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. 2007 Sep 6;27(8):1085–1095. doi: 10.1007/s10571-007-9198-1

Enhanced [3H] Glutamate Binding in the Cerebellum of Insulin-Induced Hypoglycaemic and Streptozotocin-Induced Diabetic Rats

Anu Joseph 1, Remya Robinson 1, C S Paulose 1,✉
PMCID: PMC11517331  PMID: 17805958

Abstract

Aim Energy deprivation causes neuronal death affecting the cognitive and memory ability of an individual. The kinetic parameters of glutamate dehydrogenase (GDH), the enzyme involved in the production of glutamate, was studied in the cerebellum and liver and the binding parameters of glutamate receptors in the cerebellum of insulin-induced hypoglycaemic and streptozotocin-induced diabetic rats were studied to reveal the role of glutamate excitotoxicity. Methods A single intrafemoral dose of streptozotocin was administered to induce diabetes. Hypoglycaemia was induced by appropriate doses of insulin subcutaneously in control and diabetic rats. The kinetic parameters Vmax and Km of GDH were studied spectrophotometrically at different substrate concentrations of α-ketoglutarate. Glutamate receptor binding assay was done with different concentrations of [3H] Glutamate. Results The GDH enzyme assay showed a significant increase (P < 0.001) in the Vmax of the enzyme in the cerebellum of hypoglycaemic and diabetic rat groups when compared to control. The Vmax of hypoglycaemic groups was significantly increased (P < 0.001) when compared to diabetic group. In the liver, the Vmax of GDH was significantly increased (P < 0.001) in the diabetic and diabetic hypoglycaemia group when compared to control. The Vmax of GDH increased significantly (P < 0.001) in the diabetic hypoglycaemic rats compared to diabetic group, whereas the control hypoglycaemic rats showed a significant decrease in Vmax (P < 0.001) when compared to diabetic and diabetic hypoglycaemic rats. The Km showed no significant change amongst the groups in cerebellum and liver. Scatchard analysis showed a significant increase (P < 0.001) in Bmax in the cerebellum of hypoglycaemic and diabetic rats when compared to control. The Bmax of hypoglycaemic rats significantly increased (P < 0.001) when compared to diabetic group. In hypoglycaemic groups, Bmax of the control hypoglycaemic rats showed a significant increase (P < 0.001) compared to diabetic hypoglycaemic rats. The Kd of the diabetic group decreased significantly (P < 0.01) when compared to control and control hypoglycaemic rats. There was a significant decrease (P < 0.05) in the Kd of diabetic hypoglycaemic group when compared to the control hypoglycaemic rats. Conclusion Our studies demonstrated the increased enzyme activity in the hypoglycaemic rats with increased production of extracellular glutamate. The present study also revealed increased binding parameters of glutamate receptors reflecting an increased receptor number with increase in the affinity. This increased number of receptors and the increased glutamate production will lead to glutamate excitotoxicity and neuronal degeneration which has an impact on the cognitive and memory ability. This has immense clinical significance in the management of diabetes and insulin therapy.

Keywords: Hypoglycaemia, Diabetes, Glutamate dehydrogenase, Glutamate receptors, Cerebellum, Liver

Introduction

Hypoglycaemia is a collection of symptoms brought about by an abnormally low plasma glucose level. The brain and other tissues require glucose in order to function properly. Studies suggest that acute or chronic hypoglycaemia leads to neurological dysfunction and injury. Children and adults exposed to hypoglycaemia can develop long-term impairment of cognitive function (Blattener 1968; Hawdon 1999; Karp 1989; Ryan et al. 1985; Vannucci and Vannucci 2001) and are at risk of epilepsy (Kaufman 1998). Hypoglycaemia-induced brain injury is a significant obstacle to optimal blood glucose control in diabetic patients. Prolonged insulin-induced hypoglycaemia causes widespread loss of neurons and permanent brain damage with irreversible coma. As in brain injury associated with ischaemia and neurodegenerative conditions, altered neurotransmitter action appears to play a role in hypoglycaemic brain injury (Aral et al. 1998; Auer 1991; Auer and Seisjo 1993). Attention has been focussed on glutamate as a potential mediator of hypoglycaemic brain injury (Aral et al. 1998; Cavaliere et al. 2001; Marinelli et al. 2001). Severe hypoglycaemia triggers a cascade of events in vulnerable neurons that may culminate in cell death even after glucose normalization (Sang et al. 2003, 2004, 2005, 2007). Glutamate receptor activation and excitotoxicity has long been recognized as an upstream event in this cascade (Weiloch 1985). In brain, glutamate accumulation is reported to cause neuronal degeneration (Atlante et al. 1997; Berman and Murray 1996; Budd and Nicholas 1996).

Glutamate dehydrogenase (EC 1.4.1.3) (GDH) is important in normal glucose homeostasis. Glutamate dehydrogenase, a key enzyme in metabolic interconversions of amino acids and carbohydrates, is present in both nuclear and mitochondrial fractions and has different functions (Hogeboom and Schneider 1953; Christie and Judah 1953; Beaufay et al. 1959; di Prisco et al. 1968; di Prisco and Harold 1970). GDH has been found in several mammalian tissues including liver, brain, kidney, heart, pancreas, ovaries and lymph nodes (Plaitakis et al. 1984). The importance of GDH in normal glucose homeostasis in humans is also evident from the findings that mutations in the GLUD1 gene, which encodes GDH, cause hyperinsulinism/hyperammonemia syndrome (Stanley et al. 1998, 2000; MacMullen et al. 2001). Glutamate dehydrogenase is found in significant amounts in the liver (Greenfield and Boell 2005). Developmental changes of GDH in rat liver were reported by Sokal et al. (1989). In nervous tissue, GDH appears to function in both the synthesis and the catabolism of glutamate and perhaps in ammonia detoxification (Mavrothalassitis et al. 1988). Glutamate triggers neuronal death when released in excessive concentrations by over excitation of its receptors (Vizi 2000). The extracellular accumulation of glutamate results in neuronal death by activating ionotropic glutamate receptors sensitive to NMDA or AMPA-kainite (Choi 1988).

The cerebellum is known to be resistant to hypoglycaemia, and selective cerebellar dysfunction caused by hypoglycaemia has not been reported. In a case of episodic bilateral cerebellar dysfunction caused by hypoglycaemia, quantitative dynamic PET study demonstrated decreased glucose uptake-to-utilization ratio and increased leak of glucose in the cerebellum indicating that cerebellum is not invariably resistant to hypoglycaemia (Kim et al. 2005). In the present study the kinetic parameters of glutamate dehydrogenase (GDH) in the cerebellum and liver and the binding parameters of glutamate receptors in the cerebellum of insulin-induced hypoglycaemic and streptozotocin-induced diabetic rats were investigated.

Materials and Methods

Animals

Adult male Wistar rats of 200–250 g body weight were purchased from Kerala Agriculture University, Mannuthy and Amrita Institute of Medical Sciences, Kochi and used for all experiments. They were housed in separate cages under 12-h light and 12-h dark periods and were maintained on standard food pellets and water ad libitum.

Induction of Diabetes and Hypoglycaemia

Animals were divided into the following groups as (i) control [C], (ii) diabetic [D], (iii) insulin-induced hypoglycaemia in diabetic rats [diabetic + IIH] and (iv) insulin-induced hypoglycaemia in control rats [control + IIH]. Each group consisted of 4–6 animals. Diabetes was induced by a single intrafemoral dose (55 mg/kg body weight) of STZ prepared in citrate buffer, pH 4.5 (Hohenegger and Rudas 1971; Arison et al. 1967). The diabetic + IIH group received daily 2 doses (10 Unit/kg body weight) and control + IIH group received daily 2 doses (1.5 Unit/kg body weight) of regular human insulin (Actrapid) (Flanagan et al. 2003). Diabetic + IIH and control + IIH group had daily two episodes of insulin-induced hypoglycaemia for 10 days. Control rats were injected with citrate buffer.

Tissue Preparation

Rats were sacrificed by decapitation on the 10th day of the experiment. The cerebellum and liver were dissected out quickly over ice according to the procedure of Glowinski and Iversen (1966). The tissues were stored at −70°C until assay.

Protein Determination

Protein was measured by the method of Lowry et al. (1951) using bovine serum albumin as standard.

Glutamate Dehydrogenase Assay

Glutamate dehydrogenase activity was measured in the crude extract of cerebellum and liver according to the modified procedure of Gyan and Kanungo (1970). Sample extracts were prepared by making 5% homogenate of the tissue in cold 0.04 M triethanolamine (TE) buffer and the supernatant was collected after centrifugation at 10,000 × g for 20 min. The enzyme activity was measured in supernatant. The reaction mixture in the experimental and reference cuvettes contain 0.04 M TE buffer pH 8.0, 2.6 mM EDTA, 105 mM ammonium acetate and 100 μl of the enzyme sample extract of appropriate concentrations. The reaction mixture of 1.0 ml volume was assayed at 366 nm using Milton Roy Genesis spectrophotometer by adding saturating concentrations of α-ketoglutarate and 10 mM NADH. Decrease in optical density (OD) due to the oxidation of NADH was measured at 15 s intervals for 1 min at room temperature. The decrease in absorbance was linear during the course of the assays. One unit of enzyme activity is equal to the change in OD of 0.1 in 100 s at 366 nm. Activity of enzyme was expressed as specific activity represented by Units/mg protein. Kinetic parameters V max and K m were calculated from the data of GDH activity measured at substrate concentrations of 0.5–6 mM of α-ketoglutarate.

Glutamate Receptor Analysis

Glutamate receptor binding assay was done according to the modified procedure of Timothy et al. (1984). The cerebellum was homogenized in 20 volumes of 0.32 M sucrose, 1 mM MgCl2 and 10 mM Tris–HCl, pH 7.4. The homogenate was centrifuged twice at 1,000 × g for 15 min and the pellets were discarded. The supernatant was pooled and centrifuged at 27,000 × g for 15 min. The pellet was lysed in 10 mM Tris–HCl, pH 7.4 for 30 min and centrifuged at 27,000 × g for 15 min. The resulting pellet was resuspended in 25 mM Tris–HCl, pH 7.4 and 5 mM MgCl2 and was used for the binding assay.

Glutamate receptor binding assay was done using different concentrations i.e., 20–350 nM of [3H] Glutamate in the incubation buffer, containing 25 mM Tris–HCl and 5 mM MgCl2, pH 7.4 in a total volume of 250 μl containing (0.2–0.3 mg) protein concentrations. Non-specific binding was determined using 500 μM unlabelled glutamate. The incubation was carried out at 30°C for 30 min and the reaction was stopped by centrifugation at 27,000 × g for 15 min. The pellet and the wall of the tubes were washed with ice-cold distilled water to remove unbound radioactive ligand. An aliquot of 50 μl of 1 M KOH was added and kept for overnight digestion. Bound radioactivity was counted with cocktail-T in a liquid scintillation counter (Wallac 1409). The non-specific binding determined showed 30–40% in all our experiments.

Analysis of the Receptor Binding Data

The linear regression data were analysed according to Scatchard (1949). The maximal binding (B max) and equilibrium dissociation constant (K d) were derived by plotting the specific binding of the radioligand on X-axis and bound/free on Y-axis using SIGMA PLOT (Ver. 2.03).

Statistical Analysis

Statistical evaluations were done with analysis of variance (ANOVA), using InStat (Ver. 2.04a).

Results

Glutamate Dehydrogenase Activity was Increased in the Cerebellum of Diabetic, Diabetic + IIH, Control + IIH Rats

The activity of GDH was increased in both hypoglycaemic and hyperglycaemic cerebellum of rats. Our results showed a significant increase (P < 0.001) in the V max of hypoglycaemic and diabetic rat groups when compared to control (Table 1). The V max of the enzyme in the cerebellum of hypoglycaemic rats was significantly increased (P < 0.001) when compared to diabetic group (Table 1). The K m value did not show any significant change in all experimental groups indicating that there is no change in the affinity of the enzyme (Table 1).

Table 1.

Kinetic parameters—V max and K m of glutamate dehydrogenase in the cerebellum of control, streptozotocin-induced diabetic and insulin-induced hypoglycaemic rats

Animal status Vmax (Units/mg protein) Km (mM)
Control 5.4 ± 0.12 0.5 ± 0.05
Diabetic 6.8 ± 0.11*** 0.5 ± 0.03
Diabetic + IIH 9.1 ± 0.09***††† 0.5 ± 0.03
Control + IIH 9.8 ± 0.08***††† 0.4 ± 0.03
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Values are mean ± SEM of 4–6 separate experiments. ***(P < 0.001) when compared to control, †††(P < 0.001) when compared to diabetic. IIH—Insulin-induced hypoglycaemia. The GDH activity in the cerebellum of hypoglycaemic and hyperglycaemic rats was measured in reaction mixture of 1 ml volume at 366 nm using Milton Roy Genesis spectrophotometer by adding substrate concentrations of 0.5–6 mM and 10 mM NADH

Glutamate Dehydrogenase Activity was Increased in the Liver of Diabetic, Diabetic + IIH and Decreased in Control + IIH Rats

In the liver, the V max of GDH was significantly increased (P < 0.001) in the diabetic and diabetic hypoglycaemic group when compared to control (Table 2). The V max of GDH increased significantly (P < 0.001) in the diabetic hypoglycaemic rats compared to diabetic group, whereas the control hypoglycaemic rats showed a significant decrease in V max (P < 0.001) when compared to diabetic and diabetic hypoglycaemic rats (Table 2). The K m showed no significant change amongst the groups (Table 2).

Table 2.

Kinetic parameters—V max and K m of glutamate dehydrogenase in the liver of control, streptozotocin-induced diabetic and insulin-induced hypoglycaemic rats

Animal status Vmax (Units/mg protein) Km (mM)
Control 17.60 ± 0.20 0.4 ± 0.10
Diabetic 22.73 ± 0.14*** 0.4 ± 0.10
Diabetic + IIH 29.76 ± 0.12***††† 0.3 ± 0.03
Control + IIH 15.78 ± 0.39††† 0.6 ± 0.06
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Values are mean ± SEM of 4–6 separate experiments. ***(P < 0.001) when compared to control, †††(P < 0.001) when compared to diabetic. IIH—Insulin-induced hypoglycaemia. The GDH activity in the liver of hypoglycaemic and hyperglycaemic rats was measured in reaction mixture of 1 ml volume at 366 nm using Milton Roy Genesis spectrophotometer by adding substrate concentrations of 0.5–6 mM and 10 mM NADH

Glutamate Receptors in the Cerebellum were Increased in the Diabetic, Diabetic + IIH and Control + IIH Rats

Scatchard analysis showed a significant increase (P < 0.001) in B max in the cerebellum of hypoglycaemic and diabetic rats when compared to control (Table 3; Fig. 1). The B max of hypoglycaemic rats significantly increased (P < 0.001) when compared to diabetic group (Table 3; Fig. 1). In hypoglycaemic groups, B max of the control hypoglycaemic rats showed a significant increase (P < 0.001) compared to diabetic hypoglycaemic rats (Table 3; Fig. 1). The K d of the diabetic group decreased significantly (P < 0.01) when compared to control and control hypoglycaemic rats (Table 3; Fig. 1). There was a significant decrease (P < 0.05) in the K d of diabetic hypoglycaemic group when compared to the control hypoglycaemic rats. This decrease in K d with an increase in B max reflected an increased affinity of glutamate receptors for glutamate with increase in receptor number.

Table 3.

[3H] Glutamate binding parameters in the cerebellum of control, streptozotocin-induced diabetic and insulin-induced hypoglycaemic rats

Animal status Bmax (fmol/mg protein) Kd (nM)
Control 1003.3 ± 3.3 147.67 ± 0.88
Diabetic 1493.3 ± 3.4*** 138.70 ± 1.85**
Diabetic + IIH 1726.7 ± 6.7***†††¶¶¶ 143.30 ± 0.90
Control + IIH 1826.4 ± 5.2***††† 151.33 ± 1.86††
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Values are mean ± SEM of 4–6 separate experiments. **(P < 0.01), ***(P < 0.001) when compared to control, ††(P < 0.01) †††(P < 0.001) when compared to diabetic, (P < 0.05), ¶¶¶(P < 0.001) when compared to control + IIH. IIH—Insulin-induced hypoglycaemia. B max—Binding maximum (fmol/mg protein), K d—dissociation constant (nM). Scatchard analysis of [3H] Glutamate against glutamate in the cerebellum of control, streptozotocin-induced diabetic and insulin-induced hypoglycaemic rats. Incubation was done with 40–200 nM of [3H] Glutamate in a total incubation volume of 250 μl. An aliquot of 500 μM glutamate was used to determine the specific binding. The reaction was stopped by centrifugation at 27,000 × g for 15 min

Fig. 1.

Fig. 1

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Scatchard analysis of [3H] Glutamate against glutamate in the cerebellum of control, streptozotocin-induced diabetic and insulin-induced hypoglycaemic rats (a representative graph)

Discussion

Hypoglycaemia is the major obstacle to optimal blood glucose control in diabetic patients. Clinically, hypoglycaemic unawareness is a complication of diabetes, especially in aged patients with cognitive deficits (Widom and Simonson 1994; McCall 1992; Ott et al. 1999). Severe hypoglycaemia triggers a cascade of events in vulnerable neurons that culminate in cell death even after glucose normalization (Sang et al. 2003, 2004, 2005). Hypoglycaemia is associated with increased glutamate release (Sandberg et al. 1986) and conversely, glutamate toxicity is augmented by hypoglycaemia (Novelli et al. 1988). Exposure to acute hypoglycaemia in newborn piglets showed increased glutamate binding sites of cerebral NMDA receptors (McGowan et al. 2002). Deficits in long-term potentiation during chronic diabetes arise from dysfunction of the NMDA subtype of glutamate receptors in early stages of the disease (Trudeau et al. 2004). Recent studies reported that abnormal expression of NMDA receptor is involved in the development of diabetic neuropathy (Tomiyama et al. 2005). We studied the activity of glutamate dehydrogenase and the glutamate receptor binding parameters in the cerebellum of hypoglycaemic and hyperglycaemic rats. GDH induces an increase in the extracellular glutamate levels in the central nervous system with subsequent development of excitotoxicity (Kostic et al. 1989). Malfunction of glutamate, a major excitatory transmitter in the brain, has been implicated in psychiatric disorders such as schizophrenia, drug addiction and depression (Pomara et al. 1992; Perry and Hansen 1990; Plaitakis et al. 1988; Rothstein et al. 1996). Studies have reported that up regulation of glutamate receptors and calcium-binding proteins in the diabetic retina (Ng et al. 2004). The brain is dependent on glucose for oxidative metabolism and function. In Type I diabetes mellitus, acute hypoglycaemia, a result of insulin therapy, can be severe enough to cause cognitive impairment (Rosenthal et al. 1999). Severe hypoglycaemia with brain dysfunction limits intensified therapy in patients with insulin-dependent diabetes mellitus, despite evidence that such therapy reduces the risk of chronic complications of the disease (Maran et al. 1994). It is widely accepted that energy deprivation causes a neuronal death that is mainly determined by an increase in the extracellular level of glutamate (Marinelli et al. 2001). Glutamate which causes excitotoxic neuronal damage, increases calcium influx through NMDA receptors in post synaptic neurons, leading to phospholipase A2 mediated arachidonic acid release (Miriam et al. 1996). The increase in the arachidonic acid in brain may mediate neuronal injury by inhibiting sodium channels (Fraser et al. 1993). Our previous studies also reported that GDH enzyme activity enhanced during diabetes and did not completely reverse even after insulin administration (Preetha et al. 1996; Aswathy et al. 1998). Studies using young and old diabetic rats clearly revealed that GDH activity regulation is essential to avoid diabetic associated brain glutamate toxicity (Biju and Paulose 1998). Although the deprivation of carbohydrate stores affects all brain regions, the breakdown of energy metabolism and cessation of protein synthesis occur predominantly in the cerebral cortex, caudoputamen and hippocampus. The cerebellum, brain stem and hypothalamus are largely resistant. But in a case of episodic bilateral cerebellar dysfunction caused by hypoglycaemia, quantitative dynamic PET study demonstrated decreased glucose uptake-to-utilization ratio and increased leak of glucose in the cerebellum indicating that the cerebellum is not invariably resistant to hypoglycaemia (Kim et al. 2005). Our studies showed an increased activity of GDH and glutamate binding parameters in both hypoglycaemic and hyperglycaemic condition revealing that cerebellum is not resistant to hypoglycaemia. The increased glutamate dehydrogenase activity leads to an increased production of glutamate. In the liver we observed an increase in GDH activity in diabetes and diabetic hypoglycaemic group compared to control, whereas there was a decreased in the GDH activity in the control hypoglycaemic group. Gluconeogenesis is the key process for the normal counterregulatory response to prolonged and marked hypoglycaemia (Frizzell et al. 1988). Hepatic gluconeogenesis was stimulated in response to hypoglycaemia (Magnan et al. 1998). In hypoglycaemia caused by excessive insulin, liver glycogen levels are high, but the high insulin level prevents the glycogenolysis necessary to maintain normal blood sugar levels. In intensively treated type 1 diabetes, hypoglycaemia failed to stimulate hepatic glycogen breakdown (Kishore et al. 2006). In control hypoglycaemic group, the glucose entering the liver cells have a major glycogen synthetic pathway that reduces its utilization via Krebs’s cycle. Thus the decreased α-ketoglutarate formation will be the reason for the decreased GDH activity in control hypoglycaemic rats.

Severe hypoglycaemia causes neuronal death and cognitive impairment. It is found that patients who recover from severe hypoglycaemia are left with difficulties in cognition, particularly short-term memory, out of proportion to gross motor disability (Langan et al. 1991). It has been reported from earlier studies from our lab that the glutamate dehydrogenase activity in the cerebellum of experimentally induced diabetic rats showed a significant increase leading to an increased conversion of α-ketoglutarate to glutamate (Biju and Paulose 1998; Preetha et al. 1996) leading to glutamate toxicity. Studies have reported that rats subjected to severe hypoglycaemia showed deficits in the Morris water maze test, a standard measure of learning and spatial memory (Sang et al. 2005). Evidence suggests that hypoglycaemic neuronal death involves excitotoxicity and DNA damage (Sang et al. 2003). Glutamate activation of neuronal glutamate receptors leads to production of nitric oxide and superoxide, which combine to form peroxynitrite and related species with high reactivity towards DNA and other cell constituents (Zhang et al. 1994; Beckman and Koppenol 1996; Bindokas et al. 1996; Szabo et al. 1996). Our studies on the binding parameters of glutamate receptors in the cerebellum showed an increase in B max with a decreased K d reflecting an increased affinity of glutamate receptors for glutamate with increase in receptor number. This increased number of receptors and the increased glutamate production will lead to glutamate excitotoxicity and neuronal degeneration. Our results showed that the alterations in the glutamate receptors and glutamate dehydrogenase activity is more in the hypoglycaemic than hyperglycaemic rats suggesting that hypoglycaemia cause more functional damage in the brain than hyperglycaemia. This functional damage during hypoglycaemia is suggested to contribute to cognitive and memory deficits. This has immense clinical relevance in the therapeutic management of hyperglycaemia leading to hypoglycaemia.

Acknowledgements

Dr. C. S. Paulose thanks DBT, DST, ICMR. Govt. of India and KSCSTE, Govt. of Kerala for the financial assistance. Remya Robinson thanks Cochin University For JRF.

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