Oxidative Stress Affects Synaptosomal {gamma}-Aminobutyric Acid and Glutamate Transport in Diabetic Rats

The Role of Insulin

Ana I. Duarte1,2, Maria S. Santos1,2, Raquel Seiça1,3, and Catarina R. Oliveira1,4

1 Center for Neuroscience of Coimbra, University of Coimbra, Coimbra, Portugal
2 Department of Zoology, Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal
3 Institute of Physiology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal
4 Institute of Biochemistry, Faculty of Medicine, University of Coimbra, Coimbra, Portugal


    ABSTRACT
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Evidence suggests that oxidative stress is involved in the pathophysiology of diabetic complications and that insulin has a neuroprotective role in oxidative stress conditions. In this study, we evaluated the in vitro effect of insulin in the susceptibility to oxidative stress and in the transport of the amino acid neurotransmitters {gamma}-aminobutyric acid (GABA) and glutamate in a synaptosomal fraction isolated from male type 2 diabetic Goto-Kakizaki (GK) rat brain cortex. The ascorbate/Fe2+-induced increase in thiobarbituric acid reactive substances (TBARSs) was similar in Wistar and GK rats and was not reverted by insulin (1 µmol/l), suggesting that other mechanisms, rather than a direct effect in membrane lipid peroxidation, may mediate insulin neuroprotection. Diabetes did not affect GABA and glutamate transport, despite the significant decrease in membrane potential and ATP/ADP ratio, and insulin increased the uptake of both GABA and glutamate in GK rats. Upon oxidation, there was a decrease in the uptake of both neurotransmitters and an increase in extrasynaptosomal glutamate levels and in ATP/ADP ratio in GK rats. Insulin treatment reverted the ascorbate/Fe2+-induced decrease in GABA accumulation, with a decrease in extrasynaptosomal GABA. These results suggest that insulin modulates synaptosomal GABA and/or glutamate transport, thus having a neuroprotective role under oxidizing and/or diabetic conditions.

In humans and experimentally diabetic rats, oxidative stress–mediated damage seems to be involved in the cause of diabetic complications (1). This hypothesis is based on reports of increased generation of reactive oxygen species (ROS), decreased antioxidant levels, and/or impaired generation of the reduced forms of the antioxidants (24), leading to lipid, protein, and DNA oxidation (5). Brain is one of the most important targets for ROS, because of its high levels of polyunsaturated fatty acids, high oxygen consumption, high content in transition metals (e.g., Fe2+), and poor antioxidant defenses (6).

Several authors have shown that diabetes modifies the levels of free amino acids (7,8) and the accumulation of amino acid neurotransmitters. Vilchis and Salceda (8) reported an increase in taurine and {gamma}-aminobutyric acid (GABA) uptake, whereas glycine and glutamate uptake remained unaffected in diabetic rat retina and retinal pigment epithelium. In a previous study performed in 6-month-old diabetic Goto-Kakizaki (GK) rat synaptosomes, we demonstrated a diabetes-induced decrease in glutamate accumulation, leading to the hypothesis that diabetes modifies glutamate transport and increases neuronal injury occurring under pathological conditions (3).

It has been widely accepted that peripherally synthesized insulin can be transported into the brain via the cerebrospinal fluid. However, recent molecular biological evidence suggests that it can also be synthesized de novo by neurons, because the presence of preproinsulin I and II mRNA or insulin receptor mRNA was observed in cultured neurons. Moreover, insulin immunoreactivity occurs in the endoplasmic reticulum and Golgi apparatus in vivo (9,10). In the central nervous system, insulin seems to play an important role, particularly in the complications caused by diabetes, involving the regulation of brain metabolism (11,12), neuronal growth and differentiation (9,13), or neuromodulation (13,14). Insulin may also protect against brain damage induced by stress conditions, such as oxidative stress or ischemia (12,15). Brain glucose utilization and metabolism are essential to cognitive functions, and a disturbance in both or in the desensitization of brain insulin receptors may be involved in the intellectual decline in Alzheimer’s disease and related neurodegenerative disorders (16,17), in which excitotoxicity and oxidative stress have been shown to occur (18).

GK rats are a nonobese animal model of type 2 diabetes, produced by repeated selective breeding of Wistar nondiabetic rats, using glucose intolerance as a selective index (19). In the present study, we evaluated the interactions among abnormal glucose metabolism, preincubation with insulin, and the accumulation of the amino acid neurotransmitters GABA and glutamate in synaptosomes isolated from the cerebral cortex of GK diabetic rats under oxidative stress conditions. The oxidant pair ascorbate/Fe2+ was used to induce oxidative injury, and the extent of lipid peroxidation was followed by measuring the thiobarbituric acid reactive substances (TBARSs) production.


    RESEARCH DESIGN AND METHODS
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Male spontaneously diabetic GK rats (31.9 ± 0.7 weeks of age) were obtained from our local breeding colony (Animal Research Center Laboratory, University Hospitals, Coimbra, Portugal), established in 1995 with breeding couples from the colony at the Tohoku University School of Medicine (Sendai, Japan; courtesy of Dr. K.I. Susuki). Control animals were nondiabetic male Wistar rats of similar age (28.2 ± 1.4 weeks of age) obtained from the same colony. Animals were kept under controlled light and humidity conditions, with free access to powdered rodent diet (diet C.R.F. 20; Charles River, L’Arbilesle, France) and water. Glucose tolerance tests were used as a selection index.

All chemicals used were of the highest grade of purity commercially available. [3H]GABA (65 Ci/mmol) and L-[G-3H]glutamate (49 Ci/mmol) were from Amersham International (Little Chalfont, U.K.).

Blood glucose level determination.
Blood glucose levels were determined immediately after the animals were killed, through the glucose oxidation reaction, using a glucometer (Glucometer Elite; Bayer, Carnaxide, Portugal) and compatible reactive tests.

Preparation of synaptosomes.
Crude synaptosomes were prepared from brain of male Wistar and GK rats, according to a preestablished method (20), with some modifications. After animal decapitation, the whole cerebral cortices were rapidly removed and homogenized in 10 vol of homogenization medium (0.32 mol/l sucrose, 10 mmol/l HEPES, and 0.5 mmol/l EGTA-K+, buffered with Tris at pH 7.4). The homogenate was centrifuged at 1,000g for 5 min, and the synaptosomal fraction was isolated from the supernatant by centrifugation at 12,000g for 10 min and then centrifuged again at 12,000g for 10 min in 10 ml of washing medium (0.32 mol/l sucrose, 10 mmol/l HEPES, buffered at pH 7.4 with Tris). The white and fluffy synaptosome layer without contaminant mitochondria (mitochondria free), was then resuspended, respun, and resuspended in the sucrose medium at a protein concentration of 15–20 mg/ml, as determined by the biuret method (21). Experiments were carried out within 3 h after synaptosomal fraction preparation.

Induction of oxidative stress.
The oxidizing agents ascorbic acid and iron (Fe2+, ferrous sulfate) were used to induce oxidative stress (22). Synaptosomes (1 mg/ml) were peroxidized by incubation for 15 min at 30°C in a standard medium that contained (in mmol/l) 132 NaCl, 3 KCl, 1 MgCl2, 1 NaH2PO4, 1.2 CaCl2, 10 glucose, and 20 mmol/l HEPES adjusted to pH 7.4 with Tris, supplemented with 0.8 mmol/l ascorbic acid and 2.5 µmol/l Fe2+. Ascorbic acid and Fe2+ solutions were prepared immediately before use and protected from light. Similar experiments were carried out in the presence of 1 µmol/l insulin, with synaptosomes preincubated for 15 min before the addition of ascorbate/Fe2+.

Evaluation of lipid peroxidation.
The extent of lipid peroxidation was determined by measuring TBARSs, using the thiobarbituric acid assay (23). The amount of TBARSs formed was calculated using a molar extinction coefficient of 1.56 x 105 (mol/l)–1 x cm–1 and expressed as nanomoles of TBARSs per milligram of protein.

Determination of [3H]GABA and [3H]glutamate uptake.
The uptake of [3H]GABA and [3H]glutamate was determined as previously described (24,25). Synaptosomes (0.5 mg of protein/ml) were equilibrated at 30°C in a Na+ medium that contained (in mmol/l) 125 NaCl, 3 KCl, 1.2 MgSO4, 1.2 CaCl2, 3 glucose, and 10 HEPES-Tris at pH 7.4 in the absence or presence of ascorbic acid (0.8 mmol/l) plus Fe2+ (2.5 µmol/l). The same experimental procedure was carried out in the presence or absence of 1 µmol/l insulin, with synaptosomes preincubated for 15 min before the addition of ascorbate/Fe2+. Amino-oxyacetic acid (10 µmol/l) was included in all media when studying the uptake of [3H]GABA to prevent GABA metabolism. After 15 min of incubation, the uptake reaction was started by adding [3H]GABA (final concentration 0.5 µmol/l, 0.25 µCi/ml) or [3H]glutamate (final concentration 10 µmol/l, 49 Ci/mmol). Uptake was stopped at 10 min by rapid filtration of the samples (0.2 ml) under vacuum, through glass fiber filters (Whatman GF/B), followed by a wash with 10 ml of the Na+-containing glucose-free medium (washing medium). The filters were placed in scintillation vials with 3 ml of scintillation UniverSol cocktail (ICN, Irvine, CA), and radioactivity was counted in a liquid scintillation analyzer Packard Tri-Carb 2500 TR. The quenching of radioactivity in the samples was corrected automatically by using an efficiency correlation curve obtained for 3H-quenched standards by the external standardization method. The results were presented as percentage of the control, considering the mean of Wistar rat synaptosomes not treated with ascorbate/Fe2+ as 100%.

Fluorimetric determination of extrasynaptosomal levels of amino acids after high-performance liquid chromatography separation.
The extrasynaptosomal levels of the amino acids GABA and glutamate were analyzed fluorometrically after o-phthaldialdehyde/2-mercaptoethanol derivatization and high-performance liquid chromatography (HPLC) separation, as described by Sitges et al. (26), with some modifications. The amino acids were separated by reverse-phase Gilson-ASTED HPLC system, composed of a Spherisorb ODS column (particle size, 5 µm; 150-mm long; 4.6 mm i.d.) at 25°C and a Gilson model 121 fluorescence detector set at 340 nm (excitation wavelength) and at 410 nm (emission wavelength). A linear gradient elution program carried out over 30 min was applied for amino acid elution: eluent A (30 mmol/l sodium acetate buffer [pH 6.8]) from 100 to 50%, and eluent B (methanol) from 0 to 50%, with a flow rate of 2.5 ml/min. Amino acids were detected as fluorescence derivatives after precolumn derivatization with o-phthaldialdehyde/2-mercaptoethanol. The integration of the amino acid peak area and further calculations were carried out by Gilson system software, and quantification was allowed by running standard amino acids solutions under the same conditions.

Synaptosomal membrane potential measurements.
The synaptosomal transmembrane potential ({Delta}{Psi}m) was monitored by evaluating the transmembrane distribution of tetraphenylphosphonium (TPP+) with a TPP+-sensitive electrode prepared according to Kamo et al. (27), using a calomel electrode as reference. Reactions were carried out in a chamber with magnetic stirring in 1 ml of Na+ medium that contained (in mmol/l) 125 NaCl, 3 KCl, 1.2 MgSO4, 1.2 CaCl2, 3 glucose, and 10 HEPES-Tris at pH 7.4. Synaptosomes (0.5 mg/ml) were equilibrated in Na+ medium for 15 min at 30°C in the absence or presence of 1 µmol/l insulin and then incubated for the same time with or without ascorbate/Fe2+. The experiments were started by adding 3 µmol/l TPP+, and the membrane potential was estimated from the decrease in TPP+ concentration in the reaction medium.

Determination of adenine nucleotides (ATP and ADP).
At the end of the {Delta}{psi}m measurements, each synaptosomal suspension was rapidly centrifuged at 14,000 rpm for 2 min with 0.3 mol/l perchloric acid. The supernatants were neutralized with 10 mol/l KOH in 5 mol/l Tris and centrifuged at 14,000 rpm for 2 min. The resulting supernatants were assayed for adenine nucleotides by separation in a reverse-phase HPLC, as described by Stocchi et al. (28). The chromatographic apparatus used was a Beckman-System Gold, consisting of a 126 Binary Pump Model and 166 Variable UV detector, controlled by a computer. The detection wavelength was 254 nm, and the column used was a Lichrospher 100 RP-18 (5 µm) from Merck (Darmstadt, Germany). An isocratic elution with 100 mmol/l phosphate buffer (KH2PO4; pH 6.5) and 1.0% methanol was performed with a flow rate of 1 ml/min. The required time for each analysis was 6 min. Peak identity was determined by the retention time compared with standards. The amounts of ATP and ADP were determined by a concentration standard curve, and the results were presented as percentage of the control, considering the mean of Wistar rat synaptosomes not treated with ascorbate/Fe2+ as 100%.

Data analysis and statistics.
Results are presented as mean ± SE of the indicated number of experiments. Statistical significance was determined using the t test or the one-way ANOVA test for multiple comparisons. P < 0.05 was considered significant.


    RESULTS
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
General characterization of GK rats.
When the rats were killed, blood glucose levels were significantly higher in the diabetic GK group (241.8 ± 26.5 mg/dl) as compared with control Wistar rats (93.5 ± 5.1 mg/dl).

Synaptosomal susceptibility to oxidative stress induced by ascorbate/Fe2+.
Oxidative stress was induced after 15 min of synaptosomal incubation with 0.8 mmol/l ascorbate and 2.5 µmol/l Fe2+. Under these conditions, an increase in lipid peroxidation, as determined by the formation of TBARS, has been reported (3,4,15). In the absence of ascorbate/Fe2+, synaptosomal levels of TBARS were similar in both Wistar and GK rats (2.6 ± 0.3 and 2.7 ± 0.2 nmol/mg protein, respectively; Fig. 1). In GK rat synaptosomes, insulin did not significantly change TBARS levels (2.7 ± 0.2 and 2.6 ± 0.3 nmol/mg protein in the absence and in the presence of insulin, respectively). Moreover, an 8.7-fold increase in TBARS formation induced by ascorbate/Fe2+ was observed and was not reverted by preincubation with insulin (24.2 ± 2.3 nmol/mg protein).



View larger version (37K):
[in this window]
[in a new window]
 
FIG. 1. Effect of oxidative stress induced by ascorbate/Fe2+ on the extent of lipid peroxidation on synaptosomes isolated from GK rat brain. Synaptosomes were preincubated in Na+ medium for 15 min at 30°C in the absence or in the presence of 1 µmol/l insulin and then incubated in the absence (nonoxidized) or in the presence (oxidized) of 0.8 mmol/l ascorbate and 2.5 µmol/l Fe2+ for 15 min. The extent of lipid peroxidation was evaluated by determining the production of TBARSs and expressed as nmol/mg protein. Results obtained for Wistar rat synaptosomes submitted or not to oxidative stress are also represented. Data are the means ± SE of five to seven independent experiments. ***P < 0.001 vs. the respective control (Wistar or GK rat synaptosomes) not treated with ascorbate/Fe2+; ###P < 0.001 vs. GK rat synaptosomes treated with 1 µmol/l insulin in the absence of oxidizing conditions.

 
Effect of insulin on the uptake of [3H]GABA and [3H]glutamate under oxidative stress conditions.
Under nonoxidant conditions, [3H]GABA uptake was similar in Wistar and GK rats, and preincubation with insulin increased [3H]GABA uptake in GK rat synaptosomes, to 136 ± 13.7% of Wistar controls under nonoxidant conditions (Fig. 2A). The presence of ascorbate/Fe2+ induced a 90% decrease in [3H]GABA uptake in GK rat synaptosomes, which was almost completely prevented by preincubation with insulin.



View larger version (22K):
[in this window]
[in a new window]
 
FIG. 2. Effect of insulin on the uptake of [3H]GABA (A) and [3H]glutamate (B) by GK rat synaptosomes submitted (oxidized) or not (nonoxidized) to oxidative stress induced by ascorbate/Fe2+. Synaptosomes were equilibrated in Na+ medium for 15 min at 30°C in the absence or in the presence of 1 µmol/l insulin and then incubated for 15 min with or without ascorbate/Fe2+. Results obtained for Wistar rat synaptosomes submitted or not to oxidative stress are also presented. Data, expressed as a percentage of [3H]GABA uptake or [3H]glutamate uptake in Wistar control rat synaptosomes (considering 137.9 pmol [3H]GABA/mg protein and 0.7 nmol [3H]glutamate/mg protein as 100%), are the means ± SE of three to six and of three to seven different experiments, respectively. *P < 0.05; **P < 0.01; ***P < 0.001 vs. the respective control (normal or diabetic rat synaptosomes) not treated with ascorbate/Fe2+; #P < 0.05; ###P < 0.001 vs. GK rat synaptosomes treated with 1 µmol/l insulin in the absence of ascorbate/Fe2+; ££P < 0.01 vs. synaptosomes from GK rats submitted to oxidizing conditions.

 
Under nonoxidizing conditions, there were no significant differences in synaptosomal [3H]glutamate accumulation between Wistar and GK rats. Insulin incubation increased [3H]glutamate uptake in diabetic rats by ~23% (Fig. 2B). Ascorbate/Fe2+ treatment decreased [3H]glutamate uptake to 48.0 ± 9.8% of Wistar control rat synaptosomes. However, the protective effect of insulin was lower compared with that seen in [3H]GABA. Under nonoxidizing conditions, the synaptosomal accumulation of [3H]GABA and [3H]glutamate in Wistar rat synaptosomes was 118.7 pmol/mg protein and 0.8 nmol/mg protein, respectively.

Effect of oxidative stress and/or insulin on the extrasynaptosomal levels of GABA and glutamate.
In the absence of ascorbate/Fe2+, extrasynaptosomal GABA levels were similar in both normal and diabetic rats (9.2 ± 0.6 and 8.9 ± 1.2 nmol GABA/mg protein, respectively; Fig. 3A), and insulin (1 µmol/l) treatment did not affect its levels in GK rat synaptosomes. In these animals, ascorbate/Fe2+-induced oxidation increased 1.2-fold the extrasynaptosomal levels of GABA, which was prevented by insulin treatment (5.3 ± 0.6 nmol GABA/mg protein).



View larger version (24K):
[in this window]
[in a new window]
 
FIG. 3. Effect of insulin on the release of the endogenous amino acids GABA (A) and glutamate (B) by GK rat synaptosomes submitted (oxidized) or not (nonoxidized) to oxidative stress induced by ascorbate/Fe2+. Results obtained for Wistar rat synaptosomes submitted or not to oxidative stress are also presented. The values, expressed in nmol/mg protein, are the mean ± SE of three to five independent experiments. *P < 0.05 vs. the respective control (Wistar or GK rat synaptosomes) not treated with ascorbate/Fe2+; #P < 0.05 vs. GK rat synaptosomes preincubated with insulin. £P < 0.05 vs. GK rat synaptosomes upon oxidative stress conditions.

 
Under control conditions, extrasynaptosomal levels of glutamate were similar in Wistar and GK rats (17.8 ± 0.4 and 16.4 ± 1.2 nmol glutamate/mg protein, respectively), and incubation with insulin did not affect the extrasynaptosomal content of glutamate in diabetic rats (Fig. 3B). Upon oxidizing conditions, a 1.8-fold increase in extracellular glutamate levels was observed, which was not prevented by insulin.

Effect of oxidative stress and/or insulin on synaptosomal {Delta}{Psi}m.
Synaptosomal {Delta}{Psi}m was significantly lower in diabetic rats relative to normal Wistar rat synaptosomes (–101.8 ± 4.7 and –80.4 ± 7.7 mV in Wistar and GK rat synaptosomal membranes, respectively). Insulin (1 µmol/l) incubation did not significantly affect GK rat synaptosomal {Delta}{Psi}m. In these rats, neither ascorbate/Fe2+ nor insulin treatment under oxidizing conditions significantly altered {Delta}{Psi}m values (Fig. 4).



View larger version (41K):
[in this window]
[in a new window]
 
FIG. 4. Effect of insulin on the {Delta}{Psi}m in GK rat synaptosomes submitted (oxidized) or not (nonoxidized) to oxidative stress induced by ascorbate/Fe2+. Synaptosomes were equilibrated in Na+ medium for 15 min at 30°C in the absence or in the presence of insulin and then incubated for 15 min with or without ascorbate/Fe2+. Membrane potential was determined by following the decrease of TPP+ in the medium. Results obtained for Wistar rat synaptosomes submitted or not to oxidative stress are also presented. Data, expressed as –mV, are the means ± SE of 9–13 different experiments. *P < 0.05 vs. the respective control (Wistar or GK rat synaptosomes) not treated with ascorbate/Fe2+; #P < 0.05 vs. the GK rat synaptosomes preincubated with 1 µmol/l insulin in the absence of oxidizing conditions.

 
Effect of oxidative stress and/or insulin on synaptosomal ATP/ADP ratio.
The effects of insulin and/or ascorbate/Fe2+ treatment on the synaptosomal ATP/ADP ratio are represented in Fig. 5. In the absence of oxidizing conditions, the ATP/ADP ratio was significantly lower in diabetic rats relative to normal Wistar rats, and synaptosomal incubation with 1 µmol/l insulin did not significantly change this value. Under oxidizing conditions, diabetic rats showed a 1.7-fold increase in the ATP/ADP ratio, which was not altered upon insulin incubation. The ATP/ADP ratio in Wistar rat synaptosomes under nonoxidizing conditions was 2.9 ± 0.9.



View larger version (47K):
[in this window]
[in a new window]
 
FIG. 5. Effect of insulin in the ATP/ADP ratio in GK rat synaptosomes submitted (oxidized) or not (nonoxidized) to oxidative stress induced by ascorbate/Fe2+. After the {Delta}{psi}m measurements, each synaptosomal suspension was treated with 0.3 mol/l perchloric acid and the supernatants were neutralized with 10 mol/l KOH in 5 mol/l Tris. Results obtained for Wistar rat synaptosomes submitted or not to oxidative stress are also presented. Data, expressed as a percentage of the ATP/ADP ratio in Wistar control rat synaptosomes (considering 2.9 ± 0.9 as 100%), are the means ± SE of 9–13 different experiments. ***P < 0.001 vs. the respective control (Wistar or GK rat synaptosomes) not treated with ascorbate/Fe2+; ###P < 0.001 vs. the GK rat synaptosomes preincubated with 1 µmol/l insulin in the absence of oxidizing conditions.

 

    DISCUSSION
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nonenzymatic glycation and irreversible protein cross-linking that occur under hyperglycemia may result in the formation of advanced glycation end products and ROS (29,30), supporting the idea that the increase in blood glucose levels (3,31) may render diabetic rats exposed to oxidative stress (5,29).

Aberrant synaptic signaling has been implicated in neurodegenerative disorders, such as Alzheimer’s disease (32). Synaptosomes isolated from mammalian brain constitute a useful in vitro model to study several nerve functions, because they are metabolically active and retain many properties of nerve endings, namely neurotransmitter uptake (33,34).

In previous studies (3,4), we observed that GK rat brain synaptosomes were less susceptible to ascorbate/Fe2+-induced oxidative stress than Wistar rats, suggesting a protective mechanism against ROS-induced damage. In the present study, we found that basal levels of TBARS were similar in both animal models, as previously reported (35). Our recent observation that insulin did not reverse the ascorbate/Fe2+-induced increase in TBARS levels in Wistar rats suggests that other mechanisms, rather than a direct action of insulin on lipid peroxidation, may mediate its neuroprotective role under stress conditions (15).

The controversial observations reported by some authors on alterations in brain amino acid transport using diabetic rats may be related to the diabetic model or to the experimental approach used. Morrison et al. (36) reported that streptozotocin-induced diabetes (a type 1 diabetes model) does not change the transport mechanism or the receptor sensitivity to GABA. Li et al. (37) reported an increased accumulation of extracellular glutamate in the neocortex submitted to hyperglycemic ischemia, which was correlated with the cell damage observed. In this study, we reported that diabetes had no effect on both synaptosomal GABA and glutamate transport, which is partially in accordance with the observations of Vilchis and Salceda (8). These authors demonstrated an increment in GABA uptake, without changes in glutamate uptake in diabetic rat retina and retinal pigment epithelium. These observations suggest that the modification of transmembrane electrical gradient may affect the accumulation of amino acid neurotransmitters. Our observation of a diabetes-related decrease in both synaptosomal transmembrane gradient ({Delta}{Psi}m) and ATP/ADP ratio, resulting from a decrease in ATP and/or an increase in ADP levels (data not shown), further supports this hypothesis. The decrease in ATP production is a hallmark of a disturbance in glucose metabolism, which could be partially balanced by the use of endogenous brain substrates, such as glutamate and fatty acids (17). Synaptosomal uptake of neurotransmitters is an energy-dependent process that requires the integrity of membrane systems (33) and functional mitochondria. Thus, our observation of a diabetes-mediated decrease in synaptosomal membrane potential and ATP/ADP ratio suggests a glucose-dependent protective mechanism in GK rat brain, with higher glucose levels leading to the loss of the GABA-dependent protection against glutamate neurotoxicity.

Our observation of an oxidative stress–induced decrease in synaptosomal GABA uptake, accompanied by an increase in its extrasynaptosomal accumulation, suggested that GK rats may have developed a protective mechanism against oxidative stress–related neuronal death (38,39). The decrease in glutamate uptake and increased extrasynaptosomal levels induced by ascorbate/Fe2+ in GK rats further support this hypothesis, because oxidative stress–induced damage to neuronal cell membrane leads to the release and subsequent extracellular accumulation of glutamate, contributing to excitotoxicity (38) and further promoting ROS generation (40). Sah and Schwartz-Bloom (41) reported that ROS-decreased inhibitory neurotransmission also promotes neuronal damage in oxidative stress–related diseases and aging. These effects of oxidative stress on amino acid accumulation may be due to membrane lipoperoxidation, which may impair the GABA transporter, leading to a decrease of GABA accumulation (42), and/or it may increase the release and extracellular accumulation of aspartate and glutamate (38,40). Other mechanisms that could be involved in the inhibition of glutamate uptake by ROS may be the direct oxidation of the transporter sulfhydryl groups (43,44) or the impairment of Na+,K+ ATPase activity (45).

The modulatory effect of insulin on amino acid accumulation in brain remains controversial (14,46). In some studies, 3 µmol/l insulin stimulated glutamate and GABA accumulation in rat brain synaptosomes (14), whereas in others, the same concentration was shown to inhibit GABA uptake in embryonic chick retina and newborn mouse cerebral cortex astroglial cells (11,47). According to Rhoads et al. (14), insulin dose-dependently stimulates synaptosomal amino acid uptake, and 1 µmol/l was the lowest insulin concentration that increased glutamate uptake by cortical synaptosomes, by an effect maintained during 1–20 min of in vitro incubation. In the present study, we used an insulin concentration of 1 µmol/l, incubated for 15 min with the synaptosomal fraction, in the absence or presence of the oxidizing pair ascorbate/Fe2+. In a recent article, we reported that, in Wistar rat synaptosomes, insulin reverted both the oxidative stress–related inhibition in the uptake and the increase in GABA and glutamate extrasynaptosomal accumulation (15).

In this study, we observed that 1 µmol/l insulin per se stimulated intrasynaptosomal GABA accumulation in diabetic rat brain synaptosomes, suggesting that the neuroprotective role of insulin may be exerted directly on membrane GABA transporters. Some authors (11,14) hypothesized that insulin-induced changes in GABA transport can be exerted indirectly through the stimulation of Na+,K+ ATPase activity, thus increasing the transmembrane Na+ gradient and the {Delta}{Psi}m. However, Guyot et al. (48) postulated that these changes could be related to stimulation of glycolysis and pyruvate dehydrogenase, increasing lactate and ATP levels, which stimulate ion pumps and, ultimately, the synaptosomal amino acid transport. Our results did not show insulin-induced changes in {Delta}{Psi}m or ATP/ADP ratio, suggesting that the increase in the uptake of both GABA and glutamate might be related to a direct effect of insulin in both amino acid transporters. However, the activation of insulin receptor signaling cascade via its tyrosine kinase activity, which activates the uptake of amino acids (49), cannot be ruled out. We also observed that insulin reverted the effect of ascorbate/Fe2+, increasing GABA uptake and decreasing its extrasynaptosomal levels, supporting the idea of a neuroprotective role for insulin. This hypothesis is also supported by a nonsignificant decrease in extrasynaptosomal glutamate levels observed under these conditions.

Recent evidence suggests that a dysfunction of the neuronal insulin receptor signaling cascade, with the subsequent abnormalities in glucose/energy metabolism, may affect amyloid precursor protein metabolism and induce {tau} hyperphosphorylation. These results suggest that insulin dysfunction could be involved in the cause of Alzheimer’s disease (12,17) and supports the hypothesis of a neuroprotective role of insulin under stress conditions. The data obtained in the present study support the idea that a direct effect on amino acid neurotransmitter transport system (mainly GABA) could be one of the mechanisms responsible for the neuroprotective role of insulin in oxidative stress conditions, suggested to occur in diabetes and neurodegenerative diseases.


    ACKNOWLEDGMENTS
 
This work was supported by FCT (Portuguese Research Council). A.D. is a recipient of a fellowship from FCT (PRAXIS XXI/BM/20944/99).

We thank Prof. Luís Rosário (Department of Biochemistry, University of Coimbra) for the use of GK rats and Prof. João Patrício and co-workers (Laboratory of Animal Research Center, University Hospitals, Coimbra) for help in the maintenance of animals.

Address correspondence and reprint requests to Prof. Catarina Resende de Oliveira, Center for Neuroscience of Coimbra Institute of Biochemistry, Faculty of Medicine, University of Coimbra, 3004-517 Coimbra, Portugal. E-mail: catarina{at}cnc.cj.uc.pt

Received for publication October 22, 2003 and accepted in revised form April 29, 2004

GABA,{gamma}-aminobutyric acid; HPLC, high-performance liquid chromatography; ROS, reactive oxygen species; TBARS, thiobarbituric acid reactive substance; TTP+, tetraphenylphosphonium


    REFERENCES
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Davison GW, George L, Jackson SK, Young IS, Davies B, Bailey DM, Peters JR, Ashton T: Exercise, free radicals, and lipid peroxidation in type 1 diabetes mellitus. Free Radic Biol Med33 :1543 –1551,2002[Medline]
  2. Singh S, Melkani GC, Rani C, Gaur SPS, Agrawal V, Agrawal CG: Oxidative stress and metabolic control in non-insulin dependent diabetes mellitus. Indian J Biochem Biophys34 :512 –517,1997[Medline]
  3. Duarte A, Santos M, Seiça R, Oliveira C: Effect of oxidative stress on the uptake of GABA and glutamate in synaptosomes isolated from diabetic rat brain. Neuroendocrinology72 :179 –186,2000[Medline]
  4. Santos MS, Duarte AI, Matos MJ, Proença T, Seiça R, Oliveira CR: Synaptosomes isolated from Goto-Kakizaki diabetic rat brain exhibit increased resistance to oxidative stress: role of vitamin E. Life Sci67 :3061 –3073,2000[Medline]
  5. Rösen P, Nawroth PP, King G, Möller W, Tritschler H-J, Packer L: The role of oxidative stress in the onset and progression of diabetes and its complications: a summary of a Congress Series sponsored by UNESCO-MCBN, the American Diabetes Association and the German Diabetes Society. Diabetes Metab Res Rev17 :189 –212,2001[Medline]
  6. Leutner S, Eckert A, Muller WE: ROS generation, lipid peroxidation and antioxidant enzyme activities in the aging brain. J Neural Transm108 :955 –967,2001
  7. Fernstrom MH, Volk EA, Fernstrom JD, Iuvone M: Effect of tyrosine administration on DOPA accumulation in light and dark-adapted retinas from normal and diabetic rats. Life Sci39 :2049 –2057,1986[Medline]
  8. Vilchis C, Salceda R: Effect of diabetes on levels and uptake of putative amino acid neurotransmitters in rat retina and retinal pigment epithelium. Neurochem Res21 :1167 –1171,1996[Medline]
  9. Schechter R, Yanovitch T, Abboud M, Johnson G III, Gaskins J: Effects of brain endogenous insulin on neurofilament and MAPK in fetal rat neuron cell cultures. Brain Res808 :270 –278,1998[Medline]
  10. Zhao W, Chen H, Xu H, Moore E, Meiri N, Quon MJ, Alkon D: Brain insulin receptors and spatial memory. Interrelated changes in gene expression, tyrosine phosphorylation, and signaling molecules in the hippocampus of water maze trained rats. J Biol Chem274 :34893 –34902,1999[Abstract/Free Full Text]
  11. Shah B, Hausman RE: Effect of insulin on GABAergic development in the embryonic chick retina. Dev Brain Res72 :151 –158,1993[Medline]
  12. Santos MS, Pereira EM, Carvalho AP: Stimulation of immunoreactive insulin release by glucose in rat brain synaptosomes. Neurochem Res24 :33 –36,1999[Medline]
  13. Gerozissis K: Brain insulin: regulation, mechanisms of action and functions. Cell Mol Neurobiol23 :1 –25,2003[Medline]
  14. Rhoads DE, DiRocco RJ, Osburn LD, Peterson NA, Raghupathy E: Stimulation of synaptosomal uptake of neurotransmitter amino acids by insulin: possible role of insulin as a neuromodulator. Biochem Biophys Res Commun119 :1198 –1204,1984[Medline]
  15. Duarte AI, Santos MS, Seiça R, Oliveira CR: Insulin affects synaptosomal GABA and glutamate transport under oxidative stress conditions. Brain Res977 :23 –30,2003[Medline]
  16. Biessels GJ, van der Heide LP, Kamal A, Bleys RLAW, Gispen WH: Ageing and diabetes: implications for brain function. Eur J Pharmacol441 :1 –14,2002[Medline]
  17. Hoyer S: The brain insulin signal transduction system and sporadic (type II) Alzheimer disease: an update. J Neural Transm109 :341 –360,2002[Medline]
  18. Bohr V: Repair of oxidative DNA damage in nuclear and mitochondrial DNA, and some changes with aging in mammalian cells. Free Radic Biol Med32 :804 –812,2002[Medline]
  19. Portha B, Giroix MH, Serradas P, Morin L, Saulnier C, Bailbe D: Glucose refractoriness of pancreatic b-cells in rat models of non-insulin dependent diabetes. Diabetes Metab20 :108 –115,1994
  20. Hajós F: An improved method for the preparation of synaptosomal fractions in high purity. Brain Res93 :485 –489,1975[Medline]
  21. Layne E: Spectrophotometric and turbidimetric methods for measuring proteins. In Methods in Enzymology. Colowick SP, Kaplan NO, Eds. New York, Academic Press,1957 , p.447 –451
  22. Wills E: Lipid peroxide formation in microsomes: general considerations. Biochem J113 :315 –324,1969[Medline]
  23. Ernster L, Nerdenbrand K: Microsomal lipid peroxidation. Methods Enzymol10 :574 –580,1967
  24. Santos MS, Gonçalves PP, Carvalho AP: Effect of ouabain on the {gamma}-[3H]aminobutyric acid uptake and release in the absence of Ca++ and K+-depolarization. J Pharmacol Exp Ther253 :620 –627,1990[Abstract]
  25. Volterra A, Trotti D, Cassutti P, Tromba C, Salvaggio A, Melcangi RC, Racagni G: High sensitivity of glutamate uptake to extracellular free arachidonic acid levels in rat cortical synaptosomes and astrocytes. J Neurochem59 :600 –606,1992[Medline]
  26. Sitges M, Nekrassov V, Guarneros A: Simultaneous action of MK-801 (dizclopine) on dopamine, glutamate, aspartate and GABA release from striatum isolated nerve endings. Brain Res854 :48 –56,2000[Medline]
  27. Kamo N, Muratsugu M, Hongoh R, Kobatake V: Membrane potential of mitochondria measured with an electrode sensitive to tetraphenyl phosphonium and relationship between proton electrochemical potential and phosphorylation potential in steady state. J Membr Biol49 :105 –121,1979[Medline]
  28. Stocchi V, Cucchiarini L, Magnani M, Chiarantini L, Palma P, Crescentini G: Simultaneous extraction and reverse-phase high-performance liquid chromatographic determination of adenine and pyridine nucleotides in human red blood cells. Anal Biochem146 :118 –124,1985[Medline]
  29. Kossenjans W, Eis A, Sahay R, Brockman D, Myatt L: Role of peroxynitrite in altered fetal-placental vascular reactivity in diabetes or preeclampsia. Am J Physiol Heart Circ Physiol278 :H1311 –H1319,2000[Abstract/Free Full Text]
  30. Gökkusu C, Palanduz S, Ademoglu E, Tamer S: Oxidant and antioxidant systems in NIDDM patients: influence of vitamin E supplementation. Endocr Res27 :377 –386,2001[Medline]
  31. Serradas P, Goya L, Lacorne M, Gangnerau M-N, Ramos S, Alvarez C, Pascual-Leone A-M, Portha B: Fetal insulin-like growth factor-2 production is impaired in the GK rat model of type 2 diabetes. Diabetes51 :392 –397,2002[Abstract/Free Full Text]
  32. Guo ZH, Mattson MP: Neurotrophic factors protect cortical synaptic terminals against amyloid- and oxidative stress-induced impairment of glucose transport, glutamate transport and mitochondrial function. Cereb Cortex10 :50 –57,2000[Abstract/Free Full Text]
  33. Weinberger J, Cohen G: The differential effect of ischemic on the active uptake of dopamine, {gamma}-aminobutyric acid, and glutamate by brain synaptosomes. J Neurochem38 :963 –968,1982[Medline]
  34. Nicholls DG: Release of glutamate, aspartate, and {gamma}-aminobutyric acid from isolated nerve terminals. J Neurochem52 :331 –341,1989[Medline]
  35. Kowluru RA, Engerman RL, Kern TS: Abnormalities of retinal metabolism in diabetes or experimental galactosemia. VI. Comparison of retinal and cerebral cortex metabolism, and effects of antioxidant therapy. Free Radic Biol Med26 :371 –378,1999[Medline]
  36. Morrison PD, Mackinnon MW, Bartrup JT, Skett PG, Stone TW: Changes in adenosine sensitivity in the hippocampus of rats with streptozotocin-induced diabetes. Br J Pharmacol105 :1004 –1008,1992[Abstract]
  37. Li P-A, Shuaib A, Miyashita H, He Q-P, Siesjö BK: Hyperglycemia enhances extracellular glutamate accumulation in rats subjected to forebrain ischemia. Stroke31 :183 –192,2000[Abstract/Free Full Text]
  38. Saransaari P, Oja SS: ß Alanine release from the adult and developing hippocampus is enhanced by ionotropic glutamate receptor agonists and cell-damaging conditions. Neurochem Res24 :407 –414,1999[Medline]
  39. Rego AC, Santos MS, Oliveira CR: Oxidative stress, hypoxia, and ischemia-like conditions increase the release of endogenous amino acids by distinct mechanisms in cultured retinal cells. J Neurochem66 :2506 –2516,1996[Medline]
  40. Atlante A, Calissano P, Baba A, Giannattasio S, Marra E, Passarella S: Glutamate neurotoxicity, oxidative stress and mitochondria. FEBS Lett497 :1 –5,2001[Medline]
  41. Sah R, Schwartz-Bloom RD: Optical imaging reveals elevated intracellular chloride in hippocampal pyramidal neurons after oxidative stress. J Neurosci19 :9209 –9217,1999[Abstract/Free Full Text]
  42. Palmeira CM, Santos MS, Carvalho AP, Oliveira CR: Membrane lipid peroxidation induces changes in {gamma}-aminobutyric acid transport and calcium uptake by synaptosomes. Brain Res609 :117 –123,1993[Medline]
  43. Rothstein JD, Martin LJ, Kunkl RW: Decreased glutamate transport by the brain and spinal cord in amyotrophic lateral sclerosis. N Engl J Med326 :1464 –1468,1992[Abstract]
  44. Trotti D, Danbolt NC, Volterra A: Glutamate transporters are oxidant-vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration? Trends Pharmacol Sci19 :328 –334,1998[Medline]
  45. Harris ME, Wang Y, Pedigo NW, Hensley K, Butterfield DA, Carney JM: Amyloid ß-peptide (25–35) inhibits Na+-dependent glutamate uptake in rat hippocampal astrocyte cultures. J Neurochem67 :277 –286,1996[Medline]
  46. Shuaib A, Ijaz MS, Waqar T, Voll C, Kanthan R, Miyashita H, Liu L: Insulin elevates hippocampal GABA levels during ischaemia: this is independent of its hypoglycemic effect. Neuroscience67 :809 –814,1995[Medline]
  47. Bouhaddi K, Thomopoulos P, Fages C, Khelil M, Tardy M: Insulin effect on GABA uptake in astroglial primary cultures. Neurochem Res13 :1119 –1124,1988[Medline]
  48. Guyot LL, Diaz FG, O’Regan MH, Song D, Phillis JW: Topical insulin and accumulation of excitotoxic and other amino acids in ischemic rat cerebral cortex. Proc Soc Exp Biol Med224 :28 –31,2000[Abstract/Free Full Text]
  49. Solano DC, Sironi M, Bonfini C, Solerte SB, Govoni S, Racchi M: Insulin regulates soluble amyloid precursor protein release via phosphatidyl inositol 3 kinase-dependent pathway. FASEB J14 :1015 –1022,2000[Abstract/Free Full Text]




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Request Permissions
Google Scholar
Articles by Duarte, A. I.
Articles by Oliveira, C. R.
Articles citing this Article
PubMed
PubMed Citation
Articles by Duarte, A. I.
Articles by Oliveira, C. R.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Diabetes Diabetes Care Clinical Diabetes Diabetes Spectrum DOC News