Hypoglycemia induced changes in cholinergic receptor expression in the cerebellum of diabetic rats

Abstract

Glucose homeostasis in humans is an important factor for the functioning of nervous system. Hypoglycemia and hyperglycemia is found to be associated with central and peripheral nerve system dysfunction. Changes in acetylcholine receptors have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). In the present study we showed the effects of insulin induced hypoglycemia and streptozotocin induced diabetes on the cerebellar cholinergic receptors, GLUT3 and muscle cholinergic activity. Results showed enhanced binding parameters and gene expression of Muscarinic M1, M3 receptor subtypes in cerebellum of diabetic (D) and hypoglycemic group (D + IIH and C + IIH). alpha7nAchR gene expression showed a significant upregulation in diabetic group and showed further upregulated expression in both D + IIH and C + IIH group. AchE expression significantly upregulated in hypoglycemic and diabetic group. ChAT showed downregulation and GLUT3 expression showed a significant upregulation in D + IIH and C + IIH and diabetic group. AchE activity enhanced in the muscle of hypoglycemic and diabetic rats. Our studies demonstrated a functional disturbance in the neuronal glucose transporter GLUT3 in the cerebellum during insulin induced hypoglycemia in diabetic rats. Altered expression of muscarinic M1, M3 and alpha7nAchR and increased muscle AchE activity in hypoglycemic rats in cerebellum is suggested to cause cognitive and motor dysfunction. Hypoglycemia induced changes in ChAT and AchE gene expression is suggested to cause impaired acetycholine metabolism in the cerebellum. Cerebellar dysfunction is associated with seizure generation, motor deficits and memory impairment. The results shows that cerebellar cholinergic neurotransmission is impaired during hyperglycemia and hypoglycemia and the hypoglycemia is causing more prominent imbalance in cholinergic neurotransmission which is suggested to be a cause of cerebellar dysfunction associated with hypoglycemia.

Figure 1

Representative graph showing Real Time PCR amplification of muscarinic M1 mRNA from the cerebellum of Control, Diabetic, Diabetic + IIH and Control + IIH Rats. The ΔΔCT method of relative quantification was used to determine the fold change in expression with β-actin CT value as the internal control and Control CT value as the calibrator. C- Control, D- Diabetic, D + IIH - Insulin induced hypoglycemia in diabetic, C + IIH - Insulin induced hypoglycemia in control. Values are mean ± S.D. of 4-6 separate experiments. Each group consisted of 6-8 rats. a p < 0.001 when compared to control, b p < 0.01 when compared to Diabetic, c p < 0.001 when compared to D + IIH.

Figure 2

Representative graph showing Real Time PCR amplification of muscarinic M3 mRNA from the cerebellum of Control, Diabetic, Diabetic + IIH and Control + IIH Rats. The ΔΔCT method of relative quantification was used to determine the fold change in expression with β-actin CT value as the internal control and Control CT value as the calibrator. C- Control, D- Diabetic, D + IIH - Insulin induced hypoglycemia in diabetic, C + IIH - Insulin induced hypoglycemia in control. Values are mean ± S.D. of 4-6 separate experiments. Each group consisted of 6-8 rats. a p < 0.001 when compared to control. b p < 0.001 when compared to Diabetic.

Figure 3

Representative graph showing Real Time PCR amplification of α7nAchR mRNA from the cerebellum of Control, Diabetic, Diabetic + IIH and Control + IIH Rats. The ΔΔCT method of relative quantification was used to determine the fold change in expression with β-actin CT value as the internal control and Control CT value as the calibrator. C- Control, D- Diabetic, D + IIH - Insulin induced hypoglycemia in diabetic, C + IIH - Insulin induced hypoglycaemia in control. Values are mean ± S.D. of 4-6 separate experiments. Each group consisted of 6-8 rats. a p < 0.001 when compared to control, b p < 0.001 when compared to Diabetic, c p < 0.001 when compared to D + IIH.

Figure 4

Representative graph showing Real Time PCR amplification of AchE mRNA from the cerebellum of Control, Diabetic, Diabetic + IIH and Control + IIH Rats. The ΔΔCT method of relative quantification was used to determine the fold change in expression with β-actin CT value as the internal control and Control CT value as the calibrator. C- Control, D- Diabetic, D + IIH - Insulin induced hypoglycemia in diabetic, C + IIH - Insulin induced hypoglycemia in control. Values are mean ± S.D. of 4-6 separate experiments. Each group consisted of 6-8 rats. a p < 0.001 when compared to control, b p < 0.001 when compared to Diabetic, c p < 0.001 when compared to D + IIH.

Figure 5

Representative graph showing Real Time PCR amplification of ChAT mRNA from the cerebellum of Control, Diabetic, Diabetic + IIH and Control + IIH Rats. The ΔΔCT method of relative quantification was used to determine the fold change in expression with β-actin CT value as the internal control and Control CT value as the calibrator. Values are mean ± S.D. of 4-6 separate experiments. Each group consisted of 6-8 rats. a p < 0.001 when compared to control, b p < 0.001 when compared to Diabetic.

Figure 6

Representative graph showing Real Time PCR amplification of GLUT3 mRNA in the cerebellum of Control, Diabetic, Diabetic + IIH and Control + IIH Rats. The ΔΔCT method of relative quantification was used to determine the fold change in expression with β-actin CT value as the internal control and Control CT value as the calibrator. C- Control, D- Diabetic, D + IIH - Insulin induced hypoglycemia in diabetic, C + IIH - Insulin induced hypoglycemia in control. Values are mean ± S.D. of 4-6 separate experiments. Each group consisted of 6-8 rats. a p < 0.001 when compared to control, b p < 0.001 when compared to Diabetic.