T. Peeyush Kumar

Curcumin modulates dopaminergic receptor, CREB and phospholipase c gene expression in the cerebral cortex and cerebellum of streptozotocin induced diabetic rats

Curcumin, an active principle component in rhizome of Curcuma longa, has proved its merit for diabetes through its anti-oxidative and anti-inflammatory properties. This study aims at evaluating the effect of curcumin in modulating the altered dopaminergic receptors, CREB and phospholipase C in the cerebral cortex and cerebellum of STZ induced diabetic rats. Radioreceptor binding assays and gene expression was done in the cerebral cortex and cerebellum of male Wistar rats using specific ligands and probes. Total dopaminergic receptor binding parameter, Bmax showed an increase in cerebral cortex and decrease in the cerebellum of diabetic rats. Gene expression studies using real time PCR showed an increased expression of dopamine D1 and D2 receptor in the cerebral cortex of diabetic rats. In cerebellum dopamine D1 receptor was down regulated and D2 receptor showed an up regulation. Transcription factor CREB and phospholipase C showed a significant down regulation in cerebral cortex and cerebellum of diabetic rats. We report that curcumin supplementation reduces diabetes induced alteration of dopamine D1, D2 receptors, transcription factor CREB and phospholipase C to near control. Our results indicate that curcumin has a potential to regulate diabetes induced malfunctions of dopaminergic signalling, CREB and Phospholipase C expression in cerebral cortex and cerebellum and thereby improving the cognitive and emotional functions associated with these regions. Furthermore, in line with these studies an interaction between curcumin and dopaminergic receptors, CREB and phospholipase C is suggested, which attenuates the cortical and cerebellar dysfunction in diabetes. These results suggest that curcumin holds promise as an agent to prevent or treat CNS complications in diabetes.

Effect of vitamin D3 in reducing metabolic and oxidative stress in the liver of streptozotocin-induced diabetic rats.

Diabetes mellitus is a growing health problem worldwide and is associated with severe liver complications. The aim of the present study is to analyse the status of metabolic and free-radical-scavenging enzymes and second messengers in the liver of streptozotocin (STZ)-induced diabetic rats, and to determine the hepatoprotective role of vitamin D(3). All studies were performed using the liver of adult male Wistar rats. Gene expression studies were carried out using real-time PCR with specific probes. Second messenger levels were determined using (3)H-labelled Biotrak assay kits, and glucose uptake assay with D-[(14)C]glucose. The present results show that there was a decrease in hepatic glucose uptake, malate dehydrogenase activity, glycogen content, inositol triphosphate (IP(3)) and cyclic GMP levels, and superoxide dismutase, glutathione peroxidase, phospholipase C, cyclic AMP-responsive element-binding protein, vitamin D receptor (VDR) and insulin receptor (INSR) gene expression in the diabetic rats when compared with the controls (all P < 0·05), while cyclic AMP levels and GLUT2 expression were increased (P < 0·05). Treatment of the diabetic rats with vitamin D(3) and insulin reversed the altered parameters to near control values. In conclusion, the data suggest a novel role of vitamin D(3) in restoring impaired liver metabolism in STZ-induced diabetic rats by regulating glucose uptake, storage and metabolism. We demonstrated that the restoring effect of vitamin D(3) is mediated through VDR modulation, thereby improving signal transduction and controlling free radicals in the liver of diabetic rats. These data suggest a potential role for vitamin D(3) in the treatment of diabetes-associated hepatic complications.

Enhanced NMDAR1, NMDA2B and mGlu5 receptors gene expression in the cerebellum of insulin induced hypoglycaemic and streptozotocin induced diabetic rats.

Glucose homeostasis in humans is an important factor for the functioning of the nervous system. A decrease in glucose content below a minimal level or hypoglycemia is dangerous for cells of the central and peripheral nerve system. In the present study we showed the effects of insulin induced hypoglycaemia and streptozotocin induced diabetes on the cerebellar glutamate receptor subunits and glutamate transporter. Cerebellar dysfunction is associated with seizure generation, motor deficits and memory impairment. We found an up regulation in NMDA receptor number and gene expression of N-methyl-d-aspartic acid (NMDA(R1)), NMDA(2B), metabotrophic glutamate 5 (mGlu(5)) glutamate receptor subunits in experimental rats. The glutamate content was shown to be increased with decreased glutamate aspartate transporter (GLAST) gene expressions indicating lower reuptake of glutamate. The enhanced gene expression of NMDA(R1), NMDA(2B), mGlu(5) glutamate receptors were confirmed by immunohistochemistry studies. At the second messenger level, the IP3 content and IP3 receptors were enhanced in the cerebellum of both hypoglycaemic and diabetic rats increased. The present study showed that the enhanced glutamate content activates NMDA receptors, increasing the inositol triphosphate (IP3) content which mediates Ca(2+) overload in cells causing cell damage and neurodegeneration. Our results also showed that the enhanced glutamate receptor activity were more prominent in hypoglycaemic group compared to diabetic group. Further the neurodegeneration by the up regulation of glutamate receptor activity causing motor dysfunction was demonstrated by the Rotarod test. Thus our results suggest that enhanced NMDA receptor mediated neurodegeneration affect the motor learning and memory ability of an individual.

Decreased GABA Receptor Binding in the Cerebral Cortex of Insulin Induced Hypoglycemic and Streptozotocin Induced Diabetic Rats

Hypoglycemia is the major problem to blood glucose homeostasis in treatment of diabetes and is associated with severe irreversible consequences including seizures, coma and death. GABAergic inhibitory function in the cerebral cortex plays an important role in controlling the excitability and responsiveness of cortical neurons. Present study analysed effects of insulin induced hypoglycemia and streptozotocin induced diabetes on the cortical GABA receptor binding, GABAAά1, GABAB receptor subtype expression, GAD and GLUT3 expression. Diabetic rats showed decreased [3H] GABA binding in the cerebral cortex compared to control while hypoglycemia exacerbated the decrease. GABA receptor subunits; GABAAά1, GABAB and GAD expression significantly decreased in diabetic rats whereas hypoglycemia significanly decreased the expression compared to diabetic. GLUT3 expression significantly up regulated during both hypo and hyperglycemia. Our results showed that hypoglycemia and hyperglycemia decreased GABAergic neuroprotective function in the cerebral cortex, which account for the increased vulnerability of cerebral cortex to subsequent neuronal damage during hypo/hyperglycemia.

Role of curcumin in the prevention of cholinergic mediated cortical dysfunctions in streptozotocin-induced diabetic rats

Diabetes exacerbates neuronal injury mediated through neurotransmitters deregulation in cerebral cortex. Our study analyzed the neuroprotective effect of curcumin to prevent cortical dysfunction associated with diabetes. Our study revealed decreased gene expression of muscarinic M1, insulin receptor, SOD, choline acetyl transferase and increased gene expression of muscarinic M3, α7-nicotinic acetylcholine receptor, acetylcholine esterase and GLUT3 in cerebral cortex of diabetic rats. Curcumin and insulin treatment reversed this altered parameters to near control. Immunohistochemistry studies of muscarinic M1 and M3 confirmed the gene expression at protein level. Decreased novel arm entry of diabetic rats in Y-maze test, improved in treatment group. These results suggest that cholinergic dysfunction, impaired glucose transport and oxidative stress contributes to learning and memory deficits in diabetes and further suggest that antioxidant curcumin has potential therapeutic role in preventing and/or delaying the diabetic complications associated with brain.

Behavioral deficit and decreased GABA receptor functional regulation in the cerebellum of epileptic rats: Effect of Bacopa monnieri and bacoside A

In the present study, the effects of Bacopa monnieri and its active component, bacoside A, on motor deficit and alterations of GABA receptor functional regulation in the cerebellum of epileptic rats were investigated. Scatchard analysis of [(3)H]GABA and [(3)H]bicuculline in the cerebellum of epileptic rats revealed a significant decrease in B(max) compared with control. Real-time polymerase chain reaction amplification of GABA(A) receptor subunits-GABA(Aalpha1), GABA(Aalpha5,) and GABA(Adelta)-was downregulated (P<0.001) in the cerebellum of epileptic rats compared with control rats. Epileptic rats exhibit deficits in radial arm and Y-maze performance. Treatment with B. monnieri and bacoside A reversed these changes to near-control levels. Our results suggest that changes in GABAergic activity, motor learning, and memory deficit are induced by the occurrence of repetitive seizures. Treatment with B. monnieri and bacoside A prevents the occurrence of seizures thereby reducing the impairment of GABAergic activity, motor learning, and memory deficit.

Decreased GABAA Receptors Functional Regulation in the Cerebral Cortex and Brainstem of Hypoxic Neonatal Rats: Effect of Glucose and Oxygen Supplementation

Hypoxia in neonates can lead to biochemical and molecular alterations mediated through changes in neurotransmitters resulting in permanent damage to brain. In this study, we evaluated the changes in the receptor status of GABAA in the cerebral cortex and brainstem of hypoxic neonatal rats and hypoxic rats supplemented with glucose and oxygen using binding assays and gene expression of GABAAα1 and GABAAγ5. In the cerebral cortex and brainstem of hypoxic neonatal rats, a significant decrease in GABAA receptors was observed, which accounts for the respiratory inhibition. Hypoxic rats supplemented with glucose alone and with glucose and oxygen showed a reversal of the GABAA receptors, andGABAAα1 and GABAAγ5 gene expression to control. Glucose acts as an immediate energy source thereby reducing the ATP-depletion-induced increase in GABA and oxygenation, which helps in encountering anoxia. Resuscitation with oxygen alone was less effective in reversing the receptor alterations. Thus, the results of this study suggest that reduction in the GABAA receptors functional regulation during hypoxia plays an important role in mediating the brain damage. Glucose alone and glucose and oxygen supplementation to hypoxic neonatal rats helps in protecting the brain from severe hypoxic damage.

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

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.

Acetylcholine and muscarinic receptor function in cerebral cortex of diabetic young and old male Wistar rats and the role of muscarinic receptors in calcium release from pancreatic islets

We investigated acetylcholine esterase (AChE) activity, acetylcholine and muscarinic M1, M3 receptors kinetics in the cerebral cortex of young and old streptozotocin induced and insulin treated diabetic rats. The role of muscarinic receptors in intracellular calcium release from pancreatic islets was studied in vitro. Wistar rats of 7 and 90-weeks old were used. All studies were done in cerebral cortex. AChE assay was done by spectrophotometric method. Radioreceptor binding assays were done for Acetylcholine, Muscarinic M1 and M3 receptors using specific ligands. Calcium imaging was done using fluo4-AM in pancreatic cells. Ninety-weeks old control rats showed significantly decreased Vmax and increased Km for AChE compared to 7-weeks old control rats. An increased Vmax observed in both 7 and 90-weeks old diabetic groups with significant decrease in Km. Scatchard analysis using specific agonists showed significant decrease in the Bmax and Kd of acetylcholine and muscarinic M1 receptors in 90-weeks old control rats compared to 7-weeks old control. Binding studies for M3 receptors showed no significant change compared to 7-weeks old control. Acetylcholine, muscarinic M1 and M3 receptor number significantly increased in 90-weeks old diabetic rat groups compared to their respective controls. Insulin treatment significantly reversed the binding parameters to near control compared to diabetic group. In vitro studies showed that acetylcholine through muscarinic M1 and M3 receptors’ stimulated calcium release from the pancreatic islets. Thus our studies suggest that Insulin signaling play an important part in differentially regulating pancreatic cholinergic activity, and the diabetes mediated cortical dysfunctions with age.