Limits...
Enhanced muscarinic M1 receptor gene expression in the corpus striatum of streptozotocin-induced diabetic rats.

Gireesh G, Kumar TP, Mathew J, Paulose C - J. Biomed. Sci. (2009)

Bottom Line: We observed that insulin treatment brought back the decreased maximal velocity (Vmax) of acetylcholine esterase in the corpus striatum during diabetes to near control state.Real-time PCR experiment confirmed the increase in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment.These results suggest the diabetes-induced changes of the cholinergic activity in the corpus striatum and the regulatory role of insulin on binding parameters and gene expression of total and muscarinic M1 receptors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Neurobiology and Cell Biology Unit, Centre for Neuroscience, Cochin University of Science and Technology, Cochin-682022, Kerala, India. giri_ganga2000@yahoo.co.in

ABSTRACT
Acetylcholine (ACh), the first neurotransmitter to be identified, regulate the activities of central and peripheral functions through interactions with muscarinic receptors. Changes in muscarinic acetylcholine receptor (mAChR) have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). Previous reports from our laboratory on streptozotocin (STZ) induced diabetic rats showed down regulation of muscarinic M1 receptors in the brainstem, hypothalamus, cerebral cortex and pancreatic islets. In this study, we have investigated the changes of acetylcholine esterase (AChE) enzyme activity, total muscarinic and muscarinic M1 receptor binding and gene expression in the corpus striatum of STZ--diabetic rats and the insulin treated diabetic rats. The striatum, a neuronal nucleus intimately involved in motor behaviour, is one of the brain regions with the highest acetylcholine content. ACh has complex and clinically important actions in the striatum that are mediated predominantly by muscarinic receptors. We observed that insulin treatment brought back the decreased maximal velocity (Vmax) of acetylcholine esterase in the corpus striatum during diabetes to near control state. In diabetic rats there was a decrease in maximal number (Bmax) and affinity (Kd) of total muscarinic receptors whereas muscarinic M1 receptors were increased with decrease in affinity in diabetic rats. We observed that, in all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR experiment confirmed the increase in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. These results suggest the diabetes-induced changes of the cholinergic activity in the corpus striatum and the regulatory role of insulin on binding parameters and gene expression of total and muscarinic M1 receptors.

Show MeSH

Related in: MedlinePlus

Representative graph showing Real-Time amplification of muscarinic M1 mRNA from the corpus striatum of control, diabetic and insulin treated diabetic rats. Control, Diabetic, Insulin treated diabetic rats. The ΔΔCT method of relative quantification was used to determine the fold change in expression. This was done by first normalizing the resulting threshold cycle (CT) values of the target mRNAs to the CT values of the internal control β-actin in the same samples (ΔCT = CTTarget – CT β-actin). It was further normalize with the control (ΔΔCT = ΔCT – CTControl). The fold change in expression was then obtained (2-ΔΔCT). The graph was plotted using log 2-ΔΔCT. Values are mean ± S.D of 4–6 separate experiments. Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔCT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2682793&req=5

Figure 5: Representative graph showing Real-Time amplification of muscarinic M1 mRNA from the corpus striatum of control, diabetic and insulin treated diabetic rats. Control, Diabetic, Insulin treated diabetic rats. The ΔΔCT method of relative quantification was used to determine the fold change in expression. This was done by first normalizing the resulting threshold cycle (CT) values of the target mRNAs to the CT values of the internal control β-actin in the same samples (ΔCT = CTTarget – CT β-actin). It was further normalize with the control (ΔΔCT = ΔCT – CTControl). The fold change in expression was then obtained (2-ΔΔCT). The graph was plotted using log 2-ΔΔCT. Values are mean ± S.D of 4–6 separate experiments. Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔCT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator.

Mentions: Real Time-PCR analysis showed that the muscarinic M1 receptor gene expression was increased significantly (p < 0.01) in diabetic condition and it reversed to near control value in insulin treated diabetic rats (Fig 5 & Table 7).


Enhanced muscarinic M1 receptor gene expression in the corpus striatum of streptozotocin-induced diabetic rats.

Gireesh G, Kumar TP, Mathew J, Paulose C - J. Biomed. Sci. (2009)

Representative graph showing Real-Time amplification of muscarinic M1 mRNA from the corpus striatum of control, diabetic and insulin treated diabetic rats. Control, Diabetic, Insulin treated diabetic rats. The ΔΔCT method of relative quantification was used to determine the fold change in expression. This was done by first normalizing the resulting threshold cycle (CT) values of the target mRNAs to the CT values of the internal control β-actin in the same samples (ΔCT = CTTarget – CT β-actin). It was further normalize with the control (ΔΔCT = ΔCT – CTControl). The fold change in expression was then obtained (2-ΔΔCT). The graph was plotted using log 2-ΔΔCT. Values are mean ± S.D of 4–6 separate experiments. Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔCT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2682793&req=5

Figure 5: Representative graph showing Real-Time amplification of muscarinic M1 mRNA from the corpus striatum of control, diabetic and insulin treated diabetic rats. Control, Diabetic, Insulin treated diabetic rats. The ΔΔCT method of relative quantification was used to determine the fold change in expression. This was done by first normalizing the resulting threshold cycle (CT) values of the target mRNAs to the CT values of the internal control β-actin in the same samples (ΔCT = CTTarget – CT β-actin). It was further normalize with the control (ΔΔCT = ΔCT – CTControl). The fold change in expression was then obtained (2-ΔΔCT). The graph was plotted using log 2-ΔΔCT. Values are mean ± S.D of 4–6 separate experiments. Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔCT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator.
Mentions: Real Time-PCR analysis showed that the muscarinic M1 receptor gene expression was increased significantly (p < 0.01) in diabetic condition and it reversed to near control value in insulin treated diabetic rats (Fig 5 & Table 7).

Bottom Line: We observed that insulin treatment brought back the decreased maximal velocity (Vmax) of acetylcholine esterase in the corpus striatum during diabetes to near control state.Real-time PCR experiment confirmed the increase in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment.These results suggest the diabetes-induced changes of the cholinergic activity in the corpus striatum and the regulatory role of insulin on binding parameters and gene expression of total and muscarinic M1 receptors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Neurobiology and Cell Biology Unit, Centre for Neuroscience, Cochin University of Science and Technology, Cochin-682022, Kerala, India. giri_ganga2000@yahoo.co.in

ABSTRACT
Acetylcholine (ACh), the first neurotransmitter to be identified, regulate the activities of central and peripheral functions through interactions with muscarinic receptors. Changes in muscarinic acetylcholine receptor (mAChR) have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). Previous reports from our laboratory on streptozotocin (STZ) induced diabetic rats showed down regulation of muscarinic M1 receptors in the brainstem, hypothalamus, cerebral cortex and pancreatic islets. In this study, we have investigated the changes of acetylcholine esterase (AChE) enzyme activity, total muscarinic and muscarinic M1 receptor binding and gene expression in the corpus striatum of STZ--diabetic rats and the insulin treated diabetic rats. The striatum, a neuronal nucleus intimately involved in motor behaviour, is one of the brain regions with the highest acetylcholine content. ACh has complex and clinically important actions in the striatum that are mediated predominantly by muscarinic receptors. We observed that insulin treatment brought back the decreased maximal velocity (Vmax) of acetylcholine esterase in the corpus striatum during diabetes to near control state. In diabetic rats there was a decrease in maximal number (Bmax) and affinity (Kd) of total muscarinic receptors whereas muscarinic M1 receptors were increased with decrease in affinity in diabetic rats. We observed that, in all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR experiment confirmed the increase in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. These results suggest the diabetes-induced changes of the cholinergic activity in the corpus striatum and the regulatory role of insulin on binding parameters and gene expression of total and muscarinic M1 receptors.

Show MeSH
Related in: MedlinePlus