Intern. J. Neuroscience, 114:391–401, 2004 Copyright Taylor & Francis Inc. ISSN: 0020-7454 / 1543-5245 online DOI: 10.1080/00207450490270893
NMDA RECEPTOR SUBUNITS 2A AND 2B DECREASE AND LIPID PEROXIDATION INCREASE IN THE HIPPOCAMPUS OF STREPTOZOTOCIN-DIABETIC RATS: EFFECTS OF INSULIN AND GLICLAZIDE TREATMENTS NAMIK DELIBAS IBRAHIM KILINC ZAFER YONDEN RECEP SUTCU FATIH GULTEKIN HALIS KOYLU Suleyman Demirel University Faculty of Medicine Department of Biochemistry and Physiology Isparta, Turkey Recent studies indicate that diabetes mellitus changes N-methyl-D-aspartate (NMDA) receptor subunit composition and impairs cognitive functions. It also has been known that diabetes mellitus causes lipid peroxidation. This study examined the effects of streptozotocin-diabetes and insulin or gliclazide treatment on the hippocampal NMDA receptor subunit 2A and 2B (NR2A and NR2B) concentrations. In addition, malondialdehyde (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 gliclazide treatment partially prevented the reduction of NR2A and NR2B expression and prevented the elevation of MDA levels. There was no significant 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:
[email protected]
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N. Delibas et al. suggest that the elevation of lipid peroxidation can be the primary biochemical disturbances in diabetes progression, and that changes in NMDA receptor subunit compositions can be involved in cognitive decline in diabetes. Keywords diabetes, hippocampus, insulin, lipid peroxidation, NMDA receptors, streptozotocin
Diabetes is a state of increased oxidant stress, and there is accumulating evidence that oxidative damage may play a role in the development of diabetic complications. During hyperglycemia, an increased glucose load leads to elevated nonenzymatic glycosylation, an irreversible cross-linking of certain proteins and lipid peroxidation (Makar et al., 1995). Diabetes mellitus is known to be associated with neurological complications in both the peripheral and the central nervous system (CNS). Manifestations of cerebral disorders in diabetic patients include alterations in neurotransmission, electrophysiological abnormalities, structural changes, and cognitive deficits (Volterra et al., 1994; Biessels et al., 1994). In animal models of diabetic pathology, such as the streptozotocin-diabetic rat, spatial learning impairments have been reported (Biessels et al., 1998). This cognitive deficit is associated with changes in hippocampal synaptic plasticity, including an impaired expression of long-term potentiation (LIP) (Biessels et al., 1998; Chabot et al., 1997) and an enhanced expression of long-term depression (LTD) (Kamal et al., 1999). It is known that excitatory transmission in the brain is largely mediated by glutamate, acting through different classes of receptors: ionotropic and metabotropic. In excitatory synapses the frequency-dependent Ca2+ 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 three classes of subunits, NR1, NR2, and NR3. Four separate genes encode NR2 subunits, NR2A to NR2D (Monyer, Sprengel, & Schoepfer, 1992). This receptor is involved in a wide variety of processes in the CNS, including synaptogenesis and synaptic plasticity. Additionally, 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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species (ROS). The NMDA receptor stimulation has been shown to produce ROS, including the superoxide (O2–.), in hippocampal slices (Bindokas et al., 1996; Lafon-Cazal et al., 1993). The subunit composition largely affects the pharmacological and physiological characteristics of NMDA receptors (Monyer et al., 1994). Thus, a greater understanding of the modulation of this receptor is likely to be important in understanding the physiology and pathophysiology of these processes. In this study, we investigated the effect of two different theropatic agents, namely insulin and gliclazide, on the hippocampal NMDA receptor subunit composition in STZ-diabetic rats. In addition, how these antidiabetic agents affected lipid peroxidation was also studied.
MATERIALS AND METHODS Animals Spraque Dawley rats (240–250 g body weight) were housed (2 rats per 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 and weighed weekly. Diabetes mellitus was induced by a single intraperitoneal injection of streptozotocin (STZ) (Sigma Chemical Co., St. Louis, MO, USA) at a dose of 30 mg/kg body weight dissolved 1% citrate buffer (pH 4.5) after overnight fasting. Four days after the 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-injected animals. In insulin and gliclazide-treated rats, blood glucose levels were measured every week. The experiments reported here complied with the current laws and regulations of the Turkish Republic on 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-treated diabetic group (INS; n = 6); (4) gliclazide treated diabetic group (GLC; n = 6). Insulin and gliclazide treatments were initiated directly after confirmation of diabetes and continued throughout the
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experiment. Insulin (Lilly, USA) was given through subcutaneous injection 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 hippocampus were measured in week 9 after diabetes induction. Lipid Peroxidation Assay After sacrificing the animal, the brain was removed and both hippocampi were then dissected, washed in ice-cold phosphate buffered saline (PBS), and frozen immediately in a deep freezer, until further use. One of them was homogenized (1/10, w/v) in a glass-teflon homogenizer in an ice-cold buffer (0.05 M potassium phosphate buffer, pH 7.8). The homogenate was centrifuged at 10,000 × g for 15 min at 4°C and used for determining the MDA concentration. MDA, an end product of lipid peroxidation, was assayed using the method of Drapper and Hadley (1990). Protein in tissue homogenate was assayed by the Lowry et al.’s method (1951). Data were given as mean ± standard deviation. Statistical analysis of data was performed on the computer, using the SPSS Version 10.0. The Kruskal Wallis test was used for the comparison of the four groups. If a difference was detected using Kruskal Wallis test, Bonferroni-corrected MannWhitney U test was used to find out which two groups were the causes of this difference. Significant level was set at p < .05. Western Blot Analyses Antibodies against NR2A and NR2B were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were of analytical grade or the highest grade available. The other hippocampi (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 mM EDTA, 1 mM EGTA, 25 µg/ml leupeptin, 25 µg/ml aprotinin, 1 mM sodium orthovanadate, 10 µM benzamidine, and 4 mM p-nitrophenyl phosphate], and an aliquot was taken for protein determination. Equal amounts of protein for each sample (20 µg of protein per lane) were separated by SDS/PAGE on 7.5% minigels, blotted electrophoretically to immobilon membrane, and incubated in tris-buffered saline
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with Tween 20 (TBST) [50 mm Tris-HCl (pH 7.5-8.0), 150 mM NaCl, 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 subjected to three additional 10-min washings in TBST. Blots were incubated with alkaline phosphatase conjugated monoclonal antirabbit IgG (1:10000) in 1% BSA for lb at room temperature, and three 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 were analyzed with a computerized image analysis system (Uviphoto MW V.99, Ultra-Violet Products Ltd, Cambridge, UK). SDS-PAGE and Western blot analyses were done on three independent hippocampus preparations (3 animals/group). The data were subjected to a one-way analysis of variance (ANOVA) with subsequent group comparisons using the Dunnett test at the 0.05 level of significance.
RESULTS Comparisons of the groups were given in Table 1. As shown in Table 1, diabetes mellitus caused an increase in MDA levels. Both insulin and gliclazide treatments successfully reduced MDA levels to 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 Western blot analyses were done on hippocampal homogenates. Western blot analysis of NR2A and NR2B were shown in Figures 1 and 2, respectively. The density of the protein band in the control group was accepted as 100%, and data from other groups were calculated as TABLE 1. Comparison of the groups C MDA (nmol/mg protein)
17 ± 1a
DM
INS
GLC
23 ± 2b
18 ± 0.6a
18 ± 0.8a
a,b p < .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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FIGURE 1. Representative Western blot of NR2A in hippocampus from four groups of rats. 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 treatment group. The asterisk indicates significant changes compared to other groups, using an ANOVA with subsequent group comparisons, using the Dunnett test at the 0.05 level of significance.
percentages of the control value. The Western blot analysis for NR2A and NR2B showed a significant (n = 3, p < .05, for both) reduction of approximately 53 and 45% in diabetic rats, compared with control rats, respectively. We then tested the possible effect of insulin and gliclazide treatment on the NMDA receptor subunit concentrations. The insulin and gliclazide treatment partially restored NR2A and NR2B concentrations towards control values (Figures 1 and 2). There was no significant difference between the two treatment regimens (n = 3, p > .05).
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FIGURE 2. Representative Western blot of NR2B in hippocampus from four groups of rats. 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 treatment group. The asterisk indicates significant changes compared to other groups using an ANOVA with subsequent group comparisons using the Dunnett test at the 0.05 level of significance.
DISCUSSION The main findings of this study were that 8 weeks of STZ-diabetes reduced hippocampal NR2A and NR2B subunit protein concentrations and increased hippocampal lipid peroxidation, compared to controls. Insulin and gliclazide treatments partially prevented NR2A and NR2B expression and effectively reduced lipid peroxidation. The question of how chronic diabetic pathology could change the expression of a specific NMDA receptor subunit is currently under investigation. It has been suggested that growth factors can
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influence the expression of NMDA receptor subunits. A decrease in insulin-like growth factor-I (IGF-1) and IGF-2 has been shown in diabetic patients and in STZ-diabetic rats (Brewster et al., 1994). In this respect, the possibility that IGF-/IGF-2 might influence the activity of the promoter of different NR2 subunits is particularly intriguing. It observed that after a prolonged treatment of diabetic rats with insulin, the concentration of NR2A and NR2B protein is partially restored, possibly supporting such a hypothesis. We also found a similar result in gliclazide treatment. It was thought that other factor(s) should be considered. Is there a relation between hippocampal MDA levels and NR subunit expression rate? Concomitantly of the reduced NR2A and NR2B concentration in the hippocampus, MDA levels in the hippocampus were high in diabetic rats, when compared to control rats. The possibility that reactive oxygen species (ROS) can influence the expression and/or degradation of NMDA receptor subunits is likely. However, it has been shown that NR2B mRNA levels in the hippocampus of diabetic rats were reduced, when compared with control rats (Di Luca et al., 1999). Long-term potentiation (LTP), the most intensively studied cellular and molecular model for learning and memory, is generally dependent on NMDA receptor activation in the hippocampus (Klann, 1998; Kanterewicz, Knapp, & Klann, 1998). Induction of LTP m the hippocampus is generally dependent on postsynaptic Ca2+ influx, after the activation of NMDA receptors. Furthermore, NMDA receptor stimulation has been shown to produce ROS, including the superoxide (O2–.), in hippocampal slices (Bindokas et al., 1996; LafonCazal et al., 1993). It was reported that ROS might modulate the activity of protein kinases and phosphtases during LTP (Klann & Thiels, 1999). Taken together, these data suggest the possibility that ROS may be required for the NMDA receptor-dependent pathways in the hippocampus. However, prolonged activation of NMDA receptors under pathological conditions, such as cerebral ischemia and traumatic injury, causes neuronal cell death (Choi & Rothman, 1990). It has been hypothesized that NMDA receptor-mediated excitotoxicity may contribute to the etiology or progression of numerous neurodegenerative diseases (Heintz & Zoghbi, 2000). Excitotoxicity refers to the excessive activation of glutamate receptors that result in neuronal death. Growing data implicate oxidative stress as a mediator
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of excitotoxic cell death (Coyle & Puttfarcken, 1993). In addition, free radicals themselves can increase the release and decrease the re-uptake of glutamate, thus, leading to increased glutamate in the synaptic cleft (Volterra et al., 1994). As a result, ROS are closely related with NMDA receptor functions. Di Luca et al. (1999) have shown that the NR2B subunit of the NMDA receptor was reduced in the hippocampus of streptozotocindiabetic rats. Insulin intervention partially restored NR2B levels. We found a decrease in both NR2A and NR2B concentration in streptozotocin-diabetic rat hippocampi. Our results showed that both insulin and gliclazide restore the NR2A and NR2B. It has been suggested that gliclazide recovered diabetic endothelial dysfunction by its antioxidant, but not metabolic properties (Vallejo et al., 2000). Mamputu and Renier (2002) have reported that gliclazide has an inhibitor effect on AGE production. Okuda et al. (2002) have reported that gliclazide potentialized the effects of insulin on glucose metabolism. Gliclazide has antioxidant properties, but insulin has not. But, two agents reduced MDA levels similarly. Gliclazide may have shown this result via the antioxidant effect, in addition to its antihyperglcaemic effect. It was thought that the effect of insulin on MDA levels might be due to the control of blood glucose levels. Diabetes is known to induce alterations of second messenger systems, such as cAMP, phosphoinositide, and protein kinases, such as calcium calmodulin dependent protein kinase (CAMK II) and protein kinase C (PKC). Moreover, diabetic pathology is known to produce a dysfunction in calcium homeostasis (Biessels et al., 2002). Calcium increase can cause free radical production. Therefore, MDA increase may occur in diabetic patients. The increase in ROS and decrease in NMDA receptor subunits may be due to the lack of insulin. However, the similar changes in MDA and NMDA receptor subunits, with the treatments of both insulin and gliclazide, may imply the effect of hyperglycemic control. In the beginning, the lack of insulin may cause a decrease in NMDA receptor subunits, and these changes in composition may lead to an increase in ROS. However, increase in ROS, induced by diabetes mellitus, might also cause NMDA receptor changes. Gliclazide treatment both decreased MDA and increased NMDA receptor. Another possibility is that insulin can also act via decreasing blood glucose
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levels, resulting in reduced MDA. But, different effects of insulin can be expected, due to it having different pathways, in addition to glucoregulation in brain. These changes may be independent of or secondary to a glucoregulatory effect of insulin. Diabetes and its treatment with insulin are likely to affect cerebral insulin levels and insulin signaling. It is difficult, however, to separate these “direct” effects of alterations in insulin homeostasis on the brain from the consequences of the accompanying alterations in peripheral and central glucose homeostasis, which can affect the brain. In conclusion, both insulin and gliclazide were decreased in hippocampal MDA levels and similarly restored NR2A and NR2B concentrations in STZ-diabetic rats. We thought that a decrease in NMDA receptor subunits might occur as a result of diabetic state. Free radical production also is related with hyperglcemia. The effect of lowering blood glucose levels on lipid peroxidation seems to be effective, as much as antioxidant treatment. It can be thought that gliclazide, which is an antidiabetic and antioxidant agent, may be added to insulin for decreasing the dose of insulin. Changes in NMDA receptor subunit composition may have a role in cognitive dysfunctions, as shown in patients with diabetes mellitus. Insulin and antioxidant treatment partially restores NMDA receptor subunit decrease.
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