or 12-week normal control rats (Con, n = 6), taurine treated normal control rats (Con ? Tau, n = 6), untreated diabetic rats (DM, n = 8) and taurine treated diabetic rats (DM ? Tau, n = 8). Bars represent the means ± SD of the densitometric reading of the band intensity of TauT as a ratio to that of b-actin. * P \ 0.05, ** P \ 0.01 and *** P \ 0.001 versus normal control group; ### P \ 0.001 versus untreated diabetic group
group. TauT protein expression in normal glucose controls was also significantly increased by 1 mmol/l and 10 mmol/ l taurine treatment (P \ 0.01).
Discussion In the present study, we have shown that diabetes-induced or high glucose-induced retinal glial apoptosis can be inhibited by taurine, and that taurine reverses the diabetesinduced or high glucose-induced decrease in TauT expression. Taurine influences various biological and physiological functions, including brain and retinal development, cell membrane stabilization, antioxidation, detoxification, osmoregulation, hypoglycemic action and neuromodulation. Taurine has been reported to be cytoprotective in
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several model systems, for example, protecting renal tubular cells, endothelial cells, neurones, and hepatocytes from apoptosis [2, 9, 30]. Dietary supplementation with taurine has been reported to reduce the risk of a number of diseases, including inflammatory bowel disease, ischemia, and cardiovascular disease, diabetes, and also perhaps to reduce neurodegeneration in the elderly [22, 24]. Mammals synthesize taurine from sulfur precursors, but the ability of different species to do so varies greatly. Dietary sources of taurine are thus necessary for those animals that cannot synthesize sufficient taurine, for example, the cat and human. There is evidence that plasma and tissue levels of taurine are reduced in diabetes [11, 12, 14]. We have previously shown that dietary taurine supplementation could increase the level of taurine in retina of diabetic rats, but the levels of blood glucose in 2–12 weeks taurine treatment diabetic rats had no statistical difference
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Fig. 5 Effect of taurine on the protein expression of TauT in retinal glial cells of rats. Glial cells were cultured in DMEM in the presence of 5 mmol/l D-glucose (normal glucose), 5 mmol/l D-glucose plus 0.1, 1 and 10 mmol/l taurine, 25 mmol/l D-glucose (high glucose), 25 mmol/l D-glucose plus 0.1, 1 and 10 mmol/l taurine, and 5 mmol/l D-glucose plus 20 mmol/l mannitol. Three days after cultured, TauT and b-actin expression were examined by westernblotting. Bars represent the means ± SD of the densitometric reading of the band intensity of TauT as a ratio to that of b-actin. ** P \ 0.01 versus normal glucose; @ P \ 0.05 and @@ P \ 0.01 versus high glucose
compared to their respective age-matched untreated diabetes ([35]. Diabetic retinopathy, a microvascular complication, is currently being viewed as a neurovascular complication in light of several recent reports. Some of these reports suggest that changes in neuronal components occur soon after the onset of diabetes and precedes vascular complications associated with diabetic retinopathy. A recent study has indicated retinal neuronal dysfunction to occur as early as 2 days after onset of experimental diabetes [19, 20]. We had shown that retinal glial cells undergo diabetes-induced glial reactivity and glutamate dysmetabolism at 2 weeks after onset of experimental diabetes [35]. To determine whether diabetes increases retinal glial apoptosis, we adapted the method of TUNEL labeling to retina. We found that the number of apoptotic nuclei was significantly elevated after 8 weeks of streptozotocin-induced diabetes. TUNEL-positive nuclei were detected mainly in the INL in untreated diabetic rats. At taurine treated diabetic rats, TUNEL-positive nuclei were still observed in the INL. The number of TUNEL-positive cells in taurine treated diabetic retina was significantly lower than those of untreated diabetic rats over the 8-, and 12-week time courses,
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respectively (all P \ 0.001). Similar increases in apoptosis were also observed in retinal glial cells incubated with high glucose in vitro. The percentage of early apoptotic cells was reduced by treatment with taurine. This study suggested that taurine can inhibit apoptosis in retinal and cultured retinal glial cells induced by diabetes or high glucose. The cellular taurine content is a balance between active uptake of taurine via the saturable, Na?- and Cl--dependent taurine transporter (TauT), and passive release through a volume-sensitive taurine leak pathway [29]. In the intracellular space taurine is present in millimolar concentrations, whereas taurine is found at the concentration of 2–100 nmol/l in plasma, suggesting that TauT plays an important role in maintaining a high concentration of taurine in tissues. Deficiency in TauT expression/activity and/or changes in the cellular taurine content has been associated with a plethora of mammalian disorders such as destruction of skeletal muscle function, degeneration of retinal photoreceptors and abnormal development of kidney, heart and central nervous system [5, 18, 25, 28]. Recently, a TauT knockout mouse has been established. The animals exhibited retinal degeneration and a marked impairment of reproduction [33]. These phenomena suggest that TauT is a functional protein in maintaining cell physiological function in vitro and in vivo. To determine whether protein expression of TauT is downregulated by diabetes conditions, we used western-blotting to measure the protein expression of TauT in retina and cultured retinal glial cells. Our study suggested that the protein expression of TauT was reduced in diabetic conditions and could be stimulated expression by taurine supplementation. In conclusion, we suggest that: (a) diabetes-induced or high glucose-induced retinal glial apoptosis can be inhibited by taurine; and (b) taurine reverses the diabetes-induced or high glucose-induced down-regulated expression in TauT. Acknowledgments This work is supported by the National Natural Science Foundation of China (grant 30571570).
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