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Intern. J. Neuroscience, 114:391–401, 2004Copyright Taylor & Francis Inc.ISSN: 0020-7454 / 1543-5245 onlineDOI: 10.1080/00207450490270893

NMDA RECEPTOR SUBUNITS 2A AND 2BDECREASE AND LIPID PEROXIDATION

INCREASE IN THE HIPPOCAMPUSOF STREPTOZOTOCIN-DIABETIC

RATS: EFFECTS OF INSULINAND GLICLAZIDE TREATMENTS

NAMIK DELIBASIBRAHIM KILINCZAFER YONDENRECEP SUTCUFATIH GULTEKINHALIS KOYLUSuleyman Demirel UniversityFaculty of MedicineDepartment of Biochemistry and PhysiologyIsparta, Turkey

Recent studies indicate that diabetes mellitus changes N-methyl-D-as-partate (NMDA) receptor subunit composition and impairs cognitivefunctions. It also has been known that diabetes mellitus causes lipidperoxidation. This study examined the effects of streptozotocin-diabetesand insulin or gliclazide treatment on the hippocampal NMDA receptorsubunit 2A and 2B (NR2A and NR2B) concentrations. In addition, malondial-dehyde (MDA) levels were measured as a marker for lipid peroxidation.Eight weeks after the induction of diabetes MDA, levels were increased,and NR2A and NR2B concentrations were reduced. Insulin and gliclazidetreatment partially prevented the reduction of NR2A and NR2B expres-sion and prevented the elevation of MDA levels. There was no signifi-cant difference between the effects of insulin and gliclazide. The results

Received 5 July 2003.Address correspondence to Dr. Namik Delibas, SDU Arastirma ve Uygulama Hastanesi, Klinik

Biyokimya Lab. 32260 Isparta, Turkey. E-mail: ndelibas@hotmail.com

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suggest that the elevation of lipid peroxidation can be the primary bio-chemical disturbances in diabetes progression, and that changes in NMDAreceptor subunit compositions can be involved in cognitive decline indiabetes.

Keywords diabetes, hippocampus, insulin, lipid peroxidation, NMDA re-ceptors, streptozotocin

Diabetes is a state of increased oxidant stress, and there is accumu-lating evidence that oxidative damage may play a role in the devel-opment of diabetic complications. During hyperglycemia, an increasedglucose load leads to elevated nonenzymatic glycosylation, an irre-versible cross-linking of certain proteins and lipid peroxidation (Makaret al., 1995). Diabetes mellitus is known to be associated with neu-rological complications in both the peripheral and the central ner-vous system (CNS). Manifestations of cerebral disorders in diabeticpatients include alterations in neurotransmission, electrophysiologi-cal abnormalities, structural changes, and cognitive deficits (Volterraet al., 1994; Biessels et al., 1994). In animal models of diabeticpathology, such as the streptozotocin-diabetic rat, spatial learningimpairments have been reported (Biessels et al., 1998). This cogni-tive deficit is associated with changes in hippocampal synaptic plas-ticity, including an impaired expression of long-term potentiation(LIP) (Biessels et al., 1998; Chabot et al., 1997) and an enhancedexpression of long-term depression (LTD) (Kamal et al., 1999). It isknown that excitatory transmission in the brain is largely mediatedby glutamate, acting through different classes of receptors: ionotropicand metabotropic. In excitatory synapses the frequency-dependentCa2+ influx is mediated largely by the N-Methyl-D-Aspartate (NMDA)receptors.

The NMDA receptor (NR) is an ionotropic glutamate receptor.The NMDA receptor is a heteromeric protein composed of threeclasses of subunits, NR1, NR2, and NR3. Four separate genes en-code NR2 subunits, NR2A to NR2D (Monyer, Sprengel, & Schoepfer,1992). This receptor is involved in a wide variety of processes inthe CNS, including synaptogenesis and synaptic plasticity. Addi-tionally, the NMDA receptors have been implicated in excitotoxicity,neurodegenerative disorders, and aging (Eckles, Clayton, & Bickford,2000). The NMDA receptor is closely related to the reactive oxygen

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Effects of Insulin and Gliclazide Treatments 393

species (ROS). The NMDA receptor stimulation has been shown toproduce ROS, including the superoxide (O

2–.), in hippocampal slices

(Bindokas et al., 1996; Lafon-Cazal et al., 1993). The subunit com-position largely affects the pharmacological and physiological char-acteristics of NMDA receptors (Monyer et al., 1994). Thus, a greaterunderstanding of the modulation of this receptor is likely to be im-portant in understanding the physiology and pathophysiology of theseprocesses. In this study, we investigated the effect of two differenttheropatic agents, namely insulin and gliclazide, on the hippocam-pal NMDA receptor subunit composition in STZ-diabetic rats. Inaddition, how these antidiabetic agents affected lipid peroxidationwas also studied.

MATERIALS AND METHODS

Animals

Spraque Dawley rats (240–250 g body weight) were housed (2 ratsper cage) on sawdust and maintained on a 12 h/12 h light/dark cycle(light on at 06:00 h). Rats were given food and water ad libitum andweighed weekly. Diabetes mellitus was induced by a single intrap-eritoneal injection of streptozotocin (STZ) (Sigma Chemical Co.,St. Louis, MO, USA) at a dose of 30 mg/kg body weight dissolved1% citrate buffer (pH 4.5) after overnight fasting. Four days afterthe STZ injection, blood glucose was determined in blood samples,obtained by tail prick, using a strip-operated blood glucose sensor(Prestige Lx, Home Diagnostics, Inc., Ft. Lauderdale, Florida, USA).Blood glucose levels were measured >250 mg/dl in all STZ-injectedanimals. In insulin and gliclazide-treated rats, blood glucose levelswere measured every week. The experiments reported here com-plied with the current laws and regulations of the Turkish Republicon the care and handling of experimental animals.

Four groups of rats were used: (1) non-diabetic control group (C;n = 9); (2); untreated diabetic group (DM; n = 6) (3) insulin-treateddiabetic group (INS; n = 6); (4) gliclazide treated diabetic group(GLC; n = 6). Insulin and gliclazide treatments were initiated di-rectly after confirmation of diabetes and continued throughout the

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394 N. Delibas et al.

experiment. Insulin (Lilly, USA) was given through subcutaneousinjection at a dose of 4 IU/kg per day. Gliclazide (Servier, France)was given orally in drinking water at a dose of 10 mg/kg/per day.The concentrations of NR2A, NR2B, and MDA in the hippocampuswere measured in week 9 after diabetes induction.

Lipid Peroxidation Assay

After sacrificing the animal, the brain was removed and both hippoc-ampi were then dissected, washed in ice-cold phosphate bufferedsaline (PBS), and frozen immediately in a deep freezer, until furtheruse. One of them was homogenized (1/10, w/v) in a glass-teflonhomogenizer in an ice-cold buffer (0.05 M potassium phosphatebuffer, pH 7.8). The homogenate was centrifuged at 10,000 × g for 15min at 4°C and used for determining the MDA concentration.

MDA, an end product of lipid peroxidation, was assayed using themethod of Drapper and Hadley (1990). Protein in tissue homogenatewas assayed by the Lowry et al.’s method (1951). Data were given asmean ± standard deviation. Statistical analysis of data was performedon the computer, using the SPSS Version 10.0. The Kruskal Wallistest was used for the comparison of the four groups. If a differencewas detected using Kruskal Wallis test, Bonferroni-corrected Mann-Whitney U test was used to find out which two groups were thecauses of this difference. Significant level was set at p < .05.

Western Blot Analyses

Antibodies against NR2A and NR2B were purchased from SigmaChemical Co. (St. Louis, MO, USA). All other reagents were ofanalytical grade or the highest grade available. The other hippoc-ampi (3-4 animals/preparations) were homogenized in ice-cold buffer[50 mm Tris-HCl (pH 7.5), 0.15 M NaCl, 1% Triton X-100, 1 mMEDTA, 1 mM EGTA, 25 µg/ml leupeptin, 25 µg/ml aprotinin, 1 mMsodium orthovanadate, 10 µM benzamidine, and 4 mM p-nitrophenylphosphate], and an aliquot was taken for protein determination. Equalamounts of protein for each sample (20 µg of protein per lane) wereseparated by SDS/PAGE on 7.5% minigels, blotted electrophoreti-cally to immobilon membrane, and incubated in tris-buffered saline

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Effects of Insulin and Gliclazide Treatments 395

with Tween 20 (TBST) [50 mm Tris-HCl (pH 7.5-8.0), 150 mMNaCl, and 0.1% Tween 20], containing 3% bovine serum albumin(BSA) for 30 min. Blots were incubated overnight with anti-NR2A(1:3000) or anti-NR2B (1:5000) in 1% BSA. Blots were then sub-jected to three additional 10-min washings in TBST. Blots wereincubated with alkaline phosphatase conjugated monoclonal anti-rabbit IgG (1:10000) in 1% BSA for lb at room temperature, andthree additional washings were performed with TBST for 10 min.The membrane was incubated in 20 ml of fresh reagent solution(BCIP/NBT), until color development. Images of immunoblots wereanalyzed with a computerized image analysis system (Uviphoto MWV.99, Ultra-Violet Products Ltd, Cambridge, UK). SDS-PAGE andWestern blot analyses were done on three independent hippocam-pus preparations (3 animals/group). The data were subjected to aone-way analysis of variance (ANOVA) with subsequent group com-parisons using the Dunnett test at the 0.05 level of significance.

RESULTS

Comparisons of the groups were given in Table 1. As shown inTable 1, diabetes mellitus caused an increase in MDA levels. Bothinsulin and gliclazide treatments successfully reduced MDA levelsto the control levels. Blood glucose levels were 148 ± 9 and 323 ±39 mg/dl in INS and GLC groups, respectively.

To evaluate protein concentrations of NMDA receptors Westernblot analyses were done on hippocampal homogenates. Western blotanalysis of NR2A and NR2B were shown in Figures 1 and 2, re-spectively. The density of the protein band in the control group wasaccepted as 100%, and data from other groups were calculated as

TABLE 1. Comparison of the groups

C DM INS GLC

MDA (nmol/mg protein) 17 ± 1a 23 ± 2b 18 ± 0.6a 18 ± 0.8a

a,bp < .05, Mann Whitney U Test.C: Control group; DM = untreated diabetes mellitus group; INS = insulin treatment gruop; GLC =gliclazide treatment group; MDA = malondialdehyde.

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396 N. Delibas et al.

percentages of the control value. The Western blot analysis for NR2Aand NR2B showed a significant (n = 3, p < .05, for both) reductionof approximately 53 and 45% in diabetic rats, compared with con-trol rats, respectively. We then tested the possible effect of insulinand gliclazide treatment on the NMDA receptor subunit concentra-tions. The insulin and gliclazide treatment partially restored NR2Aand NR2B concentrations towards control values (Figures 1 and 2).There was no significant difference between the two treatment regi-mens (n = 3, p > .05).

FIGURE 1. Representative Western blot of NR2A in hippocampus from four groups ofrats. Experiments were done on three independent hippocampus preparations (2 animals/group). Size marker is indicated on the left (myosin, 205 kDa). C: Control, DM:Untreated diabetes mellitus group, INS: Insulin treatment group, GLC: Gliclazide treat-ment group. The asterisk indicates significant changes compared to other groups, usingan ANOVA with subsequent group comparisons, using the Dunnett test at the 0.05 levelof significance.

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Effects of Insulin and Gliclazide Treatments 397

DISCUSSION

The main findings of this study were that 8 weeks of STZ-diabetesreduced hippocampal NR2A and NR2B subunit protein concentra-tions and increased hippocampal lipid peroxidation, compared tocontrols. Insulin and gliclazide treatments partially prevented NR2Aand NR2B expression and effectively reduced lipid peroxidation.

The question of how chronic diabetic pathology could changethe expression of a specific NMDA receptor subunit is currentlyunder investigation. It has been suggested that growth factors can

FIGURE 2. Representative Western blot of NR2B in hippocampus from four groups ofrats. Experiments were done on three independent hippocampus preparations (2 animals/group). Size marker is indicated on the left (myosin, 205 kDa). C: Control, DM:Untreated diabetes mellitus group, INS: Insulin treatment group, GLC: Gliclazide treat-ment group. The asterisk indicates significant changes compared to other groups usingan ANOVA with subsequent group comparisons using the Dunnett test at the 0.05 levelof significance.

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398 N. Delibas et al.

influence the expression of NMDA receptor subunits. A decrease ininsulin-like growth factor-I (IGF-1) and IGF-2 has been shown indiabetic patients and in STZ-diabetic rats (Brewster et al., 1994). Inthis respect, the possibility that IGF-/IGF-2 might influence the ac-tivity of the promoter of different NR2 subunits is particularly in-triguing. It observed that after a prolonged treatment of diabetic ratswith insulin, the concentration of NR2A and NR2B protein is par-tially restored, possibly supporting such a hypothesis. We also founda similar result in gliclazide treatment. It was thought that otherfactor(s) should be considered. Is there a relation between hippo-campal MDA levels and NR subunit expression rate? Concomi-tantly of the reduced NR2A and NR2B concentration in the hippo-campus, MDA levels in the hippocampus were high in diabetic rats,when compared to control rats. The possibility that reactive oxygenspecies (ROS) can influence the expression and/or degradation ofNMDA receptor subunits is likely. However, it has been shown thatNR2B mRNA levels in the hippocampus of diabetic rats were re-duced, when compared with control rats (Di Luca et al., 1999).

Long-term potentiation (LTP), the most intensively studied cellu-lar and molecular model for learning and memory, is generally de-pendent on NMDA receptor activation in the hippocampus (Klann,1998; Kanterewicz, Knapp, & Klann, 1998). Induction of LTP mthe hippocampus is generally dependent on postsynaptic Ca2+ influx,after the activation of NMDA receptors. Furthermore, NMDA re-ceptor stimulation has been shown to produce ROS, including thesuperoxide (O

2–.), in hippocampal slices (Bindokas et al., 1996; Lafon-

Cazal et al., 1993). It was reported that ROS might modulate theactivity of protein kinases and phosphtases during LTP (Klann &Thiels, 1999). Taken together, these data suggest the possibility thatROS may be required for the NMDA receptor-dependent pathwaysin the hippocampus. However, prolonged activation of NMDA re-ceptors under pathological conditions, such as cerebral ischemia andtraumatic injury, causes neuronal cell death (Choi & Rothman, 1990).It has been hypothesized that NMDA receptor-mediated excitotoxicitymay contribute to the etiology or progression of numerous neurode-generative diseases (Heintz & Zoghbi, 2000). Excitotoxicity refersto the excessive activation of glutamate receptors that result in neu-ronal death. Growing data implicate oxidative stress as a mediator

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Effects of Insulin and Gliclazide Treatments 399

of excitotoxic cell death (Coyle & Puttfarcken, 1993). In addition,free radicals themselves can increase the release and decrease there-uptake of glutamate, thus, leading to increased glutamate in thesynaptic cleft (Volterra et al., 1994). As a result, ROS are closelyrelated with NMDA receptor functions.

Di Luca et al. (1999) have shown that the NR2B subunit of theNMDA receptor was reduced in the hippocampus of streptozotocin-diabetic rats. Insulin intervention partially restored NR2B levels.We found a decrease in both NR2A and NR2B concentration instreptozotocin-diabetic rat hippocampi. Our results showed that bothinsulin and gliclazide restore the NR2A and NR2B. It has beensuggested that gliclazide recovered diabetic endothelial dysfunctionby its antioxidant, but not metabolic properties (Vallejo et al., 2000).Mamputu and Renier (2002) have reported that gliclazide has aninhibitor effect on AGE production. Okuda et al. (2002) have re-ported that gliclazide potentialized the effects of insulin on glucosemetabolism. Gliclazide has antioxidant properties, but insulin hasnot. But, two agents reduced MDA levels similarly. Gliclazide mayhave shown this result via the antioxidant effect, in addition to itsantihyperglcaemic effect. It was thought that the effect of insulin onMDA levels might be due to the control of blood glucose levels.

Diabetes is known to induce alterations of second messenger sys-tems, such as cAMP, phosphoinositide, and protein kinases, such ascalcium calmodulin dependent protein kinase (CAMK II) and pro-tein kinase C (PKC). Moreover, diabetic pathology is known toproduce a dysfunction in calcium homeostasis (Biessels et al., 2002).Calcium increase can cause free radical production. Therefore, MDAincrease may occur in diabetic patients.

The increase in ROS and decrease in NMDA receptor subunitsmay be due to the lack of insulin. However, the similar changes inMDA and NMDA receptor subunits, with the treatments of bothinsulin and gliclazide, may imply the effect of hyperglycemic con-trol. In the beginning, the lack of insulin may cause a decrease inNMDA receptor subunits, and these changes in composition maylead to an increase in ROS. However, increase in ROS, induced bydiabetes mellitus, might also cause NMDA receptor changes. Gliclazidetreatment both decreased MDA and increased NMDA receptor. Anotherpossibility is that insulin can also act via decreasing blood glucose

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levels, resulting in reduced MDA. But, different effects of insulincan be expected, due to it having different pathways, in addition toglucoregulation in brain. These changes may be independent of orsecondary to a glucoregulatory effect of insulin. Diabetes and itstreatment with insulin are likely to affect cerebral insulin levels andinsulin signaling. It is difficult, however, to separate these “direct”effects of alterations in insulin homeostasis on the brain from theconsequences of the accompanying alterations in peripheral and centralglucose homeostasis, which can affect the brain.

In conclusion, both insulin and gliclazide were decreased in hip-pocampal MDA levels and similarly restored NR2A and NR2B con-centrations in STZ-diabetic rats. We thought that a decrease in NMDAreceptor subunits might occur as a result of diabetic state. Freeradical production also is related with hyperglcemia. The effect oflowering blood glucose levels on lipid peroxidation seems to beeffective, as much as antioxidant treatment. It can be thought thatgliclazide, which is an antidiabetic and antioxidant agent, may beadded to insulin for decreasing the dose of insulin. Changes in NMDAreceptor subunit composition may have a role in cognitive dysfunc-tions, as shown in patients with diabetes mellitus. Insulin and anti-oxidant treatment partially restores NMDA receptor subunit decrease.

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